Improved Autotetraploid Induction Methods in Four Diploid Wild Peanut Species: Arachis duranensis , A . stenosperma , A. magna , and A . batizocoi

Peanut ( Arachis hypogaea L., 2 n = 4 x = 40, AABB) is an important legume crop worldwide. Wild peanuts ( Arachis spp.) represent a valuable secondary gene pool with diverse disease resistances. However, most wild peanut species are diploid (2 n = 2 x = 20) and this ploidy barrier is a major obstacle to hybridization and gene introgression. Ploidy manipulation has been used to overcome this problem but requires a rapid, efficient chromosome doubling method to facilitate gene introgressions. Our previously reported colchicine buffer submergence method successfully induced tetraploidy in diploid peanut. However, induction efficiency was low and seedling mortality was high. In this study, four modified colchicine‐buffer‐submergence methods were evaluated, including three substrate‐based and one semi‐suspension colchicine application methods. Pre‐germinated seeds of four wild peanut species, A . duranensis (A‐genome) , A. stenosperma (A‐genome), A. magna (B‐genome), and A . batizocoi (K‐genome)—were used in this study. To obtain optimal results, the treatment period for A. magna and the colchicine concentration for A. stenosperma were further adjusted. Ploidy of surviving treated plants was validated by flow cytometry. With the optimal treatment of each species, the highest tetraploid conversion rates were 57%, 14%, 60%, and 50% among surviving A. batizocoi , A. duranensis , A. magna , and A. stenosperma seedlings, respectively. The stomatal size and density results show that all diploids have smaller stomata and higher stomatal density than the tetraploids. These results provide an improved protocol for chromosome doubling in wild peanut species and will facilitate gene introgressions for future peanut breeding.