Studies on soybean tissue culture and transformation

Kim, JooHag
Major Professor
Clifford E. LaMotte
Committee Member
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Several aspects of soybean (Glycine max (L) Merr.) tissue culture and its transformation were examined in this study. A protocol for regeneration of soybean plants from excised parts of 7-day-old Peking seedlings has been developed. Two kinds of explants were used: excised cotyledons and explants each consisting of cotyledonary and primary leaf nodes, the internode that joins them, and one attached cotyledon. When both explants were cultured in vitro on the highly effective shoot-induction medium devised in this study, 35 and 37 shoots per explant on the average were produced from the petiol of the excised cotyledon and from the primary leaf node, respectively. The shoot-induction medium is a modified Murashige and Skoog (MS) medium containing the normal level of MS inorganic macronutrients, fourfold inorganic micronutrients except cobalt, B5 vitamins, 3 g · 1[superscript]-1 sucrose, 2 g · 1[superscript]-1 L-proline, 2 mg · 1[superscript]-1 N[superscript]6-benzyladenine, and 0.02 mg · 1[superscript]-1 [alpha]-naphthaleneacetic acid. Added proline and the increased inorganic micronutrients interact synergistically on shoot regeneration from both kinds of explant, but increasing the micronutrient supply alone has little or no effect. This latter finding seems to explain why earlier attempts to increase shoot regeneration by raising micronutrient did not succeed;The effect of each element of MS micronutrients on shoot regeneration was tested by either singly omitting or adding each element, or raising or lowering the concentration of each element in the medium. Among the seven elements of the MS micronutrients mixture, zinc is the most limiting and all except cobalt are in less than optimal concentration when tested in the proline-containing medium;Small, apparently transformed shoots have been obtained by using Agrobacterium-mediated gene transfer and the productive regeneration system involving shoot regeneration from primary leaf node explants. Agrobacterium strain A281, LBA4404, and A208 caused shoot transformation judging from kanamycin resistance and [beta]-glucuronidase (GUS) activity.