In vitro Rescue of Nucellar Embryos and Micropropagation of Citrus Rootstocks

Nucellar embryony is a boon for the clonal propagation of Citrus rootstocks. Poly-embryonic seeds of Citrus rootstocks possess several non-zygotic embryos of nucellar origin that are identical to the maternal genotype and is a heritable trait. Since Citrus seeds are recalcitrant the nucellar embryos need to be rescued immediately after harvest and put for germination. In the present study, rescued nucellar embryos were germinated in vitro on Murashige and Skoog medium (MS) supplemented with cytokinins, Benzyl-amino purine (BAP) or Kinetin (Kn) at various concentrations. In vitro germinated nucellar seedlings were micro-propagated further. Best nucellar embryo germination was obtained on MS medium fortified with Kn [Troyer Citrange (1.0 mgL–1), Rough Lemon collection-6 (0.5 mgL–1) and Sour Orange (0.75 mgL–1)] or BAP [Trifoliate Orange (0.75 mgL-1)]. Among the four Citrus rootstocks, germination percentage was maximum in Troyer Citrange and minimum in Rough Lemon Collection-6. Epicotyl segments from two-week-old axenic nucellar seedlings were cultured on MS medium supplemented with different concentrations of BAP alone and in combination with NAA (α-Naphthalene acetic acid) and IAA (Indole-3-acetic acid). In all the Citrus cultivars used, de novo shoot regeneration was obtained within two weeks of culture with highest shoot buds formed in Troyer Citrange followed by Trifoliate Orange, Rough Lemon Collection-6 and least in Sour Orange. Shoots of all the rootstocks rooted best on MS + NAA (0.5 mgL–1) and were acclimatized and transferred to pots with almost 90% survival.