In vitro selection and regeneration methods for wheat improvement
Manoj K. Yadav1, Manoj K. Tripathi2, Dinesh Yadav1, Sundeep Kumar3, Nagendra K. Singh4, Anil Kumar1 and Govind Krishna Garg1
1 Department of Molecular Biology & Genetic Engineering, G.B. Pant University of Agriculture & Technology, Pantnagar -263145 (Uttarakhand) INDIA
2 Deaprtment of Biotechnology, Institute of Science & Technology (ICFAI University), Dehradun (INDIA)
3 Department of Biotechnology, Sardar Vallabh Bhai Patel University of Agriculture & Technology, Meerut (UP) INDIA 250110
4 National Research Centre for Plant Biotechnology, Indian Agricultural Research Institute, New Delhi -100011
Correspondent author: Manoj K. Yadav
Department of Biotechnology, Sardar Vallabh Bhai Patel University of Agriculture & Technology, Meerut (UP) INDIA 250110
E-mail: mky711@rediffmail.com
Abstract
Wheat is an important staple food crop of world. Work on improvement of wheat is being done since long back using various methods and techniques. Emerging the field of biotechnology especially plant tissue culture has revolutionized the entire scenario of crop improvement including wheat. Regeneration of plants from callus and in vitro selection is the most important strategies that have been used to obtain efficient regenerated lines and subsequently genetic manipulation of wheat using in vitro techniques. Despite the significance of the problem for the successful implementation of in vitro techniques, the factors which control the establishment and regeneration of callus, the relative importance and the role of explants source, media, growth regulators, genotype, somatic embryogenesis, in vitro selection and in vitro induced variations are being discussed in the present paper.
Introduction
Wheat is the dominant grain of world commerce and is the staple food of millions of people globally. It is often used to produce a large variety of foods that include many kinds and types of breads, cakes, noodles, crackers, breakfast foods, biscuits, cookies, and confectionary items. Wheat, after rice, is the second most important crop in India and the World. It is a staple food for one-third of the global population. The present wheat is believed to have originated in Euphrates Valley as early as 10,000BC, making it one of the world’s oldest cereal crops. It is also believed that wheat developed from a type of wild grass native to the arid lands of Asia Minor. Initially, Schulz (1913) grouped all the species of wheat in three natural groups, einkorn, emmer and dinkel wheat. Further, Sakamura (1918) established the three natural groups of wheat form a polyploidy series with haploid chromosome number n = 7, 14 and 21, respectively. During their agricultural selection, modern wheat strains which are hexaploid, with six sets of chromosomes per cell, evolved from their wild diploid relative parallels, in terms of chromosomes. Einkorn wheat has 14 chromosomes, which are designated AA. This species is believed to have hybridized with a wild grass species to produce emmer wheat. Later it was observed that wheat has the AA chromosome sets and the BB sets of the wild grass. This tetraploid, AABB, now mated with yet another wild grass species, which had the chromosome complement designated DD, to yield AA BB DD, our modern hexaploid wheat, which has 42 chromosomes. An important application of polyploidy breeding has been the development of the wheat rye hybrid called Triticale. This resulted from an attempt to combine the high yield and high seed protein content of wheat (Triticum) with the adaptability to adverse environmental conditions (such as cold and drought) and high-lysine content of rye.
Wheat is fairly rich source of vitamins, niacin, thiamine, energy and mineral suitability of wheat for many food products depends on its unique protein. Elevated grain protein content (GPC) and its quality influences the bread making properties of wheat (Mesfin et al. 2000). Industrial uses of wheat include production of starch, gluten, distilled sprits and malts, etc. Wheat bran is rich in protein (14-18%) and vitamins. It is also used to feed livestock. Wheat straw is used as feed, animal bedding, compost and for making corrugated paper.
The wheat grain contains starch (60-70%), protein (10-17%), fiber (2-2.5%), fat (1.5-2.0%), sugar (2-3%) and mineral matter (1.5-2.0%). Worldwide, wheat is cultivated in an area of about 232 million hectares with an annual production of about 640 million tonnes. Globally, for the last five years, total wheat production is on the decline and during 2006-07 only 587 million tonnes of wheat was produced (Table 1).
Wheat improvement through classical breeding approaches in the 20th century
Wheat improvement work on systematic lines commenced only during the first decade of 20th Century. The period between “1904-1962” can be divided into three distinct phases and is well documented by Pal (1966). During first phase, beginning from 1904, the pioneering work was done by Albert Howard and Gabriella Howard who utilized a system of selection from single plants which enabled them to produce several wheat varieties like Pusa 4, Pusa 6 and Pusa 12. These varieties were popular not only in India but also in South Africa, Rhodesia and Hungary (Howard and Howard 1910). The semi-dwarf wheat varieties developed in Mexico by Norman Borlaug and his colleagues using the Norin 10 dwarfing genes, Rht-B1 and Rht-D1 attracted the attention of Indian wheat breeders.
The low input traditional agriculture, traditional tall wheat, inadequate irrigation facilities and the overall stagnate rural economy kept wheat production at a very low level. Suddenly, the wheat growing areas of India was bifurcated between India and Pakistan in 1947. Up to 1965-66, breeding efforts did not result in substantial yield improvement due to lack of suitable genotypes which can respond to fertilizer and irrigation. India became dependent on large-scale imports to feed its growing population, a situation which was described as “ship to mouth”. Then era of semi-dwarf wheat came, which became an instant success and contributed to the “wheat revolution”. The new genotypes triggered off complementary package of technologies, services and public policies which gave birth to what was termed as “green revolution” by Willium Gaud of U.S.A. in 1968. India never looked back from this stage. Nagarajan et al. (1998) observed that small gain has been obtained in wheat after every 5-10 years. At the time of release of Kalayan Sona, the yield level was about 4.2 t/ha. The next jump was made by the release of varieties like WL 711 and Arjun (HD 2009) in 1975, which resulted in a marginal yield gain of five percent.
Current scenario of wheat production
World wheat production in 2006-07 is 31 million tonnes lower than in the previous season. Harvesting of northern hemisphere wheat crops is complete, while in the southern hemisphere, harvesting is approaching completion. Many of the world’s key producing countries have lower production in 2006-07 than in 2005-06 (Table1). Combining 2006-07 production with beginning season stocks, global wheat supplies were 722 million tones, 34 million tonnes less than in 2005-06. The lower wheat supplies have been driving up world wheat prices (US hard red winter, fob Gulf ports), which have averaged above US$200 a tonne. The world wheat indicator price is forecast to increase by US$32 a tonne in 2006-07 to average US$208 a tonne.
The success story of wheat in India during the last 30-35 years is unparalleled in the history of any other country. The wheat production in India, which was just 6.4 million tones in 1950s, has progressively increased to 76.6 million tones in 2000. The average productivity has increased from 6.5 quintal per hectare to 27 quintal per hectare. The state average of Punjab and Haryana are close to 40 quintal per hectare. The more recent genotypes like PBW 299, UP 2338, UP 2425 and PBW 343 derived from further crosses the various genotypes have higher yield potential of up to 55-60 q/ha.
The post-green revolution breeding programme were mostly concentrated towards further improvement in yield, selection for adaptability to different agro climatic condition and improvement of disease resistance particularly for rusts. The strategy was to cross some of the Indian wheat varieties with the semi-dwarf wheat varieties from Mexico to combine the grain characters and Chapati making qualities of local wheat with the photo-insensitivity, disease resistance and input responsiveness to dwarf types.
Constraints in wheat productivity
Today the wheat production may look satisfactory but the fact remains that the sudden surge in population and the First World War saw India becoming a food grain deficit nation. In fact, today we suffer from overflow of go-down with stored grains. India needs a buffer stock of 14-16 million tones for food security, but today it has more than 50 million tones of grains in buffer stock. National food security has not ensured food and nutritional security for individuals. India has 25 percent of world population below poverty line who cannot get two square meals per day. The calorie intake per day is less than 1500. Main reasons for this dichotomy of famine within population are poor socio-economic condition clubbed with high cost of wheat production. If India has to avert mass scale hunger despite green revolution, it has to face challenge to ensure equitable distribution of food grains at an affordable price. The later can be achieved only by replacing varieties of green revolution that require costly input of energy, water, fertilizers and pesticides with varieties that were, besides being efficient in utilization of water and fertilizers, also resistant to biotic and abiotic stresses. To achieve this objective at accelerated pace; the conventional methods of breeding will need to be complemented with modern tools of biotechnology such as plant tissue culture and genetic engineering techniques.
Biotechnological approaches in wheat improvement
Earlier the production of wheat lines with improved quality characteristics relied on traditional plant breeding techniques which has a limitation with the complex transfer of multiple associated traits, relating to both agronomic and end-use quality attributes. Certain trait like the amino acid composition of storage proteins for nutritional quality improvement is difficult to alter through conventional breeding techniques or at least not without adverse effects on other quality traits. The advent of recombinant DNA technology led to the specific gene transfer methodology with a chance to alter a trait specifically while retaining the superior qualities of a known cultivar. The transgenic technology adopted for wheat improvement needs identification of desired gene conferring the said trait, optimal method of gene transfer, in vitro regeneration and selection.
For development of transgenic wheat, the regeneration capacity during in vitro culture has to be very efficient so that stable transgenic with useful gene(s) can be developed. Regeneration frequency of wheat during in vitro culture is a limiting factor for the creation of transgenic. Tissue culture methods using mature embryo culture followed by callusing/somatic embryogenesis and selection of callus for high regeneration capacity may speed up varietals development through rapid attainment of homozygosity. Because of the limitation of early generation testing for yield, wheat biotechnologists have been interested in ways that can cut down on the time taken to reach homozygosity so that emphasis can be placed on testing the homozygous lines rather than the efforts on producing them. Three approaches are (i) single seed descent (ii) selection of high regenerated calluses using mature/immature embryo culture and (iii) production of haploid plants either by wide crosses or anther culture, followed by chromosome doubling.
The applicability of biotechnology to crop improvement induces non-conventional plant breeding methodology. These new technologies will not replace the traditional breeding techniques but should mutually complement them by enhancing efficiency, trait transfer precision and recovery of useful, value added variation (Karp 1995). Useful variability can be generated without sexual recombination by in vitro selection/ variation (Larkin 1987).
In vitro regeneration and selection in wheat: present status
Regeneration of plants from callus is central to most of the strategies that have been proposed to obtain efficient regenerated lines and subsequently used for the development of transgenic wheat with desired trait. Genetic manipulations of wheat using in vitro techniques have been performed by several coworkers. The various factors influencing the in vitro regeneration via callus are discussed below.
Explant source
Explants sources vary in their ability to generate variation (Skirvin et al. 1994). Highly differentiated tissue (root, leaves and stems) produce more variation than explants with pre-existing meristems (axillary’s buds, shoot tips and leaf bases). Cultures give rise to normal or near-normal regenerated plants when no significant dedifferentiation (callus) step occurred (Peschke and Phillips 1992). Organogenesis can involve more than one cell. Stability differences in tissue culture originating from different explants sources often are caused by pre-existing variability in the explants source. More chlorophyll deficient variants are recovered from immature embryo cultures than from mature embryo cultures (Cai et al. 1990). The highest rate of callus induction (9.1%) and green plant production (0.8%) were obtained with wheat cultivar Apollo using anther culture (Konieczny et al. 2003).
Various explants sources, such as immature embryos, immature leaves, immature inflorescences, mature embryos, mesocotyls and apical meristems have been used for callus culture in wheat. Use of different explants sources in wheat have been given in Table 2.
Callus culture
The isolation and successful establishment of callus cultures depends on several factors like explants source and culture conditions. Callus, which shows stable characteristics under specific conditions after sub-culture through many successive passages, is a suitable material for cytodifferentiation. The advantage of using such callus is that it is composed of a fairly homogenous mass of cells and can be proliferated in large amounts under known culture conditions.
Effect of media composition and growth regulators on wheat callus
Callus is initiated in vitro on cut or exposed cell surfaces in contact with a growth medium. Callus proliferation is a wound response. Excision of the explants stimulates the wound response in vivo (induction of stress-induced enzymes and other chemical by-products, and activation of transposable elements) which can be enhanced by growth regulators (McClintock 1984). Most plant growth regulators and specifically 2, 4-dichlorophenoxy-acetic acid (2, 4-D) and 6-benzylaminopurine have been implicated in tissue culture-induced variability (Evans 1988; Shoemaker et al. 1991). Racz et al. (1993) studied the effect of sucrose, 2, 4-D and different vitamins on callus induction in winter wheat mature embryo explants. Solid MS medium was optimum for mature embryo culture (Leon et al. 1995; Dzhas and Kalashnikova 1998; Varshney et al. 1999; Sayar et al. 1999; Mendoza and Kaeppler 2002; El-Bahr et al. 2000; Delporte et al. 2001) of wheat supplemented with different combinations of plant growth regulators. Immature embryo culture supplement with 2, 4-D (Subhadra et al. 1995; Taghvaii et al. 1998; Rao and Chawla 1998; Kothari et al. 1998; El-Sherbeny et al. 2001; Shan et al. 2000) gave good callus growth. The effect of B5 (Rao and Chawla 1998), LS and potato medium (P1, P2 and P4) (Chaghamirza and Arzani 1999) were tested for callus induction. Immature embryos were cultured in liquid MS medium supplemented with 2 mg l-1 2, 4-D to obtain variation (Ahmed and Sagi 1993).
Immature embryos of 1.0-1.5 mm (14 d after anthesis were cultured on MS medium supplemented with 1.5 or 2.0 mg l -1 2, 4-D and found that 90-100% of these embryos formed callus (Arun et al. 1994). The callus induction frequency ranged from 51 to 81% and compact, nodular and morphogenic callus frequency ranged from 35 to 50%. The medium x cultivar x 2, 4-D level interaction were significant (Rao and Chawla 1998). Addition of 2, 4-D at 3 mg l -1 in combination with zeatin at 0.5 mg l -1 was beneficial for the formation of morphogenic callus (Dzhas and Kalashnikova 1988).
Ozgen et al. (1998) compared the responses of mature and immature embryo cultures. Immature embryos were aseptically dissected from seeds and placed with the scutellum upward on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) medium and 2 mg l -1 2, 4-D. Mature embryos were placed furrow downwards in dishes containing 8 mg l -1 2, 4-D for callus induction. The immature embryos formed the highest amount of callus on MS media supplemented with 2, 4-D and Kinetin while mature embryos formed the highest amount of callus with 2,4 D. Anthers of a wheat F1 hybrid (Alondrax sumai 3) on W14 media with different concentrations of salicylic acid (SA) and epi-brassinolide (epi-BR) were cultures and observed the significant differentiation and increased rates of callus development (Yang et al. 1999). Cai et al. (1999) obtained calluses of wheat from seminal roots grown in media containing 2-6 mg l -1 2, 4-D. Anthers from a doubled haploid line of spring wheat cv. Pavon 76, plated in liquid P-4 medium supplemented with 2.0 or 4.0 mg l -1 2, 4-D developed good calluses (Zheng and Konzak 1999). Immature embryos of two wheat genotypes were used to establish callus cultures (Jimenez and Bangerth 2001) for the study of endogenous effects of growth regulators (Marcinska et al. 2001). Calluses were induced on MS medium with 1-2 mg l -1 , 2, 4-D and 1.5 mg l -1 BA using hypocotyls segment (Jin et al. 2002).
The primary event causing tissue culture induced variability may be cell cycle disturbance caused by exogenous hormone (Peschke and Phillips 1992) or nucleotide pool imbalances (Jacky et al. 1983). Sister chromatid exchange frequency can increase with a low concentration of 2, 4-D (Dolezel et al. 1987).
Somatic embryogenesis in wheat
Somatic embryogenesis is the process of a single cell or a group of cells initiating the developmental pathway that leads to reproducible regeneration of non-zygotic embryos capable of germinating to form complete plants. According to Sharp et al. (1982), somatic embryogenesis is initiated either by pre-embryogenesis determined cells (PEDCs) or by induced embryogenic determined cells (IEDCs). The embryogenesis process can be divided into two main and fundamental phases: a first ‘morphogenetic’ stage where the embryo proper is formed and organs and tissues of the future plant are specified and a second ‘metabolic’ stage characterized by the storage of reserves (carbohydrates, proteins, lipids) in preparation for germination. Excellent reviews are available which focused on zygotic and somatic embryogenesis and comparisons between them (Goldberg et al. 1989; Wilde et al. 1995; Laux and Jurgens 1997; Dodeman et al. 1997; Rojas-Herrera et al. 2002). Exposure of the suspension cultures to high concentrations of 2, 4-D produces clusters of small isodiametric cells, known as pro-embryogenic mass (PEMs) (Halperin 1966). The subsequent elimination of auxins from the culture medium allows the expression of embryogenesis. One of the basic components of a medium influencing somatic embryogenesis of cereals is the type of auxin (Przetakiewicz et al. 2003). It is now accepted that, in the continuous presence of auxins, proembryogenic masses synthesize the gene products required to complete the stage of embryogenesis as well as the mRNA that can inhibit the somatic program. The depletion of auxins will result in the inactivation of a certain number of genes, permitting the embryogentic program to proceed; the transition to further developmental stages may require new gene products (Zimmerman 1993). It has been observed that the exposure of cell cultures to high concentrations of auxins can provoke hypermethylation of DNA (Lo -Schiavo et al. 1989) which, in turn, may cause transcriptional inactivation of methylated genes (Carman 1990). In the recent past, considerable success was achieved in obtaining reproducible regeneration of plants from embryogenic cultures of all major cereals.
Arun et al. (1994) reported that about 53-91% of the calluses produced somatic embryos and the frequency of embryogenesis depends mainly on the cultivar. Subhadra et al. (1995) obtained SEs from cell suspension culture of wheat. Marciniak et al. (1997) showed the effect of media and genotypes by anther culture on embryoid induction frequency (from 2.4 to 22.5 per 100 anthers). Barro et al. (1999) developed media (supplemented with picloram) for somatic embryogenesis from immature inflorescences and immature scutellum of elite cultivars of wheat and barley. Kunz et al. (2000) standardized an efficient protocol for the production of embryos in isolated wheat microspore culture. The scutellum cultures gave higher frequency of embryogenesis as compared to inflorescence culture (He and Lazzeri 2001). The effect of ovary-conditioned medium on microspore embryogenesis in wheat was well documented by Zheng et al. (2002). Ahmed et al. (2002) obtained somatic embryos from shoot apical meristem of wheat on media containing BA and 2, 4-D (0.5 mg L -1 ) with high frequencies of embryogenic calluses (48.6%) from immature embryos of wheat (El-Sherbeny et al. 2001) and 47% embryogenic callus induction rate using fragmented mature embryo cultured on MS media supplemented with 5M 2, 4-D (Delporte et al. 2001). Secondary somatic embryos were produced from somatic embryos which attained somatic clonal capability and proved beneficial for improving the transgenic system and promoting the genetic transgenic efficiency in wheat (Wu et al. 2005).
In vitro selection in wheat
The frequency of total somaclonal variation should be high enough for selection of derivable traits. The in vitro x genotype x explant source interactions contribute to one level of variation. When in vitro selective agents are added to the protocol, additional interactions are encountered (Handro 1981).
Selection in culture
A major challenge for exploitation of somaclonal variation is the identification of useful variation (Evans 1988; Smith et al. 1993). Because cells are genetically variable in culture, specific traits can be selected using in vitro induced spontaneous mutations. Cells may be selected in either a positive or negative manner using direct (cells cultured with selective agents followed by isolation of surviving cells), rescue (cell exposure to culture conditions, capable of killing or inhibiting susceptible cells followed by culture under different conditions to recover survivors) and gradual increase in selection pressure over time (Conner and Meredith 1989). In vitro selective agents have been used to detect useful variants, for example, NaCl (Arzani and Mirodjagh 1999; Zair et al. 2003; Yadav et al. 2004) for salt tolerance, AlCl3 for the Aluminum toxicity portion of acid soil tolerance (Conner and Meredith 1985; Bamabas et al. 2000), grain polyphenol variants (Cai et al. 1995), polyethylene glycol (Adkins et al. 1995) as an osmo-regulator to stimulate drought stress, in vitro regeneration of wheat under different stress of mannitol , mannose (Yao et al. 2007), in vitro screening of scab resistant wheat lines tolerant to deoxynivalenol (vomitoxin) (Lu et al. 1998); ABA for freezing tolerance (Sapina et al. 1994).
In vitro selection technique has been used to improve cold hardiness (Galiba 1994); drought tolerance (Gawande et al. 2006; Bajji et al. 2004), Salt tolerance improvement (Zair et al. 2003; Yadav et al. 2004), disease resistance (Svabova and Lebeda 2005); herbicide resistance can also evolve from somatic cell selection (Saunders et al. 1992); for resistance against Helminthosporium sativum in wheat and barley (Chawla and Wenzel 1987); and fusaric acid resistant barley plants . In vitro induced selection for high frequency regeneration (Yadav et al. 2000; Ye et al. 1998; Machii et al. 1998; Cai et al. 1999) in wheat; incorporation of useful agronomic traits in rice (Jain 2001) agronomic performance and quality characteristics of tissue culture derived lines of wheat (Villareal et al. 1999) and for increase the gliadin and glutenin subunits and protein content in seed of progenies from regenerated plants of wheat has been successfully implemented (Hu et al. 1998). Shi et al. (1998) developed six new wheat lines for resistance to powdery mildew through in vitro culture selection
In vitro regeneration in wheat
There are only a few reports on plant regeneration from cultured mature embryos of wheat. Early reports on regeneration showed a low frequency (7% of one culture) of plant regeneration (Chin and Scott 1977). Twenty percent regeneration was obtained through the use of NAA (1 mg l -1 ) plus kinetin (5 mg l -1 ) in the regeneration medium. In general a downshift in auxin concentration results in organogenesis and plant regeneration (root and shoot induction) after transfer of the callus either on a medium containing certain combination of plant growth regulators or no regulator at all (Shimada et al. 1969; Sharma et al. 1981; Bajaj 1986; Ovesna and Lhotovia 1987; Elena and Ginzo 1988; Kintzios et al. 1996; Delporte et al. 2001).
Eapan and Rao (1982) observed increased plant regeneration rates from wheat mature embryo explants cultured on media supplemented with IAA and cytokinin, mostly zeatin. A low exogenous auxin application (such as NAA at 1 mg l -1 ) initiates only roots (Chin and Scott 1977; Ahloowalia 1982).
Racz et al. (1993) used mature embryos of winter wheat cultured in MS-B medium supplemented with AgNO3 (20 mg l -1 ) and found enhanced regeneration. Leon et al. (1995) studied the effect of various media constituents (like ammonium, calcium, sucrose) and different medium (Taghvaii et al. 1998) on regeneration of mature embryo culture.
Varshney et al. (1999) reported regeneration frequency as high as 94% on the regeneration medium containing NAA (0.2 mg l -1 ) plus BAP (1 mg l -1 ) in 17 cultivars of T. aestivum and 3 cultivars of T. durum through mature embryo culture. Wang et al. (2002) achieved 70% plant regeneration frequency through embryogenic calluses of wild rye using mature embryos. El-Bahr et al. (2000) cultured mature embryos in MS media with 2, 4-D with different concentrations of NaCl for 12 weeks to select the salt tolerant wheat lines. Mendoza and Kaeppler (2002) used 2, 4-D and dicamba to enhance the number of regenerated plants per embryo and reduce the amount of time required for plant regeneration by 3-4 weeks. Some new techniques such as the endosperm supported callus induction method have been successfully used in callus induction from mature embryos (Bartok and Sagi 1990; Ahmed et al. 1992; Ozgen et al. 1996; Sayar et al. 1999). Nayal et al. (2002) obtained regeneration in wheat var. Sonalika using Kinetin (0.5 - 1.5 mg l -1 ) with IAA (0.5 mgl -1 ) in MS media. Yadav et al. (2000) selected R1 lines from wheat genotype UP2338 which gave a maximum of 19 shoots from a single callus in the MS medium supplemented with IAA and kinetin (Fig. 1 A-D).
Factors influencing in vitro regeneration potential of different explants of wheat presoaking time
The length of the imbibition period could cover different phases in the release from inactivity of the embryo, rehydration of the tissues, hormonal activation, transformation of reserves etc and might therefore influence the reactivity of the explants in culture. Chlyah et al. (1990) used several durations (16 to 64 h of imbibitions) and reported that with 20 h soaking time of mature embryos, nearly 100% of explants formed somatic embryos.
Effect of genotype
The importance of genotype in determining the in vitro response of wheat tissues has been recognized and the efficiency of callus induction, callus growth rate and plant regeneration frequency have all been reported to be genotype dependent (Zhou and Lee 1983; Racz et al. 1993; Ozgen et al. 1998; Yadav et al. 2000; Anapiiaev 2000; Schween and Schwenkel 2003; Yadav and Chawla 2001; El-Sherbeny et al., 2001). Varshney et al. (1999) evaluated 17 cultivars for their ability to produce embryogenic callus from mature embryos and reported that percentage regeneration varied widely from cultivar to cultivar.
Fragmentation, orientation and size of embryos
Chlyah et al. (1990) attempted to determine the most embryogenic zones of the wheat embryo by fragmenting it in various ways prior to culture. It was observed that fragmentation of the embryo could bring about the formation of as many embyogenic calli as explants and a large number of cells in different parts of the embryos became capable of initiating embryogenic callus by fragmented original mature embryos (Delporte et al. 2001).
Eapen and Rao (1982) studied the effect of orientation of embryos on callus induction. The embryos were placed on the medium in three different ways: (a) scutellum facing up (b) scutellum in contact with the medium and (c) one side of scutellum and embryonic axis in contact with the medium. Frequency and intensity of callus development were found to be best in orientation (a) and the least in orientation (c). Chang et al. (2003) showed the effect of embryo size for the establishment of embryogenic callus in barley cultivar (Hordeum vulgare L cv. Morex). Small size embryo was found excellent for embryogenesis and regeneration.
Other physiological factors
Recent studies suggest that genotype determined ratios of endogenous growth regulators were responsible for rapid in vitro regeneration (Marcinska et al. 2001; Jimenez and Benegerth 2001). The influence of genotype and cold pre-treatment was well documented on production of embyoids and regeneration of wheat (Mentewab and Sarrafi 1997; Xynias et al. 2001).
Effect of low temperature treatment (Hau and Teng 1994) and combined effect of colchicine, L-proline and post inoculation low temperature (Redha et al. 1998) was associated with fast differentiation rate in wheat. The effect of low intensity laser radiation (632.8 nm, 12 mW) on morphogenetic and regeneration processes in wheat (cv. Skala) callus culture were investigated by Salyaev et al. (2001). They observed a marked increase in the number of regenerated plants when the callus was subjected to irradiation. The final yield of regenerated plants comprised 38% in irradiated samples vs. 25% in controls ones.
Lange et al. (1998) attempted to study the genetics of in vitro organogenesis and precocious germination of wheat embryos. The results indicated a complex gene action controlling both traits, with additive, dominance and epistatic effects. High broad-sense heritability values were found, indicating genetic determination. In view of the complex gene control of these traits, it is suggested that genetic improvements can be achieved by selecting for the traits in advanced generations of the segregating population.
Occurrence of somatic variation
Somatic variation refers to all variability observed among tissue culture regenerated plants (Larkin and Scowcroft 1981). Tissue culture regenerated variants have also been called calliclones (Skirvin and Janick 1976), phenovariants (Sibi 1976), protoclones (Shepard et al. 1980) and subclones (Cassells et al. 1991). Chromosome multiplication and structural observations induced during culture may constitute the basis of gametoclonal and somaclonal variations (Dogramaci et al. 2001). In vitro culture per se can be extremely stressful on plant cells and involve highly mutagenic processes during explants establishment or callus induction, maintenance, embryo induction and plant regeneration (Lorz et al. 1988). Two types of somaclonal variation can result: epigenetic (developmental) and heritable variation (Skirvin et al. 1994).
Epigenetic variation can be transient or temporary in later generations even when the material is sexually propagated. This variation includes phenotypic changes that involve expression of specific genes (Hartman and Kester 1983). Because explants adapt to an in vitro environment in step wise fashion by becoming more juvenile, the resulting calluses may vary in maturity from juvenile to fully mature. Plants regenerated from these tissues also vary depending on the developmental stage progression of the tissue when the stimulus to regenerate is applied (Skirvin et al. 1994). Shoot regeneration from dedifferentiated callus can produce an immature, unstable clone that may eventually revert to the original parental clone. Broad spectrum variation is available through either nuclear or cytoplasmic sources; all types of variation could potentially be recovered and used for crop improvement (Elkonin et al. 1994; Skirvin et al. 1994; Khanna and Garg 1997). Heritable variation is stable through the sexual cycle and with repeated asexual propagation (Skirvin et al. 1994). Stable genetic alterations involve phenotypic and biochemical traits due to karyological variations (gene or chromosome aberrations) (Lorz et al. 1988). Heritable somaclonal variation involves either single or multiple gene changes, including alterations in DNA bases, chromosome or the entire genome (Orton 1994), single base pair changes, paracentric and pericentric inversions, deletions, translocations and ploidy changes (D’Amato 1991; Garcia et al. 1994). Cytoplasmic variation has been found, including mitochondrial controlled male sterility (Elkonin et al. 1994). Chimeral plants subjected to tissue culture can produce a high percentage of variants (Skirvin and Janick 1976; Skirvin et al. 1994). Chimeric regenerant frequencies range from 10-70% in sorghum and 54-79% in maize (Cai et al. 1990). However, chimeric regenerants in some plant species are rarely observed (Vasil 1983). Variation may represent pre-existing variation or variation induced during callus formation and not during the shoot formation process (Skirvin et al. 1994).
Culture age enhances variability of regenerated plants (Symillides et al. 1995; Kothari et al. 1998). This age effect can be attributed to (i) increased mutation rate per cell generation (Murashige and Nakano 1965) and accumulation of mutations over time (ii) a lag period of apparent culture stability caused by early generation mutations that are not detected until sufficient mutant cells have accumulated (Benzoin and Phillips 1988), (iii) active selection of early culture mutations that increase in number over time (Amstrong and Phllips, 1988) or (iv) increased ploidy (Colijin-Hooymans et al. 1994). As callus induction time increases, the morphogenesis potential decreases, whereas the frequency of albino shoots and callus producing only roots increases (Wen et al. 1991). Some mutations may occur in sequence throughout the callus phase rather than occurring randomly or all together at an early culture stage (Fukui 1983).
Regeneration competence in Gramineae may be rapidly lost during differentiation and senescence (Ozias-Akins and Vasil 1988). This loss may be due to the endogenous concentrations of growth regulators (Vasil 1987). Tissue culture variability rate can increase as growth regulator concentration increases (Skirvin et al. 1994).
In vitro genetic control of regeneration could be functioning specifically on response to plant growth regulators (Komamine et al. 1990). Genes controlling phytohormone signals are directly involved in plant regeneration competence (Henry et al. 1994), negating the concept that ‘dominant’ genes govern regeneration and that all cultivars have genes for regeneration. Heritability ranges of 9 to 60% (embryo germination); 30-55% (callus induction) and 15 to 49% (embryogenic callus formation) have been documented in wheat (Chevrier et al. 1990). Recently, some somaclonal variants were screened for resistance to scab (Yang et al. 1998; Liu et al. 1997), improvement of agronomic traits, salt and drought tolerance in wheat (Villareal et al. 1999), Somaclones (R3 and R4 generations) regenerated from five winter wheat genotypes were evaluated for variation in five agronomic and morphological characters (Ivanov et al. 1998).
Field selection of variants
Haploidization, somatic embryogenesis, cell suspension culture and protoplast technology all require plant regeneration techniques for genetic manipulation and subsequent selection in plant improving programs (Henry et al. 1994). The entire in vitro process from genotype and explants selection to media induced callus, embryo and plantlet regeneration is extremely productive in creating variation (Hossain et al. 2003). Somaclonal variations for a wide range of agronomically important traits and yield have been observed in sorghum, sugarcane, maize and wheat etc. In case of wheat somaclonal variations for grain colour, height, and tillers per plant and seed storage proteins were reported by Ahloowalia (1982). Nevertheless, somaclonal variation has not been incorporated as a standard protocol in most plant breeding selection schemes. Perhaps this limited integration can be traced to the segregation of biotechnology and traditional breeding laboratories or the limited understanding of molecular events that trigger in vitro induced variation coupled with strong beliefs in traditional breeding methodology.
Phenotypic variation
Phenotypic variation in somaclones resulting from emrbyogenic callus regeneration is increased with increasing time in the callus and regeneration phases (Cai et al. 1990). Phenotypically similar variations can segregate in progeny of different callus lines or reappear in later generations. Positive variations in height, maturity, biomass, grain yield, tillering, ploidy level and kernel oil content (Cai et al. 1990; Encheva 1993; Bhaskaran et al. 1987) have been documented from various in vitro programs.
Field screening techniques
A major failure for the release of more useful regenerants may be due to inappropriate germplasm/segregating progeny selection techniques or improper field screening/evaluation methods. Many of the field tests that have evaluated somaclonal variants have been conducted with small, unreplicated trials because of low seed availability (Mitchell et al. 1992; Maddock 1985). Inadequate or negative phenotypic/genetic variation between regenerants and their donor parents has discouraged wide scale adoption into many in vitro selection and breeding programs (Baille et al. 1992). It is felt that agronomic performance of somaclonal variants lines need to be confirmed in relevant field tests before they are incorporated in production programs (Arnoldo et al. 1992; Encheva et al. 2003).
Conclusion and future prospects
There is a need for novel and innovative approaches for increasing the crop productivity involving traditional plant breeding and modern biotechnological approaches. In wheat there is a need for developing superior genetic stocks with high quality grain, high fertile tillering, and more spikelets/spike using in vitro regeneration and selection methods.
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