TOBACCO AND TOMATO TISSUE CULTURE, GENE ISOLATION...

CHAPTER 4

TOBACCO AND TOMATO TISSUE CULTURE, GENE ISOLATION

AND GENETIC TRANSFORMATION

4.0 ABSTRACT

The aim of this study was to isolate the Antifreeze protein (AFP) gene from carrot

and genetic transformation of tobacco and tomato. The carrot AFP gene was isolated and

cloned to the pBI121 binary vector. The AFP gene was confirmed by DNA sequencing.

For genetic transformation studies, standardization of tissue culture techniques was

carried out for tomato. Plant regeneration efficiency varied with different explants,

cotyledonary leaves showed maximum regeneration capacity and hence used as the

explants in the present study. The cotyledonary leaves of tomato and leaf discs of tobacco

were used for Agrobacterium mediated genetic transformation. The transgenic plants

were hardened to soil. The seeds of both tobacco and tomato plants were collected and

used for further studies.

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4.1 INTRODUCTION

Cold and freezing temperature significantly confines the geographical

distribution of crops and frequently causes significant loss in the agriculture productivity.

The alterations in morphological, physiological, biochemical and molecular responses

due to cold stress allow some plants to overcome the extreme conditions. Unfortunately,

these adpatations are lacking for most of the economically important crops on which

countless people depend for survival. This situation is likely to worsen in future in view

of the climate change and global warming resulting in unpredictable climatic conditions

(Singh et al., 2010a). Hence, many strategies have been developed to withstand the low

temperature stress without affecting the yield of the crops.

The conventional plant breeding strategies have met with limited success in

improving the specific characteristics like freezing tolerance of important vegetables and

other cultivated crops (Kumar & Bhatt 2006). Hence, the application of modern

biotechnological tools along with the breeding methods was suggested for the

development of stress tolerant crop plants (Singh et al., 2010b). Understanding the

complex physiological, biochemical and molecular mechanisms and their engineering

provides enormous possibilities for improving the low temperature tolerance in plants.

The ability to transfer the genes from one plant to other (by genetic transformation

methods) without crossing provides new opportunities to improve the efficiency of crops

performance, especially during unfavourable conditions. Several attempts were made to

express diverse gene candidates to confer cold tolerance. Potential benefits (other than

low temperature tolerance) includes plants with better yield, vegetables with enhanced

nutritional values, reduction in use of fertilizers and pesticides, prolonged shelf life of


fruits and flowers, tolerance to other abiotic stress factors and biotic agents, etc., Many of

the useful traits like herbicide resistance, improved quality of fruits and flowers for high

protein/vitamin, biodegradable plastics, vaccines and antibodies have been incorporated

in the genetically engineered plants. However, the basic prerequisite for the genetic

transformation is an efficient tissue culture system. For this, an efficient system for the

callus induction and organogenesis are essential to raise the transgenic plant successfully.

4.1.1 Isolation of Cold Responsive Genes- In response to cold stress, plants respond and

adapt by altering the expression levels of many genes, whereby cellular, physiological

and biochemical processes are modified. Transcriptome analysis by microarray

technology offers a potential platform to determine the expression of genes at the global

level. The identification of genes would not have been possible without technical

advancement in the area of (a) gene isolation and discovery, (b) high throughput transcript

analysis, (c) altering gene expression by genetic transformation technologies, (d) functional

characterization of genes through knockout techniques for gene inactivation, etc.,

4.1.1.1 Gene Discovery- An important genomic approach to identify cold stress related

genes is based on ESTs generated from different cDNA libraries representing cold inducible

genes. Detailed information on the type of different libraries and number of ESTs generated

from each library is indexed in NCBI dbEST database (http:// www.ncbi.nlm.nih.

gov/dbEST/dbEST_summary.html). In order to enrich plant EST datasets, specific EST

sequencing programs based on cDNA libraries from cold treated tissues and organs at

different developmental points are necessary. As the EST data sets generated from

control as well as stressed sample, provide information on relative expression levels of


novel stress responsive genes. Putative functions can be assigned to such stress-responsive

genes by comparing with the sequence exists in the NCBI database. These studies will

provide us a valuable information regarding genes associated with stress in different

species. However, outcome of such studies indicate 20-30% of sequences in cDNA

libraries of stress exposed plants are of unknown function. Therefore, they need to be

further annotated in order to determine the possible role and to get a comprehensive

picture of the low temperature tolerance mechanisms. In this regard, an attempt was made

to determine the abundantly expressed ESTs in libraries of a salinity stress treated

halophyte Thellungiella halophila (Wang et al., 2004 a, b) as well as in monocots like

wheat, barley, maize and rice (Sreenivasulu et al., 2004). A full-length cDNA encoding a

wheat cysteine protease was recently characterised from wheat and its expression was

shown to be induced by PEG (Zang et al., 2010). A novel CRT binding factor (CBF)

gene CbCBF25 was characterised from Capsella bursa-pastoris by Rapid Amplification

of cDNA Ends (RACE) and the full length cDNA of 898bp with 669bp ORF encoding a

putative DRE/CRT (LTRE) binding protein was induced by low temperature stress

(Wang et al., 2004a).

4.1.1.2 Identification of a Stress-Responsive Gene by Transcript Profiling- In contrast to

in silico analysis of ESTs, techniques like Serial Analysis of Gene Expression (SAGE),

Massively Parallel Signature Sequencing (MPSS), semi-quantitative and quantitative

real- time PCR helps us to assess the transcripts. Insights into gene expression levels and

functions coupled with stress tolerance can be studied by EST-based cDNA macroarrays.

Transcript profiling using cDNA macroarrays or microarrays is a novel tool to identify


numerous transcripts and pathways related to stress regulatory mechanisms (Chen et al., 2002;

Sreenivasulu et al., 2006). Many reports related to abiotic stress transcriptome profiling

in model species such as Arabidopsis, maize, wheat and rice revealed new stress related

pathways (Desikan et al., 2001; Seki et al., 2001; Chen et al., 2002; Kreps et al., 2002;

Oh et al., 2005; Yang et al., 2012).

4.1.1.3 Functional Characterization of Stress Tolerance Mechanisms Using Transgenics-

Research involving gene isolation for low temperature tolerance was limited in the

pre-genomics era due to the inadequate availability of genes and specific promoters

(Zhu et al., 1997). It is now possible to study multiple transcripts simultaneously on a

genome-wide scale due to the availability of modern molecular tools. Thus, the aim of the

stress-biology research is to use large-scale genomic data to dissect and revalidate the

regulatory mechanisms using transgenic approaches. Thus, traditional breeding and

genomic studies can identify a good correlation among the gene sets. Moreover, many

important components identified of abiotic stress tolerance was tested through transgenic

technologies and confirmed the importance of the genes associated with the biosynthesis

of osmolytes, stress tolerant proteins, antioxidant enzymes, aquaporins and ion

homeostasis (Kishor et al., 2005; Sangam et al., 2005). Genetic engineering of glyoxalase

pathway leading to glutathione-based detoxification process resulted in limited yield

under extreme salinity and heavy metal stress in tobacco (Pareek et al., 2003;

Pareek et al., 2006). DNA helicases have shown to overcome salinity-induced reduction

in plant productivity and yield in transgenic tobacco plants (Mishra et al., 2005). Many of

the antifreeze proteins are reported to have the activity of PR proteins; heterologous


expression of such proteins in plants should possibly provide tolerance to both low

temperature and various biotic stresses. Table 4.1 show some of the significant reports of

the cold tolerant transgenic plants produced till date.

Sl. No Gene/Protein Host Cellular role Reference

1 SCOF 1 (cold-

inducible zinc

finger protein)

Glycine max Regulator of SGBF-1

as a transcription

factor

Kim et al., 2001

2 gpat Glycerol

3-phosphate

acyltransferase

N.tabacum Fatty acid

unsaturation

Murata et al., 1992

3 sacB Levan

sucrase

N.tabacum Fructan biosynthesis Pilon- Smits

et al., 1995

4 cbf1 CRT/DRE

binding factor

A.thaliana Transcription factor Jaglo-Ottosen

et al., 1998

5 dreb1 and dreb2

DRE-binding

Protein

A.thaliana Transcription factor Liu et al., 1998

6 CuCOR19 citrus

dehydrin

N.tabacum Inhibition of lipid

peroxidation

Hara et al., 2003

7 OSISAP1 Zinc-

finger protein

N.tabacum Transcription factor Mukhopadhyay

et al., 2004

8 At CSP3 Cold

shock protein

A.thaliana RNA chaperon Kim et al., 2009

9 ACBP6 Acyl-

CoA-binding

protein

A.thaliana Decline in

phosphatidylcholine

and elevation of

phosphatidic acid

Chen et al., 2008

10 cor15a Cold

regulated gene

A.thaliana Promotes freezing

tolerance

Artus et al., 1996

Table 4.1- Reports on Cold Tolerance in Transgenic Crops


A large number of functionally characterized genes were expressed in different

plants for tolerance against various biotic and abiotic stresses. Most studies reported an

increase in stress tolerance level when compared to the WT, as transgenic plants

exhibited higher accumulation of osmolytes and various stress tolerant proteins, leading

to higher productivity. However, these studies are only confined to the laboratory but field

tests were not carried out. In many instances, constitutive expression of stress-tolerant genes

is likely to cause unwanted effects in the transgenic lines. Therefore, it is better to go for

organ-specific or inducible gene expression of the transgenes using specific promoters.

In this regard, different stress-inducible promoters are available and thoroughly tested for its

specificity. Another advantage of inducible promoters is to overcome the gene silencing, when

gene pyramiding is carried out (multiple genes in same host) to obtain higher level of

tolerance. The advancement in genomic approaches resulted in identifying many new

pathways involved in abiotic stress response and also their relationships with the entire

metabolic network. The genetic transformation studies are a must for all the approaches and

an efficient and standardised tissue culture system is the basic pre-requisite for this.

4.1.2 Plant Tissue Culture and Genetic Transformation- The science of in vitro

culture of plants takes its root from the path breaking research in plant science by the

discovery of plant cells and proposal of cell theory. It was later proposed that, if proper

environmental conditions and essential nutrients are given, a single cell should develop

into a whole fertile plant. Based on this theory, Haberlandt tried to separate cells from

different tissues from different plants and cultured in in vitro conditions in Knoop‘s salt


solution using glucose as the carbon source. Due to the contributions, he is regarded as

the Father of Plant Tissue Culture. The importance of vitamins and plant growth

regulators were proposed by White (1937) also accelerated the tissue culture studies.

One of the most important developments was the formulation of a complete nutrient

media for the culture of plant cells, tissues and organs. Murashige & Skoog (1962), made a

very significant contribution in the formulation of defined Murashige and Skoog growth

medium (MS) suitable for the wide range of species. The MS salt formulation with

various modification and additives now forms the basis of most of the tissue culture

medium (Thorpe 2007).

The ability of a single plant cell to regenerate into a complete plant and the rapid

advancement in gene isolation has helped the development of plant genetic transformation

studies. The rapid technological advancement in this area has led to the production of

transgenic crops and has become key to many applications in Plant Biotechnology. The

isolation of novel chimeric genes, construction of plant transformation based binary vectors,

DNA delivery methods along with the regeneration of transgenic plants have led to the

expansion of plant transgenic technologies to many different species. The plant transformation

vectors and the transformation methods have been improved not only to increase the efficiency

of transformation but also for the stable expression of foreign gene (Veluthambi et al., 2003).

Due to the precise integration of the foreign gene and simplicity of the transformation method,

Agrobacterium based binary vectors continue to offer best vector of choice for the

transformation. The Ti plasmid based vectors have been improved by the incorporation of

supervirulent vir genes, matrix attachment regions, insertion of introns in the marker genes, etc.


Two main strategies have been used to transfer the genes to plant cells, the first

and foremost using Agrobacterium based genetic transformation methods and other is

direct gene transfer using gene gun or electroporation. Among the various methods,

Agrobacterium mediated transformation (a natural genetic engineering system) is the

most commonly used system, which involves the delivery of a portion of the Ti plasmid

from Agrobacterium into wide variety of dicotyledonous and monocotyledonous plants.

The naturally evolved unique ability of Agrobacterium to precisely transfer the DNA

sequences to the plant cells has been very successfully utilised to transfer the foreign

genes. Agrobacterium is a rod shaped Gram-negative soil bacteria, which interacts with

plants by means of chemotaxis. The bacteria have also the capability of transferring and

integrating a part of its genome to the host genome during infection.

Agrobacterium Ti plasmid is responsible for the transfer of a part of its T-DNA

(transfer DNA) to the host genome. The T-DNA is flanked by 25bp-inverted repeats

known as left and right border sequences. The DNA inserted between these left and right

border sequences can be transferred to the plant cells by the combined action of various

vir proteins and some of the proteins encoded by the chromosomal DNA of the bacteria.

In host, the T-DNA is integrated to the genome by illegitimate recombination (Gelvin 2003;

Cheng et al., 2004; McCullen & Binns 2006).

In last two decades, the use of Agrobacterium to genetically transform the plants has

advanced from a dream to a reality (Gelvin 2003). There are many reports of Agrobacterium

mediated genetic transformation to crop plants (Hess et al., 1990; Deng et al., 1990;

Mooney et al., 1991; Peters et al., 1999; Hiei et al., 1997; Mohanty et al., 1999; Rashid

& Bal 2011; Wang et al., 2012) In spite of all these, the host specificity of Agrobacterium


was confined only to dicots, many of the economically important plants including cereals,

pulses remained accessible for transformation by other methods. However the genetic

transformation by Agrobacterium have many advantages like preferential integration of

T-DNA into euchromatin regions which are transcriptionally active, with exclusion of

vector DNA (Hiei et al., 1997; Fang et al., 2002), unlinked integration of co-transformed

T-DNA (McKnight et al., 1987; Komari et al., 1996; Hamilton 1997; Olhoft &

Somers 2001). The transgenic plants developed are generally fertile and often the genes

are transmitted to the progeny in Mendelian ratio.

Many different genes play role in cold tolerance (McKersie & Desker 1994) and

success of improving it, using traditional breeding approaches has its own limitation.

Various genes have been already exploited and utilized to produce transgenic plants

(Chen & Murata 2002; Yuwansiri et al., 2002; Park et al., 2004; Cabello et al., 2012) for

enhancing both abiotic and biotic stress tolerance in plants. As abiotic stresses are

multigenic phenomena in plants, introduction of regulatory genes (transcription factors)

in place of gene responsible for one or few characters has become more popular.

As mentioned above, one of the major problems of Agrobacterium is the host

specificity as it is recalcitrant to monocots. In these instances, alternative transformation

methods like high velocity microprojectile bombardment; gene gun and electroporation

technologies have been employed (Klein et al., 1987). The mircoprojectile bombardment

can be performed to any intact tissue irrespective of the plant group. In this method, the

plasmid DNA containing the desirable gene/genes are coated with gold or tungsten

particles of specific size and bombarded to the cell with high velocity. As the particles

enters the cell, the DNA dissolves and get integrated with the host genome. Several


groups of plants like cereals, legumes and many monocots have been transformed with various

genes using this method (Mc Cabe et al., 1988; Klein et al., 1989; Christou et al., 1991;

Bower & Birch 1992).

4.1.2.1 Tobacco- Tobacco is a member of the Solanaceae family with more than

70 species reported worldwide. Nicotiana tabacum L. is one of the important species of

the genera Nicotiana and is an allotetraploid (4X = 48) formed by the hybridisation of

two different diploid parental species N.sylvestris and N.tomentosiforms 6 million years

ago (Okamura & Goldberg 1985). Since the beginning of the in vitro culture along with

carrot, tobacco was also used as a model crop for various studies (Skoog & Miller 1957).

The ability of plant cell to develop into a complete plant (later termed as totipotency) was

first demonstrated in tobacco. Since then, N.tabacum has become a model system for

tissue culture and genetic engineering studies over the past several decades and continues

to remain the ‗Cinderella of Plant Biotechnology’.

It was when using tobacco, Murashige and Skoog in 1962, revised the culture

media for the propagation of plants and this nutrient formulation was later used for the

in vitro culture of other crops. Isolation, culture and regeneration of protoplast into

a complete plant and somatic hybridisation were studied successfully in tobacco.

Burow et al., (1990) developed a simple genetic transformation method to tobacco based

on leaf disc method, which has become standard for producing transgenic plants.

Later employing tobacco as a model system, several genes have been transferred for

insect resistance, herbicide resistance, stress tolerance for producing recombinant

proteins and recently for even expressing antibodies.


4.1.2.2 Tomato- Tomato is one of the major vegetable crops that has achieved

tremendous popularity and is grown, consumed in almost all part of the World. Tomato

(Solanum lycopersicum formerly Lycopersicon esculentum) is an economically important

crop and a preeminent model system for genetic transformation studies. It is also the most

intensively investigated solanaceous species (after tobacco) due to its simple diploid

genetics, short generation time and availability of rich genetic and genomic resources.

The plant has a diploid genome with size of 950Mb encoding approximately 35,000 genes.

The genetic information is contained in 12 pairs of chromosomes (Barone et al., 2008).

Tomato is rich in vitamins A, Vitamin C, fibre and also it is cholesterol free. Tomato

contains approximately 20-50mg of lycopene/100g of fruit. Lycopene is one of the most

powerful antioxidant and it protects the cells from the action of many free radicals.

In vitro studies in tomato have been carried out for selection of cell lines for stress

tolerance, development of haploids, production of somatic hybrids, mass propagation, etc.

(Bhatia et al., 2004; Bhatia et al., 2005). Introduction of traits into the commercial cultivars

of tomato by conventional breeding often encounter difficulties due to high incompatibility

barriers during hybridization. Genetic engineering techniques are being used in tomato crop

improvement programmes to combat the challenges. Tomato also served as model plant for

transferring many agronomically important genes (Wing et al., 1994). There are many

reports of genetic transformation in tomato (McCormick et al., 1986; Fillatti et al., 1987;

Delannay et al., 1989; Sharma et al., 2009; Paramesh et al., 2010). PKM1, a popular

local cultivar gives higher yield and larger fruit and also resistant to many bacterial and

fungal diseases.


However, the genetic transformation is still a problem for the tomato varieties that

has low regeneration capacity (Fuentes et al., 2004). The genotype response to tissue

culture conditions is believed to drive the frequency of transgenic plant regeneration

(McCormick et al., 1986; Davis et al., 1991), whereas the capacity of cell proliferation could

determine the transformation efficiency (Sangwan et al., 1992; Villemont et al., 1997).

Therefore, the development of an efficient and genotype independent genetic

transformation method for tomato is essential.

The objectives of this Chapter are as follows:

1. To isolate and clone the Daucus carota Antifreeze Protein (AFP) gene in pBI121

binary vector

2. To standardize the tissue culture protocols for different tomato variety PKM1.

3. To produce transgenic tobacco and tomato using Dc AFP.


4.2 MATERIALS AND METHODS

4.2.1 Gene Isolation

4.2.1.1 Plant Growth Conditions- D.carota var Kuroda seeds were procured from the

Horticulture training centre, Ooty, Tamil Nadu, India. The seeds were germinated in

commercial soil mix (Keltech Energies, Karnataka, India) and plants were maintained in

controlled conditions at 25°C with 16/8 light/dark regime in growth chamber with 80-85%

humidity. The germinated plants were nourished with 1/10 strength sterile MS salt solution.

4.2.1.2 Genomic DNA Isolation- Genomic DNA was isolated from one-month-old

plants as per Doyle & Doyle (1990), with slight modification. The leaves or taproots were

ground with preheated extraction buffer (1.4M NaCl, 20mM EDTA, 100mM Tris HCl,

0.2% β-Mercaptoethanol, 2% PVP and 2% CTAB) and incubated at 65°C for 1h with

intermittent shaking. The sample was centrifuged at 12900g after adding equal volume of

24:1 Chloroform: Isoamylalcohol. The DNA was precipitated by adding 0.6 volume of

ice-cold isopropanol to the aqueous phase and incubated at -20°C for overnight.

The DNA was then washed with 70% ethanol and finally resuspended in TE buffer.

RNase A (DNase free) 10mg/mL (Fermentas INC, Maryland, USA) was added and

incubated at 37°C for 30 min in a water bath. The samples were then purified by salt

ethanol precipitation. The DNA samples were separated in 1% agarose gel containing

80ng/μL ethidium bromide and the integrity was checked using an AlphaDigiDoc® RT

Gel Documentation System®, Alpha Innotech, USA. Genomic DNA was used as the

template for the PCR. Specific primers were designed to obtain full length AFP gene

(as the gene lacks introns, genomic DNA was directly used for cloning).


4.2.1.3 Retrieval of Sequence from NCBI Database and Standardization of PCR

conditions Dc AFP - The sequence of carrot Antifreeze gene (Accession Number AJ131340)

was retrieved from NCBI GenBank database (http://www.ncbi.nlm.nih.gov/nucleotide).

The primers specific for the AFP gene were designed using the online software Primer 3

(http://primer3.sourceforge.net/). Restriction sites of XmaI (CCC GGG) and SacI (GAG

CTC) were introduced in the forward and reverse primers respectively for further cloning.

AFP FL Fw: 5‘-CAA TCC CGG ATG GTA ATA TTG AAT CA -3‘

AFP FL Rev: 5‘-AGT AGT GAG CTC CTA GCA TTC TGG CAA TGG-3‘

PCR to obtain carrot AFP gene was performed as follows to obtain the full-length

gene. The PCR programme comprised an initial denaturation at 94°C for 5 min, followed

by denaturation at 94°C for 30s, gradient annealing temperatures were tried from

50-60°C for 30s, extension at 72°C for 1 min for 30 cycles and a final extension at 72°C

for 7 min in Eppendorf Mastercycler Gradient®, Eppendorf Deutschland, Germany.

All the reagents for the PCR reaction were procured from Fermentas (Fermentas INC,

Maryland, USA). The reaction mix contained 10mM Tris HCl (pH 8.8), MgCl2

(2.5mM), dNTP mix (1mM each), AFP forward primer and reverse primer (10pM),

genomic DNA (~100ng), Taq DNA polymerase (5U/μL) and the volume was made with

PCR grade water. The PCR products were separated in 1.4% agarose gel containing

ethidium bromide and the bands were visualized and documented. The strategy followed

for cloning Dc AFP to pBI121 is shown in the figure 4.1.


http://primer3.sourceforge.net/

Figure 4.1- Strategy used for Cloning of AFP into pBI121 Binary Vector

The AFP PCR product was cloned in commercially available TA vector (pDRIVE cloning

vector). Later the AFP from TA was cloned to pBI121 to get pBI121-AFP.

pDRIVE TA

Vector

pDRIVE TA- AFP


The specific band of Dc AFP was excised from the gel and purified using

AxyprepTM

DNA gel extraction kit (Axygen Biosciences, CA, USA). The purified PCR

product was cloned to (The A overhang in the PCR product was ligated with the U

overhang of the vector) commercially available pDrive TA vector, Qiagen PCR Cloning

Kit, (Qiagen, Valencia, CA, USA), according to manufacturer‘s instruction. The ligation

mix is shown in the following table 4.2. The ligation reaction was performed at 4°C for

16h using a thermal cycler.

Sl. No Components Volume for 10μL reaction

1 pDrive cloning vector (50ng/ μL) 1μL

2 Purified PCR product (50ng/ μL) 2μL

3 Nuclease free water 2μL

4 Ligation mix 2X 5μL

Total volume of the ligation reaction was 10μL

Table 4.2- Preparation of Ligation Mixture for Cloning of Dc AFP to pDrive Vector

The ligated product was transformed to Escherichia coli strain DH5α -T1 R

(genotype: F Φ80lacZΔM15 Δ(lacZYAargF)U169 recA1 endA1 hsdR17(rk , mk ) phoA

supE44 thi-1 gyrA96 relA1 tonA) by calcium chloride method. The competence cells of

E.coli were prepared according to Sambrook & Russell (2001) with minor modification.

Luria-Bertani (LB) medium (Himedia Laboratories, Bangalore, India) was routinely used

to culture E. coli or Agrobacterium. Transformation was performed as described in the

section 2.2.6.5. Antibiotic plates for selection of transformants contained Ampicillin

(Himedia Laboratories, Bangalore, India) at a final concentration of 100mg/L + 80μg/mL


X-gal (Fermentas INC, Maryland, USA) and 50μM IPTG (Fermentas INC, Maryland, USA).

These plates were incubated at 37°C overnight and the white colonies were screened by

colony PCR presence of AFP gene using the same PCR conditions with AFP primers.

4.2.1.4 Cloning of AFP in pBI121 Binary Vector- The pBI121 plasmid DNA was a gift

from Dr. Kin-Ying To, Institute of Bio-Agricultural Sciences, Taipei, Taiwan. The carrot

AFP was cloned in pBI121 binary vector and the recombinant pBI121-AFP was used for

the genetic transformation of tobacco and tomato. The details are as follows:

The pBI121 plasmid DNA received was resuspended in TE buffer and

transformed to E.coli DH5α. The transformants were selected in LB media supplemented

with 50mg/L Kanamycin. The colonies in the selection media were confirmed by colony

PCR with GUS specific primers, F 5‘- GGT GGG AAA GCG CGT TAG AAG - 3‘ and R

5‘- GTT TAC GCG TTG CTT CCG CCA - 3‘. The PCR programme comprised, initial

denaturation at 94°C for 5 min, followed by denaturation at 94°C for 1 min, annealing at

60°C for1 min, extension at 72°C for 1 min for 30 cycles and a final extension at 72°C

for 7 min. The PCR reaction mixture was same as mentioned above except that GUS

primers were used. The PCR products were separated in 1.4% agarose gel containing

ethidium bromide and the bands were visualized and documented.

4.2.1.5 Plasmid Isolation from E.coli (harboring TA Vector with AFP and pBI121)-

The strategy of cloning AFP to pBI121 binary vector is shown in the fig. 4.1. The E.coli

harboring TA vector with AFP was inoculated in LB broth supplemented with 100mg/L

Ampicillin and E.coli harboring pBI121 binary vector was inoculated in LB broth

supplemented with 50mg/L Kanamycin. The cultures were incubated at 37°C in an


orbital shaker overnight. The plasmid DNA (from TA vector harboring the Carrot AFP

gene and pBI121) was isolated from overnight grown culture using HiPurA Plasmid

DNA isolation kit, Himedia Laboratories, India.

The plasmid DNA were separately digested with XmaI (Fermentas INC,

Maryland, USA) and SacI (Fermentas INC, Maryland, USA) to release the GUS

fragment from pBI121 and AFP from TA vector at 37°C for 6h. The digestion mix is

shown in the table 4.3.

Sl. No Components Volume for 50μL reaction

1 Plasmid DNA (10μg) 10μL

2 Restriction buffer 10 X

Universal Tango buffer

5μL


4 Restriction enzyme

XmaI (2000U)

SacI (2000U)

1 + 1μL

Total volume of the digestion reaction was 50μL

Table 4.3– Restriction Enzyme Digestion Reaction mix for Releasing AFP from TA

Vector and GUS from pB121

The digestion products were loaded onto 1.4% agarose gel and the bands were

visualized and documented. The insert (Dc AFP) released from TA and the pBI121

binary vector (except the released GUS gene portion) was excised from the gel and

purified using AxyprepTM

DNA gel extraction kit (Axygen Biosciences, CA, USA).

The GUS fragment was released from the pBI121 binary vector and Dc AFP was cloned


in place of that. The vector and insert were mixed in the ration of 1:3 and left overnight

for ligation at 4°C in a thermal cycler. The component of the ligation reaction is shown in

the following table 4.4.

Sl. No Components Volume for 20μL

reaction

1 Vector (pBI121 digested with XmaI and SacI) 2μL

2 Insert

(Dc AFP digested from TA vector with XmaI and SacI )

2μL

2 Ligation buffer (5X) 4μL


4 T4 DNA ligase (1000U) 1μL

Total volume of the ligation reaction was 20μL

Table 4.4– Restriction Enzyme Digestion Reaction for Releasing AFP from TA and

GUS from pBI121

Two microlitre of the ligated product was transformed to E.coli DH5α.

The transformed cultures were spread in LB media supplemented with 50mg/L

Kanamycin. The plates were incubated overnight at 37°C. The transformed colonies

were screened by colony PCR using AFP specific primers.

The colonies were also simultaneously inoculated in LB broth supplemented with

50mg/L Kanamycin and incubated at 37°C over night in an orbital shaker. Plasmid DNA

was isolated from the overnight grown culture. The plasmid DNA was resolved in 1.4%

agarose gel and the bands were visualized and documented. The plasmid DNA was also

used as the template for PCR with AFP specific primers.

154

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The plasmid DNA from the positive colonies was sent for sequencing to

Chromous Biotech, Bangalore, India. Both the forward and reverse read for sequencing

was performed. The sequence obtained was subjected to BLASTn programme

(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

4.2.1.6 Transformation of Recombinant Binary vector to the Agrobacterium LBA

4404 by Freeze Thaw method- A hyper virulent Agrobacterium tumefaciens strain

EHA105 was used in the present study. The Agrobacterium was propagated in LB broth

supplemented with 60mg/L of Rifampicin. Freshly cultured Agrobacterium LBA 4404

culture (1mL) was inoculated in 100 mL LB with 60mg/L of Rifampicin and grown at

28°C. The overnight grown cells were chilled in ice for 15 min after reaching the OD of

0.5-0.8 OD600. The cells were pelleted at 2500g for 10 min at 4°C and washed with sterile

1X TE and cells were centrifuged at 2500g for 10 min at 4°C and resuspended in 0.1X of

original volume using LB. The cells were aliquoted in microfuge tubes and immediately

frozen in liquid Nitrogen (Hofgen & Willmister 1988).

The Agrobacterium competent cells were thawed in ice and 10μL (1-2μg) of

pBI121- AFP was used for transformation. The mixture was incubated in ice for 5 min

and immediately the tubes were transferred to liquid Nitrogen for 5 min. The tubes were

then incubated for 5 min at 28°C and 1mL LB broth was added. The tubes were

incubated in shaker for 6h at 28°C and the cells were collected by spinning and discarded

700μL of media and cells were resuspended in remaining media and spread in LB agar

with 50mg/L Kanamycin and 60mg/L Rifampicin. The plates were incubated at 28°C for

2 days (Hofgen & Willmitzer 1988).

155


4.2.1.7 Confirmation of Agrobacterium Transformants - The colonies from the plates

were picked randomly and inoculated in LB broth with 50 mg/L Kanamycin and 60 mg/L

Rifampicin and incubated overnight at 28°C in orbital shaker. Plasmid DNA was isolated

from the overnight grown culture and used as template for PCR with Dc AFP specific

primers.

4.2.2 Tissue culture and Genetic Transformation

4.2.2.1 Transformation System - The aim is to genetically transform the Dc AFP to

model system N.tabacum (Tobacco), L.esculentum (Tomato) cv PKM 1. PKM 1 variety

was developed in 1978 by the Tamil Nadu Agricultural University, Coimbatore, India,

which was derived from a local variety Annanji with a yield of 32 t/Ha in duration of 135

days. The fruits are flat round and attractive with capsicum red color after ripening.

A fruit weigh about 70-80g. This fruit contains 23.7 mg vitamin C per 100g of fruit pulp.

4.2.2.2 Plant Material - The seeds of N.tabacum cultivar (cv) Xanthi were kindly

provided by Prof. Julian Ma, St. George‘s University of London, UK. The seeds of

L.esculentum cv PKM1 used were procured from the Division of Vegetable Crops,

Department of Horticulture, Tamil Nadu Agricultural University, Coimbatore, India.

The seeds were used for raising the in vitro plants, which was further used as the explants

for tissue culture and genetic transformation studies.

4.2.2.3 Surface Sterilization and Seed Germination- The seeds of tobacco were

washed in 5% Teepol (commercial detergent) solution for 10 min. It was then washed

with sterile distilled water thrice. The seeds were then surface sterilized using sodium

hypochlorite solution for 30s and further 5 washes were given with sterile distilled water

156


to remove traces of sodium hypochlorite. The seeds were aseptically inoculated in culture

bottles with half strength MS medium using sterile brush. The bottles were incubated in

dark at 24-27°C for seed germination.

The tomato seeds were washed well in running tap water. The seeds were then

soaked in sterile distilled water and incubated at 60°C for 2h to overnight in hot air oven.

The seeds were then washed in 5% Teepol solution for 10 min. They were then washed

well with sterile distilled water thrice. The seeds were then soaked in sodium

hypochlorite for 30 min and further 5 washes were given with sterile distilled water to

remove traces of sodium hypochlorite. The surface sterilized tomato seeds were

inoculated in culture bottles with half strength MS medium. The bottles were incubated at

24-27°C dark for seed germination.

4.2.2.4 Culture Medium and Experimental Conditions- MS basal medium and MS

media supplemented with B5 vitamins were used for the propagation of the tobacco and

tomato plants respectively. The readymade media were purchased from Himedia

Laboratories, India. The components of the media are shown in appendix I. The basal

media were supplemented with various growth regulators (The concentration of

hormones are expressed in mg/L), 3% sucrose as Carbon source (if not supplemented in

media) and 0.8% agar or 0.2% Gelrite were used throughout the study. The pH of the

media was adjusted to 5.8 with 1N NaOH or 1N HCl before sterilization. The culture

media were sterilized at 121°C for 15 min in autoclave. The explants were inoculated

appropriately and cultures were maintained at 25 + 2°C and 55 + 5% relative humidity

under continuous light (1000-2000 lux) provided by cool day light fluorescent tubes for

16/8 light/dark cycle.


4.2.2.5 In vitro propagation of Tobacco

4.2.2.5.1 Callus Induction from Leaf- The in vitro grown tobacco leaves were used as

the explants for the induction of callus. The leaves were excised aseptically and the mid

rib of the leaves was separated. The leaves were cut into small square pieces of about

1cm. About 15-20 leaf discs were inoculated upside down in petriplates containing

MS media supplemented with 0.1 IAA + 1 BAP for the induction of callus. The plates

were sealed with parafilm and incubated upside down at 22- 24°C. The explants were

subcultured every 2 weeks in fresh media.

4.2.2.5.2 Indirect Regeneration of Plants- The small green shoots of 2-3 cm with leaves

emerged from the calli was excised and the basal portion was trimmed to expose the

meristamatic tissue. The shoots were inoculated in the same media (MS media with 0.1 IAA +

1 BAP) in culture bottles or tubes. The plants were subcultured in fresh media in every 10 days.

4.2.2.6 In vitro Propagation of Tomato

4.2.2.6.1 Callus Induction from Cotyledonary Leaves or Hypocotyls Explants-

The germinated seedlings served as explants source for tissue culture of tomato.

The hypocotyl segments (1-2 cm) and the cotyledonary leaves of 7-10 days old in vitro

plants were excised under aseptic conditions. The cotyledonary leaves were trimmed on

both the ends and inoculated upside down in callusing media with different combination

of hormones as shown in the following table (table 4.5) The media was also

supplemented with 100mg/L of Ampicillin (to avoid bacterial contamination). About

50 to 60 explants were inoculated in petriplates containing the callusing media.

The plates were sealed with parafilm and the plates were incubated upside down at 22-

24°C. The explants were subcultured every 2 weeks in fresh media.

158


Sl. No Combination of Hormones Concentration (mg/L)

01 NAA with BAP 0.1 NAA + 1.0 BAP

0.25 NAA + 2.0 BAP

0.5 NAA + 2.5 BAP

0.75 NAA + 3.0 BAP

1 NAA + 3.5 BAP

02 IAA with BAP 0.1 IAA + 1.0 BAP

0.25 IAA + 2.0 BAP

0.5 IAA + 2.5 BAP

0.75 IAA + 3.0 BAP

1.0 IAA + 3.5 BAP

Table 4.5– Different Combination and Concentration of Hormones used for Callus

Induction in Tomato

4.2.2.6.2 Indirect Regeneration of Plants- The green or pink coloured small shoots

emerged from the calli were excised and the basal portion of the shoots was trimmed.

The shoots were inoculated aseptically in regeneration media. Different concentrations of

BAP or BAP in combination with IAA and BAP in combination with Zeatin were tried

for regeneration (table 4.6). The shoots were subcultured every ten days to fresh media.

The experiments were designed in such a way that each concentration and

different combination of hormones had triplicates. Uniform pieces from the same type of

explants were used in combinatorial treatments. Observations were recorded for

percentage of callus induction and plantlet regeneration. The data shown represent the

mean of three replicates + the standard error which were subjected to ANOVA followed

by Duncan‘s Multiple Range Test (DMRT) using the SPSS V13. Mean values with

different alphabets differ significantly at P<0.05.


Sl. No Hormones/ Combination of

hormones Concentration (mg/L)

01 BAP 1.0 BAP

2.0 BAP

3.0 BAP

4.0 BAP

02 IAA with BAP 0.1 IAA + 1.0 BAP

0.25 IAA + 2.0 BAP

0.5 IAA + 2.5 BAP

0.75 IAA + 3.0 BAP

03 IAA with Zeatin 0.1 IAA + 1.0 Zeatin

0.25 IAA + 2.0 Zeatin



Table 4.6- Different Combination and Concentration of Hormones Tried for Tomato

Tissue Culture System

4.2.3 Genetic Transformation of pBI121-AFP to Tobacco and Tomato cultivar-

The pBI121 harboring AFP was used for the genetic transformation of N.tabacum cv

Xanthi and L.esculentum cv PKM1. Agrobacterium mediated genetic transformation was

performed for tobacco and tomato.

4.2.3.1 Preculture of Explants of Tobacco and Tomato for Agrobacterium-mediated

Genetic Transformation- The in vitro grown tobacco leaves were excised aseptically

and cut into small leaf discs of approximately 1cm2 size. The leaf discs were inoculated

upside down in MS medium with 0.1 IAA + 1 BAP for 7 days prior to transformation.

About 15-20 leaf discs were inoculated in each plate.

The in vitro grown tomato cotyledonary leaves were excised and the cotyledonary

leaves were separated from other part of the seedling. The leaves were trimmed on both


the ends. About 30-40 explants were inoculated in petriplates containing in MS media

supplemented with Gamborg‘s B5 vitamins, 0.1 IAA + 2 Zeatin + 100mg/mL Ampicillin

for 3 days. The precultured leaves were used as explants for Agrobacterium mediated

genetic transformation.

4.2.3.2 Culture conditions for Agrobacterium LBA4404 harboring pBI121-AFP-

The glycerol stock of Agrobacterium harboring the pBI121-AFP was inoculated in LB

broth containing 50mg/L Kanamycin and 60mg/L Rifampicin and the cultures were

maintained at 28°C for 2 days in an orbital shaker. The culture was further subcultured in

fresh media and used for genetic transformation.

Agrobacterium AFP was pelleted at 9800g for 10 min and the cells were resuspended

in MS salt solution to get an OD of 0.2 at 600nm. The explants were immersed in this

suspension and incubated for 5-10 min. The explants were blotted dried using filter paper and

inoculated back in the pre-culture media, incubated for 3 more days in dark.

4.2.3.3 Selection of Transgenic Plants- After the co-cultivation period, the leaves were

blot dried for 30 min and then inoculated in selection media. Tobacco leaves were

inoculated in MS media supplemented with 0.1 IAA + 1 BAP + 100 mg/L Kanamycin +

300mg/L Carbenicillin. Tomato cotyledonary leaves were initially inoculated in MS

supplemented with Gamborg‘s B5 vitamins + 0.1 IAA + 2 Zeatin + 400mg/L Carbenicillin.

The explants were further subcultured in same selection media with 75mg/L Kanamycin

after 7 days. The explants were subcultured for every 2 weeks in fresh selection media.

4.2.3.4 Regeneration of the Putative Transformants- The putative transformants were

regenerated in selection media (same as mentioned above) and the regenerated shoots

from the organogenic calli were then transferred to shoot elongation medium.


The explants were initially inoculated in culture tubes, after the first subculture they were

transferred to culture bottles. The plants were subcultured to fresh media in every 10 days.

4.2.3.5 Rooting and Hardening- The shoots of around 5-8 cm height of tobacco were

transferred to half strength MS media with 300mg/L Carbenicillin + 75mg/L Kanamycin

and 0.2% gelrite for rooting. The shoots regenerated from tomato plants of around 5 cm

were transferred MS media with 0.1 IAA + 300 mg/L Carbenicillin+ 75mg/L Kanamycin

and 0.2% gelrite for rooting.

The well-rooted plants were transferred to commercially available soil: manure:

perlite mixture. Sterile distilled water was poured and kept for 30 min to loosen the

agar/gelrite. The plants were gently removed using a forceps (without disturbing the

roots) and the agar or gelrite adhering to the roots were washed thoroughly in running tap

water in order to avoid the fungal growth. The plants were transferred to sterile soil:

manure: perlite mixture and the cups were covered with a polythene bag with pores in it.

The plants were maintained initially for 2 days in the culture room and were later

transferred to growth chamber or containment room. The plants were daily nourished

with sterile 1/10 sterile MS liquid media.

4.2.3.6 Seed collection in transgenic plants- The flowers of tobacco and tomato were

pollinated manually and the flowers were covered with a bag in order to avoid

cross-pollination. The seeds were collected from dried tobacco fruits by pressing the pod

and squeezing the dried fruits. The seeds from same plants were pooled together. The

seeds from tomato were collected, when the fruits started changing colour from green to

red. The fruits were plucked from the plants and cut horizontally in order to access the


seeds in the fruit. The fruits were gently squeezed to remove the seeds along with the

pulp and were allowed to dry in a plastic container. Later the seeds were removed and

pooled. The seeds of both tobacco and tomato were stored in a dry place and were used

for further studies.


4.3 RESULTS AND DISCUSSION

4.3.1 Gene Isolation

4.3.1.1 Carrot DNA Isolation and Optimisation of PCR for Dc AFP- Carrot genomic

DNA was isolated from the leaves and the DNA yield was higher, when the precipitation

was left overnight in isopropanol at 4ºC. The DNA yield was also more from the leaves

as compared to the tap root (fig. 4.2). This might be due to the presence of more amounts

of polysaccharides present in the carrot tap root. Extraction of highly purified genomic

DNA from plant tissues is a tedious task due to the presence of rigid cell wall composed

of higher amounts of complex carbohydrates (Hattori et al., 1987). Contamination of

polysaccharides in genomic DNA preparation has been reported as the most common

problem affecting the purity of plant DNA (Demeke & Adams 1992; Murray &

Thompson 1980). Some classes of polysaccharides interfere and reduce the activity of

polymerases and restriction endonucleases (Furokawa & Bhavadna 1983; Shioda &

Muofushi 1987; Do & Adams 1991; Fang et al., 1992). Several factors present in plant

genomic DNA preparations inhibit Taq polymerase activity (Gelfand & White 1990).

Hence, the false negative polymorphic bands were reported in PCR-based finger printing

as a result of contamination by polysaccharides or other DNA-binding substances, which

may confound the interpretation and validation of data.


Figure 4.2 – Isolated Genomic DNA from Carrot Plants

Lane M- Marker DNA, Lane 1- 2 DNA from leaves, Lane 3- 5 DNA from carrot tap root

The genomic DNA was used the template directly, as there are no introns in carrot

AFP and PCR was performed with AFP specific primers were used. The PCR amplicons

obtained using cDNA and genomic DNA of carrot as a template showed similar sizes.

Amplification was obtained in all the temperature tried 50.5, 51.5, 52.9, 54.8, 56.4, 57.4,

58.6 and 59.2 ºC. The annealing temperature for the PCR was optimized and the

amplification was found to be best at 50°C with an annealing time of 30s. The amplicon

size was 1.1kb as expected (fig. 4.3).

500bp

100bp

10kb Genomic DNA

M 1 2 3 4 5


Figure 4.3 – Gradient PCR for Carrot AFP

Lane 1- TA* 59.2°C, Lane 2- 58.6°C, Lane 3- 57.4°C, Lane 4- 56.4°C, Lane 5- 54.8°C,

Lane 6- 52.9°C, Lane 7- 51.5°C, Lane 8- 50.5°C, Lane M – Marker DNA

*(TA) Temperature Annealing

4.3.1.2 TA cloning of Dc AFP - The purified product was cloned in TA vector as per the

manufacturer‘s instruction. Blue white screening strategy was followed to select the

transformed colonies. The colonies obtained were patch streaked in LB agar with

100mg/L Ampicillin. Colonies were selected randomly for colony PCR to determine the

positive colonies using AFP specific primers. A band at expected size of 1.1kb obtained

from most of the white colonies. These colonies were further subcultured and used for

further analysis. The DNA sequencing result showed homology with the reported carrot

AFP sequence available in GenBank database. Hence the recombinant pBI121-AFP was

further used for the genetic transformation of tobacco and tomato.

250 bp

500 bp

1 kb

10kb

1 2 3 4 5 6 7 8 M

AFP amplicon -1.1kb


4.3.1.3 Transformation of pBI121 and Plasmid Isolation- The pBI121 binary vector

was transformed to E.coli cells and colony PCR was performed to confirm the presence

of pBI121 using GUS specific primers. A single band at 1.2 kb was observed indicating

the presence of GUS gene. The positive colonies were used for further studies (fig. 4.4).

Figure 4.4 – Colony PCR to Confirm GUS Fragment in pBI121

Lane M- Marker DNA, Lane 1-8- GUS amplicon from transformed colonies

4.3.1.4 Digestion of pBI121 and TA with AFP & Ligation of AFP to pBI121-

The amount of plasmid DNA was critical for the digestion and cloning. Plasmid DNA

(5μg) was used for the digestion reaction. The digestion of pBI121 with XbaI and SacI

resulted in the release of GUS fragment (of 1.2kb) from the vector backbone. The digestion

of pDRIVE vector with same set of enzymes resulted in the release of 1.1kb AFP fragment

(as XbaI and SacI sites were added in the primers of AFP, fig. 4.5). The bands were

excised, purified from the gel and quantified. Ligation was efficient when the vector and

the insert were taken in the ratio of 1:3 and when the ligation was performed at 16°C for

1.2 kb

500bp

1 kb

10kb

M 1 2 3 4 5 6 7 8 10


18h. Colony PCR from the transformed colonies revealed the presence of 1.1kb band of

AFP (fig. 4.6). To further confirm the results, plasmid DNA was isolated from the

positive colonies and digested with XmaI and SacI to release the AFP fragment.

Figure 4.5- Restriction Digestion of TA- AFP

Lane 1- AFP in TA, Lane 2- 4- AFP TA digested with XmaI and SacI, Lane M- Marker

DNA, Lane 5- No sample

Figure 4.6 – Colony PCR of pBI121-AFP

Lane 1- 13- AFP amplicons from different colonies, Lane M- Marker DNA

1.1kb

10kb

1 2 3 4 5 6 7 8 9 10 11 12 13 M

1 kb

100 bp

100bp

1 kb

10kb

1.1kb of AFP

1 2 3 4 5 M


4.3.1.5 Transformation of Agrobacterium by Freeze Thaw Method- The plasmid

DNA (pBI121-AFP) was moved to Agrobacterium LBA 4404 and plasmid was isolated

from randomly selected putative recombinant colonies. The colonies that gave amplicon

size of 1.1kb were considered as positive (fig. 4.7) and used for Agrobacterium mediated

genetic transformation of plant. pBI121 has been widely used to clone the different genes

and to genetically transform to different plants (Rajasekaran et al., 2000; Liu et al., 2009;

Wang et al., 2009; Wang et al., 2011)

Figure 4.7- Confirmation of pBI121 AFP in Agrobacterium

Lane 1- 7- AFP amplicons from different Agrobacterium colonies, Lane M- Marker DNA

1.1kb

100bp

1kb

10kb

1 2 3 4 5 6 7 M


4.3.2 Tissue Culture and Genetic Transformation

4.3.2.1 In vitro Culture of Tobacco

4.3.2.1.1 Tobacco Seed Germination and Plantlet Development- The seeds of tobacco

were germinated in half strength MS media and the in vitro grown leaves were used as

the explants for inducing calli and further regeneration of plants from the calli.

4.3.2.1.2 Callus Induction and Indirect Regeneration in Tobacco- The in vitro grown

leaves were precultured in MS media supplemented with 1 BAP + 0.1 IAA. Green,

fragile organogenic calli were produced from in vitro grown leaves after 3 weeks of

culture. Later the shoots developed from the calli were excised and inoculated in the

same media for raising the plantlets (fig. 4.8 a, b, c, d).

The callus induction, indirect regeneration and rooting has been well studied for

Nicotiana tabacum (Thorpe & Murashige 1970; Maeda & Thorpe 1979). Skoog and co

workers demonstrated the clear differentiation of shooting and rooting in tobacco by

changing the auxin: cytokinin ratio. The presence of cytokinin increased the shoot bud

regeneration, while the effect of cytokinin was further enhanced when auxins were

supplemented in the media. The auxins in the media induced rooting in tobacco. Skoog &

Miller (1957) reported that, a low ratio auxin to high ratio of cytokinin induced calli and

regeneration in tobacco. In our present study, the similar combination and ratio of

hormones were used for the callus induction and plant regeneration. However, there are

also reports that other combinations and concentration of hormones were used to induce calli

and raise plants from different explants in tobacco (Colak et al., 1998; Zhou et al., 2008).

4.3.2.1.3 Rooting and Hardening of Tobacco Plants- Each shoots of 5-6 cm were

excised separately and inoculated in rooting media (half strength MS media). Rooting


was observed after 10 days of inoculation. The endogenous hormones present in the

plants might have induced rooting in tobacco. Puchooa et al., (1999) also reported that no

exogenous phytohormones were required for rooting in tobacco (fig. 4.8 e). The plants

with well-developed roots were transferred to soil and maintained under controlled

conditions in growth chamber. Later the plants were transferred to the containment

facility.

4.3.2.2 In vitro culture of Tomato

4.3.2.2.1 Tomato Seed Germination and Plantlet Development- The seeds started

germinating after 4-5 days and fully expanded cotyledonary leaves were seen after

10 days. Germination rate was 73% and remaining either failed to germinate or the plants

were not healthy. The efficiency of seed germination was found to be more, when the

seeds were incubated in warm water for 2-3h before surface sterilization.

4.3.2.2.2 Callus Induction and Indirect Regeneration in Tomato- The in vitro

morphogenetic responses of explants are influenced by different components of the

culture media and hence, it is necessary to assess their effects on callus induction and

plant regeneration (Gubis et al., 2004). Cotyledonary leaves were cut at the base and tip,

middle piece were used for inoculation in the callusing media. The explants were placed

with their adaxial side in contact with the media. More than 90% of the explants

responded to the pre-culture conditions by expanding in size within 48h. Callus was

initiated within 8-10 days directly from the cut surfaces of both hypocotyl and cotyledon

explants cultured (meristematic cells are exposed in this region and hence the

proliferation rate was higher) on MS basal medium supplemented with auxins (NAA and

IAA in combination with cytokinins (BAP) in different combinations. Difference in


initiation of callus formation like bulging and curling of leaves, and formation of mass of

cell aggregates were visible at the initial periods. Well developed calli were observed

between 18-22 days (fig. 4.8 f, g, h). The calli were subcultured in fresh media at regular

intervals of time.


.

Figure 4.8- In vitro Cultures of Tobacco and Tomato

a- preculture of tobacco b - bulging of leaves in pre-culture media c- callus induction in tobacco

d - regeneration of shoots from callus e - Rooting in half strength MS f- preculture of tomato

g- callus induction in tomato h, i- regeneration in tomato j, k- rooting in tomato

d e

f h g

i j k

b c a


Figure 4.9- Percentage of Callus Induction Response under the Influence of Different

Hormone Concentration and Combination for Cotyledonary or Hypocotyls

Explants

0.5 NAA+ 2.5 BAP and 0.5 IAA + 2.5 BAP show maximum response in both the explants. Graph

represents the mean + SE of three replicates (n=3), ** represents the values are significantly

different at 5 % level by DMRT.

*

*

*

*

*

*

*

*


Sl No

Hormone

combinations

(mg/L)

Cotyledonary leaves

(Response in %)

Hypocotyl

(Response in %)

1 0.1 IAA + 1 BAP 55.00 d 55.00

d

2 0.25 IAA + 2 BAP 66.66 c 68.33

bc

3 0.5 IAA + 2.5 BAP 78.33 a 85.33 a

4 0.75 IAA + 3 BAP 75.33 ab

72.33 b

5 1 IAA + 3.5 BAP 64.33 c 67.33

bc

6 0.1 NAA + 1 BAP 55.66 d 55.33

d

7 0.25 NAA + 2 BAP 64.33 c 65.67

c

8 0.5 NAA + 2.5 BAP 78.66 a 85.33

a

9 0.75 NAA + 3 BAP 72.66 b 70.66

bc

10 1 NAA + 3.5 BAP 64.00 c 65.33

c

Table 4.7- Percentage of Callus Induction Response under the Influence of Different

Hormone Concentration and Combination for Cotyledonary or Hypocotyls

Explants

The value represents mean + SE of three replicates (n=3). Among the different combinations

tried, 0.5NAA+ 2.5BAP and 0.5IAA + 2.5BAP show maximum response in both the explants.

In a column, mean followed by a different alphabet are significantly different at 5% level by

DMRT.


Figure 4.10 - Percentage of Plant Regeneration under the Influence of Different

Hormone Concentration and Combination for Cotyledonary Leaf

Induced Callus

3 BAP, 0.5 IAA + 2.5 BAP and 0.5 IAA + 2.0 Zeatin shows maximum response in both the

explants. Graph represents the mean + SE of three replicates (n=3), ** represents the values are

significantly different at 5 % level by DMRT.

**

**

**

*

*

*

*

176


Sl No Hormone combinations (mg/L) Regeneration from callus induced

cotyledonary leaves (Response in %)

1 1 BAP 13.00 d

2 2 BAP 36.00 c

3 3 BAP 75.00 a

4 4 BAP 65.00 b

Table 4.8



1 0.1 IAA + 1 BAP 26.00 d

2 0.25 IAA + 2 BAP 39.00 c

3 0.5 IAA + 2.5 BAP 67.33 a

4 0.75 IAA + 3 BAP 65.00 b

Table 4.9



1 0.1 IAA + 1 Zeatin 31.33 d

2 0.25 IAA + 2 Zeatin 40.66 c

3 0.5 IAA + 2.0 Zeatin 75.66 a

4 0.75 IAA + 3 Zeatin 53.33 b

Table 4.10

Table 4.8, 4.9, 4.10- Percentage of Regeneration Under the Influence of Different

Hormone Concentration and Combination for Cotyledonary Leaf

Induced Callus

The value represents mean + SE of three replicates (n=3). Among the different combinations

tried, 3 BAP, 0.5IAA+ 2.5BAP and 0.5IAA + 2.0 Zeatin shows maximum response. In a column,

mean followed by a different alphabet are significantly different at 5% level by DMRT.


The response of callus and callusing index were influenced by, both explant type

and phytohormones used. Different concentrations of auxins and cytokinins in

combinations had a distinct effect on callus induction from both explants and visible

results were observed. The in vitro callus induction is not only influenced by the

exogenously supplied growth regulator, but also depends on the endogenous

concentration of plant growth regulator (Pal et al., 2007). Also the requirements of

phytohormones vary for callus induction depending on the source of explants (Nikam &

Shitole 1998). A higher frequency of callus induction was observed in hypocotyls

followed by cotyledonary leaves in MS media fortified with either 0.5 IAA or NAA in

combination with 2.5 BAP (fig. 4.9, table 4.7). The morphology of calli was different in

both the cases. The calli were green and hard in medium supplemented with 0.5 NAA +

2.5 BAP whereas, it was soft, fragile and greenish yellow when NAA was replaced by

same concentration of IAA (without altering the concentration of BAP). The efficiency of

callus formation was high in MS supplemented with 2.0 NAA alone (Gulshan et al., 1981).

The effect of thiamine concentration in the shoot regeneration medium was also

studied. Tomato cotyledon explants on MS media without Thiamine supplementation

presented a dramatic decrease of chlorophyll content and increase in necrotic areas.

However, with 4 times higher thiamine concentration in the medium (0.4mg /L) healthy

and green tissue developed, which was apparently essential for cotyledon explants

survival prior shoot differentiation.

Although sufficient callus was induced on both explants (0.5 NAA or 0.5 IAA in

combination with 2.5 BAP) subsequent organogenesis was observed only on the callus


induced on medium containing Zeatin (fig. 4.10, table 4.8, 4.9, 4.10). The regeneration

frequency was low when BAP alone or in combination with auxins was used. The best

shoot formation (76%) was obtained for cotyledonary induced callus explants and

sub-cultured on MS medium containing 0.5 IAA with 2.0 Zeatin (fig. 4.8 i). However, it

was only 68%, when BAP was used alone (3 BAP). The replacement of Zeatin by single

synthetic cytokines, such as BAP was not very successful as they did not induce higher

number of shoots or helped in elongation. It can be presumed that addition of Zeatin in

the media elicits the shoot initiation in a higher rate. The role of cytokinin like Zeatin in

vascular morphogenesis is already a known fact. The necessity of cytokinin for shoot

initiation and elongation is well established (Beck & Coponetti 1983; Evans et al., 1984).

Three to four shoots per explant were obtained when the calli was subcultured on

MS medium supplemented with 0.5 IAA with 2.0 Zeatin. The choice of zeatin for shoot

differentiation and elongation was based on previous studies about growth regulators of

tomato explants morphogenesis (Cortina & Culianez-Maci 2004). In this study, Zeatin in

combination with IAA showed the best result for shoot organogenesis than other growth

regulators tested. Eventhough, the best callus induction was obtained on hypocotyls

explants, the cotyledon induced calli had better performance in regeneration through

callus indicating that cotyledonary explants of tomato is an excellent explant for plant

regeneration. Previous reports also demonstrated that cotyledon derived explants of

tomato were superior to other sources including hypocotyls, stems and leaves for promoting

shoot organogenesis of tomato (Hamza & Chupeau 1993; van Roekel et al., 1993;

Ling et al., 1998). Hence for the genetic transformation studies, MS media supplemented

with 2.0 Zeatin in combination with 0.5 IAA and B5 hormones was used for callus

179


induction and regeneration. Optimization of callus induction and indirect plant

regeneration for cultivated varieties of tomato will aid in the development of an efficient

genetic transformation system, which is also one of the objective of this chapter.

4.3.2.2.3 Rooting and Hardening of Tomato- Induction of roots on regenerated shoots

is very essential for successful establishment of the plantlet in the soil. Rooting (80%)

was obtained, when supplemented with half strength MS medium along with B5

hormones. The phytohormones were not necessary for the rooting of the in vitro regenerated

tomato shoots. Devi et al., (2008) reported that, the tomato rooting was obtained on half

strength MS basal medium. Rooting started after in 3-4 days and well-developed roots were

seen after 10-12 days of inoculation (fig. 4.8 j, k). The endogenous auxin present in the

plants might be sufficient for the rooting. Since in vitro regenerated plants are raised in

the most congenial conditions, hardening is very important to ensure survival of the

micropropagated plants upon transfer to soil. Hence, the rooted plantlets were transferred

to plastic pots containing sterile soil manure mix and were covered with polythene bags

to maintain humidity. The plants were initially kept under culture room conditions. Later

the polythene bag was removed and transferred to growth chamber maintaining

controlled conditions. After 2 weeks, the plants were transferred to containment facility.

4.3.3 Genetic Transformation of Tobacco and Tomato- Development of an effective

plant regeneration and genetic transformation protocol is a pre-requisite for successful

application of genetic engineering for improvement of various agronomic traits in crop

plants. There is no universal applicable protocol for the in vitro culture and genetic

transformation system for all the crop species. Choice of explants and age of the seedling

plays an important role in inducing in vitro regeneration and genetic transformation


(Goyary et al., 2010). The transformation efficiency of tomato depends on many factors like

the cultivar used, age and type of explant, density of Agrobacterium used, co-cultivation

time, regeneration medium, concentration of antibiotics used, etc. (Davis et al., 1991;

Madhulatha et al., 2007). As there are reports that cotyledonary explants show better

regeneration in tomato (Frary & van Eck 1996; Ellul et al., 2003), same were used as

explants for transformation in our studies. In this study, 10 days old in vitro grown

tomato seedlings and 2 weeks old leaves of tobacco were used as explants for genetic

transformation experiment. The use of cotyledonary leaf resulted in efficient

transformation and regeneration of both tomato and tobacco plantlets. The preculture of

explants was very essential for the transformation. The precultured leaves of tobacco and

tomato showed curling and bulging which can be considered as the cellular

reprogramming for the initiation of callus induction (fig. 4.11 a, b, c).

Fragile, green calli was formed at or near the wounded site in tomato after

2 weeks of transformation. MS media supplemented with B5 vitamins, 0.1 IAA + 2.0

Zeatin + 75mg/L Kanamycin + 400mg/L Carbenicillin was used for selection of tomato

transgenics (fig. 4.11 d). Zeatin in combination with IAA improved the frequency of

callus formation and shoot regeneration in tomato (Park et al., 2003). The tomato calli

obtained in selection medium after a month was subcultured and transferred for shoot

regeneration on the same media for 8-10 weeks. These regenerated shoots were

transferred to shoot elongation medium (fig. 4.11 e, f, g, h, i). After 12 weeks, the

elongated shoots of 5-6 cm were transferred to rooting media with antibiotics. About

50-60% rhizogenesis was observed in 20 days after inoculation to rooting media

(fig. 4.11 j, k, l). Regenerated plants with well-developed roots were hardened in soil


mix. The plants were then transferred to containment facility for further growth (fig. 4.12

a, b, c). It was also observed that, in vitro regeneration of plantlets from cotyledonary

leaf explants (WT) took only 2 months, whereas regeneration of transgenics required

3-4 months. The difference in regeneration time may be due to antibiotic selection

pressure in the regeneration medium. Singh et al., (2011) also reported that the transgenic

tomato plants took longer regeneration time when compared to the WT plants.

The flowers were pollinated manually and were covered with polythene bags. Later seeds

were collected from fruits and were used for further studies (fig. 4.12 d, e, f, g, h).


Figure 4.11- Agrobacterium Mediated Genetic Transformation in Tomato

a- preculture of tomato b- bulging of leaves in pre-culture media after infection c, d- callus

induction in tomato e, f, g, h,i- indirect regeneration in plants j, k, l-rooting in tomato

a b c

d e f

g h i

j k l


Figure 4.12 – Hardening and Fruiting in Tomato

a, b, c – Hardening of tomato in soil, d- fully mature plant in flowering stage e, f, g, h- fruiting in

transgenic plants.

a b c

d e f

g h


Fragile, dark green cell aggregates were formed in the infected leaves of tobacco,

which later developed to calli after 2 weeks of infection. MS media supplemented with

1 BAP + 0.1 IAA + 75mg/L kanamycin + 400mg/L carbenicillin was used for the

selection of tobacco (fig. 4.13 c). Multiple shoots were developed from the calli after

3-4 weeks (fig. 4.13 d). The WT plants got bleached and died. The shoots from the calli

were excised after a month and inoculated for shoot regeneration on the same media.

These regenerated shoots were transferred to shoot elongation medium (fig. 4.13 e, f).

The well-developed shoots of 5-6 cm were transferred to rooting media with antibiotics

(fig. 4.13 g, h, i). About 80% rooting was observed, 20 days after inoculation to rooting

media. Regenerated plants with well-developed roots were hardened in soil mix. The plants

were then transferred to containment facility for further growth (fig. 4.14 a, b, c). The flowers

were manually pollinated and covered with paper bag to avoid cross-pollination (fig. 4.14 d, e, f)

and seeds were collected from the transgenic plants. The seeds were further analysis,

Overall the transformation efficiency was high as expected in tobacco (68%) and

in tomato (32%). Also the adaptation of the hardened plants was higher in tobacco and

nearly 50-60% of the tomato plants were lost during acclimation. The efficiency of

tomato transformation depended on many factors like the, genotype, explants, hormones

and many other factors. Sharma et al., (2009) reported a highly improved transformation

efficiency of 41.4% for the tomato variety Pusa Ruby, whereas it was only 14.2% for

the same variety, when tomato leaf curl virus coat protein gene was expressed

(Raj et al., 2005).

185


Figure 4.13 – Agrobacterium-Mediated Genetic Transformation in Tobacco

a- preculture of tomato b- bulging of leaves in pre-culture media after infection c,d- callus

induction in tomato e, f- indirect regeneration in plants g, h, i- rooting in tobacco

a b c

d e f

h i j

186


Figure 4.14 – Hardening and Fruiting in Tobacco

a, b, c, - Hardening of transgenic plants in soil, d, e- flowering in transgenic tobacco, f, - fruiting

in transgenic plants

b a

e d f

c


4.4 CONCLUSION

The carrot AFP gene was isolated and cloned into Agrobacterium binary vector

pBI121. The pBI121-AFP was moved to E. coli and later to Agrobacterium. Tissue

culture conditions were standardized for PKM1 variety of tomato. The gene was

successfully transformed to tobacco and tomato. The seeds were collected and used for

further studies.


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