artificial pollination and seed germination for dendrobium candidum

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, Ya’an 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 o’clock 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. It’s 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 human’s 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: [email protected].

groundwork for the reproduce technique of

Dendrobium candidum.

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

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

396

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).

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

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.




397


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.

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

Table 1 The pollination successful number and rates of Dendrobium candidum in different time and groups.

Flower time (1) (2) (3) (4) (5)

A B A B A B A B A B

2 d 1 0 0 0 1 0 1 0 0 0

3 d 5 3 3 0 1 0 0 0 0 0

4 d 5 2 4 2 3 0 0 1 0 0

5 d 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.).

Table 2 Dendrobium candidum seeds’ germination rates in different media (%).

Medium Culture in light Culture in darkness Culture in darkness 7 days and then culture in light

MS 87 41 91

White 81 33 80

B5 78 32 79

N6 91 37 92

VW 72 28 75

The results were 30 d after sowed.


398

Fig. 5 Proembryo began to turn green.

Fig. 7 Induce into protocorm.

Fig. 6 Proembryo turn green.

Fig. 8 Protocorm differentiation.

Table 3 Effect of different hormones on Dendrobium candidum seeds’ germination.

IAA NAA KT 6-BA C (mg/L) GR※ (%) C GR C GR C GR 0 69 0 69 0 69 0 69 0.1 81 0.1 72 0.1 71 0.1 84 0.2 86 0.2 77 0.2 79 0.2 89 0.5 82 0.5 93 0.5 82 0.5 83 1.0 80 1.0 86 1.0 77 1.0 76 2.0 68 2.0 64 2.0 66 2.0 33

※ C = Concentration; GR = Germination Rates.

Table 4 Effect of different additives on Dendrobium candidum seeds’ germination.

Medium number Potato juice Banana juice Coconut juice Apple juice Active carbon Germination rates (%) CK 0 0 0 0 0 71 1 10% 0 0 0 0 91 2 10% 0 0 0 0.5% 91 3 0 10% 0 0 0 90 4 0 10% 0 0 0.5% 89 5 0 0 10% 0 0 89 6 0 0 10% 0 0.5% 91 7 0 0 0 10% 0 87 8 0 0 0 10% 0.5% 86

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,


399

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 it’s quicker and

greener than control when protocorm propagation, this

maybe because active carbon absorbed metabolite in

the period of propagation.

4. Conclusions

This study indicated that the percentage of

fruit-setting reaches the highest under xenogamy

before 10 o’clock 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.

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 potato’s

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.

[2] K.Z. Wang, W.Y. Gao, Research advance in Dendroblum medicinal plants, Chinese Traditional and Herbal Drugs 28 (10) (1997) 633-635.

[3] X.Y. Wei, M. Zhang, H.R. Huang, Medicinal research advance of Dendroblum candidum, Chinese Traditional and Herbal Drugs 31 (Supplement) (2000) 189.

[4] 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.

[5] S.J. Cheng, Artificial pollination of dendrobium, colloquium of South China botanical garden, Chinese Academy of Sciences 3 (1986) 46-49.

[6] Q. Lian, J. Li, Artificial pollination of Dendrobium devoniaum, Tropical Agricultural Science & Technology 26 (2) (2003) 44-45.

[7] J. Bo, Incompatibility in Dendrobium (Orchidaceae), Botanical Journal of the Linnean Society 103 (2) (1989) 165-196.

[8] 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.

artificial pollination and seed germination for dendrobium candidum

Documents