Optimization Of Regeneration System For Phalaenopsis Aphrodite(Orchidaceae) And Transgenic Research About F3'5'H Gene And OOMT2 Gene

Phalaenopsis aphrodite is one kind of precious flowers,which has very high ornamental and commercial value.By transgenic methods of applying structural gene and regulator gene to p.aphrodite is one of the most effective means to create new varieties of p.aphrodite.In the society it lacks of not only blue kinds of p.aphrodite,but also other kinds which has unique flavor.So it is very important to create a new kind of p.aphrodite with new colour and unique flavor to improve its ornamental value.F3 '5'H gene is also called blue gene,it controls the pigments which blue flowers require—the synthesis of delphinium pigment,which makes flowers blue.The expression of rose fragrance OOMT 2 gene promote the formation of key enzyme of the aromatic fragrance volatiles 1,3,5-trimethylbenzene,to control the synthesis of rose fragrance.In the experiment,we choose F3'5'H gene and OOMT2 gene of the clone from Viola tricolor and R.chinensis as plant expression vector,using Agrobacterium turnefaciens transformation to study genetic transformation,takeing protocorm of p.aphrodite as acceptor material,to build a stable and high-efficient transgenosis system of p.aphrodite.The main results of this experiment are as follows.1.The optimizing of regeneration system of Phalaenopsis Aphrodite:take two commercial kinds of p.aphrodite named 'red dragon' and 'shine' as acceptor material,through different groups of plant growth regulator,different concentration ratios of plant growth regulator,the diameter of protocorm diced and activated carbon content and so on,optimizing transgenosis acceptor system of p.aphrodite.The results showed that:inoculate the seed of p.aphrodite to substrate of 3g/L hyponex 1+2g/L AC+2g/L LH+15g/L S+2mg/L 6-BA+0.2mg/L NAA+6.5g/L agar,cultivated about 60 days to choose the larger protocorm(diameter 0.4cm)to enrichment culture.The cutted protocorm ofp.aphrodite was the genetic transformation direct acceptor.Make sure the tangent plane of the protocorm diced small and the diameter of the protocorm diced largerer than 0.3cm to reduce the negative effect of the excessive damage of the protocorm diced;The proliferation of protocorm diced was in the substrate of 3g/L hyponex 1+3mg/L 6-BA+0.6mg/L 2,4-D+2g/L AC+15g/L S+6.5g/L agar.The intact protocorm in the proliferation medium continued to develop buds,then shift to the substrate of 3g/L hyponex 1+1.5mg/L IBA+20g/L S+lg/L AC+6.5g/L agar to promote rooting,contrast to kanamycin,the protocorm is more sensitive to homomycin.Only 8mg/L homomycin can restrain the proliferation of protocorm,but 10mg/L kanamycin can restrain the proliferation of protocorm,so we choose 8mg/L homomycin as acceptor material for resistance selection.2.Taking protocorm named 'shine' as acceptor material for Agrobacterium-medidted transformation,and optimize the transformation system.The results showed that:The protocorm which cultivated 3 days infected 15 minutes in the heacy suspension of OD600=0.4-0.6 is the best,then cultivater 3 days in the dark conditions,eluted acceptor material,later shifted to substrate of 8mg/L homomycin for cultivated selection.In this way to make sure keep acceptor material proper growth speed and effect of the selection.The protocorm of p.amabilis is not sensitive to cephamycin,but the concentration of cephamycin reached to 300mg/L will have a bad effect for proliferation of protocorm.Through the effect of bacteriostatic agent to protocorm and agrobacterium,choose 300mg/L cephamycin for Elution of Agrobacterium tumefaciens.Add 50mg/L cephamycin to the selected substrate.3.In this study,we got the protocorm that the Butterfly orchid 'shine' through the multiplication culture as the receptor material.It was cutted patch of the diameter>0.3cm,cultured 3days in the multiplication medium,after used Agrobacterium tumefaciens(Strains GV3101,contain plant expression vector pCAMBIAI301-OOMT2,pCAMBIA plasmid,GUS reporter gene and hygromycin resistance screening gene)heavy suspension infect receptor material.The receptor material was infected 15 minutes in heavy suspension of OD600 equal 0.4-0.6 so that the material transformation was finished.The material was dried on sterile filter paper and transferred in the multiplication medium of dark condition cultured 3d.Then took out the material,we used the sterile water of 300mg/L cephalosporins drug elution 2-3 times,after transferred the filter medium of 3g/L hyponex 1+3mg/L 6-BA+0.6mg/L 2,4-D+2g/L AC+15g/L S+8 mg/L Hyg+50 mg/L Cef+6.5g/L agar and filtrated 4 times,every time 15d.The resistant material of filter culture was transferred the multiplication medium of 50mg/L cephalosporins drug,until formed sterile sprout and grew 2-3cm,then transferred rooting medium of 50mg/L cephalosporins drug.At last we got 40 resistant sprout of resistant hygromycin,through the PCR detection,There were seven positive(2 for OOMT2 gene,5 for F3'5'H gene),combined with GUS tissue staining test results,we preliminary think the purpose gene has been integrated into the Phalaenopsis aphrodite.