Creation Of Maize New Germplasms With High Starch Or High Amylose Content By Pyramiding Transgenes

Maize is the largest yield crop in the world,its grain can be used as food,feed and industrial raw materials.Starch content and quality determine its economic value in the most situation.High-starch maize has industry value,and can increase the output value of per unit area.High-amylose maize can be used to produce high-amylose starch,which is the material of food packaging film,biodegradable plastics and others.At present,China's high-amylose starch still need to be imported.Therefore,it is of great significance to strengthen the breading of high-starch and high-amylose maize.In the previous study in our lab,we found that:the co-expression of mutated Sh2r6hs gene and Bt2 gene in different inbred lines,the transgenic kernels have shown higher starch content and the heavier 100-grain weight when compare with the non-transgenic control.The over-expression of ZmWx gene could significantly improve the total starch content,and the amylose content and the 100-grain weight was also increased in the transgenic lines(to be published).In our laboratory,the SBEIIRNAi structure was transferred into the maize inbred line Chang7-2.The transgenic maize is that interfered with SBEIIa gene and SBEIIb gene at the same time.The amylose content was increased from 28%to 50%.Total starch content decreased from 66%of the control to less than 60%.The observation of starch granules and transfer cells in transgenic maize endospermTo explain the phenotype(the increased starch content and seed size)of the transgenic kernels,the longitudinal section of the kernels were obtained by paraffin section stained by PAS reaction(Periodic acid Schiff reaction)or Toluidine blue-O.The microscopic structure of the transfer cells and starch granules were observed by an optical microscope.The morphology and size of starch granules at the center of the mature endosperm were studied by scanning electron microscopy(SEM).The diameter of starch granules was measured.As the paraffin sections shown,the number of the starch granules in maize endosperm in the plants of Sh2r6hsBt2 and ZmWx gene overexpression were significantly higher than that of controls(inbred line Chang7-2,Zheng5 8),and the result was opposite in SBEIIRNAi transgenic lines.The transfer cells have shown no significant difference between the transgenic and the controls.The average of starch diameter in the over-expression line of Sh2r6hsBt2 and ZmWx gene were higher than those of inbred lines when it were observed by the scanning electron microscopy(SEM).Compared with the controls,the surface of starch granules of SBEIIRNAi transgenic lines of Chang7-2 was irregularly collapsed or depressed.As shown of the starch granules diameter distribution,the SBEIIRNAi structure transgenic lines had significantly reduced the number of starch granules which diameter distribution at the 8-13?m range,and the proportions of small and large starch granules were higher than that of Chang7-2.Therefore,the changes of the starch content and kernels size of Sh2r6hsBt2,ZmWx and SBEIINAi transgenic lines were related to the change of the number and size of starch granules,but there was no obvious relationship with the change of the number and of transfer cells.It is indicated that over-expression of Sh2r6hsBt2 gene and ZmWx gene mainly increased the activity of AGPase and GBSSI,and the efficiency of starch synthetic was improved.SBEIIRNAi structure decreased the activity of SBEII and affected the synthesis of starch.The variation of amylose contents in the hybrids of overexpressed GBSSI maize with overexpressed mutant AGPase maizeThe maize elite hybrid cultivar of ZhengDan 958 was combined with Zheng58 as the female parent and Chang7-2 as the male parent.For the transgenic hybrid,the lines with over-expressed ZmWx gene produced from Zheng58 as the female parent and the lines with transgenic mutant AGPase gene derived from Chang7-2 as the male parent,we hope breeding the transgenic ZhengDan958 with high-starch and high-amylose,and understanding the application value of the transgenic hybrid in agricultural production.The total starch and amylose content of transgenic maize were determined,and the kernel traits,ear traits,100-grain weight and yield were analyzed.Compared with the control hybrid maize,the starch and amylose content of the transgenic pyramiding maize was increased,and the plant height and ear height of the transgenic pyramiding lines did not change significantly,but the ear length,the number of kernels per ear,the 100-grain weight and the yield of per plant were increased.The transgenic hybrid showed significantly increased yield when compared with the control due to the increase of number of grains and 100-grain weight.The yield was increased from 169.4±6.23g of non-transgenic ZhengDan958 to 200.0±4.56g.Compared to the control hybrid,the transgenic hybrid kernels had increased the starch content and the amylose content.When the transgenic hybrid was applied to agricultural production improved maize yields and increased farmers' income could be produced.The analysis of the hybrids of ae mutant and different transgenic linesIn order to improve the content of amylose in maize,maize new germplasm with high-amylose must be created.In this experiment,ae mutant maize was hybrid with SBEIIRNAi structure transgenic maize,ZmWx gene overexpressing maize,mutant AGPase overexpressing maize,respectively.The total starch and amylase content of the transgenic pyramiding seeds were determined.The amylose content of ae mutant in the background of Chang7-2 reached 56.9%,and the amylose content of ae mutant in the background of Zheng58 reached 55.1%.The amylose contents of the hybrid of ae mutants and transgenic lines with SBEIIRNAi structure,were about 33.9%except of two exceptional lines.The amylose contents of the hybrids of ae mutant and GBSSI over-expression lines,were about 37.1%,that was lower than the ae mutant.The amylose contents of the hybrids of ae mutant and over-expression of mutated AGPase,were also significantly lower than that of parental ae mutant.Moreover,the amylose contents of the pyramiding lines of the ae mutant as female parent were higher than that of the transgenic maize as female parent,which showed the maternal effect existed.From the above results,the amylose contents of the hybrid kernels were lower than that of parental ae mutants,which could be inferred that the presence of wild-type SBEIIb gene in the endosperm of hybrids decreased the amylose content.For the enough generations was not achieved,homozygous ae lines for determination the amylose contents were not obtained in the experiment,the result was very preliminary.The hybridization of transgenic lines with overexpressed AGPase and mutant AGPase increases the starch content of kernelsIn this study,the hybridization of Sh2Bt2 over-expression line and Sh2r6hsBt2 over-expression line creased the heterozygosity of ADP-glucose pyrophosphorylase,and tried to further improve the maize starch content.The starch content and 100-grain weight of maize were measured,and the phenotypes of ear and kernel were analyzed.When used the Sh2Bt2 over-expression lines as female parents,respectively,the starch contents of hybrids were 76.7%,78.6%,and the 100-grain weight were 24.9±1.74g,26.2±0.65g.When used the Sh2r6hsBt2 over-expression lines as female parents,respectively,the hybrids had the starch content of 78.0%,81.1%and the 100-grain weight of 26.7 ± 0.51g,27.9 ±0.60g.These showed the higher starch contents and 100-grain weight were produced by the hybrids compared with their parents.The phenotypic analysis of ears and kernels from different lines indicated larger kernels born on the ears of the hybrids,and the ears were longer than that of parent lines.From the results,it was concluded that the hybrids of over-expressed Sh2Bt2 gene and over-expressed Sh2r6hsBt2 gene had significantly increased starch content and 100-grain weight,produced larger kernels and the longer ears compared with the parents,the heterozygous AGPase with Sh2r6hs subunit might have higher activity for starch synthesis.In conclusion,based on the observation of starch granules,transfer cells and starch synthesis in transgenic maize endosperm from different lines,it could be concluded that the correlation of the size,number of starch granules and starch content exist,but no relationship with the number and architecture of transfer cells.We bred a new maize hybrid material with high-starch and high-amylose starch by the hybridization of GBSSI over-expression line and mutant AGPase over-expression line.Although the hybridization of ae mutant lines and different transgenic lines produced the hybrid kernels of lower amylose content when compared with its parents,the result enlightened us on a new idea for the breading of high-amylose starch maize new germplasm.And the homozygous pyramiding lines of the ae gene would be selected in the future.The hybrids of overexpressed AGPase lines and overexpressed mutant AGPase lines showed an increased starch content and 100-grain weight compared to the parent,which offered a scheme for the improvement of maize yield.From this study some of strategies and methods for the breeding of transgenic high-starch or high-amylose cultivars of maize were established,and the high-starch or high-amylose maize germplasm that have been selected displayed a good prospect for application.