Gene transfer can provide plants with a novel source of disease resistance. Two different antibacterial peptides, Shiva-1 and lactoferrin, were tested in vitro for antibacterial activity. The former is from cecropin B in insects, and the latter from human or mammal fluids such as milk. Both peptides exhibited high antibacterial activity against all tested gram-negative phytopathogenic bacterial strains. Lactoferrin was more lethal than Shiva-1. A particular lactoferrin domain showed a much higher activity against bacterial strains. A gene encoding lactoferrin was then transferred to Nicotinia tabacum L. xanthi-nc to evaluate the gene expression using Agrobacterium. Stable transformation was confirmed by Southern, Northern, and Western blot analysis. Delayed wilting of the transgenic plants inoculated with Pseudomonas solanacearum was observed. A significant positive relationship between the gene expression levels and resistance was also found by either Northern or Western blotting. Biolistic transformation using a gene gun is currently underway to transfer this novel gene to common beans.
Zhanyuan Zhang, D.P. Coyne, and A. Mitra
C.S. Prakash, U. Varadarajan, and A. S. Kumar
Development of a gene transfer system will enable rapid introduction of agronomically useful genes into elite cultivars of sweet potato. We compared microprojectile bombardment and Agrobacterium cocultivation approaches to introduce foreign genes into the genome of two sweet potato cultivars. Chimeric marker genes (gusA and kan) were successfully introduced into cvs. Jewel and TIS-70357 using both approaches. However, transgenic plants were generated in vitro using only the Agrobacterium approach. Callus and root isolates with stable expression of gusA gene were obtained using the microprojectile method. Expression of the screenable marker gusA gene was detected by histochemical assays. Integration of the introduced gene into the genome of sweet potato was confirmed by polymerase chain reaction (PCR) amplification of the kan gene and Southern blot analyses. Transgenic sweet potato plants from two cultivars are being raised and studied for quantitative expression and localization of the introduced genes. These results show that foreign genes can be successfully introduced and expressed in sweet potato. Current efforts are directed at optimizing several variables to increase the transformation efficiencies and to generate transgenic cultivars with foreign genes of agricultural importance.
Jim Sellmer, Dave Ellis, Brent McCown, Dennis McCabe, Dave Russell, and Brian Martinell
Successful recovery of plants transformed by direct gene transfer techniques relies on 3 factors: 1) a regenerable cell/tissue culture system, 2) a foreign DNA delivery system which can be fine tuned, and 3) a cell population receptive to the transfer and integration of foreign DNA into its genome. Cell receptivity to foreign DNA incorporation is being determined by bombarding poplar cells with gold particles coated with plasmid DNA containing a CaMV35s pro–meted β-glucuronidase chimeric gene construct. Histochemical GUS expression assays conducted after bombardment show that early log phase cells are most receptive with a transient transformation rate of 0.26% compared to 0.08–0.04% for mid log and stationary phase cells. Furthermore, cell receptivity in early log phase cells is decreased when cytokinin is removed from the culture medium for 3 growth cycles prior to bombardment. This response suggests that plant growth regulators may be used to enhance cell receptivity along with cell synchronizing agents.
Sandip Mukhopadhyay and Yves Desjardins
Transient expression of electroporation-mediated DNA uptake was monitored in callus-derived protoplasts of two asparagus (Asparagus offcinalis L.) genotypes by measuring the GUS activity. The level of expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. An increased plasmid DNA concentration and the presence of polyethylene glycol (PEG) in the electroporation medium enhanced the transient expression level. A considerable increase in GUS activity was observed in the presence of both PEG and heat-shock treatments than with PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration. The two genotypes differed in their responses in vitro and also showed variable levels of transient expression. The present technique was suitable to obtain transgenic plants, as histochemical GUS assay revealed GUS activity in the protoplast-derived microcolonies as well as in callus tissues.
Cucurbita ecuadorensis is a valuable source of multiple virus resistance. It is resistant to zucchini yellow mosaic virus (ZYMV), papaya ringspot virus (PRSV), watermelon mosaic virus, tobacco ringspot virus, squash mosaic virus, and cucumber mosaic virus (CMV). Its virus resistance can be transferred to squash and pumpkin, but sterility barriers must be overcome. The cross Cucurbita maxima× C. ecuadorensis can readily be made, and there is no need for embryo culture. Pollen fertility of the hybrid is somewhat reduced, but sufficient for producing F2 seed. Segregation for sterility occurs in the F2, but selection can be made for fertile plants that are homozygous for virus resistance. Cucurbita ecuadorensis is much more distantly related to C. pepo than to C. maxima, and there are more formidable barriers in this interspecific cross. The cross is very difficult to make with some C. pepo cultivars, but other cultivars are more compatible. Viable seed were not produced, but hybrid plants were obtained by embryo culture. Although both parents were monoecious, the hybrid was gynoecious. Male flower formation was induced by treating the hybrid with Ag or GA, but they were male-sterile. F2 seed was not obtained, but backcross seed was easily produced by using the interspecific hybrid as the maternal parent in crosses with C. pepo. The most refractory barrier was achieving homozygosity for ZYMV resistance. Disturbed segregation occurred in succeeding generations and the progeny of most resistant plants segregated and were not uniform for resistance. This and other barriers to interspecific gene exchange were overcome and a summer squash variety homozygous for resistance to ZYMV, PRSV, and CMV is being released this year.
Ravindra K. Hajela, Neerja Hajela, Mark G. Bolyard, Wayne M. Barnes, and Mariam B. Sticklen
A simple gene transfer method based on Agrobacterium -mediated transformation of adventitious multiplication of Juneberry (Amelanchier laevis L.) basal shoots is described. Evidence is presented for successful integration and expression of a transformed gene in greenhouse-grown transgenic plants. This method can transform woody perennials that are difficult to regenerate from leaf disks, protoplasts, or other tissue culture regimens.
Fure-Chyi Chen and Adelheid R. Kuehnle
Several procedures were tested in development of a gene transfer protocol for anthurium. Etiolated internode segments of anthurium cultivars `Rudolph' and `UH1060' were co-cultured with Agrobacterium tumefaciens LBA4404 carrying the chimeric genes neo, for antibiotic resistance, and att encoding antibacterial attacin. Assays of genomic DNA and RNA from kanamycin-resistant `Rudolph' and DNA from `UH1060' plantlets, recovered as soon as 1 year after culture on selection media, indicated the presence of introduced genes, including neo and att, and transcription of att. Western analysis confirmed the expression of attacin protein in calli induced from laminae of regenerated kanamycinresistant `Rudolph' plantlets. Use of tobacco nurse cells during co-cultivation of internodes with Agrobacterium did not increase recovery of shoots under the regeneration conditions used. Improvements in culture and antibiotic selection conditions during plant development are suggested.
D.M. Shuh and James F. Fontenot
The inheritance of multiple flowers and leaf pubescence resulting from the crosses between accessions from pepper species Capsicum annuum L. and C. chinense Jacq. was examined. Hand cross- and self-pollinations were made in a glass greenhouse. Only eight normal F1 plants were obtained from crosses between the two species when C. annuum L. was the female parent. F2 and backcross generations obtained from the F, and the two parents were grown in the field. Two field studies indicated that multiple flowers and leaf pubescence were controlled by dominant genes. A three-gene model leading to an F2 segregation ratio of 45:9:10 and a two-gene model leading to an F2 segregation ratio of 13:3 were suggested for the inheritance of multiple flowers and leaf pubescence, respectively. Epistasis was evoked in the interpretation of the data. No linkage was found between the two characters. The inconsistencies between F2 and backcross data might be due to selective elimination of genes from one or the other parent in an Interspecific hybridization. Segregation ratios from intraspecific crosses for leaf pubescence supported a two-gene model and gave an F2 ratio of 13 pubescent leaf : 3 glabrous leaf progeny.