Somatic Embryogenesis and Plant Regeneration from Cotyledons and Young Embryos of immature Seeds of Camellia chekiangoleosa

 LI Ji-yuan*, FAN zheng-qi, LI Xinlei, YIN Hengfu, ZHOU Xinwen, SUN Yingkun, XIAO Zheng
^.Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, China;

* Author for correspondence, Email: jiyuan_li@126.com; Tel/Fax: 86 571 63346372

Introduction

Camellia chekiangoleosa Hu belongs to the family Theaceae, which is not only a rare Camellia species, but also an important ornamental plant. Somatic embryogenesis is one kind of organogenesis. The adventitious embryos were obtained as somatic embryos, which were developed from explants tissue, and were directly grown into plantlets without an additional rooting induction phase. Meanwhile, the technique of inducing somatic embryogenesis can be used for genetic improvement of Camellia species. There are many reports about the successful induction of somatic embryogenesis from cotyledons in Camellia species, such as Camellia sinensis (Balasubramanian et al, 2000), Camellia oleifera (Yan et al, 1980; Zhang et al, 2005), Camellia japonica (Barciela et al, 1993;Vieitez et al, 1990), Camellia nitidissima Chi (Gao et al, 2010), Camelliareticulata (Plata et al, 1990). However, there is still no report of somatic embryogenesis research on Camellia chekiangoleosa Hu. Therefore, the establishment of an efficient experimental system of somatic embryogenesis of Camellia chekiangoleosa Hu will play an important role in increasing the rate of multiplication, species conservation and making the base for genetic breeding.

Materials and Methods

Explant and Treatment

Immature seeds collected from July to September in Research Institute of Subtropical Forestry were washed under running water. After removing the fruit coat, then seeds were surface-sterilized in 75% ethanol for 30S and washed for two times in distilled water, followed by sterilization in 0.1% HgCl₂ for 15min and five to six rinses in autoclaved distilled water.

Inoculation

Immature embryos were cut into pieces. Fifty explants were inoculated in different culture mediums respectively. All cultures were maintained at 28℃ under a photoperiod of 16hrs with cool fluorescent lights.

Medium

The basal medium (pH5.8) were MS, ER, N6 and white, containing 30 g∙l^-1sucrose and 8g∙l^-1 agar. The primary embryos were induced on basal medium with addition of different concentration of 6-BA, NAA, 2,4-D, IBA, ZT, sucrose, casein acid hydrolysate and yeast extract. The secondary embryos that formed were placed on MS medium containing 0.1mg∙l^-16-BA,0.01mg∙l^-1NAA and 40 g∙l^-1 sucrose, followed by transfer to half strength MS germination medium.

Results

Induction of somatic embryo

Embryos in June were invisible. The seeds were full of semi-transparent and soft endosperm. In July embryos grew larger and endosperm expanded between cotyledon and seed coat. Cotyledons were full of the seed and the seed coat was still soft. Embryos collected in June were inoculated in culture medium directly since it was very small. It grew to normal plantlet. Nevertheless there was few somatic embryogenesis from the cotyledon surface. There was more somatic embryogenesis from embryos collected in July, whereas, we got the highest rate of somatic embryo induced from cotyledons collected in August.

Some cotyledons turned green after inoculating in culture medium for 3 days. The surfaces of cotyledons were uneven after a week. Three or four weeks late some yellow particles formed in the surface and turn green under light. Somatic embryos were formed from the particles after 40 days. Generally, somatic embryo grew in the surface of cotyledon, few in the cutting side and was induced easily at the base of cotyledon. Somatic embryo usually grew in cluster. Therefore, we could get many somatic embryos from only one explant.

Effect of basal medium on induction of embryogenesis

Explants were inoculated in different culture medium (MS, ER, N6 and White) with no hormone addition in order to research what kind of basal medium was suitable to somatic embryogenesis. Explants grew differently in different medium (Table 1).

Table 1:  Effect of basal medium on induction of embryogenesis

Some explants turned brown and died after two weeks. The survivors grew larger at different rates. Explants inoculated in MS culture medium grew well generally. Some small shoots grew up from the embryo. Explants in N6 culture medium grew worse. Most of explants in white culture medium died. The highest percentage of embryogenesis (40%) was observed in MS culture medium in contrast to 30% in N6 culture medium. There were two up to eleven somatic embryos on every explant. N6 culture medium was better than ER medium on the frequency of somatic embryogenesis. There were no somatic embryos observed in White culture medium except where explants expanded.

Effect of hormone on induction of embryogenesis

Somatic embryogenesis in MS culture medium with addition of different concentration of hormone was distinctly different. MS medium containing ZT and NAA was the best to induce somatic embryo formation as compared with the other combination of hormone, which got the highest multiplication rate. It was easy to get somatic embryos in culture medium containing 6-BA and NAA, yet high concentration of hormone was not good for somatic embryogenesis. The effect of IBA was worse than NAA. Callus in culture medium containing 2,4-D grew fast. However, 2,4-D was not good for somatic embryogenesis. The morphological characters of somatic embryo played an important role in the development of plantlets. Cotyledon shaped somatic embryo were favorable to normal plantlets formation. Globular shaped somatic embryo with fleshy leaves or hyperhydricity are likely to develop abnormally. Therefore, MS culture medium with addition of 1.0 mg∙l^-16-BA and 0.2mg∙l^-1 NAA got high efficiency of embryogenesis induction and were helpful to cotyledon shaped embryo formation.

Table 2: Effect of external hormone on induction of embryogenesis

Effect of additional nutrition on induction of embryogenesis

Explants were inoculated in MS culture medium containing 1 mg∙l^-16-BA, 0.2 mg∙l^-1NAA and 8 g∙l^-1agar with three different concentrations of sucrose. There were much more hyperhydricity somatic embryos in the medium containing 30 g∙l-1sucrose. Cotyledon shaped somatic embryo increased distinctly in the medium with addition of 40 g∙l^-1or 60 l^-1 sucrose. Sucrose served dual purpose of acting as a carbon source as well as an osmotic agent. Cells of explants absorbed water from culture medium since lower water potential was due to low concentration of sucrose in the medium. Then hyperhydricity plantlets formed. There was no significant effect observed in somatic embryogenesis with addition of yeast extract and casein acid hydrolysate. 

Table 3: Effect of nutrition on induction of embryogenesis

Germination of somatic embryos

Somatic embryos grew in clusters on the surface of explants. Continuous presence in the explants retarded the growth of somatic embryos. It was necessary to transfer the embryos in time from explants to subculture medium (MS+0.1mg∙l^-16-BA+0.01mg∙l^-1NAA+40g∙l^-1sucrose). A week later, two leaves grew up from one side of somatic embryos and roots elongated into the medium with many fibrous roots growing out. Shoots of 1-2cm high were transferred to half strength MS basal salts medium. Plantlets of 3-5cm high with several leaves were transferred to the green house and kept for one week, followed by transfer to field.

Conclusions

Immature cotyledons are commonly used as explants for somatic embryogenesis in vitro regeneration of Camellia species and develop to plantlets. It is difficult to get embryos of Camellia chekiangoleosa Hu in June since the seeds mature in September. Embryos collected after June can develop into normal plantlet by means of embryo rescue. Somatic embryos can be induced from immature embryo in certain culture medium in July. The surface cells of cotyledon from nearly mature embryo earn great differentiation ability, which is easy to induce somatic embryo.

Induction of somatic embryos from cotyledon of tea is easier, even in hormone-free culture medium. In this study, we found the same results as reports. It may be due to the existence of proper balance of endogenous hormone in immature embryos and foreign hormone is not a prerequisite for the induction of somatic embryogenesis, whereas certain addition of foreign hormone can change the balance of hormone in explants to induce many more somatic embryos. Pseudobulbil formed in the surface of explants is semi-transparent and nodular. It develops to somatic embryos easily. Pseudobulbil is a kind of somatic embryo, including epidermal cell, undeveloped vascular tissue and root primordium, without bud primordium (Vieitez et al, 1990). It is important to make full use of pseudobulbil to get higher rate of proliferation during the process of induction of somatic embryo.

References

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Fig. 1 Somatic Embryogenesis and Plant Regeneration from Immature Cotyledons and Young Embryos of Camellia chekiangoleosa Hu

A. The ear-shape somatic embryogenesis; B. The globe somatic embryogenesis; C. The semitransparent somatic embryogenesis; D. The cotyledon-shape somatic embryogenesis; E. The germination of somatic embryogenesis; F. The plantlets from somatic embryogenesis.