REVIEW OF LITERATURE

Tissue culture technology offers a great potential in addition to the traditional

methods of plant breeding and improvement particularly in the area of genotype

evolution with chemical tolerance and clonal propagation. Scientists all over the world

wish to apply in vitro methods for the management and breeding of economically

important plants i.e.; those involved in modification and improvement of plants for the

production of food, fodder, fiber and fuel. With increase in the world population, the

continued loss of prime agriculture land for housing and industry necessitates the use

of more marginal land for agriculture and new approaches are needed to the increasing

problems such as disease, drought and salinity. Tissue culture technology offers one

such possibility.

Plant tissue culture provides a powerful technique to assist the plant breeder in

improving the propagation and performance of agriculture and horticulture species.

This was started as an experimental novelty in the beginning but has emerged as the

most potent and internal part of modern biotechnology for the mass propagation of

desirable plants, the recovery of plants free from specific disease, production of

haploid plants and genetic engineering of cell and their metabolism.

The knowledge of this technique begain in 1900, Hebarlandt (1902) a

German botanist cultured single cell isolated from various parts of different plants in

artificial medium in controlled laboratory conditions. According to cell theory

proposed by Schleiden (1838) and Schwann (1839), every living organism consists of

cells which are capable of divisions, this inheritance potency of the cell is called

cellular totipotency is an important attribute which led to the beginning of tissue

culture A new approach to tissue culture was conceived simultaneously by Gautheret,

26
(1934) Nobecourt and White (1939) with their new line of investigation involving the

utilization of auxins that play an important role in cultivating the plant tissues lead to

the theory that establishment of to tipotency in higher plants in generally determined

by plant growth regulators like auxins, cytokinins and other metabolites.

Unto this time the main objective of tissue culture work was ascertaining the

possibilities of culturing the cell indefinitely. Once this object was achieved, attention

was directed to the possibility of using this technique for vegetative multiplication,

organogenesis, synthesis of secondary products etc. The starting point of modern in

vitro vegetative propagation methods (micropopagation) was started with Ball (1946),

as he pointed out exactly which part of a shoot meristem gave rise to a whole plant,

Skoog and Miller (1957) found that organogenesis was controlled by a balance

between auxin and cytokinin. This leads to the need of standardization of auxin and

cytokinin ratio.

The history plant tissue culture and its applications have been reviewed and

discussed from time to time Gautheret, (1983, 1985); Krikorian and Cronauer (1984);

Krikorian (1988); Thorpe, (1990, 2007) and Gamborg, (2002). The problems and

potentials of using tissue culture in micropropagation and biotechnology related to

crop improvement have been critically and elegantly analyzed in several reviews and

discussion Dunstan and Thorpe, (1986); Bonga and Durzan, (1987) and Hammatt,

(1994).

Though in the initial stages of growth of plant tissue culture as a discipline was

very slow, gradually it achieved momentum and has seen very fast developments. This

has resulted in a transfer of attention from basic research to apply. Virus eradication ,

micropopagation, production of secondary products, production and uses of

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somoclonal variation, somatic embryogenesis, haploid production, diploidization,

embryo rescue, protoplast culture, regeneration, fusion to produce somatic hybrids,

gene transfer etc. are some of the areas of plant tissue culture which have applied

significance.

The growth of cells, tissues and organs of higher plants on chemically defined

media in vitro has been made possible for many plant species. The in vitro culture of

cells, tissues and organs of plants is one of the growing areas of plant biotechnology

because of its potential to generate improvements have two components the generation

of genetic variation and the selection, maintenance and propagation of desired

variants, tissue culture technique can be exploited for the mass propagation of elite

plant species and multiplication of range of genotypes.

German plant physiologists Haberlandt (1902) isolated single cells in nutrient

medium containing inorganic salts and sucrose. Robbins (1922) successfully achieved

subcultures of cultivated maize roots, but the cultures did not survive. Steward et al.,

(1958) reported roots were capable of forming roots, shoots and finally whole through

callus.

The degree of success in any technology employing plant cell, tissue organ

culture is related to relatively few factors. A significant factor is the choice of nutrient

medium and growth regulators. The nutrient media used for most of the cultures were

number of reports have appeared on modification of about media compositions

et al.,(1968). A defined nutrient medium

consists of inorganic salts, a carbon sources, vitamins and growth regulators Cocking

(1978). Other components such as organic nitrogen compounds, organic acids and

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plant extracts, may be added for specific purposes Gamberg and Shylock (1981). The

most widly used media for plant tissue culture studies are those of (MS) Murashige

5) Gambo et al.,

(1968).

The various aspects involved in obtaining successful in vitro plant regeneration

by organogenesis are physical and chemical factors Vasil, (1984); Murashige, 1974;

Thorpe, (1990). Fairly large number of plants species have been propagated by in vitro

techniques Narayan Swamy (1977), Vasil and Vasil (1986), Evans et al., (1983).

Micropropogated plants can be exploited commercially Conger (1981), Mantell et

al.,(1985), Pierk (1987), Chu and Kurtz (1992) and means of germ plasm storage for

maintaining diseases free stocks Wilklins and Dodds, (1983); Withers, (1989). The

various factors and different aspects of plant regeneration by tissue culture has been

reviewed Mroginski and Kartha (1984); Parott et al., (1992).

Salinity

Salinity is a major environmental factor determining plant productivity and

plant distribution. It affects more than 10 percent of arable land and salinization is

rapidly increasing on a global scale, declining average yield for most major crop

plants by more than 50 percent reported by Steppuhn, et al., (2005).

Despite decades of research into the effects of salinity on crop plants, the

causes of sodium toxicity remain controversial. Plants are stressed in two ways by the

increase in osmotic potential of the rooting medium as a result of high solute content,

and by the toxic effect of high concentration of ions (Tejera, et al., 2005).

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 Sodium chloride is the most soluble and abundant salt present in the soil and is

mainly responsible for salinity related problems in plants. According to the FAO Land

and Plant Nutrition Management Service (FAO, 2012), over 6% of the world's land

area (400 million ha) is affected by salinity. Of this 400 million ha salt-affected area,

45 and 32 million ha land is associated with irrigated and dry land agriculture,

respectively, and the remaining area is non-cultivated. In India, 6.73 million ha of land

is affected by salinity and 25% of ground water used for irrigation is saline. According

to an estimate, 11.7 million ha land in India is likely to be affected by salinity by the

year 2025.

The success of breeding programs with the ultimate goal of improving crop

productivity is limited by the lack of a clear understanding of the molecular basis of

salt tolerance. Recent advances in genetic analysis of Arabidopsis mutants defective in

salt tolerance, molecular cloning of these loci, have given some insight into salt stress

signaling and plant salt tolerance.

The response of tomato to salinity has been studied intensively (Satti et al.,

1994 and 1995; Satti and Al-Yahyai, 1995; Pasternak and De Malach, 1995).

Tomatoes are especially sensitive to salinity at the young seedling stage (Satti et al.,

1994; 1995). Chloride salts reduced plant dry weight, increased defoliation and

accumulation of Cl in the leaves, and caused a sharp reduction in photosynthesis, leaf

water potential and stomatal conductance (Pasternak and De Malach, 1995).

Different explants were used for study regeneration in tomato and another

plant in vitro under salt stress example hypocotyl (El-Meleigy et al.,2004; Mohammed

et al.,2007; Aazami et al.,2010), cotyledons (El-Meleigy et al.,2004), true leaf (El-

Meleigy et al.,2004 ) and shoot apex (Mohammed et al.,2007). El-Anany (1997)

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studied shoot regeneration from hypocotyl and cotyledon under salt stress and showed

that decrease in shoot number with increase in salinity and high level (100, 150 mM)

NaCl inhibited shoot regeneration. On the other hand, Mohammed et al., (2007) used

shoot apices explants and found the shoot length decreased with increase in salinity.

Abed- Alrahman et al., (2005) and Fayek et al., (2010) respectively showed decrease

in growth traits of cucumber (Cucumis sativus L.) and Jojoba (Simmondsia chinensis)

with increased salinity in vitro.

Effects of salts concentration on emergence and growth of tomato in tropical

areas was reported by Umara, et al., (2013). Abdalmajid et al., (2011) was reported by

In vitro performances of hypocotyl and cotyledon explants of tomato cultivars under

sodium chloride stress. Shibli et al, (2007) found that growth parameters were

adversely affected by increased salinity in the medium in both Roma and Patio tomato

cultivars. Similar results was reported by Hassan et al., (2008) where they noted

decrease in fresh and dry weight of tomato and mung bean shoots strongly related

with salt stress. Siler et al., (2007) studied the effects of salinity on growth and

morphogenesis of Centaurium erythraea in vitro; they found that the high salt

concentrations were effective in induction of auxiliary and adventitious buds on

shoots. Similar results were reported by Potluri and Prasad (1994) in potato, Kashyap

and Sharma (2006) in Morus alba and Erturk et al., (2007) in cherry, where they

found degradation in shoot length, shoots number and dry matter with increased

salinity in growth medium.

Tissue culture in Solanaceae family

The most successful species to respond tissue culture are from Solanaceae

family, which have been used as model system for in vitro studies. Tobacco has used

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as model system from in vitro studies of regeneration since the classical studies of

Skoog and Miller (1957). MS medium was formulated from results of growth

experiments with Nicotina tobacum Murashige and Skoog (1962). The first successful

production of haploid plant by the in vitro cultures of excised anthers was achieved by

using Datura innoxia Guha and Maheshwari, (1964). Totipotency was first

demonstrated with Nicotina tobacum by regeneration of mature plants from single cell

vasil and Helderbrandt, (1965).

Plant regeneration through intermodal segments of Datura metel was reported

by Aroikiasamy et al., (1999). Recent study on micro-propagation and organogenic

studies in Withania somnifera was achieved by Pankaj (2001).

Organogenesis and somatic embryogenesis in Datura alba reported by Manju

Madhavan and Joy Joseph (2001), and axillary shoot proliferation in potato was

achieved by Debabrata et al., (2002). Effect of explant age, hormones on somatic

embryogenesis and production of multiple shoot from cotyledonary leaf explants of

Solanum trilobatum reported by Chakravarti et al., (2009).

The Solanaceae have also attracted interest because they produce a number of

specialized metabolites that have medicinal properties Schijlen et al., (2006);

Oksman-Caldentey, (2007). The Solanaceae are remarkable in that the gene content of

the different species remains similar despite the highly varied phenotypic outcomes

Tanksley et al.,(1992); Knapp et al.,(2004). This makes Solanaceae an excellent

model for the study of plant adaptation to natural and agriculture environments Knapp

et al., (2004). Most species of the Solanaceae are diploid and basic set of

chromosomes, Olmstead et al., (1999).

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Tissue culture in tomato

Tomato is considered as a member of the family Solanaceae. The botanical

name of tomato is Lycopersicon esculentum Mill. It is diploid plant with 2n=24

chromosomes. Naturally, the tomato is a perennial plant, but it is cultivate annually

because of having a great economical and commercial advantages. Moreover, tomato

is considered as the second greatest significant vegetable crops in the world after

potato Mohamed et al., (2010).

Over the last century, tomato (Lycopersicon esculentum Mill.) as an important

vegetable crop has attained a tremendous popularity. It can be grown in most places

all over the world, like growing in the field, greenhouses and net houses. The tomato

crop is grown and used for both fresh market and processing and it is an adaptable

crop. Over the past 25 years, the demand on tomato recorded highly data in both

producing and consuming and it has grown quite rapidly.

In vitro regeneration of cultivated tomato has been a topic of research because

of the commercial worth of the crop and its flexibility for further improvement via

genetic manipulation. Consequently, many studies on plant regeneration from a varied

range of tissue and organs of wild and cultivated tomato germplasm have been

conducted (Evans, 1989). Moreover, tomato breeding programs can extremely benefit

of biotechnological tools, such as gene transfer technology, which accepts the

introduction of foreign genes into a germplasm, without modifying the genetic

background of elite varieties. However, a breeding program associated to

biotechnological tools depends upon the development of an efficient in vitro plant

regeneration system (Abu-E1-Heba et al., 2008).

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 In addition, the success in tomato regeneration response has been found to

depend largely on explants, genotype and plant growth regulator used in culture

medium. Plant growth regulators have effect on morphogenesis of tomato cultures.

Many kinds of plant growth regulators have been used with varying concentrations for

tomato regeneration. Different types of cytokinins and auxin combinations could

induce callus in tomato from different explants (Mohamed et al., 2010). Furthermore,

auxins and cytokinins have a multitude of complex interactions, which control plant

development. The hormonal balance between auxins and cytokinins can regulate the

formation of roots, shoots, and callus tissue in vitro. The way of interaction between

auxins and cytokinins mostly depend upon the type of tissue and on the plant species

in which the interaction occurs (Abu-E1-Heba et al., 2008). Another kind of PGRs is

known as gibberellins, which are naturally occurring in plants, they have affect on cell

enlargement and division which leads to internodes elongation in stem. They have a

dwarf reversing response e.g., it allows certain dwarf cultivars to grow normal height

when treated with gibberellins. It has also a different influence on developmental

processes, particularly those controlled by temperature and light for instance seed and

plant dormancy, germination, seed stalk and fruit development are controlled by

gibberellins (Afroz et al.,2009).

Many studies were conducted on tomato to establish an efficient system of

shoot multiplication and regeneration with growth regulators. The tissue culture has

been done on tomato covering various aspects from excised root culture, regeneration,

somaclonal variations, salt tolerance, disease resistance and genetic transformation.

Namita and Negi (2013) A highly reproducible in vitro regeneration method for

tomato (Lycopersicon esculentum

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using hypocotyl, leaf and cotyledon explants from in vitro raised seedlings on

Murashige and Skoog medium supplemented with different concentrations and

combinations of hormones BAP (2 to 4 mg/l) and IAA (0.1 to 1 mg/l). The medium

supplemented with 2 mg/l BAP and 0.1 mg/l was found to be the best for inducing

direct shoot regeneration and multiple shoots per explant from hypocotyl explants.

Callus induction was observed in all the explants and regeneration of shoots was also

promoted by all these combinations.

Mohammad et al., (2013) reported to develop an efficient protocol of shoot

organogenesis for Lycopersicon esculentum Mill. (Var. cerasiforme) through using of

different types of explants and growth regulators. Generally, all explants respond

significantly to presence of BAP. Best shoot regeneration for leaf (100%) was

achieved on MS supplemented with BAP (2 mg/l) + IAA (0.1 mg/l), whereas it was

recorded on MS supplemented with BAP (2 mg/l) and BAP (2 mg/l) + IAA (0.5 mg/l)

for cotyledons (95%). In addition, hypocotyls (77%) showed the best shoot response

on MS supplemented BAP (3 mg/l). Highest number of shoots per explant was 13.33,

12.25, 7.94 respectively for hypocotyls, leaves, cotyledons.

Cotyledonary leaf explants were used by Chaudhary and Islam while working

with popular Bangladeshi varieties (BINA tomato 3, BINA tomato 5, Bahar) and

Indian variety Pusa Ruby, they found MS medium supplemented with 2mg/l BAP for

best shoot regeneration of all four varieties (Chaudhary and Islam, 2012). Das

mentioned MS media containing 1.5mg/I BAP and 0.2mg/I IAA showed best result

with lowest callus formation and increased number of shoot in BINA Tomato 3, BARI

tomato 3, Bahar and Pussa Rubby varieties (Das, 2011). Ferdous, also mentioned that

MS media supplemented with 2 mg/l BAP found to be best for shoot regeneration for

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BARI Tomato 2, BARI Tomato 3, BARI Tomato 14, BARI Tomato 15 and BINA

Tomato 3 variety (Ferdous, 2012). It was found by (Sarker, 2013) that MS medium

containing 2 mg/l BAP showed the best result with highest number of shoots in BARI

Tomato-3 and BINA Tomato-3 and 1 mg/l BAP containing MS medium showed the

best result with highest number of shoots in BARI Tomato-7 variety.

Pritesh Pawar et al., (2012) reported direct regeneration the use of 1.5 mg/l

Zeatin + 1.5 mg/l IAA with MS medium for the shoot development of cv. Pusa ruby.

Godishala Vikram et al., (2011) reported highest % of response for callus

induction was recorded in (cv. S-22 ) cotyledonary leaf explant at 3.0 mg/l BAP where

multiple shoots were formed at 0.1 mg/l IAA +2.5-5.0 mg/l B AP containing medium.

Kilanaje Ashakiran et al., (2011) mentioned MS media supplement with

0.75mg/l TDZ showed best for shoot regeneration for tomato variety MHTM and

Shalimar.

Bhatia and Ashwath (2008) reported activated charcoal and ascorbic acid

produced longer shoots and addition of casein hydrolysate significantly reduced callus

induction response in shoots of tomato cv. Red Coat.

Majoul et al., (2007) reported the regeneration in tomato var (Justar and

Nemador) from leaf and cotyledon explants. 3-week-old seedlings were used and best

regeneration was observed on MS basal medium supplemented with 1 mg/1 zeatin/ 1

mg/1 IAA and 2 mg/1 of BAP and rooted with 0.1 mg/1 of IAA. Leaf explants

showed the most important organogenesis capacity in comparison to cotyledon

explants.

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 Plevnes et al., (2006) reported MS media supplemented with 2 mg/1 of IAA

and 1 mg/1of BAP best for callus induction in cotyledons of tomato cultivar

L. peruvianum.

Effects of genotype and explants type on in vitro shoot regeneration were

reported in tomato (Jabeen et al., 2005). In vitro regeneration frequency of hypocotyls,

leaf disc and shoot tip of five tomato cultivars was investigated and maximum

regeneration was reported with regeneration medium supplemented with 1 mg/1 zeatin

and 0.1 mg/l IAA. The highest regeneration capacity was observed in cultivar Rio

Grande (80% by using shoot tip, 64.5% by using hypocotyls and 56% by using leaf

disc) from all types of explant.

Callus induction and regeneration was previously reported by several workers

Devi et al, (2008); Ishag et al., (2009); Lima et al., (2009); Osman et al., (2010). In

most cases regeneration of shoots has been obtained through callus Singh et al.,

(2010); Kaur and Bansal, (2010).

Moghaieb et al., (1999) studied the effect of genotype on the regeneration

efficiency callus and regeneration hypocotyl and cotyledonary leaf. Bulk et al., (1990)

reported the effect of explants source, Plants were regenerated from leaf, cotyledon,

and hypocotyl of the cultivar. Schnapp and Preece (1986) reported regeneration using

shoot tip as explants.

Somatic embryogenesis

Somatic embryogenesis is an efficient method of plant regeneration allowing

plants derived from somatic embryos are usually unicellular in origin and hence

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genetically uniform. Somatic embryogenesis is useful in many ways viz., large scale

propagation of plants, production of synthetic seeds, and as target tissue for genetic

transformation. The study of somatic embryogenesis has become more diverse, still

lacking a unifying conceptual framework. The reason, this process of somatic

embryogenesis is so incredible is because; one can produce a plant which is

genetically identical to its parent using nonsexual reproduction. Somatic

embryogenesis is a complex process as a biopolar structure processing shoot and root

resembling zygotic embryo is produced from somatic cells.

Effect of culture manipulation, nutritional, physical and chemical factors for

the induction of somatic embryogenesis has been well documented by Ammirato

(1987); Ammirato (1983a); Lazzeri et al., (1987); Reddy and Reddy (1997). Auxin

compounds has usually well effected for induction of somatic embryos (Ranchi et al.,

1985); Rani and Padmaja (1997).

Somatic embryogenesis is potentially one of the most efficient methods for in

vitro mass propagation. Unlimited production of single individual with functional root

and shoot poles is possible through somatic embryogenesis (Luttman et al., 1994).

Somatic embryogenesis in tomato was reported by Godishala, V. and Nanna S,

(2011); Kaparakis and Alderson, (2002); Philip, et al., (1996); Chen and Adachi,

(1994); Gill et al., (1995).

Tomato transformation

Tomato is one of the most important vegetable crops and a genetic model for

improving other dicotyledonous crop plants (McCormick et al., 1986; Ling et al.,

1998). Transformation of plants has become an integral tool in plant biotechnology for

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both basic and applied research including functional genomics. Transgenic technology

has tremendous potential to address various biological issues related to crop

improvement, including higher yield, tolerance to abiotic and biotic stress, and

expression of desired traits of diverse origin which are otherwise difficult to achieve

by conventional breeding approaches (Liénard et al.,2007; James, 2010; Park et

al.,2011).

The first report of tomato transformation was by McCormick et al., (1986).

There was various reports transformation of tomato cultivars are reviewed below. In

tomato regeneration capacity and transformation frequencies show variation among

cultivars. Transformation frequencies have ranged from 6%-49% (Qiu et al., 2007),

49% (Jabeen et al., 2009) to 49.5% (Raj et al., 2005). Efficiency of transformation is

affected by many factors like Agrobacterium strain, inoculation duration, co-

cultivation time, pre- selection period, concentration of acetosyringone or other

antibiotics in the media, type of explants and genotype (Ahsan et al., 2007). Many

reports were related to only optimization of transformation protocol and other related

to transform for production of resistance against bacterial, viral and bacterial

pathogens in tomato from various sources.

Koul et al., (2014) reported factors affecting the Agrobacterium mediated gene

transformation of tomato cv. PED using cotyledonary leaf and leaf disc with selection

based on either nptII, hptII or bar gene. Approximately 21.8% for cotyledonary leaf

and 35.70% leaf disc explant transformation frequency was achieved. Optimal

hormone additive were 2.5 mg/l BAP + 0.5 mg/l IAA in SIM, and 1.0 mg/l GA3 in

SEM for leaf disc; versus 1.0 mg/l Zeatin and 0.2 mg/l IAA for cotyledonary leaf

39
explants. MS medium with 0.5 mg/l IBA in RIM was optimum or rooting, from both

explants sources.

Janani C et al., (2013) reported among the different concentration of hormones

tested, the maximum percentage (90%) of multiple shoot in MS medium containing

BAP (2 mg/l). The callus induction (100%) was observed in 2, 4 D (2 mg/l)

combination with NAA (1 mg/l). Then the preliminary protocol for the efficient

transformation of cv. Shalimar was standardized, using the binary vector pBI121 into

the Agrobacterium strain LBA4404. And transient GUS expression was used as the

basis for identifying the most appropriate conditions for transformation. The

transformed leaf explants survived in selection medium (MS medium with 2 mg/l

zeatin) fortified with 50 mg/l of each Kanamycin and Cefotoxime. The transient GUS

gene was confirmed by which showed blue color formation through GUS assay.

Ruma Devi et al., (2012) a reproducible transformation system for the tomato

genotype IPA-3 using Agrobacterium tumefaciens and cotyledon explants were

developed. Disarmed Agrobacterium tumefaciens - GV 3101 carrying binary vector

p35S-2-SFR was used in the genetic transformation study. The binary vector

contained GUS under the control of cauliflower mosaic 35S (CaMV35S) promoter.

Factors governing the efficiency of Agrobacterium-mediated transformation include

age of explants; bacterial concentration, preculture period and co-cultivation time

were optimized. The frequency of GUS expression varied from 2.66 to 23.16 % under

different treatments. The Agrobacterium (OD 600=1) inoculum when diluted to 1:20

recorded maximum GUS expression on treatment duration of 30 minutes. The 10 day

old seedling explants i.e. cotyledons also proved superior to transformation over 7 and

12 day old. The effect of 1 day pre-culture was also significant over 2 and 3-day.

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 Guruprasad M et al., (2011) a procedure for in vitro regeneration was

developed by using cotyledon explants of commercially grown tomato cultivar. This

regeneration procedure was used to facilitate gene transfer through Agrobacterium

tumefactions in tomato Var.S-16 containing binary vector pCAMBRA 2301 harboring

Gus as reporter gene and neomycin phospho transferase II (npt II) as selectable

marker. The cotyledon explants were co-cultivated with Agrobacterium tumefactions

and the shoots were regenerated on MS-Medium containing 1mg/l BAP and 1mg/l

NAA and root regeneration was obtained when MS-medium supplemented with 1mg/l

IBA respectively and higher plantlets survival (86%) was obtained in coconut peat and

vermiculate mixture in the ratio 1:1 within 10-15days. Stable integration of the

transgene in the putative transfermate was confirmed by using PCR.

Paramesh et al., (2010) reported an efficient and reproducible transformation

protocol for tomato Var (L15) using Agrobacterium strain GV 2260 carrying

pCAMBIA -GUS and hpt gene. The use of pre-cultured

cotyledon, leaf and hypocotyl, bacterial density (OD600) and co-cultivation time of 48

hours and a cefotoxime concentration of 300 mg l-1 were found to be ideal to keep the

Agrobacterium under control during the transformation experiments. 2.83 % of

selection was observed and Gus assay of the explants were used to evaluate

transformation efficiency in early steps.

Abu-El-Heba et al., (2008) reported Agrobacterium mediated transformation

(binary vector pISV2678 with GUS-intron and bar genes) and biolistic gun

(pMONRTG harboring the GUS gene). Maximum and quickest regeneration were

obtained on MS medium supplemented with 1 mg/1 BAP, 1 mg/1 zeatin ripozide, and

5 mg/1 AgNO3 and Nitch & Nitch vitamin. Regeneration percentage of 92% was

41
obtained within 10-12 days using cotyledons and hypocotyls. Plants were analyzed

using GUS assay and the PCR.

Hasan et al., (2008) reported the genetic transformation of tomato with

Agrobacterium contains Arabidopsis early flowering gene AP1, infiltrated to ripened

fruits of tomato. Among kanamycin resistant plants 87.9% -94.9% showed stable GUS

expression, while 5.1% to 12.07% were escapes. The effect of incubation period was

highly significant, with 48 hours incubation period having maximum efficiency

(68%). Transformation was confirmed by analyzing the PCR amplified product of

AP1, GUS and NPT-II genes.

Qiu et al., (2007) reported improved protocol for Agrobacterium mediated

Transformation of tomato (Micro-Tom) for incorporation of the carotenoid

biosynthetic gene. Cotyledons used as explants source were cultured for 1 day on the

medium containing zeatin 2 mg/1 and IAA 0.1 mg/1, submerged in Agrobacterium

(OD600 = 0.2) for 20 min and co cultivated for 3 days on the same medium.

Cotyledons were shifted to pre-selection medium with 500 mg/1 cefotoxime for 3

days and shifted to selection medium with 100 mg/1 kanamycin and 500 mg/1

carabenicillin for 6-8 weeks. 20% transformation efficiency was observed.

Sun et al., (2006) transformed cotyledon explants of tomato, Micro Tom

cultivar with A. tumefaciens (Rhizobium radiobacter) C58C1RifR harboring the

binary vector pIG121Hm, resulted in generation of mass of chimeric non-transgenic

and transgenic adventitious buds. Repeated shoot elongation from the mass of

adventitious buds on selection media resulted in the production of multiple transgenic

plants with transformation efficiency of 40%.

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 Roy et al., (2006) reported Agrobacterium mediated transformation utilizing

with hygromycin as selection marker. Stable integration of the T-DNA in to nuclear

genome was confirmed by PCR and southern hybridization. Northern blot analysis

detected the different levels of transcripts of bspA gene in individual transgenic lines.

43