Immunoaffinity techniques applied to the purification of gibberellins from plant extracts.

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA(1), GA(3), GA(4), GA(5), GA(7), and GA(9) conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA(7) was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA(20) and GA(29) were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA(9) were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA(9) was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA(20) and GA(51) were also observed in testae.

Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue.

A reproducible method for regeneration of plants from primary leaf tissue of 27 varieties of soybean (Glycine max), encompassing maturity groups 00 to VIII, has been developed. Progeny from seeds recovered from regenerated plants appear normal. Best regeneration was from leaf explants (2.1-4.0 mm) obtained from 5 day old seedlings. While 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was demonstrated to be essential for regeneration, addition of benzyladenine (BA) was found to enhance regeneration. Of the 6 other auxins tested, only picloram induced any regenerative response. Using identical volumes of medium and other conditions, regeneration could be obtained in 95 × 25 mm glass culture tubes but not in 60 × 15 mm Petri dishes.The regeneration of soybeans from primary leaf tissue was shown to be greatly enhanced by pyroglutamic acid (5-oxoproline). Stimulatory effects were attained if pyroglutamic acid was added directly to the medium or if it was formed in situ as a result of chemical transformation of glutamine during autoclaving. The "active" component produced by autoclaving glutamine was not a conjugate of glutamine with inorganic salts or another organic component of the medium. Filter-sterilized glutamine was shown to be inhibitory to regeneration.Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) basal media were compared to Gamborg B5 medium. All contained 0.1 mg/l 2,4,5-T, 40 mg/l adenine sulfate and 10 mM pyroglutamic acid. No regeneration occurred when MS medium was used. Growth and appearance of callus growing on SH and B5 media with the additives were similar. The incidence of regeneration among cultures growing on SH medium was only one third compared to cultures grown on B5 medium.

An improved enzymatic synthesis of labeled gibberellin A12-aldehyde and gibberellin A12.

The biochemical synthesis of labeled gibberellin A12-7-aldehyde (GA12ald) and and gibberellin A12 (GA12) from labeled R,S-mevalonic acid (MVA) using cell-free extracts from pumpkin (Cucurbita maxima) endosperm has been improved over the previously reported procedure. Three major improvements were developing a one-step HPLC procedure to isolate GA12ald and GA12 in radiochemically pure form; adjusting the pH of the reaction mix to pH 6.9 which increased the GA12ald/GA12 ratio over that at pH 7.8 by ca. 18-fold while reducing the combined yield of these two compounds by less than 17%; and developing a technique that permitted sampling of the fruits without interrupting growth; this doubled the fraction of extracts with "high activity." Conversion of MVA into GA12ald or GA12 displayed Michaelis-Menten kinetics with half-maximal synthesis at 0.4 mM MVA. Four-hour incubations afforded the highest yields from 0.25 mM MVA. Up to 15 and 7% of the MVA was incorporated into GA12ald and GA12, respectively. About one-quarter of the extracts incorporated at least 10% of the 0.25 mM MVA into GA12ald. One pumpkin fruit can provide sufficient endosperm to synthesize ca. 0.6 mumol of GA12ald.

Plant regeneration by organogenesis in Glycine max.

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5µM BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.

Culture of soybean mesophyll protoplasts in alginate beads.

Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.