Int. J. Med. Arom. Plants, ISSN 2249 – 4340

RESEARCH ARTICLE

Vol. 1, No. 3, pp. 195-202, December 2011

In Vitro Plant Regeneration of Ginger (Zingiber officinale Rosc.) with Emphasis on Initial Culture Establishment S. SATHYAGOWRI, Thayamini H. SERAN* Department of Crop Science, Faculty of Agriculture, Eastern University, Chenkalady, Sri Lanka *Corresponding Author, Tel: +94 65 2240760, +94 65 2240490, Fax: +94 65 2240740 Article History: Received 5th September 2011, Revised 26th October 2011, Accepted 27th October 2011.

Abstract: Ginger (Zingiber officinale Rosc.) is one of the oriental spices which is widely used in Sri Lanka for culinary purposes and as an important ingredient in Ayurvedic preparations from time immemorial. The conventional ginger propagation method is by rhizomes which have many limitations. A study was conducted to select the suitable explant size for initial culture establishment of Sri Lankan local variety of ginger and their subsequent plant regeneration. Field grown ginger rhizomes with buds were rinsed thoroughly in running water then dipped in 70% ethanol for 1 min. They were surface sterilized using Captan 0.3% followed by Doxycycline 0.2% for 10 min and finally with commercial bleach 20% of CloroxTM. The sterilized rhizome buds of various sizes viz., 0.5 cm, 1.0 cm and 2.0 cm length were excised and cultured on MS medium with 3.0 mg L-1 BAP and 0.5 mg L-1 NAA to select the suitable size of explants for the establishment of culture. Results revealed that explants of 0.5 cm length exhibited high rate of survival (66.67%) and morphogenic response (44.44%) among the explant sizes tested. Consequently the selected explants (0.5 cm long) cultured were transferred to MS medium with 5.0 mg L-1 BAP and 0.5 mg L-1 NAA for shoot multiplication after 6 weeks of cultures. Thus microshoots were placed on the medium containing 3.0 mg L-1 BAP and 0.5 mg L-1 NAA to regenerate in vitro plantlets. The regenerated plantlets were successfully acclimatized. This might be helpful to obtain large number of in vitro planting material of ginger rapidly for cultivation. Keywords: Ginger; in vitro plantlets; morphogenic response; rhizome buds.

Introduction Ginger (Zingiber officinale Rosc), a herbaceous perennial belonging to the family Zingiberaceae is grown commercially in many tropical regions and is native to tropical South East Asia (Pieris 1982). It is a common condiment for various foods and beverages. It has a long history of medicinal use dating back 2500 years (Shukla and Singh 2007). Breeding of ginger is handicapped by poor flowering and seed set therefore it is propagated vegetatively through rhizome (Kambaska and Santilata 2009). Conventional propagation through seed rhizomes produces 10-15 lateral buds in a season of 8-10 months (Bhagyalakshmi and Singh 1988). This crop also heavily attacked by bacterial wilt (Pseudomonas solanacearum), fusarium yellows (Fusarium oxysporum f.sp.zingiberi) and root knot nematode (Meloidoryne incognita) (Dohroo 1989). *Corresponding author: (E-mail) [email protected] ©2011 Open Access Science Research Publisher

Studies revealed that there were three main varieties (Local, Chinese and Rangoon) of ginger commercially grown in Sri Lanka. Local ginger rhizomes are small and the fibrous flesh is ash white in color. The local ginger’s taste and aroma is comparatively higher than other species and it is largely used for beverages (Anon 2010). Although ginger is cultivated throughout the country, the local farmers were unable to manage the demand within the country until the near past hence it is imported from South India which is one of the biggest exporters of ginger (ITI 2009). In Sri Lanka, there is a need to increase the production of local ginger and more planting material will be required. Propagation by conventional means is hampered due to slow propagation rate and the risk of disease transmittance through rhizomes therefore in vitro technique is considered the best alternative method that may supply a large number of planting materials for commercial planting and further studies (Hamirah et al. 2007). Millions http://www.openaccessscience.com [email protected]

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of plantlets can be produced within 1-2 years from a single explant and tissue of ginger has been cultured in vitro and consequently freed of pathogens (Sahavacharin 1995). High frequency regeneration of plants from in vitro cultured tissues is a pre-requisite for successful application of tissue culture technique for crop improvement (Akter 2001). There was frequently an optimum size for explants used to initiate tissue cultures. Very small explants do not survive well in culture but large explants may be difficult to decontaminate effectively or are less easily manipulated (George et al. 2009). Several workers have used different sized explants in tissue culture of ginger and other related species but there is limited information on particular explant size of ginger to be used for in vitro culture establishment. Although there are several reports presented for in vitro techniques for propagation of ginger but no recent reports from Sri Lanka which concern about the efficient in vitro establishment and propagation of economically valuable local ginger variety exist in the literature survey. Therefore the present study is an attempt to develop successful and reproducible protocol for in vitro plant regeneration of local ginger with emphasis on effective initial culture establishment with ideal explant size. Materials and Methods The experiments were conducted during 2010-2011 at the Tissue Culture Laboratory of the Department of Crop Science of the Eastern University of Sri Lanka. Field grown Sri Lankan local variety was used in this experiment. For the initiation of cultures and regeneration of plantlets, MS (Murashige and Skoog 1962) medium was used in the present investigation. Culture media with two different concentrations of BAP were used in the present study.

1 minute. They were then surface sterilized with Captan 0.3% followed by Doxycycline 0.2% for 10 minute. Subsequently the rhizome pieces were immersed in 20% of Clorox™ (Sodium hypochlorite, 5.25% active ingredient) with 2-3 drops of tween 20 for 20 minute. Finally, they were washed three times thoroughly with sterile distilled water. The sterilized rhizome buds were then aseptically trimmed to 0.5, 1.0 and 2.0 cm long pieces respectively under sterile laminar flow with the flame sterilized scalpel and forceps. Culture establishment MS (Murashige and Skoog’s 1962) medium containing 30% sucrose as carbon source and growth regulators [3.0 mg L-1 BAP (6-benzylamino-purine) and 0.5 mg L-1 NAA (α- naphthalene acetic acid)] were solidified with 8 g L-1 agar for initial culture establishment. The pH of the medium was adjusted to 5.8 with 0.1N NaOH or 0.1N HCl before autoclaving at 15 psi pressure and 121 °C temperatures for 20 minute. Molten medium of 20 mL was dispensed into every sterilized culture bottle (125 mL capacity), covered by plastic lid and labeled. Aseptically excised explants of various sizes were placed on the MS medium. All cultures were incubated at 16 hrs light/8 hrs dark photoperiod (cool, white fluorescent light) and 25±2oC temperature. The cultures were observed daily. Each treatment had 24 replicates and the experiment was repeated thrice. Induction of multiple shoots After six weeks of culture establishment, the survived explants were transferred to fresh MS medium supplemented with 5.0 mgL-1 BAP and 0.5 mg L-1 NAA (Inden et al. 1988) for the formation of microshoot multiplication. The cultures were maintained by the regular subculture on fresh medium.

Explant source and sterilization Healthy rhizomes were collected from the field and rhizome pieces with buds were excised. These rhizome pieces were rinsed several times with running tap water followed by distilled water and then dipped in 70% ethanol for Sathyagowri and Seran

In vitro plantlets Microshoots were collected from the cultures containing 5.0 mg L-1 BAP and 0.5 mg L-1 NAA and then placed on MS medium containhttp://www.openaccessscience.com [email protected]

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ing 3.0 mg L-1 BAP and 0.5 mg L-1 NAA for the in vitro plant regeneration. Rooted micropropagules with shoots about 4-5 cm in length were removed from the culture bottles and the roots were washed under running tap water to remove agar. Then the plantlets were transferred to sterile poly pots (small plastic cups) containing pre-soaked coir dust and sand (1:1) maintained inside the growth chamber made by the polythene sheet. After acclimatization of plantlets, they were transplanted to pots containing the mixture composed of sand, red soil and cattle manure (1:1:1).

The results exhibited high survival rate (66.67%) of cultured explants which were aseptically trimmed upto 0.5 cm pieces.

Observation of cultures

Figure 1: The percentage survival of various sizes of explants cultured on MS medium at weekly intervals (Data based on 72 total explants).

Data were analysed using the SAS software. The number of days taken to shoot bud initiation was transferred to square root transformation and analysis of variance done. The percentage data were analysed using the Chi-square test. The significant difference between means was estimated using Tukey’s Studentized Range (HSD) Test at 5% significant level. Results Initial culture establishment Explant survival The experiment results (Figure 1) indicated that the survival rate was high as 100% in treatment with 0.5 cm long explants until 5th week and 1.0 cm long explants showed the second highest survival rate. The least survival pattern observed in explants of 2.0 cm length as they showed 100% explants death earlier at the 3rd week due to contamination. The explants of 1.0 cm length survival began to reduce after 3rd week and reached the lower level (18.06%) during the 6th week due to endogenous microbial contamination (Figure 1). Meanwhile the 0.5 cm explants showed comparatively very low rate of contaminations. Therefore, they were able to maintain higher survival rate. Some of the explants turned brown and died. The 0.5 cm and 1.0 cm long explant treatments indicated a drop in survival only after the 5th week of culture. Treatment containing 0.5 cm long explants significantly differed (P