High Efficiency Peanut Regeneration Using a Nonimbibed Immature Leaflet Culture Method

Abstract

Efficient plant regeneration is an essential part of gene transfer systems using recombinant DNA technol ogy. Efficiency of regeneration from immature leaflets of peanut (Arachis hypogaea L.) was compared among several explant treatments in an effort to maximize recovery of plants from culture. In one experiment, explants were derived from leaflets of cv. NC 7 from dry mature seeds or from mature seeds which had been imbibed for 1 or 4 d. To avoid confounding treatment effects with variation among individual seeds, both nonimbibed and imbibed leaflets originated from a single seed. For each seed, four nonimbibed leaflets from a single leaf were excised, sterilized, rinsed, and plated on MS-based medium amended with 4 mg Lr 1 benzylaminopurine and 2 mg L' 1 naphthaleneacetic acid. The embryonic axis with the other leaf (four remaining leaflets) and one cotyledon attached was then imbibed in water for 1 or 4 d. After 4 wk in culture, 53% of nonimbibed leaflets, 37% of leaflets imbibed for 1 d, and 6% of leaflets imbibed for 4 d produced shoots. Subsequendy, regeneration efficiency was compared among immature leaflet cultures from nonimbibed seeds of four cultivars representing three botanical varieties from two subspecies. Shoot frequency after 4 wk in culture averaged 9% for Peruvian introduction A 2 (NC Ac 17090) representing subsp.fastigiata var. peruviana versus 53% for cv. NC 7 and Bolivian introduction B 2 (PI 274191), both representing subsp. hypogaea var. hypogaea. At 6 wk after plating, these frequencies increased to 28 and 61%, respectively. The response of Argentine introduc tion C 2 (PI 262000) representing subsp. fastigiata var. •Corresponding author.greater than in the other varieties, whereas it was signifi cantly lower in var. peruviana. Regenerated plants developed normal flowers and pods in the greenhouse. The study indicated that A. hypogaea can be cultured efficiently from nonimbibed leaflets.

Progress in the Development of Tissue Culture and Transformation Methods Applicable to the Production of Transgenic Peanut

“…This limitation was particularly obvious with mesophyll cell (41) and mesophyll protoplast (67) cultures of A. hypogaea, where the only regenerable protoplast cultures reported thus far have been derived from immature cotyledons (51). Juvenile leaves, on the other hand, are much more regeneration-competent in vitro; however, the competent cells are localized primarily to the region surrounding the central vein and not throughout the leaf lamina (1,14,82).…”