To produce true-to-type and rapid multiplication micropropagation technique was utilized to the nucellar tissue with ovular halves of C. jambhiri. Nucellar tissues were cultured in a modified MS medium supplemented with different concentrations of 2ip viz. 0.25,0.50 mg l-1 alone and in combination of 0.50 mg l-1 NAA. Initiation of cell division and differentiation of proembryogenic tissue became apparent in first 80 days.These proembryos developed into embryos through subculturing in fresh medium. Low concentration of 0.25 2ip was found more suitable for the development of embryo producing large number of fully developed embryo in comparision to the embryo produced in the high concentration of 2ip (0.50 mg l-1) and in combination of 0.25 2ip and 0.50 mg l-1 NAA. Normally developed embryos in 0.25 2ip showed best germination in the fresh medium supplemented with 0.25 mg l-1 IAA, 100 ME mg l-1 and 5mg mg l-1 amino acids within 30 days as compared to other treatments. These germinated embryos were utilized for producing disease free saplings after hardening and nurturing in laboratory conditions. The disease free saplings thus produced can be used to establish new Citrus orchards within short time.
Citrus aurantifolia (lime) has been selected as explant for nucellar embryogenesis. Nucellus is a non-vascularized tissue being true-to-type same as mother plant, meristematic cells have no plasmodesmata connection, no virus can pass through nucellus, thus it seems to be a good material for production of virus freeplantlet.Putrescine at 0.25 or 0.5 mg1-1 and anapthaleneacetic acid at 0.10 mg1-1 supplemented to nutrient formulation were most effective in alleviating cotyledonary proliferation and fasciation while promoting embryo-to-embryo proliferation producing numerous whitish globular embryos were formed. For further development of globular embryos to well-differentiated cotyledonary embryos, additional presence of 2-isopentenyladenine at concentrations of 0.10 or 0.25 mg 1-1 was essential, contrary to incorporation of 0.10 or 0.25 mg 1-1 6benzylaminopurine, which promoted excessive proliferation of cotyledonary structures and their fasciation while zeatin at the same concentrations produced intermediate response. In the optimum treatment containing 0.25 mg l-1 putrescine, 0.10 mg 1-1 isopentenyladenine, 0.10 mg 1-1 indole-3-acetic acid and 100 mg l-1 malt extract, an average 10 well-developed embryos per culture were formed, besides some abnormal cotyledonary structures. Well-developed embryos measuring ca. 2 cm. in length (leaving the root) germinated 100% into plantlets, during 60 days, in the additional presence of amino acid supplement comprising, 5 mg 1-1 each of L-arginine, L-asparagine, L-histidine, L-cysteine, L-lysine and 10 mg l-1 L-glutamine. Such plantlets nurtured in a different medium attained a height of ca. 4 cm in 45 days before they were taken out for ex vitro growth. There was 100% transplant success and the plants grew normally.
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