| Perennial ryegrass(Lolium perenne L.) is not only a kind of high-quality forage, but also a kind of turfgrass used for ornamental lawn and sport turf.Based on the difficulty of weed control on turf establishment and management,disadvantages of drought sensitivity and short green stage of cool-season turfgrass,bivalent herbicide-resistant gene and drought-resistant gene were transformed into perennial ryegrass to obtain new lines with two resistance.In this thesis,taking some good varieties of perennial ryegrass such as "Accent" and "Kutter" as explants,the initiation and regeneration system at very high frequency for embryogenic callus and the selection scheme for tolerant callus were established and modified,and the transformation method mediated by Agrobacterium for callus of these perennial ryegrass were optimized too.By using the Agrobacterium-mediated transformation system,bivalent herbicide-resistant gene(bar) and nicotianamine synthase gene(nas) were introduced into the callus of perennial ryegrass and 20 tentative transgenic lines were obtained.PCR,RT-PCR and Western blot analysis showed that target genes were integrated into the genome of these transgenic plants. Through identification,the transgenic plants were more resistant to Basta and drought than non-transgenic plants.1.The initiation and regeneration system for embryogenic callus of perennial ryegrass were established and optimized at very high frequencyThe result showed that the half-seeds with embryo were the best explants for callus induction.Compared to other explants such as mature seeds,the frequency of calli induction and buds differentiation from half-seeds with embryo could reach to 86%and 85%.MS medium was the best basic medium for induction and subculture of the calli among all the media tested.Calli induction frequency raised with the rise of 2,4-D concentration,and 8.0 mg/L was suitable.In order to reducing the damage of high 2,4-D concentration on the growth of calli and buds differentiation,2,4-D concentration was decreased on the calli subculture medium.Mannitol could improve the quality of calli and promote the formation of embryonic calli.Buds induced on the MS medium were strong and robust,while those induced on the NB medium were thin and close.The whole regeneration process was:the calli induced on medium of MS+2.4-D8.0 mg/L(with mannitol 25 g/L or not) were subcultured on medium of MS+2.4-D8.0 mg/L+ mannitol 25 g/L for 1 to 2 months,then cultured on medium of NB+6-BA2.0 mg/L to induce adventitious buds.Adventitious buds can be extending propagated on MS+6-BA2.0 mg/L or induced roots on medium of 1/2MS+NAA0.5mg/L+IAA0.5mg/L.2.Transgenic perennial ryegrass strains were obtained by Agrobacteriummediated transformation with bivalent bar and has genesPlant expressing vector with bivalent bar gene and nas gene was constructed.Nas gene was cloned into a prokaryotic expression vector pGEM-KG to express the recombinant protein pKG-NAS,the polyclonal antibody of NAS was prepared.The transformed methods mediated by Agrobacterium tumefaciens were optimized.In the experiment,the Agrobacterium tumefaciens strain EHA105 was more efficient than LBA4404 for perennial ryegrass transformation.The quality of receptor could be improved through subculturing the calli on subculture medium with mannitol.Taking filter papers dipped with liquid co-culture medium as co-culture carrier instead of solid co-culture medium,it was beneficial to inhibit the growth of Agrobacterium,maintain the quality of calli,increase transformation efficiency and simplify the procedure.The selection scheme for tolerant calli and buds of perennial ryegrass was established.The suitable selection scheme was culturing the callui and buds on the medium containing 7.5-10 mg/L Basta for 40-60 days.By using the Agrobacterium-mediated transformation system,bivalent bialaphos resistance gene(bar) and nicotianamine synthase gene(nas) were introduced into the calli of perennial ryegrass and 20 transgenic lines were obtained through selection with Basta.PCR,RT-PCR and Western blot analysis showed that target genes were integrated into the genome of these transgenic plants.3.Resistance identification of transgenic plants to herbicide and droughtThrough identification by spraying Basta solution on the leaves,the transgenic plants were more resistant to Basta than non-transgenic plants,could resist 150-200mg/L Basta.Drought resistance identification with repeated drought showed:The transgenic plants were more resistant to drought than non- transgenic plants,having longer green stage;the non- transgenic plants lost water seriously,died at last.Under natural drought conditions,the carrying-water ability,whole plant growth speed,root growth speed,relative water content of leaves,chlorophyll content of transgenic plants were higher than those of non-transgenic plants.On the process of drought treatment,the malondiadehyde(MDA) content,Proline(Pro) content,superoxide dismutase (SOD) activity,peroxidase(POD) activity,catalase(CAT) activity,leaf water potential(LWP) and relative electrical conductivity(LEC)showed certain variation laws and changing trends. |
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