Phalaenopsis In Vitro Culture

Phalaenopsis (Orchidaceae) were cultured as cut flowers and potted plants. In this study, the tissue culture of Phalaenopsis in vitro was researched. The axillary buds of floral stalk in Phalaenopsis were pre-cultured in vitro to germinate the dormancy buds. The adventitious buds derived from pre-cultured flower stalks were used as explants to induce protocorm-like body (PLB) . Then, the multiplication of PLBs, plant regeneration and seedlings rooting were performed. Additional, somatic embryogenesis of Phalaenopsis and explants browning in vitro were investigated. The detailed works and results were as followed.The axenic adventitious bud was gained by inducing the germination of the dormancy buds. For dormancy buds sprouting, ex-hormones were necessary. The axillary buds of floral stalk in middle part were easy to sprout than that in top part. Murashige and Skoog (MS) medium supplemented with 5.0 mg/L BA and 0.5 mg/L NAA were fit to induce sprouting.For induction of PLB form leaf segments, leaf segments were cultured in the dark for 2 weeks, followed in the light. This method could ease the degree of browning and raise the rate of induction. The different effects of medium, 6-BA, NAA, and active carbon (AC) on PLB formation from leaves were compared by the orthogonal design. The results showed that 6-BA played the best important role in inducing. The effects of medium were stronger than NAA and AC. The best combination was MS + 5.0 mg/L BA + 0.5 mg/L NAA + 0.1 g/L AC + 150 ml/L coconut milk (CM).For multiplication of PLBs, the effects of medium, 6-BA, NAA and organic compounds were examined by the orthogonal design. The results revealed that 3 g/L Hyponex No.1 + 5 mg/L BA + 1 mg/L NAA +150 ml/L CM medium were suitable for multiplication of PLBs. The highest ratio of multiplication of PLBs was 5.5 in theory.NAA played a beneficial role in root differentiation. Hyponex No.1 medium supplemented with NAA and 6-BA could induce root differentiation of PLBs. Appending 0.5 g/L AC can promote the average amount of roots and average root length. The suitable media inducing rooting was 3 g/L Hyponex No.1 + 0.1 mg/L BA + 1 mg/L NAA + 0.5 g/L AC + 100 ml/L potato juice.The study of callus induction and somatic embryogenesis indicated that sucrose played the best important role in inducing callus. The medium with VW + 0.3 mg/L BA + 0.1 mg/L NAA + 40 g/L sucrose or VW + 0.1 mg/L BA + 0.1 mg/L 2,4-D + 60 g/L sucrose showed the best effects. Histological of cultures showed that the embryonic cells were emerged from the outer cell of callus. As the differentiation progressing, the embryos started to initiate and topmost cells differentiated protrusion. Then these protrusions turned into PLBs without any vascular connection with each other and callus tissue. The somatic embryogenesis in vitro cultures were proved by the histological observation.Tissue browning is major impediments of in vitro culture. Explants browning occurred after 6 days' culture. The study on the enzyme involved in browning showed that the activity of PPO and POD became high in the first stage, while their activities declined in the later stage. The study on PPO and POD isoenzyme revealed that the four bands of POD in browning cell appeared and the activity was strong after 3 days' culture. They exhibited no obvious change as the culture period extended. For PPO isoenzyme, the activity of the band which its mobility was 0.311 was strong and the band all appeared during the browning period.As explants turning black, the activity of PAL was increasing and the content of chlorophyll a and b decreased, in accordance with the colour change of explants. The activity of PAL had a very important relationship with explants browning.