Institute of Biological Sciences (IBSc)

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    Improvement of Abiotic Stress Tolerant Rice (Oryza Sativa L.) Through Biotechnological Techniques
    (University of Rajshahi, 2016) Siddique, Md. Abu Baker; Islam, S. M. Shahinul
    The present study was conducted to improve rice (Oryza sativa L.) cultivars considering the features related to tolerance in abiotic stresses, through biotechnological approaches. Seventeen Bangladeshi indica rice varieties were tested to evaluate the efficiency on somatic embryogenesis (SE), organogenesis, relative growth rate (RGR), tolerance index (TI), relative water content (RWC), test of viability of calli in salt stress, effect of partial air desiccation to enhance regeneration. Under this study regeneration has been enhanced by applying desiccation as stress pre-treatment factors. To develop transgenic plants tolerance to major abiotic stress (salt) Agro bacterium-mediated genetic transformation has been conducted. For SE, callus induction (CI) and its subsequent regeneration were done to screen a suitable rice cultivar. For this purpose, effects of fifteen different concentration and combination (T1-T15) of plant growth regulators (PGRs) singly and/ or combined were tested for CI. Out of seventeen varieties, BRRI dhan29 performed the highest number of CI (78.67%) in T5 (MS + 2.5 mg/l 2,4-D) and the lowest was for both BR11 and BRRI dhan27 (33.33%) in T1 (MS + 0.5 mg/l 2,4-D). Effects of four basal media i.e. MS, N6, LS and SK1 were examined to CI where maximum callusing was recorded for BRRI dhan29 (77.33%) in MS; while the lowest was in SK1 for BR23 (36.00%). Fifteen types of PGRs (H1-H15) individually and/ or combined were examined for regeneration; the maximum performance was found for BRRI dhan29 (70.67%) when 3 weeks old calli were transferred to MS that supplemented with 2.0 mg/l BAP + 1.0 mg/l Kin + 1.0 NAA (H11); and BRRI dhan32 (22.67%) gave the lowest plant regeneration in H15 (MS + 4.0 mg/l BAP + 0.5 mg/l Kin + 0.5 mg/l NAA). Considering 2.0 mg/l BAP + 1.0 mg/l Kin + 1.0 NAA (H11) as constant, highest plant regeneration was recorded for BRRI dhan29 (69.33%) in MS, out of four basal media (MS, N6, LS and SK1) tested; and lowest in LS for BR10 (24.00%). To observe the viability of calli in salt stress, four concentrations of NaCl (50, 100, 150 and 200 mM) were tested. The varieties BRRI dhan47, BR10 and BRRI dhan32 gave 53.33, 14.67 and 2.67% viable calli after one week cultured in 200 mM NaCl levels respectively. When the calli of BRRI dhan32 were cultured for 4 weeks no calli were viable; while in case of BRRI dhan47 45.33% calli were alive at 200 mM salt level. Three weeks old calli were cultured in 50, 100, 150 and 200 mM NaCl levels also for 3 weeks; and RGR (relative growth rate), TI (tolerance index) and RWC (relative water content) were determined. The recorded RGRs were 1.03, 0.23 and 0.11 at 200 mM for BRRI dhan47, BR10 and BRRI dhan32; while in control condition (without salt) RGRs were 5.12, 4.02 and 4.61 respectively. BRRI dhan47 carried the highest ability to grow in salt stress showing TI of 0.20 in 200 mM salt level. Comparatively lower TI values were recorded for BR10 (0.06) and BRRI dhan32 (0.02). The WRC were BRRI dhan47 (10.23%), BR10 (7.22%) and BRRI dhan32 (7.03%) in 200 mM salt. The growth pattern of BRRI dhan47 was determined in 0, 50, 150 and 200 mM salt stress. In this case RGR was increased up to two weeks at all NaCl levels. While after 2 weeks RGR was restricted at 100, 150 and 200 mM stress levels. In 50 mM, RGRs were gradually increased up to 4 weeks. Applying air desiccation pretreatment (15, 30, 45 and 60 hrs) to the calli of four age groups (3, 4, 5 and 6 w), around 2 folds enhanced regeneration was gained for BRRI dhan32 when the calli age of 4 weeks was desiccated by 45 hrs. Desiccated calli at optimal level (45 hrs) gave enhanced plant regeneration in salt stress. In this case, BRRI dhan47 gave 1.98 folds higher regeneration (26.98%) than undedicated (control) calli (14.29%) at 200 mM NaCl. In the same stress level (200 mM), BR10 and BRRI dhan32 could not be regenerated from undedicated calli; whereas, after desiccation pretreatment they were been capable to regenerate plants with frequency of 11.11% and 4.76%, respectively. The experiments on organogenesis were done for investigating regeneration efficiency of nine explants viz. mature seeds (zygotic embryos, ZE), radicle, adventitious root (AT), adventitious root tips (ART), mesocotyl nodal segments (MNOS), mesocotyl internodal segments (MINS), coleoptiles, primary leaf (PL) and secondary leaf (SL) of BRRI dhan29. The highest callusing was found in ZE (70.63%) and lowest in coleoptile (12.50%) in MS + 2.5 mg/l 2, 4-D + 1.0 mg/l kin + 300 mg/l L-proline + 400 mg/l. It was observed that maximum primary calli of ZE couldn’t be proliferated for producing secondary callus, while almost all the primary calli of MNOS and MINS were proliferated and produced secondary callus. The calli of four age groups (3, 4, 5 and 6 weeks) induced from different explants were separately transferred to regeneration medium (MS + 2.0 mg/l BAP + 0.5 mg/l NAA + 1.0 mg/l Kin) for plant regeneration. Among nine explants, 6 weeks old calli of MNOS gave the highest regeneration (90.63%), and lowest in AR (32.50%) for 3 weeks old calli. ZE gave maximum 55.00% regeneration by 4 weeks old calli which was lower than MNOS (90.63%), MINS (89.06%), PL (75.00%), SL (72.92%) and coleoptile (71.88%). Agrobacterium-mediated transformation was conducted using BRRI dhan29 by targeted gene of PDH47 (pea DNA helicase 47). The co-cultured calli derived from ZE and MNOS produced 23.33 and 18.33% putatively transgenic plants respectively after infection of Agro bacterium tumefaciens strain of LBA4404. Out of eleven putatively transgenic plants, four showed PCR positive which obtained from ZE derived calli, analyzed through genomic DNA isolation. Further studied have been done and all four showed positive results that confirmed gene integration by southern hybridization analysis. It was observed that the potentiality of explants to regenerate transgenic plants were varied 6.67% for ZE and 3.33% for MNOS. In the experiment of leaf disk senescence (LDS), the transgenics were able to hold chlorophyll contents in their mesophyll tissues and remaining healthy with green in color.