Development Of The Regeneration And Genetic Transformation System In Citron

Citrus is a perennial woody plant and it is often used to verify the gene function and accelerate the breeding process through transgenic technology.However,there are still several problems in citrus tissue culturing and genetic transformation,such as high manipulation requirement,complex operation,long period,low transformation efficiency and hard mature transformation.The preliminary study of the research group found that the original citrus germplasm Citron C-05 not only has unique resistance to citrus canker disease,but also has excellent regeneration ability.Therefore,it is an ideal material for citrus genetic transformation.This study carried out a systematic study of stem cuttage,stem tissue culture regeneration,leaf cuttage regeneration,agrobacterium-mediated stem cuttage genetic transformation and stem tissue culture genetic transformation of Citron C-05.The main results are as follows:1.The regeneration system for germinating adventitious buds of Citron C-05 was established.According to the difference between the degree of semi-lignification and lignification of branches,branches with diameters below 3.5 mm,3.5 to 4.5 mm and4.5 mm above were trimmed into stem segments about 7 cm.And they were cultured at different temperatures(22℃,25℃,28℃,32℃,36℃).The statistical results of adventitious bud germination showed that the ratio of adventitious bud germination was the highest at 46.2% when Citron C-05 stems with a diameter of 3.5 to 4.5 mm were selected as cuttings,and the number of adventitious buds was less than 1 in each stem segment.In addition,the cuttage stems grew best at 28℃.The top of the cuttage was moistened with cotton wool soaked in MT(1/4)+ 6-BA(3 mg/L)solution.Cultivate the stems in an indoor light-filled artificial climate room with the temperature of 28 ℃and light intensity of 9000 lux.The average number of adventitious buds at the top of stem can up to 2.2.The regeneration system of Citron C-05 adult stem segments was constructed and optimized.Studies on the in vitro disinfection method and regeneration culture system of the adult stem segments of Citron C-05 showed that: the new shoots of Citron C-05 grown in a clean plant culture room were selected and disinfected with75% alcohol for 30 seconds and 1% sodium hypochlorite solution for 15 minutes,the disinfection effect is the best,and the success rate can reach 85.2%.However,it is difficult to disinfect the stem material cultivated in the field or greenhouse in vitro.When no exogenous hormones were added to the stem segments,adventitious buds could also be formed on MT basic medium,and the rate of adventitious buds was33.52%.In addition,adventitious buds could be directly rooted on MT medium without micro-bud grafting or using rooting medium,which shortened the time to obtain regeneration plants.3.The regeneration system of Citron C-05 leaf cuttings was determined.The leaves of Citron C-05 with different leaf ages and types(with or without bud)were cut and cultured on sterilized vermiculite,and it was found that the leaves of Citron C-05 could be rooting,among which the root development of mature leaves was the best.Leaves with bud could be developed into complete plants.Leaves without bud could not germinate adventitious buds,and the conditions for germinating adventitious buds were being further explored.4.The genetic transformation system of adult stem tissue culture of Citron C-05 and the efficient genetic transformation system of stem cuttage of Citron C-05 preliminarily explored.With ‘Bingtang’ orange adult stems for comparison,through the above-mentioned Citron C-05 adult stem segments disinfection method,using agrobacterium-mediated genetic transformation of Citron C-05 stem segments,the results showed that: The growth rate of agrobacterium during co-cultivation of Citron C-05 stem segments was significantly higher than that of ‘Bingtang’ orange,and the bacteriostatic antibiotic Cefotaxime(600 mg/L)was difficult to inhibit the growth of agrobacterium during the transformation of Citron C-05.The relevant conditions need to be further optimized.Using the stem section of Citron C-05 with the largest number of germinated adventitious buds determined in this study as a material,the method of keeping the top of the stem segments not easy to dry or die was determined.Agrobacterium suspension with 10% glycerol instead of sucrose,the top of the stem segments were soaked in the resuspending for 15 minutes,and then MT(1/4)+ 6-BA(3 mg/L)+ Hyg(25 mg/L)solution was used to moisturize the top of the stem segments to make the stem segments generate better callus.Transgenic plants were currently undergoing further screening and identification.This method is simple and easy to operate without specific tissue culture conditions,and can be developed as an efficient genetic transformation pathway for citrus adult tissue.