CULTURE OF VERY YOUNG PHASEOLUS VULGARIS L. PODS AND PLANTLET REGENERATION

Abstract

Introduction First results on the development of an in vitro pod culture technique for young pods of P. vulgaris (PV) are reported by Geerts et al (2000). Different culture techniques for 2-dlay old Phaseolus pods were described using a modified Phillips ^i al (1982) medium for maturation. The authors showed that the application of high and variable osmolality conditions, similar to those observed in vivo, during pod culture and before extracting the embryos, gave the best results in terms of ovule and embryo development (Geerts et a/. 1999). However, the plantlet survival remained very low and no regeneration was described. In this paper, we report the method used to obtain the first regeneration of plantlets from 2 days-old Phaseolus embryos using an in vitro pod culture technique. Material and methods A P. vulgaris genotype (NI 637) was grown in a growth chamber under the following controlled conditions: day/night temperature of 24/20°C, light intensity of 580 |imol,m*^s^ and a day length of 11 h 30 min. Pod culture technique was adapted fi-om Geerts et al (2000). Basal medium contains Phillips et al. (1982) mineral salts, 80 g.F^ sucrose, 1 mg.F^ thiamin HCl, 5 mg.F^ nicotinic acid, 0.5 mg.r' pyridoxme, lOOmg.r' myoinositol, 1,000 mg.I'^ L-glutamine, 1,000 mg.F^ casein hydrolysate, 0.1 jLiM NAA, 10 \xM adenine and 1.0 |iM Benzylaminopurine. Seven pod culture methods were compared using three different osmoticum agents (Table I): four solid media (SI, S2, S3 and S4) and three liquid media (LI, L2 and L3). For each of the solid cultures, a sequence of three media with a decreasing osmotic pressure were used. Pods were cultured on 580 mosm of osmolality during I day, transferred to 450 mosm for 2 days more, and lastly transferred for 4 days more to basal medium (350 mosm). For S4 technique, pods were directly transferred to basal medium for 1 week. Table L Type and quantity of osmoticum added to the basal medium. Techniques Osmoticum added Osmolalities of media 580 mosm ^*^ 450 mosm 350 mosm SV''' LI Sucrose 63 g.r^ 22 g.r' S2 L2 Mannitol 36 g.r' 16 g.r ^ S3 L3 Polyethylene glycol 8000 128 g.r' 55 g.r' _ S4 ^*^ at this osmolality, vitamins, amino acids and growth regulators of basal medium are modified : 0.25 mg.f' thiamin HGl, 1.25 mg.T^ nicotinic acid, 0.125 mg.r' pyridoxine, 25 mg.r' myoinositol, 250 mg.r' Lglutamine, 250 mg.r' casein hydrolysate, 0.095 ^M abscisic acid, 5.5 |iM Tryptophan and 0.1 ^M NAA. ^**^ 7 g.r'Select Agar-Gihco BRL.