Improvement of Androgenesis and Somatic Embryogenesis Using Physical and Chemical Pretreatment Factors in Barley (Hordeum Vulgare L.)

Abstract

In vitro androgenesis and somatic embryogenesis are an essential tools for advance biotechnological research for barley and other crops improvement. To evaluate the growth and yield contributing characters six BARI barley and eight European genotypes were considered for this study. The major goals of this study was to screen a suitable genotype; optimization of media, plant growth regulators (PGRs), to evaluate the effect of salt and heat pretreatment factors on regeneration; various concentrations of copper sulphate and cobalt chloride added in medium to improve somatic embryogenesis. Various carbon sources were also evaluated with MS and other medium and standardized silver nitrate and amino acids concentration. Callus age and sizes were considered for their effects on plant regeneration. Different physical pretreatment factors such as cold and drought stresses also applied directly to the targeted explants (anthers, spikes) to improve anther culture responses of barley in Bangladesh. To assess the performance of the genotypes, variability and influence of sowing times on yield and yield contributing characters, six local and eight European barley genotypes were considered. Among the studied genotypes BB-5 showed earliest booting (59 days) and the maturity was found after 101 days from the date of sowing. Most yield contributing traits were found in Hor-9465, BB-5, Hor-291, Hor-9580 and BB-6 promising with good yield compared to other studied genotypes. Six barley genotypes were tested and among them four (BB-6, BB-3, BB-1 and BB-2) showed significant results on callus induced and regeneration. Twelve different combinations of MS medium (CIM1 – CIM12) used for primary callus induction. It was observed that CIM8 (4.0 mg/l 2,4-D, 200 mg/l L-proline and 300 mg/l casein) showed better performance on callus induction (38.17%) in BB-6. Nine different concentrations of plant growth regulators were used in MS medium and among them RM7 (MS + 1.5 mg/l BAP + 30 g/l sucrose) showed better performance on plant regeneration in BB-6 (9.26%). Another attempt was taken about heat pretreatment factor along with various concentration of NaCl. For this experiment, mature embryos were used that derived from three barley genotypes (BARI barley-3, BARI barley-6 and BHL-18). In this case BB-6 showed highest viability on callus (14.72%) induction and regeneration (7.69%) with high concentrations of NaCl (6.5 g/l). With high amount of NaCl (6.5 g/l) BB-6 exhibited maximum relative growth rate (0.91) and tolerance index (0.42) among the three studied genotypes. The result revealed that calli of BHL-18 performed highest desiccation (59.70%) when it was incubated at 40℃ and BB-6 gave the best regeneration (41.66%) when the calli incubated at 35℃ temperature. To evaluate the effect of copper sulphate and cobalt chloride in medium, mature embryos of BARI barley-3 and BARI barley-6 were considered. The concentration of both chemicals at 2.5 to 7.5 mg/l was suitable for callus induction. In the case of plant regeneration T10 showed maximum plant regeneration (53.25%) in BARI barley-6 that was around 3 fold higher in comparison with control. For another experiment seeds of BARI barley-6 were pretreated with different concentrations of 2,4-D and various durations prior to culture. The highest frequency of callus induction (71.38%) was recorded with 3.5 mg/l 2,4-D that pretreated up to 4 days. To optimize a suitable carbon sources for callus induction various amount of sucrose, maltose and D-sorbitol were used either single or in combination with three media. The maximum percentage of primary callus (89.16%) was recorded in MS medium that supplemented with 60 g/l D-sorbitol (T6). For another experiment various concentrations of PGRs were evaluated and the maximum embryogenic calli (70.0%) obtained when 2,4-D (2.0 mg/l) + BAP (0.5 mg/l) were used. Highest plant regeneration (47.40%) was recorded when the MS medium was supplemented with 0.5 mg/l NAA + 1.0 mg/l BAP. For rooting GM showed better performance in addition with 1.0 mg/l IAA. In Exp. 6 (section 4.5) an efficient protocol has been established for callus induction and regeneration. In this case AgNO3 and two different types of amino acids were used in addition to the medium. As explants immature embryos were used that derived from three genotypes viz. BB‐1, BB‐3 and BB‐6. Here five doses of AgNO3 either single or in combination were used along with two amino acids (L-proline, L‐glutamine). The maximum callus were recorded for BB‐6 (49.20%) and BB‐3 (32.66%) when 2.0 mg/l AgNO3 and 200 mg/l L‐glutamine were added. Plant regeneration (37.20%) remarkably increased on MS medium that supplemented with 1.0 mg/l BAP + 1.5 mg/l AgNO3 + 150 mg/l L‐glutamine in BB‐6. Other attempts (Exp. 7; section. 4.6) have been done using callus size, age and their fresh weight to improve regeneration using mature embryos that derived from immature embryos (milky phases of seeds) in BB-6. It was observed that callusing and plant regeneration was better when 1.6-2.0 mm size of embryos, 4-6 weeks old calli and 151-200 mg weight were used. To identify a suitable androgenetic genotype two BARI barley cultivars (BB-3 and BB-6) were used for anther culture. Under this study cold pretreatment for 8-12 days showed better performance on embryos formation in FHG medium. The highest level of embryoids (14.6%) was found at 10 days cold pretreatment and finally produced 13.8% plantlets and 10.72% green plants in BB-6. MSR medium showed better results on green plant regeneration. Another attempt was done for drought stress pretreatment factors that applied to the excised anthers of BB-3 and BB-6 (Expt. 9; section 5.2). It was observed that drought stress for 150 min (T5) showed highest percentage of embryoids (26.50%) and green plants (13.58%) in BB-6. For all cases the number of androgenic embryos and regenerated green plants increased in liquid induction (FHG) medium. From these experimental findings it might be concluded that the cold and drought stress pretreatment factors enhanced anther culture responses. The protocol established under this study might be helpful for future advance level of biotechnological research for barley and other cereal crops in Bangladesh.