Study On Callus Induction And Plant Regeneration Of Camellia Japonica L.’Naidong’

Camellia japonica L.’Naidong’, an evergreen broad-leaved shrub or smalltree of genus Camellia under Theaceae, is mainly distributed in Laoshan of Qingdaopeninsula and Changmenyan island in Shandong Province where it grows in the northernmostnatural distribution of Camellias in China. It is well-known for its unique features ofevergreen leaves, gorgeous colors, beautiful flowers as well as long flowering period. It is themost important woody flower in Qingdao city, and it was chosen as city flower of Qingdao in1988. Its seedlings are mainly still propagated by conventional way of seed propagation.However, due to its narrow distribution, sources of seeds and seedlings are of a seriousshortage so that it cannot meet demand of seedling market.In this experiment, callus induction, proliferation and organogenesis were studied withcotyledons of C. japonica L.’Naidong’ to obtain the best methods and optimum timing ofsterilization, effective ways in preventing browning of treated materials, such as epicotyl,hypocotyl, nodal segment and root segment, culture conditions, optimum hormonecombinations in callus induction, proliferation and differentiation of adventitious buds, andbetter means of regenerated plantlet rooting, domestication and transplanting. The results aresummarized as follow:⑴The best manner and optimum timing of sterilization in C. japonica L.’Naidong’young fruitsImmature Camellia fruits were sterilized together with its peel, mature Camellia embryoswere disinfected;10mL Tween20and2³drops of1mol·L^-1HCl solution were added into500mL0.1%（w/v） HgCl2when prepared. Immature fruits or mature seeds were soaked in themodified HgCl2solution for8min, rinsed5⁶times with sterile distilled water, and then putin a dish placed with a filter paper drying for next experimental operation.⑵Efficient methods of C. japonica L.’Naidong’ explant anti-browning 0.2%（w/v） PVP was added into MS medium, and explants were cultured under weaklight for the first5days, which could prevent browning efficiently.⑶Culture conditions and optimum hormone combinations of callus induction in C.japonica L.’Naidong’With DPS software and with L43orthogonal experimental design, the effects of sucrose,auxin and cytokinin combinations on the rate of cotyledons callus induction of C. japonica L.’Naidong’ were studied. The experiment results showed that: in lightness culture, the optimumcombination of sucrose and hormone was MS+30g·L^-1sucrose+0.5mg·L^-12,4-D+0.5mg·L^-16-BA, with the highest rate of callus induction up to94.5%. In darkness culture, thebetter combination of those three factors was MS+50g·L^-1sucrose+1.0mg·L^-12,4-D+2.0mg·L^-16-BA, with the highest ratio up to98.57%. The days of callus induction in darknessculture was less than that in lightness culture. The growth of callus was higher, and the rate ofcallus induction under dark conditions was generally higher than that under lightingconditions. At last cultured in light conditions with MS+30g·L^-1sucrose+0.5mg·L^-12,4-D+0.5mg·L^-16-BA was adopted.⑷Better hormone combination for callus proliferation and adventitious budsdifferentiationAfter2times subcultured in MS+0.1mg·L^-1NAA+2.0mg·L^-16-BA, the creamy yellow orlight yellow callus in firm texture was transferred to MS+0.5mg·L^-1NAA+10.0mg·L^-16-BAfor differentiation of adventitious buds.⑸Rooting of C. japonica L.’Naidong’ adventitious budsThe shoot base was immersed in500mg·L^-1IBA solution for60min, then transferred tofilter paper bridge received MW liquid medium cultured in low light (100²00μmol·m-2·s^-1).The rate of rooting is about50%.