Studies On The Introduction Of Antisense ACS Gene To Narcissus Tazetta Var. Chinensis Mediated By Agrobacterium Tumefaciens

In this experiment,Chinese narcissus(Narcissus tazetta var.chinensis)as well as Agrobacterium tumefaciens strain LBA4404 were used as materials for first studies on the introduction of antisense ACS gene to Narcissus tazetta vat.chinensis mediated by Agrobacterium tumefaciens,which mainly included the selection of high-frequency in vitro regeneration approaches,the establishment and optimization of two classes of transgenic receptor system,both regenerated bulblets and calli,and the introduction of antisense ACS gene into Chinese narcissus mediated by Agrobacterium tumefaciens and the optimization of that transformation system,and resistant bulblets screening and plant regeneration,transient expression of GUS of gene-transformed slivers of regenerated bulblets and calli,and stable expression of GUS of regenerated leaves from gene-transformed slivers of regenerated bulblets and calli,and the gus gene PCR assay of leaves from transformed bulbs.The main results were as follows:1.The selection of high-frequency in vitro regeneration approaches of Narcissus tazetta var. chinensis.The slivers of bulbs,slices of ovaries as well as anthers were used as explants to carry out studies on the in vitro regeneration of Narcissus tazetta var.chinensis and choose the suitable explants as well as their high-frequency regeneration approaches.The preferable sterilization way for the bulbs was 0.1%mercuric chloride added with 3-5 drops of Tween-20.The ordinal effect of direct bulblet regeneration was slivers of bulb,slices of ovary and anthers on the all and the relative optimized medium of MS added with 0.1 mg·L^-12,4-D and 5.0 mg·L^-1BA,the ordinal effect of callus induction was slivers of bulbs,anthers and slices of ovaries on the all and the relative optimized medium MS added with 2.0 mg·L^-12,4-D and 0.1 mg·L^-1BA,and the ordinal effect of bulblet regeneration from calli was slivers of bulbs,slices of ovaries and anthers on the all and the relative optimized medium of MS added with 0.1 mg·L^-12,4-D and 5.0 mg·L^-1BA.2.The establishment and optimization of transgenic receptor systems for Narcissus tazetta var.chinensis.Integrating the former screening results with regeneration frequency as well as infection effects,both regenerated bulblets and calli were established as transgenic receptor systems.100 mg·L^-1and 110 mg·L^-1kanamycin concentrations were respectively suitable for transgenic operations on slivers of regenerated bulblets and calli,both whose bulblet differentiation rates were stably higher than 80.00%where the cepha1ycin concentration range was 0-300 mg·L^-1.There were three inoculation suitable phases for transgenic operations,namely September-October,November-December and January-March in the next year.The optimized growth regulator combinations for stably high-frequency regeneration of system slivers of regenerated bulblets was 5.0 mg·L^-1BA added with 0.1 mg·L^-12,4-D,while the effect - ranking factors on calli was KT afterward 2,4-D and BA,of which the bulblet differentiation rate was evaluated almost 6.00%presented in the combination of 5.0 mg·L^-1KT and 2.0 mg·L^-1BA, added with 0.1 mg·L^-12,4-D,where the efficient propagation coefficient is more than 12.00.The subculture period for system slivers of regenerated bulblets was 30 d and that of system calli was 15 d.1/2 bulblet(with slight-cut in the exterior edge)treatment was relatively suitable for the receptor system of slivers of regenerated bulblets used in transgenic operations,and the receptor system of calli was provided with strong ability of differentiation and regeneration,showing favorable genetic stability by cytology observation and analysis.3.The introduction of antisense ACS gene into Chinese narcissus mediated by Agrobacterium tumefaciens.Based on the above results,new transgenic strategies of both regenerated bulblets and calli were performed.The technical parameters of transgenic operations on two systems showed that the sameness was:1/2 MS liquid medium for diluents,a certain concentration of glucose for the activation of Agrobacterium tumefaciens,time,temperature,and pH values of coculture were respectively 4 d,25℃and 5.8,etc.On the other hand,the differences represented in preculture time(the former 6 d while the latter 10 d),pretreatment modes(the former partly acupunctured while the latter air-dried for 30 min and meanwhile partly acupunctured), suspension concentration OD₆₀₀values(the former 0.5 while the latter 0.3),the sucrose concentrations in suspension(the former 100 g·L^-1while the latter 80 g·L^-1),and components of infection liquid(the former suspension while the latter suspension added with some quartz), Agrobacterium inoculation time(the former 20 min while the latter 15 min),etc.The asepsis water with 300 mg·L^-1cephamycin was used in removing Agrobacterium turnefaciens.4.The resistant bulblets screening,plant regeneration as well as assays.During the screening course,the ordinal differentiation rate of resistant bulblets in different screening modes was intermittent mode,afterward gradual-adding mode and direct mode.While the ordinal stable expression rate of GUS in resistant bulblets from both slivers of regenerated bulblets and calli presented to direct mode,afterward gradual-adding mode and intermittent mode.As a whole,both differentiation rate and GUS stable expression rate of resistant bulblets in the receptor system of calli were higher than that in regenerated bulblets.After screening a total of six antisense ACS gene-transformed resistant bulblet lines were obtained.Moreover,the gus gene PCR assay of leaves from transformed bulblets showed that one of the lines appeared clearly visible strip,which might indicate gene transformed and conformed into the genome.Furthermore,the identified transgenic plants of Narcissus tazetta var.chinensis survived after and seedling training and transplant.