Apr. 2013, Vol. 7, No. 4, pp.

395-399 Journal of Life Sciences, ISSN 1934-7391, USA

Artificial Pollination and Seed Germination of Dendrobium candidum Wall. ex Lindl.

Ke Xu, Yi Tang, Jia Lai, Ze-Sheng Yan, Qian Luo and Huan-Xiu Li
College of Horticulture, Sichuan Agricultural University, Yaan 62504, Sichuan, China

Received: April 7, 2013 / Accepted: April 17, 2013 / Published: April 30, 2013. Abstract: The artificial pollination and the seed germination on Dendrobium candidum were conducted. Results showed that the percentage of fruit-setting reaches the highest under cross pollination before 10 oclock in the morning after blooming 2 d to 4 d. The optimal medium for seeds germination was MS+ NAA 0.5 mg/L + potato juice 10% + active charcoal 0.5%, and cultured for 7 d in the dark then under light. Key words: Dendrobium candidum, artificial pollination, seed germination.

1. Introduction
Dendrobium candidum Wall. ex Lindl. is a perennial herbaceous orchidaceous plant and is distributed over Yunnan, Guizhou, Sichuan and Guangxi provinces of China. Its a famous traditional Chinese medicine which is used to replenish yin and nourish stomach, clear heat and engender liquid, moisten lung and control cough [1-3]. Seeds of Dendrobium are as small as dust, and there are ten thousand seeds in a capsule. Because the seed has no endosperm, it can only germinate in natural conditions when the seed is accrete with fungi. Its germination percentage is usually lower than 5% [4]. The traditional production is difficult because the plant needs a strict live condition, and due to humans over exploitation, Dendrobium candidum resources are going to dry up. So it is imperative to reinforce the research on reproduce methods of Dendrobium candidum. In Dendrobium candidum reproduce system, the most difficult is to attain seeds and make them germinated. The author did a preliminary study on these two techniques, expecting to supply
Corresponding author: Huan-Xiu Li, professor, research field: application of biological technology in horticulture plant. E-mail: hxli62@163.com.

groundwork for the Dendrobium candidum.

reproduce

technique

of

2. Materials and Methods

2.1 Materials Dendrobium candidum materials were from Jingdong county, Simao city, Yunnan Province and they have been planted more than 3 years in Sichuan Province. Chose some healthy and strong plants to pollinate artificially [5], then they were cultured in plastic green house for 8 months, finally the seeds were sowed on the medium before the seed capsule cracking. 2.2 Methods 2.2.1 Artificial Pollination Chose healthy Dendrobium candidum plants, sacked the flowers for 2 d before blooming and pollinated artificially in the second days after blooming. There were 5 groups of artificial pollination test: (1) xenogamy of different flowers in the different plants; (2) geitonogamy of different flowers in the different inflorescence; (3) pollination of different flowers in the same inflorescence; (4) self-pollination in the same flower; (5) natural pollination after sacked

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Artificial Pollination and Seed Germination of Dendrobium candidum Wall. ex Lindl.

as control group. The flowers were pollinated twice a day for 4 d (before 10 a.m. and after 6 p.m.), and every time 5 flowers were pollinated in each group. Technique of artificial pollination: At first removed the labella of female parents by tweezers, then removed the anther cap of male parents carefully, and using tip of stud dipped mucus in the stigma of female parents to absorb the pollen grain of male parents, at last put the pollen grain in the stigma. Changing the tools of pollination every time and bagging the flowers after pollination to avoid wrong mating. The plants were fertilized and watered as normal. The results of pollinations were studied after pollination 2 weeks. 2.2.2 Seed Germination Chose healthy capsule produced by xenogamy, then sowed the seeds as following steps: (1) Rinsed off the smudginess from seeds; (2) Sterilized 10 seconds with 75% alcohol; (3) Disinfected 10 minutes with 0.1% mercuric chloride, washed 5-6 times with sterile water; (4) Dried the seeds with bacteria free filter paper and cut open the capsule and get seeds with bacteria free scalpel; (5) Sowed the embryos on different medium with inoculating needle, and changed to the rapid propagation medium after seed germination. The research used five basic culture media (MS, White, B5, N6 and VW) and three illumination modes (culture in darkness; culture in darkness for 7 d and then culture in light with illumination intensity of 1600-2000lx; culture in light with illumination intensity of 1600-2000lx).

of the 5 groups Dendrobium candidum in different time. Pollination before 10 a.m. was better than that after 6 p.m.; the pollination success rates among different groups were: control group < self-pollination < different flowers in the same

Fig. 1

The fruits of 2 months after pollinated successfully.

Fig. 2

The fruits of 5 months after pollinated successfully.

3. Results
3.1 Artificial Pollination The petals of flowers pollinated successfully began to contract after pollinated 2 days, the ovaries began to bulge quickly 10 days later. The results showed in Figs. 1-4. The petals and receptacles of flowers pollinated unsuccessfully turned yellow after pollinated 2 days, and the whole flower fell off 7 days later. Table 1 showed the artificial pollination results

Fig. 3

The fruits of 6 months after pollinated successfully.

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3.2 Dendrobium candidum Seeds Germination in 5 Media Table 2 showed that germination rates of seeds in MS and N6 medium were higher than that in the other media in all the three treatments. This results was similar to the observation in Ref. [9]. The original buff proembryo changed into green protocorm after Dendrobium candidum seeds have been sowed in medium for 10 days, which were shown in Figs. 5 and 6. Among the three treatments, the germination rate of darkness culture was lower than others, and the germination rates of the other two treatments are similar. Comparing with the treatment of culture in light, the proembryo cultured in darkness for 7 d and then in light changed into green 5-10 d later, but they germinated evenly, and the protocorm was green and sparkling (Fig. 7) and they are better in differentiation (Fig. 8) and rooting. 3.3 Effect of Different Hormones on Dendrobium candidum Seeds Germination Chose 8-month seeds of self-pollination and sowed them in MS basal medium with 4 hormones, survey the germination results after cultured in darkness 7d

Fig. 4

The fruits of 7 months after pollinated successfully.

inflorescence pollinated < geitonogamy < xenogamy. The control group had no successful pollination, and the successful rate of xenogamy was much higher than self-pollination. It was similar to the results observed by Lian [6] and Bo [7]. The pollination success rates increased distinctly 3 days after the flower came into bloom and it was decreased in the 5th day, which indicated that pollen has a higher activity on the 3rd and 4th day after Dendrobium candidum have come into bloom. We observed that stigma secrete the most mucus before 10 a.m. and it was the best time to accept pollens.
Table 1

The pollination successful number and rates of Dendrobium candidum in different time and groups.

(1) (2) (3) (4) (5) A B A B A B A B A B 2d 1 0 0 0 1 0 1 0 0 0 3d 5 3 3 0 1 0 0 0 0 0 4d 5 2 4 2 3 0 0 1 0 0 5d 4 3 2 1 0 1 1 0 0 0 Total 15 8 9 3 5 1 2 1 0 0 (1) xenogamy of different flowers in the different plants; (2) geitonogamy of different flowers in the different inflorescence; (3) pollination of different flowers in the same inflorescence; (4) self-pollination in the same flower; (5) natural pollination after sacked as control group. (A. The flowers were pollinated before 10 a.m.; B. The flowers were pollinated after 6 p.m.). Flower time Table 2 Medium MS White B5 N6 VW Dendrobium candidum seeds germination rates in different media (%). Culture in light 87 81 78 91 72 Culture in darkness 41 33 32 37 28 Culture in darkness 7 days and then culture in light 91 80 79 92 75

The results were 30 d after sowed.

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Artificial Pollination and Seed Germination of Dendrobium candidum Wall. ex Lindl.

Fig. 5

Proembryo began to turn green.

Fig. 6

Proembryo turn green.

Fig. 7 Table 3

Induce into protocorm.

Fig. 8

Protocorm differentiation.

Effect of different hormones on Dendrobium candidum seeds germination. IAA GR (%) 69 81 86 82 80 68 C 0 0.1 0.2 0.5 1.0 2.0 NAA GR 69 72 77 93 86 64 C 0 0.1 0.2 0.5 1.0 2.0 KT GR 69 71 79 82 77 66 C 0 0.1 0.2 0.5 1.0 2.0 6-BA GR 69 84 89 83 76 33

C (mg/L) 0 0.1 0.2 0.5 1.0 2.0

C = Concentration; GR = Germination Rates. Table 4 Effect of different additives on Dendrobium candidum seeds germination. Potato juice 0 10% 10% 0 0 0 0 0 0 Banana juice 0 0 0 10% 10% 0 0 0 0 Coconut juice 0 0 0 0 0 10% 10% 0 0 Apple juice 0 0 0 0 0 0 0 10% 10% Active carbon 0 0 0.5% 0 0.5% 0 0.5% 0 0.5% Germination rates (%) 71 91 91 90 89 89 91 87 86

Medium number CK 1 2 3 4 5 6 7 8

and then cultured under light 30 d. We can found from Table 3 that the germination rates of MS basal

medium with 0.5 mg/L NAA was the highest; When the 4 hormones concentration were below 1.0 mg/L,

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the germination rates were higher than blank control.. When the concentration of IAA, NAA and KT were above 1.0 mg/L, the seeds germination was restrained. When the concentration of 6-BA was 1.0 mg/L, the seeds germination was significantly restrained. The blank control MS basal medium with no hormone also can induce seeds become protocorm, but need longer time and the induction rates was lower, the induction time was delayed about 10 d. 3.4 Effect of Different Additives on Dendrobium candidum Seeds Germination Chose 8-month seeds of xenogamy and sowed them in MS basal medium with different additives, survey the germination results after cultured in darkness 7d and then cultured under light 30 d. We can found from Table 4 that the germination rates were increased by adding additives. Adding active carbon had little influence to the germination rates, but its quicker and greener than control when protocorm propagation, this maybe because active carbon absorbed metabolite in the period of propagation.

Compared to culture in light, the proembryo cultured in dark 7 days then under light became green later, but the germination rates was higher, and the growth potential of protocorm was more stronger in the later culture. Compared the five culture media, MS medium had a significant higher germination rates, the seeds can germinate as well in MS medium with no hormones and additives. The germination rates of different additives were well-matched and potatos price is lower, so potato is better to be used in factory production. The optimum culture medium sifted for seed germination was MS + NAA 0.5 mg/L + potato juice 10% + active charcoal 0.5%, and cultured in the dark 7 d then under light.

Acknowledgments This work was supported by Sichuan Agricultural

University Shuang-Zhi Plan.

References
[1] X.L. Ye, S.J. Cheng, F.X. Wang, N.F. Qian, Morphology of immature seeds and development in vitro of Dendroblum candidum, Acta Botanica Yunnanica 10 (3) (1988) 285-290. K.Z. Wang, W.Y. Gao, Research advance in Dendroblum medicinal plants, Chinese Traditional and Herbal Drugs 28 (10) (1997) 633-635. X.Y. Wei, M. Zhang, H.R. Huang, Medicinal research advance of Dendroblum candidum, Chinese Traditional and Herbal Drugs 31 (Supplement) (2000) 189. M. Vellupillai, S. Swamp, J.H. Chong, Histological and protein change during early stages of seed germination in the Orchid Dendrobium Erumenatum, Horticulture Science 7 (26) (1997) 941. S.J. Cheng, Artificial pollination of dendrobium, colloquium of South China botanical garden, Chinese Academy of Sciences 3 (1986) 46-49. Q. Lian, J. Li, Artificial pollination of Dendrobium devoniaum, Tropical Agricultural Science & Technology 26 (2) (2003) 44-45. J. Bo, Incompatibility in Dendrobium (Orchidaceae), Botanical Journal of the Linnean Society 103 (2) (1989) 165-196. S.J. Zeng, S.J. Cheng, J.L. Zhang, F.P. Zhao, Embryo culture and propagation of Dendrobium in vitro, Acta Horticulture Sinica 25 (1) (1998) 75-80.

4. Conclusions
This study indicated that the percentage of fruit-setting reaches the highest under xenogamy before 10 oclock in the morning after blooming 2 d to 4 d, which was done in the last two years, and got the same results. It can be inferred that the pollen activity of Dendrobium candidum was high in that period. Contrasted with self-pollation, the way of xenogamy showed that the fruit-setting was higher, the capsule of optimun maturity was bigger, the protocorm was fuller, which may because the descendants genetic dominance caused by the great difference among different individuals, that should be proved by further research of anatomy and genetics. The most suitable maturity of capsule appeared when it grew for 8 months, some capsule may be abscission in that period, the reason was not found yet and need further study.

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