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.
Helena Mathews, C. Cohen, W. Wagoner, J. Kellogg, V. Dewey, and R. Bestwick
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.
Shin Je Kim, Kyung-Hee Paek, and Byung-Dong Kim
A cDNA clone of cucumber mosaic virus (CMV) 117 N-satellite RNA driven by the cauliflower mosaic virus (CaMV) 35S transcript promoter, was stably integrated into the genome of Petunia hybrida `Bluepicoti' tissues by Agrobacterium tumefaciens Ti plasmid-mediated transformation. Transgenic plants producing CMV satellite RNA showed delayed disease development when inoculated with CMV-Y, a helper virus for the I17N-satellite RNA. Furthermore, transgenic petunia plants showed delayed disease development against tobacco mosaic virus (TMV), a tobamovirus not related to CMV. Northern blot analysis revealed that large amounts of unit length satellite RNA (335 bp) were produced in CMV-infected transgenic petunia plants; whereas, mainly transcripts driven by the CaMV 35S promoter (approximately 1 kb) were produced in TMV-infected transgenic plants. SDS-PAGE and Western blotting showed that symptom reduction was correlated with a reduction in the amount of viral coat protein in transgenic plants.
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.
Mark E. Lewis and Fred A. Bliss
Ten common bean (Phaseolus vulgaris L.) lines—including cultivars, breeding lines, and one wild line—were evaluated for susceptibility to Agrobacterium tumefaciens strain C58 by stab-inoculating intact shoot tips of germinating seeds. Significant differences for tumor frequency and size were found on the resulting 3-week-old seedlings. UW 325, a wild bean, had the highest rate of tumorigenesis; `Olathe', a dry bean cultivar, had the lowest. Uninoculated excised shoot tips cultured in media with BA or BA plus NAA exhibited differences in phytohormone sensitivity, as evidenced by callusing and root initiation. The cultivar Montcalm seemed to be highly sensitive, while `Olathe' was relatively insensitive. Fluorometric GUS assays of shoot tips from germinating seeds inoculated with the disarmed GUS-containing A. tumefaciens strain C58C1(pGV3850/pKIWI105) showed that UW 325 had the highest level of GUS activity. `Montcalm' had a high rate of tumorigenesis but a low level of GUS activity; this anomaly was attributed to its high phytohormone sensitivity. The use of the virulence-inducing compound acetosyringone in the inoculum culture medium did not alter genotypic differences (ranks) in susceptibility. Histochemical GUS assays of inoculated UW 325 shoot tips showed that 60% of the apexes exhibited one or more transformation events. Chemical names used: β-glucuronidase (GUS); α-naphthaleneacetic acid (NAA); N-(phenylmethyl)-1H-purin-6-amine (BA).
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.
Mingbo Qin, Chiwon W. Lee, Alex Y. Borovkov, and Murray E. Duysen
A study was initiated to characterize key enzymes that influence sweetness in carrot (Daucus carota L.) roots. Sucrose synthase (SS), sucrose phosphate synthase (SPS), and UDP-glucose pyrophosphorylase (UDPL) genes were isolated from potato (Solanum tuberosum L.) and cloned in an anti-sense orientation into Agrobacterium tumefaciens Bin19, which has a CaMV 35S promoter. Seedling hypocotyl sections of selected carrot lines were pre-incubated on B5 medium for 2 days, co-cultivated with A. tumefaciens Bin 19 for additional 3 days, and then transferred to a modified B5 medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin. In 4 weeks, 18.6%, 33.3%, and 26.7% of the cultures from a breeding line (W204-C) were found to be transformed, respectively, with SS, SPS, and UDPL as determined by kanamycin resistance. In contrast, no kanamycin-resistant calli were obtained from a commercial cultivar (Navajo) in these transformation studies. The transformed calli proliferated in the medium containing 50 g/mL kanamycin and 400 g/mL carbenicillin, whereas non-transformed calli died in the same medium. These transformed calli are currently being used to regenerate plants via asexual embryogenesis using a suspension culture. The influence of these additional genes on sugar metabolism and accumulation in root tissues of transformed carrots will be characterized in the future.
J.D. Chung, B.J. Lee, H.S. Lee, and C.K. Kim
Lettuce (Lactuca sativa L.) were transformed using microparticle bombardment with two different genes, alpha-glucuronidase (GUS) gene and Chinese cabbage Glutathione Reductase (GR) gene. The adventitious shoots of cotyledonary explant from 4-day-old seedlings were formed (46.7%) in MS basal media supplemented with 5.0 μm IAA and 1.0 μm 2ip. When 1100 psi helium pressure, 9 target distance, and coating with tungsten 10 microparticles were used and explants were treated with osmoticum-conditioning medium (0.6M sorbitol/mannitol), 4 h prior to and 16 h after bombardment, it was identified by GUS assay that these conditions were the most efficient for transformation of foreign genes into cotyledon tissue of lettuce with particle bombardment. PCR confirmed that the band observed in the transgenic plants were originated from T-DNA tranfer with strong hybridization. The genomic Southern analysis showed that the 1.5-kbp fragment was hybridized with radiolabeled 1.5-kbp GR probe. To know whether the expression of the GR gene can be stably maintained in the next generation, when T2 selfing seeds that were obtained from the transformed mother plants were sowed on MS medium supplemented with 200 μm kanamycin, 70% of seedlings were revealed resistance to kanamycin.