Author: acer

A preliminary study on in vitro seed germination and rooted callus formation of
Tetrastigma leucostaphylum a host plant for Rafflesia
Netty W. Surya and M. Idris*
*email : uwakidris@gmail.com
Plant Physiology Laboratory, Biology Department
Faculty of Mathematics and Natural Sciences, Andalas University - Padang
West Sumatera, Indonesia 25163

8th Flora Malesiana Symposium 23-27 August 2010 Singapore Botanic Gardens,
Singapore

Abstract
Propagation in vitro of Tetrastigma leucostaphylum (Dennst.) Alston ex. Mabb. as a host plant for Rafflesia is one of the alternatives that must be explored in a sustainable concervation effort for Rafflesia. This research was conducted as a preliminary study on propagation of T. leucostaphylum including in vitro seed germination on several media types and environmental conditions necessary for germination as well as inducing rooted callus formation on hypocotyl pieces treated with two types of auxin with adding cytokinin.
The result show that T. leucostaphylum seed germination is epigeal with seedling emergence period ranging from 30-60 days after planting, regardless of presence or absence of light. Percentage of seed germinated after 60 days was between 54-60% of the total seed that germinated in media treatments. Murashige-Skoog (MS) medium + 0,5 mg/L Kinetin was a better medium to promote seed germination. Callus formation began 7 days after planting on MS medium + 2 mg/L 1-Naphtalene Acetic Acid (NAA). While, on MS medium + 2 mg/L 2,4-Dichlorophenoxyacetic Acid (2,4-D), callus formation began 21 days after planting. In this medium, a cutting part of hypocotyl resulted some phenolic that make the medium browning. Rooted callus formation was seen after 21 days treatment of MS medium + 2 mg/L NAA and it was not evident with the addition of 2 mg/L 2,4-D. Callus producing roots is a better source for germination and infection of Rafflesia seed for the next research phase.
Key words : in vitro germination, Tetrastigma leucostaphylum, rooted callus

1. Introduction
Tetrastigma (family : Vitaceae) comparises about 100 species and occurs in tropical and subtropical Asia and Northern Australia. The Malesian region is richest in species (about 60), followed by Indo-China (about 20) and Thailand (about 15). Some Tetrastigma are used in traditional medicine in Indonesia and Malaysia, mainly in the form of Leaf poultice to treat pussy inflammatios, boils and fever (Lemmens, 2003).
Tetrastigma is well known as the exclucive host of the parasitic Rafflesia with its spectacular flowers ((Nais, 2001). In Sumatera, Tetrastigma leucostaphylum is one of the main host plant for Rafflesia arnoldii and Rafflesia gadutensis (Meijer, 1997). The high destruction of habitat in Sumatera make the population of Rafflesia and its host become limited, only in the conservation area (Zuhud, Hernidiah dan Hikmat, 1997).
In vitro conservation is the most important step and allowed to maintain the existence of Rafflesia in Sumatera with preparing the host plant in in vitro technique. The limitation information about microprogation of this plant is the main reason that this research is more important and must to do it
The preliminary study aimed to know about germination of T. leucostaphylum seed and induction of rooted callus as the main sources for Rafflesia seed in vitro infection for the next research phase.

3. Result and Discussion

Based on the results obtained during the research revealed that the blackout did not affect the effectiveness of germination. This means that the seed germination is not affected by lighting conditions. Plant growth regulator (Kinetin) promoted seed germination (Table 1). The type of seed germination is epigeal in which elongated hypocotyls and cotyledons taken from the growing media (Figure 1)

Table 1. Range days of germinated and percentage of germination after 60 day in lighting condition 12 HL/12HD (dap; day after planted)

No	Treatment	Range days of germination (dap)	Percentage of germination (%)
1	MS	30-60	54
2	MS + 0,5 mg/L kinetin	30-54	60
3	MS + 1 g/L active charcoal	30-52	56
4	MS + 1 g/L active charcoal + 0,5 mg/L kinetin	30-52	56

MS medium + 2mg/L NAA is better to promote rooted callus formation as compared with MS + 2mg/L 2,4-D (Figure 1. and Table 2.). Jaskani et al (2008) have rooted callus formation of Vitis vinifera with addition of 2 mg/L NAA to the medium.
Rooted callus formation is the main media as a sources of infection for Rafflesia seed in vitro. Zhou et al (2004) using rooted callus of Brassica napus as the primary source to in vitro infected of Orobanche seed.

Table 2. Times of callus and rooted callus formation and percentage of callus and rooted callus formation (dap; day after planted).

Observation	MS + 2mg/L NAA + 0,5 mg/L Kinetin	MS + 2 mg/L 2,4-D + 0,5mg/L Kinetin
Times callus formation (dap)	7-10	15-21
Time rooted callus formation (dap)	21-30	21-45
Percentage of callus formation (%)	100	100
Percentage of rooted callus formation (%)	100	0

4. Conclusion
Based on this research can be concludes as :
MS + 0,5 mg/L Kinetin is the best medium for promoting seed germination.
Rooted callus formation can be produced in MS + 2 mg/L NAA + 0,5 mg/L Kinetin and MS + 2 mg/L 2,4-D + 0,5 mg/L Kinetin produce callus without roots

Acknowledgements
The Authors thanks to The Herbarium of Andalas (ANDA) Andalas University for identifying of T. leucostaphiylum samples used in this research.

Autobiography
Jaskani, M. J., H. Abbas, R. Sultana, M. M. Khan, M. Qasim and I. A. Khan. 2008. Effect of Growth Hormones on Micropropagation of Vitis vinifera L. cv. Perlette. Pak. J. Bot. 40 (1) : 105-109.
Lemmens, R. H. M. J. 2003. Tetrastigma (Miq.) Planchon. In Plant Resources of South – East Asia No. 12 (3) Medicinal and Poisonous Plants 3. R. H. M. J. lemmens and N. Bunyapraphatsara (Eds). Prosea Foundation. Bogor – Indonesia. Pp : 400 – 401.
Meijer, W. 1997. Rafflesiaceae. Flora Malesiana Series I, Vol. 13 : 1-42.
Nais, J. 2001. Rafflesia of The World. Sabah Parks. Kota KInabalu. Sabah
Zuhud, E. A. M., N. Hernidiah and A. Hikmat. 1999. Pelestarian Rafflesia hasseltii Suringar di Taman Nasional Bukit Tiga Puluh Riau-Jambi. Media Konservasi VI (1) : 23-26.
Zhou, W. J. , K. Yoneyama, Y. Takeuchi, S. Iso, S. Rungmekarat, S. H. Che, D. Sato and D. M. Joel. 2004. In Vitro Infection of Host Roots By Differentiated Calli of The Parasitic Plant Orobanche. Journal of Experimental Botany 55 (398) : 899-907.

Figure 1. Morphological performance of seed germination and callus; A. MS, B. MS + 1g/L active charcoal, C. MS + 0,5mg/L Kin, D. MS + 0,5mg/L Kin + 1g/L active charcoal, E. MS + 2 mg/L NAA + 0,5mg/L Kin and F. MS + 2 mg/L 2,4-D + 0,5mg/L Kin