A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both beta-glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.
For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.
Leaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of β-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 μF; and using 125 μg of calf thymus carrier DNA and 75 μ of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.
The raffinose family of oligosaccharides (RFO) is a series of complex carbohydrates stored in seeds of many plant families, especially in legumes. The digestive system of nonruminant animals, including that of humans, cannot break down all of the chemical bonds in these carbohydrates; therefore, catabolism is achieved anaerobically by intestinal flora. The resulting digestive problems reduce acceptance and limit the widespread consumption of these otherwise nutritious seeds. To demonstrate a solution to this problem, transgenic lines of pea ( Pisum sativum L.) expressing the α-galactosidase gene from coffee ( Coffea arabica L.) were developed. Plants with a single copy of the inserted gene were selected, and two of these lines showed significant reductions of up to 40% in oligosaccharide content (raffinose, stachyose). Quantitative RT-PCR confirmed the presence of the α-galactosidase RNA in both leaves and cotyledons. Sugars were analyzed using whole seeds or only a portion of a seed; in the latter case, germination rates for each of the seeds analyzed were determined. The reduced raffinose contents did not affect germination rates, which remained very high (96%). The relative oligosaccharide contents of tissues within a seed also were determined; these were highest in the embryo axis, lower in the cotyledon and lowest in the seed coat.
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