In Vitro Screening of Callus Cultures and

Research Journal of Biotechnology

Vol. 10 (6) June (2015) Res. J. Biotech

In Vitro Screening of Callus Cultures and Regenerants for Drought Tolerance in Upland Rice Tripathy Swapan K. Sinha Molecular Breeding Laboratory, Department of Plant Breeding and Genetics, College of Agriculture, OUAT, Bhubaneswar-751 003, INDIA [email protected]

genetic changes could be attributed to chromosome rearrangements17, modification in DNA methylation pattern24 and transposons mediated mutations3,6. In vitro screening of cell lines, somaclonal variants and somatic mutants is possible using various selection agents20 in a small area under controlled condition. Generally, the tolerance at the callus level is positively correlated with that at the plant level. The calli and regenerants which sustain the selection pressure are retained while others fail to grow and develop with the desired character4. Thanh and Tuyen35 selected and regenerated rice plants in vitro for tolerance against acidic condition. Moreover, in vitro selection largely depends upon extent of tissue culture induced genomic variation, in vitro selection method, regeneration method of tolerant calli38 and the desired character to be inherited.

Abstract Proliferated calli from mature caryopsis culture of six upland rice cultivars (cv. Khadagiri, Mandakini, Annada, Sahbhagidhan , N22 Selection and Vandana) were cultured on selection medium supplemented with polyethylene glycol (PEG6000:0.0, 0.5, 1.0, 1.5 and 2.0M) / mannitol / sorbitol (0.0, 2.5, 5.0, 7.5 and 10M) used as selective agents for osmotolerance. The drought tolerant calli were selected and also cultured for two cycles, each of two weeks on regeneration medium supplemented with PEG6000/ mannitol /sorbitol. Increased concentration of PEG resulted in a progressive reduction in callus fresh weight whereas it was more than double in no stress or control after four weeks of sub-culture in the callus induction medium. Significant reduction in callus fresh weight was observed in response to PEG at 1.52.0M concentration. Increased concentration of PEG/mannitol/sorbitol in the medium resulted in decreased plant regeneration frequency.

PEG (polyethylene glycol), mannitol and sorbitol are known to cause osmotic stress which alters the osmotic potential of the cell and hence these serve as useful selection agents for drought tolerance. PEG with molecular mass of 6000 and above are non-ionic, non-toxic, water soluble polymers which do not penetrate intact plant tissues rapidly. It forms hydrogen bonds with water, decreasing the water potential of culture medium and finally inhibits both water and mineral uptake by root32. Thus, such a osmotic agent acts in lowering the water potential in a way similar to soil drying15.

However, the effect of drought stress was more prevalent in PEG at even lower concentration(s) than mannitol and sorbitol. Therefore, calli of rice genotypes could be tested for drought tolerance at 1.5-2.0M PEG. N22 selection was tested as highly drought tolerant while Khandagiri and Annada were highly sensitive to PEG based on callusing ability and plant regeneration among the present set of six upland genotypes. A number of putative drought tolerant plants were obtained from N 22 sel., Sahbhagidhan and Mandakini and those were forwarded for field evaluation following a sequence of hardening process. Besides, the method adopted for in vitro screening of plant regenerants may pave the way for improvement of drought tolerance.

Short et al31 stated that in the in vitro culture, PEG is capable to induce water stress and is positively correlated with that in the field or the green house7. Besides, sugar alcohols e.g. mannitol and sorbitol are frequently employed as osmotica to modify the water potential of a culture medium as these are not usually metabolized by plant tissues for use as carbon sources. Significant improvement in drought tolerance has been achieved in maize8, sugarcane33, rice18, wheat12, soybean28 and mungbean11 by exploiting somaclonal variation. Mahmood et al19 tested the progeny of selected wheat plants (R1-generation) and their donor parent in pots for drought tolerance under simulated water stress (by PEG) and it was found that selected somaclones were less affected by water stress than their donor parent. Prakash et al25 successfully obtained drought tolerant plants in rice using PEG as selection agent in the regeneration medium.

Keywords: Callus proliferation, plant regeneration, in vitro screening, drought tolerance, upland rice.

Introduction Somaclonal variation expressed among regenerants and their progeny is a manifestation of genetic changes mostly induced in the callus culture level9. Prolonged period of tissue culture increases the frequency of gross chromosomal aberrations29 and regenerants from such long term cultures are expected to have one or more heritable changes2. The most commonly observed in vitro induced

Rice is usually susceptible to water stress34,39. It is estimated that 50% of world rice production is affected by drought5. Genetic improvement of rice for drought tolerance through conventional breeding is slow owing to a

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variety of abiotic interferences, low heritability of yield under water stress, limited inherent variation in the field and uncertainty in drought timing, intensity and duration27.

Results and Discussion Plant tissue cultures have been used extensively to produce genetic variants in crop improvement programme. Callus cultures grown for several weeks are in vogue harbour cytological instability and induced genetic changes which may result in wide spectrum of somaclonal variation. Therefore, long term regenerative calli were subjected to osmotic stress with a view to obtain drought tolerant regenerants. Joshi et al13 screened calli by sub-culturing for 15 days on MS basal medium23 supplemented with different concentrations of PEG. Volume and size of calli progressively decreased while total proline content was increased significantly with increase in PEG concentration. In this study, an attempt was taken for selection of drought tolerant regenerative cell lines and to follow up plant regeneration from such calli using the optimized in vitro culture protocol for callus induction and regeneration30. PEG, mannitol and sorbitol were used as selective agents for osmotolerance. Different researchers preferred to use concentration of selection agents in different units e.g. molar conc.(M), gm/l(w/v), -Mpa (osmotic potential), -bar or percentage (% in soln.).

However, in vitro selection in an artificially created waterstress environment is relatively simple, practicable and easy technique that can provide opportunity to select superior drought tolerant genotypes out of a large population or where genes of interest are not available for crop improvement22. Therefore, an experiment was designed for in vitro selection for drought tolerance and to broaden opportunities for genetic manipulation of rice through tissue culture.

Material and Methods Fully matured healthy and well dried kernels of six genetically pure cultivars e.g. Khadagiri, Mandakini, Annada, Sahbhagidhan, N22 Selection and Vandana were pre-sterilized with 70 % ethanol for 70 sec and surface sterilized with bavistin (fungicide) followed by HgCl2 (0.1 %w/v). Kernels were washed (3-5x) with double distilled water after each treatment. Five dehusked seeds in each of the flasks and two such explants per culture tube were inoculated on an optimized callus induction medium30 for callus formation.

In this study, concentrations of stress inducing agents are expressed in molar (M) concentration for ease of comparison. Proline was knowingly omitted from the selection medium as it broadly acts as osmotic stabilizer and may reduce the effect of drought stress. Instead glycine (10mg/l) was added to the callus and regeneration medium. Extent of callus growth was assessed in terms of its fresh weight after sub-culture. Increased concentration of PEG caused a progressive reduction in callus fresh weight whereas it was more than double in no stress or control after four weeks of sub-culture in the callus induction medium (Table 1).

The proliferated calli were divided into small clumps of 100±10 mg and transferred to callus selection medium (callus induction medium containing basal salts + 3% sucrose, 0.3% agar + 0.2% phytagel, 500mg/l casein hydrolysate + 10mg/l glycine and 2.0mg/l 2, 4-D + 0.5mg/l Kn + PEG6000/ mannitol /sorbitol) to assess in vitro stress response for drought tolerance over a period of four weeks. Highly globular, white to yellowish in colour, nodular and friable calli were selected. These tolerant calli were shifted onto an optimized regeneration medium (basal salts +10mg/l Glycine + 2.0mg/l Kn + 2.0mg/l BAP + 0.5mg/l NAA for organogenic regeneration and basal salts +10mg/l Glycine + 2.0mg/l Kn + 0.5mg/l NAA for somatic embryogenic regeneration) added with PEG6000/ mannitol /sorbitol and cultured for two cycles, each of two weeks for plantlet formation. For rooting, the regenerated plantlets (organogenic) were shifted onto half strength R medium supplemented with NAA (1.0mg/l) + BAP (0.2mg/l).

Significant reduction in callus fresh weight was observed in response to PEG at 1.5-2.0M concentration. To verify the efficiency of calli to regenerate in the presence of drought stress, the regenerative calli were exposed to elevated levels of PEG (0.5 to 2.0M) in the plant regeneration medium. There was normal plant regeneration in the nostress medium but increased PEG concentration in the medium decreased plant regeneration frequency. The presence of PEG in the regeneration medium might have a detrimental effect upon most parameters associated with plantlet regeneration. Similar was the case for mannitol and sorbitol used to test osmotolerance at 2.5 to 10.0M concentration.

For plant establishment, the plantlets with defined root and shoot were initially transferred to half-strength liquid R basal medium for one week before transfer to pots filled with soil mixture of peat moss: perlite 2:1 (v/v). Thereafter, plantlets were successfully acclimatized and cultivated in glasshouse under partial shade. The experiment was set up in a completely randomized block design and each treatment was replicated thrice and the average values were estimated. An analysis of variance was performed and significant differences among treatment means were calculated at a probability level of 0.05. The experiment comprising the test treatments was repeated at least twice.

However, the effect of drought stress was more prevalent in PEG at even lower concentration(s) than mannitol and sorbitol in order for both organogenic and somatic embryogenic response. Therefore, calli of rice genotypes could be tested for regenerability under drought stress and screened for drought tolerance at the sub-optimal concentration of 1.5-2.0M PEG in the regeneration medium. In fact, PEG stimulates water stress in a way that

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Table 1 Effect of different concentrations of water stress inducing agents (PEG6000, Manitol and Sorbitol) on proliferation and regeneration frequencies of cv. Sahbhagidhan in the optimized media. Water stress Molar Growth of calli Plant Regeneration inducing concentration Callus wt.(mg) Callus wt. (mg) after Organogenic Somatic agents (M) before stress four weeks of stress response {%) embryogenic treatment treatment response (%) 0.0 100 + 10 217.5+ 3.15*(00.0) 83.8+ 2.17 80.2+ 2.05 Control 0.5 100 + 10 145.2+ 2.52 (33.3) 47.5+ 1.23 42.2+ 1.41 PEG 1.0 100 + 10 107.1+ 1.45 (50.7) 23.8+ 0.85 18.1+ 1.02 1.5 100 + 10 78.0+ 1.33 (64.1) 13.8+ 0.59 8.2+ 0.58 2.0 100 + 10 68.0+ 1.05 (68.7) 8.0+ 0.48 3.0+ 0.04 2.5 100 + 10 177.3+2.50 (18.5) 53.8+ 1.34 50.2+ 2.03 Mannitol 5.0 100 + 10 127.0+1.35 (41.6) 38.8+ 0.81 34.2+ 1.05 7.5 100 + 10 105.0+1.08 (51.7) 28.8+ 0.77 23.2+ 0.21 10.0 100 + 10 102.0+2.01 (53.1) 9.8+ 0.68 15.2+ 0.29 2.5 100 + 10 188.0+1.95 (13.5) 65.1+ 2.37 60.8+ 2.15 Sorbitol 5.0 100 + 10 152.0+1.51 (30.1) 43.5+ 1.29 53.6+ 2.08 7.5 100 + 10 118.0+2.01 (45.7) 35.7+ 1.08 30.1+ 1.32 10.0 100 + 10 106.0+1.22 (51.2) 15.3+ 0.52 20.0+ 1.01 Figures in the parenthesis denotes % reduction over control, * Values are mean + S.E. inhibits entry of water to somatic cells except those which have particular mechanism for absorbing water. Only somatic cells or calli competent to tolerate selective agent survive in the long run and are subsequently regenerated into plantlets due to their „totipotency‟ in the regeneration medium.

vulgare L.). Addition of PEG to the media resulted reduced in callus fresh weight as compared to control irrespective of the genotypes used. Joshi et al13 recorded decreased callus volume and increased total proline content with increase in PEG concentration in all genotypes. Since, the genotypes under study revealed a varied genetic response for callus proliferation (in control), 50% reduction in growth of calli as compared to control may be suitably used as a criterion for study of genotypic response for drought tolerance imposed by PEG. Annada and Khandagiri attained 50% reduction in growth of calli at around 0.75 and 1.0M PEG respectively while Vandana, Sahbhagidhan, Mandakini and N22 Selection showed the similar effect of PEG at around 1.10, 1.25, 1.5 and 1.75M molar concentrations. This evidenced that N22 Selection was highly drought tolerant while Annada was the most sensitive cultivar based on callusing ability among the present set of six upland genotypes.

Verma et al36 reported decrease in callus growth, proliferation and calli volume (in first two sub-cultures in callus culture medium) and reduced plantlet regeneration (in follow-up regeneration medium) to a certain level to acquire tolerance to PEG-induced drought. Kavi Kishor and Reddy14 and Adkins et al1 obtained promising somaclonal variants after in vitro selection for PEG tolerance in rice. The selected somaclones for drought tolerance may be genetically stable and helpful for crop improvement26. Genetic factors are considered to be the prime factor for in vitro response of cultured tissues. In addition, osmotic stress can affect physiological status and morphogenetic potential of calli16,21. Therefore, a follow-up experiment was conducted to study genotypic response of six upland rice varieties for callus culture and regeneration under drought stress induced by PEG (0.5-2.0M). High callus proliferation was noticed in N 22- Selection followed by Mandakini, Sahbhagidhan and Khandagiri under no stress treatment (control). After four weeks of sub-culture, growth of callus was approximately double or more than double in these genotypes (Table 2) while Annada and Vandana had shown comparatively poor callus proliferation and most of the calli became necrotic. Ganeshan et al10 observed genotypic differences in callus growth, colony formation and production of embryogenic calli and the regenerated plantlets in barley (Hordium

The cell lines that sustained water deficit induced by PEG were transferred to regeneration medium and cultured for two cycles, each of two weeks to test for regenerability as compared to the control. Regeneration frequency decreased with increase in PEG concentration irrespective of the genotypes tested (Table 2). However, the decrease in regeneration frequency was much prevalent in drought sensitive varieties than the tolerant ones (Sahbhagidhan, Mandakini and N22 Selection). Calli derived from Annada and Khandagiri were found to lose regenerability at higher concentrations of PEG (1.5-2.0M). Joshi et al13 recorded the highest percentage of somatic embryogenesis among selected calli in Pusa Basmati 1

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Genotype

Khandagiri

Mandakini

Sahbhagi Dhan

Concentration of PEG (M)

0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 0.0


Table 2 Genotypic response to drought stress induced by PEG. Growth of calli Plant Regeneration Callus wt.(mg) Callus wt. (mg) after 4 Organogenic Somatic before stress weeks of stress response {%) embryogenic treatment treatment response (%) 100 + 10 192.2+1.15 *(0.00) 70.0+ 2.12 76.4+ 2.02 100 + 10 132.1+ 1.23 (31.2) 30.2+ 1.23 35.2+ 1.33 100 + 10 98.4+ 1.08 (48.8) 10.1+ 1.28 11.2+ 0.43 100 + 10 57.0+ 1.67 (70.3) 2.3+ 1.26 0.0 100 + 10 40.0+ 1.27 (79.2) 0.0 0.0 100 + 10 216.0+ 2.12 (0.00) 85.0+ 2.56 52.8+ 2.07 100 + 10 157.3+ 1.15 (27.2) 40.0+ 1.23 20.4+ 1.28 100 + 10 147.4+ 1.30 (31.7) 17.5+ 1.24 15.2+ 0.60 100 + 10 107.6+ 1.50 (50.2) 12.8+ 0.33 12.0+ 0.03 100 + 10 67.2+ 0.85 (68.9) 8.0 6.6+ 0.01 100 + 10 210.5+ 1.15 (0.00) 83.5+ 1.86 80.6+ 2.08

0.5 100 + 10 166.0+ 2.42 (21.1) 47.8+ 1.26 1.0 100 + 10 136.0+ 1.45 (35.4) 23.9+ 1.27 1.5 100 + 10 76.1+ 0.93 (63.8) 13.5+ 0.91 2.0 100 + 10 70.8+ 1.25 (66.3) 8.0+ 0.98 N 22 Sel. 0.0 100 + 10 237.3+ 3.52 (0.00) 78.6+ 1.24 0.5 100 + 10 203.6+ 3.32 (14.2) 48.0+ 2.13 1.0 100 + 10 133.4+ 2.46 (43.8) 32.0+ 1.09 1.5 100 + 10 128.8+ 1.36 (45.7) 28.0+ 1.23 2.0 100 + 10 102.0+ 1.02 (57.0) 12.0 +0.98 Annada 0.0 100 + 10 148.0+ 2.51 (0.00) 45.2+ 2.02 0.5 100 + 10 92.4+ 1.32 (37.5) 15.0+ 0.83 1.0 100 + 10 48.8+ 1.30 (67.0) 3.0+ 0.52 1.5 100 + 10 32.6+ 1.29 (77.9) 0.0 2.0 100 + 10 23.3+ 0.89 (84.2) 0.0 Vandana 0.0 100 + 10 167.3+ 2.23 (0.00) 55.8+ 1.93 0.5 100 + 10 128.8+ 2.53 (23.0) 23.0+ 1.41 1.0 100 + 10 87.3+ 1.63 (47.8) 10.2+ 0.43 1.5 100 + 10 69.8+ 1.26 (58.3) 2.0+ 0.04 2.0 100 + 10 55.5+ 0.99 (66.8) 0.5+ 0.01 Figures within the parenthesis denotes % reduction over control, *Values are mean + S.E. and the lowest in Pant Sugandh Dhan 17 under drought stress induced by PEG.

42.3+ 1.40 18.1+ 0.98 8.3+ 0.50 3.0+ 0.07 61.8+ 2.36 35.0+ 1.61 27.8+ 1.03 20.0+ 1.34 10.0+ 1.25 23.1+ 1.03 3.0+ 0.03 0.0 0.0 0.0 33.2+ 1.82 26.0+ 1.43 18.4+ 1.00 10.6+ 0.41 5.5+ 0.09

resulted in satisfactory plant regeneration to the tune of 75% (organogenic) and 70% (somatic embryogenic) which is about only 10% reduction as compared to control (83.8+ 0.27 and 80.2+ 0.05%). This is indicative of the fact that these cell lines are adapted to the sub-optimal drought stress and the regenerants derived from such calli could harbor high level of drought tolerance. In the present pursuit, a number of putative drought tolerant plants were obtained in Sahbhagidhan, N 22 selection and Mandakini and forwarded for field evaluation following a sequence of hardening process.

Wani et al37 reported reduction in callus induction ability and plant regeneration efficiency with increased levels of PEG stress. They reported that PEG can be used as water stress inducing agent under in vitro conditions and rice variety PR 116 was relatively tolerant to drought stress as compared to PAU 201. Biswas et al4 verified the status of drought tolerance under in vitro culture in a set of known salt tolerant genotypes e.g. SR 26B, Nonabokra and C14-8. Considering all in vitro parameters, C 14-8 was found to be most appropriate in developing drought tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra.

Conclusion The following are the concluding remarks from the present pursuit:

In the present pursuit, some of the cell lines derived from tolerant cultivars e.g. Sahbhagidhan and N 22- selection

1. In vitro stress imposed by PEG/mannitol/sorbitol led to progressive decrease in callus proliferation and

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Vol. 10 (6) June (2015) Res. J. Biotech and induced mutants from haploid cultures, Theor. Appl. Genet., 61, 225-232 (1982)

regeneration frequency. However, the effect of drought stress was more prevalent in PEG than mannitol and sorbitol.

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2. Selection medium supplemented with 1.5-2.0M PEG could be used for in vitro screening of callus cultures and plantlets.

11. Gulati A. and Jaiwal P. K., In vitro selection and characterization of Vigna radiata cell line resistant to PEGinduced drought stress, Acta Physiol Plant, 16(1), 53-60 (1994)

3. N22 Selection and Sahbhagidhan expressed high level of drought tolerance while Khandagiri and Annada were sensitive to PEG based on callusing ability and plant regeneration.

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4. In vitro screening of somaclonal variants using different selection agents could produce genetically new variants with desired characteristics.

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5. The in vitro screened drought tolerant plantlets recovered from Sahbhagidhan, N 22 selection and Mandakini callus cultures could have higher level of stress tolerance than the source cultivars.

14. Kavi Kishor P. B. K. and Reddy G. M., Resistance of rice callus tissues to sodium chloride and polyethylene glycol, Curr Sci, 54, 1129–1131 (1985)

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(Received 13th October 2014, accepted 10th December 2014)

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