A novel visual marker to distinguish haploids from doubled haploids in rice (Oryza sativa, L) at early growth stages

Doubled haploid technology, which enables the generation of homozygous lines in a single step, is one of the modern tools being employed for accelerating breeding processes in different crops. In rice, a globally important staple food crop, doubled haploid production through androgenesis is increasingly being employed in breeding programs. Amongst the androgenic rice lines, doubled haploids are formed spontaneously at about 50-60%, while the remaining 40-50% of plants remain as haploids. As haploids cannot be easily identified, it is routine to grow all the rice androgenic lines till maturity and harvest the seeds from the fertile doubled haploids. Therefore, the methods that facilitate easy identification of haploids at an early developmental stage in rice would enable treatment of such haploid lines with colchicine, to increase the efficiency of doubled haploid production. Further, it would also help in eliminating the operational cost involved in maintaining them till maturity. In the above context, a systematic study to identify easily observable physiological and morphological differences between haploid and doubled haploid rice lines was undertaken. Rice haploids were found to be noticeably different from doubled haploids in photosynthetic rate, transpiration rate, stomatal conductance, and morphology of lodicules, stigma and style, features which have not been reported before. Most importantly, rice haploids invariably have acute leaf apex which is easily distinguishable from the doubled haploids that have attenuated leaf apex shape. Very high per cent accuracy in the prediction of ploidy level was observed when haploids were identified at an early developmental stage based on leaf apex shape, and the results verified with flow cytometry perfectly matches with leaf apex shape. The study establishes 'acute leaf apex' shape as an accurate visual marker to rapidly identify haploid rice lines at an early developmental stage in a cost-effective manner.