Initiation of microcalli in culture of pea (Pisum sativum L.) isolated microspores

Abstract

Innovative biotechnologies based on use of double haploids enables developing new varieties considerably faster compared to conventional plant breeding approaches. In pea, reliable methods of haploid plants production are not fully elaborated yet. The current research aimed at testing different conditions (genotype, medium, and stress treatment) for initiation of sporophytic developmental shift in culture of pea isolated microspores. Reprogramming pea microspores towards a sporophytic development was stimulated with temperature stress. Cold (+4°С) and heat (+35°С) stress treatments were applied to pea isolated flower buds and microspores, respectively. Microspores were isolated from plants of 6 pea genotypes, treated at 18 temperature regimes and cultivated in 8 liquid nutrient media with various chemical compounds including growth regulators, vitamins, sugars, glutamine, casein hydrolysate, and osmotic agents. Microcalli were produced from isolated microspores of pea breeding line 109b and variety Stabil in conditions of nutrient media KM-ар1 and MSB-M3 after cold (+4°С) stress treatment for 16 and 10 days, respectively. The media KM-ар1 and MSB-M3 contained a relatively low concentration of sugar (10 and 6 g L-1, respectively), and were supplemented with polyethylene glycol 6000 or mannitol as osmotic agents.