The Establishment Of Open Tissue Culture System Of Gerbera Jamesonii Bolus And The Cloning And Analysis Of Its Ferredoxin Genes

Gerbera(Gerbera jamesonii Bolus),one of the five most famous cutting flowers,its demand is huge in the world.Plant tissue culture is commonly used method for the rapid propagation of gerbera.However,with the expanded production scale of gerbera tissue culture,the high cost problem is still difficult to solve.The open tissue culture,achieve high efficiency of large-scale commodity production,which can greater reduce the cost compared with plant tissue culture.In recent years,facilities cultivation provide a good environment for the growth of gerbera at the same time,also provide favorable conditions for cold resistance,breeding of plant diseases and insect pests,leading to the outbreak of large-scale disease and pests.A direct result of the production of gerbera cut flowers is low,and poor quality,leading to the huge blow of the gerbera industry.Ferredoxin(Fd)gene,as a large gene family,is prevalent in plants and is closely related to the accumulation of ROS in plants,which helps plants to achieve broad-spectrum resistance.In this study,we established the open tissue culture system of gerbera,and analyzed the genetic stability of gerbera plantlets under open tissue culture and traditional tissue culture systems by ISSR.In addition,cloning and expression analysis of Fd genes family based on the database of gerbera transcriptome to improve the disease resistance of gerbera.The principal results were as follows:1 Establishment of open tissue culture system of gerberaFour kinds of antimicrobial agents,including sodium hypochlorite,mancozeb,carbendazim,potassium sorbate,were firstly selected for testing in the gerbera open tissue culture.Results showed that 0.008%～0.014%sodium hypochlorite is the most suitable bacteriostatic agent.And the best proliferation medium of gerbera open tissue culture is MS+0.5 mg/L 6-BA+0.05 mg/L NAA+25 g/L sucrose+ 0.008%sodium hypochlorite,its proliferation coefficient of gerbera plantlets is 3.98,which higher than that in the traditional tissue culture seedling of 3.87.The best rooting medium of gerbera open tissue culture is 1/2 MS+0.1 mg/L NAA+30 g/L sugar+0.008%～0.014%sodium hypochlorite,its root number is 3.73,slightly lower than that in the traditional tissue culture system(3.90).After acclimatization for two days,more than 98.8%of the hardened gerbera plantlets survived after transplanting.And substrate with perlite and vermiculite by 1 to 1 was the optimal.Gerbera open tissue culture system can save 39,808.5 RMB per year compared with traditional tissue culture,accounting for 3.3%of the total cost of tissue culture.2 Analysis of genetic stability of gerbera plantlets under open tissue culture and traditional tissue cultureIn this study,the genetic stability of gerbera plantlets from the open tissue culture and the traditional tissue culture were studied.And seven ISSR primers were selected to analyze the DNA of these plantlets.The results showed that the genetic similarity coefficient of 12 gerbera DNA was between 0.7083-1.0000.The first generation of the traditional culture has the highest similarity coefficient with the sixth generation of the open culture,which is 1.0000;the first generation of the traditional culture has the lowest similarity coefficient of the eleventh generation of the open culture,which is 0.7083.Comparison analysis showed that gerbera plantlet of open proliferation and traditional proliferation did not show significant differences;and the similarity coefficient of gerbera after several generations culture did not change significantly.These results proved that the antimicrobial agent(sodium hypochlorite)could not affect the genetic variation of plantlets in DNA molecular level,and also showed that the gerbera plantlets maintained stable genetic characteristics under the process of successive transfer.Thus,open tissue culture technology is feasible for gerbera factory seedling.3 Cloning of Fd family gene based on the database of gerbera transcriptome and expression analysis of gerbera plantlets in different subculturesThe resistance of the tissue culture of gerbera has important theoretical and practical significance,and the ferredoxin gene has broad spectrum resistance.Gerbera leaves of ’Linglong’ was used as material in this study.Five fragments of Fd gene were screened according to the transcriptome data of gerbera,named as MFDX1(KY914548),Fd3(KY914549),Fld3(KY914550),Fd1(KY914551),Fld(KY914552),and their full-length sequences were cloned by RACE technique.Their full length sequences were 774 bp,882 bp,767 bp,648 bp and 929 bp,including 546 bp,465 bp,465 bp,444 bp,and 585 bp ORF length,encoding 235,329,329,212,294 amino acid residues,respectively.BLAST analysis of these gene sequences by NCBI showed that these genes had high homology with Fd gene of other species,and had fer2 superfamily structural domain.Bioinformatics analysis was conducted for these five Fd genes.Expression analysis results showed that the relative expression of Fd genes in different gerbera varieties was significantly different,suggesting that different gerbera varieties may be has different resistance;and the relative expression of Fd genes in diseased plants was higher than that in healthy plants(P<0.05),suggesting that Fd gene maybe play a potential in disease-resistant in gerbera.The expression of Fd3 in the fifth generations of the open proliferation culture of gerbera was much higher than that of the traditional proliferation culture,and the other types of Fd genes did not change significantly.Fd gene under different proliferation generations did not change trend.Fd3 was known to improve plant broad-spectrum resistance.Up-regulation of Fd3 gene in open plantlets of gerbera may enhance its broad-spectrum resistance.These results further suggest that open tissue culture could be used in the industrial breeding of gerbera.