N6 Medium Research Articles

English

  Five elite Indian maize inbreds namely; HKI1105, HKI1105, HKI335, CM300 and LM5 were evaluated for callus induction and regeneration. Immature embryos obtained 14 days after pollination were used as explants. Genotype, medium, type of auxin and their concentrations influenced callus induction. N6 medium supplemented with different concentration of 2,4-D (1, 2 and 3 mg/l) and Dicamba (1, 2 and 3 mg/l) were used for callus induction. N6 supplemented with 2 mg/l of 2,4-D has shown highest percentage of embryogenic callus induction. Among the five genotypes tested, CM300 gave highest percentage of embryogenic calli. CM300 and LM5 both have shown higher regeneration percentage of 12.22%.    Key words: Maize, in-vitro culture and regeneration.

High frequency plant regeneration from mature seed of elite, recalcitrant Malaysian indica rice (Oryza sativaL.) CV. MR 219

An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.

Regulation and improvement of triterpene formation in plant cultured cells of Eriobotrya japonica Lindl

Loquat (Eriobotrya japonica Lindl) is a traditional Chinese medicinal plant that contains triterpenes, which have been shown to exhibit pharmaceutical activities. In this study, we investigated various different culture conditions for cultured cells of loquat to produce triterpenes, including illumination, carbon source, nutrient composition and culture system. When cultured on 2.5mg/l of 6-benzyladenine, 1mg/l of naphthalene acetic acid and 30 g/l of sucrose at 25 ± 2 °C in the dark for 30 days, the nutrient composition significantly regulated the cell growth and triterpene production. Supplied with the Murashige and Skoog medium reached higher level of dry weight (1.27 ± 0.09 g per flask) and total triterpene production (151.54 ± 12.58 mg/g of cultured cells), and the N6 medium produced tormentic acid but inhibited other triterpene products, while the B5 medium produced relatively high corosolic acid. Also found, suspension cultures of loquat cell could achieve high productivity as callus culture.

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L−1 picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L−1 2,4-D, 0.16 g L−1 putrescine, 0.5 g L−1 casein amino acids and 2.0 g L−1 activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L−1) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.

In vitro Clonal Propagation of Anthurium (Anthurium andraeanum L.) Using Callus Culture

High frequency of calli was obtained from leaf and spadix segments of Anthurium andraeanum L. when cultured on N6 medium containing 2.5 mg/l BAP and 0.2 mg/l 2,4-D in dark condition. The calli were maintained in dark condition on the same medium containing same hormonal supplements by sub-culturing up to three months but no shoot formation occurred during this period. Leaf and spadix segments derived calli were then tried for multiple shoot regeneration by culturing onto MS fortified with BAP and Kn singly or in combination with NAA in light condition. Best response towards multiple shoot regeneration was observed from both leaf and spadix segments derived calli on MS fortified with 1.0 mg/l BAP. In this combination an average of 18 shoot buds regenerated from leaf segment derived callus while 14 shoot buds regenerated from spadix segment derived callus. The number of shoot buds increased three to four-folds after subculturing in the same medium. Regenerated multiple shoots were excised and cultured onto half strength of MS with different concentrations of IBA and IAA for root induction. Best root development was obtained in half strength MS containing 1.0 mg/l IBA. About 85 per cent of the regenerated plantlets survived in natural conditions. Key words: Clonal propagation, In vitro, Anthurium andraeanum D.O.I. 10.3329/ptcb.v19i1.4961 Plant Tissue Cult. & Biotech. 19(1): 61-69, 2009 (June)

In vitro production of double haploid plants from two rice species (Oryza sativa L. and Oryza glaberrima Steudt.) for the rapid development of new breeding material

Eight genotypes of two rice species (Oryza sativa and Oryza glaberrima) were studied for their response to anther culture in terms of callus induction and frequency of plant regeneration. N6 medium (Chu et al., 1975) was used for callus induction, and MS medium (Murashige and Skoog, 1962) with 1 mg/l BAP and 0.5 mg/l NAA for plant regeneration. Generally, O. glaberrima genotypes produced more callus than O. sativa genotypes. All O. glaberrima genotypes regenerated plants. Among O. sativa genotypes only japonica variety IKP produced plants. Other O. sativa genotypes showed low frequency of callus induction without plant regeneration. Many albino plants were obtained from the culture. Only one O. glaberrima genotype (6202 Tog) produced green plants. In this experiment, a total of 93 plants were regenerated with 14 green plants and 79 albino plants. Anther culture response is largely species and genotype dependent. Key words: Rice species, Oryza sativa (L.), Oryza glaberrima (Steudt.), anther culture, medium, callus induction, plant regeneration.

Effect of Culture Media for Anther Culture of Indica Rice Varieties and Hybrids of Indica and Japonica

Pollen plants from anther culture in rice breeding allows selection of fine strains in early generations. Exploitation of anther culture technique in research is limited due to very low regeneration frequency of indica cultivars and the transfer of anther culture traits from japonica to indica varieties is important in improving these rice varieties. In this study the optimization of media requirements and culture conditions for high frequency callus induction and plant regeneration of several indica × japonica F1 hybrids were evaluated using improved anther culture media. Five indica rice varieties (BG 90-2, BG 379-2, BG 94-1, Dahanala and Suduru samba) and two japonica varieties (Hu Lo Tao and Chuan 4) were selected and F1 hybrids were taken with all possible crosses between them. Panicles were cold pre treated at 5°C for 7 days and anthers were cultured in agar solidified modified N6, B5 and Millers media. Calli were transferred to MS medium for plant regeneration. Best callus induction frequencies (0.8 - 29.4%) were obtained in N6 medium containing 5% (w/v) sucrose, than B5 (0.5 - 19.2%) and Millers (0.2 - 19.8%) media. The F1 hybrids were more responsive to anther culture than their parents. Highest callus induction frequency of 29.4% could observe in N6 medium for F1 hybrid Hu Lo Tao × BG 90-2. The green plant regeneration frequency of calli induced on N6 medium was higher than the other two media and the highest frequency of 41.0% was occurred in F1 hybrids of Hu Lo Tao × BG 90-2. Green plants were transferred to pots with 50 - 75% survival rate. Out of 99 plants survived, 28 plants had more than 5% spikelet fertility. Modified N6 medium had positive effect on anther culture performance and the F1 hybrid Hu Low Tao × Bg 90-2 had the best performance. Key words: anther culture; indica; japonica; modified media; plant regeneration; rice DOI: 10.4038/tare.v10i0.1866 Tropical Agricultural Research & Extension Vol.10 2007 17-22

English

  Transformation efficiency enhancement in maize Agrobacterium-mediated transformation was tested using four different basal media, five levels of N6 salts, two antioxidants and copper sulfate. In the absence of the antioxidants L-cysteine and dithiothreitol (DTT), the frequencies of transient GUS expression was higher using Linsmaier and Skoog (LS) and Murashige and Skoog (MS) media as an alternative to Chu (N6) and Ducan (D). N6 basal medium exhibited better performance in the presence of antioxidants than MS, LS and D basal media. Five different levels of N6 medium salts (10, 30, 50, 70 and 100%) were tested, and the highest transformation efficiency was 15.9% under a 50% salt concentration, followed by 6.4% transformation efficiency with 70 and 3.2% under 100% salt conditions. More than 95% of infected immature embryos exhibited GUS staining under 10 and 30% salt concentrations, however none of the embryos developed into embryogenic callus, indicating that low salt levels favored T-DNA delivery, but not stable transformation. Additions of DTT or L-cysteine, or a combination of L-cysteine and DTT, showed a significant improvement in the frequency of transient GUS expression, however increases were not observed with independent CuSO4treatments. Polymerase chain reaction (PCR) and Southern-blot analysis confirmed T-DNA integration into the maize genome.   Key words: Maize (Zea mays L.), Agrobacterium tumefaciens, transformation.

In vitro regeneration and genetic fidelity of Tigridia pavonia (L.f.) DC.

Plants of Tigridia pavonia (L.f.) DC were regenerated from twin-scaling explants cultured on Murashige and Skoog and N6 basal medium. The highest formation of shoots per responding explant was obtained on N6 medium supplemented with 4.5 µM 2,4-dichlorophenoxy acetic acid in combination with 2.2 µM benzylaminopurine. Shoots rooted readily on N6 basal medium supplemented with 1 g l-1 activated charcoal and 2.6 µM naphtalenacetic acid. The rooted shoots achieved 100% survival. Inter Simple Sequence Repeat analysis was carried out to check for possible genetic alterations in plants obtained after two consecutive subcultures. The results revealed that the recovered plants did not exhibit any type of polymorphism.

The somatic embryogenesis and plant regeneration from immature embryo of sweet corn inbred line

Synthetic seed consisting of somatic embryos enclosed in protective coating are a suitable tool for clonal mass propagation of elite plant varieties. The in vitro study was aimed to evaluate the optimizing medium for sweet corn somatic embryogenesis, synthetic seed production which leads to increasing germination and seed viability percentage. The in vitro study was aimed to evaluate the optimizing medium for sweet corn somatic embryogenesis, synthetic seed production which leads to increasing germination and seed viability percentage. Sweet corn (Zea mays var. saccharata) variety FAH01 embryogenic callus were derived from culturing immature zygotic embryos at 11 days after pollination on N6 medium that contained 2, 4-D 2 mg L-1 and sucrose 60 g L-1. Somatic embryos was developed after transferred embryogenic callus to N6 medium contained with 2 mg L-1 2, 4-D and 30 g L-1 sucrose. Sweet corn synthetic seed was produced by somatic embryos encapsulated into a protective calcium-alginate matrix with provides mechanical support, protection and is coated with a wax film to prevent desiccation. Synthetic seed were produced, it was found that when synthetic seed were treated with 60 g L-1 sucrose and stored at 15 ± 2 degree Celsius for 2 weeks, the percentage of germination of synthetic seeds were 42%, percentage of normal seedling was 91% and abnormal seedling was 8%, and they germinated for 8 - 9 days and could be produced normal planlet. When the synthetic seed were dehydrated by silica gel until remained 60% of their moisture content and then stored for 2 weeks, they could germinated at level 23%, which 83% of normal seedling and 17% of abnormal seedling. The survival ratio in sweet corn synthetic seed in this investigation indicated that there is still some more research required to increase number of the survival seeds and the optimum storage technique to prolong their viability is also needed Key words: Somatic embryo, synthetic seed, sweet corn, artificial seed, tissue culture.

Multicellular genesis of leaf primordium was demonstrated via chimaeric transgenic plant of maize (Zea mays L.) regenerated from Type II calli

Type-II embryonic calli were induced from immature embryos of maize (Zea mays L.) genotype YD and bombarded with beta-glucuronidase gene. Bombarded calli were proliferated on normal N6 medium for 2 weeks at 26°C in the dark and selected on N6 medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg/l phosphinothricin (PPT) but without casamino acids and proline under the same conditions for 14 days. Regeneration was carried out on hormone-free MS medium containing 5 mg/l phosphinothricin at 26°C under 3000 lux illumination. Plants over 8 cm were transplanted into soil and sprayed with 250 mg/l phosphinothricin when two new leaves appeared. Except normal transgenic plants, chimaeric transgenics also were regenerated in the present work. The expression pattern of beta-glucuronidase gene in leaves of chimaeric transgenic plant revealed that more than one cell formed leaf primordium at the initial stage, and filial cells stemed from each cell in leaf primordium arranged in a row longitudinally from leaf base to leaf apex. There was a clear boundary as a straight line between the area formed by transformed cells and the area formed by normal cells. A hypothesis was put forward that the primitive cells in leaf primordium divided in a longitudinal style, resulted in leaf elongation, then the filial cells divided transversally and synchronously toward the outside to broaden the leaf.

Effect of Low Temperature Pre-Treatment on Anther Culture in Selected Indica, Japonica Rice Varieties and their Inter Sub-Specific Hybrids

Effect of cold pre-treatment of panicles with reduced temperatures (of 5°C, 7°C, 8°C and 10°C) and exposure for different time durations (at weekly intervals up to 4 weeks) on anther culture of Japonica rice variety Hu lo tao, Indica rice varieties Bg 90-2, Bg 379-2 and their F1 hybrids for high frequency callus induction and plant regeneration was investigated. Anthers were cultured in N6 medium with 5% sucrose, 2 mg l-1 2,4 D and 0.5 mg l-1 Kinetin. The cultures were kept in the dark at 28 ± 2°C for callus induction. Induced calli of 1-2 mm diameter were transferred to half strength Murashige and Skoog (MS) medium supplemented with 2.0 mg l-1 Kinetin and 0.5 mg l-1 NAA. The effect of variety and cold pre-treatment was significant at 5% probability level. The Indica varieties had the lowest callus induction frequency (0.3% - 7.0%). Japonica and Japonica × Indica hybrids had high callus induction (12.3% - 70.0%). The F1 hybrid Hu lo tao × Bg 90-2 had comparatively high callus induction (31.0% - 70.0%) than the other genotypes. Japonica variety Hu lo tao and F1 hybrids had highest callus development at cold pre-treatment at 8°C for 14 and 21 days. The highest green plant regeneration was observed in F1 hybrid, Hu lo tao × Bg 90-2 followed by Japonica variety subjected to 8°C for 14 days. Very poor green plant regeneration was observed in Indica variety, Bg 90-2 when it was exposed to 7°C for 21 and 28 days. Variety Bg 379-2 did not produce any green plant at any treatment. The F1 hybrid Hu lo tao × Bg 379-2 produced lower green plants than F1 hybrid Hu lo tao × Bg 90-2. Cold pre treatment at 8°C temperature for 14 days was found to be most effective for anther culture in experimental genotypes used in the study. Key words: variety, cold pre-treatment, callus induction, plant regeneration, rice DOI: 10.4038/cjsbs.v38i1.1322 Cey. J. Sci. (Bio. Sci.) 38 (1): 11-16, 2009

Callus induction and whole plant regeneration in elite Indian maize (Zea mays L.) inbreds

Callus induction and regeneration ability of five elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations influenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l−1 showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l−1 Dicamba induced the highest frequency of organogenic callus. Among the five genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l−1 benzyladenine and 0.5 mg l−1 indole acetic acid promoted the highest frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l−1 naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66.

Possibility of Sweet Corn Synthetic Seed Production

Somatic embryogenesis in sweet corn has been reported by a number of workers. However, the knowledge maintaining storage life, vigor and viability of these somatic embryos are limited. A model system of synchronous somatic embryos production combined with encapsulation to synthetic seed was studied in sweet corn (Zea mays var. saccharata). In this study immature zygotic embryo cultured on N6 medium, contained 2, 4-D 2 mg L(-1) and sucrose 60 g L(-1) form the embryogenic callus. Higher 2, 4-D levels did not show increasing in inducing embryogenic callus. If the concentration of 2, 4-D decreased globular-stage, somatic zygote form the roots. Somatic embryo develop without surrounding nutritive tissues and protective seed coat has been devoted to causing somatic embryos to functionally mimic embryo, then was encapsulated by 3% (w/v) sodium alginate with 4-6 mm in diameter. It was found that when synthetic seed were treated with 60 g L(-1) sucrose and stored at 15+/-2 degree Celsius for 2 weeks, the survival rate of synthetic seed were 44%, after 8 days of germination test, it was found that there were 91% of which were normal seedling and 9% were abnormal seedling. This result indicated that there is a possibility in sweet corn synthetic seed production. Anyhow, more research for better technique are further required.

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in Zoysia japonica

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N6 medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L−1 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L−1 2,4-D, 0.5 mg L−1 kinetin, 500 mg L−1 casein hydrolysate, 500 mg L−1 proline, and 500 mg L−1 myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO4 to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.

Plant regeneration via somatic embryogenesis of tropical maize (<i>Zea mays</i> L.) commercial hybrid lines

Four commercial tropical maize genotypes (H627, H625, H513 and DLC1) were screened in vitro for callus initiation and plant regeneration from immature embryos. Callus was initiated on N6 medium supplemented with varying concentrations of 2,4-D, 3% sucrose, 10 mg l-1 silver nitrate, 100 mg l-1 casein hydrolysate and 2.875 mg l-1 proline. The induction of embryogenic and primary callus highly dependend on the concentration of 2,4-D, genotype and day after pollination of immature embryos. H627 displayed the highest percentage of embryogenic callus formation from immature embryos 16 days after pollination (45.1%) and 20 days after pollination (28.3%). The percentage of embryogenic and primay callus initiated from immature embryos 16 days after pollination was significantly higher compared to 20 days after pollination (p

Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.)

Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

식물생장조절물질이 레드 톱 밴트그래스 (Agrostis alba L.)의 종자로부터 캘러스 유도와 식물계 재분화에 미치는 영향

In order to develop a simple and reproducible protocol for red top bentgrass (Agrostis alba L.), effect of different growth regulators was investigated for embrogenic calli induction and subsequent plant regeneration using mature seeds. MS medium containing 2 mg/L 2,4-D was optimal for embryogenic callus induction from mature seeds. The highest plant regeneration frequency (64.4%) was showed when the embryogenic callus tissues were cultured on N6 medium supplemented with 0.5 mg/L 2,4-D and 2 mg/L BA. Regenerated plantlets were grown normally when shoots transplanted to the soil. A high-frequency and efficient regeneration system from mature seeds would be helpful for molecular breeding of new variety of red top bentgrass through Agrobacterium-mediated genetic transformation.

티모시 성숙종자로부터 캘러스 유도 및 식물체 재분화

ABSTRACT Timothy ( Phleum pratense L.) is an important grass species as forage. In order to optimize tissue culture conditions of timothy, the effects of plant growth regulators on callus induction and plant regeneration was investigated with mature seeds of colt cultivar. The optimal concentration of 2,4-D for the induction of primary callus from mature seeds was 3 mg/L. The highest embryogenic callus frequency (25%) was observed when the mature seed were cultured on MS medium supplemented with 3 mg/L 2,4-D and 0.1 mg/L BA. The highest plant regeneration frequency was observed when type B callus was transferred to N6 medium supplemented with 1 mg/L 2,4-D and 3 mg/L BA. Regenerated plants were grown normally when shoots were transplanted to the soil. A short tissue culture period and regeneration system would be beneficial for molecular breeding of timothy by the production of transgenic plant.( Key words : Timothy, Callus, Plant regeneration, Tissue culture) Ⅰ. 서 론 티모시( Phleum pratense

In vitro Selection and Characterization of Drought Tolerant Somaclones of Tropical Maize (Zea mays L.)

Somaclonal variation for drought tolerances of two Kenyan maize genotypes, KAT and PH01, were obtained from in vitro selection. Three-month-old calli derived from immature embryos were grown on N6 medium supplemented with mannitol or polyethylene glycol (PEG-6000) to find-out the appropriate concentrations of selection pressure. Calli were sequentially transferred to 3.28 and 5.28% mannitol or 18 and 20% PEG and maintained for 9 weeks. Survival percentage and regeneration capacities of selected and unselected calli were determined. It was observed that selected calli had lower survival and regeneration capacities than unselected calli. The result showed that survival percentages of maize calli grown in mannitol and PEG were 8.4 and 4.2%, respectively. Six plantlets were regenerated from mannitol-tolerant calli, while, only two plantlets were regenerated from the PEG tolerant calli. Plantlets were regenerated from selected calli at lower frequencies than those from unselected calli. To investigate the putative genetic basis of water stress tolerance, the mannitol tolerant PH01 somaclones designated MT JM1-01, MT JM2-01, MT JM3-01 and MT JM4-01 and the PEG tolerant clone, PT JM1-01, were further evaluated using RAPD. The assay with the decamers OP-07 and OPD-08 revealed that the water stress tolerant somaclones were genetically different from the controlled, unselected plants.