| Citrus is one of the most widely cultivated fruits among the world.But the research of its functional genomics is limited by some weakness biological characteristics such as long juvenile phase and large size.The Hongkong kumquat(Fortunella hindsii)is a member of the Fortunella genus.It has been acknowledged that Hongkong kumquat shows short juvenile period,dwarf stature,and strong resistance,which makes it an excellent material for genetic transformation and to be a model plant for basic research in citrus.The efficiency of genetic transformation in kumquat is low,which needs to be further improved.In this study,different strains of Kumquat collected by our group were used as experimental materials.Based on the statistics of seed number in single fruit,epicotyl diameter and morphological characters of seedlings,we selected some candidate strains.The segment of Kumquat epicotyl was further used as explants to carry out genetic transformation experiments and the best genotype which is suitable for Kumquat genetic transformationwas screened out.The transformation system was optimized and supplemented by vacuum duration,agrobacterium infection duration,ultrasonic treatment or not.The main results are as follows:1.Screening of efficient genotypes for genetic transformation of F.hindsiiThe most suitable genotypes were screened from the aspects of morphological traits and genetic transformation efficiency.Through morphological preliminary screening,15 materials with strong growth and large yield were selected from 33 polyembryonic F.hindsii strains.The number of single fruit seeds,the epicotyl diameter of 45 d seedling and the higher morphological characters of 45 d seedling were statistically analyzed,and the genetic transformation experiment was carried out with the above epicotyl stem segment as the explants.The results showed that the efficiency and genetic transformation efficiency of polyembryonic gamut lines were 60.85～96.03% and 2.00～10.66%,respectively,with significant differences among different lines.The three polyembryonic lines with the highest transformation efficiency were QK-3,RYS02-18 and QK02-2.The conversion efficiency was 10.66%,8.93% and 8.41%,respectively,and the regeneration efficiency of the three strains was above 90%,which could be used as the most suitable genotypes for subsequent genetic transformation experiments.The results showed that seed number of single fruit,epicotyl diameter and regeneration efficiency were moderately and significantly correlated with transformation efficiency,indicating that these indexes could be used as the basis for predicting genetic transformation efficiency of newly collected lines of Kumquat in the future.2.Optimization of genetic transformation system of F.hindsiiIn this study,the effects of vacuum treatment time,Agrobacterium infection time and ultrasonic treatment on the genetic transformation efficiency were investigated by using YSY02 as the experimental material.The results showed that when the vacuum treatment time was 10 min and Agrobacterium infection time was 30 min,the conversion efficiency was the highest.The conversion efficiency was significantly improved after ultrasound treatment was added.When the ultrasonic treatment time was 25 min,the conversion efficiency reached the highest,increasing from 12.18% to 21.11% in the control group without ultrasound treatment.The high efficiency polyembryonic receptor material QK-3and the low conversion efficiency polyembryonic receptor material DYT01-5 were further used to verify the ultrasonic treatment.The results showed that the conversion efficiency of epicotyl stem segment of QK-3 seedling was significantly reduced after15 min and 30 min ultrasonic treatment.The conversion efficiency of DYT01-5 under the condition of ultrasonic treatment for 5 min increased slightly,from 4.79% to 5.52% in the control group,while the conversion efficiency under other ultrasonic treatment conditions decreased,and there was no significant difference among all treatments.The results showed that the improvement effect of ultrasonic treatment on genetic transformation efficiency varied with genotype.In summary,this study optimized the genetic transformation systems of polyembryonic and monoembryonic gambit from the screening of high-efficiency genotypes for genetic transformation and the treatment of explants,respectively,which will promote the research progress of gambit as a citrus model plant and provide technical support for the research and development of gambit and the study of citrus gene function. |
PDF Full Text Request