Establishment Of Efficient Reproduction System Of Phalaenopsis Amabilis Cv V31and Screening Of Novel Type Anti-browning Natural Additives

Phalaenopsis is one of the currently popular high-grade flowers. China owns six kinds.It has many ornamental parts, including changeable petals, wonderful color, thick andbright blade and intricacies of pedicel，etc. As the Spring Festival flower and the materialof cut flower, Phalaenopsis is in large marketable demand every year. Although breedingof new varieties is increasingly faster, industrialized production of Phalaenopsis still hasmany burning questions. Problems in actual cultivation process are as follows: Firstly,output of Phalaenopsis is far less than demand due to difficulty of its large reproduction;Secondly, because growth cycle of Phalaenopsis is long, it is difficult to meet to thedemand of supply time; Thirdly, good character of female parent is difficult to keep underextensive cultivation mode; Fourthly, traditional means to prevent Browning are toosingle, and the effect is limited. It needs to constantly change the culture medium toachieve inhibition effect; Fifthly, cultivating method of Phalaenopsis is single, resulting inno plant in good quality to supply market.Phalaenopsis cv V31was used as research material in this study. we proceeded in twodifferent tissue culture line to seek rapid propagation system of Phalaenopsis, at the sametime improve the tissue culture system.One line was peduncle slices and axillary buds of peduncle were used as explant. Theexplant directly generated differentiation of multiple shoots.then roots generated. Wescreened out the best system of rapid propagation and optimal culture medium ofPhalaenopsis, as a result we shortened the breeding period.The other line was we used blade, pedicel section, and tender leaves, stem tip of tissuecultured seedling as explant. The explant engendered differentiation of cluster buds afterproducing induction and proliferation of Protocorm, finally formed complete system oftissue culture through rooting.Furthermore, with the green environmental protection idea we found new alternative totraditional methods of preventing Browning, using biochemical characteristics of plantitself.We obtained the following results:（1） Filtering out the best disinfection solution by full-factorial experiment.Pedicel and pedicel slice of Phalaenopsis cv V31: After preliminary cleaning and flowingwater treatment, explants were placed on clean bechtop. Pedicels and pedicel slices wereimmersed in70%alcohol for25s, and then in0.2%HgCl₂（containing0.1%twain-20）for11min； leaves were disposed using70%alcohol for15s, and then in0.1%HgCl₂ （containing0.1%twain’s-20） for14min. Explants were flushed by sterile water thenvaccinated.While, compared with leaves, time of former explants soaked by detergent shortened tohalf and water flushing time shortened to0.5h in earlier stage processing.（2） Establishing rapid propagation system of Phalaenopsis cv V31, selecting out theoptimum medium formula of each stage by orthogonal experimental design of four factorsin three levels.The optimum culture medium in stage of inducing multiple buds: MS+8.0mg/L6-BA+2.0mg/L NAA+15g/L powder of coconut.The optimum culture medium in proliferation phase of Multiple buds: MS+10mg/L KT+0.6mg/L NAA+0.2mg/L2,4-D;The optimum culture medium in rooting and seedling stage:1/2MS+0.1mg/L NAA+1mg/L6-BA+20g/L sucrose+10g/L powder of banana.（3） Screening best concentration for inhibiting Browning and perfecting the Phalaenopsiscv V31tissue culture system. We conducted full-factorial experiment by setting threeconcentration gradient of natural additives（marigold extract, lemon extract, tomato extract androsemary juice） and conmbining with culture medium（MS、B5and N6）.（4） Improving tissue culture system of the Phalaenopsis cv V31.Sifting the optimum culture medium inducing protocorm to become proliferate byuniform experimental design of four factors in six levels:The optimal Culture medium of protocorm proliferating: N6+2.0mg/L6-BA+0.1mg/LNAA+150mg/L citric acid+12g/L powder of banana;The optimal culture medium of protocorm differentiating to become multiple shoot: weused the optimal culture medium in the rapid propagation system, but the different cultureconditions with illumination time shortened to half.We also found differentiation rate of seedling lighted6h was greater than that of seedlinglighted12h.Medium of Rooting and seedling stage was same with that of the rapid propagation system.Bolting out the optimized cultivation substrate: Sphagnum soaked by0.1%KMnO4for0.5h+corn cob.The rapid propagation and integrated tissue culture system of Phalaenopsis cv V31established by this experiment will lie bases of large production of P.amabilis in highquality for the future. Meanwhile, it also provides new springboad and new research idea for future inhibiting Browning of natural additives. It has practical significance tissueculture research in the future.