| Dendrobium officinale is a kind of herb with high medicinal,ornamental and commercial value,which belongs to Dendrobium genus,Orchidaceae family,and rich in polysaccharides,astragalus,bibenzyls and alkaloids.Polyploid breeding is an important breeding method of medicinal plants,and there have been a lot of reports concerning autopolyploid induction in D.officinale,however,there are few researches on allopolyploid induction and the effects of polyploidization on micropropagation characteristics till today,also there are no reports concerning the molecular mechanism of the increase of polysaccharide content caused by polyploidization.In the study,D.officinale,D.nobile and D.aphyllum were employed to establish the technical system of creating polyploid by colchicine treatment combined with tissue culture technique and develop autopolyploid and allopolyploid in D.officinale,on the basis,the effect of polyploidization on micropropagation characteristics and the molecular mechanism of the increase of polysaccharide content caused by polyploidization in D.officinale are investigated.The main results are as follows:1.Colchicine treatments had significant effect on PLB survival rates of intraspecific and interspecific hybrids.In general,the higher the colchicine concentration was,the longer the duration of treatment was,the lower the PLB survival rate was,the mean PLB survival rate of intraspecific hybrids was higher than that of interspecific hybrids.The efficiency of polyploid induction depended on colchicine concentration,time duration and genotype.Within the duration adopted,no autopolyploids were produced at 0.05 mg·L-1 colchicine,in contrast,allopolyploids were obtained,the mean induction rate of allopolyploids was higher than that of autopolyploids.The autotetraploid induction rate of DO.201-1 was the highest（47.00%）when the PLBs cultured in 0.1 mg·L-1 colchicine solution for 3 d,the autotetraploid induction rate of DO.201-3 was the highest（48.89%）when the PLBs cultured in 0.15 mg·L-1 colchicine solution for 3 d,the autotetraploid induction rate of DO.202-7 was the highest（46.67%）when the PLBs cultured in 0.1 mg·L-1 colchicine solution for 5 d and the autotetraploid induction rate of DO.202-8 was the highest（50.00%）when the PLBs cultured in 0.1 mg·L-1 colchicine solution for 4 d.The allotetraploid induction rate of the PLBs of interspecific hybrids‘107-6’and‘107-13’coming from the combination of D.officinale?D.nobile and‘200-4’coming from the combination of D.officinale?D.aphyllum were all the highest,which was 46.67%,37.17%and 55.00%,respectively,when the PLBs cultured 0.1 mg·L-1 colchicine solution for 4 d,and when the PLBs cultured in 0.05 mg·L-1 colchicine solution for 7 d,the allotetraploid induction rate of the PLBs of hybrid‘200-7’coming from the combination of D.officinale?D.aphyllum was the highest（50.00%）.The results indicated that the suitable regimes for polyploid induction of different genotypes were not the same in D.officinale.2.Four autotetraploids and four allotetraploids were obtained.The morphological characters of autotetraploids and allotetraploids were obviously changed after chromosome doubling,all of tetraploid seedlings had shorter but thicker stems and roots,the number of stromata cell decreased,but the surface area became larger.Mean fresh weight of autopolyploid was heavier than that of diploid,while mean fresh weight of allopolyploid was lighter than that of diploid.3.Chromosome doubling had significant effect on micropropagation characteristics of D.officinale.The PLBs induction rates of autotetraploids were all significant higher than that of the counterpart diploids except for DO.202-7.T;Proliferation coefficiencies of autotetraploid DO.201-1.T and DO.201-3.T were higher than that of diploids,while proliferation coefficiencies of autopolyploid DO.202-7.T and DO.202-8.T were lower than that of diploids;The mean number of bud regenerated of DO.201-1.T was less than that of diploid,the mean number of root regenerated and fresh weight were similar to that of diploid,the mean number of bud regenerated of DO.201-3.T was more than that of diploid,its mean number of root regenerated and fresh weight were similar to that of diploid,the mean number of bud regenerated of DO.202-7.T was more than that of diploid,so did fresh weight,there were no significant differences in the mean number of bud regenerated and fresh weight between DO.202-8.T and DO.202-8.D.The PLBs induction rates of allopolyploids were all significant higher than that of the counterpart diploids except for DON.107-6.T,proliferation coefficiencies were also higher than that of diploids except for DON.200-7.T,the mean number of shoot and root regenerated were all less than that of diploids except for DON.107-13.T.4.The polysaccharide contents in roots,stems and leaves of DO.201-1.D and DO.201-1.T six-month-old seedlings were measured.The results showed the polysaccharide contents in roots,stems and leaves of autopolyploid DO.201-1.T were all significantly higher than that of diploid DO.201-1.D,indicating that autopolyploidization resulted in the increase of polysaccharide content in D.officinale.5.PLBs,bud,four-month-old and six-month-old seedlings of autotetraploid DO.201-1.T and diploid DO.201-1.D were subjected for transcriptome sequencing by using of Illumina Hiseq 2500 platform,274403 unigenes were generated.There were 33428differential expressed genes（DEGs）in PLBs of autotetraploid DO.201-1.T and diploid DO.201-1.D,among them,13446 DEGs were upregulated,which were significantly enriched in metabolic pathways including Spliceosome,m RNA surveillance pathway and Glycolysis/Gluconeogenesis,19982 DEGs were downregulated,which were significantly enriched in metabolic pathways including Ribosome,Biosynthesis of amino acids and Protein processing endoplasmic reticulum.There were 13465 DEGs in buds and seedlings of autotetraploid DO.201-1.T and diploid DO.201-1.D,among them,2164 DEGs were upregulated,which were significantly enriched in metabolic pathways including Protein processing in endoplasmic reticulum,Ubiquitin mediated proteolysis and Amino sugar and nucleotide sugar metabolism,11301 DEGs were downregulated,which were significantly enriched in metabolic pathways including Ribosome,Protein processing in endoplasmic reticulum and Glycolysis/Gluconeogenesis.6.The express of eleven genes related to polysaccharides including Do Man2-2,Dog1gc1,Dog1gc3-1,Do PGM1,Do PGM2-2,Doi PGM1-2,Do PMM2-1,Do GMP1-3,Do CSLA3-1,Do CSLA2-1 and Do GMPP was analyzed by the use of q RT-PCR.The results indicated compared with the counterpart diploid,the express of Dog1gc1,Dog1gc3-1,Do GMP1-3,Do GMPP,Doi PGM1-2,Do Man2-2,Do PGM2-2 and Do CSLA2-1 in autotetraploid DO.201-1.T was significantly upregulated,in contrast,the express of Do CSLA3-1,Do PGM1 and Do PMM2-1 was downregulated.In autotetraploid DO.201-3.T,DO.202-7.T and DO.202-8.T,the upregulated genes were Do CSLA2-1,Dog1gc1,Do PGM2-2 and Do PGM1,Do CSLA2-1,Do CSLA3-1,Dog1gc1,Do GMPP,Doi PGM1-2and Do PGM1,and Do CSLA2-1,Dog1gc1,Dog1gc3-1,Do Man2-2 and Do PGM1,respectively.In allotetraploid DON.107-6.T,the upregulated genes involved Do CSLA2-1,Dog1gc1 and Do PMM2-1,the rest eight genes were downregulated.In allotetraploid DON.107-13.T,DON.200-4.T and DON.200-7.T,the upregulated genes were Do CSLA2-1,Do Man2-2 and Do PGM2-2,Do GMPP,Do GMP1-3,Doi PGM1-2,Do PGM1 and Do CSLA2-1,and Do CSLA2-1,Do CSLA3-1,Do GMP1-3,Do GMPP,Doi PGM1-2,Do Man2-2,Do PGM1 and Do PMM2-1.Among the eleven genes,the express of Do CSLA2-1 was significantly upregulated in all autotetraploid and allotetraploid,indicating that Do CSLA2-1was the possible key gene resulted in the increase of polysaccharide content in medicinal Dendrobium.Through the above research,the technical system of creating polyploid was established in D.officinale,four allopolyploids and four allopolyploids were obtained,the effect of chromosome doubling on micropropagation characteristics was elucidated,and the molecular mechanism of the increase of polysaccharide content caused by chromosome doubling was preliminarily clarified.These lay a solid foundation for further investigating on the effects of polyploidization and their molecular mechanism and developing new polyploid cultivars in D.officinale. |
PDF Full Text Request