Development of in vitro multiplication and acclimatization protocols for melia volkensii.

Abstract

Melia volkensii is a medicinal and ornamental tree plant which contributes to the farmer’s income generation in dryland areas of Eastern Africa. It is normally propagated using seed which has been reported to be hard and expensive during seed extraction and seeds germination. The purpose of this study was to develop protocols for micropropagation, rooting and acclimatization of M. volkensii. Different concentrations of 6-benzylaminopurine (BAP) and derived cytokinins supplemented in Murashige and Skoog’s (MS) were evaluated for shoot regeneration from nodal and shoot tip sections. Thidiazuron (TDZ) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) were used to induce callus from leaf disc while 6-benzylaminopurine, α-Naphthalene acetic acid (NAA) and Gibberellic acid (GA3) at different concentrations were used during shoot regeneration from callus. Thidiazuron and 6-benzylaminopurine were used to proliferate shoots from cotyledon and shoots were rooted using Indole-3-butric acid (IBA) supplemented in MS or McCown woody plant medium. Four concentrations (0 μM, 5 μM, 10 μM and 15 μM) of abscisic acid (ABA) and pyrabactin (PYR) added in sterile soil substrate used during M. volkensii acclimatization. Cultures in growth chamber and greenhouse were arranged in completely randomized design replicated four times. Analysis variance showed that shoot formation from nodal and shoot tip sections significantly (p<0.05) increased by supplementing BAP, meta-topolin (mT), meta-topolin riboside (mTR) and meta-Methoxy topolin (memTR) in MS medium. Up to 90 to 100% of shoot formation was achieved in MS medium supplemented with BAP (1 μM or 2 μM). No differences was observed in shoot formation frequency among 1 μM BAP, 2 μM BAP, 5 μM mTR, 5 μM memTR and 5 μM mT supplemented in MS culture medium. The highest mean number of shoots per explant (4.17 shoots), highest mean shoot length (2.64 cm) and number of leaves per shoots (8.08 leaves) recorded in shoot tips explant inoculated in MS media supplemented with 2 μM BAP. The combinations of BAP and NAA supplemented in MS medium significantly (p<0.05) enhanced in vitro microshoots growth. Supplementation of 2, 4-D and TDZ in MS medium had increased callus induction frequency and fresh weight of callus from leaf disc. The maximum shoot formation frequency (100%) from callus was observed in MS medium + 2.2 μM BAP + 1 μM NAA + 0.9 μM GA3. The longest shoot (1.45 cm) and maximum number of shoots (5.17) was recorded in MS + 2.2 μM BAP. The results showed that 50% of cotyledon inoculated in MS medium supplemented with 2 μM BAP induced highest number of shoots per cotyledon (11.5) than TDZ concentrations. McCown woody plant medium supplemented with IBA significantly (p<0.05) increased rooting percentages of M. Volkensii shoots from in vitro than MS medium and control. The highest root formation (78.75%) were recorded in McCown woody plant medium supplemented with 1 μM IBA. McCown woody plant medium increased shoot rooting more than 50% compared to MS medium. Pyrabactin added in sterile soil showed a significant (p<0.05) effect on survival rates, number of leaves, plant height and chlorophyll content. Current study has demonstrated that the use of 2 μM BAP is recommended for M. volkensii multiple shoot proliferation while 5 μM mTR should be adopted in M. volkensii micropropagation. The simple M. volkensii protocols developed in current study could be exploited for commercial M. volkensii micropropagation and germplasm conservation.