Tissue cultures were established from newly expanded leaves and axillary buds of rubber trees (Hevea brasiliensis Muell. Arg.). Calli formed from these explants, but no regeneration occurred. Shoots were obtained from axillary buds cultured on Murashige and Skoog's (MS) medium (Physiol. Plant. 15: 473-497, 1962) supplemented with 1.0 mg/l kinetin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose and 4 g/l Difco agar. Formation of a root similar to a tap root was induced on MS medium supplemented with 5.0 mg/l naphthaleneacetic acid (NAA), 3.0 mg/l indolylbutyric acid (IBA), 50 g/l sucrose and 4 g/l Difco agar. Several types of explants were used in attempts to recover complete rubber tree plants with well-developed tap roots. Leaf explants and axillary buds formed calli on MS basic medium with different combinations of kinetin, benzylaminopurine (BAP), 2,4-D, IBA, NAA and indolylacetic acid (IAA). The antibiotic tetracycline was also used to control possible bacterial infections. However, no antibiotic effect was noted. Calli formation was abundant, but no regeneration was observed when the calli from different media was transferred to MS medium without growth hormones. On this basic medium, callus cultures became necrotic and died. Shoots developed from axillary buds, rooted vigorously when cultured on MS medium with NAA, IAA, and IBA. Based on these results, further studies with commercially important clones should lead to a feasible micropropagation technique.
[Show abstract][Hide abstract] ABSTRACT: A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl(2) concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl(2) before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl(2) concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl(2) concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl(2) concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.
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