Maximizing Hybrid Seedlings Recovery and Early Identification of Highly Polyembryonic Acid Lime (Citrus aurantifolia Swing.) × Lemon (Citrus limon Burm.) Hybrids using SSR Markers

Nucellar embryony is the major obstacle in getting hybrid seedlings in interspecific crosses in citrus. Hence, the present study was conducted to standardize embryo age, culture media for maximizing germination, and subsequent identification of simple sequence repeat (SSR) markers to differentiate the hybrids. A factorial experiment was conducted with three embryo ages – 80–90, 110–120, and 130–140 days after pollination (DAP). The germination of the rescued immature embryos of acid lime × lemon crosses was initiated on three different growing media. The fruits harvested at 130–140 and 80–90 DAP had a higher number of functional seeds (90%). Older embryos (130–140 DAP) and middle-aged embryos (110–120 DAP) germinated in 100% on Murashige and Skoog (MS) medium supplemented with 500 mg·l⁻¹ malt extract, 1.5 mg·l⁻¹ gibberellic acid (GA3), and 0.02 mg·l⁻¹ naphthalene acetic acid (NAA). MS medium supplemented with 500 mg·l⁻¹ malt extract proved better for the germination of embryos taken at 80–90 DAP. Plantlet survival was the highest in younger embryos (80–90 DAP) cultured on MS basal medium (84.21%) and the lowest in older embryos cultured on MS medium supplemented with 500 mg·l⁻¹ malt extract plus 1.5 mg·l⁻¹ GA₃ and 0.02 mg·l⁻¹ NAA. The seedlings obtained from the culture 80–90 DAP had the highest root length (4.9 mm) and shoot length (5.3 mm) at 60 days after inoculation (DAI) on the above medium. SSR marker analysis revealed that CCSM-4 and CAC-33 markers expressed polymorphism between female and male parents, proving their ability to identify the hybrids of ‘Kagzi’ acid lime × ‘Konkan Seedless’ lemon.