Development and characterization of salt tolerant somaclones in rice cultivar pokkali

Transcription

1 Indian Journal of Experimental Biology Vol. 38, January 2000, pp Development and characterization of salt tolerant somaclones in rice cultivar pokkali Asit B Mandai, Bikash Chowdhury & T E Sheeja Biotechnology Laboratory, Central Agricultural Research Institute, Port Blair I, India Received 13 November 1998; revised 24 August 1999 Primary regenerants (1190) of a tall traditional salt tolerant rice cultivar pokkali were produced through in vilro culture from mature seed derived calli of fourth subculture. Out of SC 2 regenerants, 26 promising lines with superior agronomic traits were chosen initially for evaluation. SC) and SC 4 generations were stringently evaluated under hydroponics with excess salt stress as well as under field conditions across two growing seasons in Bay Islands. A set of 10 promising somaclones was further evaluated at SCs and SC 6 of which BTS 2, BTS 13, BTS 18 and BTS 24 were found promising. In SC 7 and SCx yield trials in research farm, BTS 24 was found to produce a mean yield of 36.3 and 45.9 q ha- I undcr salinc and normal soil conditions, respectively. Somaclones varied significantly from the parent with respect to yield and yicld attributes. Grain quality and biochemical parameters of all elite somaclones were different from the parent. However. somaclones did not deviate much from their parent in respect of disease and insect pest resistance pattern. Somaclonal variations (SV) are usually generated in a tissue culture system while culturing explants in vitro on synthetic media with varied levels of diverse phytohormones l. Somaclonal variants general1y possess one or a few altered trait/s in the parental background. This variation was found to be wide spread 2 ; heritable in many occasions in diverse crops and ostensibly used in crop improvement, as the problem of havoc genetic rearrangements encountered in recombination breeding gets minimized). SV is well evident in rice, has been successful1y exploited to isolate plants with improved agrobotanical traits4-ll. The present study was thematically aimed at exploiting SV to evolve promising short statured lines in pokkali en route somaculture without much altering yield potential, disease and pest resistance traits and salt tolerance character. Materials and Methods Rice (Oryza sativa L.) cultivar pokkali, a tall traditional salt tolerant photosensitive cultivar obtained from INGER, IRRI (International Rice Research Institute), Philippines was employed for this study. Healthy, mature seeds were washed in aqueous teepol solution (5%) for 5 min, surface sterilised in freshly prepared HgCI2 solution (0.01 %) with 2-3 drops of tween 20 as wetting agent in vacuo followed by four times wash with sterile double distil1ed water. Axenic seeds were blot dried under laminar air flow and placed onto the surface of cal1us induction medium (eim) constituting of MS salts l2, sucrose (3%) and agar (0.8%) (Sigma Chemical Company, US). Different levels of various phytohormones viz. 2,4-D (1-3 mg rl) and kinetin ( mg rl) singly or in combination were employed to obtain efficient callus induction. ph of the medium was adjusted to 5.7 before autoclaving and cultures were incubated at 25 ±2 C in dark. After! 0 days, the primary loose calli that appeared on scuttelar surface were excised and kept onto CIM in the same culture tube for another 18 days. Cal1i were subcultured on freshly prepared medium of the same composition at 28 days interval. Calli from the fourth subculture were transferred onto the regeneration medium (RM) containing MS with sucrose (3%) and agar (0.8%) supplemented with different levels of kinetin (I - 3 mg rl).and NAA ( mg rl) singly or in combination. Cultures were kept at 25 ±2 C under illumination from cool fluorescent tubes (-2500 lux) in 16/8 hr light/dark cycle. Green spots started appearing within days on calli surface and subsequently green plants with roots developed following somatic embryogenesis. The plants of 6-8 cm height were transplanted in plastic pots and covered with polyethylene bags initially for 3-4 days and watered regularly. The established plants after 20 days were shifted to big cement pots in the net house. At maturity, the seeds from individual plants were collected, threshed, cleaned and kept separately. SC 2 somaclones were evaluated under hydroponics in Hoagland solution fortified with excess salt (8 ds m- I ) in the form of NaCI and CaCh: The survivors were field evaluated

2 MANDAL et at. : DEVELOPMENT AND CHARACTERIZATION OF SALT TOLERANT RICE CUL TIV AR 75 under rainfed condition in the institute's research farm at Bloomsdale, Port Blair ( ' 13.04"N latitude; '30. 16"E longitude). Out of plants from 1190 somaclone families evaluated during the rainy season of 1993, 26 lines were selected. They were further evaluated during the second cropping season of the same year under intense disease and insect pest pressure at SC 3 During the rainy season of 1994, 26 selected entries were again field evaluated. Based on the performance in respect of major agronomic traits including disease and insect pest tolerance norms, 10 lines were identified as promising. Each year, selected somaclones were grown both under saline (EC dsm- l ; ph during cropping season) and normal soil conditions to assess the tolerance norms to major insect and disease pests prevailing in Bay Islands. Grain quality and salt soluble grain protein analysis l3 were performed to monitor any genetic divergence in the somaclones from parental stock at polypeptide level as well as to determine the consumer acceptability at SC 6. Having considered yield and component characters, grain quality and pest tolerance profile, only four lines viz. BTS 2, BTS 13, BTS 18 and BTS 24 were found to be promising. They were forwarded for yield trials at SC 7 and SCs under rainfed condition in normal and saline soils in research farm and farmer's field across north and south Andamans. Results and Discussion Pokkali somaclones varied significantly with each other and the parent at SC 6 in respect of yield and yield attributing characters (Table I). Days to 50% flowering were reduced significantly in majority of the somaclones. BTS flowered earliest. Plant height reduced in all than the parent Pokkali. BTS 13 and BTS 24, respectively had maximum and minimum height. Among somaclones, BTS I 1-16 and BTS 11-7 had maximum panicles and BTS 13 had the longest panicle. Spikelet sterility of somaclones was found to be reduced in comparison to parent, BTS having the minimum. In general, reduction in days to 50 % flowering (earliness by 6-4 days), plant height, number of tiller per plant, spikelet sterility and increase in number of panicle per plant, panicle length and yield were discernible in most of selected somaclones. A similar high frequency of SV was reported earlier in rice l4. Grain yield of all selected somaclones both under normal and saline soil conditions increased significantly over the parent. BTS 2 had the maximum yield followed by BTS 24 in normal soil. However, BTS 24 produced maximum yield under saline soil (Table I). The significant yield improvement of somaclones over parent both under normal and saline soils might be due to gross increase in length and number of panicle along with reduced spikelet sterility and apparently major alteration in harvest index. The elite performance of BTS 2 and BTS 24 is probably due to inherent improvisation of yield attributing traits or higher accumulation of culture induced variation facilitating more yield. Similar observations were also made in rice l ). Occurrence of short culmed, early heading and high tj '11" enng nce samac I ones as reporte d ear I' ler reaffirms our observation of SV in pokkali rice. Grain quality of all selected somaclones varied widely in respect of alkali spreading value. In many somaclones grains became very soft, broken surfaced and colourless unlike pokkali which remained hard, intact and purple. Except BTS 2, BTS 11-3, BTS 11-7 Table I-Performance of selected pokkali somaclones at SC 6 generation Somaclone Days to Plant height No. of No. of Panicle Spikelet Yield 9 ha 1 Yield 50% (cm) panicle/plant tiller/plant length sterility Normal soil Saline soil reduction flowering (cm) (%) ( 0/0 ) BTS2 86(-2.3) (-30.8) 9.5 (+7.95) 9.5 (-16.7) 25.2 (+14.0) 45.5 (-28.0) 44.9 (+92.7) 34.5 (+70.8) 22.3 BTS (0.0) 96.5 (-40.4) 9.6 (+9.1) 10.6 (-7.0) 24.1 (+9.0) 46.6 (-26.3) 44.7 (+91.8) 37.2(+84.1) 16.8 BTS (-9.1) 99.8 (-38.4) 10.5 (+19.3) 11.4 (0.0) 23.8 (+7.7) (-43.5) 41.0 (+76.0) 33.7 (+66.8) 17.8 BTSI3 83 (-6.8) (-27.0) 7.9 (-10.2) 9.0 (-21.0) 25.4 (+14.9) 55.9 (-11.5) 39.5 (+69.5) 32.8 (+62.4) 17.0 BTS (-6.8) 97.2 (-40.0) 9.9 (+12.5) 11.1 (-2.6) 24.9 (+12.7) 28.7 (-54.6) 38.5 (+65.2) 34.7(+71.8) 09.0 BTS II-II 83 (-6.8) 98.5 (-39.2) 9.1 (+3.4) 10.6 (-7.0) 22.4 (+1.3) 38.7 (-38.8) 37.7 (+61.8) 31.3 (+54.1) 16.9 BTS (-6.8) (-355) 10.5 (+19.3) 10.5 (-7.9) 23.6 (+6.8) 44.4 (-29.7) 42.9 (+84. 1) 27.4 (+35.6) 16.7 BTS (-7.9) 98.9 (-38.9) 7.9 (-10.2) 11.5 (-2.6) 21.4 (-3.2) 46.4 (-26.6) 32.2 (+38.2) 28.6 (+41.6) 11.2 BTS (-6.8) (-36.6) 7.6 (-13.6) 8.6 (-24.6) 21.1 (-4.5) 36.1 (-42.9) 32.0 (+37.3) 28.3 (+40.1) 11.6 BTS (-7.9) (-32.2) 7.4 (-15.9) 7.2 (-36.8) 24.0 (+8.6) 52.1 (-17.6) 31.6 (+35.6) 26.5 (+31.2) 16.1 Pokkali (parent) C.D. (P=0.05) *Values in parentheses indicate per cent increase (+) or decrease (-) over parent pokkali

3 76 INDIAN J EXP BIOL, JANUARY 2000 and BTS 24, in the remaining, viscosity and density of grains increased considerably over pokkali with BTS showing maximum value (Table 2). Alteration in biochemical properties like alkali spreading value, gel consistency and colour of grains is in accordance. h l' 18 Wit our ear ler report. Hardly any improvement was discernible in the tolerance profile of somaclones to major disease and insect pests. Though BTS 2 and BTS 24 were relatively more tolerant to sheath blight (ShB) and bacterial leaf blight (BLB), they were less or equally tolerant to Helminthosporium leaf spot (HLS). BTS 11-16, BTS and BTS 10-9 were found to be relatively more tolerant to HLS than pokkali. BTS 2, BTS 24, BTS 11-16, BTS , BTS II-II, BTS 11-7 and BTS 10-9 were tolerant to stem borer (SB) than their highly susceptible parent. Our observation of low frequency of occurrence of SV for improved disease and pest resistance is corroborative with previous findingsl9. This might be owing to low inherent resistance of parent to disease and insect pests or high insularity of such traits to alteration by SV. It is mentionable that the frequency of variants tolerant to diseases could be increased only if the parent material possess at least some level of inherent tolerance 2o Significant level of tolerance to ShB as Table 2-Grain quality parameter of a few selected pokkali somaclones Somaclone Whole grain (Brown rice) 100 Dehusked rice 100 seed Length (L) Breadth (B) UB grain Length (L) Breadth (B) UB wt. (g) (cm) wt. (g) (cm) BTS2 0.8I±O ±O ±O ±O BTS ± ± o.4o;to ± BTS ± ± I ±O ±O BTS ±O I±O ±O ±O BTS ±O ± ± ± BTS ±O ± ±O ± BTS ±O ±O ±O ±O BTS ±O.0 I ±O ±O.OOI 0.2I±O.OI I I.H BTS ± ± O.50±0.001 O.13±O BTS ±O ±O ±O ±O Pokkali (parent) 0.70± ±O.OO ±O ±O Chemical characteristic" Alkali Gel spreading consistency value h vallie" 3 (C) I (0.4).'i(C) I (0.5) 5 (C) 9 (D.5) 5 (C) 2 (0 H) 5 (C) 1 (0.5) 3 (P) 4 (1.5) 3 (LP) 1(0.4) '!(C) 5 (1.7) 9(C) 6 (2.0) 3 (C) 3 ( 1.0) I (P) I (0.5) By the SES for ricelrtp( 1988), IRRI, Philippines 23 h scale represented as 9- very soft & disintegrated; 5- soft with surface broken; 3- soft and 1- intact hard grain. C-colourless, P- purple, LP- light purple "-scale represented as 1- maximum viscous & maximum density; 9- minimum viscous & minimum density; Figures within parentheses indicate migration values in cm.. Table 3-Tolerance profile of selected pokkaji somacjones to major insect and disease pests in Andamans SomacJone Disease and insect J2ests tolerance (0-9 scale)* Additional character *** ShB BLB HLS SB** LOD PI BTS BTS BTS BTS BTS BTS II-II I BTS BTS BTS 10: BTS Pokkali (parent) *By SES for Rice "trtp (1988), IRRI, Philippines 23 ** ShB- Sheath blight; BLB- Bacterial leaf blight; HLS- Helmillthosporiwll leaf spot; SB- Stem borer *** LOD - Lodging and PI - Phenotypic index

4 MANDAL et al.: DEVELOPMENT AND CHARACTERIZATION OF SALT TOLERANT RICE CULTIY AR 77 shown by BTS 2 and BTS 24 might be culture induced SV. ShB and BLB tolerant rice somaclones were recovered earlier a1s0 4. While assessing lodging tolerance, BTS 2, BTS 24, BTS 13, BTS , BTS 11-11, BTS 10-8 and BTS 18 were found more tolerant than pokkali. This character was remarkably improved in all somac1ones plausibly due to their short plant stature. Phenotypic indexin~ based on over all agronomic performance was much better due to direct selection for elite somaclones under well-defined environment. BTS was rated as phenotypically excellent while BTS 24, BTS 11-16, BTS 13, BTS and BTS 18 as very good (Table 3). Salt soluble grain polypeptide profiling through SOS-PAGE showed that none of the somaclones matched cent per cent with each other or with the parent (Fig. 1). A maximum of 34 bands was observed, of which 16 were found to be polymorphic. Pokkali displayed 6 intense bands, which were common in all somaclones. Somac1ones had common bands of high molecular weight of ko. Ten new bands of different molecular weight appeared and a few disappeared too in selected somaclones. Somaclones also showed wide variation in themselves with respect to presence or absence of bands of different molecular weights. Absence of bands viz. 17 and 23 \n BTS 18 and presence of 15 in BTS 2 were diagnostic to those genotypes. Intra somaclonal affinity through estimation of similarity index (Table 4) revealed maximum similarity between BTS and BTS sharing 29 common barids. On an average, BS showed maximum resemblance with sister somac1ones sharing a maximum of common bands. BTS 13 aifld 24 showed Table 4-Similarity indices and number of shared bands between different somac\ones and their parent pokkaji BTS BTS BTS BTS BTS BTS BTS BTS BTS BTS Pokkal i ll-ii BTS BTS BTS BTSI BTS BTS BTS BTS BTS BTSI Pokkali (parent) *Yalues below diagonal represent number of shared bands and values above diagonal denote similarity indices. KD Fig. 1-SDS-PAGE of salt soluble polypeptides of grain proteins in selected pokkali somac\ones. [Lane (I)-SOS-PAGE marker; (2)-BTS 18; (3)- pokkali ; (4)-BTS 2 ; (5)-BTS 24; (6)-BTS 11-16; (7)-BTS 13; (8)-BTS ; (9)-BTS ; (lo)-bts 11-7; (l\)-bts 10-9; (12)-BTS 10-8; and (l3)-sos-page marker; 1

5 78 INDIAN J EXP BIOL, JANUARY 2000 Table 5 - Mean performance of a few promising somaclones at SC 7 and SC x yield trials Seed yield (q ha l) Farmers field Somaclone SC 7 (saline soil only) Normal soil Saline soil Normal soil Saline soil *South ** North Andaman And aman BTS H Not tested BTS lS BTS BTS Pokkali (parent) C.D.(P=O.05) H * South Andaman - ph: , EC: ds m 1 ** North Andaman (Diglipur) - ph: , EC: ds m 1 during cropping season a very high band homology. Between BTS 10-8 and 10-9, high similarity was observed along with disappearance of diagnostic bands 25 and 32 in BTS Inter-genotype affinity between pokkali and somaclones revealed a very low synteny. Seed protein electrophoretic pattern analysis is a reliable approach to classifying genotypes or in demarcating genome characteristics The present study revealed a clear existence of SV, at salt soluble polypeptide level in SDS-PAGE. Maximum bands of somac1ones and pokkali were common because of their origin. Similarities among the somac1ones may be due to execution of similar selection pressure during their genesis, development and recovery process. Similarity indices among somac1ones were more than parent, plausibly due to direct selection of semi tall selectants with similar plant types from somaclones derived from tall parent pokkali. Only four very promising somaclones viz. BTS 2, BTS 13, BTS 18 and BTS 24 were selected after extensive evaluation for 6 generations. Yield trials conducted with those lines at SC 7 and SCg revealed that BTS 2 and BTS 24 produced maximum yield under both normal and saline soils apart from their tolerances to major disease and insect pests prevailing in Bay Islands. Somac1ones without segregation and consistent performance at advanced generations are assumed to bear gene or chromosome variations, which deems to be stable and heritable 2. However, BTS 2 was found to be susceptible to lodging and nitrogen application, thus discarded. BTS 24 had a maximum mean yield of 37.3 and 46.8 q ha- ' under saline and normal soils, respectively and an average yield of 38.7 q ha- ' under farmer's field in saline soil across north and south Andamans (Table 5). In nutshell, the present study reaffirmed existence of de novo SV in rice, which may profitably be exploited in rice genetic improvement. This offers ample scope in employing ill vitro selection for recovering promising variants, besides prospecting utility of culture induced variation in genetic upgradation. BTS 24, an elite, medium duration, short statured, high yielding and salt tolerant somac1one, with moderate level of field tolerance to yellow stem borer (YSB), may be recommended for cultivation under normal soil in general and under saline soil in particular in Bay Islands. It may be tested for adaptability and yield attributes in multilocation trials across the country along with superior sister selections viz. BTS 13 and BTS 18 to identify location specific genotypes to exploit low lying salt prone econiches. References I Larkin P J & Scowcroft W R. Th('or App/ Gellet. 60( 19X I) 197: 2 Karp A, Euphytica, 85( 1995) Scowcroft W R & Larkin P J, Applicatiolls olp/allt cell tissue and organ culture- Ciba foundation symposium /07 (Willey Eastern Ltd., Chichester, U.K.) Cao J, Rush M C, Nabors M W, Xie Q J, Croughan T P & Nowick E, BNF associated with rice productioll. edited by S K DUlla and Charles Slogger (Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi) 1991, Lutts S, Kinet J M & Bourhamont J. P/allt Phvsio/. I 52( 1(98) Mandai A B,Veenakumari K. Prashanth Mohanraj & Bandyopadhyay A K Rice Biotechllo/. 20 (1994) Mandai A B, Pramanik S C, Illdiall Agrie. 42( 1(98) Oono K, Biology of rice edited by S Tsanoda and N Takashashi (Japan Science Society Press. Tokyo) Rueb S, Leneman M, Schilperoort R A & Hensgens LAM, Plant Cell Tissue Organ Cult. 36 (1994) Yoshida S, Ogawa M, Suenagar K & Ye H C. Induction and selection of salt tolerant mutant lines by tissue culture techniques for cereal crop improvement (Science Press. Beijing, China) 1983,237. II Zakri A H, Rice genetics- Proceedings of the international rice genetics symposium (International Rice Research Institute. Philippines) Murashige T & Skoog F, Physiol Plant. 15 (1962) 473.

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