Prasad M. P., Invitro optimization of growth hormones in the micropropagation of Gerbera species, Int.J.Curr.Biotechnol., 2014, 2(2):15. International Journal of Current Biotechnology ISSN: 2321 8371 Journal Homepage : http://ijcb.mainspringer.com Invitro optimization of growth hormones in the micropropagation of Gerbera species Prasad M. P.* Sangenomics Research Lab, Domlur Layout, Bangalore560071, India. A R T I C L E I N F O Article History: Received 15 January 2014 Received in revised form 28 February 2014 Accepted 28 February 2014 Available online 28 February 2014 Key words: Gerbera jamesonii, Growth regulators, Hormones, Callus, Micropropagation. A B S T R A C T Introduction Plant tissue culture is being widely accepted for its potentials in mass multiplication and preservation of elite plants. The most important aspect of plant tissue culture technique is the ability of cultured cells and tissues to regenerate in to complete plants which is being used commercially on a large scale in the ornamental flower industry (Chu 1992; Huetteman et al. 1993;, Mantell et al., 1985; Pierik et al., 1987). Gerbera is a valuable ornamental species grown as a potted plant as well as for its cut flowers. Since the genetic variability within Gerbera genus is relatively limited. There is also limited genetic material that can be used to improve resistance to biotic and abiotic stress (Orlikowska et al., 1999). The genus Gerbera was named honour of a German naturalist Traugott Gerber, who traveled in Russia in 1743. The genus consists of about 40 species out of the recorded species, only one species Gerbera jamesonii is under cultivation (Das et al., 1989). The production of Gerbera was approximately US $ 220 million in 2001 representing 70 million stems sold in US alone (Broek et al., 2004). Gerbera plants can be propagated either sexually or asexually. The vegetative propagation through divisions of shoot tip culture is possible, however plant multiplication by this method is too slow to be *Corresponding author. Email address: Prasad_m_p@hotmail.com Mobile no: +919844357929. Gerbera is used as cut flower in many countries and has gained popularity as an ornamental flower and is in great demand in the floral industry. To commercialize Gerbera jamesonii, alternative methods of micropropagation like tissue and organ culture and other techniques are used to meet the demand for the planting material. In the present study, callus induction, shoot initiation, shoot multiplication and formation of roots were optimized by manipulating the growth regulators. Various kinds of growth regulators such as 2,4 Dichlorophenoxy acetic acid (2,4 D), 6 Benzyl Aminopurine (BAP), Kinetin, Indole3acetic acid (IAA) and alphanaphthalene acetic acid (NAA). The growth regulator hormones were added to MS medium in different combinations and concentrations. Callus were obtained from leaf and flower stalk explants cultured on the MS medium supplemented with 1.5mg L 1 2,4D. Effectiveness of shoot generation medium, the different type of growth regulators used and the duration of the induction period were studied. Shoot regeneration and multiplication was seen in MS medium which was supplemented with 0.3 mg L 1 IAA. The root explants did not regenerate in all the experiments conducted. commercially viable (Murashige et al., 1974). The physiological status of the donor is determined by environmental conditions such as temperature, light intensity, day length and light wave length. Plant regeneration via direct organogenesis is much preferred over regeneration through somatic embryogenesis and callus (Arockiasamy et al., 2002). The propagation rates via organogenesis can be much higher than auxiliary shoot proliferation, (Chun, 1993; Kumar et al., 2004) were able to regenerate adventitious shoots from leaf and petiole pieces of Gerbera jamesonii. They reported that about 75 77% of the calli from both types of the explants which produced 1215 shoots/callus. Gerbera plant has been regenerated from calli derived from a variety of explants (Zheng et al., 2002; Modh et al., 2002; Tyagi et al., 2004; Ray et al., 2005; Kumar et al., 2006). Agrobacterium mediated gene transfer method have been established before for a commercial gerbera variety, but still, to date, general transformation protocols suitable for a range of elite varieties have not been developed. Gerbera jamesonii is also used in the preparation of traditional Chinese medicine, tuerfeng, for curing cold and also for treating rheumatism (Ye et al., 1990). In the present study, experiments were conducted to investigate organogenesis from various sources of Gerbera explants. The effect of various concentrations of growth regulators on the initiation, multiplication and root induction were examined. 1 Int.J.Curr.Biotechnol. Volume 2; Issue 2; Feb, 2014
Materials and Methods Callus culture Pot gerbera (Gerbera Jamesonii) was grown in the department of Biotechnology in University of GKVK, Bangalore (Karnataka), India. Leaves, flower stalk and root explants were collected and washed in running tap water. The explants were surface sterilized with teepol for 20 min and with 0.1% bavistin for 30 min. Explants were cut in to small pieces under sterile conditions. Explants were treated with 0.1% HgCl 2 for 23 min, rinsed with double distilled water for 45 times and culture on MS medium (Murashige et al., 1962). The medium was supplemented with 25gm/l of sucrose and solidified with 8gm/l of agar. The ph of medium was adjusted to 5.65.8 before autoclaving. Explants cultured without growth regulators served as the control. The following growth regulators were also added to the medium, IBA (Indol3butyric acid): 0.5, 1.0, 1.5 and 2 mg/l. NAA (Naphthalene acetic acid): 0.5, 1.0, 1.5 and 2 mg/l. 2, 4D (Dichlorophenoxyacetic acid): 0.5, 1.0, 1.5 and 2mg/l. Each treatment was applied in three replications of 10 explants each. The explants were cultured in a growth room at 25 o C in the dark to encourage the formation and the growth of callus. When the calli were one month old, were transferred to a growth chamber and maintained less than 16 h photo period. After 30 d, the following data were recorded percentage of explants producing callus; callus growth and callus type. Shoot regeneration/initiation Shoot regeneration was encouraged by culturing the calli on MS medium supplemented with 20gm/l sucrose and solidified 10gm/l of agar. The following growth regulators were added to the medium BAP (Benzyl amino purine): 0.3, 0.5, 1.0, 1.5, and 2mg/l, Kinetin: 0.3, 0.5, 1.0, 1.5 and 2 mg/l BAP and Kinetin: 0.3+0.3 and 0.5+0.5 mg/l. Subculture was done two times within 60 days with the same hormone concentrations and was maintain less than 16 hours photoperiod at 23 o C. After 60 days the following data were recorded percentage of calli producing shoots and average number of shoots per callus. Multiplication and rooting Subsequently the shoot multiplications were experimented with various concentrations of hormone BAP (Benzyl Amino purine); 0.3, 0.5 and 1 mg/l. Individual shoots were separated and rooted on MS medium supplemented with IAA (Indole acetic acid):0.1, 0.2, 0.3, 0.4 and 0.5mg/l with and without charcoal. Results Various explants have been used for callus induction and regeneration of shoot formation was studied. Different types of auxin and cytokinin combination were used in order to obtain complete regeneration of Gerbera jamesonii invitro. Calli did not form on plain MS medium, but it forms on medium supplemented with any of the growth regulators tested (Figure1 & Table1). The best result was observed in MS media containing 1.5 mg/l of 2, 4D which is friable from leaf explant The calli from leaf explant differentiated into shoot after 68 weeks of incubation on the MS containing BAP, Kinetin and its combination, about 76.2% of calli produced shoots in MS medium supplemented with 1mg/ l BAP (Figure2). Rapid multiplication of shoots was achieved on MS supplemented with 1mg/l BAP (Figure3). Rooting was achieved in MS with 0.3 mg/l IAA (Figure4). Root explant were found to be non regenerative in all experiment conducted. Discussion Plant hormone and type of explants plays very important roles in determining the regeneration of Gerbera jamesonii invitro. Many commercial ornamental plants are being propagated by in vitro culture on the culture medium containing auxin and cytokinin, (Preil, 2003; Rout et al., 2004). Ornamental plants and woody plant species are also propagated by auxiliary bud proliferation (Mantell et al., 1985; Pierik 1987; Chu 1992). The callus induction was best with 1.5mg/l 2, 4 D. In a previous study on Gerbera jamesonii callus growth was best when explants from fully expanded leaves were Table 1: Effect of growth regulators on the percentage of explants producing callus and callus type in Gerbera jamesonii G ro w th R eg ula to rs m g /l IBA N A A 2,4 D P ercen ta g e of exp lan ts prod ucing ca llus L eaf 1 1.7 1 4.6 1 8.0 1 7.2 3 5.0 6 3.8 7 8.0 7 6.8 9 2.5 9 3.8 9 6.2 9 2.7 F low er Stalk 6.2 7.7 12.2 11.3 63.0 67.0 75.2 75.0 89.4 90.2 9 92.2 C all us Ty pe Volume 2; Issue 2; Feb, 2014 Int.J.Curr.Biotechnol. 2
Table 3: Effect of growth regulators on the percentage of calli producing shoots in Gerbera jamesonii Growth Regulators Percent of Calli producing shoots (mg/l) Leaf Flower stalk 0.3 65.4 66.8 0.5 68.6 69.3 BAP 1.0 76.2 72.4 1.5 74.8 73.8 2.0 70.0 73.3 0.3 2.2 0.5 2.8 Kinetin 1.0 3.9 1.5 3.2 2.2 2.0 2.8 2.6 BAP + Kinetin 0.3+0.3 0.5+0.5 58.9 63.2 43.2 51.0 induced on MS medium containing 1.0 mg/l of IBA, NAA or BA (Parthasarathy et al., 1997). In another study, callus translucency and regeneration were best when capitulum explants were induced on medium containing both 2 mg/ l BA and 0.5 mg/l IAA (Arello et al., 1991). The current study describes a protocol including one month of callus induction stage, six to eight weeks of shoot formation stage and 20 25 days of root formation stage. Aswath et al., (2002); Kumar et al., (2004) reported that BAP produced compact nodular callus from gerbera leaf or petioles. In the present study, callus from leaf explants grow on MS medium containing 2 mg/l 2,4D. In both the explants callus type were friable. In a previous study, an average of 58 shoots were formed by calli from Gerbera leaf explant that were induced in MS medium containing 4mg/l Kinetin and 0.1 mg/l IAA (Tyagi et al., 2004). In this study, an average of 912 shoots were formed by calli from Gerbera leaf explant that were induced in MS medium containing 1mg/l BAP. Regenerated shoots were inoculated in the MS medium containing various concentration of IAA (0.1, 0.2, 0.3, 0.4 and 0.5mg/l with and without charcoal). Maximum rooting was observed in the MS medium containing 0.3 IAA without charcoal. It is concluded that plant regenerated from leaf and flower stalk via adventitious shoot formation may be very useful for mutation breeding as the chances of mutants arising from adventitious shoots are very high. Further studies using biotechnology tools, protoplast fusion and genetic transformation may results in producing a new variety. References Arello E F, Pasqual J E, Pinto B P & Barbosa M H P 1991. Invitro establishment of explants and seedling regeneration in Gerbera jamesonii Bolus ex Hook by tissue culture. Pe quisa Agropecuarian Brasileria 26: 269273. Arockiasamy S, Prakash S & Ignacimuthu S 2002. Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium foetidum. L Biol plant 45: 129 132. Aswath and Choudhari 2002. Rapid plant regeneration from Gerbera jamesonii Bolus callus culture. Acta Bot Croat. 61: 125134. Boek van den L, Haydu J J, Hodges A W and Neves E M 2004. Production, marketing and distribution of cut flowers in the United state and Brazil. Annual Report of Florida Agricultural experiment station University of Florida. 1 19 Chu I Y E., 1992. Perspectives of Micro propagation Industry In Transport production systems Kurata K & T Kozai (eds). Kluwer Academic, Amsterdam. 137150. Chun Y W., 1993. Clonal propagation in Non Aspen Poplar Hybrids In Micropropagation of woody plants. Dordrecht. 209222. 3 Int.J.Curr.Biotechnol. Volume 2; Issue 2; Feb, 2014
Figure1: Callus from Leaf explants of Gerbera Jamesonii growing on MS medium supplemented with 1.5mg/l of 2,4D Figure2: Shoot regeneration from leaf callus with 1mg/l of BAP Figure3: Multiplied shoots growing on MS medium supplemented with 1 mg/l of BAP Figure4. Rooting of shoots on MS medium supplemented with 0.3 mg/l of IAA Das P & Singh P K S., 1989. Gerbera In Bose T K & Yadav L P (eds) Commercial Flowers Calcutta. Naya Prokash. 601 622. Murashige T, Serpa M and Jones J B., 1974: Clonal Multiplication of Gerbera through tissue cultures. Hort Sci. 9: 175180. Huetteman C A and Preece J E., 1993. Thidiazuron A Potent Cytokinin for Woody Plant Tissue Culture. Plant Cell Tissue Organ Culture. 33: 105150. Mureshige T and Skoog F., 1962. A devised medium for rapid growth and bioassay with tobacco tissue cultures. Plant physiology. 15: 473497. Kumar S and Kanwar J K., 2006. Regeneration ability of petiole, leaf and petal explants in gerbera cut flower cultured in vitro. Folia Hort. 18: 5764. Parthasarathy V A, Parthasarathy U, Nagaraju V A and Mishra M., 1997. Callus Induction and subsequent plant regeneration from leaf explants of Gerbera jamesonii. Folia Hort. 9: 8386. Kumar S, Kanwar J K and Sharma D R., 2004. Invitro regeneration of Gerbera jamesonii Bolus from leaf and petiole explants. J Plant Biotechnology. 13: 7375. Pierik R L M., 1987. Invitro culture of higher plants Martinus Nijhoff. Dodrecht. 183230. Mantell S H, Matthews J A and Mckee R A., 1985. Principles of Plant Biotechnology. 1st Edn Blackwell Scientific, Boston U K. 130157. Pierik R L M, Jansen J L M, Maasdam A and Binnendijk C M., 1975. Optimization of Gerbera Plantlet Production from Excised Capitulum Explants. Sci Hortic. 3: 351357. Modh F K, Dhaduk B K and Shah R R., 2002. Factors affecting Micropropagation of Gerbera from capitulum explants. J Ornm Hort. 5: 46. Preil W., 2003. Micro propagation of ornamental plants In Plant Tissue Culture 100 years since Gottlieb. Haberlandt Laimer M & Rucker W (eds), Springer Verlag, New York. 115133 Volume 2; Issue 2; Feb, 2014 Int.J.Curr.Biotechnol. 4
Ray T, Saha P and Roy S C., 2005. Invitro plant regeneration from young capitulum explants of Gerbera jamesonii. Plant Cell Biotech Mol Biol. 6: 3540. Rout G R and Jain S M., 2004. Micropropagation of ornamental plants cut flowers Propagation. Ornamental plants. 4: 328. Tyagi P and Kothari S L., 2004. Invitro regeneration of Gerbera jamesonii from different explants. Indian J Biotechnol. 3: 584586. YE J N, Wang T Z, Cai S Q, Komatsu, K, Mikage, M and Namba T., 1990. Pharmacological studies on folk medicine in Sichuan province China. II On Tuer fang derived from Gerbera plants. J Pharmac Soc Jap. 110: 374382. Zheng X F, Wang J H and Li M Y., 2002. Factors affecting organogenesis in Gerbera jamesonii Bolus cultures. Invitro J Jlangsu Forest Sci Technol. 29: 2931. 5 Int.J.Curr.Biotechnol. Volume 2; Issue 2; Feb, 2014