Conditions for Agrobacterium transformation of asparagus embryogenic suspension cells were investigated using an intron-containing GUS gene in pCNL56 to detect transformation events. Embryogenic suspension cultures of Rutgers 22 were maintained on LS medium with 5 mg NAA/liter, subcultured weekly, and used 5 days thereafter. For initial experiments, cells were inoculated at 5 x 108 cfu/ml for 15 min and cocultivated for 4 days on LS medium with 10 g Phytagel/liter (EM1 medium). Subsequently, the cells were transferred to EM1 with 200 mg Timentin/liter and tested for GUS expression after a total of 6 days. The effect of acetosyringone (AS) on four A. tumefaciens strains was tested. With or without AS induction, EHA105 and C58C1(pMP90) produced a significantly greater number of GUS foci on embryogenic cells than C58C1(pGV2260) and LBA4404. Transient expression was very low from cells inoculated with C58C1(pGV2260) and nonexistent from LBA4404. AS induced significantly more GUS foci from EHA105 and C58C1(pMP90) than from the same noninduced bacteria; however, it had no effect on C58C1(pGV2260). Upon repeating the experiment using EHA105 and C58C1(pMP90) only, no differences in response were observed, although AS induced more GUS foci from both strains and EHA105 outperformed C58C1. Inoculum density was investigated using induced EHA105. 5 x 107 cfu/ml significantly increased the number of GUS foci than 108, 5 x 108, or 109, although it was not statistically different from 107, which produced slightly fewer foci.
Roger A. May and Kenneth C. Sink
Wendy Wagoner, J. Stamp, H. Matthews, J. Kellogg, and R. Bestwick
Ethylene is a known causal factor in the decay and senescence of fruits and vegetables. The aim of the present study was to incorporate a gene for control of ethylene biosynthesis in order to prevent or delay the senescence of the cauliflower curds. We first developed a reproducible transformation system using marker genes for beta glucuronidase (GUS) and antibiotic resistance. Brassica oleraceae L. var. botrytis was transformed by inoculating hypocotyl explants with the Agrobacterium tumefaciens strains C58 or EHA101 containing plasmids pAG5110, pAG5420, or pAG5520. The plasmid pAG5110 contains the genes for neomycin phosphotransferase II (NPTII) and GUS. The plasmids pAG5420 and pAG5520 contain a functional gene for S-adenosylmethionine hydrolase (SAMase) under an ethylene or wound inducible promoter, respectively. Hypocotyl explants were screened on regeneration medium with kanamycin for selection of transformants. Shoot regeneration occured within 4-6 weeks and morphologically normal plants developed within 3-4 months. The transgenic nature of the plants was confirmed by histochemical GUS assay, an ELISA based NPTII assay and Southern blot analysis. Transgenic plants outplanted in the greenhouse are being evaluated and selfed to study expression and inheritance pattern of the introduced trait.
Helena Mathews, C. Cohen, W. Wagoner, J. Kellogg, V. Dewey, and R. Bestwick
We have developed efficient plant rageneration and transformation systems for red raspberry (Rubus idaeus L.). We have successfully introduced a gene for controlling biosynthesis of ethylene into raspberry for the first time. Leaf and petiole segments were co-cultivated with disarmed Agrobacterium strains EHA 101 or 105 containing plasmids pAG5420, pAG 1452 or pAG1552. The plasmids encoded gene sequences for S-adenosylmethionine hydrolase (SAM ase) driven by the fruit specific or wound and fruit specific tomato SE8 or E4 promoters. SAM ase catalyses the conversion of S-adenosylmethionine (SAM) to methylthioadenosine (MTA) and homoserine which can reenter the methionine recycling pathway. SAM is therefore not available for the synthesis of 1-am inocyclopropane carboxylic acid (ACC), the metabolic precursor for ethylene biosynthesis. Initial shoot regenerants were mostly chimeras containing transformed and non-transformed cells. Solid clones of pure transgenics were developed by repeated culture of leaf, petiole and nodal explants of primary regenerants on higher stringency selection medium. Transformants were screened on medium with kanamycin, geneticin or hygromycin depending on the selection marker gene NPTII or hpt. Genomic integration of transgenes were confirmed by Southern hybridization. Transgenic plants of cultivars Canby, Meeker and Chilliwack have been transplanted to the greenhouse for fruit set and further evaluation of transgenic traits.
Jyothi Prakash Bolar, Susan K. Brown, John L. Norelli, and Herb S. Aldwinckle
The overall goal of our research is to develop an efficient transformation and regeneration system for `McIntosh' apple. The first objective was to determine the optimum combination of Gelrite (G) and agar (A) to maximize regeneration and minimize vitrification. Treatments included the following combinations of agar (in g–liter–1) and Gelrite (in g–liter–1): 1) 7 and 0; 2) 5.25 and 0.625; 3) 3.5 and 1.875; 4) 1.75 and 1.875; and 5) 0 and 2.5. There were 10 replications, and a single petri plate containing six leaf pieces was the unit of replication. Both 5.25(A) and 0.62(G) and 3.5(A) and 1.25(G) provided high regeneration of healthy, nonvitrified shoots. Since modification of media affects the concentration of antibiotics used in selection due to precipitation of antibiotics, the second objective was to determine the optimal concentration of antibiotic for the selection and regeneration of transformed `McIntosh' on gelrite–agar-based media. Kanamycin was tested at 0, 10, 25, 50, 75, and 100 μg–ml–1 and paromomycin was tested at 0, 50, 100, 150, 200, and 250 μg–ml–1. Antibiotic selection will be discussed relative to optimum concentration and efficiency of selection.
Hak Tae Lim, Y.S. You, and E.J. Park
In order to regenerate explants of Brassica campestris ssp. pekinensis, known to be one of the most difficult crops to regenerate via organogenesis, three different explants, cotyledon, hypocotyl, and leaf, were cultured on MS basal medium supplemented with several plant growth regulators. In the medium containing NAA at 0.5 mg/L and BAP at 3.0 mg/L, the shoot regeneration, when hypocotyl was used as explant, was found to be quite effective. In the case of cotyledon, the most suitable combination of plant growth regulators was NAA at 1.0 mg/L and BAP at 3.0 mg/L. Treatment of AgNO3 (1.0 mg/L) for shoot regeneration gave positive results in general. Zeatin at 2.0 mg/L was very effective in shoot induction of leaf explant, especially when combined with BAP at 2.0 mg/L, NAA at 1.0 mg/L, and AgNO3 at 0.5 mg/L. A system to produce transgenic plants in Brassica spp. has also been developed using hypocotyl and cotyledonary-petiole segments and shoot-tips. An explants from 4-day-old seedlings were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimaeric bacterial gene encoding hygromycin and kanamycin resistance, along with other genes of interests. The explants were co-cultured for 2 to 6 days before transfer to hygromycin and kanamycin selection media. Shoots regenerated directly from the explants in 1 to 4 weeks and were excised, transferred to shoot elongation medium, rooted in root induction medium, and planted in soil. Genetic transformation was confirmed by kanamycin or hygromycin resistance, GUS activities, and Southern blotting.
Elvia C. Palacios-Torres, M. Alejandra Gutièrrez-Espinosa, Gloria A. Moore, Gustavo Mora-Aguilera, Daniel L. Ochoa-Martínez, and Angel Villegas-Monter
Citrus Tristeza Closterovirus (CTV) induces mild and/or severe symptoms on Citrus species. It may cause death of trees if the rootstock-scion combination is susceptible. It has been found in other plant/virus combinations that transformation with partial or complete viral genes (e.g., coat protein genes) can confer resistance to the resulting transgenic plants. We previously reported A. tumefaciens mediated transformation and production of two sour orange (C. aurantium L.) plants expressing the coat protein gene of CTV, which was the first report of production of transgenic Citrus using a viral gene. However, in order to properly evaluate resistance, it is necessary to obtain as many transgenic Citrus plants from single transformation events as possible. Therefore, we are currently transforming grapefruit (Citrus paradisi) `Marsh' and `Star Ruby' and sweet orange (C. sinensis) `Valencia' with CTV coat protein genes. These species are susceptible to CTV and more amenable to transformation than sour orange. Epicotyl segments of etiolated seedlings were inoculated with A. tumefaciens strain EHA101 harboring binary plasmid pGA482GG containing the coat protein gene of mild Florida CTV strain T30 (CP-T30) or severe Florida strain T36 (CP-T36). Putatively transformed shoots were regenerated on selection medium containing kanamycin. Regenerated shoots were evaluated with GUS assays; those shoots positively identified by GUS were then evaluated with PCR. We have currently identified 17 `Marsh' grapefruit, 20 `Star Ruby' grapefruit, and seven sweet orange putatively transformed plants.
Hak-Tae Lim, Kei-youn Lee, Yeoung-Sook Yoo, and Duck-Chun Yang
Since in vitro regeneration and transformation systems in hot pepper (Capsicum annuum L.) have not been available, the application of new genetic manipulations has been limited. Here we report an efficient procedure to regenerate whole pepper plants and to generate transgenic plants expressing a foreign gene was established. High frequency of plant regeneration was observed when hypocotyl and cotyledon explants were cultured on MS/B5 medium supplemented with NAA 0.05 mg·L–1 plus zeatin 2.0 mg·L–1, NAA 0.05 mg·L–1 plus zeatin 2.0 mg·L–1, IBA 10.0 mg·L–1 plus BA 1.0 mg·L–1, IAA 0.02 mg·L–1 plus zeatin 3.0 mg·L–1. An addition of AgNO3 5–10 μm to these media improved the regeneration rate by about 10%. For plant transformation, hypocotyl and cotyledon explants of pepper were preconditioned on kanamycin-free shoot induction medium for 48 hours. Then, co-cultivation with Agrobacterium tumeaacience was done on the co-culture medium for 2 days. The explants were then blotted in sterile filter paper and placed on shoot induction and selection medium containing kanamycin sulfate (100 mg·L–1) and carbenicillin (500 mg·L–1). PCR showed that the introduced ADA gene was integrated and stably expressed in the regenerated plants. ADA enzyme activities were checked by spectrophotometric analysis.
Violeta Colova-Tsolova, Rachel Gollop, Sharon Farchie, Sylvie Even, Nahman Shahar, and Avi Perl
Embryogenic cell suspension was developed from in vivo anthers of seedless grape cv. Sugarone. Agrobacterium genetic co-transformation was realized with two vectors carried respectively two different reporter genes: hpt and nptII, and three (1+2) agronomicaly beneficial genes encoding for proteins that are involved in fungus disease resistance. The effciency of transformation procedure and integraton of foreign genes was verified by hystochemical assay as a first step after insertion in embryogenic suspension two different constructs with Gus-reporter gene under control of different promotors. PCR assay and Southern blot analysis were used to confirm the co-transformation in regenerated grape plants.
Hassan Salehi, Zahra Seddighi, Alexandra N. Kravchenko, and Mariam B. Sticklen
Bermudagrass (Cynodon L.C. Rich.) is grown on more than 4 million ha in the southern United States. The black cutworm (Agrotis ipsilon Hufnagel) is the most commonly encountered pest of bermudagrass, especially on golf course greens. Developing insect-resistant cultivars is a very desirable substitute, both environmentally and economically, to using current synthetic pesticides. Here we report, for the first time, Agrobacterium-mediated transformation of `Arizona Common' common bermudagrass [Cynodon dactylon (L.) Pers.] with the Bacillus thuringiensis Berliner cry1Ac gene encoding an endotoxin active against black cutworm. Mature seeds were used for producing embryogenic callus, and calli were transformed with a plasmid containing a synthetic cry1Ac and the kanamycin resistance (nptII) genes. Putative transgenic calli and plantlets were selected on media containing 100 and 50 mg·L-1 G418, respectively. RNA-blot analysis of PCR-positive lines revealed the expression of the cry1Ac transgene in three out of five putative transgenic lines. The larvae fed on transgenic plant leaves experienced highly significant (over 80%) mortality.
Ana Cristina M. Brasileiro, Francisco J. Lima Aragão, Sílvia Rossi, Diva Maria A. Dusi, Leila M. Gomes Barros, and Elíbio L. Rech
To develop an efficient protocol for Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.) and tepary bean (P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.