Selangor, Malaysia. Keywords: Murashige and Skoog, growth regulators, in vitro, regeneration, plantlet, acclimatization. Abstract. Adventitious shoots of Gerbera ...
Establishment of In Vitro Plantlets and Acclimatization of Gerbera jamesonii Bolus ex. Hook f. R.M. Taha1, N.A. Hasbullah1, A.H. Abdul Aziz1 and A. Awal2 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Faculty of Applied Science, Mara University of Technology, 40450 Shah Alam Selangor, Malaysia Keywords: Murashige and Skoog, growth regulators, in vitro, regeneration, plantlet, acclimatization Abstract Adventitious shoots of Gerbera jamesonii Bolus ex. Hook f. were successfully obtained when petiole explants from aseptic seedlings were cultured on MS media supplemented with 2.0 mg/L 6-Benzylaminopurine (BAP) and 0.5 mg/L αNaphthalene acetic acid (NAA). The shoots were subcultured every 2 weeks on the same culture media to obtain in vitro plantlets. Mature plantlets were achieved after 8 weeks of subculture. These plantlets were transferred to MS Basal media for further root elongation for 2 weeks. Complete plantlets were transferred to several growth media for acclimatization process. These growth substrates were garden soil, black soil, vermiculite and autoclaved garden soil. Before being transferred to the greenhouse, these plantlets were first placed in the culture room for 3 weeks to allow adaptation of plantlets to the new environment. Survival of the plantlets in the greenhouse was observed. It was found that acclimatized gerbera plantlets in garden soil (black soil: red soil, 2:1) gave best result with 86.0±0.9% survival rates, followed by vermiculite with 73.0±1.3 survival rates. Gerbera plantlets were maintained in the greenhouse for further flowering phase. INTRODUCTION Gerbera jamesonii is an ornamental flowering perennial which belongs to the Asteraceae family. This plant is very well known to be planted as cut flowers, bedding plant and also pot crops. Gerbera jamesonii produce leafless flower stalk with beautiful flowers. Gerbera jamesonii is also known as ‘Barberton Daisy’, ‘Traansval Daisy’ or African daisy. Gerberas are mostly found in temperate countries. However, through improvements and continuous research, this plant could be successfully planted in tropical countries such as Malaysia. In the present study, experiments were conducted to establish plantlet regeneration from various sources of gerbera explants and also to determine an efficient system for plantlet acclimatization in the greenhouse. The effects of various concentrations of plant growth regulators on the multiplication of shoots were also investigated. MATERIALS AND METHODS Establishment of In Vitro Plantlets Petioles obtained from aseptically grown young plantlets of Gerbera jamesonii were used as source of explants. Leaves and petioles were cultured for shoot induction on Murashige and Skoog (MS, 1962) media supplemented with 3% sucrose and 0.8% technical agar containing 6-benzlaminopurine (BAP) and α-naphthalene acetic acid (NAA) at various concentrations. Shoots developed were then transferred to MS basal media for further plantlet growth and elongation of roots. Acclimatization of In vitro Plantlets Plantlets were removed safely from the culture vessels and the roots were rinsed with sterile distilled water to remove excess agar on the roots. The plantlets were first Proc. IVth IS on Acclim. and Establt. of Micropropagated Plants Ed.: J. Prakash Acta Hort. 865, ISHS 2010
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kept in the culture room at 25±1°C with 16 h light and 8 h dark 3 weeks before being transferred to the greenhouse and the plant pots were covered with plastic covers with small holes to allow adaptation and adjustment process of plantlets to the new environment. All plantlets were watered everyday. After 3 weeks in the culture room, plantlets were ready for the next step of growth and transferred to the greenhouse. Plantlets were transplanted into 4 various scheme of growth substrates: 1) Combination of black soil: red soil at 2:1 ratio 2) Combination of black soil: red soil at 1:1:2 ratio (autoclaved) 3) Vermiculite 4) Red soil Based on all experiments done, the best acclimatization technique which gave the highest survival rates was identified. RESULTS AND DISCUSSION In vitro propagation is an alternative method to vegetative propagation and a suitable means for regeneration and multiplication of elite clones. There are several methods to achieve in vitro multiplication such as multiplication of shoots from various explants such as shoot tips, petioles, axillary buds or from the formation of somatic and zygotic embryos. The first significant success of plant tissue culture in ornamental was reported in 1920s when orchid seeds were germinated under sterile conditions (Knudson, 1922). In vitro propagation normally involves four different stages which are initiation of cultures, multiplication and rooting of shoots and acclimatization. These four stages are vital in ensuring the success of the whole in vitro propagation process. There are a few factors that affect growth conditions of in vitro propagation such as the amount of organic, inorganic nutrients and plant growth regulators in the culture media, carbon source, humidity, photoperiod and gaseous exchange. Although these factors may result in plant regeneration and growth, these factors may cause physiological and morphological changes in plants and could effect the survival of plants when transplanted to the environment. Thus, acclimatization of regenerated plants from tissue culture system to the field needs to be done systematically. These plants need to adapt and adjust to the changes of environment from high humidity and low irradiance in the culture system to low humidity and high irradiance in the field. In the present study, the effect of plant growth regulators on in vitro regeneration of shoots of Gerbera jamesonii Bolus ex. Hook f. was studied. Plantlets established from in vitro cultures were then acclimatized and transferred to the field. The effects of various sowing media were studied to investigate the adaptation of these plantlets to the outside environment. Many commercial ornamental plants were cultured on culture media containing auxins and cytokinins (Preil, 2003; Rout and Jain, 2004). The right combination of auxin and cytokinin in the culture media determined the effectiveness of in vitro regeneration of gerbera shoots. Several different explants have been used for in vitro shoot regeneration. In the present study, petiole explant was found to be the most suitable explant for shoot regeneration. Highest shoot formation was obtained when petiole explants were cultured on MS media supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA with the percentage of 94.3±2.5 (Table 1). Meanwhile, the lowest shoot formation was observed when petiole explants were cultured on MS media added with 0.1 mg/L BAP and 2.0 mg/L NAA with percentage of 4.6±0.8 (Table 1). Pierik et al. (1973) reported that the addition of strong auxin (NAA) with BAP promoted better shoot formation compared to weak auxin (IAA). Gerbera shoots were regenerated from flower buds of greenhouse grown plants (Pierik et al., 1973, 1975; Laliberte et al., 1985). Regeneration of shoots from Begonia was obtained when leaf explants were culture on Ms media supplemented with 1.0 mg/L BAP with the addition of 1.0 mg/L NAA (Awal, et al., 2007). Daud et al. (2008) reported that MS media supplemented with 1.0 mg/L IAA and 2.0 mg/L zeatin resulted in 100% shoot regeneration with the highest number of shoots (15.0±0.8 shoots per explant) in Saintpaulia ionantha Wendl. Plantlets derived could be hardened and transferred to the greenhouse with 84.0±1.6% success rate. Vijaya 402
et al. (1991) reported that BAP was the most effective growth regulator in stimulating shoot ploriferation of Rosa hybrida. Plantlets derived from in vitro regeneration need to be transplanted to the greenhouse for further growth and development process. In this study, various sowing media were used in order to study the adaptation and survival of plantlets in the field. It reported that plantlets acclimatized in garden soil (combination of black and red soil 2:1) gave the optimum plantlets survival in the greenhouse with 86.0±0.9% of survival rate (Table 2). This followed by the usage of vermiculite as the sowing media with 73.0±1.3% of survival rate. However, plantlets transferred to autoclaved garden soil failed to survive due to the lack of nutrients in the soil. Roest and Bokelmann (1975) successfully induced roots in the adventitious shoots of chrysanthemum in the liquid MS media containing 1.0 mg/L IAA. Rooted in vitro cultured plants of chrysanthemum were also reported to be successfully established in the soil (Rout et al., 1996; Roberts and Smith, 1990). Kumar et al. (1998) established micropropagation protocol for Ficus carica cv. Gular by using apical buds and succeeded in getting multiple shoots and rooting in half strength liquid MS media supplemented with 2.0 mg/L IAA and 0.2% activated charcoal. The micropropagated plantlets were successfully acclimatized in soil with 68.0% survival rate. In this study, plantlets established from in vitro regeneration of Gerbera jamesonii were morphologically identical to the mother plant and developed normally and produced flowers after 6 months being transplanted to the greenhouse. Literature Cited Awal, A., Taha, R.M. and Hasbullah, N.A. 2007. In vitro formation of synthetic seed from microshoots of Begonia x hiemalis Fotch. CATRINA Intl. J. of Environmental Sciences 2(2):189–192. Daud, N., Taha, R.M. and Hasbullah, N.A. 2008. Studies on plant regeneration and somaclonal variation in Saintpaulia ionantha Wendl. (African violet). Pakistan J. of Biological Sciences 11(9):1240–1245. Debergh, P.C. and Read, P.E. 1991. Micropropagation. p.1–13. In: P.C. Debergh and R.H. Zimmerman (eds.), The Netherlands: Kluwer Academic Publishers. Knudson, L. 1922. Flower production by orchid grown non-symbiotically. Bot Gaz. 89:192. Kumar, V., Radha, A. and Kumar Chitta, S. 1998. In vitro plant regeneration of fig (Ficus carica L. cv. Gular) using apical buds from mature trees. Plant Cell Reports. 17(9):717–720. Laliberte, S., Chretien, L. and Vieth, J. 1985. In vitro plantlet production from young capitulum explants of Gerbera jamesonii. HortScience 20(2):137–139. Murashige, T. and Skoog, F. 1962. A revised media for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology 15:473–497. Pierik, R.L.M., Jansen, J.L.M., Maasdam, A. and Binnendijk, C.M. 1975. Optimilization of Gerbera plantlet production from excised capitulum explants. Scientia Horticulturae 3(4):351–357. Pierik, R.L.M., Steegmans, H.H.M. and Marelis, J.J. 1973. Gerbera plantlets from in vitro cultivated capitulum explants. Scientia Horticulturae 1(1):117–119. Preil, W. 2003. Micropropagation of ornamental plants. p.115-130. In: M. Laimer and W. Rucker (eds.), Plant Tissue Culture 100 years since Gottlieb Haberlandt. New York: Springer-Verlag. Roberts, A.V. and Smith, E.F. 1990. The propagation in vitro of chrysanthemum for transplantation to soil: I. Protection of roots by cellulose plugs. Plant Cell Tissue Organ Culture 21:129–132. Roest, S. and Bokelmann G.S. 1975. Vegetative propagation of Chrysanthemum morifolium Ramat. tn vitro. Sci Hort. 3:317–330. Rout, G.R. and Jain, S.M. 2004. Micropropagation of ornamental plants-cut flowers. Propagation of Ornamental Plants 4(2):3–28. Rout, G.R., Palai, S.K., Panday, P. and Das, P. 1996. Direct plant regeneration of 403
Chrysanthemum morifolium Ramat. cv. Deep pink: influence of explant source, age of explants, culture environment, carbohydrates, nutritional factors and hormone regime. Proc. Natl Acad Sci (India) 67:57–66. Vijaya, N., Satyanarayana, G., Prakash, J. and Pierik, R.L.M. 1991. Effect of culture media and growth regulators on in vitro propagation of rose. Curr. Plant Sci. Biotechnol. Agric. 12:209–214. Tables Table 1. Percentage of shoot formation and number of shoots per explant produced on MS media supplemented with different hormones and concentrations after 8 weeks in culture. MS + hormone (mg/L)
Explants
2.0 mg/L BAP + 0.5 mg/L NAA 2.0 mg/L BAP + 1.5 NAA 0.1 mg/L BAP + 2.0 mg/L NAA 1.0 mg/L BAP + 2.0 mg/L NAA 1.5 mg/L BAP + 1.0 mg/L NAA 0.5 mg/L BAP + 0.5 mg/L NAA 3.0 mg/L BAP
Petiole Petiole Petiole Petiole Petiole Petiole Petiole
Shoot regeneration (%) 94.3±2.5 79.2±2.0 4.6±0.8 15.4±2.7 83.1±0.5 33.4±1.2 80.4±1.6
Number of shoots per explant 9.3±0.6 5.2±1.2 1.6±0.7 3.5±1.8 8.3±1.1 4.7±0.5 8.8±0.9
Mean values are significantly different at p= 0.05 using Duncan’s Multiple Range Test.
Table 2. Response showed by in vitro gerbera plantlet after being acclimatized in various growths substrates. Method Plantlets were acclimatized in garden soil (combination of black soil and red soil at 2:1 ratio) Plantlets were acclimatized in ‘vermiculite’ Plantlets were acclimatized in red soil Plantlets were acclimatized in autoclaved garden soil (combination of black soil and red soil at 2:1 ratio)
Observation
Survival of Gerbera plantlet (%)
Plantlets survived and showed healthy growth
86.0±0.9
Plantlets survived and showed healthy growth Plantlets survived but showed slow growth Plantlets failed to survive
Mean values are significantly different at p=0.05 using Duncan’s Multiple Range Test.
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73.0±1.3 37.5±1.0 0
