Study of polyembryony and development of molecular markers for identification of zygotic and nucellar seedlings in Khasi mandarin (Citrus reticulata Blanco)

The objective of this work was to evaluate the occurrence of polyembryonic seedlings and other morphological parameters in Khasi mandarin during three harvest years and to identify zygotic (sexual) seedlings from nucellar (asexual) ones grown under in-vitro conditions using molecular markers. Embryos from 27 polyembryonic and 7 monoembryonic seeds of Khasi mandarin were grown in-vitro. DNA from seedlings and mother parent was analyzed using 16 ISSR and 5 RAPD primers, of which 4 ISSR and a set of 3 RAPD primers were effective to identify zygotic or nucellar origin of the seedlings. In-vitro culture enables maximum embryos of each seed to grow, favouring the origin of seedlings to be identified as zygotic. Among 69 tested individuals, 37 zygotic and 32 nucellar seedlings were recognized. In polyembryonic and monoembryonic seeds, 59.6% and 42.8% of the seedlings, respectively, have the sexual origin. Morphological characteristics of seeds and the seedlings generated varied significantly and were not correlated with polyembryony except for the clutch size and the number of branches. Polyembryonic seeds in the cultivar are high, ranging from 50.0%, 55.5% to 83.3% over three harvest years with more clutch size and the possibility of obtaining zygotic plants from them is high. In polyembryonic seeds not all zygotic seedlings were produced by small embryos located at the micropyle. Identification of zygotic seedlings by ISSR and RAPD markers in Khasi mandarin cultivar is efficient and reliable at an early developmental stage.