Transforming immature somatic plant, esp. maize, embryos - by treating, in dry state, with nucleic acid soln., esp. for introducing resistance to phosphinothricin

摘要

Immature somatic embryos are transformed by being incubated, in the dried state, with a soln. contg. DNA or RNA. Also described are (1) transformed somatic embryos of monocotyledonous plants and (2) plants regenerated from these embryos. Embryos are pref. derived from callus or organ cultures, partic. of Gramineae, specifically maize. Before incubation with the nucleic acid soln., the embryos are cultured in a nutrient medium contg. sugar or sugar alcohol. Embryos (which have at most 75% of the size of mature embryos) are pref. dried, to 5-15% moisture content, under gentle conditions over a 10-18 day period. They can then be stored at room temp. for over a year. Rehydration is in a medium contg. 2-6% sucrose (and opt. agar), and the nucleic acid is supplied as an aq. soln., opt. also contg. medium components and buffer salts. Nucleic acid can express e.g. a selection marker (such a sPAT); Bacillus thuringiensis endotoxin; a protein for virus resistance; protein which improves photosynthetic capacity, etc.. Gene can be present or incorporated in a cloning vector, and the embryos can be treated with UV light or X-rays to break DNA strands. USE/ADVANTAGE - Dried, monocotyledonous somatic (opt. transgenic) embryos are useful in prodn. of 'artificial seeds', or they can be regenerated and grown to fertile transgenic plants. Esp. maize plants are modified by incorporation of the phosphinothricin acetyl transferase (PAT) gene (opt. modified) from Streptomyces viridochromogenes. This imparts resistance to phosphinothricin (I) and bialaphos to the plants and their derivs..