Factors affecting Agrobacterium-mediated transformation efficiency of kumquat seedling internodal stem segments

Abstract

Kumquat (Fortunella crassifolia Swingle.) has a short juvenile phase of 2–3 years and it can flower and bear fruit several times per year. It is, therefore, helpful for citrus to evaluate functional genes rapidly. In the present experimentation, a comprehensive and systematic study was performed to improve the transformation efficiency of kumquat, which was usually less than 4.1% in previous reports. Several factors, including explant status, Agrobacterium infection procedure, and co-cultivation conditions, were tested by using single factors or orthogonal experimental design. The higher regeneration and transformation rates were obtained from the seedlings cultured for 25days in darkness followed by 10days under light. The Agrobacterium cells suspended with co-cultivation medium to a final concentration 1×109 cfu/ml (OD600 value of 0.5) and immersing explants in Agrobacterium inoculum for 30min, resulted in better infection efficiency. The results indicated that the following conditions could improve the transformation efficiency. These treatments included adding 100μM AS into inoculum and co-cultivation media, placing internodal stem segments by end to end on co-cultivation medium for 3days at 25°C and placing co-cultivation medium in pH 5.4 with sterile filter paper support. With all these improvements, GUS-positive buds were recovered at the positive rate of 23.6% on selection medium. Polymerase chain reaction (PCR) analysis using gene-specific primers further confirmed the integration of the transgene for in vitro grafted plantlets at the transformation rate of 10.35%. Normal expression of nptⅡ and FLP genes was observed by RT-PCR and Real time q-PCR analysis.