Glenn B. Collins

A procedure for plant regeneration from immature cotyledon tissue of soybean

For some time, soybean (Glycine max (L.) Merr.) has been conspicuous among major crops in lacking an in vitro regeneration system. This deficiency has precluded the application of many nonconventional techniques of crop improvement (See Abelson, 1983; Lea and Stewart, 1984; Hildebrand et al., 1985 for reviews). Although soybean is recalcitrant in vitro, some wild Glycine species are more responsive. Of these, Glycine canescens F. J. Herm appears most amenable; shoot production has been obtained from hypocotyl (Kameya and Widholm, 1981; Widholm and Rick, 1983), cotyledon (Widholm and Rick, 1983; Grant, 1984), leaf and flower tissues (Lazzeri, Hildebrand and Collins, unpublished) and also from protoplasts (Newell and Luu, 1985; Myers, Lazzeri and Collins, unpublished). Shoot regeneration has also been reported in two other wild species, G. tomentella Mayata (Kameya and Widholm, 1981) and G.clandestina Wendl. (Hymowitz et al., 1986).

Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene

Soybean (Glycine max. Merrill. cv. Fayette) cotyledonary nodes were transformed with bean pod mottle virus (BPMV) coat protein precursor (CP-P) gene via Agrobacterium-mediated transformation. The transformation rate was low, and only five primary transformants derived from five different cotyledons were obtained from 400 original cotyledons. Southern blot hybridization verified the integration of the BPMV CP-P gene. Inheritance and expression of this gene in R1 plants were also demonstrated. About 30% of R2 plants derived from one transgenic line showed complete resistance to BPMV infection, as assessed by symptomatology and ELISA, suggesting that homozygous, but not hemizygous, plants exhibit the resistant phenotype.

Recovery of primary transformants of soybean

Three transformants of soybean, Glycine max (L.) Merr., have been recovered among a total of 18 plants regenerated by somatic embryogenesis from immature cotyledon tissues after cocultivation with Agrobacterium strains carrying a 15 kD zein gene (pH5PZ3D). DNA from upper leaves hybridized to a synthetic RNA probe specific for the zein sequence at a level equivalent to at least one copy per haploid genome. Hybridization to a vir G/C probe, however, was negligible, indicating that sequestration of whole bacteria or even persistence of plasmids within the tissues could not account for the zein hybridization signals. Progeny of all plants were uniformly untransformed. Since most somatic embryos have a multicellular origin in the regeneration system used, it is believed that the primary transformants were chimeric. The results indicate that somatic embryogenesis may be adaptable to Agrobacterium-mediated transformation in soybean, but that greater numbers of mitotic cycles under selection before embryo initiation will be required if somatic embryogenesis is to be used efficiently for production of plants with transformed germ-line cells.

Factors affecting soybean cotyledonary node transformation

Abstract Cotyledonary node transformation efficiency was evaluated using a sonication assisted Agrobacterium-mediated transformation (SAAT) protocol, three dissimilar A. tumefaciens strains, and explants derived from 28 diverse cultivars and/or genotypes of soybean [Glycine max (L.) Merr.]. The explants were evaluated at 10 and 45 days after co-cultivation for transformation with a binary vector containing both a GUS-intron gene and an NPTII selectable marker. The best overall strain of A. tumefaciens was determined to be KYRT1 based on stable GUS transformation of soybean cotyledonary node explants measured at the terminal 45 day evaluation point. SAAT did not increase stable transformation at 45 days post co-cultivation. SAAT was determined to significantly decrease shoot proliferation of some genotypes, but it is unclear what effect this may have on the recovery of transformed shoots. Significant differences were also detected between genotypes for transformation and shoot proliferation frequency.

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 μM), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 μM) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.

OPTIMIZATION OF SOMATIC EMBRYOGENESIS AND EMBRYO GERMINATION IN SOYBEAN

SummarySomatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container.

Recent Advances in Soybean Transformation

Soybean (Glycine max (L.) Merrill) transformation continues to be problematic even with the efforts of numerous researchers in the field. In spite of this low efficiency, soybean transformation is now routine utilizing the somatic embryogenie and meristematic tissue culture methods. A comparison, including the advantages and disadvantages, of each method are presented in this review. Soybean transformation is currently done using both Agrobacterium tumefaciens and particle bombardment. We review some of the current selectable marker genes in use, and we also describe additional non-standard transformation techniques that have been, or are being attempted, to produce transgenic soybeans.

Effect of genotype on somatic embryogenesis from immature cotyledons of soybean

Genotype has a large effect on the ability of immature soybean cotyledons to undergo auxin-stimulated somatic embryogenesis. Among 33 soybean lines, all those showing good regeneration were found to have in their pedigrees one or both of the highly regenerative ancestral lines, ‘Manchu’ or ‘A.K. Harrow’. When ‘Manchu’ was crossed with ‘Shiro’, a genotype showing extremely poor regeneration, F1 hybrid cotyledons showed intermediate regeneration capacity.

Auxin-orientation effects on somatic embryogenesis from immature soybean cotyledons

SummaryDevelopment of somatic embryos of soybeanGlycine max (L.) Merr. has been studied using cultivars J103 and McCall and five auxin-sucrose treatment: naphthalene acetic acid at 10 mg/liter with 1.5% sucrose (N10); 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.25, 0.5 or 1.0 mg/liter with 1.5% sucrose (D.25, D.5 D1); and 2,4-D at 25 mg/liter with 3% sucrose (D25). Cotyledons were excised aseptically from immature embryos 3 to 5 mm in length, and placed on the media with either the adaxial or abaxial side down (adaxial or abaxial orientiation, respectively). After 30 d, numbers of normal or total somatic embryos, or both, were counted. For J103 explants on D25 or N10 media, the greatest number of embryos was produced from the central region of abaxially oriented explants on D25, but only 5% of these embryos were normal. The greatest number of normal embryos was produced from the marginal region of adaxially oriented explants on D25. For McCall explants on D.25, D.5, D1, or N10 media, the greatest numbers of normal embryos were produced from the marginal regions of abaxially oriented explants on D1 or N10. Embryo induction was examined histologically for the N10, D25, and D.5 treatment, using serial section of paraffin-embedded cotyledons stained with hematoxylin and safranin. On N10 medium, embryos were produced unicellularly, or more frauently, multicellularly, from homogeneous embryogenic tissue arising from epidermal and subepidermal cells at the distal periphery of the cotyledon. On D25 medium, embryos were produced multicellularly from a geterogeneous embryogenic tissue formed in the central region of the cytoledon, and consisting of embryogenic cells interspersed with large, vacuolate cells. The D.5 treatment showed an intermediate response. Embryo initiation in this soybean system is predominantly multicellular.

Soybean somatic embryogenesis: Effects of nutritional, physical and chemical factors

Immature soybean (Glycine max (L.) Merr) embryos, or cotyledons isolated from them, were cultured on modified MS medium containing B5 vitamins and NAA (50 μM) to induce somatic embryogenesis. The effects of media variables, dissection treatments and light conditions were investigated in this system. The efficiency of embryogenesis increased as sugar concentration decreased from 12 to 1.5%; sucrose and glucose were similarly effective as carbon sources. In an examination of the effects of medium pH, values between pH 5.0 and 7.0 gave similar embryogenesis efficiencies, but the frequency of normal embryos was greater in media with low pH values. In buffered medium (10 mM MES), a pH of 5.0 was inhibitory to embryogenesis, and most normal embryos were produced at pH 5.5. Under various dissection treatments, embryogenesis efficiency and root and callus production were increased by tissue damage. Somatic embryogenesis was observed both in darkness and in light, although embryo development was impaired under high light (80 μE m-2 s-1) conditions. The ability of somatic embryos to germinate was closely correlated with embryo normality, and was influenced little by the hormone content of germination media. Of various media tested for their ability to support the growth of germinated embryos, a medium based on hydroponic nutrient salts, supplemented with yeast extract, and gelled with Difco-Bacto agar gave the best plantlet growth.