Capsule development and seed production: In vitro artificial pollination could lead to successful capsule development. The capsules developed quickly after pollination, the length and diameter of them saw a gradual growth over the prior 60 days, reaching at 36.88 and 7.71mm respectively (Fig. 1f). After that, the figures remained at a stable level. The size and shape of the fruit In vitro were similar to those grown In vivo. The mature capsules with fertile seeds formed after 120 d of culture, when the capsules had turned yellowish. These capsules were harvested upon maturation, and seeds were sown on 1/2 MS medium (Fig. 1g). After 60 days, 86.6% of the seeds germinated into PLBs or green shoots and grew into seedlings (Fig. 1h) (Table 3).
Discussion
The results presented here describe an efficient protocol for multiple-plantlet regeneration from shoot- tip explants of D. officinate, followed by flowering and fruiting of regenerated plantlets In vitro. In vitro flowering of D. officinate has previously been documented when seeds were cultured on MS medium (Wang et al., 1997), but reports on fruiting and seed set were lacking. The present study offered another regenerative system for In vitro flowering, which allowed the formation of multiple shoots, flowering plantlets, and fertile seed production. This reproductive system might play an important role in orchid breeding because shoot-tip explants can be easily obtained.
The cytokinin requirement for the growth and development of flower buds has been reported in several plants (Heylen & Vendrig, 1988; Huang et al., 1999; Zhang, 2007), and BA was found to be effective for early floral induction in orchids (Chang & Chang, 2003; Wang et al., 1997; Sim et al., 2007). However, TDZ has been considered to be more potent than most of the commonly used cytokinins (Sajid & Aftab, 2009; Naz et al., 2009). The importance of TDZ for In vitro flower induction has been reported in D. nobile and Cymbidium ensifolium, where TDZ had a stronger flower-inducing effect than BA (Chang & Chang, 2003; Wang et al., 2009). The results in this study agreed with those findings. However, abnormal flowers occurred in the culture with higher TDZ concentrations (Table 2), so the cytokinins may influence a delicate regulative system in floral development. Some genes controlling shoot apical meristem activity may be related to floral regulation (Lindsay et al., 2006).
The cultures with optimal BA and TDZ concentrations produced normal flowers; these flowers resembled the flowers of field-grown plants and had complete structures. However, the In vitro plantlets produced fewer and smaller inflorescences than the field-grown plants (Table 2). This could be due to the smaller size of the In vitro plantlets, since reproductive output may be affected by plant size (Sletvold, 2002).
The morphology of pollen and female organs in the In vitro flowers was normal. The morphological and anatomical examination showed that a normal column with pollen and stigma formed in the In vitro- developed flowers (Fig. 2b), and the pollen could germinate on the culturing medium (Fig. 3b). Furthermore, the success of In vitro pollination and viable seed formation demonstrated that the gametes produced from In vitro flowers were functional. In addition, the technology of seed production in culture could have tremendous applications in the breeding of D. officinate (Hee et al., 2007).
Conclusion
In summary, D. officinate flowered and produced viable seeds in culture in this study. Such a reproduction system is ideally suited for the study of the molecular basis and hormonal regulation of flowering and development, while also having practical implications for effective plant breeding.