| The great progresses have been made for Citrus transgenic researches in recent decades. But as most of the woody plants, the hysteretic problems during citrus industrialization development are increasingly exposed, as well as the relative low-efficiency in citrus transformation. The main reasons are described as follows:1) the genetic transformation system is not perfect enough; and2) seedling materials used as explants extended the period of transgenic breeding to large degree. However, above-mentioned problems could be solved effectively by optimizing the transgenic technology system and using the mature materials as explants, which could be thus of significant importance to citrus transgenic breeding process.In this present research, to obtain the high-efficiency in citrus transformation system, the internode stem segments of seedling kumquat (Fortunella spp. Swingle) and sucarri orange(Citrus sinensis (L.) Osbeck) were used as explants and various factors (including explants materials, agrobactium infection conditions, medium and culture conditions) that might affect the regeneration and transformation efficiency were investigated. The rejuvenation treated materials were obtained by adult buds re-grafting on seedling rootstock in vitro or in vivo, which could effectively reduce contaminate rate and improve the regeneration efficiency of adventitious buds, and therefore resulted in yielding the relative higher transformation efficiency. Parallel analyses (containing microstructure, physiological characteristic and the molecular level) were conducted to understand the possible mechanisms underlying rejuvenation process. Based on the optimized genetic transformation technology system, the ’Gene-deletor’ and ’seedless fruit’ genes were successfully transferred into the seedling kumquat and sucarri orange. The elimination situation of exogenous genes in transgenic clones was analyzed and the specificity of pollen and seed special promoter in citrus was detected, which provided a foundation for citrus biosafety research. The main results were as follows:1. Establishment of seedling kumquat efficient regeneration and transformation technology systemThe internode stem segments of seedling kumquat (Fortunella spp. Swingle) were used as explants and main factors (including seedling age, seedling illumination settings, explants set orientation, regeneration and rooting medium) that might affected the regeneration were investigated to establish an efficient regeneration technology system. The results showed that the internode stem segments from35days completely dark cultured seedling. were level set on regeneration medium consisting of MS basal medium supplemented with3mg/16BA for50days, then cut the adventitious buds from explants and transferred on rooting medium consisted of1/2basal medium supplemented with3mg/1IBA and1g/1AC, which canfinally contribute to90%of adventitious bud regeneration rate and86%of the rooting rate.The internode stem segments of seedling kumquat (Fortunella spp. Swingle) were used as explants and main factors that might affect the transformation efficiency were investigated to establish an efficient transformation technology system. The results showed that internode stem segments from35days age of seedling which cultured for25days in dark and10days in light were immersed infected for30min by agrobacterium which was resuspended by the co-cultivation liquid medium consisting of MS basal medium supplemented with2mg/16BA,1mg/1NAA,1mg/1KT and100μM AS when its OD value reached0.5, then the infected internode stem segments were cultivated horizontally side-by-side in co-cultivation medium (CCM) whose pH value was adjusted to5.8prior to be autoclaved for3days under dim light at25℃, finally, transferred it intothe selection medium consisted of MS basal medium supplemented with3mg/16BA,50mg/1Kan and400mg/1Cef,which can result in optimum GUS positive rate. Based on this efficient genetic transformation technology system, the ’Gene-deletor’ and ’seedless fruit’ genes were successfully transferred into the kumquat with6.07%of the transformation efficiency.2. Optimization of seedling succari orange transformation technology systemThe internode stem segments of seedling sucarri orange (Citrus sinensis (L.) Osbeck) were used as explants and main factors that might affected the transformation efficiency were investigated to further optimize the transformation technology system. The results revealed that, when using internode stem segments from30days age of seedling which cultured20days in dark and10days in light as explants,100μ M as AS concentration,5.7as co-cultivation medium pH value and19℃as co-cultivation temperature, the GUS positive rate could reached at 29.4%. When resistant buds were cultured on stem elongation medium consisting of MS basal medium with0.2mg/16BA,0.2mg/1IAA and0.2mg/1GA3,95%of the resistance buds appeared elongation and the elongation length reached at an average of2.3cm. When resistant buds were transferred on the rooting medium consisting of1/2MS basal medium with0.5mg/1NAA,0.1mg/1IBA and1g/1AC,80.2%of the resistance buds appeared rooting and the rooting length reached at an average of5.83cm. On the basis of this optimized transgenic technology system, the’seedless fruit’ gene was successfully transferred into the sucarri orange and the transgenic lines were grafted in greenhouse with superb growth potential.3. Rejuvenation treatment of citrus adult materialsIn our study, adult buds were grafted on juvenile rootstocks in vivo or in vitro. For the former (in vivo), the adventitious bud regeneration (ABR) efficiency of mature internode stem segments from sucarri orange and’Bingtang’sweet orange reached72.4%and67.7%, respectively. Interestingly, we found the higher rate of ABR could be obtained using sour orange(Citrus. aurantium L.) as rootstock when compared with trifoliate orange (Poncirus. trifoliata L. Raf.); and showed higher rate of ABR in the first and second flushes after grafting than the third flushes. Furthermore, the repeated grafting could promote rejuvenation effect. For the latter (in vitro),’Bingtang’ sweet orange after being transferred in greenhouse could result in53.2%of the ABR rate.Through the observation of micro structure, we found the microstructure of rejuvenation treated stem had no significant differences to untreated stem, which indicated that organizational structure could not be the main cause of the regeneration differences. But the extent of cell proliferation in cambium from the rejuvenation treated materials was higher than untreated materials after15days. The result showed cambium cells meristematic ability was enhanced by rejuvenation. By further analysis endogenous hormone change between the rejuvenation treated material and untreated material, we found that the contents of endogenous hormones IAA, GA3and ZR, but ABA decreased. These results showed that changes of endogenous hormone contents or ratio after rejuvenation treatment could promote adventitious regeneration. Through MSAP analysis (Methylation sensitive amplified polymorphism), we found that the level of DNA methylation in rejuvenation treated materials was less than untreated materials, and the reduced type was much more than increased type in DNA methylation change type.4. Transformation of mature explants by rejuvenation treatment and introduction of’Gene-deletor’ geneUsing mature materials by rejuvenation treatment as explants, the ’Gene-deletor’gene were successfully transferred into the mature ’Bingtang’ sweet orange and4.63%of the transformation efficiency could be obtained in this study. Meanwhile, the untreated materials couldn’t get the transgenic plants. PCR detection confirmed npt II gene driven by35S promoter and recombinase gene FLP driven by the pollen and seeds specific promoter PAB5exsited in transgenic plants leaves, but RT-PCR and quantitative PCR detection found that npt II gene expressed in leaves but recombinase gene FLP not, which preliminary verified there was a specificity of the pollen and seeds specific promoter PAB5in citrus.Based on the GUS histochemical assay, GUS gene activity wasn’t detected in all the nucellus embryo and zygote embryo. The result preliminary showed that exogenous genes had been clear in seed. In order to further understand the condition of organization specific promoter PAB5starting and the exogenous genes clearing in citrus, each part of transgenic fruit was analyzed by GUS histochemical assay. The results showed, albedo and juice vesicles compared with embryogenesis belonging to apical histogenic layer LⅡ weren’t observed GUS staining. Flavedo from the L layer and the segment membrane and central core from LⅢ layer were showed GUS activity, which showed that exogenous genes were not clear in these organizations. The results confirmed the organization specific promoter can be derived the clearing of exogenous genes in the organs and tissues from LⅡ layer, but LⅠand LⅢ layers.The application of ’Gene-deletor’ technology in citrus provided a solid foundation for citrus environment safety transgenic research. But the edible part in citrus fruit still mixed with the segment membrane where the exogenous gens still be within. This is therefore also to be a safety problem in transgenic food. We need to screen fruit specific promoter in the next step of work, and clear all exogenous genes from pollen, seeds and fruits, aiming to thoroughly solve safety problems in the transgenic citrus. |
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