Induction of haploid plants in citrus through gamma-irradiated pollen and ascertainment of ovule age for maximum recovery of haploid plantlets

Authors: MANOJ KUNDU, ANIL DUBEY, MANISH SRIVASTAV, SURENDRA KUMAR MALIK

Abstract: The present investigation was carried out for the induction of haploid plants in Citrus grandis through in situ parthenogenesis by pollination with gamma-irradiated pollen of C. limetta and C. sinensis, treated with 50, 100, 200, 300, and 400 Gy gamma ray doses followed by in vitro ovule culture. Ovule culture at 50 days after pollination (DAP) was found optimum for the maximum recovery of in vitro-raised plantlets as compared to 35 and 20 DAP. At 50 DAP, irrespective of the pollen parent, plantlet regeneration capacity decreased at 300 and 400 Gy irradiation treatment (0.62% and 0.60%, respectively) with maximum recovery in the nonirradiated control (3.07%). Chromosome counting in actively growing root tips of all the in vitro-raised ovule cultured plantlets revealed that two haploid plants with chromosome number of nine were induced in C. grandis from the ovule culture that was cultured at 50 DAP following pollination with irradiated pollen of C. sinensis at 400 Gy and C. limetta pollen at 300 Gy. However, irradiation doses below 300 Gy were incapable of inducing any haploids in either cross combination. Similarly, all the plantlets regenerated from in vitro embryo culture of mature seeds were found to be diploid in nature, irrespective of the pollen parent and irradiation dose. Molecular analysis of the in vitro-raised haploid and diploid plants using SSR markers confirmed the maternal origins of the haploid plants; however, the diploid plants were found zygotic in nature with one allele from the seed parent and the other one from the pollen parent.

Keywords: Embryo culture, irradiation, ovule culture, ploidy level, pummelo, SSR marker

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