An Improved Protocol for Regeneration of Sorghum bicolor from Isolated Shoot Apices.

Abstract

An improved and producible protocol for in vitro regeneration of sorghum [Sorghum bicolor (L.) Moench] from shoot meristem explant is reported. By striking an optimal balance between a weak auxin like naphthalene acetic acid (NAA) and the cytokinin thidiazuron (TDZ), the isolated shoot meristems were manipulated to follow either organogenic or embryogenic pathway. There was no intermediate callus formation. Multiple buds were induced on enlarged meristems using MS medium with 5.0μM of TDZ, 17.72μM Benzylaminopurine (BAP), and 1.074μM NAA. To maximize the number of bud initials per explant, three parameters (seed size, germination technique, and age of explant) were studied. Five to 7-day-old shoot meristems responded best with ≥80% induction of bud initials. Seed size was not significant in influencing induction potential of the shoot meristems. Six weeks after in vitro culture, each meristem produced 35-40 shoot buds. Direct somatic embryogenesis (≥80% induction) was accomplished following a two-step culture procedure consisting of induction of multiple buds and formation of somatic embryos. A high frequency (700-1000) of somatic embryos per explant was obtained on MS medium with 17.72μM BAP and 2.69 μM NAA. Shoots from both organogenic and embryogenic pathways rooted on half-strength Murashige and Skoog (MS) medium with 8.28μM Indole 3-butyric acid (IBA) and 1.14μM Indole acetic acid (IAA). After one month on rooting medium, plants with well-developed roots were transferred to jiffy cups. Such plants were subsequently acclimatized in the glasshouse, and were grown till maturity; they showed normal seed set. Random amplified polymorphic DNA (RAPD) analysis of regenerants did not detect any DNA polymorphism.