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Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of 10-12 shoots were induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog’s (MS) medium containing 0.1 mgl-1 indole- 3-acetic acid (IAA) + 2.5 mgl-1 benzylaminopurine (BAP) + additives. Higher temperature (31 +/- 2-degrees-C) and mixed (fluorescent and incandescent) light of 50 mumol m-2 s-1 photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants from thornless trees produced 6-8 shoots per explant on MS medium containing 0.1 mgl-1 IAA + 5.0 mgl-1 BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgl-1 indolebutyric acid (IBA) + 2.5 mgl-1 BAP.

Differentiated shoots multiplied best on MS medium containing 0.1 mgl-1 naphthalene acetic acid (NAA) + 1.0 mgl-1 BAP + additives. To yield multiple shoots the original explant was transferred 6 times on fresh medium after harvesting the differentiated shoots. Shoots were rooted by pulsing with 100 mgl-1 IBA for 4 h and then culturing on hormone-free half strength MS medium. Initial dark incubation for 5 days at high temperature (33 +/- 2-degrees-C) was found essential for root induction from shoots which was 63% within two weeks. The rooted plantlets contained a consistent number of chromosomes (2 n = 28). It is suggested that the protocol developed could be useful for cloning of mature and tested trees of P. cineraria.