| Supersweet corn, also known as fruit or vegetable corn, is one of the mostimportant crops in the world. However, yield loss and quality decrease of thesupersweet corn are getting more and more serious owing to the damages causedby an insect species, corn borer. Due to the shortage of germplasm resourcesfor insect resistance, resistant varieties are not easily obtained in thesupersweet corn by conventional breeding in the near future. Recently, plantgenetic engineering has been a novel way in crop genetic improvement. In thisstudy, a stable, high efficient transfergenic-receptor system has beenestablished. Multiple insect-resistant genes were co-transformed successfullyinto an elite inbred line and a cultivar by using the particle bombardment andovery microinjection. Transgene integration, expression and inheritance wereinvestigated based on molecular analyses as well as the phenotypic performancesof the target traits. The main results are as follows:1 The key factors influencing callus induction and somatic-embryoidformation were tested, and the callus-receptor system for gene transformationhas been established in the supersweet corn. The results showed that, for callusinduction, genotype was the critical determining factor;the best length ofimmature embryos was 1.0-2.0mm;and the most suitable concentration of 2,4-Dwas 2mg·L-1. The highest rates of embryoid-callus formation of the inbred line,1132 , and a cultivar , Yuetian 3, are 45.88% and 35.00%, respectively. Theinbred line 1132 demonstrated the highest inducing frequency of the type Ⅱembryo callus. The addition of 9mg·L-1 of AgNO3 to the N6 medium significantlystimulated embryoid formation and the rate of embryogeny was increased 31.25%compared to that of control, indicating that AgNO3 plays a significant role incallus induction. Furthermore, the addition of L-proline (700mg·L-1), whichregulates osmotic pressure and promotes the process of embryogeny, enabled thefrequency of embryoid formation to be increased 12.13% compared to that ofcontrol. The results also indicated that, for selection, the most suitableconcentration of the herbicide Basta was 5mg·L-1.2 Through optimizing the conditions in regenerating, rooting andtransplanting, the frequency of regeneration and the viability of transgenicplants were improved along with reducing somaclonal variations. Thus, a stable,efficient plant post-transformation regenerating system has been achieved. Itwas shown that calli lost their regenerating ability after long-term subculturesand could be well maintained with high osmotic treatments in a medium containing40g·L-1 of sucrose, 20g·L-1 of mannitol, and 5.0mg·L-1 of ABA. As a result, thepresent study has provided an efficient, stable plant regeneration method byscreening various concentration-combinations of sucrose and cytokinins. Thehighest frequencies of differentiation and regeneration were 81.13% and 35.85%,respectively, when 20g·L-1 of sucrose, 2.0mg·L-1 of 6-BA, and 0.1mg·L-1 of NAAwere supplied in the medium. It was also revealed that rooting frequency was57.72% when 0.1mg·L-1 of NAA was used, and that it could reach 98.66% when theconcentrations of NAA and IBA were increased up to 0.2mg·L-1 and 3mg·L-1,respectively.3 The calli, derived from immature embryos, of the inbred line 1132 weretransformed with the plasmid DNA containing Bt, PinⅡ, RIP and GNA genes viathe particle bombardment-mediated method. A total of 31 plants were regenerated,of which 26 were fertile. Genomic PCR and Southern hybridization analyses ofthe T0 individuals demonstrated that, Bt gene was integrated into the genomesof plants 1,5,6,9, and 10, RIP gene was into plants 1,9,13,23,24,26,27, and 29,and GNA gene was into plants 1,5,6,9,and 10, and PinⅡgene was into plants8,11,12,13,and 15～29. It was further concluded that three insect-resistantgenes were co-integrated into the genomes of some transgenic plants, or one genewere into the genomes of other transgenic plants as a sigle copy. Follow-upmolecular analyses showed that RIP and GNA genes were inherited successfullyto the T1 progeny. In addition, the analysis of RT-PCR proved that the PinⅡandGNA genes were expressed in the T2 progeny , respectively.4 The insect resistance has been investigated in the T1 plantsG9,G24,G25,G26,and G27 by feeding the corn borers in a lab. The results showedthat great differences exist among different T0-derived T1 families as well asamong individuals within a family. On average, the mortality of the 5 pedigreeswas 74.25%, which was much higher than that of control (46.67%). Although mostT1 plants highly resisted to the corn borers, a few lines demonstrated low orno resistance to the pests at all. This phenomenon may be caused by genesegregation, which results in the loss of gene expression or the gene itself.5 Foreign insecticidal genes were successfully transferred into an elitesupersweet corn hybrid by overy injection in a field.Overall, the transformationfrequency could reach as high as 9.09%. One transgenic plant, ZT11, containingthe Bt gene has been obtained. PCR and Southern hybridization analyses not onlyconfirmed the existence of the alien gene, but also indicated that only asingle-copy of the Bt gene had been integrated into the host genome. It wasalso shown that most of the transgenic plants could inherit the Bt genesuccessfully with a pattern of segregation following the Mendelian ratio 1:1.Insect-resistant bioassay indicated that some plants exhibited great resistanceto the corn borer. However, large variations exist among the individuals withinthe T1 generation, ranging from very high to no resistance to the insect.As a whole, the present research has provided much fundamental knowledgeof the transgenic insect-resistance regarding the methods of transformation andidentification, the patterns of transgene expression and inheritance, as wellas the breeding methodologies in the supersweet corn. |
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