Regeneration System Optimization And Genetic Transformation System Establishment And Tetraploid Induction Of Drumstick (Moringa Oleifera Lam.)

Moringa oleifera Lam.,commonly known as drumstick,belongs to the Moringaceae family and Moringa Adans.,and is a perennial woody plants with a range of potential commercial uses.So far,drumstick is still planted mainly by traditional seedling-raising way,but it's very difficult to norm manage as seedling group has serious segregation of character.Fortunately,the technology of plant tissue culture is becoming mature and sequencing and annotating the whole genome is finished.These provide the preconditions for cloning uniform drumstick plantlets and launching molecular breeding.In the present study,on the one hand,complete WRKY genes in drumstick have been searched based on the whole genome.Subsequently,classify and name,phylogenetic analysis,motif analysis,gene structure,selective pressure and expression patterns of WRKY genes were also investigated using the methods of bioinformatics.On the other hand,an efficient regeneration protocol by direct shoot-buds regenerating approach has been developed firstly,then a reliable and stable Agrobacterium tumefaciens-mediated transformation system and tetraploid induction system have been developed on the basis of this regeneration protocol.The main results of this study are as follows:Search,comparision and selection were performed in drumstick genome and protein sequences using various bioinformation softwares.A total of 54 WRKY genes were predicted and named as MoWRKY1-54.A multiple sequence alignment of 54 MoWRKY domains were performed.Two phylogenetic trees on the basis of WRKY domains and genes respectively were constructed after multiple sequence alignment and similar evolutionary relationship were found indicating that WRKY domains were the important evolutionary unit of MoWRKY genes.Three major groups(I,II and III)were categorized as described by Somssich.Additionaly,five subgroups(IIa,IIb,IIc,IId and IIe)were further categorized in group II.Conserved MoWRKY domains of 54 MoWRKYs were analysied and a kind of variation was found in MoWRKY24 that WRKYGQK in core sequences were mutated into WRKYGKK.The conserved motifs of WRKY family proteins in drumstick were investigated using MEME online software.Twenty distinct motifs were identified and proteins with similar motif compositions were clustered in the same class generally,indicating similar functions among members of the same class.Selection pressure analysis indicated that strong purifying selection has played a key role in most MoWRKYs,little MoWRKYs underwent adaptive selection and little MoWRKYs underwent neutral selection.After compared with the rate of evolution between sub-groups further,we found different sub-groups had different rate and II c evolved fastest.The results of gene expression patterns indicated that there were large differences of expression level between MoWRKYs under different conditions and different tissues.An efficient regeneration protocol has been developed using leaf segments as explants.Genotype,developmental stage and inoculation orientation of the explants,and the combination of plant growth regulators(PGRs)in the culture medium all have effects on the regeneration frequency.It was found that Murashige and Skoog(MS)basal elements supplemented with 0.8 mg/L 6-benzyladenine(BA),0.2 mg/L kinetin(KT)and 0.05 mg/L a-naphthaleneacetic acid(NAA)provided the most suitable medium for shoot-bud regeneration,delivering a maximum regeneration frequency of 93.3% and a mean of 4.4 shoots per explant.In addition,orienting the proximal ends of the explants down towards the medium was much better than them facing upwards.We were able to root approximately 90% of the regenerated shoots on MS medium with 0.1 mg/L NAA.Even though success of this protocol varied according to genotype,it still has high applicability at a large scale and could be used in the production of high quality plantlets to meet large scale cultivation needs.In addition,it may be useful for genetic transformation studies.Based on the regeneration system,the present study was undertaken to develop a reliable and stable Agrobacterium tumefaciens-mediated transformation system for the genetic improvement of drumstick.A.tumefaciens strain EHA105,harbouring the plasmid pCAMBIA1301 containing the hygromycin phosphotransferase II and ?-glucuronidase genes driven by the CaMV 35 S promoter,was used to establish a transformation system in drumstick.The use of vacuum infiltration-assisted Agrobacterium infection(excluding a pre-culture step)and co-cultivation for two days significantly increased the transformation frequency.Genomic integration and transgene expression were confirmed by ?-glucuronidase(GUS)assays,polymerase chain reaction(PCR),Southern blot analysis and Real-time quantitative PCR(RTqPCR).This transformation protocol provides a basis for the future development of genetic engineering techniques to improve the performance of drumstick.Based on the regeneration system,in vitro-induced tetraploid of drumstick was carried out by treating explants with colchicine.The regeneration frequency was greatly declined after treated with colchicine as it is harmful for explants.Taking regeneration frequency and tetraploid induction ratio into consideration,treatment with 500 mg/L colchicine for 3 days was the most efficient for induction of tetraploid yielding 21% of tetraploids among all the regenerated shoots.In comparison with diploid plants,tetraploid plants had larger leaf and stomata.At the same time,the content of crude protein,crude fat,crude ash content,acid detergent fiber,neutral detergent fiber,calcium and phosphorus in tetraploid drumstick plants all higher than diploid plants to varying degrees.