Gametic embryogenesis and callogenesis in Isolated microspore culture of Jatropha curcas L. a recalcitrant bioenergy crop

Jatropha curcas is an undomesticated crop and its plantations did not meet commercial expectation due to absence of high yielding commercial line with desired agronomic characters. Earlier, breeding efforts did not pay much attention on yield and disease improving traits due to lack of desired genetic variability. Isolated microspore culture is one of the most widely used in vitro techniques to induce gametic embryogenesis with wide degree of genetic variability for the development of haploids and doubled haploids. In this study, an efficient isolated microspore culture system was established for different genotypes with the optimization of various factors that affect microspore embryogenesis. After 15–20 days of culture at 25 °C, tetrads, mid, early un-vacuolated and vacuolated late stage uninucleate microspores, which were preincubated at 4 °C for 7 days under shaking conditions, induced the formation of embryo like structures (ELSs) in a modified MS medium with 2.0 mg/l, 2,4-d, 0.1 mg/l kinetin, 300 mg/l casein hydrolysate, 1 g/l glutamine, 0.5 mg/l folic acid, 0.05 mg/l biotin and 5% sucrose. It is observed that the shock at 4 °C for 7 days, subsequently incubation of microspore cultures at 25 °C for 15 days and then at 15 °C for 10 days played a significant role in induction and accelerated creation of ELSs in three different genotypes. Microscopic analyses confirmed the different developmental stages of microspore embryogenesis including cell division and multicellular embryo like structures. Additionally, flow cytometric analyses verified the calli and ELSs were haploids in nature and strengthened that the origin was from microspores. This study supports the evidence of gametic embryogenesis in Jatropha microspore culture.