A Simple Novel Expedited Spike Culture-derived Variation Creation Strategy in Wheat

A wheat (Triticum aestivum L.) immature spike culture system was used to expeditiously generate mutations for use in wheat improvement programs. Wheat immature spikes in culture were treated with three concentrations of ethylmethane sulphonate (EMS) to generate a spike culture derived variant (SCDV) population. EMS in a concentration dependent manner affected seed development in wheat immature spike cultures. Based on the number of seeds produced, inclusion of EMS (25 mM) for three hours in immature spike culture medium generated variants in wheat cv. AC Nanda. The wheat AC Nanda SCDV population showed variation in several phenotypic characters. Flag leaf (length, angle and sheath length), length of first and second internode, spike length, number of spikes, number of seeds per spike and seed weight, showed variation below and above the non-treated controls. A molecular screening technique combining simple sequence repeat (SSR) oligonucleotide primers with high resolution melt (HRM) PCR with EvaGreen was used to identify the variants. Screening for starch branching enzyme IIb (SbeIIb) revealed 75 lines with point mutations. Combining SSR and SbeIIb, a total of 100 Kbp portion of wheat DNA was screened. The estimated mutation frequency in SbeIIb was one per 20.8 Kbp. The spike culture system utilizes very small amounts of EMS for a brief period, thus needs minimal handling of EMS and saves one generation of plant growth in a greenhouse. The morphological variants observed are similar to those reported for seed-derived variants using EMS.