2007. 9 Science of Plant and Animal Production United...

The Establishment of in vitro Culture and Genetic Transformation of the Wheat Glu-1Dx5 Gene to Rice Plants by Gene Gun Bombardment

2007. 9

Science of Plant and Animal Production United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology

Nono Carsono

This research was conducted from October 2004 to May 2007 at Utsunomiya University, Japan. The work carried out during the course of this research has resulted in the following publications: 1) Nono Carsono and Tomohiko Yoshida (2006)

Identification of Callus Induction Potential of 15 Indonesian Rice Genotypes Plant Production Science 9 (1) : 65-70.

2) Nono Carsono and Tomohiko Yoshida (2006) Plant Regeneration Capacity of Calluses Derived from Mature Seed of Five Indonesian Rice Genotypes Plant Production Science 9 (1) : 71-77.

3) Nono Carsono and Tomohiko Yoshida (2007) Variation in Spikelet-Related T raits of Rice Plants Regenerated from Mature Seed-derived Callus Culture Plant Production Science 10 (1) : 86-90.

4) Nono Carsono and Tomohiko Yoshida Transient Expression of Green Fluorescent Protein Gene in Rice Calluses: Optimization of Parameters for Helios Gene Gun Bombardment Reviewed in Plant Production Science

5) Nono Carsono and Tomohiko Yoshida Genetic T ransformation of the Wheat Glu-1Dx5 Gene into Rice cv. Fatmawati In preparation

Contents

General Summary 1 Summary 2

I Introduction

5

II Establishment of in vitro Culture for the Success of Genetic Transformation 2.1 Callus Induction Potential of 15 Indonesian Rice Genotypes 2.2 Plant Regeneration Capacity of Calluses Derived from Mature Seeds of Five

Indonesian Rice Genotypes

13 26

III Assessment of Somaclonal Variants from Primary Callus-derived Plants Variation in the Spikelet-related Traits of Rice Plants Regenerated from Mature Seed-derived Callus Culture

42

IV Genetic Transformation of Rice Plants Using Helios Gene Gun for Developing Transgenic Rice Lines 4.1 Transient Expression of Green Fluorescent Protein in Rice Calluses:

Optimization of Parameters for Helios Gene Gun Bombardment 4.2 Genetic Transformation of the Wheat Glu-1Dx5 Gene to Rice cv. Fatmawati

53 70

V Gene Transfer to Rice Mediated by Agrobacterium tumefaciens Transient Expression of Green Fluorescent Protein in Rice Calluses of cvs. Fatmawati and Nipponbare

81

VI General Discussion

91

References Acknowledgment

96 106

1

General Summary

The work carried out and presented in this thesis was mainly aimed to establish a suitable in vitro

culture system as well as to introduce the wheat Glu-1Dx5 gene encoding a high molecular weight glutenin

subunit 1Dx5 into rice genome using HeliosTM gene gun bombardment.

Reliable phenotypic assessment on callus performance was able to identify promising genotypes

with high quality callus production. Genotype, medium and explant are considered to be essential factors

for inducing high quality calluses. Five selected Indonesian rice genotypes were capable in regenerating

whole green plants. Genotypes Fatmawati and BP-140 consistently performed best in callus subculture as

well as in plant regeneration. Culture media D1 and NB5 were the most suitable media for callus subculture

and plant regeneration, respectively. Phenotypic evaluation in the spikelet-related traits of primary callus-

regenerated plants (T 0) showed that the occurrence of somaclonal variants varied with the genotype. The

spikelet fertility of the regenerated rice plants was not significantly lower than that of the initial plants except

in Ciapus and BP-140.

Optimization of parameters of HeliosTM gene gun for rice callus bombardment based on synthetic

green fluorescent protein (sgfp) expression has been successfully conducted. Parameters found to be the

most favorable conditions were as follows: 250 psi helium pressure, 0.6 m gold particle size, 0.25 mg gold

particles per shot, and 1.5 g plasmid-DNA per shot.

The introduction of the wheat Glu-1Dx5 gene in combination with either selectable marker bar or hpt

gene into rice cv. Fatmawati has been performed well. PCR analysis confirmed the presence of transgene

in the genome of some transgenic plants. These plants will be incorporated into breeding program for

further assessment of their benefits.

The significant differences in transient expression of sgfp of two genotypes mediated by

Agrobacterium were found with regard to the osmotic treatment (0.4 M mannitol), solid-subcultured callus,

and 10 min. air drying. The highest sgfp expression was achieved in Nipponbare callus treated with 10 min

air drying. The level of green fluorescent spots was higher in Nipponbare than that in Fatmawati.

2

Summary

Rice is one of the important food crops that serves energy for human life, thus attempts on

genetic improvement of rice crop would have a significant contribution to the benefits of humankind. This thesis was mainly devoted to introduce the wheat Glu-1Dx5 gene encoding a

high molecular weight glutenin subunit 1Dx5 which is one of the major determinants of bread-making quality in bread wheat (Triticum aesticum L.) into rice genome. Steps in establishing callus

induction, proliferation and in vitro plant regeneration, assessing somaclonal variants of primary callus-regenerated plants, optimizing the parameters of HeliosTM gene gun device and

transforming the rice plants with this valuable gene accompanied with selectable marker bar gene conferring resistance to herbicide bialaphos or hpt gene harboring resistance to hygromycin B

using Helios gene gun were also presented. Finally, transient expression of synthetic green fluorescent protein (sgfp) mediated by Agrobacterium in two cvs. Fatmawati and Nipponbare was

described. This present study has highlighted some interesting findings as described bellow:

1. Callus induction is one of the substantial steps for selecting the suitability of genotypes for

tissue culture-based research and for plant improvement program, particularly for genetic

transformation. Genotype x medium x explant interaction effect was found for inducing high quality callus in terms of white/cream/yellow callus with an organized structure (callus type I

and II) and for callus browning, but not for callus induction ability and diameter of callus. Genotypes significantly differed in inducing high quality of calluses depending on medium and

explant used. Four indica types, Fatmawati, Ciapus, BP-23 and BP-360-3, had callus quality-related traits similar to those of Nipponbare. Culturing seed explant on MS was more suitable

for callus induction than either root explant on MS or both explants on CI medium.

2. Establishment of a suitable system for multiplication and plant regeneration of rice calluses is

a prerequisite for the success of genetic transformation using callus as the target tissue. The experiment with five Indonesian rice genotypes showed that callus-growth capacity was

significantly affected by genotype and callus-proliferation capacity by medium. The interaction effect between genotype and medium was found on both shoot-regeneration and plantlet-

regeneration capacities. However for green plant-regeneration capacity, it was affected

independently by genotype and medium. Culture media D1 and NB5 were the most suitable media for callus subculture and plant regeneration, respectively. Genotypes Fatmawati and

BP-140 consistently performed best in the callus subculture and plant regeneration.

3

3. Callus is an excellent source for in vitro plant regeneration, but plants regenerated from callus

sometimes show phenotypic and genotypic variation from the initial plants. Phenotypic

variation in the spikelet-related traits of the callus-regenerated plants was not always in a reduction in their mean value. For instance, panicle length, spikelet number and fertile

spikelet number of Indonesian rice genotypes Ciapus and BP-23 in the regenerated plants were significantly greater than those of the seed-grown plants. The spikelet fertility of the

regenerated rice plants was not significantly lower than that of the initial plants, except in Ciapus and BP-140. The occurrence of somaclonal variants varied with the genotype. Ciapus,

BP-23 and BP-140, which induce many somaclonal variants, are suggested to be valuable for genetic, breeding or functional genomic studies, while the opposite, Fatmawati could be used

for genetic transformation study.

4. An optimized condition for particle bombardment is necessary for efficient genetic

transformation. The parameters for HeliosTM gene gun, another device for cell transformation, were optimized based on transient expression of synthetic green fluorescent protein (sgfp) in

rice calluses of indica cv. Fatmawati and japonica cv. Nipponbare. Parameters found to be the

most favorable conditions for transient expression of sgfp in rice callus cells were as follows:

250 psi helium pressure, 0.6 m gold particle size, 0.25 mg gold particles per shot, and 1.5

g plasmid-DNA per shot. Desiccation of callus cells for eight min. was found also

appropriate. The level of transient sgfp expression was not significantly influenced by the pre-culture for 4 to 12 days before bombardment or by callus age between 10 and 33-weeks old

in Fatmawati. Optimized parameters for this particular device should improve the transient

expression, thus enabling stable expression of introduced genes via HeliosTM gene gun using callus as a target tissue.

5. The wheat Glu-1Dx5 gene encoding a high molecular weight (HMW) glutenin subunit 1Dx5 from bread wheat, Triticum aesticum L. and either bar gene conferring resistance to herbicide

bialaphos or hpt gene conferring resistance to hygromycin B was co-transformed to rice callus cells of cv. Fatmawati using Helios gene gun device. Nine plants regenerated from bialaphos-

containing medium and ten plants from hygromycin-containing medium were molecularly characterized. By means of PCR analysis, the Glu-1Dx5 gene had been integrated into some

transgenic rice plants. These plants will be incorporated into breeding program for further assessment of their benefits.

4

6. Transient expression of synthetic green fluorescent protein (sgfp) gene mediated by

Agrobacterium is rapid and useful approach for visual monitoring the genetic transformation

event in transformed cells/tissues of examined genotype. The significant differences in transient expression of two genotypes were found with regard to the osmotic treatment (0.4 M

mannitol), solid-subcultured callus, and 10 min. air drying. While, no significant differences in sgfp expression were observed in two genotypes on without air-drying and 5 min. air-drying of

calluses prior immersed in Agrobacterium suspension. Surprisingly, the sgfp expression could not be detected in suspension-subcultured callus of both cultivars. The highest sgfp

expression was achieved in Nipponbare callus treated with 10 min. air drying. The level of green fluorescent spots was higher in Nipponbare than that in Fatmawati, however, plants

regenerated from Fatmawati were considerably comparable with those of Nipponbare. Seventeen and 16 transgenic rice plants expressing sgfp transgene were obtained from

Nipponbare and Fatmawati, respectively.

5

Chapter 1

Introduction

6

Rice (Oryza sativa L.) is a major staple food and one of the most important crops worldwide.

More than half of the world’s population depends on rice for its major daily source of energy and

protein. Twenty three percent of the total calories consumed globally are supplied by rice (Khus,

2001). There are two cultivated and twenty–one wild species of genus Oryza. O. sativa, the Asian

cultivated rice is grown all over the world. The African cultivated rice, O. glaberrima is planted in

small scale of West Africa (Khush, 1997). The majority of Asian rice cultivars can be classified as

either japonica or indica, based on agro morphological traits (Oka and Morishima, 1997) and

supported by a number of studies based on the use of molecular marker, such as isozymes

(Glaszmann, 1987), restriction fragment length polymorphisms (RFLP; Wang and Tanksley, 1989),

amplicon length polymorphisms (ALP; Xu et al., 1998), inter-simple sequence repeat (ISSR; Blair

et al., 1999) and short interspersed elements (SINE; Cheng et al., 2003). These studies show a

clear genetic differentiation between two subspecies groups, indica and japonica, arose from two

independent domestication events in Asia.

Currently, rice is recognized as a model plant for the study of cereals genomes (Shimamoto

and Kyozuka, 2002), due to its small genome size (~430 Mb), the availability of complete genome

sequence (Sasaki et al., 2002), the largeness of public germplasm collection and the development

of several key genomic mapping resources (McCouch et al., 2002) and synteny with other cereals

crops (Sasaki et al., 2002).

At present, with the advent of recombinant DNA and genetic transformation techniques, it is

now possible to transfer genes from other organisms into the plant genome. The genetic

transformation has offered many advantages for basic research and plant improvement program.

Those advantages are to find unknown function of the genes/cDNA sequence (Pereira, 2000), to

introduce new gene pool (Gepts, 2002) and many desirable genes in a single event, to improve

the crop with more precisely and in a relatively short time. This approach has been applied to

complement traditional plant breeding methods in rice genetic improvement. Recently, it has

7

become a valuable approach for introduction of useful genes for rice quality improvement (Anai et

al., 2003; Datta et al., 2003), molecular farming (Takagi et al., 2005; Yang et al., 2007), transposon

tagging (Greco et al., 2001), developing rice plant resistant to biotic stress: insect pests (Tu et al.

1998; Shu et al., 2000), fungi (Patkar and Chattoo, 2006), bacteria (Patkar and Chattoo, 2006),

virus (Sivamani et al., 1999) and rice plant tolerant to abiotic stress: drought (Sawahel, 2003),

salinity (Oh et al., 2005), cold (Takesawa et al., 2002; Ozawa et al.,2006), and among others. It is

clear that for many desirable traits from unrelated plants or other organisms, genetic

transformation is the only source of variation for breeding programs. However, the application of

genetic transformation to rice breeding requires efficient and reproducible in vitro culture systems

that allow production of high number of regenerative tissues and recovery of fertile transgenic rice

plants.

Callus, comprising mainly masses of undifferentiated cells, is a good starting material for in

vitro manipulation. Moreover, calluses, induced from scutellar tissue of mature seeds, are the

excellent source of cells for in vitro regeneration as stated in this thesis, and even for the

production of transgenic rice (Hiei et al., 1994; Rashid et al., 1996). Genotypes, medium

composition and explant sources are often considered to be essential factors in callus induction as

well as in plant regeneration. Therefore, identification of high quality callus with regard to its

appearance, such as good-looking, healthy/no browning and actively growing, which is supposed

to be embryogenic calluses induced by some rice genotypes, is one of the most essential factors

for efficient plant regeneration.

Plant regeneration has been known as a major bottleneck on the successful application of

genetic transformation of valuable genes into rice genome. Rice genotypes display a wide range in

regeneration capacity depending on their genetic background and their interaction with the culture

media (Khanna and Raina, 1998; Lee et al., 2002; Lin and Zhang, 2005). Many leading varieties,

such as Koshihikari in Japan and IR-64 in tropical country, show low regeneration capacities

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(Nishimura et al., 2005), resulting in serious obstacle to efficient production of transgenic plants. In

order to obtain high totipotency (i.e. capable of giving rise of a whole plant) and to facilitate

recovery of stable and fertile transformants, appropriate genotype and medium culture should

therefore be examined for the establishment of regeneration system.

Based on many earlier experiments, tissue culture may possibly induce variations (Larkin

and Scowcroft, 1981; Karp, 1995), known as somaclonal variations which could be very valuable

for rice breeding. However, the occurrence of somaclonal variants hampers the use of in vitro

culture for the development of transgenic rice plants since somaclonal variants will complicate the

evaluation of the effects of inserted genes. In addition, they have potential to alter agronomic

characteristics of rice, such as, reducing the spikelet fertility, thus constitute a major problem

especially for rice that generatively propagated by seeds. A preliminary assessment on the

possibility of somaclonal variants from primary callus-regenerated plants is therefore considered

necessary for evaluating the occurrence of tissue culture-derived variations in order to develop

transgenic rice plants expressing the genes of particular interest without altering their phenotypes.

The introduction of genes into plant genome by means of genetic engineering is becoming

an accepted technique in plant breeding. Genes derived from unrelated plant species and even

other kingdoms (bacteria, fungi, animals), which would otherwise be inaccessible for plant breeder,

can be introduced into plant breeding programs by this technology (De Block, 1993). The first

transgenic rice plants were obtained by direct DNA uptake from protoplast (Toriyama et al., 1988).

Shortly thereafter, transformation of several indica and japonica either by particle bombardment

(Christou et al., 1991) or by Agrobacterium-mediated transformation (Hiei et al., 1994; Rashid et

al., 1996; Kumria et al., 2001) was reported. Currently, production of transgenic rice plants become

routine experiment in many laboratories in the world with various purposes. Even more, transgenic

crops are grown at present on more than 40 million hectares in 13 countries around the world.

Transgenic technology promises the great improvement for food and non food with regard to

9

quantity, quality, diversity, nutrient and environmental friendly products (Dunwell, 2002).

Genetic transformation mediated by particle bombardment-based technology offers the

means of selective introducing of valuable genes to our source of energy, rice. Particle

bombardment (bombarding nucleic acids-coated particles into the target cells) is now emerging as

a new tool for delivering nucleic acids into target organisms. This innovative technique proved to

work quite efficiently regardless of the genotype used, and is of particular utility for the

transformation of recalcitrant species.

Genetic transformation of rice using particle bombardment has offered some attrac tive

objectives. Those are as following: to obtain a large number of transformed plants (Dai et al.,

2001), to get high transgene copy number (Dai et al., 2001; Shou et al., 2004), to introduce

simultaneous multiple genes which are valuable for designing the metabolic pathway (Maqbool

and Christou, 1999; Agrawal et al., 2005), plastid or chloroplast transformation (Daniell et al.,

2001; Kanamoto et al., 2006), and to introduce the gene cassettes (promoter-coding sequence-

terminator) without the sequence of vector (Breitler et al., 2002; Agrawal et al., 2005). Moreover,

no biological constraints or host limitations, diverse cell types can be targeted efficiently and

capable for delivery of high molecular weight DNA are other advantages of using particle

bombardment (Alpeter et al., 2005). However, particle bombardment is still not devoid of all the

disadvantages connected to direct DNA transfer, like multiple DNA integrations promoting

rearrangements and transgene silencing (Dai et al., 2001; Taylor and Fauquet, 2002).

Particle bombardment has been fruitfully applied for genetic enhancement of rice crop.

However, to date, most bombardment experiments for rice transformation utilize the PDS-1000/He

Biolistic Particle Delivery System (for example, Bec et al., 1998; Ghosh-Biswas et al., 1998; Jiang

et al., 2000; Martinez-Trujillo et al., 2003) and currently there is no protocol for rice callus cells

bombarded using the HeliosTM gene gun device (Bio-Rad Laboratories, USA) and furthermore,

transgenic plants obtained using this particular tool have not been reported yet. Moreover, some

10

physical and biological parameters of HeliosTM gene gun would affect the efficiency of

transformation. Therefore, optimization of parameters for HeliosTM gene gun based on transient

expression of green fluorescent protein (gfp) is considered necessary for efficient genetic

transformation.

The use of gfp as a reporter gene is much valuable for confirming the expression of the

inserted gene or transformation event in transformed cells or tissues. In plant science, gfp has

been extensively applied for testing the efficacy of the promoter (Newell et al. 2003), detection of

virus activity (Oparka et al. 1997), protein localization (Hibberd et al. 1998; Jang et al. 1999) and

optimization of transformation methods (Tee and Maziah, 2005).

Other essential requirement for genetic transformation technology is the utilization of

selectable marker genes, such as hpt (hygromycin phosphotransfrease gene) conferring

resistance to Hygromycin B and bar (phosphinothricin N-acetyl transferase gene) harboring

resistance to herbicide bialaphos. They could facilitate the selection and the recovery of

transformed tissues. This is because the chances of recovering transgenic lines without selection

are extremely low due to only a very small proportion of cells are transformed in most experiments.

The selectable marker gene is usually co-transformed with a gene of interest. Once the transgenic

plant has been generated, characterized and bred through classical genetic crosses, the

selectable marker gene generally no longer serves an essential purpose (Miki and McHugh, 2004).

Genetic transformation of rice has been successfully employed for quality improvement

program. Such successful examples are rice with high iron content (Goto et al., 1999), human-

lactoferrin (Nandi et al., 2002), low amylose content (Liu et al., 2005), and ß-carotene (Golden

Rice, Datta et al., 2003). Other improvement on the rice grain quality, especially on rice flour

quality, is of great relevance since Japan and Indonesia are rice producer countries in which rice is

abundant available and rice flour has been used for many food products. However, dough from

rice flour lacks extensibility and elasticity. A probable cause for this is that rice endosperm lacks the

11

proteins responsible for this trait. In contrast, dough made from wheat flour is elastic and

extensible making it suitable for many food products particularly for bread (Sangtong et al., 2002).

Wheat flour is different from other cereal flours, including rice, because it contains gluten

that gives it elasticity and extensity required for breadmaking quality in bread wheat (Triticum

aesticum L.; Payne et al., 1987; Barro et al. 1997). Gluten mainly consists of two types of seed

storage proteins, the glutenins and the gliadins. Glutenins are classified into high-molecular-weight

(HMW) subunits and low-molecular-weight (LMW) subunits. They are encoded by Glu-A1 loci, Glu-

B1 loci, Glu-D1 loci, located in the long arms of the chromosome of homoeologous group 1 (Payne

et al., 1987).

Genes encoding six HMW glutenins have been cloned (Anderson et al., 2002). The

availability of cloned HMW glutenin genes allows plant transformation approaches to altering HMW

glutenin content. Cloned HMW glutenin genes have been shown to be functional when introduced

into tobacco (Roberts et al., 1989), wheat (Alvarez et al., 2000), tritordeum (Rooke et al., 1999),

maize (Sangtong et al., 2002) and more recently into rye (Altpeter et al., 2004).

Although the HMW glutenins contribute only about 5% of the total protein in mature wheat

kernels (Shewry et al., 1989), the elasticity of wheat dough depends mainly on the HMW glutenins,

thus they are important determinants of bread-making quality (Payne et al., 1987). Moreover,

Payne et al. (1987) reported that most good bread-wheat cultivars contain the 1Dx5-1Dy10 HMW

glutenin combination. This suggests that bread-making quality is associated with the presence of

this combination of HMW glutenins. It would be useful to set out the experiment to determine

whether a wheat HMW glutenin gene could be used to develop rice with novel dough

characteristics which could possibly substitute the wheat flour.

The scope of the thesis

The scope of the research work described in this thesis was to establish a suitable in vitro

culture protocol and to develop transgenic rice lines with the wheat Glu-1Dx5 gene. This thesis

12

describes five major activities and one additional activity. Those are as following:

1. Study on callus induction potential of 15 Indonesian rice genotypes,

2. Establishment on plant regeneration system of mature seed-derived callus culture of five

Indonesian rice genotypes,

3. Preliminary assessment on the spikelet fertility of primary callus-regenerated rice plants,

4. Optimization of parameters for HeliosTM gene gun bombardment for rice calluses based on

transient expression of green fluorescent protein,

5. Genetic transformation of the wheat Glu-1Dx5 gene into rice cv. Fatmawati using HeliosTM

gene gun bombardment,

6. Transient expression of synthetic green fluorescent protein in calluses of cvs. Fatmawati and

Nipponbare mediated by Agrobacterium tumefaciens

13

Chapter 2

Establishment of in vitro Culture for the Success of Genetic Transformation

2.1. Callus Induction Potential of 15 Indonesian Rice Genotypes

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Abstract

Callus induction potential of 15 indica rice genotypes from Indonesia was examined in comparison

with that of the japonica rice Nipponbare. Callus was induced from embryos of mature seeds and root segments on MS and CI media. There was genotype x medium x explant interaction effect for

inducing white/cream/yellow callus with an organized structure (callus type I and II) and for callus browning, but not for callus induction ability and diameter of callus. Genotypes significantly differed

in inducing high quality of calluses depending on medium and explant used. Four indica types, Fatmawati, Ciapus, BP-23 and BP-360-3, had callus induction-related traits similar to those of

Nipponbare. These genotypes would be useful for tissue-culture based research and for crop improvement program, particularly for genetic transformation. Culturing seed explant on MS was

more suitable for callus induction than either root explant on MS or both explants on CI medium.

15

Introduction Callus induction is one of the substantial steps for selecting the suitability of genotype for

tissue-culture-based research and for plant improvement program, particularly for genetic

transformation. In rice, different callus types, i.e., type I, II, III and IV, can be induced (Visarada et al., 2002). Type I callus is white and cream colored compact organized callus, type II is yellow

organized callus, type III is yellow or brown unorganized callus, and the type IV is highly

unorganized white, yellow or brown callus. Type-I and-II calluses are embryogenic and can be induced from tissues of various organs such as immature seeds (Masuda et al., 1989), immature

embryos (Koetje et al., 1989), and roots (Abe and Futsuhara, 1985; Hoque and Mansfield, 2004). The type-III callus is dark and necrotic. In general, immature embryos and meristematic tissues,

having undifferentiated cells, are suitable for callus induction and plant regeneration than mature tissues (Morrish et al., 1987). However, such explants are available only in a restricted period of

the growth cycle in the rice plant (Hoque and Mansfield, 2004), and to obtain such explants all year round, we have to grow the plants in a greenhouse. However, embryos of mature seeds are

available throughout the year, and are more suitable for rice callus culture. The embryogenic calluses induced by culture of mature seeds are effectively used for genetic transformation by

particle bombardment (Jiang et al., 2000) or Agrobacterium-mediated transformation (Kumria et al., 2001). The aseptic culture of root explants is also useful since it is relatively easy to provide in any

season.

In the in vitro culture of rice, a significant difference in callus induction has been found among the different genotypes of indica rice (Abe and Futsuhara, 1986; Peng and Hodges, 1989;

Seraj et al., 1997) and japonica rice (Yoshida and Oosato, 1998; Ogawa et al., 1999). Indica rice cultivars generally show less induction of callus formation than japonica (Abe and Futsuhara,

1986). Not only callus production, but also somatic embryogenesis and subsequent plant regeneration (Chu and Croughan, 1990) are also inferior in indica rice, which may limit the

success in the transformation for indica rice. Callus induction as well as regeneration potential is affected not only by genotype and the

type of explant but also by the composition of the culture medium including plant growth regulators, and by the culture conditions. However, in particular, genotype and type of explant are important

factors for the successful embryogenic callus induction and regeneration of rice plants (Rueb et al., 1994). In this study, genotypic differences among 15 Indonesian rice genotypes in callus induction

potential was examined, combined with two types of explants (embryos of mature seed and root

segment) and two culture media: MS (Murashige and Skoog, 1962) and CI (Potrykus et al., 1979).

16

The success using MS medium (Seraj et al., 1997; Khanna and Raina, 1998; Lee et al., 2002), as

well as CI medium (Jiang et al., 2000) for callus induction has already been reported.

The objective of the present study was to identify callus induction potential of 15 Indonesian rice genotypes. The genotypes identified to have a good induction potential could then be utilized

in tissue culture-based studies directed for the coming genetic transformation studies with the valuable genes of agricultural interest.

Materials and Methods

Fifteen indica rice genotypes from Indonesia (Table 2-1-1) and one japonica rice cv. Nipponbare as a check were used in the experiment since this genotype has been widely used as

model plant in tissue culture and plant improvement studies (Ogawa et al., 1999; Sasaki and Burr, 2000; Kawahigashi et al., 2003). The first five genotypes listed in the table are broadly cultivated in

Indonesia and the following ten genotypes are promising ones bred by Indonesia Institute for Rice Research (Sukamandi, Subang, West Java).

Mature healthy seeds were dehusked manually and soaked in 70% ethanol for 3 min. with

gentle agitation followed by rinsing with sterile distilled water. The seeds were then surface sterilized in 50% commercial bleach (5% sodium hypochlorite) for 30 min. with agitation and rinsed

with sterile distilled water three times. The root explants were obtained from the seedlings grown on MS-basal medium. The embryos of mature seeds (seed explants) and root segment (root

explants, from 5- to 7-day-old seedling) were aseptically cultured on callus induction medium; eight explants were placed on a Petri dish (9 cm in diameter) containing 20 mL MS or CI medium

supplemented with 2 mg L-1 2,4 dichlorophenoxyacetic acid (2,4-D) (Table 2-1-2). To anticipate the contamination that would arise, culturing additional explants of each genotype on both media was

supplied. The pH of medium was adjusted to 5.8 before autoclaving, and the cultures were kept at 25±10C under dark condition.

17

Table 2-1-1. Genotypes used in this experiment. No. Genotypes Pedigree 1. Fatmawati BP68C-MR-4-3-2/Maros 2. Gilirang B6672/Memberamo 3. Ciapus Memberamo//IR-66154-221-2-2/Memberamo 4. Cimelati Memberamo//IR-66160/Memberamo 5. IR-64 IR-5657/IR-2061 6. BP-23 IR-64/IRBB7//IR64 7. BP-140 IR-66738-18-1-2//Barumun/IRBB7 8. BP-360-2 B10182/Memberamo///IR-66160/Memberamo//Memberamo 9. BP-360-3 B10182/Memberamo///IR-66160/Memberamo//Memberamo 10. BP-205D-KN-78-1-8 Dacava line 85/Memberamo 11. BP-135 IR-65598-27-3-1/Suban//Barumun 12. BP-138 Muncul//IR-64/TB154E-TB-1 13. BP-143 Memberamo/IR-65598//Memberamo 14. B10597F IR-BB7/Cincklonik 15. BP-355E Memberamo//IR-665600/Memberamo///IR-65598/Cibodas 16. Nipponbare (check) Yamabiko/Sachikaze

After a 30-day culture, explants were examined for callus induction ability (CIA : total number of explants with calluses per total number of explants cultured x 100%), average diameter of callus

(ADC in cm), % of white or cream or yellow calluses that belong to callus type I or II (WYC : number of white or cream or yellow calluses with compact or friable structure per number of

explants cultured x 100%), and % of callus browning (CBW : number of callus browning per

number of explants cultured x 100%). A completely randomized factorial design with three replications was employed in this study.

Analysis of variance (ANOVA) and Dunnett’s pairwise multiple comparison t-test for genotypes were performed by using SPSS 10 software (SPSS for Windows, 1999; SPSS Inc., USA).

18

Table 2-1-2. Composition of media used in this study.

Components Medium MS* Medium CI* Inorganic salts Macro elements NH4NO3 1650 640 KNO3 1900 1212 CaCl2.2H2O 440 588 MgSO4.7H2O 370 247 KH2PO4 170 136 Micro elements KI 0.83 0.83 H3BO3 6.2 3.1 MnSO4.4H2O 22.3 11.15 ZnSO4.7H2O 8.6 5.76 Na2MoO4. 2H2O 0.25 0.24 CuSO4. 5H2O 0.025 0.025 CoCl2. 6H2O 0.025 - CoSO4. 7H2O - 0.028 FeSO4. 7H2O 27.85 27.8 Na2EDTA. 2H2O 37.25 37.3 Organic supplement Nicotinic acid 0.5 6.0 Pyridoxine-HCl 0.5 1.0

Thiamine-HCl 0.5 8.5 Coconut water - 100 mLL-1 Glycine 2.0 2.0

Carbon source Sucrose 30000 20000 Mannitol - 36430 Phytohormone (2,4-D) 2.0 2.0

* : Concentration in mgL -1. Both media d id not contain Myoinositol.

19

Results All genotypes induced calluses, but callus induction ability (CIA) varied with the genotypes.

Total number of explants with calluses was 76 for Nipponbare; on the average per dish were 6.3 (79.17%). Fig. 2-1-1. shows the induced callus of some genotypes. B10597F had poor callus

induction ability. Phenotypically, calluses derived from mature seed were relatively hard, and sometimes dry,

but those derived from root segment were highly mucilaginous, soft and covered with a translucent sticky substance. In some roots, calluses were produced on a few sites of the segment. Most of

the root segments with hairs had a high ability to induce callus. A significant interaction was detected for genotype x medium x explant (p<0.01 for WYC and

CBW), medium x explant (p<0.01 for CIA, WYC and CBW), genotype x explant (p<0.01 for CIA, WYC and p<0.05 for CBW), and genotype x medium (p<0.05 for WYC) (Table 2-1-3). For WYC

and similarly for CBW, except for the genotype x medium interaction, main- and interaction effects were significant. For CIA, the medium x explant interaction accounted for the greatest variation. In

general, explant accounted for the greatest variation in ADC, WYC, and CBW, but not in CIA. The

author found no significant interaction among the genotype x medium x explant for CIA and ADC. Medium x explant and genotype x explant interaction for ADC, genotype x medium interaction for

both CIA and ADC were also not significant. The main effect of each factor for these traits was significant (p<0.01, Table 2-1-3).

20

A1 B1 C1 A2 B2 C2 A3 B3 C3 A4 B4 C4 Fig. 2-1-1. Calluses of the genotypes B10597F (A), BP-360-3 (B) and Nipponbare (C), derived from seed explant (A, B,

C fo llowed by subscript number 1 and 2) and root explant (with subscript number 3 and 4), cultured on MS (with subscript number 1 and 3) and CI (with subscript number 2 and 4) media.

21

Although a significant genotype x medium x explant interaction was obtained for WYC and

CBW as previously mentioned, the author mainly analyzed the genotypic difference among the

traits observed. In comparison with Nipponbare, Fatmawati, Ciapus, BP-23, and BP-360-3 had similarly superior callus induction-related traits irrespective of the medium and explants used

according to Dunnett’s pairwise multiple comparison t-test at 0.05 probability level (Table 2-1-4). On the other hand, BP-135 was the only genotype showing significantly inferior performance in all

traits as compared with Nipponbare. In the overall analysis, MS medium and seed explant were superior for CIA, ADC, and WYC,

whereas CI medium and root explant were superior for CBW.

Table 2-1-3. Analysis of variance for callus induction performances.

Source of variation

df

Callus induction

ability (CIA)

Average diameter of

callus (ADC)

Callus white/ yellow (WYC)

Callus browning

(CBW) Genotype (g) 15 2669.92** 0.04** 2337.67** 557.29** Medium (m) 1 8138.02** 0.16** 2929.69** 3987.63** Explants (e) 1 14179.69** 1.87** 19300.13** 9847.00** Genotype x Medium (g x m) 15 276.91ns 0.01ns 462.67* 291.45ns Genotype x Explants (g x e) 15 617.19** 0.02ns 1079.64** 397.35* Medium x Explants (m x e) 1 17347.00** 0.02ns 6018.88** 6302.08** Genotype x Medium x Explants (g x m x e)

15 319.23ns 0.01 ns 610.89** 435.76**

Error 128 243.33 0.01 263.67 191.24 Total 191 Data show mean square value, ns : nonsignificant, * : significant at p = 0.05, ** : significant at p = 0.01, respectively.

22

Table 2-1-4. Comparison for callus induction-related traits of 15 genotypes with those of Nipponbare and overall means for media and explants.

No.

Genotype

Callus induction

ability (%,CIA)

Average diameter of

callus (cm,ADC)

Callus white/ yellow

(%,WYC)

Callus browning (%,CBW)

1. Fatmawati 69.79ns 0.29ns 44.79ns 11.46ns 2. Gilirang 59.38* 0.19* 34.38* 19.79ns 3. Ciapus 80.21ns 0.26ns 50.00ns 14.58ns 4. Cimelati 67.71ns 0.24* 36.46* 20.83ns 5 IR-64 79.17ns 0.24* 57.29ns 8.33ns 6. BP-23 86.46ns 0.30ns 65.63ns 6.25ns 7. BP-140 90.63ns 0.33ns 64.58ns 25.00* 8. BP-360-2 67.71ns 0.20* 33.33* 25.00* 9. BP-360-3 82.29ns 0.33ns 52.08ns 17.71ns 10. BP-205D-KN-78-1-8 69.79ns 0.25ns 44.79ns 23.96* 11. BP-135 46.88* 0.19* 28.13* 22.92* 12. BP-138 57.29* 0.26ns 31.25* 17.71ns 13. BP-143 61.46* 0.20* 34.38* 13.54ns 14. B10597F 33.33* 0.15* 15.63* 14.58ns 15. BP-355E 70.83ns 0.29ns 45.83ns 27.08* 16. Nipponbare (check) 79.17 0.38 57.29 6.25 Seed explant 77.47 0.36 53.52 24.35 Root explant 60.29 0.16 33.46 10.03 MS medium 75.39 0.29 47.40 21.75 CI medium 62.37 0.23 39.58 12.63 ns : nonsignificant, * : significant compared to Nipponbare’s per for mance using Dunnett’s pairwise multip le comparison t-test at 0.05 probability level with right- sided (>check) for CIA, ADC and WYC, and left-sided (<check) for CBW. Values are means of combination of 2 media and 2 explants with 3 replicates. The difference between seed explant and root explant or between MS medium and CI medium are significant (Table 2-1-3).

23

Discussion

It is well known that the genotype (Abe and Futsuhara, 1986), the interaction between

genotype and medium (Khanna and Rainna, 1998) and also the interaction between genotype and explant (Hoque and Mansfield, 2004) have significant effects on callus induction. In this study, the

effect of genotype x medium x explant interaction on WYC and CBW was significant, but that on CIA and ADC was not (Table 2-1-3). The WYC and CBW are believed to be related to the quality of

callus. White/cream/yellow calluses as reported by Visarada et al. (2002) are embryogenic calluses and also associated with at least two other types of calluses, i.e. 1) a soft, transparent,

unorganized callus, which is nonmorphogenic or may form only roots, and 2) soft mucilaginous callus, which often form roots. Maeda (2000) and Maeda et al. (2002) reported that white and

green patches are often seen on the surface stratum of callus with high regeneration ability. Moreover, in sorghum, Kaeppler and Pedersen (1997) reported that the production of a high

quality callus depends mainly on the genotype. Lee et al. (2002) found that the number, color, size, shape and appearance of the embryogenic calluses varied among the rice genotypes depending

on the type of basal medium, indicating that induction of high-quality rice callus is influenced by

genotype, medium, and the kind of explant as well as by their interactions. Callus browning is one of the major problems in an in vitro culture. Abe and Futsuhara

(1991) and Ogawa et al. (1999) argued that callus browning is genetically controlled, which correspond to the present study. A decrease in the rate of callus growth is occasionally related to

the appearance of a brown colored area (Maeda et al., 2002). A low 2,4-D concentration (Mitsuoka et al., 1994) and high temperature (Hoque and Mansfield, 2004) seem to cause callus browning or

regeneration of albino plants. However, in the present study, 2 mg L-1 2,4-D and 25oC were used for callus induction, which are supposed to be an optimum for callus induction. However, the result

of callus browning in this study should be considered carefully, since explants of the genotype that could not produce the calluses were not examined. If the browning of the culture including the

browning of explants was examined, the result would be slightly different, particularly in the genotypes that had low ability in callus induction ability, i.e., Gilirang, BP-135, BP-138, BP-143,

and B10597F (Table 2-1-4).

In the present study, the author provided root explants from 5- to 7-day-old seedlings; although Hoque and Mansfield (2004) found that the younger explants (3- to 5-day-old seedlings)

of indica rice were most efficient in both callus induction and plant regeneration. This is because a large variation was found in root length in the 3-to 5-day-old seedlings, but in some genotypes root

24

length was only a few centimeters.

Medium composition plays an important role in callus induction (Khanna and Raina, 1998;

Ogawa et al., 1999). The nutrient, particularly the nitrogen source, affects the callus induction of somatic embryos, particularly in monocots (Leifert et al., 1995). MS medium was suitable for

obtaining high CIA, ADC and WYC, while CI medium was suitable for obtaining low CBW, although the difference was slight (Table 2-14). MS medium, compared with CI medium, has a higher

concentration of macro and micro elements, but lower content of organic supplement, only one carbon source (i.e. sucrose) and no coconut water (Table 2-1-2). Such differences might cause the

differences in tissue culture response. Although genotype significantly contributed to the variation of traits observed, the

contribution was not as large as expected (Table 2-1-3). This may be due to the similar genetic background of the materials used. Eight of them are derived from the same parent, i.e., cv.

Memberamo (from crossing between B6555 and IR-19661), and twelve of them are also derived from the IR-series (breeding lines) developed by IRRI (Table 2-1-1), thus all genotypes carry a

genetic composition (female/male parent) from IR-series.

In Table 2-1-3, the author found a high contribution of the explant to the variation of traits observed. The seed explant was suitable for obtaining high CIA, ADC, and WYC, and root explant

for low CBW (Table 2-1-4). In addition, four genotypes i.e., Fatmawati, Ciapus, BP-23, and BP-360-3 have been identified as excellent genotypes for high CAI, ADC, WYC and low CBW

compared with Nipponbare (Table 2-1-4). This shows that the seed explants of these superior genotypes have a high callus induction potential which depends on the activity of genes that

determine and maintain the meristematic activity of the cells, level of hormones, and sensitivity to hormones, as well as on the activity of other genes that control different stages of plant

morphogenesis (Ezhova, 2003). Fatmawati, released as a new cultivar in Indonesia in 2003, and BP-360-3 are new plant type (NPT) with low tillering capacity and large panicles. Ciapus is also a

new cultivar released in 2003 and BP-23 is a promising line, though not released yet. These genotypes should be further examined for their plant regeneration ability or may be used as

genetic transformation materials. The author believed that these genotypes with good callus

induction-related traits would be useful for plant regeneration, though Visarada et al. (2002) found that genotypes showing moderate callus induction showed high regeneration ability. Since these

genotypes are similar to Nipponbare in callus induction ability, further works especially on plant regeneration and genetic transformation experiments may be promising.

25

Concluding remarks It has been shown that reliable assessment on callus performance i.e., good-looking callus

with white/cream/yellow appearance, no browning spot/healthy, actively growing that supposed to be embryogenic induced by some rice genotypes may lead to identify genotypes with high quality

callus production. These selected genotypes were further examined for in vitro plant regeneration since regeneration capacity is thus far a bottle neck in the success of genetic transformation

experiments.

26

Chapter 2

Establishment of in vitro Culture for the Success of Genetic Transformation

2.2. Plant Regeneration Capacity of Calluses Derived from Mature

Seeds of Five Indonesian Rice Genotypes

27

Abstract

Establishment of a suitable system for plant regeneration of rice calluses derived from mature

seed is a prerequisite for the success of genetic transformation using callus as the target tissue. Selecting the most suitable medium and assessing the genotype performance for in vitro response

are essential requirement for this purpose. The experiment with five Indonesian rice genotypes showed that callus-proliferation capacity (CPC) was affected only by medium and callus-growth

capacity were significantly affected by genotype and by medium. The shoot-regeneration capacity and plantlet-regeneration capacity were affected by the interaction effect between genotype and

medium. However for green plant-regeneration capacity, it was affected independently by genotype and medium. Culture media D1 and NB5 were the most suitable media for callus

subculture and plant regeneration, respectively. Genotypes Fatmawati and BP-140 consistently performed best in the callus subculture and plant regeneration.

28

Introduction

Plant regeneration capacity is one of the primary properties of totipotency of plant cells, which has been widely used for application of plant biotechnology including genetic transformation.

For efficient genetic transformation, a highly efficient and robust tissue culture system is a prerequisite since the efficiency of transformation mainly depends on regeneration capacity of

genotypes, among others (Alfonso-Rubi et al., 1999). To date, efficient plant regeneration for rice

has been reported by some authors (Abe and Futsuhara, 1986; Higuchi and Maeda, 1990; Lee et al., 2002), but despite the availability of protocols for rice regeneration, no procedure appears to

be universally adaptable when a new genotype is to be considered for in vitro manipulation (Visarada et al., 2002).

In rice, plant regeneration capacity is affected by genotype (Abe and Futsuhara, 1986; Seraj et al., 1997), age and type of explant (Hoque and Mansfield, 2004), nutrient media such as, basal

media (Khanna and Raina, 1998; Lee et al., 2002; Lin and Zhang, 2005), plant growth regulators (Pons et al., 2000; Lee et al., 2002), passage in subculture (Lin and Zhang, 2005) and culture

conditions. Numerous efforts have been undertaken to improve plant regeneration capacity of rice callus with varying degrees of success. Amino acids such as proline (Yang et al., 1999; Chowdhry

et al., 2000) and glutamine (Pons et al., 2000), a specific growth regulator, such as, abscisic acid (ABA) (Higuchi and Maeda, 1990; Kobayashi et al., 1992; Yang et al., 1999) and high

concentration of gelling agent (Lai and Liu, 1988; Lee et al., 2002) have been used for improving

the regenerability of rice callus culture. Furthermore, genetic transformation with a ferredoxin- nitri te reductase gene isolated from indica Kasalath (Nishimura et al., 2005) and cDNA Os22A

isolated from indica Konansou (Ozawa, et al., 2003) have been applied for improving regeneration capacity of rice plants.

For in vitro regeneration, the use of mature embryos rather than immature tissues as initial explants has distinctive advantages since embryogenic calluses induced from mature seeds are

suitable for gene delivery and genetic transformation, actively dividing and capable of regenerating into fertile plants (Jiang et al., 2000). Thus, proliferation of the embryogenic calluses with high

regeneration capacity through subculture is prerequisite for the successful development of transgenic rice plants via callus culture. In addition, establishing an efficient plant regeneration

system will therefore facilitate the development of transgenic and other biotechnological aims. In this study, the author employed five culture media in combination with five Indonesian rice

genotypes for examining callus proliferation (subculture) and plant regeneration capacities. The

29

objective of this study was to establish a suitable plant regeneration system for five selected

Indonesian rice genotypes using a mature seed as the initial explant.


1. Plant materials Mature healthy dehusked seeds of Fatmawati, Ciapus, BP-23, BP-140 and BP-360-3 (all

indica subspecies) were soaked with 70% ethanol for 3 min. and surface sterilized in 50% commercial bleach (4.5% sodium hypochlorite) supplemented with 2-3 drops of Tween20 for 45

min. with gently shaking, and then rinsed thoroughly with sterile distilled water three times. The seeds were cultured in Petri dishes (9 cm in diameter) containing 20 mL of callus-induction

medium and incubated at 270C in the dark for 30 days.

2. Callus induction, subculture and regeneration MS (Murashige and Skoog, 1962) medium, gelled with 0.8% agar, supplemented with 3

mgL-1 of 2,4-D and 3% sucrose was used for callus induction. Five basal media, MS, CI (Potrykus

et al., 1979), D1 and L3 (Lin and Zhang, 2005), and NB5 (micronutrient and organic components of

Gamborg et al., 1968; macronutrient of Chu et al., 1975) (Table 2-2-1), were used for both subculture of calluses (simply called subculture hereafter) and plant regeneration. All media for

subculture were supplemented with 3 mgL-1 2,4-D, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 3% maltose and semi solidified with 0.25% Phytagel (Gellan gum, Kanto Chemical Co., Inc.).

30

Table 2-2-1. Composition of media for callus induction, subculture and plant regeneration*.

Components Medium D1a Medium L3b Medium NB5c Macro elements NH4NO3 1,700 (NH4)2SO4 450 463 KNO3 2,900 1,900 2,830 CaCl2.2H2O 170 400 166 MgSO4.7H2O 185 370 185 KH2PO4 400 170 400 Micro elements KI 7.5 7.5 0.75 H3BO3 30 30 3 MnSO4.4H2O 100 100 10 ZnSO4.7H2O 20 20 2 Na2MoO4. 2H2O 2.5 2.5 0.25 CuSO4. 5H2O 0.25 0.25 0.025 CoCl2. 6H2O 0.25 0.25 0.025 CoSO4. 7H2O FeSO4. 7H2O 55.9 41.8 27.8 Na2EDTA. 2H2O 74.5 55.9 37.3 Organic supplement Nicotinic acid 1.0 1.0 1.0 Pyridoxine-HCl 1.0 1.0 1.0

Thiamine-HCl 1.0 1.0 10 Glycine 2.0 2.0 Coconut water Myo-inositol 100 100 100

* : Concentration in mgL-1 and for MS (Murashige and Skoog, 1962) and CI (Potrykus et al., 1979) presented in page 18; a Lin and Zhang (2005); b Lin and Zhang 2005; c Chu et al. (1975) for macronutr ient and Gamborg et al. (1968) for micronutrient and organic components. Callus induction medium: MS + 3 mgL-1 2,4-D + 3% sucrose + 0.8% agar, pH 5.8. Callus proliferation/subculture medium: a ll five media + 3 mgL-1 2,4-D, 500 mgL -1 l-proline, 500 mgL-1 l-glutamine, maltose 3% + Phytagel 0.25% , pH 5.8. Regeneration medium: All five media + 3 mgL-1 Kinetin + 3 mgL-1 BA + 0.5 mgL-1 IAA + 0.5 mgL -1 NAA + 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate + maltose 3% + Phytagel 0.3% , pH 5.8. Rooting medium: MS + sucrose 3% + agar 0.6% , pH 5.8.

31

Fine, friable, white/cream/yellow pale globular embryogenic calluses, 2-3 mm in diameter,

were selected, and 20 calluses were transferred into each Petri dish containing 20 mL subculture

medium (Fig. 2-2-1a, 1b) with three replications. They were subcultured at 270C in the dark for 3 weeks.

For plant regeneration, the above-mentioned media supplemented with 3 mgL-1 Kinetin, 3 mgL-1 BA, 0.5 mgL-1 IAA, 0.5 mgL-1 NAA, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1

casein hydrolysate, 3% maltose and 0.3% Phytagel was used. The pH of all media was adjusted to 5.8 before autoclaving. Calluses grown and proliferated from the same medium were transferred to

the corresponding regeneration medium. Subcultured clusters of calluses ca. 10-13 mm in diameter were transferred into Petri dish containing 20 mL regeneration medium, 10 clusters in

each dish (Fig. 2-2-1c) with five replications, and incubated under a 16-h photoperiod of fluorescent light, with 8-h darkness, at 270C, for 3-5 weeks.

Regenerated shoots, when their height reached over 2 cm, were transferred to a plant growth regulator-free MS medium for root development (Fig. 2-2-1d). When leaf height reached 10

cm, the green plants were transferred onto the soil and grown in a greenhouse for further growth.

3. Evaluation and experimental design For the callus subculture, two variables: a) callus-proliferation capacity (CPC in %): (number

of callus clusters proliferated)(number of callus clusters on the subculture medium)-1 x 100); and b) callus-growth capacity (CGC in mm2) shown by the size of callus clusters after a 21-day culture,

were examined. The size of callus clusters is the average of multiplication of the longest and the shortest diameter of the cluster of callus.

For plant regeneration, three variables were examined: a) shoot-regeneration capacity (SRC in %): (number of callus clusters developing green shoot buds)(number of callus clusters on the

regeneration medium)-1 x 100; examined after a 3-week culture; b) plant-regeneration capacity (PRC in %): (number of callus clusters developing green plantlets)(number of callus clusters on

the regeneration medium)-1 x 100; examined after 4-5-week culture; and c) green plant-regeneration capacity (GRC) shown by the number of green plantlets per replication; examined

when plantlets were transferred to soil.

A completely randomized factorial design was used in this study. Data were then analyzed statistically by the analysis of variance (ANOVA) and differences among means were evaluated by

Duncan’s multiple range test (DMRT).

32

a ba

c

d

e f

Fig. 2-2-1. Plant regeneration from mature seed-derived calluses. a) an embryogenic calluses of genotype Fatmawati

on the D1 subculture medium; b) 20 clusters of calluses of Fatmawati on the NB5 subculture medium after a 21-day culture; c) 10 clusters of calluses of Fatmawati on the NB5 subculture medium showing shoot regeneration after a 14-day culture; d) plantlets of BP-140 growing on D1 regeneration medium; e) and f) plantlets of Fatmawati regenerated on medium NB5 and D1, respectively. Scale bars in a) to d) correspond to 0.5 cm, while in e) and f) are 1 cm.

33

Results

In the subculture, callus-growth capacity (CGC) was affected independently by medium

(p<0.01) and genotype (p<0.01), but callus-proliferation capacity (CPC) was affected independently by genotype (p<0.01) (Table 2-2-2). The interaction effect between genotype and

medium was not significantly detected for these traits (CGC and CPC). In the five genotypes, CGC varied with the genotype but CPC did not. In addition, medium accounted for the greatest variation

in the proliferation and growth of calluses (Table 2-2-2). Since the interaction effect is not significant for both traits, the effect of genotype on CGC and that of medium on CPC and CGC are

presented in Fig. 2-2-2 and 2-2-3, separately. Among five genotypes, Fatmawati and BP-140 had the highest CGC (Fig. 2-2-2). All media except for L3 had high CPC (Fig. 2-2-3i). Medium D1 had

the highest CGC among the five media and L3 had the lowest CGC (Fig. 2-2-3ii). In the plant regeneration experiment, all five genotypes tested were capable of generating

shoot bud and subsequently regenerating plantlets. However, the regeneration capacity significantly varied with the genotype (Table 2-2-2). Irrespective of medium used, plantlet

regeneration was early in Fatmawati. In general, shoot regeneration was poorly synchronized; it

took 15- to 20 days for rice calluses to regenerate shoots on regeneration medium. SRC and PRC were significantly affected by the interaction effect between genotype and medium (p<0.01) but

GRC was not (Table 2-2-2). Genotype accounted for the greatest variation in SRC and PRC. Conversely, medium contributed to give the highest variation in GRC (Table 2-2-2). Fatmawati

showed no difference in SRC on different media and had a high PRC on NB5 (Table 2-2-3). Fatmawati and BP-140 showed the highest PRC on NB5 (Table 2-2-3) and the highest GRC on the

average (Fig. 2-2-4i). Ciapus cultured on CI and NB5; BP-23 on MS; BP-140 on NB5, CI, and DI; BP-360-3 on DI and CI, had high SRC (Table 2-2-3). However, Fatmawati and BP-140 cultured on

NB5, and BP-360-3 cultured on DI had high PRC. Ciapus, BP-23 and BP-360-3 produced relatively low PRC (Table 2-2-3) and low GRC (Fig. 2-2-4i). Medium NB5 gave the highest GRC for all five

genotypes and MS, CI and D1 gave similarly low GRC (Fig. 2-2-4ii). The appearance of plantlets greatly varied with the medium culture. For instance, on

medium NB5, Fatmawati produced numerous green healthy plantlets with many tillers (Fig. 2-2-1e),

while, on D1, it produced light yellow/brownish plantlets with few tillers (Fig. 2-2-1f).

34

AAB

BC

CC

0

10

20

30

40

50

60

Fatmawati Ciapus BP-23 BP-140 BP-360-3

Genotype

The s

ize o

f clu

ster

of

cal

lus

(mm

2)

Fig. 2-2-2. Comparison for genotype on CGC in the subculture after 21-day culture. Values w ith the same alphabet

show no difference by DMRT at the 0.05 probability level. Each value is the average of genotype perfor mance across five media with three replicates and each consists of 20 clusters of seed-derived callus. CGC ( callus-growth capacity) is shown by the size of callus clusters in mm2. The size of callus cluster is the average of the multip lication of the longest and the shor test diameter of the cluster of callus.

36

Table 2-2-2. Analysis of variance for callus proliferation (CPC, CGC) and plant regeneration variables (SRC, PRC, GRC). Source of variation dfa dfb CPC CGC SRC PRC GRC Genotype (g) 4 4 259.50 ns 2202.08 ** 7893.50 ** 3381.50 ** 2383.82 * Medium (m) 4 3 5786.17 ** 4855.48 ** 1260.00 ns 2366.33 ** 4964.35 ** Genotype x Medium (g x m) 16 12 39.92 ns 267.25 ns 2667.50 ** 1075.50 ** 1142.02 ns Error 50 80 191.33 298.77 715.50 215.50 762.67 Total 75 100

Data show mean square value. ns: non-significant, *: significant at p = 0.05, **: significant at p = 0.01, respectively. a: for callus subculture, calculated on the basis of combination of 5 genotypes and 5 media with 3 replications. b: for regeneration, calculated on the basis of combination of 5 genotypes and 4 media with 5 replications ( medium L3 was excluded from the analysis because of no variation over

replications).

CPC (callus-proliferation capacity in % ): number of callus clusters proliferated)(number of callus clusters on the subculture medium)-1 x 100.

CGC (callus-growth capacity in mm2) is the average of the multip lication of the longest and the shor test diameter of the cluster of callus after a 21-day culture.

SRC (shoot-regeneration capacity in % ): (number of callus clusters developing green shoot buds)(number of callus clusters on the regeneration medium)-1 x 100.

PRC (plant-regeneration capacity in % ): (number of calluses clusters producing green plantlet)(number of callus clusters on the regeneration medium)-1 x 100.

GRC (green plant-regeneration capacity in % ): number of green plantlets per replication.

Chapter 3 – Assessment of Somaclonal Variants

37

A A A

B

A

0

20

40

60

80

100

120

MS CI D1 L3 NB5

Medium

Cal

lus-

pro

life

rati

ng

cap

acit

y (%

)

i

B

C

A

BB

0

10

20

30

40

50

60

70

MS CI D1 L3 NB5

Medium

The

siz

e o

f c

lust

er

of

cal

lus

(mm

2)

ii

Fig. 2-2-3. Comparison for medium on (i) CPC, (ii) CGC in the subculture after 21-day culture. Values w ith the same

alphabet show no difference by DMRT at the 0.05 probability level. Each value is the average of the value from five genotypes with three replicates and each consists of 20 clusters of seed-derived calluses. CPC (callus-proliferation capacity in % ): number of callus clusters proliferated) (number of callus clusters on the subculture medium)-1 x 100. CGC (callus-growth capacity in mm2) is the average of the multiplication of the longest and the shor test d iameter of the cluster of callus.


38

Table 2-2-3. Comparison for genotypes and media on SRC and PRC using DMRT. SRC (%) PRC (%) Genotype

MS CI D1 NB5 MS CI D1 NB5 Fatmawati 56A

a 54AB

a 80A

a 98A

a 20A

b 32A

b 22AB

b 60A

a Ciapus 20A

b 68AB

a 12 C

b 40 B ab

6A a

4 B a

6 B a

14 B a

BP-23 52A a

20 C b

26 BC b

30 B b

4A a

4 B a

2 B a

10 B a

BP-140 44A b

84A a

62AB ab

96A a

8A b

22AB b

18AB b

70A a

BP-360-3 34A b

46 BC ab

80A a

20 B b

8A b

6 B b

34A a

4 B b

Data are the means of five replicates, each with 10 clusters of callus. The same capita l alphabet (between genotypes with in a medium) and small a lphabet (between media within a genotype) in column and row, respectively, show no difference by DMRT at the 0.05 probability level. SRC (shoot-regeneration capacity in % ): (number of callus clusters developing green shoot buds)(number of callus clusters on the regeneration medium) -1 x 100. PRC (plant-regeneration capacity in % ): (number of calluses cluster s producing green plantlet)(number of callus clusters on the regeneration medium) -1 x 100.


39

A

AB

B

A

B

0

5

10

15

20

25

30

35

Fatmawati Ciapus BP-23 BP-140 BP-360-3

Genotype

Nu

mbe

r o

f pl

antl

ets

pe

r re

plic

atio

n i

A

BB

B

0

5

10

15

20

25

30

35

40

MS CI D1 NB5

Medium

Nu

mbe

r o

f pl

antl

ets

pe

r re

plic

atio

n ii

Fig. 2-2-4. Comparison for genotype (i) and medium (ii) on GRC in the regeneration. Values w ith the same alphabet show no difference by DMRT at the 0.05 probability level. Values in (i) show the mean for four media, and the values in (ii) show the means for five genotypes w ith five replicates and each consists of 10 clusters of seed-derived calluses. GRC (green plant-regeneration capacity in % ): number of green plantlets per replication. Medium L3 was excluded from the analysis because of no variation over replication.


40

Discussion Plant regeneration is indispensable for plant transformation technology and other

biotechnology aims. In this study, a suitable regeneration system for mature seed derived calluses of five indica subspecies has been established. Stages from callus proliferation till regeneration

have been investigated. After the step of callus induction, in my view, selecting the most suitable medium and assessing the genotype’s capacity for callus proliferation and regeneration in order to

improve its regenerability are essential for the success of genetic transformation using callus as target material. The result of the present experiment indicated that the callus-proliferation capacity

(CPC) was affected only by medium, while the callus-growth capacity (CGC) was independently influenced by genotype and by medium (Table 2-2-2). The significant effect of genotype on CPC

was not detected (Table 2-2-2) due to the fact that the genotypes used excluding BP-140 had been selected as the most responsive ones in callus induction and quality-related callus traits as stated

in the previous section. However, the genotypes and media tested significantly differed in CGC, with Fatmawati and BP-140 were the most responsive genotypes (Fig. 2-2-2) and D1 was the

most suitable medium for callus subculture (Fig. 2-2-3ii). The genotypic differences in CGC may

have resulted from the difference of the activity of the genes that control the callus proliferation process, such as those involved in plant hormone metabolism (Henry et al., 1994; Ezhova, 2003).

Some differences in composition existed among five media seem to cause the difference in promoting proliferation and growth of callus (Table 2-2-1).

For callus proliferation, 2,4-D is an essential element as reported by Inoue and Maeda (1980) and Mitsuoka et al. (1994). In the present study, however, 3 mg L-1 of 2,4-D was supplied

equal to all media tested. Suggesting the differences in genotype’s response may not be associated with 2,4-D. This also suggests that the genetic and non–genetic factors (medium,

growth regulator, and culture condition) clearly influence both CPC and CGC. However, author could not determine exactly what nutrient is a critical fac tor for callus proliferation/propagation,

since the nutrients are too numerous and demands as well as uptake by the rice cell are diverse. It is likely that the basal composition of medium influenced embryogenic callus proliferation, as has

been previously reported by some researchers (Rueb et al., 1994; Lin and Zhang, 2005). The

result of present experiment, however, is not in accordance with the result of Lin and Zhang (2005), who reported that L3 was the best medium for subculture of three indica genotypes.

For plant regeneration, the author identified a significant interaction between genotype and medium in SRC and PRC, but not in GRC that was affected independently by genotype and by

medium (Table 2-2-2). This indicates that, for SRC and PRC of each genotype shows a different


41

response with regard to regeneration media used, with Fatmawati and BP-140 being the most

responsive (Table 2-2-3). Fatmawati is a new plant type, released in 2003 in Indonesia, with many

valuable agronomic characters such as low tillering capacity, large panicle and high palatability. On the other hand, BP-140 is a promising line, thought not released yet. Both genotypes would be

useful for breeding program through in vitro approach. Each genotype had nearly the same GRC on the four regeneration media tested. Khanna

and Raina (1998) found the interaction effect between genotype and medium in SRC and plant regeneration frequency of mature seed-derived calluses of three indica types, although the author

did not. Such different findings may be attributed to the differences in genotypes, media and method of calculation used.

Generally, medium NB5 was the most suitable medium for plant regeneration in this study. Medium NB5 probably may promote the propagation of embryogenic calluses and thus improve the

regeneration capacity. This result is in accordance with Sivamani et al. (1996) and Visarada et al. (2002), who used NB5 with a slight different in composition of plant growth regulators.

From this experiment, it is clear that medium is only suitable for specific developmental

stage of callus. It also indicates that the composition of basal medium used for callus proliferation is not always optimal for plant regeneration since the nutritional requirements of the two phases of

development (callus proliferation and regeneration) may vary. This finding corresponds to Khanna and Raina (1998) and, finally, the author concluded that both callus proliferation and plant

regeneration capacities were genotype- and medium-dependent.

Concluding remarks

The author has examined different combinations of genotype and medium culture for callus

proliferation as well as plant regeneration. These studies clearly indicate that genotypes inducing high-quality callus, selected from previous experiment, were capable in regenerating whole green-

plantlets. This well-established regeneration system will open a wide opportunity for the application of genetic transformation and other biotechnological aims for improving agronomical and

economical important rice traits.


42

Chapter 3

Assessment of Somaclonal Variants from Primary Callus-derived Plants

Variation in the Spikelet-related Traits of Rice Plants Regenerated

from Mature Seed-derived Callus Culture


43

Abstract

Callus is an excellent source for in vitro plant regeneration, but plants regenerated from callus sometimes show phenotypic and genotypic variation from the initial plants. In this study, the variation in spikelet-related traits of the rice plants regenerated from calluses and their performance in the paddy field were examined.

The phenotypic variation in spikelet-related traits of the regenerated plants was not always in a reduction in their mean value. For instance, panicle length, spikelet number and fertile spikelet number of Indonesian rice genotypes Ciapus and BP-23 in the regenerated plants were significantly greater than those of the initial plants (developed from the seeds). The spikelet fertility of the regenerated rice plants was not

significantly lower than that of the initial plants except in Ciapus and BP-140. The occurrence of somaclonal variants varied with the genotype. Ciapus, BP-23 and BP-140, which induce many somaclonal variants, are suggested to be valuable for genetic, breeding or functional genomic studies, while the opposite, Fatmawati could be used for genetic transformation study.


44

Introduction

The major goal of plant breeding is to improve existing cultivars and to develop new or elite cultivars. The genetic variability induced by in vitro culture has been exploited to serve such breeding purposes due to the fact that the plants obtained from in vitro culture sometimes show phenotypic and genotypic

variations from the initial plant. This phenomenon is called ‘somaclonal variation’ (Larkin and Scowcroft, 1981), which refers to the variation arising in cell cultures, regenerated plants and their progenies (Larkin and Scowcroft, 1981; Karp, 1995). The genetic basis of somaclonal variation is not fully understood yet. However, it is postulated that the events such as large-scale deletions and gross changes in chromosome

structure/number and directed and undirected point mutations, transposon activation and epigenetic changes (Kaeppler et al., 2000; Jain, 2001) such as DNA methylation, hystone acetylation and chromatin remodeling (Joyce et al., 2003), contribute to the in vitro variations. Somaclonal variation is considered to be a useful source of variation and has been demonstrated to be valuable in rice (Zheng et al., 1989; Ling et al., 1989; Yamamoto et al., 1997). Somaclonal variants tolerant to sheath blight, Rhizoctonia solani (Xie

et al., 1992); drought (Adkins et al., 1995); chilling (Bertin and Bouharmont, 1997); aluminium (Jan et al., 1997); salinity (Lutts et al., 2001); and blast, Pyricularia grisea (de Araujo and Prabhu, 2002) are successful examples of rice plants obtained by in vitro selection.

A wide range of altered phenotypic expression in regenerated plants can be found, such as,

chlorophyll deficiency, dwarfs, seed characteristics, reproductive structures, and necrotic leaves (Phillips et al., 1994). A reduction in spikelet fertility or changes in agronomic traits has been reported to occur in in vitro-regenerated plants (Lee et al., 1999b; Rongbai et al., 1999). However, in contrast, somaclonal variants are undesired when the tissue culture protocols applied for developing transgenic plants since the

occurrence of somaclonal variants will complicate the evaluation of the effects of inserted gene (s), thus constitutes a major problems (Lutts et al. 2001). Jiang et al. (2000) reported that particle bombardment using callus as a target tissue produced 8% aberrant phenotypes among the transgenic plants. To develop stable transgenic rice plants expressing the genes of interest while retaining the genomic and phenotypic

integrity, a preliminary test on the possibility of somaclonal variants of callus-regenerated plants among rice genotypes is therefore considered necessary. The present chapter was designed to evaluate the spikelet-related traits of R0 plants in comparison with those of the initial genotypes and to evaluate the performance of regenerated plants in the field. Spikelet fertility and its related traits are the main concern of in vitro

regenerated plants especially for rice in which reproductive parts (i.e. grains) are harvested for many valuable uses.


Five Indonesian rice genotypes Fatmawati, Ciapus, BP-23, BP-140, BP-360-3 (all indica ssp), and

one japonica Nipponbare were regenerated from calluses following the methods described in the Chapter 2.


45

Briefly, mature seeds were cultured on callus induction medium (4 weeks), calluses were then subcultured

(3 weeks) and regenerated (3-5 weeks). After acclimatization for 3-4 weeks, 542 regenerated plants randomly selected were transplanted to the pot (17.2 cm in diameter, 19.2 cm in high) containing Andosol soil with 5 to 7 seedlings per pot and grown in a glasshouse. Another 308 regenerated plants were grown in the paddy field. The initial genotypes, developed from the seeds, were also grown in the glasshouse.

At the time of sowing, pots were fertilized with equal to 60 kg N, 60 kg P2O5 and 60 kg K2O ha-1. Additional top-dressing with the same dosage was applied at panicle initiation. In the paddy field experiment, planting density was 15 x 30 cm spacing and 16 kg N, 16 kg P2O5 and 16 kg K2O ha-1 plus 16 kg ha-1 of organic fertilizer was applied. The experiment was conducted at 113.26 m above sea level. In the

glasshouse, plants were irrigated at 2 ~ 3-d intervals for maintaining the water availability by direct watering to the pots, while in the paddy field, water was maintained approximately 5 to 10 cm from the soil surface. The daytime temperature during the experiment ranged from 250C to 380C in a glasshouse and from 200C to 330C in the field, and the nighttime temperature from 230C to 250C in the glasshouse and 150C to 250C in

the field. Phenotypic traits were then recorded for plant height (from ground level to tip of the tallest panicle),

spikelet fertility (percentage of filled and half-filled seeds per total spikelets), panicle length, ratio of filled or half-filled spikelets to unfilled spikelets, spikelet number per panicle, fertile spikelet number per panicle, and

100-grain weight. Spikelets were characterized as unfilled when the palea and lemma folded easily when pressed with fingers (IRRI, 2002). T illers were excluded from the analysis. Panicles were carefully harvested by hand to prevent the loss of spikelets and they were removed manually and subsequently divided into groups of filled and unfilled spikelets. Measured values of the regenerated plants were

compared with those of the initial genotypes by Student t-test with taking the homogeneity of variance into account using Levene’s test (SPSS Inc.). Analysis of variance (anova) was performed for the regenerated plants grown in the paddy field. To achieve a normal distribution, data were transformed; however, transformation did not result in marked improvement of the distribution. Therefore, nontransformed data

were used throughout (Steel and Torrie, 1980).

Results and Discussion

1. Comparison of spikelet-related traits of regenerated plants grown in the glasshouse with those of the initial plants

Spikelet-related traits of rice plants regenerated from mature seed-derived callus culture are important to be explored since it is a crucial determinant of seed set, which is a basis for both transmission

to the next generation and a yield per se. In this study, 542 regenerated plants grown in a glasshouse were scored for plant height and spikelet-related traits (Table 3-1). The spikelet fertility of callus-regenerated plants varied with the genotype ranging from 44.8% (partially fertile) in Fatmawati to 92.2% (fertile) in Nipponbare (Table 3-1). Low spikelet fertility observed in Fatmawati and BP-360-3 is possibly due to a high


46

air temperature (above 350C) which frequently observed in a glasshouse during flowering stage in the

summer (mid-August 2005) and most likely these genotypes are two of the heat-sensitive ones as formerly reported by Satake and Yoshida (1978) and more recently by Prasad et al. (2006).


47

Table 3-1. Comparative performance of the plants regenerated from callus and the initial genotypes grown in a glasshouse.

Genotype Origin Number of plants

Plant height (cm)

Panicle length (cm)

Spikelet fertility (%)

Ratio of filled to unfilled

spikelets Spikelet number

Fertile spikelet number 100-grain

weight (g)1 Fatmawati Seed 21 94.4 1.4 26.7 0.5 44.3 3.3 0.92 0.1 154 14.9 73.6 8.9 2.88 0.05 Callus 166 92.6 0.8ns 25.9 0.2ns 44.8 1.6ns 1.13 0.1ns 149 4.2ns 64.6 2.8ns 2.90 0.04ns Ciapus Seed 36 87.4 1.3 20.1 0.6 75.6 2.3 5.61 1.5 80 5.5 58.6 3.8 3.15 0.03 Callus 122 89.1 6.5ns 21.9 0.2** 67.9 1.1** 2.80 1.2ns 102 2.5** 69.1 1.8** 3.23 0.02* BP-23 Seed 41 83.7 0.7 20.8 0.4 80.3 2.8 11.21 2.0 68 3.6 52.3 2.7 2.71 0.02 Callus 36 88.0 1.2** 23.7 0.4** 75.3 3.3ns 4.13 0.5** 92 4.1** 67.8 3.9** 2.63 0.03* BP-140 Seed 32 94.9 0.9 23.7 0.7 65.4 3.2 3.53 0.8 106 8.7 64.2 6.1 3.55 0.02 Callus 157 90.3 0.7** 23.8 0.2ns 47.0 1.1** 1.03 0.1** 116 2.3ns 55.6 1.8ns 3.62 0.03* BP-360-3 Seed 29 90.3 1.5 23.5 0.5 59.4 3.8 2.78 0.7 109 8.0 65.3 6.6 2.93 0.01 Callus 42 96.8 1.7** 23.9 0.5ns 53.8 2.3ns 1.40 0.1ns 129 6.0* 71.3 5.2ns 2.86 0.02** Nipponbare Seed 31 83.6 1.1 16.3 0.5 92.1 1.7 25.24 3.3 68 3.3 62.8 3.4 2.84 0.02 Callus 19 79.9 1.8ns 16.7 0.3ns 92.2 0.9ns 17.38 2.9ns 70 4.3ns 64.8 4.1ns 2.74 0.01**

Differences between mean values of plants regenerated from callus and those of initial p lants of the corresponding genotype are non-significant (ns) or significant at P = 0.05 (*) or P = 0.01 (**) by a Student t-test with considering homogeneity of variance by Levene’s test. Data show mean values w ith standard error of means. 1) Data were taken from 15 samples of each genotype at a water content of 15% .


48

The mean spikelet fertility of callus-regenerated plants was significantly lower (P = 0.01) than that of

the initial plants in Ciapus and BP-140, but not in other genotypes (Table 3-1). This particular trait in the

regenerated plants tended to be lower than that in the initial plants. Lee et al. (1999b) and Rongbai et al.

(1999) reported a decrease of spikelet fertility in in vitro-regenerated plants. Thus, it is suggested that the

genetic instability of callus culture of Ciapus and BP-140 in regenerating fertile plants is greater than that of

the other four genotypes since the occurrence of somaclonal variation is strictly affected by genotype (Karp,

1995; Lutts et al., 1998; Joyce et al., 2003), cultivated explants (Lutts et al., 2001), medium, duration of

culture (Jain, 2001) and the survival of plantlets transplanted into soil (Rongbai et al., 1999).

Genetic instability of callus culture occurs due to the cells face traumatic experiences from isolation,

proliferation, and may reprogram during plant regeneration which are different from natural condition. Such

reprogramming or restructuring of events can create genetic and epigenetic changes resulting in a wide

range of phenotypic expression (Phillips et al., 1994; Jain, 2001) as shown in the significant difference in

spikelet fertility of Ciapus and BP-140. Such a phenomenon was also observed in plant height in BP-23,

BP-140, and BP-360-3; panicle length in Ciapus and BP-23; ratio of filled to unfilled spikelets in BP-23 and

BP-140; spikelet number in Ciapus, BP-23 and BP-360-3; fertile spikelet number in Ciapus and BP-23, and

in 100-grain weight in five genotypes except Fatmawati (Table 3-1). These data suggest that phenotypic

alteration in the primary callus-regenerated plants is not always in a negative direction (reduction in their

mean value). Possible reasons for these differences can be attributed to the genotype with regard to its

sensitivity to generate genetic alteration during in vitro culture in the nucleus or chromosome (from a point

mutation, large scale deletion, aneuploidy and or polyploidy) (Brown et al., 1990; Chatterjee and Das Gupta,

1997), in the cytoplasmic genome such as chloroplast deletion (Abe et al., 2002) or activation of

transposable elements such as retrotransposons which are activated as tissue culture get older (Hirochika

et al., 1996) or epigenetic mechanism such as DNA methylation event (Brown et al., 1990; Kaeppler et al.,

2000). The latter even can enhance quantitative traits because several genes can be affected

simultaneously (Jain, 2001), as found in this study. Moreover, it seems likely that no single event or factor


49

controls an altered phenotype obtained from in vitro culture (Jain, 2001). The genotypes found to induce

somaclonal variants in the present study should be then further investigated for genetic, breeding or

functional genomic studies. On the other hand, genotypes that are relatively stable would be valuable for

genetic transformation studies.

Judging from the significance of t-test on given traits in Table 3-1, the genetic instability of callus

culture among the six genotypes was the highest in BP-23 followed by Ciapus, BP-140, BP-360-3,

Nipponbare and Fatmawati in this order. Table 3-1 also shows that all phenotypic traits of the regenerated

plants were not significantly different from those of the initial plants in Fatmawati. This suggests that

Fatmawati has a high genetic stability in regenerating normal plants from callus, although for other traits

that were not evaluated, variations are still possible to be found, because callus culture acts as a mutagenic

(Phillips et al., 1994; Jain, 2001) and most likely changes in tissue culture occurs by a cellular stress-

response mechanism (Phillips et al., 1994; Kaeppler et al., 2000; Joyce et al., 2003). Thompson et al.

(1986) reported that callus DNA showed many discrete bands. Banerjee et al. (1997) and Chowdari et al.

(1998) also reported that many DNA variations were detected in the regenerated plants thus more

variations would be obtained. Further assessment on the next progeny using cytogenetic or molecular tools

is needed to detect such variations at the chromosome or DNA levels.

2. Performance of regenerated plants grown in the paddy field

Primary callus-derived plants of six genotypes were sown in a paddy field, and 40 plants of each

genotype were randomly sampled and examined for plant height and spikelet-related traits. Analysis of

variance showed that all traits of the regenerated plants significantly varied with the genotype (P = 0.01)

(Table 3-2), indicating that those genotypes varied widely in the traits evaluated. Spikelet number showed

the greatest mean square value while 100-grain weight showed the lowest. Spikelet number may be greatly

influenced by callus culture or it may vary widely in nature. Grain weight is thought to be quite constant due

to a rigid hull whose size is genetically determined (Fabre et al., 2005). Further comparison using Duncan’s


50

Multiple Range Test showed that the regenerated plants of Fatmawati had the highest mean value in plant

height, panicle length, spikelet number and fertile spikelet number (Table 3-3). Nipponbare was the most

fertile genotype showing high spikelet fertility and high ratio of filled to unfilled spikelets, although BP-140

showed the lowest fertility (Table 3-3). This is not surprising since Nipponbare has been grown for a long

period of time in Kanto Region in which Utsunomiya is located, and it has been well adapted to climatic

condition in this region. The high fertility of Nipponbare or low fertility in BP-140, Ciapus, BP-23 and

Fatmawati could be attributed to genotypic differences in source-sink relationship which plays a significant

role in rice yield potential, or it could be possible that trait has been influenced by in vitro culture (Lee et al.,

1999b; Rongbai et al., 1999). BP-140 had the heaviest 100-grain weight of the others (Table 3-3). From

these results, the author considers that Fatmawati, BP-140 and Ciapus are promising parents for creating

the high-yielding variety.

In general, in vitro culture increased the variability of regenerated rice plants as already shown in

other works (Adkins et al., 1995; Chatterjee and Das Gupta, 1997; Lutts et al., 1998). In this study, callus

culture did not promote a high phenotypic variation as anticipated, but some callus-derived plants of

Indonesian genotypes were found to be promising for further studies.

Concluding remarks

The data presented here demonstrate that genotype which induces somaclonal variants in spikelet-

related traits was observed in some rice genotypes. In order to minimize the occurrence of somaclonal

variants which is unwanted when callus culture applied for development of transgenic plants, selection of

an appropriate genotype and minimum sub-culture should be maintained.


51

Table 3-2. Analysis of variance for the regenerated plants grown in the paddy field.

Source of variation

df1 df2 Plant height (cm)

Panicle length (cm)


Ratio of filled to unfilled spikelets

Spikelet number

Fertile spikelet number

100-grain weight (g)1

Genotype 5 5 1324.4 ** 501.6 ** 8486.5 ** 1884.98 ** 312559 ** 116771 ** 0.819 ** Error 234 84 55.8 3.4 178.6 58.12 2879 1375 0.003 Total 239 89

Data show mean square value. **: significant at 0.01 probability level. df1 and df2: degree of freedom for the first six traits and 100-grain weight, respectively. 1) Data were taken from 15 samples of each genotype at a water content of 15% .


52

Table 3.3. Performance of the regenerated plants in spikelet-related traits grown in the paddy field.

Genotype Plant height (cm)

Panicle length (cm)


Ratio of filled to unfilled spikelets

Spikelet number Fertile spikelet number

100-grain weight (g)1)

Fatmawati 105.1 1.8 a 31.4 0.5 a 65.8 1.8 c 2.34 0.2 b 358 13.9 a 233 9.9 a 2.71 0.01 e Ciapus 91.9 0.9 c 25.9 0.3 bc 60.8 1.5 c 1.71 0.1 b 147 5.0 d 89 3.7 d 3.10 0.01 b BP-23 89.3 0.7 c 25.6 0.2 c 61.0 3.7 c 3.12 0.5 b 157 5.6 d 91 4.5 d 2.68 0.01 e BP-140 100.5 1.2 b 26.6 0.3 b 48.8 1.9 d 1.07 0.1 b 251 12.5 b 121 6.7 c 3.27 0.02 a BP-360-3 98.6 1.3 b 26.0 1.2 bc 72.1 1.9 b 3.24 0.3 b 197 4.3 c 142 4.8 b 2.87 0.02 c Nipponbare 98.0 0.8 b 20.3 0.2 d 92.1 0.7 a 18.99 2.9 a 117 3.0 e 107 2.4 c 2.80 0.01 d

Data were taken from 40 regenerated plants of each genotype grown in the field. Means followed by the same letter in the column were not significantly different according to Duncan’s Multip le Range Test at 0.05 probability level. Data show mean values with standard error of means. 1) Data were taken from 15 samples of each genotype at a water content of 15% .

53

Chapter 4

Genetic Transformation of the Rice Plants Using Helios Gene Gun for Developing Transgenic Rice Lines

4.1. Transient Expression of Green Fluorescent Protein in Rice

Calluses: Optimization of Parameters for Helios Gene Gun Bombardment

54

Abstract

An optimized condition for particle bombardment is necessary for efficient genetic transformation.

Parameters for HeliosTM gene gun, another device for cell transformation which is hand-held device sold by Bio-Rad Laboratories (California USA), were optimized based on transient

expression of synthetic green fluorescent protein (sgfp) in rice calluses of indica cv. Fatmawati and japonica cv. Nipponbare. Parameters found to be the most favorable conditions for transient

expression of sgfp in rice callus cells were as follows: 250 psi helium pressure, 0.6 m gold

particle size, 0.25 mg gold particles per shot, and 1.5 g plasmid-DNA per shot. Desiccation of

callus cells for eight min. was found also appropriate. The level of transient sgfp expression was not significantly influenced by the pre-culture for 4 to 12 days before bombardment or by callus

age between 10 and 33-weeks old in Fatmawati. Optimized parameters for this particular device should improve the transient expression, thus enabling stable expression of introduced genes via

HeliosTM gene gun using callus as a target tissue.

55

Introduction

Among the well-known reporter genes, the green fluorescent protein (gfp) from the jellyfish

(Aequorea victoria) is the most widely used reporter in biotechnology or molecular biology due to many advantages such as no necessity of any additional co-factors (Heim et al. 1994), non-

invasive detection (Chalfie et al. 1994), broad spectrum of host cells (Cubitt et al. 1995) and even without toxicity to the cells (Niwa, 2003). Moreover, in plant science, gfp has been extensively

applied for testing the efficacy of the promoter (Newell et al. 2003), detection of virus activity (Oparka et al. 1997), protein localization (Hibberd et al. 1998; Jang et al. 1999) and optimization of

transformation methods (Tee and Maziah, 2005). Recently, many types of gfp are available; one of them is a synthetic gfp [sgfp (S65T)], replacement of the serine in position 65 with a threonine

which is 100-fold brighter than wild-type gfp in plants (Chiu et al. 1996; Niwa et al. 1999). The sgfp has been used as an efficient reporter system for direct gene transfer in maize (Chiu et al. 1996),

oat (Cho et al. 2003), soybean (El-Shemy et al. 2006) and others. Transient expression of reporter genes is useful in both plant and animal sciences

particularly for improving the efficiency of transformation technology. Physical and biological

parameters of transformation technology including particle bombardment could be optimized for efficient genetic transformation. Schopke et al. (1997) pointed out that the establishment of optimal

parameters in particle bombardment for any plant tissues is necessary. Particle bombardment is now emerging as the method of choice for introduction of useful

agricultural genes into rice and other cereal crops. However, most bombardment methods for rice transformation utilize the PDS-1000/He Biolistic Particle Delivery System (for example, Bec et al.,

1998; Ghosh-Biswas et al., 1998; Jiang et al., 2000; Martinez-Trujillo et al., 2003) and currently there is no protocol for rice callus cells bombarded using the HeliosTM gene gun device (Bio-Rad

Laboratories, USA) and transgenic plants obtained using this particular tool have not been reported yet. In this study, the author used the HeliosTM gene gun that differs from PDS-1000/He

device. It does not use a vacuum chamber and thus can be used to deliver nucleic acids into tissues, organs, and even whole organisms (Taylor and Fauquet, 2002), thereby removing

limitations to the target and its size. Other advantages of the HeliosTM gene gun are portable in its

size, wide application to plant and animal cells, and also the cartridge can be stored for several months.

In this study, transient expression of sgfp (Chiu et al. 1996) was used as a reporter system for rapid assay to optimize the parameters of HeliosTM gene gun for calluses of two distinct

56

cultivars, indica Fatmawati and japonica Nipponbare. These parameters are helium pressure,

particle gold size, amount of gold particles per shot (MLQ), and amount of plasmid-DNA delivered

per shot (DLR). In addition, the effects of desiccation treatment, pre-culture of calluses before bombardment, and callus age, which are considered as the biological parameters, were also

evaluated. The objective of this section was to optimize parameters for HeliosTM gene gun bombardment by evaluating the transient expression of sgfp using calluses of two cultivars as

target materials.


1. Plant material and proliferation of embryogenic callus cells Embryogenic callus cells induced from scutellar tissues of mature seeds of two cvs. Fatmawati

and Nipponbare and proliferated as described in the previous chapters, were used as target

materials in bombardments.

2. Plasmid used for bombardment Plasmid pIG001 with the sgfp gene and the hygromycin phosphotransferase (hpt) gene

conferring resistant to hygromycin B was used in all bombardment experiments (Fig. 4-1-1). The sgfp gene was driven by a maize ubiquitin-1 promoter (Christensen and Quail, 1996), while the

hpt gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The plasmid was amplified in E. coli DH5αcells and purified by the alkaline-SDS lysis method (Sambrook et al.

1989), followed by PEG/NaCl treatment. The concentration of plasmid-DNA was checked by Spectrophotometer (Gene Spec III, Naka Ins truments Co., Ltd., Japan).

Figure 4-1-1. Schematic map of p lasmid pIG001. hpt, hygromycin phosphotransferase gene, conferring resistance to

hygromycin B; npt II, neomycin phosphotransferase II gene, harboring resistance to kanamycin; sgfp, synthetic green fluorescent protein gene; PUbi, promoter of a maize Ubiquitin-1; Pnos, promoter of nos (nopaline synthase) gene; P35S, the 35S promoter of cauliflower mosaic virus; Tnos, ter minator of nos; RB, right border; LB, left border.

RB LB

Pnos Tnos

nptII

PUbi

sgfp

hpt

Tnos Tnos P35S

57

3. HeliosTM gene gun bombardment procedures The preparation of bombardment for HeliosTM gene gun was done according to the

manufacturer’s instruction and method of Helenius et al. (2000) with a minor modification for rice

callus cells. In brief, 40 L of 50 mM spermidine was added into a microcentrifuge tube containing

12.5-25.0 mg gold particles, then sonicated for 5-8 s, and subsequently, 40 L of plasmid DNA

(50-100 g) in TE buffer was added and gently vortexed. While mixing, 40 L of 1M CaCl2 was

added drop by drop, to associate the DNA with the gold particles. The suspension was allowed to

settle at room temperature for 10 min. The gold-DNA pellets were washed three times by vortexing in 1 mL of absolute ethanol,

microcentrifuged at 12000 rpm for 1 min. and the supernatant was removed. The pellet was then resuspended in 3 mL absolute ethanol-PVP (polyvinylpyrollidone; 0.05 mg mL-1). By using a

syringe attached to an adapter, this suspension was drawn into a Tefzel tubing (Bio-Rad

58

Laboratories, USA) 76.2-cm in length. The tubing was then transferred into a tubing preparation

station. After gold beads were allowed to settle, ethanol was slowly drawn off and the turner was

rotated for 30 s, smearing the gold-DNA around the inside of the tubing. The residual ethanol was removed by passing nitrogen through the tubing for 3-5 min. The tubing was cut into 2.54 cm-long

tubes and then loaded into the HeliosTM gene gun device.

Figure 4-1-2. (a) Eight embryogenic calluses ready for HeliosTM gene gun bombardment. (b) Bombardment of rice calluses with a p lastic bowl to avoid jumping the target tissues by bombardment. Transient sgfp gene expression in rice calluses cvs. (c) Fatmawati and (d) Nipponbare (64X). The parameters applied were 250 psi, 0.6 m gold size, 0.25 mg gold par ticles per shot, 1 g plasmid-DNA delivered per shot, 0.4 M mannito l osmotic medium (4-6 h before and 16 h after bombardment), and 4-day pre-culture before bombardment. Scale bars in c) and d) correspond to 50 m.

a b

c d

59

Eight embryogenic calluses, 4-5 mm in diameter, were placed in the center of Petri dish with

filter papers (Fig. 4-1-2a), and covered with a plastic bowl with cavity that suites the spacer of

HeliosTM gene gun (Fig. 4-1-2b). These calluses were bombarded once, then immediately transferred to osmotic medium and incubated in the dark at 260C. Osmotic treatment of callus cells

with 0.4 M mannitol on medium NB5 was applied for 4-6 h before and 16 h after bombardment.

4. GFP monitoring and transient assessment sgfp-expressing cells were detected using a stereo-fluorescence microscope (MZFL III,

Leica) assisted with the GFP2 filter to mask the red fluorescence of chlorophylls resulting from the

non-transformed cells, thus allowing visualization of distinctive differences for the sgfp-expressing cells. The number of sgfp positive cells emitting green fluorescent spots was recorded. The sgfp-

expressing cells were always counted at the same magnification, i.e., 80X at the back and upper sides of the Petri dish, 2-3 days after bombardment. Photographs were developed by using

SnapShot software. Because some calluses were broken into pieces after bombardment, data on the average of green fluorescent spots for eight calluses were not available, and only the total

green fluorescent spots per bombardment was recorded.

In this experiment, the HeliosTM gene gun parameters were varied while all other parameters

were maintained as standard procedure as follows: 200 psi (equal to 1,379 Kpa), 0.6 m gold

particle diameter, 0.250 g gold particles per shot, 1.0 g plasmid-DNA per shot, 4-day pre-culture,

and 0.4 M mannitol of osmotic medium. Desiccation of callus cells was done using a vacuum drying oven (Yamato Scientific Co., Ltd., Japan) at 76 cm Hg without osmotic treatment. Callus

cells in the same age were used for parameters tested.

Data were then analyzed using one-way analysis of variance and the differences were analyzed using Duncan’s multiple range test (DMRT). Standard errors of the means were

calculated for four replications (bombardments).

60


Transient sgfp gene expression in rice calluses of two cultivars was observed within 48 to 72

h after bombardment with the HeliosTM gene gun (Fig. 4-1-2c, d). In total, 162 bombardments were performed and 1,344 embryogenic calluses from two genotypes were bombarded. The highest

level of transient expressions of sgfp was obtained with a helium acceleration pressure of 250 psi (1,724Kpa) in both cultivars (Figure 4-1-3). However, no significant difference was detected in the

number of sgfp spots between 200 psi (1,379 Kpa) and 250 psi in Fatmawati. A low acceleration pressure resulted in low number of sgfp spots. The high helium pressure seems to be very

powerful to drive plasmid DNA from the inner surface of tubing, so that i t can penetrate into deeper cell layers. It is presumed that acceleration pressure affects the depth of penetration and

distribution of gold particles into callus cells as already reported by Rasco-Gaunt et al. (1999) and Tee and Maziah (2005) using a PDS-1000/He device. They assumed that a high acceleration

pressure bombarded a smaller area than a low pressure. Moreover, Sanford et al. (1993) explained that the highest transient expression is generally achieved with rather violent treatments

giving better particle penetration, but these conditions may affect the cell division or growth. Extra

tissue damage or injury (Rasco-Gaunt et al., 1999; Tee and Maziah, 2005), more necrotic, no formation of new calluses or somatic embryos (Tadesse et al., 2003) are some adverse effects of

bombarded tissues with high acceleration pressure due to high velocity of gold particles. Tissue damage may be due not only by helium acceleration pressure, but also by some other parameters

such as gold particles size and amount of gold particles per shot.

Gold particles, 0.6 m in diameter, produced significantly a higher level of sgfp expression

than 1.0 m particles in Fatmawati, but not significant in Nipponbare (Fig. 4-1-4). It seems that a

0.6 m gold particle is more suitable than a 1.0 m particle to carry plasmid pIG001, which is

around 18 kbp. This result is in agreement with a previous report that the 0.6 m gold particle is

best for transient expression of the uidA gene ( -glucuronidase, GUS) in the leaves of arabidopsis,

tobacco and birch bombarded when using a HeliosTM gene gun (Helenius et al., 2000). A possible

explanation for this condition is that a small particle size (0.6 m) may cause less cell damage of

bombarded target tissues due to low particle mass, thus the cells may be capable of transiently

expressing sgfp gene, even capable of undergoing further division or growth.

61

Fatmawati

0

50

100

150

200

250

100 150 200 250

Acceleration pressure (psi)

Num

ber

of sg

fp s

pots

b

cc

a

Nipponbare

0

50

100

150

200

250

300

350

400

450

100 150 200 250

Acceleration pressure (psi)

Num

ber

of sg

fp s

pots

aa

a

b

Figure 4-1-3. Effect of acceleration pressure on sgfp expression in calluses of two cultivars. The bars represent the

average with standard error of the means from 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to Duncan’s multiple range test (DMRT, p = 0.05).

62

Fatmawati

0

10

20

30

40

50

60

70

80

90

100

0.6 1.0

Gold size (micron)

Num

ber

of s

gfp s

pots

b

a

Nipponbare

0

50

100

150

200

250

300

0.6 1.0

Gold size (micron)

Num

ber

of s

gfp s

pots

a

a

Figure 4-1-4. Effect of gold particles size on sgfp expression in calluses of two cultivars. The bars represent the

average with standard error of the means from 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to DMRT (p = 0.05).

A small amount of gold particles or microcarriers delivered per shot (0.25 mg) led to a

significant increase in transient expression of sgfp, particularly in Fatmawati (Fig. 4-1-5). No significant difference in the number of sgfp expression was found when Nipponbare calluses were

bombarded with different amounts of gold particles. Gold particles at more than 0.25 mg per shot produced no improvement in sgfp expression in either cultivar. A large number of cells are

probably damaged or killed by the large amount of gold particles delivered to the callus cells, resulting in a reduced amount of surviving cells, therefore undesirable for developing stable

transformation. Hunold et al. (1994) found that the viability of particle-containing cells decreased

63

within two days after bombardment, which supports this result. However, a minor different was

reported by Helenius et al. (2000), who found that 0.125 mg gold particles delivered per shot was

effective for transient luciferase activity in arabidopsis and tobacco leaves, but for uidA expression in birch leaves, 0.25 mg gold particles per shot were optimum.

Fatmawati

0

50

100

150

200

250

300

0.250 0.375 0.500

Amount of gold (mg shot-1

)

Num

ber of sg

fp s

pots

b

a a

Nipponbare

0

50

100

150

200

250

0.250 0.375 0.500

Amount of gold (mg shot-1

)

Num

ber of sg

fp s

pots

aa

a

Figure 4-1-5. Effect of the amount of gold par ticles delivered per shot on sgfp expression in calluses of two cultivars.

The bars represent the average with standard error of the means from 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to DMRT (p = 0.05).

The use of an appropriate concentration of DNA is important for efficient DNA-microcarrier

binding (Parveez et al., 1998). Plasmid-DNA delivered with 1.5 g per shot produced the largest

number of sgfp spots in Fatmawati, while in Nipponbare, 1.5 and 2.0 g plasmid-DNA per shot

were not significantly different in producing sgfp spots (Fig. 4-1-6). A similar observation has been

observed in oil palm (Parveez et al., 1998), in which 1.5 g plasmid-DNA per shot gave the

highest level of the GUS expression in embryogenic callus. Increasing plasmid-DNA concentration

64

improved the sgfp expression in Nipponbare, but not in Fatmawati calluses. It appears that the

sgfp expression was genotype-dependent in terms of differences in callus cell types they produced,

which has been described by Bec et al. (1998). Moreover, differences in the sgfp expression in calluses of the two cultivars bombarded using a HeliosTM gene gun might be attributed to the

difference in biological constraint such as the activity of endogenous nuclease (Barandiaran et al., 1998), local RNA silencing which blocks expression of transgene (Baulcombe, 2004; Miki et al.,

2005) and with physical constraint such as callus cell density or callus compactness or callus friability that might not be exactly identical in the two cultivars.

The limitation of using HeliosTM gene gun bombardment is the tubing that sometimes contains unequally distributed plasmid-DNA resulting in unequally spread plasmid-DNA in the

target tissues. In the present experiment, the sgfp expression was mostly limited to 2 to 5 calluses out of 8 bombarded calluses. In addition, Harwood et al. (2000) found that this device was not

suitable for barley transformation because the tissues jumped to other places by bombardment. However, by covering target tissues (callus cells) using a plastic bowl, this demerit could be

overcome.

65

Fatmawati

0

100

200

300

400

500

600

1.0 1.5 2.0

Amount of plasmid-DNA per shot (ug shot-1)

Num

ber

of s

gfp s

pots

a

b

a

Nipponbare

0

100

200

300

400

500

600

700

1.0 1.5 2.0

Amount of plasmid-DNA per shot (ug shot-1)

Num

ber

of s

gfp s

pots

a

b

b

Figure 4-1-6. Effect of the amount of plasmid-DNA delivered per shot on sgfp expression in calluses of two cultivars.


Bombarding target tissue at the right developmental stage is important because actively

dividing cells are most receptive to the particle bombardment (Moore et al., 1994). Pre-culture of callus cells prior to bombardment did not affect the sgfp expression as shown in Fig. 4-1-7.

However, it was reported that 7 day pre-culture and 4 day pre-culture gave the highest GUS expression in immature embryo of oil palm (Parveez et al., 1998) and in microspores of wheat

(Folling and Olesen, 2002). Moreover, in two types of orchid Dendrobium calluses, 2 day pre-culture period was suitable for sgfp expression (Tee and Maziah, 2005), indicating that the effect of

pre-culture duration for the efficiency of bombardment varies with the target tissue, though such

variation was not significantly found in rice callus cells. However, in order to minimize the

66

occurrence of somaclonal variations in the transgenic regenerants, 4 day pre-culture or less would

be beneficial.

Fatmawati

0

50

100

150

200

250

300

4 8 12

Pre-culture (d)

Num

er

of s

gfp s

pots

a

a

a

Nipponbare

0

50

100

150

200

250

4 8 12

Pre-culture (d)

Num

ber

of s

gfp s

pots a

a

a

Figure 4-1-7. Effect of pre-culture prior to bombardment on sgfp expression in calluses of two cultivars. The bars

represent the average with standard error of the means from 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to DMRT (p = 0.05).

Removing partial humidity of rice callus cells via desiccation treatment for 8 and 12 min. prior to bombardment gave a larger number of sgfp spots in Fatmawati (Fig. 4-1-8), but not in

Nipponbare. Desiccation treatment for more than 8 min. was not effective due to a reduction in sgfp expression. It is likely that prolonged exposure to desiccation leads to cell stress due to an

excessive leakage of protoplasm. Optimal desiccation time makes callus cells to be properly

plasmolyzed, which is apparently as the same effect of osmotic treatment, thus the cells may be

67

less likely to extrude their protoplasm following penetration of the cell by gold particles (Vain et al.,

1993).

The effect of age of target tissues for bombardment experiment should be considered for obtaining highly efficient transformation. Ramesh and Gupta (2005) discovered that maximum

GUS expression in rice calluses was observed when calluses were bombarded after 44 days old (6.3 wk) and tended to decrease in older callus age (68 days old or 9.7 wk). However, in

Fatmawati in this study, callus age did not significantly influence the number of sgfp spots, although the sgfp expression tended to decrease in older callus (Fig. 4-1-9), indicating the same

tendency as previously observed. A decrease in sgfp expression in older callus cells might be associated with physiological state of deterioration of callus cells due to the genetic alteration,

such as point or single-gene mutation, large-scale deletion, gross changes in chromosome structure/number (Kaeppler et al., 2000), transposable elements activation i.e., retrotransposons

(Hirochika et al., 1996), chloroplast deletion (Abe et al., 2002), or epigenetic change, such as DNA methylation (Kaeppler et al., 2000; Joyce et al., 2003) which is most frequently observed when in

vitro culture get older.

68

Fatmawati

0

50

100

150

200

250

300

350

400

0 4 8 12

Desiccation treatment (min)

Num

ber

of s

gfp s

pots

a

ab

c

bc

Nipponbare

0

20

40

60

80

100

120

140

160

180

0 4 8 12

Desiccation treatment (min)

Num

ber

of s

gfp s

pots

a a

a

a

Figure 4-1-8. Effect of desiccation treatment prior to bombardment on sgfp gene expression in calluses of two cultivars.


69

For stable expression of inserted genes for rice callus bombarded using HeliosTM gene gun,

use of helium at a pressure of 200 to 250 psi with 0.6 m gold particles size, 0.25 mg gold

particles delivered per shot, 4 day pre-culture, desiccation for 8 min. prior to bombardment, and

younger callus age (4 to 6 wk), is therefore recommended. Optimized parameters for HeliosTM gene gun device would be tremendously helpful for producing stable transformation of rice and

other cereal crops.

Fatmawati

0

50

100

150

200

250

300

10 33

Callus age (wk)

Num

ber

of s

gfp s

pots

a

a

Figure 4-1-9. Effect of callus age of Fatmawati on sgfp expression. The bars represent the average with standard error

of the means from 4 replicates, each with 8 embryogenic calluses. Same letters indicate values are not significantly different according to DMRT (p = 0.05).

Concluding remarks

Transient expression of sgfp as tailored in this section may be possible to extend to other applications like testing the gene expression constructs and producing the foreign proteins in very

short period of time as well as developing stable expression of transgenes, thus optimized parameters of HeliosTM gene gun will be valuable for both development of transient as well as

stable transformation.

70

Chapter 4

Genetic Transformation of Rice Plants Using Helios Gene Gun for Developing Transgenic Rice Lines

4.2. Genetic Transformation of the Wheat Glu-1Dx5 Gene to Rice

cv. Fatmawati

71

Abstract

The wheat Glu-1Dx5 gene encoding a high molecular weight (HMW) glutenin subunit 1Dx5 from

bread wheat, Triticum aesticum L. and either bar gene conferring resistance to herbicide bialaphos or hpt gene conferring resistance to hygromycin B was co-transformed to rice callus cells of cv.

Fatmawati using Helios gene gun device. Nine plants regenerated from bialaphos-containing medium and ten plants from hygromycin-containing medium were molecularly characterized. By

means of PCR analysis, the Glu-1Dx5 gene had been integrated into some transgenic rice plants. These plants will be incorporated into breeding program for further assessment of their benefits.

72

Genetic transformation offers an attractive opportunity to speed up the development of

genetically improved genotypes of crops and currently it has been utilized for improving

agronomically important traits of rice. This approach has been applied to complement classical plant breeding methods in rice genetic improvement. Recently it has been successfully employed

for rice quality improvement. These successful examples are rice with high iron content (Goto et al., 1999), human-lactoferrin (Nandi et al., 2002), low amylose content (Liu et al., 2005), and ß-

carotene (Golden Rice, Datta et al., 2003). Improvement on rice quality, especially on rice flour is of great relevance since rice flour has been used for many food products. However, dough made

from rice flour lacks extensibility and elasticity. A probable cause for this is that rice endosperm lacks the proteins responsible for this trait. On the other hand, dough made from wheat flour is

elastic and extensible making it suitable for many food products particularly for bread (Sangtong et al., 2002).

Wheat flour is different from other cereal flours, including rice, because it contains gluten that gives it elasticity and extensity required for breadmaking quality in bread wheat (Triticum

aesticum L.) (Payne et al., 1987; Barro et al., 1997). Gluten consists mainly of two types of seed

storage proteins, the glutenins and the gliadins. Glutenins are classified into high-molecular-weight (HMW) subunits and low-molecular-weight (LMW) subunits. They are encoded by Glu-A1 loci, Glu-

B1 loci, Glu-D1 loci, located in the long arms of the chromosome of homoeologous group 1 (Payne et al., 1987).

Genes encoding six HMW glutenins have been cloned (Anderson et al., 2002). The availability of cloned HMW glutenin genes allows plant transformation approaches to altering HMW

glutenin content. Cloned HMW glutenin genes have been shown to be functional when introduced into tobacco (Roberts et al., 1989), wheat (Alvarez et al., 2000), tritordeum (Rooke et al., 1999),

maize (Sangtong et al., 2002) and more recently into rye (Altpeter et al., 2004). Therefore, it is an attractive challenge to transfer the HMW glutenin into rice plants for a long term objective, i.e., to

improve rice flour quality traits. The objective of this chapter was to introduce the wheat Glu-1Dx5 gene into rice cv.

Fatmawati. This gene either with bar gene conferring resistance to herbicide bialaphos or with hpt

gene encoding a resistance to hygromycin B as selectable marker have been co-transformed to rice callus cells of cv. Fatmawati using HeliosTM gene gun bombardment device.

73


1. Preparation of target tissues Callus induction and maintenance were followed previous chapters and callus derived from

scutellar tissues of mature seed of cv. Fatmawati was chosen for genetic transformation

experiments based on its high regeneration capacity and not induce somaclonal variants in the spikelet-related traits. Culture media used for genetic transformation using HeliosTM gene gun is

presented in Table 4-2-1.

2. Plasmid DNA used in bombardment Plasmid pK+Dx5B (Anderson et al., 2002) and either pAHC25 (Christensen et al., 1996) or

pIG001 were used for bombardments. The pK+Dx5B contains an 8.2 kb genomic DNA fragment

from hexaploid bread wheat (cv. Cheyenne) that includes the Glu-1Dx5 coding sequence (GenBank Accession Number X12928; Fig. 4-2-1), 3.2 kb of 5' flanking sequence and 1.2 kb of 3'

flanking sequence (Halford et al., 1992). Plasmid pAHC25 contains bar gene (Thompson et al., 1987) from Streptococus hygroscopicus, conferring resistance to herbicide bialaphos/

phosphinotricin acetyl transferase (PAT), while pIG001 contains hpt gene conferring resistance to

hygromycin B. Previously UidA gene in pAHC25 has been removed by cleaved with BamHI then self-ligated, and resulting plasmid named pBar25. The schematic map for plasmid pIG001 is

presented in Fig. 4-1-1 (Chapter 4.1). The Glu-1Dx5 gene was driven by its own promoter, while promoter of maize ubiquitin-1 (Christensen and Quail, 1996) and the 35S promoter of cauliflower

mosaic virus were used to drive bar gene and hpt gene, respectively.

Figure 4-2-1. Schematic diagram of the pK+Dx5B which is consisted of coding sequence of the wheat Glu-1Dx5 gene

that used for transfor mation. Black areas represent the wheat genomic DNA, and the cross-hatched arrow represent the coding sequence of the wheat Glu-1Dx5 gene with arrow showing its d irection of transcription. The polylinker region of vector pBluescript is denoted with a clear region. The size of fragment generated upon digestion with EcoRI is shown bellow restriction map.

vector vector 1Dx5 coding

region

EcoRI EcoRI

8248 bp EcoRI

2519 bp

74

The plasmid was amplified in E. coli DH5αcells and purified by the alkaline-SDS lysis method

(Sambrook et al. 1989), followed by PEG/NaCl treatment. Confirmation of plasmids used for

bombardment was done by restriction digestion analysis and checked by agarose gel electrophoresis. The concentration of plasmid-DNA was checked by Spectrophotometer (Gene

Spec III, Naka Instruments Co., Ltd., Japan).

3. Helios gene gun bombardment procedures The bombardment procedure for HeliosTM gene gun (BioRad) followed method in the

previous section. The parameters applied in this study were 250 psi Helium pressure (equal to

1,724 Kpa), 0.6 m gold particle diameter, 0.25 g gold particles per shot, and 0.4 M mannitol of

osmotic medium. The amount of plasmid-DNA per shot was 1.0 g for pk+Dx5B and pBar25, while

1.5 for g pk+Dx5B and pIG001. The molar ratio used was 1:1 for both co-transformation events.

Eight embryogenic calluses were placed in the center of Petri dish with filter papers, and

covered with a plastic bowl with cavity that suites the spacer of HeliosTM gene gun. These calluses were bombarded once, then immediately transferred to osmotic medium and incubated in the dark

at 260C. Osmotic treatment of callus cells with 0.4 M mannitol on medium NB5 was applied for 4-6 h before and 16 h after bombardment.

4. Selection and regeneration of transformed calluses After exposure to osmotic treatment, immediately those calluses were then transferred to

NB5 subculture medium for one wk (Table 4-2-1). The same medium with 4 mg bialaphos or 50mgL-1 hygromycin was used for callus selection. Two to three successive selections were

applied. Resistant calluses as shown by arrows in Fig. 4-2-2a, b, were then transferred to pre-regeneration and subsequently to regeneration media.

5. Molecular characterization of transgenic rice plants Genomic DNA was extracted from leaves of primary transformed plants and untransformed

plants using Plant DNA extraction kit (Nucleon Phyto Pure, Amersham, USA). The primer design,

in silico PCR and restriction analysis were always carried out using FastPCR software (Kalendar, 2007).

PCR was carried out with 20-50 ng of genomic DNA, in reaction mixture containing 5 L

KOD Dash 10xPCR buffer, 5 L dNTP, 1 L each primer and 1 L KOD DNA polymerase (Toyobo

Co., LTD, Osaka, Japan).

75

Table 4-2-1. Medium used for HeliosTM gene gun transformation. Culture media Composition

Callus induction MS* medium, 3 mgL-1 2,4-D, 30 gL-1 sucrose, 2.5 gL-1

Phytagel, pH 5.8 Callus subculture NB5** medium, 3 mgL-1 2,4-D, 500 mgL-1 l-proline, 500

mgL-1 l-glutamine, 30 gL-1 maltose, pH 5.8

Callus selection NB5 medium, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 4 mg bialaphos or 50mgL-1 hygromycin, pH 5.8

Plant regeneration

a) pre-regeneration (1 wk): NB5 medium, 3 mgL-1 Kinetin, 3 mg L-1 BA, 10 mgL-1 ABA, 0.3 mgL-1 IAA, 0.3 mgL-1 NAA, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate, 30 gL-1 maltose, 4 mgL-1 bialaphos or 50mgL-1 hygromycin, 8 gL-1 agarose, pH 5.9

b) regeneration: NB5 medium, 3 mgL-1 Kinetin, 3 mgL-1 BA, 0.3 mgL-1 IAA, 0.3 mgL-1 NAA, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate, 30 gL-1 maltose, 4 mgL-1 bialaphos or 50mgL-1 hygromycin, 8 gL-1 agarose, pH 5.9

Plantlet rooting Half strength of MS medium with 4 mgL-1 bialaphos or 50mgL-1 hygromycin, 2 gL-1 Phytagel, pH 5.9

Note: *Medium MS from Murashige and Skoog, (1962); ** Medium NB5: adapted from Chu et al. (1975) for macronutrient, and Gamborg et al. (1968) for micronutrient and organic components.

76

PCR amplification was carried out for the Glu-1Dx5 coding sequence (upper primer 5'-

CTGGATCCATGCAGGCTACC-3'; lower primer 5'-AAGGATCCGAGACATGCAGC-3'). The PCR

conditions were as follows: 30 cycles for expected amplified product 3.2 kb in size (940C for 30 s, 590C for 4 s, 940C for 1 min. 25 s, and final extension 940C for 5 min.). DNA amplification was

carried out in a final volume of 50 L. PCR products were examined by electrophoresis through

1% agarose in 1x TBE buffer.


1. Selection of putative transgenic rice calluses A major problem during the course of transformation was a necrotic response which was

developed in calluses after transfer to selection medium containing 4 mgL-1 bialaphos (Fig.4-2-2a) or 50 mg mgL-1 hygromycin (Fig.4-2-2b). The growth of the bombarded callus cluster was severely

inhibited and they gradually turned brown. Mostly, their growth became retarded after exposure to bialaphos or hygromycin for 2-3 wk in the selection medium. In appearance, calluses become

mixed between necrotic or browning and pale yellow or whitish which was varied in some regions of calluses (Fig. 4-2-2a, b). This could be due to some callus cells already died and some survive,

which can be hypothesized that transgenes already integrated into rice genome. This is because the medium is very toxic for other callus cells that not expressing the bar or hpt gene. In addition,

alteration in callus color was frequently found, from embryogenic pale yellow to a bit brownish,

suggested some browning calluses were non-transformed callus cells or they were weak in physiological development due to nutrient deficiency. Some category of callus has been detected

during the course of bombardment following its exposure with bialaphos or hygromycin: 1. Callus that are in turn browning, dark-browning after 24 to 72 h in the selection medium;

2. Calluses that are mix between yellow pale and browning in some part of the callus surface; 3. Calluses that are fast growing, highly friable, not nodular/globular in appearance, with slightly

yellow, however, they can not induce shoot or plantlets if they are regenerated. During regeneration experiment, shoots were abundantly obtained, however green plantlets

were obviously rare, especially on calluses that bombarded with pK+Dx5B and pBar25 (growing on bialaphos-containing medium). It has been known that transformation efficiencies are

suboptimal with toxic substrates like bialaphos or hyrgomycin, because dying untransformed cells

may inhibit transformed cells from proliferating by secreting inhibitors or preventing transport of essential nutrients to the living transformed cells (Haldrup et al., 1998).

77

Fig. 4-2-2. Growth and appearance of calluses of cv. Fatmawati after two successive selections on: a) medium

contain ing 4 mgL-1 bia laphos, bombarded with pK+Dx5B and pBar25, and b) medium containing 50mgL-1 hygromycin, bombarded w ith pK+Dx5B and pIG001. Arrows show the survived calluses.

Fig. 4-2-3. Plantlets of putative transgenic rice plants in rooting medium contain ing a) bia laphos, b) hygromycin.

a b

a b

78

After two till three subcultures on bialaphos-containing NB5 medium, 11 putative transgenic rice

plants were regenerated, but only 9 plants were able to grow further (Fig. 4-2-3a). But, in contrast,

around 70 plants were obtained from hygromycin-containing NB5 medium and were able to grow further (Fig. 4-2-3b). They were grown in the glass house of Genomic Center, Utsunomiya

University. 2. Molecular detection of the wheat Glu-1Dx5 gene in transgenic plants

Molecular screening by means of PCR revealed that some transgenic lines contain the Glu-

1Dx5 coding sequence, though some not (Fig. 4-2-4). Using primers that designed specifically

amplify the 3.2 kb of expected DNA fragment for the Glu-1Dx5 gene was successfully detected. However, this fragment was not detected in untransformed plant (negative control), while this band

was able to appear in positive control using plasmid pK+Dx5 as template (Fig. 4-2-4). From this result, it is shown that a 3.2 kb of 5' flanking sequence of the Glu-1Dx5 provides a

promoter that can drive the gene for transcription and translation. However, further characterization regarding this topic should be carried out by the gene expression analysis, such

as using northern blot or RT (reverse-transcription)-PCR.

To predict the existence of promoter in the full-length of 8.2 kb Glu-1Dx5 DNA sequence was then examined by TSSP-Prediction of Plant Promoter Software (Softberry Inc.), which revealed

that at least 4 promoters or enhancers were found on the basis of TATA box prediction. It is obvious also that such promoter did not act as a specific promoter, which drives the

gene specifically expressed in specific tissues, such as in the endosperm of rice seed. It is due to Sangtong et al. (2002), were not able to detect the Glu-1Dx5 protein in the leaves of transgenic

maize. Success in introducing this valuable gene (Glu-1Dx5) into rice cultivar will open the

possibility to improve rice flour quality traits. These promising transgenic rice plants will be examined further and then will be incorporated into rice breeding program for assessing their benefits.

79

1 2 3 4 5 6 7 8 9 M C+ C-

Fig. 4-2-4. PCR amplifications of 3.2 kb fragment of the Glu-1Dx5 coding sequence from transgenic rice plants cv. Fatmawati. Lanes 1 – 9: transgenic plant line numbers, lane M: 1 kb DNA Ladder (Invitrogen), C+: positive control (pK+Dx5 as template), C-: negative control (untransfor med plant genomic DNA as template).

3054 bp 1636 bp

80

Concluding remarks

It has been shown that the Glu-1Dx5 gene from bread wheat has been able to be transferred into rice cv. Fatmawati. Some transgenic plants selected from this research will be

valuable materials for breeding purposes and for further research in transgene expression, transgene stability or other topics that related to application and assessment of transgenic plants

derived from particle bombardment-based method.

81

Chapter 5

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens

Transient Expression of Green Fluorescent Protein in Rice Calluses of cvs. Fatmawati and Nipponbare

82

Abstract

Transient expression of synthetic green fluorescent protein (sgfp) gene mediated by

Agrobacterium is rapid and useful approach for visual monitoring the genetic transformation event in transformed cells/tissues of examined genotype. The significant differences in transient

expression of two genotypes were found with regard to the osmotic treatment (0.4 M mannitol), solid-subcultured callus, and 10 min. air drying. While, no significant differences in sgfp expression

were observed in two genotypes on without air-drying and 5 min. air-drying of calluses prior immersed in Agrobacterium suspension. Surprisingly, the sgfp expression could not be detected in

suspension-subcultured callus of both cultivars. The highest sgfp expression was achieved in Nipponbare callus treated with 10 min. air drying. The level of green fluorescent spots was higher

in Nipponbare than that in Fatmawati, however, plants regenerated from Fatmawati were considerably comparable with those of Nipponbare. Seventeen and 16 transgenic rice plants

expressing sgfp transgene were obtained from Nipponbare and Fatmawati, respectively.

83

Introduction

Genetic transformation mediated by Agrobacterium tumefaciens, a soil plant pathogenic

bacterium, is one of the methods for the introduction of the foreign genes into plant cells. This is due to Agrobacterium has the exceptional ability to transfer a particular DNA segment (T-DNA) of

the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is then stably integrated into the host genome (Binns and Thomashow, 1988). The initial studies on T-DNA transfer

process to plant cells demonstrate three important facts for the practical use of this process for plant transformation. Firstly, the tumor formation is a transformation process of plant cells resulted

from transfer and integration of T-DNA and the subsequent expression of T-DNA genes. Secondly, the T-DNA genes are transcribed only in plant cells and do not play any role during the transfer

process. Thirdly, any foreign DNA placed between the T-DNA borders can be transferred to plant cells, no matter it comes from. These well-established facts allowed the construction of the first

vector and bacterial strain systems for plant genetic transformation (Torisky et al., 1997). Agrobacterium-mediated transformation has been established for many years in

dicotyledonous plants, and recently has been adapted to monocotyledonous plants including rice

(Hiei et al., 1994; Rashid et al., 1996). In order to visualize the genetic transformation event mediated by Agrobacterium, transient expression of reporter gene i.e. synthetic green fluorescent

protein (sgfp) is rapid and useful method for this purpose. Transient expression occurs almost immediately after gene transfer, it does not require the regeneration of whole plants, and it occurs

at much higher frequency than stable integration (Altpeter et al., 2005). Thus, in this chapter, the author applied a sgfp gene as reporter and non-destructive markers to visualize the genetic

transformation event mediated by Agrobacterium in transformed callus cells of cvs. Fatmawati and Nipponbare.

84


A. tumefaciens strain EHA105 harboring plasmid pIG001 was used. Plasmid pIG001 was a

binary vector that contains a neomycin phosphotransferase (npt II) gene, a hygromycin-resistance gene (hpt) and sgfp gene as described in Chapter 4.1. (Fig. 4-1-1). The Agrobacterium strain

EHA105 (pIG001) was a kind gift from Dr. Hiroyuki Anzai, Gene Research Center, Ibaraki University, Japan. Agrobacterium was grown in LB medium (Tabel 5-1) at 270C in a gyratory

shaker for 3 days. Agrobacterium cells were collected and resuspended in NB5 liquid medium with

200 M acetosyringone. Six weeks old calluses, induced from mature seeds of Fatmawati and

Nipponbare, were placed in bacterial suspension for 10 min. After briefly draining the calluses on

sterilized paper filter, calluses were then transferred to NB5 solid medium with 200 M

acetosyringone and co-cultivated at 270C in the dark for 3 days. The co-cultivated calluses were washed three times with sterile water containing 250 mgL-1 cefotaxime and 250 mgL-1 carbenicilin

to kill Agrobacterium. The transformed calluses were then incubated in the selection medium containing

hygromycin B 50 mgL-1, cefotaxime 250 mgL-1 and carbenicilin 250 mgL-1 for 2-3 weeks. Small, good-looking, hygromycin resistant calluses were obtained, and then transferred to pre-

regeneration medium for one wk and subsequently transferred to fresh regeneration medium (Table 5-1).

Monitoring sgfp expression was performed according to previous chapter. A completely randomized design with three replications, each with 10 calluses, was used in this study.

Transformation frequency was evaluated on day 2 after co-cultivation as the total number of green

fluorescent spots (transformation events) per explant. Data were then analyzed statistically by analysis of variance and the differences between means of two genotypes in sgfp

expressions/spots were evaluated by Duncan’s multiple range test (DMRT).

85

Table 5-1. Medium used for bacterial culture, tissue culture and Agrobacterium-mediated transformation.

Culture media Composition

Callus induction MS* medium, 3 mgL-1 2,4-D, 30 gL-1 sucrose, 2.5 gL-1 Phytagel, pH 5.8 Callus subculture a) suspension: NB5** medium, 3 mgL-1 2,4-D, 500 mgL-1 l-

proline, 500 mgL-1 l-glutamine, 30 gL-1 maltose, 2.5 gL-1

Phytagel, pH 5.8 b) solid: NB5 medium, 3 mgL-1 2,4-D, 500 mgL-1 l-proline, 500

mgL-1 l-glutamine, 30 gL-1 maltose, pH 5.8

Agrobacterium culture LB medium, 10 gL-1 glucose, 50 mgL-1 s treptomycin and 50 mgL-1 hygromycin

Co-cultivation a) liquid: NB5 medium, 10 gL-1 glucose, 200 M acetosyringone, pH 5.4

b) solid: NB5 medium, 10 gL-1 glucose, 4 gL-1 phytagel, 200 M acetosyringone, pH 5.4

Callus selec tion NB5 medium, 50 mgL-1 hygromycin, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 250 mgL-1 carbenicillin, 250 mg L-1 cefotax ime, pH 5.8

Plant regeneration

c) pre-regeneration (1 wk): NB5 medium, 3 mgL-1 Kinetin, 3 mg L-1 BA, 10 mgL-1 ABA, 0.3 mgL-1 IAA, 0.3 mgL-1 NAA, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate, 30 gL-1 maltose, 50mgL-1 hygromycin, 8 gL-1 agarose, pH 5.9

d) regeneration: NB5 medium, 3 mgL-1 K inetin, 3 mgL-1 BA, 0.3 mgL-1 IAA, 0.3 mgL-1 NAA, 500 mgL-1 l-proline, 500 mgL-1 l-glutamine, 800 mgL-1 casein hydrolysate, 30 gL-1 maltose, 50mgL-1 hygromycin, 8 gL-1 agarose, pH 5.9

Plantlet rooting Half s trength of MS medium, 50mgL-1 hygromycin, 2 gL-1 Phytagel, pH 5.9

Note: *Medium MS from Murashige and Skoog, (1962); ** Medium NB5: adapted from Chu et al. (1975) for macronutrient, and Gamborg et a l. (1968) for micronutrient and organic components.

86

Results and Discussion Two days after co-cultivation of rice calluses with Agrobacterium suspension, green

fluorescent spots corresponding to transformation events were observed in both cultivars (Fig. 5-1a, b). These sectors were clearly distinguishable from untransformed parts. Around 2 to 90 sgfp-

expressing cells were observed per explant on back and upper of Petri dish. However, the fluorescent was limited only in small parts of the callus cells. It is apparent that the sgfp gene

driven by ubiquitin-1 promoter isolated from maize was working well in rice callus. Fluorescent was not observed in the callus of the control (immersed in water).

The genetic transformation mediated by Agrobacterium has been adapted for various plant species, including rice, which was formerly considered to be recalcitrant to Agrobacterium. The

studies on genetic transformation of rice suggest that numerous factors including Agrobacterium strain, vectors, plant genotype, explant, selection marker and selective agent, various conditions of

culture media, are of importance (Hiei et al., 1997). The results, here, demonstrate that transient expression of sgfp was significantly different in two genotypes with regard to osmotic treatment

(0.4 M mannitol) (Table 5-2).

Osmotic treatment, provided by 0.4 M mannitol, could act to protect the target tissue to less extrude their protoplasm following Agrobacterium infection, due to a plasmolysis effect (Vain et al.,

1993) and reducing cell turgidity. Osmotic treatment for enhancement of Agrobacterium-mediated transformation is largely dependent on genotype as found in this study. It is likely both genotypes

had discrepancy of response to osmotic treatment, in which Nipponbare was the most susceptible by Agrobacterium infection since we obtained higher expression of sgfp in japonica Nipponbare

than in indica Fatmawati (Table 5-3). Indica varieties are generally recalcitrant in both tissue culture and plant transformation, and are poor hosts for Agrobacterium (Hiei et al. 1997). A

recalcitrant of monocotyledonous plants to Agrobacterium-mediated transformation may be due to an appropriate process in the following one or more steps involved in transformation: chemotaxis

of Agrobacterium towards wounded plant cells (Parke et al., 1987), binding of Agrobacterium to plant cells (Douglas et al., 1982), inducing of vir genes by plant signal molecules (Stachel et al.,

1985; Vijayachandra et al., 1995), generation of T-DNA transfer intermediates (Veluthambi et al.,

1987), transfer of T-DNA transfer intermediates to plant cells and integration of T-DNA into plant genome (Simpson et al., 1982).

87

Fig. 5-1. The expression of sgfp in rice calluses cv. Fatmawati (a, 80X) from solid-grown-derived callus, and

Nipponbare (b, 64X) treated with 10 min. a ir drying. The transfor med callus and regenerating shoots

expressing sgfp cv. Nipponbare (d), and leaves of control (cv. Nipponbare, non-transforman). Bars on a) and

b) are 50m, while c) and d) are 0.2 mm.

a b

c d

88

Tabel 5-2. Analysis of variance for parameters tested on sgfp expression of two cultivars. Source of variation

df Osmotic (0.4 M)

Solid-grown derived callus

No-air drying 5 min. air drying

10 min. air drying

Genotype 1 421.4** 3212.0** 3.8ns 156.8 ns 1041.7*

Error 58 56.5 225.0 178.1 129.8 234.6

Total 59

Data show mean square values. ns: nonsignificant, *: significant at p = 0.05, **: significant at p = 0.01, respectively. Table 5-3. Comparison for cultivar on sgfp expression.

No Tested parameters Fatmawati Nipponbare

1. Osmotic medium (0.4 M mannitol) 0.83 2.33 A 6.13 5.68 B

2. Solid-subcultured callus 1.50 3.01 A 16.13 9.31 B

3. Liquid-subcultured callus 0.00 0.00 A 0.00 0.00 A

4. No-air drying 5.07 8.93 A 4.57 6.24 A

5. 5 min. air drying 0.23 0.74 A 3.47 9.27 A

6. 10 min. air drying 0.17 0.43 A 8.50 2.50 B

Data show means with standard error of the means. The differences were analyzed by DMRT.

89

In both cultivars, the sgfp expression was significantly influenced by callus that was

subcultured in solid-agar media (Table 5-2). Such expression was lower in Fatmawati than in

Nipponbare (Table 5-3), indicating Nipponbare is more susceptible by Agrobacterium infection

than Fatmawati. But, surprisingly, no sgfp spots were detected in the suspension-subcultured

callus of both cultivars (Table 5-3), suggesting the suspension-subcultured callus is inefficient for

Agrobacterium-mediated transformation. This may be explained by the fact that, in many

monocotyledonous, the cells (at wound site) tend to be lignified or sclerified (Hiei et al., 1997),

which may inhibit the Agrobacterium to directly infect the rice cells. Another explanation is that

inclusion of 200 M acetosyringone was not sufficient for inducing vir genes for suspension-

subcultured cells. However, transgenic rice plants had been obtained via Agrobacterium using a

callus suspension-subcultured cells (Urushibara et al., 2001; Deng et al., 2003), without clearly

stated the amount of acetosyringone used in their transformation experiments.

A significant factor that enhances transformation of crop species is desiccation of explants prior to or after Agrobacterium infection. Ten min. air drying was found to be significant in obtaining

the sgfp expression of two cultivars prior immersed in Agrobacterium suspension, but not significantly different when rice callus cells were treated with no air drying and five min. air drying

(Table 5-2 and Table 5-3). Ten min. air drying apparently improved the sgfp expression in Nipponbare, but not in Fatmawati (Table 5.3), suggesting the difference in cultivar’s response to

prolonged air-drying treatment. Urushibara et al. (2001) reported that 10 to 15 min. air-drying treatment of Nipponbare suspension culture enhanced the transformation efficiency 10-fold or

more as compared to the control without air drying (solid-subcultured callus). In this experiment, overgrowth of Agrobacterium was frequently found in calluses of

Nipponbare. At the end of the course of Agrobacterium-mediated transformation, the author

obtained around 58 calluses resistant to hygromycin in Nipponbare, while only 7 calluses in Fatmawati. However, plants regenerated from Fatmawati were considerably comparable with

those of Nipponbare. Seventeen and 16 transgenic rice plants expressing sgfp transgene (as screened by a fluorescent microscope shown in Fig. 5-1c) were obtained from Nipponbare and

Fatmawati, respectively.

90

Concluding Remarks

Gene transfer mediated by Agrobacterium offers a valuable tool for plant improvement

programs, especially for application in developing country. Transient sgfp gene expression will be of greatly helpful in testing the expression vector, optimizing some parameters for Agrobacterium-

mediated transformation and selecting the suitable genotype for Agrobacterium-mediated transformation, thus, in turn, it will improve the efficiency of genetic transformation mediated by

Agrobacterium, which is to date still far from efficient for some rice genotypes.

91

Chapter 6

General Discussion

92

Rice has evolved to humans to be a major staple food crop and has recently recognized to

be a cereal model plant (Shimamoto and Kyozuka, 2002) from which gene function discovery is

projected to contribute to genetic improvement in a variety of other cereals like maize and wheat.

The recent release of rough draft of the rice genome sequence (Sasaki et al., 2002; Yu et al.,

2002) for public research community provides a vast resource of gene sequences whose function

need to be determined by reverse genetics (’from sequenced gene to function’) or by forward

genetics approach (’from mutant phenotype to gene’) using the advent of genomics based

technologies such as genetic transformation technology. This valuable tool is not only for basic

research, such as for finding the unknown gene function of rice (Pereira, 2000), but also for rice

genetic improvement program, which recently has been successfully applied, for example, rice

transformed with human-lactoferrin gene (Nandi et al., 2002) and other, rice engineered with

phytoene synthase, lycopene β-cyclase and phytoene desaturase genes which enabling

biosynthesis of provitamin A in rice endosperm (Datta et al., 2003). These successful

breakthroughs and other functional genomic efforts on rice can only be achieved if the in vitro

culture, which allows multiplication of embryogenic target tissues as well as efficient plant

regeneration system, has been established well. The effort on the establishment of in vitro culture

is highly demanded since some rice cultivars have been known to be recalcitrant for in vitro

manipulation (Abe and Futsuhara, 1986; Seraj et al., 1997).

The establishment of in vitro culture for Indonesian rice genotypes has been successfully

accomplished in this thesis. Genotype, medium and explant are considered to be essential factors

for inducing high quality callus as genotype x medium x explant interaction effect was significantly

detected. Moreover, reliable phenotypic assessment on callus performance, i.e., good-looking

callus with white/cream/yellow appearance, no browning spot/healthy, and actively growing that

supposed to be embryogenic, may lead to identify the promising genotypes with high quality callus

production. Visual selection of embryogenic callus based on morphology has been extensively

studied by Visarada et al. (2002) which enable to enhance a number of plants regenerated.

However, they did not present the quantitative data as described in this thesis. The author does

hope this approach could be applied and further adapted to other experiments dealing with in vitro

93

culture of rice.

The explant of mature seed embryos from the selected genotypes, i.e., Fatmawati, Ciapus,

BP-23 and BP-360-3, grown on medium MS was found to be the best combination for obtaining

high quality callus as revealed by Anova analysis. This result clearly indicates the importance of

simultaneous screening for genotype, explant, and medium by reliable phenotypic assessment in

callus induction performance may direct to successful regeneration of green and fertile rice plants,

which is then proved in subsequent plant regeneration experiments.

In the study of plant regeneration capacity, the author found that subculture provides means

for multiplication of embryogenic calluses. Five Indonesian rice genotypes selected from preceding

experiments were capable in regenerating intact green plantlets. This achievement was possible

by manipulation of medium culture as suggested by those former experiments including more

gelling agent (Lai and Liu, 1988; Lee et al., 2002), inclusion of amino acids, i.e., l-glutamine (Pons

et al., 2000) and l-proline (Yang et al., 1999; Chowdhry et al., 2000), casein hydrolysate and

maltose (Lin and Zhang, 2005), and proper ratio of auxin-cytokinin (Masuda et al., 1989; Lin and

Zhang, 2005). However, only two genotypes, Fatmawati and BP-140 that consistently performed

best in the subculture as well as in the plant regeneration and culture media DI and NB5 were

selected as suitable media for subculture and plant regeneration, respectively.

Testing a number of selected genotypes in combination with media for subculture and plant

regeneration as adapted in this study, in my view, is the proper strategy for exploring the

totipotency of genotype and the suitability of medium since genotype x medium interaction effect is

often found in regeneration frequency of rice (Khanna and Raina, 1998) and plant regeneration-

capacity in this thesis, thus being more efficient in finding a suitable genotype-medium combination.

Somaclonal variations induced by in vitro culture of rice have been well studied so far

(Chatterjee and Das-Gupta, 1997; Chowdari et al., 1998; Lee at al., 1999b; Abe et al., 2002) and it

has been proven that somaclonal variants do exist in transgenic rice assessed by DNA

polymorphisms (Arencibia et al., 1998) and phenotypic assessment (Jiang et al., 2000).

Phenotypic evaluation in the spikelet-related traits of primary callus-regenerated plants (T0)

showed that the occurrence of somaclonal variants varied with the genotype as revealed by t-test.

94

Genotypes Ciapus and BP-140 were found to be significantly difference in spikelet-fertility,

suggesting abnormalities at meiosis may have occurred due to their genome instability during

cultured in vitro (Phillips et al., 1994; Arencibia et al., 1998; Jain, 2001). Due to minimum

somaclonal variants are highly desired in developing transgenic rice plants, innovative efforts

should be undertaken for shortening in vitro culture, such as by direct utilization of regenerative

tissues i.e., root segment as target in transformation. However, much effort would be needed due

to less efficiency in plant regeneration.

Optimization of parameters of HeliosTM gene gun for efficient genetic transformation for rice

calluses based on synthetic green fluorescent protein (sgfp) expression has been successfully

conducted. Helium acceleration pressure, gold particle size, amount of gold particle delivered per

shot, amount of plasmid-DNA per shot are being of importance. In addition, the use of binary

vector pIG001, containing both visual marker gene, sgfp, and selectable marker gene, hpt, in one

plasmid, enabled visual detection of transformation event as well as selection of transformed

tissues simultaneously. The sgfp expressions were slightly difference in calluses of japonica

Nipponbare and indica Fatmawati in each parameter tested which can be attributed to the

difference in biological constraint, such as the activity of endogenous nuclease (Barandiaran et al.,

1998), local RNA silencing which blocks expression of transgene (Baulcombe, 2004; Miki et al.,

2005) and physical constraint, such as callus cell density, callus compactness or callus friability

that might not be exactly identical in those two cultivars. Optimization of such parameters for

HeliosTM gene gun bombardment for rice callus will open the possibility to other applications

particularly to other cereal crops.

After establishing in vitro cultures for callus induction, proliferation, plant regeneration as

well as optimizing the gene delivery method i.e., HeliosTM gene gun bombardment, improvement

on rice quality traits via genetic transformation becomes greatly possible. The introduction of the

wheat Glu-1Dx5 gene in combination with either seletable marker bar or hpt gene into rice cv.

Fatmawati has been performed well. Nine plants regenerated from bialaphos-containing medium

and ten plants regenerated from hygromycin-containing medium were molecularly characterized.

PCR analysis confirmed the presence of transgene in their genome. Transgenic rice plants

95

obtained from this research would then be selected based on their transgene expression, and then

they will be incorporated into breeding program for further assessment of their benefits.

In the course of this thesis, beginning with callus induction and plant regeneration,

somaclonal variant assessment, HeliosTM gene gun optimization, and ending up with introducing

the wheat Glu-1Dx5 gene, would therefore significantly contribute to the further development of

rice genetic improvement endeavors.

96

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* In Portuguese with English summary. ** In Japanese with English summary

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Acknowledgements

First of all, I deeply praise Alloh, the Most Beneficent, the Most Merciful for His blessing

and His gift to fill my life with very little knowledge about His unique creativity, the gene. My deepest gratitude goes to my mother, Ibu Rokiyah (late) and my father, Bapak Sudarma (late), the

first teachers who taught me to live and love nature. In particular, I am much indebted to many

other teachers who taught me invaluable knowledge since my primary school in Indonesia until my doctorate degree in Utsunomiya University, Japan.

After almost three years, finally I came to the end of my doctoral study, here, in United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology. Although,

in the beginning, I would never imagine that I could finish this manuscript on time since I wish many things to do. However, with the completion of this dissertation, I would like to express my

sincere gratitude to all people for their special contribution at scientific and personal matters. Prof. Dr. Tomohiko Yoshida, my supervisor, contributed the most from the first day of my

arrival to today and guided me during the whole my PhD study. Your strategic suggestion to do many things in parallel and concentrate on some promising ones has enabled me to graduate in

this university. Without your sincere guidance, valuable advice and critical comments, I wouldn’t

come to this achievement. For these, I would like to thank you. I greatly acknowledge Prof. Dr. Tadashi Hirasawa, Tokyo University of Agriculture and

Technology, who has kindly supervised this thesis. I appreciate his valuable contribution to improve the thesis.

I wish to express my gratitude to Prof. Dr. Hitoshi Honjo, Professor of Utsunomiya University, for his invaluable suggestions. I really appreciate for sharing your experiences when

you were in Indonesia. I always remember when you mentioned some Indonesian words. My grateful thanks are due to Prof. Dr. Toshiaki Matsuda, Professor of Ibaraki University,

for his valuable suggestion in preparing the manuscript. I am very much indebted to Dr. Yoshiharu Wada, Laboratory of Crop Science, Utsunomiya

University, who was always available for help whenever I needed. I appreciate very much your critical advice and support during my study.

Thanks also go to Prof. Dr. Tomohide Natsuaki, who gave me a permission to do

molecular works in his lab and Genomic Center for Gene gun transformation and transgenic rice experiment.

My great thanks also to some other researchers: Dr. Buang Abdullah, Indonesia Institute

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for Rice Research, Sukamandi, Indonesia, for kindly providing indica rice seeds; Dr. Ann Blechl,

United States Department of Agriculture, Agriculture Research Service (ARS), for plasmid

pK+Dx5B; Prof. RGF Visser, Wageningen University, for plasmid pAHC25 with permission of Prof. Peter H. Quail, University of California, USA; Dr. Anzai Hiroyuki, Gene Research Center, Ibaraki

University for kindly providing Agrobacterium tumefaciens strain EHA105 harboring plasmid pIG001.

To past and present friends in Lab. of Crop Science: Takumi Anazawa (thanks for taking good care of the rice plants) and Ly Tong (thanks for helping me in daily life matters in Japan).

Michael, Komatsubara, Saiga, Yoshiizumi, Kaji, etc., I would like to thank for our friendship during my stay in the lab.

To people from Lab. of Plant Pathology: Wan and Suehiro (thanks for teaching me gene gun bombardment and gfp assessment), Asad and Abdullah (thanks for their help in molecular

works). I would like to thank my Indonesian friends for their warm friendships and kindness: Pak Arif and Pak Arien with their family, Anna, Chichi, Sidiq, and Mbak Dewi.

Appreciations are extended to the Japanese Government for three years

Monbukagakusho Scholarship. I would like to thank Prof. Dr. Achmad Baihaki for his encouragement and support, and all

staff at the Lab. of Plant Breeding, Padjadjaran University, Indonesia, for their generous support. SpeciaI thank goes to Dr. Anas, who sent me an application form.

My deepest gratitude goes to all my brothers for their love, encouragement and continuous support during my study. I would like to thank my mother in law, Ibu Hj. Noh Nurrohmah

and father, Bapak Eman Nurzaman (late), for their warm support and excellent taking care of my children.

My very special thanks and my deepest feelings go to my beloved wife Elia Nurma Rahmawati, who always amazes me with her love. Thanks for taking care of our lovely children:

Haeqal, Iqbal and Hana. Thanks for continuous support, understanding and your patience during whole my PhD period.

Time to go back home, hope to see you again in the future…….

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Curriculum Vitae

Nono Carsono was born in Indramayu, West Java, Indonesia on 10th of October 1972. He

grew up in this small coastal town until he finished high school. He completed his undergraduate study in Plant Breeding at the Faculty of Agriculture, Padjadjaran University, Indonesia on 20th of

August 1996, presenting a thesis entitled ‘Genetic Control of Red Pepper Male Sterility’ supervised by Prof. Ridwan Setiamihardja PhD, Ceciliany Permadi MS, and Dr. Nani Hermiati. After

completing his undergraduate program, he joined the same laboratory where he was studying. He attained his MSc degree from Laboratory of Plant Breeding, Department of Plant

Sciences, Wageningen University on 24th of January 2003 with a thesis entitled ‘Identification of

Differentially Expressed Genes Using cDNA-AFLP during Six Days in in vitro Tuberization of

Potato’. The experimental work was carried out under the supervision of Dr. Christian Bachem and

with support from The Netherlands Fellowship Programme (NFP-RFP). In October 2004, he had a chance to pursue doctorate degree at the United Graduate

School of Agricultural Science, Tokyo University of Agriculture and Technology with the scholarship provided by Japanese Government (Monbukagakusho) and under supervision of Prof. Dr.

Tomohiko Yoshida at the Laboratory of Crop Science, Utsunomiya University, with a research project on the development of transgenic rice lines. After obtaining his PhD degree, he will return

to work as lecturer and researcher at the Laboratory of Plant Breeding, Padjadjaran University, Bandung, Indonesia.

E-mail address: [email protected]

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