­Differentiation of adventitious buds and induction of somatic embryos derived from immature cotyledons of Camellia azalea

Wu Bin, Li Jiyuan, Fan Zhengqi
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, Zhejiang, China, 311400

Camellia azalea, also known as Camellia changii, native to China, is a unique camellia species. It is a compact, evergreen shrub with dense foliage, red flowers, and blooms in summer season. Thus this species is being widely used as an excellent breeding parent and a rare woody ornamental because of its unique flowering period among over 280 camellia species (Chang H.T., Ren, S.X. 1998). It naturally distributes only in E'huangzhang Nature Reserve in Yangchun, Guangdong Province. The individual number of wild species is so extremely low that it was even crowned ‘The Panda of Plants’ by taxonomists and camellia enthusiasts.

Nowadays, there are great dilemma between low supply of seedlings and great demand of markets, although some research have been done on traditional vegetative propagations, such as cut rooting and graft (Ni S., et al., 2008), a new effective and rapid propagation is still badly needed to be developed in current stage. A number of reports have been involved in tissue culture of regeneration from callus in Camellia recent years (Liu D. J., et al., 2011, Shi L. R., 2012), but no further progress on differentiation of adventitious buds from cotyledon of C. azalea has been published.

In this experiment, immature cotyledons of C. azaleawere used as explants, adventitious buds were aimed to be obtained through somatic embryos, whichwould play a solid basis for massive asexual reproduction and genetic transformation experiments in C. azalea.

MATERIAL AND METHODS

Immature fruits of C. azalea were collected in a greenhouse of Research Institute of Subtropical Forestry (Fuyang, Zhejiang, China) in September.

1 Treatment of Explants

Immature fruits of C. azalea were firstly washed with detergents to remove dirt and oil material on surface, then disinfested with 75% ethanol for 30 seconds, sterilized in 0.1% HgCl₂ for 8 minutes, and finally rinsed with steamed water5~6 times. The peels and the seedcoats of the immature fruits were removed, and then internal sterilized cotyledons were finally cut into tiny segments and cultured on MS medium for somatic embryo formation.

2 Culture Medium and Conditions

Certain combinations of NAA and 6-BA or 2,4-D and 6-BA added into the basal MS medium were designed for induction of somatic embryos from the initial cotyledon explants. The plant growth regulators (PGRs) were 0.2 mg·L^-1NAA and 1.0 mg·L^-16-BA, 2.0 mg·L^-1NAA and 1.0 mg·L^-16-BA, 0.1 mg·L^-12,4-D and 1.0 mg·L^-16-BA or 2.0 mg·L^-12,4-D and 1.0 mg·L^-16-BA, supplemented with 30 g·L^-1sucrose, 6.5 g·L^-1agar and 50 mL·L^-1CW. The inductionrate of somatic embryos was recorded after inoculation for 40 days.

In the second series of experiments, the somatic embryos were cultured on subculture medium containing 0.1 mg·L^-1NAA and 1.0 mg·L^-16-BA or 0.1 mg·L^-1NAA, 0.1 mg·L^-1IBA and 0.8 mg·L^-16-BA, supplemented with 30 g·L^-1sucrose, 6.5 g·L^-1agar and 100 mL·L^-1CW. After two cycles of 45-day subculture periods, the somatic embryos were transferred onto basal MS medium with 0.125 mg·L^-1NAA and 10.0 mg·L^-16-BA, plus 40 g·L^-1sucrose, 7.0 g·L^-1agar and 50 mL·L^-1CW. The cultures were evaluated and the differentiation rate of adventitious bud was recorded after cultured for 28 days.

The final pH of all culture medium was adjusted to 5.8 before 121℃ autoclaving for 16 minutes. The chamber temperature was maintained at 25±2℃, and the materials were cultured under a 12 hours photoperiod regime with light intensity from 2500 to 3000 μmol·m^-2·s^-1.

RESULTS

1 Effect of different PGRs combinations on somatic embryogenesis of C. azalea

Immature cotyledon explants were green and swollen after inoculation for 7 days. Somatic embryos continually developed on the swollen surfaces of cotyledon segments cultured on MS medium with four different PGRs combinations after 4 weeks.Up to 60.61% of the cotyledon explants on the medium containing 0.2 mg·L^-1NAA and 1.0 mg·L^-16-BA developed somatic embryos, while about 8.05% of the cotyledon explants on the medium containing 2.0 mg·L^-12,4-D and 1.0 mg·L^-16-BA(Table 1).

Numerous tight yellow-green or green translucent somatic embryos were obtained from immature cotyledons cultured on the medium with 0.2 mg·L^-1NAA and 1.0 mg·L^-16-BA. With higher concentration of NAA, the induction rate decreased so that a few deep green somatic embryos were differentiated on the surfaces of swollen cotyledons. Compared to somatic embryos formation, more callus were formed from the cotyledons cultured on the medium containing 2,4-D. Furthermore, no somatic embryos or callus were achieved from the cotyledons cultured on the medium with 2.0 mg·L^-12,4-D and 1.0 mg·L^-16-BA, but some cotyledons immersed on the medium browned visibly. High concentration of 2,4-D (2 mg·L^-1) was reported to be needed in somatic embryogenesis from cotyledons of Camellia oleifera(Bi F. C., et al., 2004, Yen M. Q. and Chen P., 1980), however, our experiment results showed that 2,4-D was not necessary for somatic embryogenesis of C. azalea , but high concentration of 2,4-D inhibited its somatic embryos formation. The similar results were reported from other related studies (Li J. Y., et al., 2003, Zhuang C. J. and Liang H. X., 1985).

2 Subculture and differentiation of adventitious bud from somatic embryos of C. azalea

Yellow and translucent primary somatic embryos showed some vacuolization, and those somatic embryos in different size partially called pseudobulbils developed together tightly after 60 daysinduction and during subculture(Fig. 1-A). Someyellow-green somatic embryos developedinto reddish brown ‘bud-like’ embryos after 2cycle subculture (Fig. 1-B,C). These embryos had differentiation capacity, and adventitious buds generally occurred on the medium supplemented with relatively high concentration of 6-BA (Fig. 1-D,E,F).

CONCLUSIONS

Nearly overall 72% of the cotyledons successfully developed the embryos cultured on MS medium with 0.2 mg·L^-1NAA and 1.0 mg·L^-16-BA, supplemented with 50 mL·L^-1 CW. Moreover, the embryogenic capacity has been obtained for over 90 days by subculture on MS medium containing 0.1 mg·L^-1NAA, 1.0 mg·L^-16-BA and 100 mL·L^-1 CW. Adventitious buds were achieved after 22 days on the medium with 0.125 mg∙L^-1NAA, 10.0 mg∙L^-16-BA and 50 mL·L^-1 CW. The differentiation rate was 63.83%.

Acknowledgments:

This work was supported by the National Key Twelfth-Five Science and Technology Program (2012BAD01B0703), International Cooperation Project of China (2011DFA30490), and Zhejiang Key Flower Breeding Program (2012C12909-6) as well as Basic research fund for Central public Scientific Research Institute (RISF6141).

Literature Cited

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Tables

Table 1 Somatic embryogenesis from cotyledon of C. azalea cultured on various induction media for 40 days

 Figures

Fig.1 The process of subculture and adventitious buds differentiation of C. azalea

A：pseudobulbils occurred from primary somatic embryos；
B：somatic embryos achieved embryogenic capacity after subculture；
C：reddish brown ‘bud-like’ embryo；
D：differentiation of adventitious bud；
E、F：more adventitious buds occurred on somatic embryos