| Rose(Rosa rugosa)is a perennial and evergreen shrub in rose species of Rose Family,Because of its variety,gorgeous petal color,rich and attractive fragrance,as special widely used ornamental flower,the position in the garden application is very important.In addition,rose also has important economic value.Rose not only can be used as natural food additive also can be used in spices,chemical and other industries.Rose essential oil extracted from the rose petals can be used special widely in cosmetics and beauty industry.For its price is very expensive,so the title of "liquid gold" is also worthy of the name.In summation,rose has viewing,medicinal,health care,food and other functions,its design and color of flowers and other secondary metabolite also have important economic value.But in the presence of the ornamental flowers in short supply,because of the rose petal colors lack of diversity,this point greatly narrowed the scope of the application in landscape greening and other industry.Chinese rose(Rosa hybrida)belongs to the rose species of Rose Family and is perennial and evergreen shru.Due to its rich design and gorgeous color,curvaceous plant type,tangy fragrant,extensive management,strong adaptability,easy breeding and long florescence,it is be called ’the queen of flowers’.Chinese rose is one of ornamental flowers plants which has highly ornamental and green value.Its application range is very wide,not just confined to the landscape,also can be used as potted plants and cut flowers,has a high application value.The main purpose of this study is to develop some new varieties of rose and Chinese rose which have more economic value and can meet the market demand.At the same time,using the somatic embryogenesis of rose and Chinese rose as materials,try to do a tentative exploration in molecular regulatory mechanism of secondary metabolites such as flower color and fragrance.Around this purpose,this study use the agrobacterium mediated method and use the somatic embryogenesis of rose ’Bao White’ and Chinese rose’Samantha’ as acceptor material to genetic transformation the target genes RrMYB7,MYBX,PAP1,PLL1 which have the possibility to regulate the color and fragrance of plant flowers.In the progress,the genetic transformation system has been further optimized and finally obtained the transgenic plants of EsMYBX,PLL1 transformation of rose ’Bao White’,from the somatic embryo germinate resistant buds to take roots,the whole plants show purple and pink color respectively.Besides,we obtained the transgenic plants of PAP1 transformation of Chinese rose ’Samantha’,compared with the controlled plants,the transgenic plants have no big difference on the phenotype.And obtained plants ofRrMYB7 transformation of rose ’Bao White’,but it is difficult to take root.The contents and results of this study are as follows:(1)EsMYBX,RrMYB7,PLL1 genetic transformation to the somatic embryo of rose ’Bao White,Roses team set up the plant regeneration system of rose using the stems of rose ’Bao White’before.Firstly induced the lateral bud to germination in the medium MS+sugar30g/L+agar8.0g/L+BA0.5mg/L+NAA0.01mg/L+GA30.5mg/L.Secondly proliferation and subculture in the medium MS+glucose30g/L+agar8.0g/L+ BA 0.5mg/L+NAA 0.01 mg/L.Finally cut the fresh leaves from the plantlets and then put them in the medium MS+ glucose30g/L+phytagel3.0g/L+2,4-D4.0mg/L+KT0.05mg/L to reduce the somatic embryos.Put the somatic embryo induced from this method in the medium MS+glucose40g/L+phytagel3.0g/L+2,4-D1.0mg/L+BA0.01mg/L to proliferation to a certain amount and then selected those grow strongly and vigorous as genetic transformation acceptor material,using the agrobacterium mediated method did the genetic transformation for EsMYBX,RrMYB7,PLL1 genes.After co-culture,selection and proliferation,selection and germination,selection and shoots elongation,selection and rooting,five stages and about 8 months resistance screening,during the selection and rooting stage,using the original genetic transformation system,resistance seedlings cannot take root.So we adjust the growth hormone concentration and ratio of the rooting medium.At last the culture medium 1/2 MS+ glucose 30 g/L + agar 7.5 g/L + IBA1.0 mg/L gained the highest rooting rate.After genetic transformation,finally we obtained 8 strains of EBMYBX transformed plantlet,121 resistance seedlings,27 of which have taken roots and placed in the ground,the germination rate and rooting rate was 4.88%,67.74%respectively;we obtained 6 strains of RrMYB7 transformed plantlet,26 resistance seedlings,the germination rate was 3.37%,cannot take root;and obtained 5 strains of PLL1 transformed plantlet,7 resistance seedlings,the germination rate was 2.64%,cannot take root.PCR detection results show that these three genes have been successfully imported to the genomes of rose ’Bao White’.The organs,such as root,stem,leave and so on,of most obtained EsMYBX transformed plantlet are purple.So we can preliminary judge that EsMYBX gene promoted the anthocyanin accumulation to a certain extent and expressed in various organs of the rose plant.This may be due to the expression of the structure genes on the anthocyanin synthesis pathway have raised.The PLL1 transgenic plants show pink color,preliminary showed that PLL1 gene play a positive role in promoting anthocyanins accumulation.RrMYB7 gene promoted the production of fragrance secondary metabolites in tobacco plants.But due to the transformed reae plants cannot take root,so it is uncertain whether it can control the generation of fragrance secondary metabolites.(2)PAP1 genetic transformation to the somatic embryo of Chinese rose ’Samantha’Our research group established the Chinese rose regeneration system in early days.Then obtained the somatic embryo induced from new leaves of tissue culture plantlets through this system of ’Samantha’.Put the somatic embryos in culture medium MS ＋glucose 60g/L + phytagel 3.0 g/L +2,4-d 1.0 mg/L + BA 0.01 mg/L to successive propagate a few generations.Then selected about 160 plump-eared,glossy and exuberant vitality somatic embryos as genetic transformation receptor material and transformed PAP1 gene to them through the agrobacterium mediated method.Finally we obtained four strains and 12 resistance plants.The germination rate and rooting rate is 3.14%,2.78%respectively.PCR detection result showed that PAP1 gene have been successfully imported into the genomes of Chinese rose ’Samantha’,but did not obtain the expected effect of design and color will be darker,may be due to the flower color of Chinese rose ’Samantha’is red,also may be affected by other factors.Because PAP1 gene have been verified that it can promote anthocyanin accumulation in a variety of other plants can,so need to do further research. |
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