Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method – plant development and surfactant are important in optimizing transformation efficiency

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the -glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.     

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 M aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.     

Performance and impact of the biological control agent Xubida infusella (Lepidoptera; Pyralidae) on the target weed Eichhornia crassipes (waterhyacinth) and on a non-target plant, Pontederia cordata (pickerelweed) in two nutrient regimes

Xubida infusella (Walker) (Lepidoptera: Pyralidae) is potentially a useful biological control agent targeting Eichhornia crassipes (waterhyacinth) in the USA but many regions infested with waterhyacinth are also inhabited by an alternative native host, Pontederia cordata (pickerelweed). Experiments were conducted in Australia to assess the impact of X. infusella on pickerelweed compared to waterhyacinth where both these plants were available and X. infusella had already been released. Overall X. infusella had a greater impact on pickerelweed than on waterhyacinth. More than one larva per plant was required to reduce the total shoot dry weight of waterhyacinth but only one larva per plant reduced the total shoot dry weight of pickerelweed. Insect feeding caused the number of secondary shoots (daughter plants) of pickerelweed to double whereas the number of daughter plants produced by waterhyacinth remained unchanged. We suggest this indicates a considerable impact on pickerelweed rather than effective compensation for insect damage because the shoots produced were very small. Waterhyacinth produced a constant number of daughter plants when fed on by up to three larvae per plant. Higher nitrogen status of both species of host plant increased the rate of larval development and pupal weight of X. infusella. The weight and fecundity of X. infusella reared on pickerelweed were lower than those reared on waterhyacinth but large numbers of progeny were produced on both plant species. This experiment demonstrates a considerable impact of X. infusella on pickerelweed suggesting this plant is at risk from this agent if released in the USA where pickerelweed is present. The considerable impact on waterhyacinth demonstrates the potential for this insect to contribute to waterhyacinth control in countries where risk assessment favours release.     

Expression of the B subunit of <Emphasis Type="Italic">E. coli</Emphasis> heat-labile enterotoxin in tobacco using a herbicide resistance gene as a selection marker

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l^-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformed transgenic triploid bermudagrass (<Emphasis Type="Italic">Cynodon dactylon</Emphasis> × <Emphasis Type="Italic">C. transvaalensis</Emphasis>) plants are highly resistant to the glufosinate herbicide Liberty

Glufosinate resistance gene isolated from Streptomyces hygromicinroscopicus (bar) that confers the resistance of herbicide Liberty, a broad-spectrum grass and broadleaf contact herbicide widely used for weed control, was introduced into triploid bermudagrass by Agrobacterium-mediated transformation. Embryogenic calluses derived from stolonous nodal segment were co-cultured with the disarmed strain EHA105 harboring the binary vector pBG1300H containing the bar gene under the control of adh-1 promoter. A total of 18 independent transgenic lines were obtained. The integration of bar gene into plant genome was confirmed by the GUS histochemical staining assay, PCR amplification, and Southern blotting. Herbicide bioassay indicated that the bar-expressing transgenic plants exhibited greater herbicide resistance than the wild type and the non-transformed tissue culture-derived plants.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.     

T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation

Summary Two lines of transgenic Nicotiana tabacum transformed to kanamycin resistance by means of a binary Agrobacterium vector containing a nos-npt gene were investigated over three generations. Southern hybridization and crossing analyses revealed that a single copy of T-DNA had integrated in each line and that the kanamycin resistance was regularly transmitted to the progeny as a monogenic dominant trait. Homozygous transgenic plants were fully fertile, morphologically normal and did not significantly differ from wild-type plants in the quantitative characters examined (plant height, flowering time). The two lines showed very low, but significantly different levels of meiotic instability: kanamycin-sensitive plants occurred among backcross progeny from homozygous transgenic plants with frequencies of 6/45,000 and 25/45,000, respectively. The sensitive plants arose independently of each other and thus resulted from meiotic rather than mitotic events. These findings demonstrate for the first time that integrated foreign genes can be transmitted to progeny with the high degree of meiotic stability required for commercial varieties of crop plants. They emphasize the importance of non-homologous integration and of avoiding co-integration of inactive gene copies for achieving meiotically stable transformants.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Efficient <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of oilseed mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.] using leaf piece explants

Leaf piece explants of five Brassica juncea (L.) Czern. cultivars were transformed with an Agrobacterium tumefaciens strain EHA105 harboring the plasmid pCAMBIA1301, which contains the β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of cauliflower mosaic virus 35S (CaMV35S) promoter. Transgenic plants were regenerated on Murashige and Skoog (MS) medium fortified with 8.87 μM 6-benzylaminopurine, 0.22 μM 2,4-dichlorophenoxyacetic acid, and 20 μM silver nitrate in the presence of 30 mg/l hygromycin. When co-culture took place in the presence of 100 μM acetosyringone, the efficiency of stable transformation was found to be approximately 19% in the T ₀ generation, with the transgenic plants and their progeny showing constitutive GUS expression in different plant organs. Southern blot hybridization of uidA and hpt genes confirmed transgene integration within the genome of transformed plants of each cultivar. Inheritance of hpt gene for single copy T-DNA inserts showed a 3:1 pattern of Mendelian segregation in progeny plants through germination of T ₁ seeds on MS medium containing 30 mg/l hygromycin. The protocol described here reports superior transformation efficiency over previously published protocols and should contribute to enhanced biotechnology applications in B. juncea.     

Genetic transformation ofLotus corniculatus withAgrobacterium tumefaciens and the analysis of the inheritance of transgenes in the T1 generation

The herbage legume,Lotus corniculatus (bird's-foot trefoil), was transformed using the disarmedAgrobacterium tumefaciens strain LBA4404 (pAL4404) carrying a binary construct, pJit73. This plasmid carries two antibiotic resistance genes,aphIV andnptII encoding resistance to hygromycin and kanamycin respectively, and the easily detectable reporter gene,uidA encoding the enzyme -glucuronidase (GUS). Transgenic plants were regenerated from two separate co-cultivations of leaves withA. tumefaciens either with or without an acetosyringone pretreatment. A total of 110 putative transformants were regenerated, 52 (47%) of which grew on selection media containing hygromycin. Twenty-five plants were analysed further for morphological variation and presence of transgenes and were used to study the inheritance of expression of the transgenes in the T₁ generation. Expression patterns of the transgenes in the T₁ progeny generated from these 25 plants differed. In the majority of plant linesaphIV anduidA transgenes segregated together, but the apparent number of copies of the transgenes varied. No expression of either transgene was detected in the progeny from three plants, while the progeny from six other plants were resistant to hygromycin but had no GUS expression. Progeny of all of the remaining 16 plants had GUS activity. For three plants, inheritance data were consistent with more than one dose ofuidA andaphIV; another two plants yielded data expected for exactly one dose of both transgenes. In the progeny of the remaining 11 plants, the percentage of seedlings expressing bothuidA andaphIV was lower than expected.     

Paternal inheritance of mitochondria in rapeseed (Brassica napus)

Summary Transfer of a mitochondrially associated plasmid following sexual crosses in Brassica napus rapeseed suggested that paternal mitochondria were being transferred to the cytoplasm of the egg. To examine this possibility further, plants carrying the chloroplast (cp) marker of triazine resistance, but which had lost the plasmid associated with the mitochondria of this cytoplasm, were crossed as females to males carrying the polima cytoplasm. The males carried a nuclear fertility restorer gene on an extra chromosome to overcome the male sterility marker conferred by the mitochondria of this cytoplasm. Approximately 10% of the F₁ progeny displayed the male sterility and flower morphology of the male parent. Mitochondrial (mt) DNA from the progeny showed the combined restriction patterns of both parents, but this rut heterogeneity did not continue into subsequent generations. All progeny retained the cp DNA restriction patterns of the maternal plant as well as resistance to the herbicide atrazine. To date, sexually mediated cybrid plants have shown no morphological abnormalities and have maintained their unique combination of cp and mt traits through several sexual generations.     

Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella

Summary Two commercial wheat cultivars with low embryogenesis efficiencies, AC Karma and Hy417, were transformed by the bombardment of isolated scutella with two gene constructs. Three AC Karma plants (433, 436, and 437) carrying plasmid pRC62 containing a gus:npt fusion gene, and one Hy417 plant (438) carrying plasmid pBARGUS containing a bar gene and a gusA gene were recovered and characterized. Presence of transgenes in T0 and T1 plants was confirmed by both PCR and Southern hybridization. Copy number of transgenes varied from one to six in these four plants. The inheritance of transgenes in the progeny was characterized. The gusA gene and its activity in AC Karma plant 436 and bar gene and its activity in Hy417 plant 438 segregated in the selfed T1 progeny in a Mendelian 3:1 ratio, but gusA gene and its activity in AC Karma plants 433 and 437 segregated in selfed T1 progeny in a non-Mendelian 1:1 ratio. The gusA activity in all three AC Karma plants was stably transmitted to selfed T2 or T3 progenies. The levels of gusA and nptII activities in nine T1 plants from AC Karma plant 437 were also determined. A GusA fluorometric assay indicated that gusA activity in the nine T1 plants increased by 2.5–7.2-fold compared with the nontransformed control, while and NptII ELISA assay detected nptII activity only in two of the nine T1 plants, suggesting the nptII gene was silenced in the other seven T1 plants.     

Engineering 2,4-D resistance into cotton

Summary To reduce damage by drift-levels of the herbicide 2,4-dichlorophenoxyacetic acid, we have engineered the 2,4-D resistance trait into cotton (Gossypium hirsutum L.). The 2,4-D monooxygenase gene tfdA from Alcaligenes eutrophus plasmid pJP5 was isolated, modified and expressed in transgenic tobacco and cotton plants. Analyses of the transgenic progeny showed stable transmission of the chimeric tfdA gene and production of active 2,4-D monooxygenase. Cotton plants obtained were tolerant to 3 times the field level of 2,4-D used for wheat, corn, sorghum and pasture crops.