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Abstract
This study was carried out in the plant tissue culture laboratory, horticulture research Institute, Agriculture research center, ministry of agriculture, during the period from 2011 to 2014. An efficient direct shoot regeneration micropropagation protocol has been established successfully for commercial in vitro propagation of holmskioldia sanguinea, a rare plant with ornamental and medicinal values. The optimized strilization condition for the single node stem cutting explants was exposure to 30% clorox for 20 min (70.83 survival %, 4.17 mortality % and 25.00 contamination %). In establishment stage full strength of murashige and Skoog medium (MS) supplemented with NAA at 0.5 mg/l was superior in in vitro growth of explant in respect of shoot length (2.13 cm) and number of leaves / explant (11.33 leaves). The multiplication stage should be carried out, with cutting the shoot apex, on MS medium supplemented with 1.0 mg/l BA which recorded 5.33 shoots at the end of the third subculture. For rooting the regenerated shoots, 1/8 strength MS medium supplemented with 2.0 mg / l IBA was effective and economically treatment, which resulted in 100% rooting percentage, 9.67 roots/plantlet, 8.00 cm root length, 14.67 leaves/plantlet and 8.00 cm plantlet length. Growing medium consised of peatmoss and vermiculite at the ratio of 2:1 (v / v) was the best for acclimatization of regenerated plantlets giving 100% survival percentage which grew into mature plants, recording highest values (after 6 and 12 weeks) for plantlet length (10.83 and 15.67 cm, respectively), number of leaves / plantlet (14.67 and 22.33 leaves, respectively), stem diameter (1.80 and 1.97 cm, respectively), and number of shoots/plantlet (2.33 shoot for each). Inter simple sequence repeats (ISSR) analysis detected similarity (13.7%) to the mother plant in the in vitro regenerated plants from the nodal explants, that the direct regeneration protocol will be useful for holmskioldia sanguinea production. This in vitro plant developmental protocol could be used for large scale regeneration of holmskioldia sanguinea