The world having a turn down in food production per capita and the growing global demand for food make it essential to produce solution for maximum consumption of available resources and improve crops to triumph over this problem. In many arid and semi arid regions, good soils are scarce with their overall productivity declining because of soil degradation and lack of proper soil and water management practices. Salt-affected soils, which are widespread in arid, semi-arid and coastal regions of sub-humid areas, have low productivity. There are 380 million hectares of saline soils on earth's land surface, and of these 140 million hectares are highly saline and have higher electrical conductivity (EC). Food production in many parts of the world is severely affected by high salt contents in soils. In southern Asia and the Near East, for example, several million hectares of agricultural area are affected by salinity (e.g. 6.3 million ha in Pakistan, 2.5 million ha in India) causing losses in food production, excessive runoff due to compaction of saline soils and progressive desertification. It is estimated that nearly 10 % of the total land of the world used for crop production is adversely affected by soil salinity.
The major solutes comprising the dissolved mineral salts that affect soil fertility are the cations Na+, K+, Ca++, Mg++ and anions Cl-, SO4--, HCO33-and SIO32-. Normally, salt-affected soils often occur under natural conditions. Salinity problems of greatest importance in agricultural areas arise when previously fertile, productive soils become salinized as a result of irrigation.
Salinity and crop production
Salinity is increasingly important constraint to crop production worldwide (Ghassemi et al., 1995) regardless of the cause (ion toxicity, water deficit and nutritional imbalance) high salinity in the root zone severely impeded normal plant growth and development, resulting in reduced crop productivity or crop failure. The main effect of salinity on plant growth and crop production are:
- Slow and insufficient germination of seeds.
- Physiological drought, wilting, desiccation of plant.
- Stunted growth, reduce branching.
- Retarded flowering, fewer flowers, sterility and small seed.
- Low yield of seed and other plant parts.
Prevention and reclamation of soil salinity
Different measures are taken to reclaim the saline land which includes physical, chemical and biological.
Physical methods of land reclamation:
The proper solution of salinity and water-logging is through engineering technology i.e. proper drainage system of all agricultural land. This technology has been used in Pakistan at national level to control salinity by draining the soil salt through a network of surface and subsurface drain and tube wells.
Chemical Method of Land reclamation:
Although reclamation of salt-affected soils by chemical means is an established technology, traditional reclamation methods have been proved to be difficult (Rafiq, 1990), inadequate (Qureshi, et al., 1992), expensive (Qureshi, 1993; Qureshi and Barrett-Lenard, 1998), and uneconomical (Rafiq, 1975) on highly impermeable dense saline-sodic soils in Pakistan. Further, under the existing circumstances not only the scope of this approach is limited, its sustainability is also questionable (Qureshi, 1993; Qureshi and Barrett-Lenard, 1998).
Biological method of salt land reclamation:
This approach is based on growing salt tolerant plant species and use of saline waters to utilize salt-affected soils has been explored to a lesser extent (Qureshi and Barrett-Lenard, 1998). However, an understanding of the plant responses to various stresses and the mechanisms that make some species/genotype more tolerant than other is essential.
Mechanism of salt tolerance in plants:
Plants survive under saline conditions by adapting some special physiological, biochemical and anatomical mechanisms, which enable them to grow under salt stress. In general, plants avoid toxic concentration of salts either by restricting ion uptake or by compromising with high salt concentration through osmotic adjustment. Some important mechanisms for salt tolerance are:
Histological changes under salt stress:
Plant transport salts to shoots (even in halophytes) the amount of salts in excess is required for turgor maintenance. Excretion of salts through special glands i.e. salt glands is one of the most important mechanisms for salt tolerance (Gorham, 1996).
Salt gland controls the salts content of leaves. The quantitative contribution of salt glands to regulation of salt concentration in leaves has been studied in relatively few species. However, a substantial portion of salt entering a leaf of Leptochloa fusca can be excreted through salt glands (Gorham, 1996). The structural details of various kinds of salt glands have also been reviewed by Thomson et. al. (1988) and Fahn (1988). They may be multicellular organs of highly specialized cells, for example Avicenna marina, or are simple type glands comprising of only two cells, e.g in Leptochloa fusca (Wieneke et.al. 1987).
Salinity causes several specific structural changes that disturb plant water balance (Robinson et. al. 1983). These structural change include fewer and smaller leaves, less number of stomata per unit leaf area, thickening of leaf cuticle and wax deposition of leaf surface, reduced differentiation and vascular tissues, increased development of tyloses, earlier lignification of roots, low chlorophyll content, higher elasticity of cell walls, fully developed water storing tissues and increased succulence (Yeo and Flowers 1984). These responses vary with plant species and the type of salinity (Aslam et. al. 1993).
Physiological mechanism under salinity
Plant has the ability to regulate the influx of salt, which determine salt tolerance. In the pathway from the rhizodermis (the point of initial entry of salts in to roots) to the xylem, the movement of ions could be controlled by exchange processes in the cortex (Staples and Toenniesson, 1984) enforced passage through membranes (and hence selectivity) at the endodermis and by selective xylem loading (Gorham, 1996). Some species recirculate sodium in the phloem, although this is mainly a feature of salt-sensitive species such as beans and lupines (Jeschke et. al. 1987). Young expanding leaves are supplied with a potassium-rich inorganic solute supply via phloem, while sodium accumulates in older leaves, often replacing potassium accumulated previously. The potassium in older leaves is thus available for recirculation via phloem to sink tissues (Gorham, 1996). All the plants are salt excluders with varying degrees of exclusion. Some important physiological mechanisms for salt tolerance are:
- Osmotic adjustment
- Exclusion/inclusion of ions
- Potassium-sodium selectivity and
Tomato (Lycopersicon esculentum L.) is one of the major vegetable crops of the world especially of the most of countries like America, Japan, Pakistan, India, Bangladesh, and China. Tomato was grown commercially in 161 countries during 2004 with a production of over 115 million metric tonnes. The leading countries in tomato production are the United States of America, Italy, Egypt, Mexico and Spain. The United States of America accounts for about one fifth of the world's production (Moresi and Liverotti, 1982).
It is a member of nightshade family (Solanaceae) along with pepper, eggplant and potato. Botanically it is classified as fruit, since it is developed from ovary, although it is commercially recognized and treated as vegetable. It include the genus with several Known wild forms of tomato, i.e., Lycopersiocon pimpinellifolium, L. hirsutum, L. peruvianum, that have been useful in breeding programs for biotic and abiotic stresses.
Tomato bloom at different latitude under a wide range of soil types, temperature and it can be cultivated any where by providing it adequate nutrients. In cool seasons the production of tomato in the tropics tend to be more successful in mountain region or in low land (Ruben, 1980).
Production of Tomato
Tomato (Lycopersicon esculentum) is the second most important vegetable crop next to potato. Present world production is about 100 million tons fresh fruit produced on 3.7 million hectares.
In Pakistan tomato production has been increased since 90s.During 2001-2002 tomato crops was grown on average area of 29-30 thousand hectares with annual production of 294 thousand tons and the average yield is 13.8 tons per hectare (Anonymous, 2001).
Tomato can be consumed either cooked or raw. It is used in a variety of ways i.e. ketchup, beverages, salad, sauces and various other products. After processing oil can be extracted from the seed and the residual seed cake used for animal feed. Tomatoes have a very high nutritive value. It contains protein, thiamine, riboflavin, vitamin C, b-carotene Ca, Fe and carbohydrates. It is the cheapest and richest source of vitamin C and A (Kanahama, 1980). The acids present in it are citric acid, malic acid, aminobutyric acid, cis-aconitic acid and formic acid along with fair amount of histidine, lysine and certain minerals (Loh and Woodroof, 1975).
Factors affecting tomato production
Biotic factorsDiseases and pathogens are important among biotic stresses. Some diseases reach epidemic proportion and causes serious crop losses which others causes only negligible crop losses. Numerous disease of tomato, caused by fungi, bacteria, viruses and nematode.
Tomato is subjected to various abiotic stresses, which are unfavorable soil, temperature and water conditions, which cause very extensive losses to the yield of tomato. Similarly salinity, drought, cold, acidity, iron toxicity and submergence under water adversely affect tomato production.
Objectives / Aims of thesis
In Pakistan lots of work had been done on trees and cereal crops with regards to salt tolerance but very little work had been reported on vegetables. Present investigation was aimed to study the effect of salt on the physiology of tomato genotypes and to transfer the salt tolerant gene in the selected tomato genotype. To achieve this goal twelve tomato genotypes were selected for screening ( ) the mechanism of salt tolerance were studied in these genotypes with respect to plant growth ionic content, water content of plant and the production of ABA- the stress hormones. The genetic diversity among these genotypes was studied by Randomly Amplified Polymorphic DNA (RAPD). The tolerant gene was transferred to selected tomat genotype through Agrobacterium mediated gene transformation.
Review of literature
Response of Plants to Salinity Stress
Salinity affects plant growth and development because of low uptake and accumulation of essential nutrients and high accumulation of toxic ions such as Na+ and Cl- (Sabir and Ashraf, 2008). It reduces the plant's ability to take up water which slows down the growth. This is the water-deficit effect of salinity on plants. Second, the salt enters into the transpiration stream and injures the transpiring leaves cells and then further inhibits the growth. This is due to ion-excess toxic effect of salinity (Munns, 1993). Cavalcanti et al., (2007) found that the salt treatment (200 mM NaCl) inhibited the relative growth rate of both leaves and roots. Fresh and dry weights of plants increased with an increase in salinity in Salicornia rubra while the optimal growth occurred at 200 mM NaCl and the growth was inhibited with a further increase in salinity (Khan, 2001). Salt stress decreased the root and shoot growth in rice and sunflower (Rodriguez et al., 2006, Noureen and Ashraf, 2008). Salinity inhibit metabolic and enzymatic activities which adversely effect growth, flowering and yield of plants (Ramoliya and Panday, 2003). Rabie, (2005) found that salinity inhibited the growth of mungbean. Ghoulam et al, (2002) found that salt treatment resulted in reduction of growth parameters such as fresh and dry weight and a decrease in the K+ concentrations but increased the proline content.
Role of proline in salt tolerance
The amino acid proline is recognized to occur extensively in higher plants and normally accumulates in large quantities in response to environmental stress (Kavi Kishore et al., 2005). Salt stress is reported to accumulate the compatible solutes such as proline. A great variation exists for solute accumulation and osmoregulation among the genotypes. Osmoregulation prevents folded protein structures against denaturation, stabilizes cell membranes by increasing phospholipids, or serves as an energy and nitrogen source (Nayyar and Walia, 2004., Claussen, 2005). Proline is a reliable indicator for the evaluation of tolerance or sensitivity of plants to stress (Patel and Vora, 1984). The accumulation of free proline under stress conditions which primarily is due to the stimulation of proline biosynthesis (Rhodes et al., 1986). These solutes being hydrophilic could compete for water at the surface of proteins, protein complexes, or membranes. Under stress conditions they act as stabilizers of proteins, protein complexes or membranes (Ramanjulu and Bartels, 2002. Proline is known to induce to expression of salt stress responsive genes, which posseses proline responsive elements (eg PRE, ACTCAT) in their promoters (Chinuusamy et al., 2005).
The callus cultures of rice, modified to grow under increasing levels of NaCl, accumulated considerable amounts of free proline than unadapted cells (Kishor, 1988). Chandler and Thorpe (1987) reported that in Brassica napus, both unselected and tolerant callus better responded to water stress by osmotic adjustment and accumulation of praline. In same way, callus culture of Medicago sativa accumulated proline in response to NaCl stress (Shah et al., 1990). The salt tolerant sugar beet callus was also associated with a significant accumulation of proline under higher levels of salinity (Le Dily et al., 1991). Moreover the proline content in a callus culture of pearl millet grown in 1% NaCl increased more than 20-fold compared with non-salinized controls (Das et al., 1990).
Aziz et al. (1999) investigated the alterations induced in the levels of proline in response to salt (NaCl) stress using tomato (Lycopersicon esculentum) grown under saline conditions with varying levels of CaCl2 and KCl. There was found marked increase in amounts of proline accumulated in the leaf subjected to higher salinity (100 - 300 mM NaCl). They also reported that the internal Na+ and Ca2+ levels exerted a positive effect on proline. The accumulation of prolin in plants is mediated by both ABA-dependent and ABA independent signaling pathways (Zhu, 2001 and Zhu, 2002).
Role of Abscisic acid in salt tolerance
Abscisic acid is a natural growth inhibitor of a 15 carbon sesquiterpenoid and generally known as stress hormone. Its biosynthesis mechanism operates in chloroplast and other plastid by mevalonic acid pathway. ABA promotes adaptation to environmental stress and improve stress tolerance (Hasson and Polijakoff, 1981). ABA accumulate as a result of salinity and water stress and mediates osmotic adaptation of plants (Shinozaki et al., 1997., Stewart and Voetberg, 1995). Abscisic acid plays an important role in plant responses to salt stress (Zhang et al., 2006), it decreases the accumulation of toxic chloride ions (GomezCadenas et al., 1998, 2002) and other adverse effects of NaCl (Popova et al., 1995). The secondary messengers like Ca+2 (Xiong et al., 2002) and reactive oxygen species (Zhao et al., 2001) are involved in stress induced ABA accumulation.
ABA is known to intervene signals in plant cells subjected to environmental stresses like salinity and the resulting signals bring about expression of certain stress-related genes followed by the synthesis of compatible solutes such as proline (Kavi Kishore, 2005., de Bruxelles et al., 1996).
ABA has been investigated to alleviate the adverse effect of NaCl on photosynthesis, growth and translocation of assimilates (Popova et al., 1995). It has been reported that ABA reduces the release of ethylene and leaf abscission under salt stress in citrus probably by decreasing the accumulation of toxic Cl− ions in leaves (GomezCadenas et al., 2002). ABA-inducible genes are predicted to play an important role in the mechanism of salt tolerance in rice (Gupta et al., 1998). Salt stress increased the levels of ABA in Citrus sinensis (Gomez et al., 1998). The increase of Ca2+ uptake is associated with the rise of ABA under salt stress and thus contributes to membrane integrity maintenance (Chen et al., 2001). The osmotic potential in leaves of plants growing in natural salt stress appear to be strongly correlated with ABA content which might contribute in maintaining osmotic potential of leaves under stress. Water deficit conditions induce endogenous ABA that is correlated with leaf water potential (Zhang et al., 2006).
Salt stress causes disturbance of ionic equilibrium like influx of Na+, dissipates the membrane potential and facilitates the uptake of Cl- and down regulate the chemical gradient. The Na+ is toxic to normal cell metabolism and has lethal effect on the activity of some enzymes (Tawfik, 2008).
The ions involved in salt stress signaling include Na+, K+, H+ and Ca+2. High concentration of ions such as Na+, Cl-, Mg2+ and SO42- in saline soils inhibit the growth and development of many plants (Lambers, 2003). High salt uptake competes with the uptake of other nutrient ions, especially K+, leading to K+ deficiency. Increased treatment of salt induces increase in Na+ and Cl- and decrease in Ca2+, K+ and Mg2+ levels in a number of plants (Khan et al., 2000; Haleem et al., 2007). Under saline conditions, due to excessive amounts of exchangeable Na+, high Na/Ca2+ ratio occur in soil. Plants subjected to such environments, take up high amounts of Na+, whereas the uptake of K+ and Ca2+ is reduced. Reasonable amounts of both K+ and Ca2+ are required to maintain the integrity and functioning of cell membrane (Wenxue et al., 2003). Decrease of Ca2+ and Mg2+ contents of leaf have been reported upon salt accumulation in Brassica Parviflora suggesting increasing membrane stability and decrease chlorophyll contents respectively (Parida et al., 2004). The underlying mechanism for maintenance of adequate K+ in plant tissue under salt stress seems to be dependent upon selective cellular K+ and Na+ compartmentation and distribution in the shoots (Carden et al., 2003). Plants use low and high affinity transporters for uptake of K+ from the growth medium (Blumwald, 2000). High K+/Na+ selectivity in plants under saline conditions has been suggested as an important selection criteria for salt tolerance (Ashraf, 2002; Wenxue et al., 2003). The concentration of Na+ and Cl- in leaf was increased when treated with NaCl stress (Gurmani et al., 2007).
Conventional plant breeding is commonly utilized in improvement of crop plants and vegetables for better yields and usually it involves the production of variability by making sexual crosses between selected genotypes with characters to be combined, to produce a population of plants that include better genotypes. This is followed by widespread selection from the offspring to identify these genotypes which will ultimately direct to the development of new varieties. This input of new characters or generation of new genetic recombination is partial to the early stages and most of the attempt is taken up by field selection over many years. Over past forty years or so significant yield increase have been achieved in developed countries, about half have contributed by genetic improvement and other half by improved agronomic practices.
The main objective of plant breeding is to obtain increase yield, improved quality, disease and pests resistance, stress tolerance and herbicide resistance. A problem of conventional breeding is that agronomically desirable characters are frequently genetically ill defined and many are polygenic. The best current varieties usually have specific defects that breeders wish to improve. Thus there may be sacrifice of quality to obtain a high yielding variety, or the variety may be susceptible to particular pathogen that limits its range of use.1.12.2 Problems of conventional breeding
The problem of conventional breeding is that the range of genes that is accessible is limited to the related species that can be crossed sexually. Variation is important for any breeding programme, but variation itself is exhausted by conventional breeding especially by extensive breeding programme in the last few decades and the gene pool of important crops is highly reduced. The main objective of conventional plant breeding is to obtain increased yield, improved quality (both nutritional and technical), disease and pest resistance, stress tolerance (e.g. drought, cold, heat) and herbicide resistance. Conventional plant breeding, however, has its own limitations. It depends on sexual compatibility and often takes 10-15 years to release a new variety due to extensive backcrossing (Pauls 1995).
Environmental stresses are usually much more important factors limiting the food production in the tropics and subtropics than in temperate zones. Salinity is serious threat to tomato production, which hinders the plant development. Resistance to salinity is low, so there is a need to improve the tomato through in vitro approaches.
In vitro approaches
Tissue culture is a technique used to produce whole plant from an explant. Plant tissue culture is used as a gross term for protoplast, cell, tissue and organ cultures grown under aseptic conditions.
Whatever transformation system would be employed, efficient systems for embroygenic callus induction and shoot regeneration have been considered the basic matter in obtaining fertile transgenic rice. The application of tissue culture has made rapid progress in dicotyledons (Yamada, 1977).
Le at al. (1991) showed that hypocotyl segments gave better results for callus induction on media containing 2 or 5 mM 2, 4-D and shoots were derived directly from hypocotyl explants cultured on MS medium with 44.4 mM IBA and 5 mM IAA.
Duzvaman et al. (1994) reported more shoot regeneration rate on MS medium supplemented with 0.20 mg/l IAA and 3.0 mg/l IAA and 1-2 mg/l kinetin. Growth of hypocotyl, cotyledons and leaf discs of two tomato cultivars were also compared. The degree of shoot regeneration was in the order of leaves, hypocotyl and cotyledons.
Newman et al. (1996) achieved invitro regeneration from 4 tomato cultivars. They used hypocotyl segment as an explant source. They obtained regeneration on basal medium without any hormone as well as on MS medium containing BAP in different concentrations. The best medium for obtaining more shoots was MS medium containing 1.0 mg/l IAA and 2.0 mg/l kinetin. BA was also used and had an inhibitory effect on explants obtained from buds.
Jatoi et al. (1995) reported the callogenesis and regenerative response of leaf explant of F1 tomato hybrids, Bornia and Royesta, were studied at different PGR levels. MS medium was supplemented with either IAA (0, 0.7, 1.5 or 2 mg/l), kinetin (0, 3, 4, 5 or 7.5 mg/l) and 2ip (0, 2, 3 and 4 mg/l). Bornia showed a higher frequency of callogenesis than those of Royesta when cultured on MS containing Kin and IAA. Leaf explant of Royesta, incontrast, cultured on MS medium containing BAP and IAA exhibited a higher callogenesis frequency. No regeneration was obtained in any hybrid on MS containing 2ip and NAA.
Zagorska et al. (1997) tested the androgenetic ability of 85 tomato genotypes. Callus was induced from anthers of 53 lines and hybrids. Regeneration f plant was obtained only from calli of 15 genotype. The invitro response of anthers from the cultivars Roma, Pearson, San Marzano, Par, Sar, Viga Pol, Day, David and Start containing the ms 1035 gene, which is responsible for the male sterility in tomato, confirm the strongly expressed dependence of both callus induction and organogenetic potential on the homozygous and heterozygous state of that gene. More than 600 regenerants have been obtained.
Geetha et al. (1998) obtained white calli from leaf explants on MS medium containing 2, 4-D while regeneration was obtained by sub culturing the calli on MS medium containing. NAA (0.20-1 mg/l) and BAP (2.5-7 mg/l). Shoot regeneration was obtained on all media containing NAA and BAP.
Shtereva (1998) observed the factors affecting the induced androgenesis in tomato cv. Roma. Anthers isolated from plants, grown in green house during winter at high humidity and in short days, possessed high androgenetic ability. MS medium were used for callogenesis, organogenesis and regeneration. The combination of 2ip and IAA showed greater % age of callus formation than Zeatin and IAA. Zeatin promote the entire plant regeneration. Treatments at 4oC (48h) and 1DoC (9 days) stimulate these processes. Combined treatment of anthers with 4 Gy and 1DoC for 9 days was most efficient.
Takashina et al. (1998) reported that the explant's nature effect callus induction and regeneration. Hypocotyl segments showed healthy calli and more regeneration rate on MS medium containing Zeatin but no regeneration from root explants on the same medium.
Costa et al. (1999) reported that cotyledon explants of tomato cv. Somta Clara, Firme, mutant 1PA-5 and 1PA-6 were excised from 8-10 days old in vitro grown seedlings. Four different shoot induction media supplemented with timentin (300 mg/l) were screened. Higher regeneration frequency and maximum number elongated shoots were obtained when MS medium supplemented with Zeatin (1 mg/l) and IAA (0.1 mg/l) and also supplemented with timentin. In two of three cultivars tested, rooting of shoots was positively influenced, both in the presence and absence of timentin in the rooting medium, among shoots regenerated from explants derived from timentin-supplemented medium.
Oktem et al. (1999) studied the regeneration conditions for leaf pieces of two tomato cultivars (ES58 and WC156). MS medium with Gamborg's vitamins, BAP (2.5 mg/l), IAA (0.2 mg/l) were used for callogenesis and regeneration. Callus formation was observed in more than 90% of the cultured leaf explants in 15-25 days. After 8th week of cultured period, 70% of the leaf pieces in c.v. ES58 developed shoots, whereas 50% from WC156. In cv. ES58, IAA seemed to be important in rooting medium whereas WC156 showed rooting in rooting medium without IAA.
Hu, W. and Phillips, G.C. (2001) used different media with different growth regulators in shoot formation and elongation of tomato c.v. UC 82. Cotyledons were used as an explant source from 8-days old invitro plants, the frequency of explants showing shoot bud induction on AB-AZ medium (64.2%) was significantly higher than that on MS101 medium (48.4%). The number of shoot buds per explant obtained using AZ medium (9.4) was significantly higher than those on MS101 (3.8) and MS85 medium (2.5). the highest frequency of shoot elongation was obtained using MS119 medium after transfer from AZ medium.
Arillaga et al. (2001) used two different accessions (LA1401 and LA530) of Lycopersicon cheesmanii (Riley) for invitro plant regeneration. Cotyledons and leafdiscs from 20 days old invitro plants were used as an explant source LA1401 showed maximum % age of callogenetic response on full strength MS major salts containing 4.4mM BA and 1.1mM IAA from leaf explants (65%). However the other hormonal combinations i.e. BA/IAA (4.4/1.1 mM) with Zeatin (9.1 mM) showed best results than any other combination. The accession LA530 showed higher organogenetic potential than LA1401 (97% vs 80%) of organogenetic explants.
Sonia et al. (2001) studied the genetic stability in tissue cultured tomato plants was examined by randomly amplified polymorphic DNA (RAPD) analysis. Picloram was used along with benzyladenine (BA) for callus induction in tomato. Calli were induced from leaf explants on MS medium supplemented with 8.8 mmM BA and 4.13 mmM picloram. Regeneration was obtained after culturing freshly induced calli on MS medium containing 17.7 mmM BA alone. Microshoots were rooted in the presence of 10 mmM IBA on MS medium.
Chaudhry et al. (2001) used two different cultivars of tomato Nagina and Feston for callogenesis and Regeneration. Hypocotyl and leaf discs from 17-18 days old invitro plants were used. More callus response and more fresh weight was obtained on MS medium with 0.5 mg/l NAA and 2 mg/l BAP. Whereas maximum shoot formation from calli was obtained on MS medium with 0.5 mg/l IAA and 4 mg/l BAP. Feston showed maximum % age of shoot formation in hypocotyl (56%) than leaf disc (42% - 30%). So hypocotyl for Feston and leaf explant from Nagina showed better results
Genetic transformation studies
Genetic manipulation of plants has been an on going science since prehistoric times, when early farmers along the Euphrates began carefully selecting and maintaining seed from their best crops to plant for the next season. Early people also bred plants, and modern crops are a result of thousands of years of genetic manipulation (O'Neal 2001). Due to unsuccessful crosses and narrow gene pool available within a species, genetic engineering is now a day, used as an additional tool to crop improvement programs being studied for increasing the qualitative and quantitative food production.
Genetic transformation is a process through which genetic materials isolated from one organism can be introduced into and expressed in another organism with different genetic background. This process involves several distinct stages, namely insertion, integration, expression and inheritance of the newly introduced gene in the host genome. In plant, this technology not only has potential to achieve crop improvement with a more rapid and precise manner than the conventional breeding programs, but also has becamean indispensable enabling tool for further dissection and understanding of the plant species.
Genetic engineering has allowed explosive expansion of our understanding in the field of plant biology and provides us with the technology to modify and improve crop plants. A remarkable progress has been made in the development of gene transfer technologies (Gasser and Fraley 1989), which ultimately have resulted in production of a large number of transgenic plants both in dicots and monocots. Potential benefits from these transgenic plants include higher yield, enhanced nutritional values, reduction in pesticides and fertilizer use and improved control of soil and water pollutants. Some of the important characters like resistance to herbicide (Smith 1994), disease (Smith 1994), insect (Perlak et al. 1990), high protein content (Habben and Larkins 1995), cold tolerance (Georges et al. 1990), fruit quality (Fray and Grierson 1993), bio degradable plastics (Poirier et al. 1995), antibodies and vaccines (Mason et al. 1992) etc. have been incorporated in the genetically engineered plants.
Methods of gene delivery
PEG-mediated and Electroporation-mediated tomato transformation
Two general methods have been used to introduce DNA into protoplast for transient assays: treatment of protoplast with polyvalent cations or electroporation.
Polyvalent cations such s polyethylene glycol (PEG) or poly -L -Ornithine (PLO) have been used extensively to induce protoplast fusion and they are thought to act to promote DNA up take precipitating the DNA ,minimizing charge repulsion between the protoplasts and stimulating DNA up take of DNA endocytosis.
With electroporation an electric pulse is used to reversibly permeabilize the cell membrane, allowing the up take of DNA.
In many dicots, plants can be regenerated from mesophyll protoplast, but in most crops like cereals there's only scant evidence that protoplast isolated from leaves are capable of sustained divisions. It is difficult to initiate and maintained cell suspension and regeneration capacity has been observed to decline during long term cultivating. So plant regeneration from most protoplast is difficult (Ayres and park, 1994). In addition low efficiency of transformation (Toriyama et al., 1988) and plants regenerated from protoplast are sterile and phenotypically abnormal (Datta et nal., 1992). Other problem include the integration of multiple copies of genes into genomes (TADA at al., 1990), the fragmentation and rearrangement (Wo at al., 1995), and occasional non-mendelian inheritance of transgenes (Peng et al., 1995)
Microprojectile bombardment -mediated gene transfer
Microprojectile (other wise known as Particle Bombardment) involves directly shooting a piece of DNA into the recipient plant tissue. This is carried out using a gene gun. Tungsten or gold beads (which are smaller than the plant cells themselves) are coated in the gene of interest and fired through a stopping screen, accelerated by Helium, into the plant tissue. The particles pass through the plant cells, leaving the DNA inside.
This method can be used on both monocotyledonous and dicotyledonous species successfully. It is again a relatively simple laboratory procedure. The transformed tissue is selected using marker genes such as those that code for antibiotic resistance. Whole plants are then regenerated from the totipotent transformed cells in culture, containing a copy of the transgene in every single cell (Nottingham, 1998)
Agrobacterium mediated gene transfer technique:
The genetic transformation of plant cells mediated by Agrobacterium tumefaciens is a well established system for gene delivery to many dicotyledonous (Binns and Howitz, 1994) and more recently some monocotyledonous species (Smith and Hood, 1995). The possibility to widen the application of Agrobacterium mediated transformation to important crops, such as cereals, represent a real advance in a plant biotechnology (Ishida et al., 1996).
Stable plant transformation is commonly achieved by Agrobacterium tumefaciens mediated procedures (Ellis, 1993). Agrobacterium is a plant pathogen which causes the formation of crown-galls or tumors in tissues infected by the bacterium (Sheng and Citovsky, 1996 and Gheysen et al., 1998).
Agrobacterium tumefaciens is a gram negative soil bacterium that causes a neoplastic plant disease by the transfer and integration into the host genome of a set of plant-expressible-genes (Zupan and Zambryski, 1995). A subset of these genes, termed oncogenes, is involved in the synthesis of plant growth regulators and caused a tumor proliferation of the affected cells (Hooykaas et al., 1988). Briefly the determinants for establishing and sustaining tumors are located mostly on large (> 200 kb) Ti (Tumor-inducing) plasmids. The T-DNA and the virulence (Vir) regions are two distinct regions of all Ti plasmids which are essential for Agrobacterium-mediated plant transformation. The T-DNA is a discrete section of the Ti plasmid bounded by 25 bp imperfect repeats termed as right (RB) and left borders (LB). The T-DNA is transferred to and integrated in the host cell nuclear genome at the onset of infection. The processing of T-DNA and its transfer to the host plant cell nucleus is achieved primarily by the concerted action of about 20 vir gene products. All the plasmid encoded vir genes reside in a region of the Ti plasmid. Ti plasmid-encoded vir genes can function in trans to promote the transfer of T-DNAs from co-resident plasmids to recipient plant cells (Hoekema et al., 1983). Such T-DNA containing plasmids are termed as Ti vectors (Guerineau and Mullineaux, 1993). Genes and sequences to be transformed into plants are inserted between the LB and RB of the Ti vector of T-DNA.
Binary Ti vectors are able to replicate in Escherischia coli and Agrobacterium species. Wild type A. tumefaciens strains are converted into transformation vectors by the deletion of either the complete set of oncogenes (Zambryski et al., 1983) or the tumorigenic (oncogene-containing) T-DNA. These processes are referred to as disarming. Ti plasmid gene that are mechanistically involved in T-DNA transfer, the so called virulence gene are not affected by the disarming process.
Gene transfer with disarmed A. tumefaciens strains has been used successfully for a large number of plant species.
- Agrobacterium mediated transformation in dicots.
- Agrobacterium mediated transformation in tomato.
- Induction of resistance by genetic manipulation.
- Salt tolerant gene.