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Establishment of in Vitro Propagation System of Phalaenopsis and Investigation on in Vitro Regeneration Pathways

Author: YangHaiZuo
Tutor: WuZhen
School: Nanjing Agricultural College
Course: Ornamental Plants and Horticulture
Keywords: Phalaenopsis.ssp In vitro propagation adventitious shoots Protocorm-like body Morphological genesis
CLC: S682.31
Type: Master's thesis
Year: 2006
Downloads: 49
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Abstract


Phalaenopsis, one of the Orchidaceae family flowers named "Queen of tropic Orchid" for its lightsome inflorescence, pealike flower color and particular flower shape, is a most popular flower in the world market. However, it is difficult to propagate phalaenopsis by dividing shoot cluster because of its simple stem and aerial root. And it cannot germinate by the tiny seeds which have no endosperm. The traditional propagation methods used before lead to low propagation rate and readily virus contamination in flowers and leaves can not satisfy the commercial need to produce phalaenopsis. However, tissue culture provides a way to solve the problems above.In recent years, many researches on phalaenopsis tissue culture is reported on PLB induction, however, PLB induction rate is very low and its development or growth needs more time. And in vitro regeneration pathway and its capabilities differ more frequently due to the difference of genotypes. However, it is easier to obtain leaf as explant. The pathway through axillary bud proliferation which makes hereditary character stable is a better propagation way. Thus, the factors impacting the in vitro adventitious buds induction、proliferation and rootage were investigated in this thesis and morphogenesis pathway and anatomical structure of PLB development were studied. The results were as follows:1. In this study, the excised leaf of Phalaenopsis RSW1 was used as explants, MS and 1/2MS were the optimal basic media, while improved KC was disadvantageous to shoot regeneration; TDZ was more effective than 6-BA in inducing shoot regeneration from leaves of Phalaenopsis RSW1; Appropriate dark treatment could increase the regeneration rate of adventitious shoot; When those leaves aged 45 days were used as explants, higher induction rate and differentiation coefficient could be achieved. Consideing all these factors, 1/2MS supplemented with l.Smg-L-1 TDZ, 0.5mg·L-1 NAA, 2% sucrose and 0.55% agar was a best media. Under the most effective condition of 20 days dark treatment, the induction rate of adventitious shoot reached 94% with the differentiation coefficient 19.4. 2. By taking adventitious shoot from Phalaenopsis cultivar RSW1 leaves as the test material, the factors of axillary buds proliferation were studied. Results showed that, improved Hypnoses were the best basic media; 6-BA was more effective than TDZ in adventitious shoots proliferation, and KT was worse. AD had an enhancement effect on 6-BA, and it promoted the induction of axillary buds. The media supplemented with coconut water was advantageous to shoots proliferation. Compared with the plantlets regeneration from floral buds or axillary buds after proliferation, adventitious shoots have more strong proliferation capacities.3. In the study of rooting and development culture of in vitro plantlets, NAA was the best to root, and when combined with 6-BA, it was advantageous to the growth of stem and leaf. AC had no significant influence on rooting, but it had an effect on the development of plantlets. Organic additives banana homogenate and peptone, at the concentration from 100 to 150ml·L-1, 2g·L-1 respectively, promoted plantlet growth and development. After hardening period, the plantlet with three or four roots and leaves was transplanted, wrapped by sedge. New roots and leaves generated after 30 days, and the survival rate reached 98%.4. Two cultivars ofphalaenopsis （RSW1 and Y） could be induced to regenerate directly through adventious shoots under suitable culture condition (1/2MS+1.5 mg·L-1TDZ+0.5 mg·L-1NAA+10 mg·L-1AD+100 ml·L-1CW, pH 5.4 ) , while the eultivar 20 regenerated indirectly from callus; The media above supplemented with 2 g·L-1 peptone, and with 100 ml·L-1CW removed was suitable for the cultivar RSW1 to regenerate through PLB. The adventitious shoots regenerated from callus could drive from either outer or inner layers. Concomitant with the mitotic activities of the subepidermal cell, many small protuberances were observed on the explants. Continual divisions within these protuberances resulted in PLB formation. After some time of culture, PLB structures began to take shape. As for the controversial question of PLB derivation, the results showed that PLB probably had two regeneration pathways: somatic embryo and callus regeneration.

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CLC: > Agricultural Sciences > Gardening > Ornamental Horticulture ( flowers and ornamental trees) > Perennial Flowers > Other Flowers > Orchids
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