High frequency plant regeneration from coleoptile tissue of barley (Hordeum vulgare L.)

Abstract

In vitro protocol for efficient plant regeneration has been developed from coleoptile segments of barley ( Hordeum vulgare L. cvs. Ratna and Bw-8). Embryogenic nodular callus was initiated from 4- and 6-day-old coleoptile segments on MSB medium (a medium consisting of organic macro and microelements of MS medium, vitamins and amino acids of B5 medium) supplemented with 2,4- d (9.1–18.2 μM) or NAA (10.7–21.4 μM) in combination with BA (2.2 μM) and sucrose (3%, w/v). The highest frequency (86.6%) of embryogenic callus induction was obtained from 4-day-old coleoptile segments of cv. Ratna on MSB medium supplemented with 11.3 μM 2,4- d . Highly organized callus induction and development of somatic embryos was achieved after transfer of callus clumps to MSB medium containing 2.2 μM BA, 3.8 μM ABA, 1.7 mM l -glutamine and 2.3 μM 2,4- d or 2.7 μM NAA alone or in combination with l -tryptophan (0.12–0.98 mM). Addition of l -tryptophan into medium enhanced significantly ( P <0.05–0.001) average number of somatic embryos, the maximum number (19.8±0.57) being obtained from 4-day-old coleoptile segments of cv. Ratna on MSB medium containing 0.49 mM l -tryptophan. Histological sections of embryogenic callus pieces revealed the presence of well-developed globular somatic embryos. Plantlet regeneration was achieved after transfer of embryogenic callus clumps to MSB medium supplemented with 2,4- d (0.09 μM) or NAA (0.11 μM) alone or in combination with BA (2.2–17.6 μM). MSB medium containing BA enhanced significantly ( P <0.05–0.001) both frequency of plantlet regeneration and mean number of plantlets. The highest frequency of plant regeneration (85.7%) and maximum average number of plantlets (15.8±0.61) were obtained from 4-day-old coleoptile segments of cv. Ratna on MSB medium containing 0.11 μM NAA and 8.8 μM BA. In comparison, response was significantly low ( P <0.05–0.001) from 6-day-old coleoptile segments of both cvs. Ratna and BW-8. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds.