As a first step in the development of a successful Agrobacterium tumefaciens mediated transformation method for kenaf, factors influencing the successful T-DNA integration and expression (as measured by the GUS expression) were investigated. Transformation was carried out using two kenaf cultivars and Agrobacterium strain EHA 105 carrying different vectors, plasmid pIG 121-Hm or pEC:gus. Pre-culturing the explants for 2 days in benzyl adenine containing medium, and wounding the explant before inoculation were found to enhance the transient GUS expression. Increasing the duration of pre-culture and co-culture period enhanced the transient GUS expression up to a threshold level. Increased transient GUS expression did not correlate with an increase in stable expression. Gene integration was confirmed by PCR analysis.Kenaf (Hibiscus cannabinus L.), an economically important industrial fiber crop, belongs to family Malvaceae. Success in in vitro manipulations of kenaf has been achieved very slowly. Banks et al. (1993) first reported the expression of trans genes in kenaf callus. However, shoot regeneration from kenaf callus has not been successful though direct shoot regeneration without a callus phase has been achieved (Srivatanakul et al., 2000;Herath et al., 2004). Srivatanakul et al. (2001) reported on the effect of Agrobacterium strain, temperature, and acetosyringone on T-DNA integration into kenaf shoot apices; no stable transformants were obtained. Hence, we further investigated the influence of some factors such as duration and cytokinin concentration during pre-culture and co-culture, and wounding the explant on successful transient and stable gene expression of kenaf.Since several factors had to be tested two independent experiments were carried out to determine the effect of the BA concentration during pre-culture and co-culture periods and to determine the effect of the duration of pre-culture and co-culture periods. Effect of wounding the explant was determined in a preliminary experiment and was carried out in all experiments. In experiment one, treatment factors were BA concentration during pre-culture (0, 4.4, and 8.8 lM) and coculture (8.8, 13.2, and 22 lM). Each of the 9 treatment combinations was replicated three times with at least 50 explants for each replicate. Number of plants inoculated differed for each treatment since some plants were damaged during wounding. In experiment 2, treatment factors were duration of pre-culture (0, 1, 2 and 4 days), and duration of coculture (2 and 3 days). All 8-treatment combinations were replicated three times with at least 60 explants for each replicate. This experiment was duplicated using the two vectors (pIG 121 (Hm) and pEC: GUS). Kenaf cultivar Tainung 2 (provided by the Oji Paper Company Ltd. Japan) was used for all experiments. Seed germination and shoot regeneration was carried out according to the previous published method (Herath et al., 2004) unless otherwise stated. After 7-10 days in the germination medium, the shoot tip of the seedling
Morphological changes in shoot apices of kenaf treated with benzyl adenine (BA) were compared with those of untreated control plants using light and scanning electron microscopy (SEM). Seven days after the start of BA treatment (day 7), meristematic regions appeared adjacent to the axillary buds, and on the abaxial surface of the primary leaf primordia. Some of these meristematic regions were later developed into shoots. Further, the cells in the primary shoot apical meristem were reprogrammed to induce several meristematic loci. At day 28 in culture, supernumerary vegetative shoot buds were observed in and near the axillary buds. In the control plants neither axillary nor adventitious buds developed. These results suggest that the treatment with BA reprogrammed the developmental fate of a large number of cells in the shoot apex of kenaf. Further, it reconfirmed the ability of BA to overcome the apical dominance of shoots.
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