LIBRO-CONGRESO-CITRUS

S05P19
Establishment of a transient expression system in citrus via agro-infiltration
Dai S.M., Li F., Yan J.M., Li D.Z., Li R.H., and Deng Z.N.
Hunan agricultural university, China. dsm531@126.com
Genetic transformation is a traditional tool for both functional genomics and molecular breeding in plants,
but it is time-consuming and low efficient in citrus due to its low transformation rate. It is essential to
establish a transient expression system to speed-up gene function identification. Here we report a gene
transient expression by using agro-infiltration for citrus. A full comparison was performed between different
parameters including Agrobacterium injection buffer solutions, expression vectors and concentrations of
the bacterial solution. Results showed that in the transient expression system with injection buffer solution
composed of 50mM MES pH5.6, 10mM MgCl 2 , 150µM acetosyringone, pCAMBIA-2301 expression vector and
Od 600 = 0.4 as ideal concentration of bacterial solution, a foreign gene could be effectively expressed in citrus.
With this system, it is possible to analyze gene function in citrus rapidly.
S05P20
Improvement of transformation efficiency in citrus
Yang L. 1 , Hu W. 1 , Xie Y.M. 1 , Li Y. 2 , and Deng Z.N. 1
1 National Center for Citrus Improvement (Changsha) (NCCI), Hunan Agricultural University, China; and 2 Department of Plant Science
(DPS), University of Connecticut, USA. yangli526526@126.com
Internodal stem segments from citrus seedlings have been widely used as explants in transformation
research, due to their ability of easy regeneration. Since 1990, genetic transformation has been applied on
different citrus species including sweet oranges (Citrus sinensis), mandarins (C. reticulata), trifoliate orange
(Poncirus trifoliata), sour orange (C. aurantium), pummelo (C. grandis), citrange (C. sinensis × P. trifoliate),
and lemon (C. limon) in our biotechnology program. But the successful transformation rates are low, thus
impeding the realization of transgenic cultivar breeding. Consequently, the development of an efficient
transformation system becomes essential. A series of factors was tested to improve transformation rates
in sweet orange and kumquat (Fortunella crassifolia). The results indicated that using seedlings grown
20 to 25 days in dark and 10 days in light as explants could provide higher frequencies of GUS positive
regenerated buds. Adding auxin and placing sterile filter paper during the co-cultivation estimulated
the growth of regenerated buds, and increased about 13% the frequency of GUS positive regenerated
buds. Lower pH and co-cultivation temperature under acetosyringone induction enhanced transformation
competence. With all the improved factors, the transformation rate was in average 18.6%, and in the best
case, the transformation rate reached 23%.
S05P21
Improvement of genetic transformation with mature explants of sweet orange (Citrus sinensis)
Xie Y.M. 1 , Yang L. 1 , Hu W. 1 , Li Y. 2 , and Deng Z.N. 1
1 National Center for Citrus Improvement (Changsha) (NCCI), Hunan Agricultural University, China; and 2 Department of Plant Science
(DPS), University of Connecticut, USA. xym9366@163.com
High level of contamination, low morphogenetic potential and recalcitrance to Agrobacterium tumefaciens
infection are the main problems in the transformation of adult citrus tissues. A series of experiments was
performed to solve these problems. ‘Newhall’, ‘Succari’ and ‘Bingtang’ sweet orange cultivars were tested.
Adult shoots from the 3 cultivars were grafted and re-grafted on Poncirus trifoliata rootstocks in the
greenhouse. Internodal stem segments of about 1 cm in length from 30-day old mature shoots of were taken
as explants. Through re-grafting mature shoots, the contamination rate of explants was much lower, even
0%. The adventitious bud regeneration rate was obviously improved, approximately 80%, 67.5% and 55% in
‘Succari’, ‘Bingtang’ and ‘Newhall’ sweet oranges, respectively. After Agrobacterium infection, the highest
adventitious bud regeneration rate of ‘Bingtang’ sweet orange reached 25%. The transformation rate was
3.8%, 4.6% and 3.2% in ‘Succari’, ‘Bingtang’ and ‘Newhall’ sweet orange cultivars, respectively. The ‘gene-
XII INTERNATIONAL CITRUS CONGRESS 2012 - 87
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