Studies Of Protoplast Culture And Microspore Embryogenesis In Tobacco

Plant protoplasts which have no cell wall can be used as an experimental system to study the basic theory of cell wall regeneration,cell division and differentiation.At the same time,it is also as an ideal material for somatic cell hybridization,clonal variation and mutant screening,isolation of organelles,and a receptor for genetic transformation.Because the microspores are single cells,easy to obtain,not affected by anthers,with advantages of haploid,synchronization,being easy to double,often used to study the plant embryonic development process.Microspore-derived embryos also provide an alternative system to study plant embryo development and plant cell totipotency without interference from the maternal tissue.In this paper,the protoplast of 12 genotypes of tobacco protoplasts was explored to explore the time of enzymatic hydrolysis between different genotypes,the time of protoplast initiation,the time of budding and the rate of budding,and the effects of different media on tobacco protoplasm Induction of callus induction,differentiation of adventitious buds and adventitious buds.NtDRP was used as a marker gene for the development of microspore embryos in tobacco by studying the process and developmental pattern of microspore embryos by culturing free microspores.The main results are as follows:1.Comparison of different genotypes of tobacco protoplast isolation hydrolysis time,as prolonged hydrolysis time,protoplast different genotypes of tobacco mesophyll cells begin to separate out.Because of the different susceptibility of different genotypes of tobacco to enzymes,the time required for different genotypes is different.The optimal enzymolysis time of G3,G4,G5,SR1 was 3h;the optimal enzymolysis time of G1 and NtDRP was 3.5h;the optimal hydrolysis time of G2 and G6 was 4.5h;and G7,G8,G9 and G10 of the enzymatic hydrolysis time of 4h,the vitality of the protoplast of the highest yield.The results showed that the same enzymatic hydrolysis conditions,the physiological state of the different genotypes required different enzymatic hydrolysis time is different.The enzymatic time required for tobacco protoplasts is related to genotype.2.The protoplasts obtained by the same enzymatic hydrolysis were isolated and cultured under the same culture conditions.The first division was found in the protoplast cells of G1,G8,G10 and SR1 for 2 days,G4 and G6 protoplasts did not undergo the first division until 5days of culture.The time of the first division of protoplast cells of other genotypes was between 2-5 days.In addition to tobacco G4 protoplast cell division less,the resulting callus less,other genotype tobacco culture of protoplast cells can be divide several times.The callus of different genotypes of the same size was transferred into the same differentiation medium to induce the buds.The callus of tobacco SR1,G1 and G10 was the earliest budding point,and the time required for differentiation budding was 16 days,and the G4 and G6 buds need the longest time for 22d.The germination rate of SR1 was the highest,reaching 97%.The germination rate of G7 was the lowest,60%,and the germination rate of other genotypes was over 80%.The results showed that the ability of bud differentiation was different under the same culture conditions,the same growth environment,physiological state,splitting rate and frequency of different genotype cells.The division and differentiation of protoplasts are related to genotype.3.The growth and differentiation of protoplast callus were studied by using 13 kinds of medium containing different hormone components and different hormone concentration,and the callus of protoplasts was amplified and induced.The results showed that the culture medium and the hormone directly affected the growth and differentiation of protoplast callus.4 culture medium is conducive to the promotion of callus induction,amplification,while the No.4 medium can also induce buds.10 and 11 medium can induce the formation of more callus,but also can produce a large number of buds.Therefore,In the early stage,callus induction and increment could be obtained by using No.4 medium,and a large number of calli were obtained.Then transfer the callus to the medium of 10 and 11 to differentiate the bud.4.In the case of well-growing tobacco SR1,the adventitious buds induced by protoplast culture of tobacco SR1 were transferred to four different media to induce rooting.It was found that No.1 medium induced rooting ratio is low,less root,root,although thick,but the root is short,induced rooting takes a long time,about 10d or so.the root of the No.2 medium has the lowest rooting rate and the root quantity is less.The root of the No.4 medium is short and the rooting rate is induced.The rooting rate of the medium is low,Up to 86%,take root time to take about 12d;to the best medium 3,the highest rate of induction of rooting,up to92%,more root,root is thick and long,induced rooting only 7d can be complete Of the plant.5.The microspores of tobacco were subjected to starvation and heat shock treatment in B medium for 5 days or 6 days,and the development pathways of microspores were changed from gametophyte pathway to sporophyte pathway.In the culture medium transfected into AT3 for 2 days,the nuclei were equally divided,resulting in two similar size of the nucleus,in the course of some small microspore cell wall rupture,resulting in a similar zygotic embryo development pattern,the formation of embryo and embryo body structure.Microspores with complete outer wall after 45-60d,eventually developed into cotyledon embryos.6.Induction of microspore embryogenesis by starvation and heat shock.The rate of microspore-induced embryogenesis under starvation and heat shock conditions is higher than that of normal temperature?starved or non-starved?or microspores cultured alone,but also reduces the dependence on stress treatment.Experiments show that the combination of the two stress treatments has a superposition effect on the induction rate of microspore embryogenesis.7.Explore whether the NtDRP gene can act as a marker gene for microspore embryogenesis.In the process of microspore culture,the microspores of mononuclear latex were cultured in starved medium?32??for 48 hours,and the microspores did not express NtDRP.After 72 hours of culture,the microspores began to express NtDRP.The microspore culture of four different strains of p_{NtDRP::NtDRP-GFP},wild type SR1,RNAi-?and RNAi-?were compared.The results showed that the microspore morphology was spherical at the initial stage of culture,The microsporogenesis of tobacco microspore was significantly decreased,and the microspores of RNAi-?strain could hardly develop into embryos and the microspores began to shrink.It was proved that NtDRP could be used as a marker gene for microspore embryos of tobacco.