A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens.

Genes encoding Class II EPSPS enzymes are disclosed. The genes are useful in producing transformed bacteria and plants which are tolerant to glyphosate herbicide. Class II EPSPS genes share little homology with known, Class I EPSPS genes, and do not hybridize to probes from Class I EPSPS's. The Class II EPSPS enzymes are characterized by being more kinetically efficient than Class I EPSPS's in the presence of glyphosate. Plants transformed with Class II EPSPS genes are also disclosed as well as a method for selectively controlling weeds in a planted transgenic crop field.

Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases

This invention relates to a reproducible system for the production of stable, genetically transformed maize cells, and to methods of selecting cells that have been transformed. One method of selection disclosed employs the Streptomyces bar gene introduced by microprojectile bombardment into embryogenic maize cells which were grown in suspension cultures, followed by exposure to the herbicide bialaphos. The methods of achieving stable transformation disclosed herein include tissue culture methods and media, methods for the bombardment of recipient cells with the desired transforming DNA, and methods of growing fertile plants from the transformed cells. This invention also relates to the transformed cells and seeds and to the fertile plants grown from the transformed cells and to their pollen.

Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof

Summary
In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative ‘model’ legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.1992.00487.x

Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.

Insect resistant cotton plants.

Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.

Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer.

The European corn borer [ECB; Ostrinia nubilalis (Hubner)] is an economically significant pest of corn (Zea mays L.). The ability to routinely transform corn has broadened the control options available to include the introduction of resistance genes from sexually incompatible species. In this study, microprojectile bombardment was used to introduce synthetic versions of cryIA insecticidal protein genes from Bacillus thuringiensis subsp. kurstaki (Btk) into embryogenic tissue of the Hi-II (A188/B73 derivative) genotype of corn. Of 715 independent transgenic calli produced, 314 (44%) had insecticidal activity against tobacco hornworm (Manduca sexta L.) larvae. Plants were regenerated, self-pollinated when possible, and crossed to B73. First-generation progeny of 173 independent Btk-protein expressing calli were evaluated under field conditions with artificial ECB infestations in 1992 or 1993. Approximately half (89/173) segregated in a single-gene manner for resistance to first-generation ECB leaf-feeding damage. All of the 89 lines evaluated in 1992 or 1993 for resistance to second-generation ECB exhibited less stalk tunneling damage then the non-transgenic controls. In 1993, 44% (37/77) of the lines tested had ≤ 2.5 cm of tunneling, compared to severe damage (mean = 45.7 cm) in the B73 × Hi-II controls. Experiments are in progress to evaluate the effect of the introduced genes on yield and other agronomic properties

Field evaluation of european corn borer control in progeny of 173 transgenic corn events expressing an insecticidal protein from Bacillus thuringiensis

The Biolistics® particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

Stable transformation via particle bombardment in two different soybean regeneration systems

Organogenesis in alfalfa callus (Medicago sativa L. cv. 'Regen S') has been obtained by the transfer of callus from an induction medium containing growth regulators to a regeneration medium lacking growth regulators. The transfer of callus from induction medium containing high levels of 2,4-D and low levels of kinetin to regeneration medium resulted in the formation of shoots. Conversely, the transfer of callus from induction medium containing low levels of 2,4-D and high levels of kinetin resulted in the formation of roots. The pattern of organogenesis on regeneration medium was modified by the nutritional composition of that medium. When Blaydes medium supplemented with inositol and yeast extract was employed as regeneration medium, root organogenesis was inhibited. Root organogenesis was not inhibited by either Shenk and Hildebrandt medium or Gamborg's B5 medium. Shoot formation occurred on all of these media. A survey of the in vitro organ-forming capacity of 14 genotypic clones from the cv. 'Regen S' was conducted. The capacity to form organs differed quantitatively among the clones analyzed. A more detailed analysis of a highly responsive clone (RA3) and a poorly responsive clone (RA5) revealed no significant qualitative difference in their organogenic responses. THE regeneration of shoots from in vitro cultures

https://www.jstor.org/stable/info/2442311

The hormonal control of organ formation in callus of medicago sativa l. cultured in vitro

A rapid and reproducible Agrobacteriummediated transformation protocol for sorghum has been developed The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin A screen of various A tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF2, designated NTL4/Chry5, was most efficient for gene transfer to sorghum immature embryos A NTL4/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlusTM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event The transformation frequencies among the successful experiments ranged from 03 to 45%, with the average transformation frequency being approximately 1% for both genotypes Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile Co-expression of GUSPlusTM occurred in 89% of the transgenic T0 events Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant Analysis of T1 progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events Communicated by P Ozias-Akins A Howe · S Sato · M Fromm · T Clemente ( ) Center for Biotechnology, University of Nebraska, N300 Beadle Center, Lincoln, NE 68588, USA e-mail: tclemente1@unledu Tel: +1-402-472-1428 Fax: +1-402-472-3139 I Dweikat · M Fromm · T Clemente Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68583, USA M Fromm · T Clemente Plant Science Initiative, University of Nebraska, N300 Beadle Center, Lincoln, NE 68588, USA

/pdf/genetic-transformation-and-hybridization-2t30g4k9wu.pdf

Shirley J. Sato

Papers

Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer.

Field evaluation of european corn borer control in progeny of 173 transgenic corn events expressing an insecticidal protein from Bacillus thuringiensis

Stable transformation via particle bombardment in two different soybean regeneration systems

The hormonal control of organ formation in callus of medicago sativa l. cultured in vitro

Genetic transformation and hybridization