Studies On Cell Suspension Culture And Protoplast Isolation Of Zanthoxylum Bungeanum

Zanthoxylum bungeanum is an important economic species of Shaanxi province. Zanthoxylum in Fengxian and Hancheng countys have the hightest economic value and most widely distribution. In recent years, with the new utilization approaches of Zanthoxylum developed, the market demand of Zanthoxylum is rising. In order to meet the current needs of the development of Zanthoxylum industry, carrying Zanthoxylum breeding work is imminent. However, Zanthoxylum belongs to apomictic plants and artificial hybridization is very difficult. Currently, Zanthoxylum genetic improvement mainly relies on natural variation, but the low variation and uncertainty mutation rate makes Zanthoxylum germplasm enhancement and breeding work has been no breakthrough. The advancement of modern biotechnology and the maturation of forest tree somatic cell hybridization technology provide new ideas and methods for the breeding of Zanthoxylum. Somatic cell hybridization technology enables to integrate good traits of Zanthoxylum in Fengxian and Hancheng countys, but must obtain high yield and viability protoplasts as a precondition. For hybrid cells screening, one of the most common methods is fusing two parental species protoplasts from non-chloroplast cells(such as callus and cell suspension culture cells) and chloroplast-containing cells(such as mesophyll cells) respectively. Our group has established tissue culture regeneration system of Zanthoxylum in Fengxian county previously, but the callus is used to induce adventitious buds and has compact structure which is not suitable for establishing cell suspension and isolating protoplast. And the medium used to culture Zanthoxylum plantlet in Fengxian county does not necessarily apply to Zanthoxylum plantlet in Hancheng county. Therefore, on the basis of our previous study, in order to meet the requirements of protoplast isolation, research has been carried out of three aspects: First, stem culture of Zanthoxylum in Hancheng county and rapid propagation of regenerated plantlet; second, callus subculture of Zanthoxylum in Fengxian county to get vigorous growth and loosely structured callus, for establishing cell suspension of Zanthoxylum; third, taken plantlets leaves of Zanthoxylum in Hancheng county, and callus and suspension cells of Zanthoxylum in Fengxian county as materials to study protoplast isolation and purification technology system of Zanthoxylum. The main results are as follows:1. Culture of Zanthoxylum bungeanum and rapid propagation of regenerated plantletTender stems with bugs of Zanthoxylum bungeanum were taken as experimental material for studying the effects of different conditions on sterilized culture. The results showed that the optimum disinfection time of 0.1% mercuric chloride was 10-13 min. The best sampling time was from April to May. The best bud germination culture medium of Zanthoxylum bungeanum was MS+6-BA 1.0 mg·L-1+IBA 0.3 mg·L-1+GA3 2.0 mg·L-1+ sucrose 30 g·L-1+ agar 6 g·L-1. The optimum medium for bud multiplication culture was MS+6-BA 0.5 mg·L-1+IBA 0.5 mg·L-1+GA3 2.0 mg·L-1+sucrose 30 g·L-1+agar 6 g·L-1 and the propagation coefficient was 4.6. The rooting culture medium was 1/4MS+IBA 0.4 mg·L-1+ sucrose 30 g·L-1+ agar 6 g·L-1 and the number of roots was1-4, in which the rooting rate was 80%.2. Cell suspension culture of Zanthoxylum bungeanumPlantlets leaves of Zanthoxylum bungeanum were taken as experimental material for studying the effects of different composition of hormone on callus induction and subculture. The crisp callus was selected to establish cell suspension culture. The results showed that the best callus subculture medium was MS+2, 4-D 1.0 mg·L-1+6-BA 0.5 mg·L-1+sucrose 30 g·L-1+agar 6 g·L-1. The best suspension culture medium for Zanthoxylum bungeanum cells was MS+2,4-D 1.0 mg·L-1+6-BA 1.0 mg·L-1+glucose 30 g·L-1.The best culturing conditions were: Inoculum size 0.05 g·m L-1, gyratory shaker rotation speed 110 rpm, temperature 25 ℃, and in dark environment. Under these conditions suspended cells grew well and the maximum fresh weight of cells was 4.52 g. Suspended cells growth of Zanthoxylum bungeanum was “S” type curve and optimal subculture cycle was 16 days.3. Protoplast isolation and purification of Zanthoxylum bungeanumUsing leaves, callus and suspension cells of Zanthoxylum bungeanum as experimental material. Through single factor experiment, studying the effects of enzyme concentration, enzymolysis time, osmoticum concentration and sucrose concentration on protoplast separating and purifying, and protoplast yield and protoplast viability were measured by using hemocytometer counting and FDA staining. The results showed that the best enzyme combinations for protoplast isolation from leaves of Zanthoxylum bungeanum were CPW-0.7 mol·L-1 mannitol+1.0%(w/v) cellulase onozuka R-10+1.5%(w/v) pectinase, the optimum enzymolysis time was 10 h and the sucrose concentration for protoplast purification was 30%(w/v). Under these conditions, protoplast yield was 82.36×105 ·g-1 and the protoplast viability was 72.74%. The suitable enzyme combinations for protoplast isolation from callus and suspension cells were CPW-0.6 mol·L-1 mannitol+2.0%(w/v) cellulase onozuka R-10+0.5%(w/v) pectinase, the optimal enzymolysis time was 12 h-14 h and the sucrose concentration for protoplast purification was 25%(w/v)-30%(w/v). The yield and viability of protoplasts were 31.26×105 ·g-1, 59.15% and 53.87×105 ·g-1, 63.92%, respectively. High sucrose concentration was beneficial to increase protoplast yield but also lead to the decrease of viability. Leaves were the best material for protoplast separating, suspension cells the second, and the yield and viability of protoplasts from callus the lowest.