Creation And Study On Mechanism Of The Leaf Color Variation Of Chinese Orchid

As one of the famous traditional flowers in China,Chinese orchid has a high cultural and economic value.The consumption demand of Chinese orchid is not only on the ornamental flowers,but also on the plant type,floral art and leaf art.At present,most of the leaf art varieties sold on the markets are the orchid which got directly from the mountains.It seriously damages the wild orchid resources.However,those orchids have been hyped crazily,disturbing the order of the orchid market,and people can't afford the expensive orchids.In addition,the rate of natural bud mutation is very low.In this situation,it is imperative to increase the market share of artificially induced new varieties of orchid.In view of the difficulty in artificial breeding of new varieties with leaf art,it is urgent to explore the causes of the formation of leaf art so as to lay a theoretical foundation for artificial induction of Chinese orchid.In this study,a new material of the leaf color variation Cymbidium longibracteaturn‘longchangsu'was obtained by combining radiation mutation with tissue culture technology.It was named'Yeyilongchangsu'.'Yeyilongchangsu'was used as a material for the first time to carry out research from cytology,Physiology,biochemistry and molecular biology technology levels.We systematically analysed the differences between‘Yeyi Longchangsu'and‘Longchangsu',and clarified the color formation mechanism in‘Yeyi Longchangsu'.The results listed here will help orchid breeding in future.The main results of this study are as follows:1.Creation of the leaf color variation Cymbidium longibracteaturn‘longchangsu'.Taking the spring sword'Longchangsu'bud as the explant,the induction and differentiation conditions were optimized.1/2 MS as the basic medium,NAA 1.4-2.0 mg/L,4 g/L activated carbon was added to complete the start-up of the rhizome.Under the culture condition of B5+6-BA 1.0 mg/L+NAA 0.1 mg/L,the rhizome was induced to differentiate and sprout,and the efficiency of rhizome bud differentiation was improved by magnetic treatment technology.On the basis of the established asexual propagation system of'Longchangsu',⁶⁰Co gamma ray treatment was performed on the rhizome of'Longchangsu'.At the dose of 20 Gy treatment,the rhizome with yellow color was obtained,and then the leaf color variation Cymbidium longibracteaturn‘longchangsu'was differentiated.It was named‘Yeyilongchangsu'.2.Study on structure of leaf and related Physiologyogical parameters.The results showed that compared with‘Longchangsu',the leaf mutant had the following changes:(1)the proliferation and differentiation ability of rhizome decreased,and tube seedlings growed slowly;(2)the contents of chlorophyll and carotenoids decreased;(3)soluble sugar content,soluble protein content,superoxide dismutase,catalase activity decreased;peroxidase activity,malondialdehyde,relative conductivity increased;(4)The chloroplast structure is incomplete,disintegrated into vesicles,and grana lamellae and thylakoids are reduced;(5)chlorophyll synthesis intermediates ALA,PBG,Urogen III,Coprogen and Proto?decreased in content,suggesting that the blocked sites may occur from Proto?to Mg-Proto.The formation of the mutant affects the synthesis,transport and transformation of substances in rhizomes and in vitro seedlings,which caused Physiologyogical dysfunction,reduced resistance and decreased differentiation ability.3.Transcriptome sequencing of rhizome.The reason why the rhizome color turned yellow was analyzed by means of transcriptome sequencing using'Yeyilongchangsu'rhizome as material.We speculate that the yellow color of rhizome is not related to the expression of chlorophyll key enzyme gene,and whether it is related to chlorophyll degradation needs to be further confirmed.The decreased expression of psbC and psbH genes may affect the structure of chloroplast substrate layer,resulting in the reduction of chlorophyll content and the yellowing of the rhizome,and decreased the proliferation and differentiation efficiency.4.Transcriptome sequencing of leaf.Using the leaf of'Yeyilongchangsu'as a material,the reason for the formation of Yeyi's character was analyzed by using transcription group sequencing technology.The expression of the gene encoding uroporphyrin decarboxylase was reduced in'Yeyilongchangsu',which affected the synthesis of Uroporphyrin III,resulting in the obstruction of chlorophyll biosynthesis and the reduction of chlorophyll content.However,the CHLM gene encoding magnesium porphyrin IX methyltransferase and the HEMA gene of glutamine-tRNA reductase were significantly increased in the'Yeyilongchangsu'.Whether it is a compensatory enhancement expression needs to be further confirmed.In addition,a gene related to chlorophyll degradation was selected.Whether the formation of'Yeyilongchangsu'is the result of chlorophyll biosynthesis and chlorophyll degradation,more research is needed to determine.The downward expression of chloroplast development and photosystem-related genes in the'Yeyilongchangsu'leaves may affect the development of thylakoid membranes,resulting in the inability to integrate chlorophyll into photosynthetic proteins after chlorophyll synthesis,which leads to the decrease of chlorophyll content.