Raspberry has very cultivar specific requirements for proliferation. Plant regeneration rates from isolated explants are inconsistent and vary widely among cultivars. As a step towards developing a viable transgenic system in red raspberry (Rubus idaeus L.) we first developed an efficient and consistent protocol for plant regeneration from isolated explants. A modified MS medium with cytokinin BA gave vigorous shoots with an average proliferation rate of 3-5 depending on the cultivar. These vigorous shoot proliferants served as an ideal explant source for plant regeneration experiments. The average rate of shoot regeneration from leaf explants was 72, 32, 68. and 72% for cvs. Canby, Chilliwack, Meeker and Heritage respectively. In addition to leaf, petiole explants were equally good sources for inducing shoot organogenesis. In all the above-mentioned cultivars, 44-57% of the petiole explants gave rise lo healthy and vigorous shoot regenerants in culture. The regenerated shoots were induced lo root on a rooting medium and were successfully transplanted to the greenhouse. This regeneration system was successfully applied in our laboratory for developing gene transfer system in red raspberry (see abstract by Mathews, et al).
Cynthia Cohen, H. Mathews, V. Dewey, and R. Bestwick
Helena Mathews, C. Cohen, W. Wagoner, J. Kellogg, V. Dewey, and R. Bestwick
We have developed efficient plant rageneration and transformation systems for red raspberry (Rubus idaeus L.). We have successfully introduced a gene for controlling biosynthesis of ethylene into raspberry for the first time. Leaf and petiole segments were co-cultivated with disarmed Agrobacterium strains EHA 101 or 105 containing plasmids pAG5420, pAG 1452 or pAG1552. The plasmids encoded gene sequences for S-adenosylmethionine hydrolase (SAM ase) driven by the fruit specific or wound and fruit specific tomato SE8 or E4 promoters. SAM ase catalyses the conversion of S-adenosylmethionine (SAM) to methylthioadenosine (MTA) and homoserine which can reenter the methionine recycling pathway. SAM is therefore not available for the synthesis of 1-am inocyclopropane carboxylic acid (ACC), the metabolic precursor for ethylene biosynthesis. Initial shoot regenerants were mostly chimeras containing transformed and non-transformed cells. Solid clones of pure transgenics were developed by repeated culture of leaf, petiole and nodal explants of primary regenerants on higher stringency selection medium. Transformants were screened on medium with kanamycin, geneticin or hygromycin depending on the selection marker gene NPTII or hpt. Genomic integration of transgenes were confirmed by Southern hybridization. Transgenic plants of cultivars Canby, Meeker and Chilliwack have been transplanted to the greenhouse for fruit set and further evaluation of transgenic traits.