Review of  Literature 

             

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REVIEW OF LITERATURE

Cereals including Sorghum form centre-piece of world agriculture by providing

more than half of the food consumed by man (Vasil 1994). The genetic improvement of

cereals has therefore been a major focus of plant breeding efforts during the past fifty

years, resulting in remarkable increases in the yield as well as improvements in the

quality of this group of food crops. However, plant breeding has severe biological

limitations, and such increase in yield and productivity may not be sustained indefinitely.

In this regard, the emerging technologies, plant tissue culture and genetic engineering,

have attracted much attention as they provide powerful and novel means to supplement as

well as complement the traditional method of plant improvement.

The concept of totipotency is important for understanding plant cell culture and

regeneration. Haberlandt in 1902 described totipotency as the ability of a plant cell to

perform all the functions of development, which are characteristic of zygote, i.e., ability

to develop into a complete plant. Plant tissue culture methods have been employed as an

important aid, which is not possible through the conventional methods. The process in

use of plant cell and tissue culture in producing pathogen free plants, synthetic seeds,

secondary metabolite production and also provides basic requirement for genetic

transformation. Until 1980s there were only few reports of reproducible plant

regeneration system from tissue cultures of Poaceae (Vasil 1990). Of late, considerable

progress has been made in biotechnology can be applied to cereal crops as well.

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In 1949, La Rue reported tissue culture work by using maize endosperm; this was

the first successful tissue culture work in cereals. Consequently Narayanaswamy (1959)

reported callus induction from immature embryo explants of pearl millet; this was the

first report on callus induction in cereals. Today, almost all major cereal crops can be

regenerated from tissue culture (Kumar et al., 2001). Furthermore, the uniqueness of crop

species requires researchers to fit new techniques into current frameworks of crop

improvement. Sorghum is one such important food and feed crop which comes up well in

difficult environments helping the poorest of the poor to realize sustainable rate from the

farm enterprise.

In this chapter, a brief review has been made on the development of cell and

tissue culture systems and the rapid achievements made in the field of genetic

transformation for the improvement of Sorghum.

Progress made in tissue culture research of Sorghum

Plant regeneration in vitro can be achieved using single cell explants like cell

suspensions and protoplast as well as multicellular explants like immature embryo,

immature inflorescence and shoot tips. The type of explants used plays a significant role

in the somatic embryogenesis and further regeneration in vitro. Regeneration in tissue

cultures rely on totipotent or somatic plant cells that can be developed into whole plants

under in vitro conditions through organogenesis or somatic embryogenesis with the help

of optimum hormonal and nutritional supply (Skoog and Miller, 1957). In Sorghum, the

earliest work on in vitro culture was reported by Strogonov et al., (1968) and they

reported callus induction from aseptically germinated Sorghum seedlings.

8

Masteller and Holden (1970) reported that, the callus growth may be the growth

of aberrant meristematic tissue and not undifferentiated cells. They also showed that, this

callus growth generally forms at the basal node of the Sorghum seedlings in response to

2,4-D, an auxin analog and the growth regulator of choice.

Gamborg et al., (1977) observed morphogenesis and plant regeneration from callus

cultures of immature embryo of Sorghum. They reported that, cultured explants released

black and purple pigmented material into the medium, which causes the growth

retardation of callus cultures. They also observed somaclonal variations i.e., variation in

leaf morphology and growth habit. This was the first report of somaclonal variation from

cell and tissue culture derived cultures of Sorghum. Shoot and embryo like structure

formation from cultured tissue of Sorghum were reported by Thomas et al., (1977).

They observed two types of calli in their immature inflorescence and immature embryo

cultures and named them as embryogenic callus and non embryogenic callus.

Dunstan et al., (1978) studied the anatomy of secondary morphogenesis in

cultured scutellum tissues of Sorghum. They observed callus initiation and development

of embryo like structures from the scutellar tissue. They made a detailed study on plantlet

production from the cultured tissue. They observed the formation of embryogenic calli

from the scutellum either by direct embryogenesis of scutellar cells or by subsequent

proliferation of these meristems induced by auxin in the medium.

Brettell et al., (1980) noticed the development of embryo like structure from less than 20

mm length immature inflorescence. They described somatic embryogenesis and efficient

regeneration in Sorghum. This group also suggested that competence in vitro, i.e., the

9

ability to regenerate plants is correlated with continued meristematic activity. Somatic

embryogenesis from Sorghum leaves were reported by Wernicke and Brettell (1982).

Boyes and Vasil (1984) reported plant regeneration by somatic embryogenesis

from cultured young inflorescence. Lower response of the youngest as well as oldest

inflorescence was also observed by them. They also observed the inhibitory effect of

higher 2, 4-D concentrations on somatic embryogenesis. In sweet Sorghum, plant

regeneration via somatic embryogenesis from immature and mature embryos were

reported by Mackinnon et al., (1986), they studied grain and sweet Sorghum cultivars and

observed that pigmentation color of the callus differed among grain and sweet Sorghum

types. Thus, in vitro somatic embryogenesis from immature embryos and immature

inflorescences is now the desired pathway for the regeneration of genetically uniform

plants (Vasil and Vasil 1986).

Kresovich et al., (1987) made detailed study on application of cell and tissue

culture techniques for the genetic improvement of Sorghum. They opined that, reports

of plant regeneration from Sorghum tissue cultures are limited due to the secretion

of phenolic compounds in the cultures. Induction and maintenance of long-term callus

cultures were found to be extremely difficult in Poaceae. The developmental stage of the

explant has been reported to be the critical factor in the establishment of totipotent cells

in these studies (Vasil 1987). The culture of the explants obtained from tissues or organs

that contain meristematic and undifferentiated cells allowed the induction and long-term

maintenance of embryogenic tissue cultures in a wide variety of Poaceae species

(Vasil 1987). The nutrient requirements for tissue culture of most of Poaceae members

are rather uniform, as MS medium (Murashige and Skoog 1962), supplemented with

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2, 4-D was proved to be adequate for callus development and subsequent plant

regeneration (Vasil 1987).

Enhanced somatic embryogenesis in Sorghum from shoot tip cultures were

reported by Bhaskaran et al., (1988). Though they reported enhanced somatic

embryogenesis, the regeneration obtained by them is limited to a number of genotypes.

Bhaskaran and Smith (1989) observed the control of morphogenesis in Sorghum

by 2, 4-D and cytokinins in the leaf segment derived cultures.

Cai and Butler (1990) established a procedure for the induction of regenerable

calli from immature inflorescence segments of high tannin cultivars of Sorghum. They

observed genotypic differences in pigment production, embryogenic callus formation,

and shoot differentiation and regeneration capacity. Among the different developmental

stages of immature inflorescence tested in their study, no critical differences were found

regarding callus development, embryogenesis and regeneration. Soft and friable,

but highly embryogenic callus cultures were described in Sorghum (Wei and Xu 1990).

The influence of genotype on in vitro growth and differentiation patterns has been

reported in a number of crop plants, including cereals (Bhaskaran and Smith 1990).

Rao et al., (2000) reported genotypic differences in callus initiation from immature

inflorescence in Sorghum. Cai and Butler (1990) also observed the increase in

embryogenic callus formation and decrease in pigmentation with the addition of amino

acids.

Isenhour et al., (1991) noticed that tissue culture derived Sorghum plants exhibits

resistance to leaf-feeding by the fall Armyworm. They also reported that, tissue culture

11

induced variations can be a viable means of generating new sources of genetic diversity

for crop improvement. The findings of Miller et al., (1992) are another indication that,

tissue culture induced variations can be a viable means of generating new sources of

genetic diversity for crop improvement. They observed acid soil stress tolerance in tissue

culture derived Sorghum lines. After several sub-cultures, most cereal cultures loose their

morphogenic capacity (Kishor et al., 1992, Pius et al., 1993) and their transfer onto

2, 4-D medium is no longer sufficient to induce embryo development.

Emons et al., (1993) reported that osmolarity of the medium is an important factor

in somatic embryogenesis. Using high concentrations of 2, 4-D. Elkonin et al., (1993)

obtained diploid plants from cultures derived from mature panicles i.e., 35 mm length

panicles. They reported that, the majority of the diploid regenerants contained mutations

which mainly affect male fertility and plant height. They also reported that, the ploidy

levels of regenerated plants were affected by the age of explants and

2, 4-D concentrations in the culture medium.

The appropriate culture type was limited to few cultivars or cell lines

(Christou 1993), which includes callus induction, somatic embryogenesis, occurrence of

somaclonal variations and regeneration. Broad range of varietal differences in callus

formation and plant regeneration was observed by Hagio (1994).

Hagio (1994) reported that, sustained varietal differences are displayed in callus

formation and plant regeneration on Sorghum as well as in other major cereals. He

suggested that mature seeds are a good source of material for in vitro culture, as they are

most readily available and free from seasonal limits which immature embryo,

inflorescence and anther have. Inclusion of certain amino acids like tryptophan, serine

12

and proline in the culture medium has been shown to increase the number of somatic

embryos in Sorghum (Rao et al., 1995), they reported enhanced plant regeneration in

grain and sweet Sorghum by asparagine, proline and Cefotaxime.

Elkonin et al., (1995) reported that presence of L-proline and L-asparagine

in tissue culture media reduces the production of phenolic compounds. Bai et al., (1995)

and Bhat et al., (1995) have shown that immature inflorescence is superior to immature

embryo for plant regeneration. Direct somatic embryogenesis and plant regeneration on

Sorghum have been reported by Gendy et al., (1996) by using transverse thin cell layers

from roots and epicotyls. They concluded that the success in controlling direct and rapid

somatic embryogenesis is a prerequisite condition for production of transgenic plants.

Explants derived from meristematic tissue at early stages of development are most

amenable to tissue culture conditions (Puddephat et al., 1996).

Kaeppler and Pederson (1997) evaluated 41 genotypes of Sorghum using

immature inflorescence as the explant and identified genotypes capable of high quality

callus production. Zhong et al., (1998) developed an efficient and reproducible plant

regeneration system from shoot apices of aseptically germinated seedlings of Sorghum;

they produced multiple shoot clumps by using 2-4 mg/L of BAP. They made a detailed

histological study by using scanning electron microscope to observe the developmental

stages of somatic embryos.

The effect of developmental stages on embryogenic callus induction was reported

by Rao et al., (2000). They found that, the inflorescence of 4-5 cm in length with pinhead

size spikelet primordial were the optimal stage for the production of efficient

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embryogenic callus production. In vitro system was developed for the production of

numerous primary and secondary somatic embryos from shoot tip cultures of Sorghum

(Seetharama et al., 2000).

Mythili et al., (2001) demonstrated the potential use of cytogenetic variants in the

cultures of Sorghum and they obtained efficient regeneration with 1 mg/ L of KN and

1 mg/L of BAP. Oldach et al., (2001) developed in vitro plant regeneration protocol from

immature embryos of Sorghum. Adventitious shoot regeneration from immature embryos

of Sorghum was reported by Hagio (2002). He studied the tissue culture response of

11 genotypes of Sorghum and also observed the position effect of proline and

polyvinylpyrrolidone on shoot formation. Direct somatic embryogenesis from isolated

shoot apex was reported by Harshavardhan et al., (2002). They developed an improved

protocol for direct somatic embryogenesis by using MS supplemented with 5 μM

of TDZ, 17.72 μM BAP and 1.074 μM of NAA and for root induction they used MS

supplemented with 8.28 μM of IBA and 1.14 μM IAA.

Visarada et al., (2003) reported that, tissue culture protocols are genotype specific

and suitable protocols need to be developed when a new variety is to be used. They made

a detailed study on callus induction and regeneration using different explants of Sorghum.

They reported multiple shoot induction and regeneration using 1-6 mg/L concentration of

BAP. Nirwan and Kothari (2004) reported that copper in callus induction and

regeneration media increases callus induction rate and regeneration frequency.

Rathus et al., (2004) reported that, the addition of casein hydrolysate, mixture

of amino acids and vitamins to the callus induction medium promoted the production

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of embryogenic callus in Sorghum. They also reported that, physiological state of the

immature embryo as the most important factor for embryogenic callus induction and

regeneration. In vitro response of different explants viz., shoot tip, immature

inflorescence and immature embryo were studied by Mythili et al., (2004). They found

that shoot tip and immature inflorescence were more totipotent than the immature

embryo. Sato et al., (2004), reported that 1 g/L KH2PO4 along with MS medium

increases the response of Sorghum explants in tissue culture.

Anju verma and Anandkumar (2005) developed an efficient plant regeneration

system from different explants of Sorghum; they reported multiple shoot induction by

using 2 mg/L of BAP in the culture medium. Transformation without regeneration and

regeneration without transformation has limited scope (Sharma et al., 2005).

Sorghum tissue culture is reported to be highly recalcitrant mainly because the release of

toxic phenolic compounds in culture media, lack of regeneration in long term in vitro

cultures, and high degree of genotype dependence. Age of the seedlings in relation to the

number of actively dividing cells that contain greater potential for in vitro response, was

reported for the first time in Sorghum with electron microscopy by Kishore et al., (2006).

Gupta et al., (2006) reported that genotypic limitations of plant regeneration could

be overcome by the use of immature inflorescence explant and inclusion of kinetin in

callus induction media. Kishore et al., (2006) suggested mature seeds as the best explants

for developing callus and regeneration because the seeds can be stored easily and

available throughout the year. Pola et al., (2006) reported highest number of somatic

embryos from leaf segments of Sorghum on MS medium supplemented with 2.0 mg/L

of 2,4,5-T and 1.0 mg/L of ZN.

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Kishore et al., (2006) evaluated 24 Sorghum genotypes using shoot tip as explant

but the results indicates that the genotypes tested are not promising for genetic

transformation. They also described a simple procedure, for handling large callus

obtained from shoot tips. Gupta et al., (2006) compared the tissue culture regeneration of

immature embryos and immature inflorescences from eight genotypes Sorghum.

They indicated that the regeneration potential of immature inflorescences was much

superior to that of immature embryos and their performance was almost equivalent across

the genotypes tested. The superiority of immature inflorescences can be due to its higher

proportion of meristematic tissues (floral meristems, rachis, rachillae, and primordial of

various floral organs) in comparison to immature embryos (mainly scutellum) according

to authors.

Girijashankar et al., (2007) reported the Sorghum plant regeneration from shoot

tips and also observed direct somatic embryogenesis from two different types of calli

simultaneously in the same explants. Jogeswar et al., (2007) developed direct somatic

embryogenesis and regeneration protocol for immature inflorescence explants

SPV-839, SPV-462 and M 35-1 Sorghum genotypes.

Grootboom et al., (2008) reported callus initiation and regeneration potential of

five Sorghum genotypes on specific nutrient media using immature zygotic embryos as

explants with the aim to identify the best genotype-nutrient medium combination that

results in satisfactory regenerability. Pola et al., (2009) developed a protocol for long

term maintenance of callus cultures. They succeeded in maintaining the embryogenic

callus cultures derived from immature embryo, up to 57 weeks in a regenerable stage by

using MS medium containing 2 mg/L 2, 4-D, 0.5 mg/L KN, 10 mg/L AgNO3, 400 mg/L

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casein hydrolysate and 200 mg/L each of L-asparagine and L-proline. They also reported

the effect of different constraints on shoot regeneration of Sorghum using mature embryo

as the starting material to identify an ideal genotype for plant tissue culture and genetic

transformation. Zhao et al., (2010) developed an efficient regeneration system using

germinated seeds of two cultivars (Yuantian No. 1 and M81E) of Sorghum bicolor. They

observed best shoot induction on MS medium supplemented with different concentrations

of IAA, BAP, and KN. Liu and Godwin (2012) standardized callus induction,

regeneration and rooting medium to reduce phenolics production.

Progress made in multiple shoot induction of Sorghum

Previously in Sorghum several authors have described callus induction and

multiple shoot induction from different explants. Kresovich et al., (1986) reported

multiple shoots from sweet Sorghum varieties; they reported less than 10 shoots in the

mature embryo cultures. George et al., (1989) reported high frequency of somatic

embryogenesis and regeneration from 10-25 mm length immature inflorescence

of Sorghum by using high concentrations of KN + BAP in the culture medium.

Low level of regeneration response in cereals including Sorghum is associated with

genotypic specificity and gradual loss of regeneration ability after several subcultures

(George et al., 1989, Cai and Butler 1990). Rao et al., (1995) reported enhanced plant

regeneration in grain and sweet Sorghum by L-asparagine, L-proline and Cefotaxime.

They reported that addition of certain amino acids like tryptophan, serine and proline in

the culture medium has been shown to increase the regeneration frequency in Sorghum.

17

High frequency of plant regeneration from embryogenic callus cultures

of Sorghum was reported by Kuruvinashetti et al., (1998) by using 0.75-1.0 mg/L BAP.

Zhong et al., (1998) developed an efficient and reproducible regeneration system from

shoot apices of aseptically germinated seedlings of Sorghum. They reported multiple

shoot clumps through an intensive differentiation of both axillary and adventitious buds

by using 2-4 mg/L BAP. Multiple shoot induction of switch grass was reported by Gupta

and Conger (1999) from the seedling tissue. Sairam et al., (1999) reported 8 shoots for

explant from mesophyll derived protoplasts of Sorghum by using

0.2 mg/L KN + 2 mg/L BAP.

Rao et al., (2000) reported 18 shoots per explant in Sorghum by using

0.25 mg/L BAP. They found that the inflorescence of 4-5 cm in length stage for the

production of efficient callus production. Devi et al., (2000) reported multiple shoot

induction from the inflorescence and shoot apices explants of pearl millet. They obtained

80 shoots from each explant by using four different concentrations of BAP. Formation

of multiple shoots in immature inflorescence of finger millet was reported by

Kumar et al., (2001). They reported 94.2 shoots from 100 mg of callus. An improved

protocol for regeneration of Sorghum has been reported by Harshavardhan et al., (2002).

They reported 35 - 40 shoots from the isolate shoot apices by using

MS + 5 μM of TDZ + 17.72 μM BAP + 1.074 μM NAA. For root induction they used

8- 28 μM of IBA and 1.14 μM IAA. Multiple shoot induction from the shoot apical

meristem of wheat was reported by Ahmed et al., (2002). They reported 40-50 shoots per

explant by using 2-4 mg/L BAP + 0.5 mg/L 2, 4-D in the culture medium.

Visarada et al., (2003) observed multiple shoot induction in different explants of

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Sorghum viz., shoot apex, immature inflorescence, immature embryo and mature embryo.

They reported 6-18 multiple shoots per explant, by using 2.0-6.0 mg/L BAP.

Mishra and Khurana (2003) reported 30 plantlets for each leaf base explant in Sorghum

by using 0.1 mg/L BAP. O`Kennedy et al., (2004) have established a highly efficient

regeneration system via somatic embryogenesis in pearl millet. They reported an average

of 80 regenerants per individual immature zygotic embryo; they also studied the effect of

IAA and AgNO3 on regeneration efficiency. Nirwan and Kothari (2004) reported 39

regenerants per explant from mature embryo cultures of Sorghum by using

2 mg/L BAP + 0.5 mg/L IAA.

Anju verma and Anandkumar (2005) reported 8.64 multiple shoots with 2 mg/L

BAP from shoot apex as a source explant. Baskaran et al., (2006) reported 35 shoot per

callus in Sorghum. Maheshwari et al., (2006) reported 42 green shoots per callus from

shoot tip explants subjected to a vertical slit from the base of SPV 462 and

M 35-1 genotypes of Sorghum. Kishore et al., (2006) reported multiple shoots by

manipulation of 2.0 mg/L BAP, 0.5 mg/L TDZ. Baskaran et al., (2006) reported

35 shoots per callus in Sorghum using 13.3 μM BAP + 2.3 μM 2,4-D. Pola et al., (2007)

reported 72 plantlets per explant from immature embryo of Sorghum by using different

concentrations of BAP, TDZ and IAA. 43.2 shoots per culture from mature embryo

explants of Sorghum by using 1.5 mg/L BAP, 1.5 mg/L TDZ and 1.0 mg/L IAA was

reported by Pola et al., (2009). Kumar and Bhat (2012) reported 20 shoots per explant

from shoot tip explant by using 3.0 mg/L TDZ.

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Progress made in direct somatic embryogenesis of Sorghum

The plants derived from direct somatic embryogenesis usually are unicellular in

origin and hence genetically uniform (Vasil, 1987). The most frequent approach for

cereal regeneration includes initiation of embryogenic calli from immature tissues and

subsequent regeneration of plantlets (Wan and Lemaux 1994). Unfortunately, callus

mediated methods induce somaclonal variation (Barcelo et al., 1994b). Moreover, for

these methods, tissue and varietal selection are important as only a few cereal plant

tissues are capable of regeneration and a few varieties are amenable to regeneration

of fertile plantlets. Thus, the callus mediated system is an inefficient method for

regeneration of most cultivars of commercial importance in all over the world.

An alternative method is a callus free development and regeneration pattern, through the

induction of direct somatic embryogenesis.

Previously, direct somatic embryogenesis has been reported in rice by

Nhut et al., (2000) by culturing the apical meristem explants on MS + 2,4-D + BAP.

Mariani et al., (2000) reported improvement of direct somatic embryogenesis in rice by

selecting the optimal developmental stage of the explant. Direct somatic embryogenesis

has also been reported in several other monocots like garlic (Sata et al., 2000),

rice (Sahasrabudhe, 2000), minor millet (Vikrant et al., 2001). In Sorghum,

Harshavardhan et al., (2002) reported direct somatic embryogenesis from the shoot apices

cultures and they successfully controlled the pathway of direct somatic embryogenesis by

striking an optimal balance between BAP and NAA concentrations.

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Eudes et al., (2003) developed direct somatic embryogenesis and secondary

embryogenesis protocol for seven cereal species, viz., Hordeum vulgare,

Triticum aestivum, T. durum, T. monococcum, T. urartu, Secale cereale and Avena sativa.

The regeneration of cereal plant cells from callus remains a major limiting step in

obtaining elevated number of cereal clones or independent transgenic cereal lines

(Eudes et al., 2003). Desai et al., (2004) developed a protocol for direct somatic

embryogenesis without intervening callus phase for Saccharum using immature

inflorescence segments. Conversion of somatic embryos into plantlets has remained

inefficient and limited because of low frequency, genotype specificity, and occurrence

of callus phase before embryogenesis (Maheswari et al., 2006). Moreover, conversion of

somatic embryos into plants is the most prolonged phase in culture (Gupta et al., 2006).

Girijashankar et al., (2007) reported direct somatic embryogenesis and

organogenesis pathway of plant regeneration simultaneously from the shoot apices

explant of Sorghum. They also reported that regeneration of Sorghum plants through

intervening callus phase is one of the possible reasons for the loss of germ line

transmission of transgenes. Jogeswar et al., (2007) reported high frequency of direct

somatic embryogenesis from immature inflorescence explant of Sorghum by using

MS medium supplemented with 2.0 mg/L 2, 4-D and 0.5 mg/L KN. They also reported

that the presence of 1.5 mg/L BAP and 1.0 mg/L KN in MS medium was most efficient

for maturation and germination of somatic embryos. Sadia et al., (2010) reported

regeneration of plants directly from shoot meristems of Sorghum without an intervening

callus phase.

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Progress made in SEM studies of Sorghum

Histological studies by using light microscopy and scanning electron microscopy

(SEM) to confirm somatic embryogenesis and to reveal the detailed pathway of somatic

embryo development were very important in tissue culture studies. Because the best stage

of explant and cell type for regeneration can be predicted in the very beginning stage

of in vitro culture. In Sorghum, limited ontogenic studies have been made on somatic

embryogenesis either with light or using a scanning electron microscope

(Aparna et al., 2004). The earliest work on histological study on callus culture

of Sorghum was that of Masteller and Holden (1970), they observed vacuoles, active

cytoplasmic streaming and inclusion bodies in single cells by using phase contrast

microscope.

Dunstan et al., (1978) studied the development of embryo like structures from the

callus cultures of Sorghum immature embryo by using light and scanning electron

microscope. Brettell et al., (1980) observed the proliferation of embryo like structures

and shoot primordial from the cultures of Sorghum immature inflorescence.

Wernicke and Brettell (1982) made detailed description on morphogenetic pathways and

morphogenesis from cultured leaf tissue of Sorghum with regard to somatic

embryogenesis from multiple cells. The detailed work on in vitro culture development

of callus cultures in cereals was reported by Morrish et al., (1987), they studied the

developmental morphogenesis and genetic manipulation in tissue and cell cultures. The

nature of embryogenic callus in Sorghum was described as white, compact and globular

with smooth shiny surface by Cai and Butler (1990).

22

Zhong et al., (1998) demonstrated embryogenesis from shoot apices by using

scanning electron microscope. They analyzed that embryogenesis began with the random

differentiation of leafy scutella and tubular coleoptile from the organized apical domes,

while the cellular integrity of the epidermis was well conserved during such folding and

budding process. Ahmed et al., (2002) studied in vitro morphogenesis of shoot apical

meristem, somatic embryogenesis and regeneration system in Triticum aestivum using

scanning electron microscopy. They reported a proliferating budding state in the shoot

apex that gave rise to somatic embryos and adventitious shoots on MS + BAP and

2, 4-D medium.

The morphology and histology of somatic embryogenesis in Sorghum bicolor

have been reported by Aparna et al., (2004) by using light and scanning electron

microscope. Histological studies made by them revealed that, the formation of somatic

embryos and their maturation form immature inflorescence with well organized bipolar

structures showing embryogenic axis, scutellum, coleoptile and coleorhizae.

Nirwan and Kothari (2004) observed the origin of shoot apices from the surface

of enlarged meristemoids from the cultures of immature embryo and mature embryo.

Phenolic compounds are generally present in the plant kingdom and more than

8,000 phenolic compounds are known (Harborne, 1994; Pietta, 2000). Amongst the

cereals, Sorghum contains the highest content of phenolic compounds and they can reach

up to 6% (w/w) in some varieties (Hahn et al., 1984; Deshpande et al., 1986;

Beta et al., 1999; Awika and Rooney, 2004). Polymeric polyphenols, antifungal proteins

(AFP) expressed naturally in plants confer resistance to many plant pathogenic fungi.

Many such proteins have been identified in different plants and also the phenols are used

23

for developing resistance against fungal diseases (Butler 1990, Ulaganathan et al., 2001).

Plant regeneration was reduced due to the phenolic compound production

(Gamborg et al., 1977; Vasil and Vasil, 1981). The amount of phenolic compounds

present in any particular variety is influenced by the genotype and the environment in

which it is grown (Dicko et al., 2006).

Progress made in Genetic transformation of Sorghum

Genetic engineering of cereal crops was not successful at the beginning because

of fundamental problems associated with the method of gene transfer into the target

tissue and their subsequent regeneration. Efficient plant regeneration systems depend

upon optimal levels of plant regeneration besides an amenable tissue culture and

regeneration system. Apart from this, the following factors are also critical for successful

transformation: (i) method of DNA delivery into cell (ii) suitable target gene in a

convenient vector with reporter and selectable marker genes, (iii) suitable target tissue

(explant), and (iv) efficient testing methods to confirm the transformed phenotype.

It is equally important to ensure consistent inheritance of transgene in the progeny,

lack of gene silencing or pleiotropic effects, before efficient transgenics are developed.

Transformation of Sorghum is difficult since the response is genotype dependent,

very low regeneration, production of phenolics and various problems in acclimatization

(Maheshwari et al., 2006).

Soon after the first foreign gene transfer in plants was successfully done in 1983,

effective transformation and regeneration protocols have been standardized in various

laboratories worldwide for different crops. Protoplast based direct gene transfer; biolistic

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transformation (Casas et al., 1993; Casas et al., 1997; Zhu et al., 1998;

Tadesse et al., 2003; Girijashankar et al., 2005) and Agrobacterium-mediated gene

transfer (Zhao et al., 2000; Carvalho et al., 2004; Gao et al., 2005; Hiei et al., 2006)

are the major techniques that are being routinely used for transgenic production (Christou

1995, Casas et al., 1993, Zhu et al., 1998).

Transgenic cereal plants were first obtained in rice from protoplast transformation

system, with DNA uptake mediated by electroporation or PEG (Toriyama et al., 1988,

Zhang et al., 1988, Zhang and Wu 1988, Shimamoto et al., 1989, Datta et al., 1990,

Peng et al., 1992). In these methods, the chances of obtaining chimeras are negligible.

But, the method is laborious, needs high skill, tends to be cultivar specific, and that too

with low regeneration frequency (Maheshwari et al., 1995). This requires delicate

manipulation of protoplasts as well as embryogenic cell suspension cultures, which are

often genotype dependent, and therefore is not readily applied to all cereals.

The first successful attempt reported the transformation of Sorghum using particle

bombardment, but the plants regenerated prove to be sterile (Battraw and Hall 1991).

The regeneration of fertile Sorghum plants following particle bombardment was first

achieved by Casas et al., (1993).

Particle bombardment is an efficient method of genetic transformation of cereals.

It offers advantages such as introduction of multiple genes, simplicity and transformation

in those plants where Agrobacterium infection is difficult. This process was initiated by

J.C. Sanford and T.M. Klein at Cornell University in 1980`s. Transient GUS expression

in cultured shoot tips of Sorghum was observed by Devi et al., (2001) they developed an

optimal micro projectile bombardment procedure for Sorghum. Apart from

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microprojectile mediated DNA transfer, there have been significant breakthroughs in

Agrobacterium-mediated transformation of monocots like rice and maize in recent years

(Chan et al., 1992, Gould et al., 1991).

Agrobacterium-mediated transformation

This method is now widely used for the production of transgenic dicotyledonous

plants and major cereals as well. Genetic transformation of plants using Agrobacterium is

believed to be more efficient, as the success rates of transformation are greater than with

biolistics. Further, complex equipment is not involved. This method needs no cell wall

removal from target cells or bombardment, and often single copies of the transgenes are

integrated in transgenic plants. Therefore, many groups attempted to establish this

relatively easy and convenient system to obtain transgenic cereals. Unfortunately, these

attempts were unsuccessful before 1990s.

For a long period of time, monocotyledons seemed to be insensitive to

Agrobacterium infection and subsequent transformation. It has been demonstrated that

the inefficiency of transformation of monocotyledonous species is caused by the lack

of production of virulence inducing substances (Usami et al., 1987, Sahi et al., 1990).

Progress in Sorghum transformation has been hampered by difficulties associated with

tissue culture, such as accumulation of phenolic pigments and low regeneration

frequencies. The long periods of selection needed for the recovery and regeneration

of putative transgenic plants often hampered optimization of conditions for Sorghum

transformation. A number of reports of cereal transformation have come particularly after

the success with rice (Hiei et al., 1994). Godwin and Chikwamba (1994) reported

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inoculation of Sorghum meristem tissue with Agrobacterium. Sorghum transformation

through Agrobacterium has been reported by Zhao et al., (2000). They used the immature

embryos of Sorghum and inoculated with LBA 4404 bacterium strain carrying a

super binary vector with bar gene as a selected marker of herbicide resistance in the plant

cells. This was the first report of the successful use of Agrobacterium for production of

stably transformed Sorghum plants.

Jeoung et al., (2002a) reported optimization of Sorghum transformation

parameters for both the Agrobacterium and Biolistic bombardment methods.

Zhao et al., (2003) reported transformation of Sorghum with agronomically important

HT12 gene for higher grain lysine content. Visarada et al., (2003) also made a detailed

study on transient GUS expression in Sorghum. They developed a simple protocol for

Agrobacterium-mediated genetic transformation. Carvalho et al., (2004) developed three

transgenic Sorghum events through the use of super binary vector with hygromycin

phosphotransferase gene (hpt) as a selectable marker. Girijashankar et al., (2005) reported

transformation of Sorghum with agronomically important cry1Ac gene for insect

resistance. Hiei et al. (2006) reported a several-fold enhancement of transformation

frequency of rice and maize by treating immature embryos (IEs) with both heat and

centrifugation before infecting with A. tumefaciens.

Howe et al., (2006) reported 37 transgenic events were generated from two

Sorghum genotypes using Agrobacterium tumefaciens having disarmed Ti plasmid

pTiKPSF2. Nguyen et al., (2007) recovered 15 transgenic Sorghum events from

300 immature embryos using improved protocol employing standard binary vector

(with hpt as the selectable marker), cold pre-treated immature embryos and activated

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charcoal. Lu et al., (2009) provide a time range of 9-12 weeks to obtain regenerated

plants with a developed root system. Gurel et al., (2009) studied the effect of temperature

on Agrobacterium-mediated frequency on Sorghum and identified 43�C as the optimum

temperature where transformation frequency increased to 7.6 % from 2.6 %

(without heat treatment).

Based on these concerns, complementary studies have been established in the

present study to produce multiple shoots, direct somatic embryogenesis and histological

studies to identity the pathway of somatic embryogenesis and genetic transformation

of Sorghum from different explants by using Agrobacterium-mediated transformation

method.