Preparation, Fusion AndCulture Of Protoplasts Of Hibiscus Cannabius L

Linum usitatissimum L. is an annual dicotyledonous herb of the family linaceae ofsubclass Rosidae. As the flax fiber has some merits such as strong tension, tenderness,excellent evenness, weak conductivity, easy wet and dry, high rate of swelling, it has earnedthe reputation of "Fiber Queen". However, there still exist some shortcomings in practicalproduction of Linum usitatissimum L. including low growth rate and low biomass. Hibiscuscannabinus L., another major source of fibers, possesses properties of tall trunk, short lifeperiod, high yield of biomass, and rapid accumulation of dry material. By means ofprotoplasts fusion, it is possible to overcome the problem of reproductive isolation betweenLinum usitatissimum L. and Hibiscus cannabius L., thereby it will produce a new variety offlax species by making using of their advantages of rapid growth rate and high-quality fibers.The seeds of Linum usitatissimum L. and Hibiscus cannabius L. were used as explantsfor the induction of callus. Through optimization and screening of culture medium, a kind ofgolden, grainy texture, fast growing and friable callus was obtained to isolate and purifyprotoplasts. The effects of some factors such as combinations of enzyme mixture, digestingtime, mannitol concentration, and pH value of digesting solution on protoplasts purification ofthe two materials were also studied, and subquently the preliminary experiment of protoplastfusion was carried out aiming to seek novel pathway for somatic hybridization betweenLinum usitatissimum L. and Hibiscus cannabius L.. The main results are as follows:1. Seeds were used as explants and MS as the basic medium, effects of2,4-D, NAA,6-BA on callus induction and growth rate were investigated by combinations of these growthregulators. The results showed that all the three regulators could induce callus successfullyunder certain concentration ranges, the optimized medium for Linum usitatissimum L.callusinduction was MS+2,4-D1.0mg/L, and for callus growth it was MS+2,4-D0.5mg/L+6-BA0.1mg/L. For the callus induction of Hibiscus cannabius L., the optimal culture medium wasMS+2,4-D1.0mg/L+6-BA0.5mg/L, and for callus growth was MS+2,4-D0.5mg/L+6-BA0.5mg/L.2.The optimized treatment for isolation of Linum usitatissimum L protoplast was2.0% Cellulase R-10,0.5%Pectinase,0.55mol/L mannitol as osmotic pressure,28℃under dark50r/min for6h, and the highest yield achieved to4.87×10⁵per gram, the protoplast vitalitywas62.2%. And for Hibiscus cannabius L., it was2.0%CellulaseR-10,1.0%Pectinase,0.55mol/L mannitol as osmotic pressure,28℃under dark50r/minfor6h, and the highestyield achieved to5.51×10⁵per gram, the protoplast vitality was63.6%.3. The effects of chemical and electric methods on the both protoplasts fusion werestudied and compared. And the results indicated that the best conditions for the fusion ofLinum usitatissimum L. and Hibiscus cannabius L. protoplasts was40%PEG （6000） withaddition of0.3mol/L Ca²⁺and pH9.5, and the protoplast fusion percentage was11.5%. Theoptimal electric parameters for protoplast fusion were100V/cm of alternating current （AC）,50s of duration time,800V/cm of direct current （DC） intensity,60μs of direct current time,and3times of pulse.