An optimized protocol for high frequency regeneration of selected groundnut (Arachis hypogaea L) varieties from East Africa using cotyledons

Abstract

An efficient protocol for regenerating high yielding groundnut varieties; ICGV-CG2 and CG2 through direct organogenesis has been optimized using 6-benzylaminopurine and 2,4- dichlorophenoxyacetic acid. Murashige and Skoog medium with B5 vitamins was supplemented with individual treatments of BAP and 2,4-D and with their combinations to induce shoot bud formation. Preliminary results showed no shoot formation on media with the individual plant growth regulators. The highest and lowest regeneration frequencies in CG2 were 93.33% and 36.67% respectively on medium supplemented with 1 mg/L 2,4-D and 4.5 mg/L BAP. In ICGV-CG2 the highest frequency was 90% on media with 1.5 mg/L 2, 4-D and 4.5mg/L BAP with a low of 46.67%. Three (3) to ten (10) shoots were recorded per explant, with a maximum of ten shoots recovered from the best performing plant growth regulator regimes. Shoots elongated on MS with reduced BAP levels (0.75-4.5mg/L), rooted on media with α-naphthalene acetic acid and later acclimatized in the glasshouse on peat moss. Hardening and growth of plants to maturity was achieved in the glasshouse on a sand-soil mixture (1:1 w/w) in pots. In vitro induction of flowers in one of the explants could have been as a result of somaclonal variation. This work has developed an optimized protocol that sets up a platform for improvement of these varieties that are adapted to East Africa with novel genes through genetic transformation Keywords: Direct organogenesis; genotype dependent; optimized protocol; somaclonal variation; vaporphase sterilization. Abbreviations: 2,4-D; 2, 4-dichlorophenoxyacetic acid, BAP; 6-benzylaminopurine, MS; Murashige and Skoog, NAA; α-naphthalene acetic acid, PGR; Plant growth regulator, SEM; Shoot elongation medium, SIM; Shoot induction medium, RIM; Root induction medium.