Isolation Of Mesophyll Protoplasts And Transient Expression Of GFP In Carica Papaya L.

Papaya(Carica papaya L.) is a popular and important economically fruit tree of tropical and subtropical countries because of its high nutritional value, medicinal and industrial applications. Since the recent sequencing of the papaya genome in 2008, the functional identification of papaya genes has become increasingly important. The yield and viability of protoplasts obtained by repeating this procedure was insufficient for gene functional analysis for papaya, It means that papaya in the model species of fruit tree will play a very important part in biological research and in other related fields. Furthermore, protoplasts also may provide a single cell model system to study the infection and replication of plant virus. Papaya ringspot virus (PRSV, genus Potyvirus, family Potyviridae) is considered to be the most destructive disease and occurs in most of the major papaya plantation areas of the world. Although transgenic papaya resistance to PRSV generally are considered the best strategy for long-term virus control, different specificities of PRSV resistance were displayed in transgenic papaya lines expressing different PRSV CP genes toward PRSV isolates from different geographic origins and the application is limited in other areas of the world because of strain-specific resistance. For understanding of molecular events associated with PRSV infection, PRSV-induced gene silencing and PRSV-host interactions to develop new strategies against PRSV, establishing an efficient protocol for high yield and viability of protoplasts from papaya leaf is essential to further study the infection and replication of PRSV. In this paper, the seedings of Carica papaya L.was served as donor material, systematic investigation was carried out on factors affecting the protoplast isolation and the transient expression of PEG-mediated DNA uptake in the protoplasts of papaya, preliminarily developed the high-throughput transient gene expression systems of protoplast in papaya, and laid the foundations for further cytobiology and molecular biology operation.Several key abiotic factors, such as enzyme mixtures, osmotic potential, incubation time, and digestion pH affect the isolation of plant protoplasts. Each plant species requires specific optimal conditions for protoplasts isolation, and there are many reports on the optimal isolation conditions for different species. However, the yield of the isolated protoplasts was low, the average yield of protoplast was 2.33±0.69×106 cells per gram (82% viability) of papaya leaves was reported by Janjira (2003) [1], and 8.4±0.6×106 cells per gram obtained by Kumrop (1998) [2], and we see that the yield and viability of protoplast are unsatisfactory, there will be great scope to boost. In this study, the effects of factors (the concentration of Cellulase R-10, Macerozyme R-10 and D-mannitol, duration of enzyme incubation) determining yield and viability of protoplasts isolation were investigated using Taguchi experimental design to optimize protoplasts isolation conditions from the leaves of Carica papaya L. seedlings. The results showed that the concentration of cellulase R-10 and incubation time were key factors affecting the protoplast yield; D-mannitol concentration played a significant role in protoplast viability. Moreover, the result of single factor tests revealed pH of the enzyme solution influenced significantly both yield and viability of protoplasts. The highest yield of 1.745x107 protoplasts g-1 fresh weight (FW) with more than 90% viability were consistently obtained by the optimum isolation conditions:enzyme combination of 1.2% cellulase R-10,0.3% macerozyme R-10 and 0.44 M D-mannitol, pH 5.8 and incubation for 13 h at 23℃with a shaker speed of 60 rpm in darkness..Based on these results, main factors influencing transient gene expression of papaya protoplast were studied, including PEG relative molecular weight, PEG-treament concentration, PEG-treament time,DNA concentration.The results showed that the optimal transformation conditions was obtained under conditions of adding amount of plasmid (HBT95-SGFP(S65T)-NOS) 16μg, taking 20% PEG8000(ultimatedensity), concentration of protoplast 2×105cells, treatment time 10 min, and incubated at 23℃for 24 h,the average transformation rate was 70%.