Callus Formation and Plant Regeneration in Camellia chekiangoleosa Hu

FAN Zheng-qi*, LI ji-yuan, LI Xinlei, YIN Hengfu
Research Institute of Subtropical Forestry, CAF, Fuyang 311400, Zhejiang, China

* Author for correspondence, E-mail: fzq_76@126.com

Introduction

Genus Camellia, a population of subtropical evergreen broadleaved plants, and is one of the most important woody oil plantation crops and ornamental plants in South China. It is difficult to get normal seeds by means of distant hybridization: this is because distinct differences in flowering period, flower organ structure and chromosome ploidy, frequently results in high infertile rates among Camellia species. Therefore, molecular breeding of camellias may hold great potential for the future. However, there has only been one successful case of genetic transformation of somatic embryo system in tea up to now (Mondal et al, 2001). The major reason is that we have not developed an effective and operational method for callus formation and plantlet regeneration ex vitro. Thus, tissue culture on organogenesis will be widely used in exploring important camellias that cannot be easily bred by traditional ways. There have been a few reports about organogenesis in Camellia japonica L (Pedroso et al 1993; Shults et al, 1996; Fedroso et al, 1993; 1995; Vietiz et al, 1991). The present study was undertaken to establish an efficient regeneration system in organogenesis in C. chekiangoleosa L.

Materials and methods

Materials

Leaves and stem segments of C.chekiangoleosa Hu from in vitro grown plantlets were used as explants.

Inoculation

Young leaves were cut into pieces (0.5x0.5cm). After removing axillary buds, the stems were cut into 0.5cm long. Explants were inoculated in different culture medium containing different concentration of different hormones for a few days, followed by the transfer to differentiation medium. When buds differentiated, callus tissues were transferred to the culture medium supplemented with lower hormone concentration. Buds of 2cm high were transferred to rooting medium.

Results

Effect of hormone on callus induction

Leaves inoculated in the induction medium began to wrinkle after a week. A few callus tissues induced at the edge of leaves, increased steadily latter, yet totally bad. In contrast, Stem segments were easier to induce callus, which grew well. Therefore, we chose stem segment as explants.

2,4-D could promote callus generation and growth effectively. In this experiment high generation frequency was induced with addition of high concentration of 2,4-D. Callus induced in low concentration was very loose, easy to break up, whereas callus induced in high concentration was too dense, and was bad for shoots differentiation. Callus in culture medium supplemented with 1.0mg l-1 2,4-D was dense, easy to differentiate. Callus in the culture medium with addition of 2,4-D alone grew to 0.3cm in diameter after one month. Culture medium containing 2,4-D and 6-BA could increased the frequency of callus induction, which grew up to 0.5cm in diameter after 4 weeks. The frequency of callus induction could reach 93% in MS culture medium containing 6-BA 0.5 mg∙L^-1 and 2,4-D 1.0 mg∙L^-1. Moreover, the callus grew fast and dense and was easy to differentiate.

Table 1  Effect of hormones on callus induction from stems of C .chekiangoleosa

Effect of hormone on callus induction from stem segments

Table 2  Effect of hormone on differentiation of shoots

After incubation for 6 weeks, stem callus that was loose or too dense did not differentiate. There were some shoots differentiated from the callus covered with prominent particles, which grew to adventitious shoots after a month. Shoots germinated in differentiation culture medium containing high concentration of 6-BA, but few grew in 20mg∙l^-16-BA and began to brown in 25mg∙l^-1 6-BA. Regeneration rate induced in the medium with the addition of IBA was higher than NAA among the combination of auxins and 6-BA. Regeneration rate increased significantly in the culture medium with addition of KT. The number of adventitious shoots differentiated from one callus could be up to 11. In this study, the best culture medium was MS＋6-BA 20 mg∙L^-1＋IBA 0.1 mg∙L^-1＋KT 0.1 mg∙L^-1, in which shoots regeneration rate was 20%.

Effect of hormone and pretreatment on rooting of adventitious shoots

In vitro rooting of adventitious shoots was one of the key factors to fast tissue culture since most of Camellia species grown in vitro are difficult to root. Shoots of 2cm high were cut down and transferred to rooting medium.

Basal medium

1/2MS culture medium just contained half-strength MS salts. MW culture medium were the same as MS except its micro-element were different, containing KNO₃80mg∙L^-1, Ca(NO₃)₂∙4H₂O150mg∙L^-1, MgSO₄∙7H₂O350mg∙L^-1, NaH₂PO₄∙H₂O100mg∙L^-1, KCl 65mg∙L^-1 and Na₂SO₄200mg∙L^-1.

Table 3 Effect of basal medium on rooting

Rate of rooting in liquid culture medium did not rise and the number of roots was few (Table 3). Solid culture media were good for rooting. Rooting rate in MW culture medium was higher than that in 1/2MS culture medium and the roots were longer and stronger.

The bases of shoots were dipped in 1000g∙l^-1 IBA, followed by the transfer to culture medium (MW+0.2g∙l^-1IBA). We got definite results after 30 days.

Table 4 Effect of IBA treatment on rooting

Rooting rate of shoots without dipping in IBA was very low. IBA treatment could increase the rate of rooting significantly. The rooting rate after IBA treatment was all up to 100%. The shoot cuttings exposed to the air were easier to root. Some roots were light pink, growing from leaves. Time of treatment with IBA played an important role in rooting. Roots dipped in IBA for 20min grew well.

Effect of auxins on rooting of shoots

Table 5 Effect of auxins on rooting of shoots

Roots of adventitious shoots incubated for 30days grew well in the culture medium with addition of some auxins (Table5). In this study combination of IBA and NAA was the best for rooting.

Discussions

Research on micropropagation of Camellia started in 1970s. There have been many reports about somatic embryogenesis. A protocol for transferring genes into tea has been developed via Agrobacterium-mediated genetic transformation of somatic embryos (Mondal et al, 2001). The limitation of taking somatic embryo as explants is that most of Camellia species produce good quality ones with difficultly. Meanwhile, cotyledon is heterozygote and variable, which is not the same as the parents in heredity. The present progress of micropropagation by organogenesis in C. japonica is rather slow. The rate of tea leaf regeneration is 7.4% and that of stem is lower (Sudripta et al, 1996). Resistant callus or hairy root was noted in the research in transformation of tea organ (Matsumoto et al, 1998; Konwar et al, 1998).

Few researches on organogenesis regeneration of ornamental C. japonica have been reported. Pedrosos et al (1993) chose mature leaf as explants, found that callus were easier to develop in the leaf petiole or main vein, and somatic embryo formed directly from the edge of leaf. Somatic embryo has been induced successfully by means of cell suspension culture (Fedroso et al, 1995), pollen culture (Fedroso et al, 1993) or root culture (Vietiz et al, 1991). It still takes a long time in somatic embryo induction, maturation and germination, and transformation would be longer. Time spent on transformation of somatic embryo of tea was at least 70 weeks (Mondal et al, 2001). Shults et al (1996) took camellia petals as explants. Callus grew well in the culture medium containing BA and NAA, but there was no regeneration of plantlets.

Genetic improvement in C. japonica L shows good prospects for its improved ornamental characters. We found that it was easier to induce callus tissues from stem segments than that from leaves. Somatic embryos differentiated to shoots in the culture medium with addition of high concentration of 6-BA. Rooting was induced easily when the base of shoots were dipped in auxins. In this study we developed an efficient transformation system in which shoots induced directly from stem segments developed the root system successfully. The present study may help micropropagation research on other Camellia species and make a base for genetic transformation of C. japonica.

References

Fedroso M. C. and Pais M. S. 1993. Regeneration from anthers of adult Camellia Japonica L. In Vitro Cell Dev. Biol., 29: 155-159.
Fedroso M. C. and Pais M. S. 1995. Plant regeneration from embryogenic suspension cultures of Camellia japonica. InVitro Cell Dev. Biol., 31: 31-35.
Konwar B. K., Das S. C., Bordoloi B. J., Dutta R. K. 1998. Hairy root development in tea though Agrobacterium rhizogenes-mediated genetic transformation. Tow and a Bud, 45(2): 12-20.
Matsumoto S, Fukui M. 1998. Agrobacterium tumefaciens-mediated genes transfer to tea plant (Camellia sinensis) cells. JARO, 32: 287-291.
Mondal T. K., Bhattacharya A., Ahuja P. S. 2001. Transgenic tea [Camellia sinensil (L.) O. Kuntze cv. Kangral Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep, 20: 712-720.
Pedroso M. C., Pais M. S. 1993. Direct embryo formation in leaves of Camellia japonica L.. Plant Cell Report, 12:639-643.
Shults A. M., Haldeman J. H. 1996. More on callus production from Camellia japonica. Camellica Journal, 51(1):19.
Sudripta D, Timir B J, Sumita J. 1996. Organogeilesis and regeneration from pigmented callus ub Camellia sinensis (L.) O. Kuntzecv Nandadevi, an elite Darjeeling tea clone. Plant Science, 121: 207-211.
Vietiz A M, San-José C, Vieitez F J, Ballester A. 1991. Somatic Embryogenesis from Roots of Camellia japonica Plantlets Cultured in vitro. J. Amer. Soc. Hort. Sci., 116 (4): 753-757.

Figure1  Callus induction, shoots differentiation, rooting in C.chekiangoleosa Hu

A. Callus tissue from leaves;B. Callus tissue from stem segment; C. Dense callus from stem segments D. Callus from stem segment differentiating shoots;E. Shoots from callus of stem segments;F. Adventitious shoots elongation; G. Rooting at base of adventitious shoots; H. Root system at early stage; I. Root system at late system