negative control ( Zheng et al., 2009 ). MdMYB4 overexpression vector construction and apple calli transformation. The complete sequence of the 35S promoter was amplified from the pBI121 vector ( Supplemental Table 2 ) and cloned into the pCAMBIA1304
Ruigang Wu, Yi Wang, Ting Wu, Xuefeng Xu, and Zhenhai Han
Kenneth R. Schroeder, Dennis P. Stimart, and Erik V. Nordheim
Nicotiana alata Link and Otto (Jasmine tobacco) was transformed with an autoregulated senescence-inhibition gene construct PSAG12-IPT encoding isopentenyl transferase via Agrobacterium-mediated transformation. Transformation was confirmed by polymerase chain reaction. Transgenic plants exhibited up to 2- to 4-fold fewer senesced leaves, 29% longer in situ flower life, 26% more shoot dry weight, and a 32% to 50% reduction in flowers per branch. Additionally, transgenics were 28% shorter and had up to 174% more branches, indicative of cytokinin overproduction and a lack of tight autoregulation of PSAG12-IPT. Variation among independent transgenics suggests selection for enhanced PSAG12-IPT is feasible. Our observations of increased branching and in situ flower longevity, as well as reduced plant height and flowers per branch provide new information on PSAG12-IPT and its potential value for biological study and horticultural application.
Jeung-Sul Han* and Chang Kil Kim
A procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAMBIA3301, which contains glufosinate ammonium-resistant (bar) and the reporter (gus) genes, is describe. Infection was the most effective (highest infection frequency and index) when explants were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.001-0.1 mg/L L-a-(2-aminoethoxyvinyl) glycine (AVG). Transgenic plants were obtained with frequencies of about 0.2% when the explants were cultured on selection medium (MS medium supplemented with 3.0 mg/L BAP, 0.5 mg/L AgNO3, 500 mg/L cefotaxime, 2.0 mg/L DL-phosphinothricin, 0.3% sucrose and 0.8% Plant Agar. A histochemical gus assay, PCR and Southern blot analyses confirmed that transformation had occurred. Genetic analysis of T1 progenies showed that the transgenes were inherited in a Mendelian fashion. To our knowlege, this study represents the first report for Agrobacterium-mediated transformation in bottle gourd, rootstock for watermelon and other cucurbit crops in many countries.
Taly Trainin, Alexander Lipsky, Avraham A. Levy, and Doron Holland
The maize transposable element Activator (Ac) has been shown to be active in a number of dicots, including arabidopsis [Arabidopsis thaliana (L.) Heynh.], tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), and aspen (Populus tremuloides Michx.). However, no information is available on somatic transposition in any plant during several years of growth and development. It is not known how transposition affects genetic variability among vegetative parts that have developed during a long period of growth. In order to explore the possibility of using somatic Ac transposition for gene tagging and mutagenesis in fruit trees, a derivative of the maize Ac transposable element was introduced into `Duncan' grapefruit (Citrus paradisi Macf.) by Agrobacterium tumefaciens (Smith & Towns.) Conn.-mediated stable transformation. Genetically identical 4-year-old sibling trees were established by grafting one of the transformants on Troyer citrange [Citrus sinensis (L.) Osbec. × Poncirus trifoliate (L.) Ras.] rootstocks. We demonstrated that the Ac element was active upon transformation in citrus (Citrus L.) trees and that transposition can create genetic variability among tree siblings and among leaves collected from different parts of the same tree. Ac was still active among propagated plants 4 years after transformation, clearly indicating that it is capable of maintaining itself in citrus trees for a long period of time. The observation of different integration patterns in different parts of the same tree and within tree siblings originating from the same transformant suggests that an Ac-based mutagenesis system could be very useful in creating somatic mutations in citrus trees.
Charleen M. Baker and William E. Dyer
Our goal was to develop efficient regeneration protocols for safflower that could be used in conjunction with Agrobacterium tumefaciens -mediated transformation to introduce genes conferring economically important traits. Direct regeneration of whole plants has been achieved from cotyledon and hypocotyl explants of 30-day-old `Centennial' and `Montola' seedlings. Explants transformed with Ti plasmids containing NPTII and the β-glucuronidase (GUS) reporter gene produced kanamycin-resistant calli and shoots testing positive for GUS activity. Current work is incorporating the bar gene into appropriate Ti plasmids that will be used to confer glufosinate herbicide resistance to elite safflower cultivars. An esterase gene from Bacillus subtilis will be introduced to confer resistance to Alternaria carthami leaf spot disease.
A. Virginia Freire, David A. Lightfoot, and John E. Preece
Efficient genetic transformation could enhance coffee breeding, which is limited by its long generation time and narrow genetic base. Three explant types of three coffee cultivars were inoculated with 14 strains of Agrobacterium spp. Callus and hairy roots were produced with 13 of the 14 strains tested. With A. tumefaciens, nopaline strains were more effective than octopine strains. Cucumopine and mannopine strains of A. rhizogenes were both effective in inducing hairy roots and callus. PCR amplification of a 0.72 Kb fragment of T-DNA encoding a portion of the ipt gene was achieved with DNA from A. tumefaciens strain A208 and with putatively transformed tissue inoculated with A208. No amplification was observed with virB in putatively transformed tissue which indicates it was not contaminated with Agrobacterium. We conclude that coffee can be genetically transformed by some Agrobacterium strains.
Hak-Tae Lim, Haeng-Soon Lee, and Tage Eriksson
Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.
Yali Liu, Fuling Hao, Rui Meng, and Weirong Xu
The enzyme ACC oxidase (ACO), encoded by a small multigene family in many plants, catalyzes the terminal step in the ethylene biosynthesis pathway. In this research, based on the total RNA isolated from the flowers of Asia hybrids `Pollyanna' and Oriental hybrids `Sorbonne', we obtained two cDNA fragments of ACO genes (Genbank accession DQ062133 and DQ062134) by RT-PCR technique. The two cDNA fragments were reversely inserted into plant expression vector pWR306 respectively, and constructed two antisense ACO gene expression binary vectors harboring hygromycin phosphotransferase (hptII), glucuronidase (uid A), and a green fluorescent protein (GFP) gene in the T-DNA region. We have developed a system to produce transgenic plants in LiLium via Agrobacterium tumefaciens-mediated transformation of calli. Transformants were subjected to GFP expression analysis, PCR assay, and Southern hybridization to confirm gene integration.
Roger A. May and Kenneth C. Sink
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.
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.