K.I. Kandasamy, Siti Suhaila A.R. and Rosilah A.A.

Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor, Malaysia.

Tel: +603-62797154, Fax: +603-62801614; Email: kodiswaran@frim.gov.my

Abstract

Shorea leprosula belongs to the family Dipterocarpaceae, and its synonyms are Hopea maranti Miq., Shorea maranti Burck, Shorea astrostricta Scort. Ex. Foxw, and the common name (in Malaysia) is meranti tembaga. This dipterocarp species has been propagated mostly through seeds and cuttings; however the seed production is erratic and infrequent. Furthermore, the seeds deteriorate rapidly in storage (a behavior associated with recalcitrant seeds). In addition, production of cuttings from mature trees is difficult and plagiotropic growth of shoots from cuttings is common. In recent years, many researchers have been reporting on the use of tissue culture techniques to address these problems, however most used embryo or axillary buds as starting material. Nevertheless, there is an increasing interest in clonal propagation of dipterocarp species by tissue culture because the forest of Dipterocarpaceae has been disappearing rapidly. Thus, the development of an efficient culture method for mass production of dipterocarp is important. Although, it was reported that this species does not tolerate watery soil, but it is worth noting that the water uptake by this plant is quite high. Thus, we believe that this plant could be manipulated (for propagation via micropropagation) using a suitable temporary-immersion system. In this paper, we will be reporting the ability to successfully micropropagate Shorea leprosula by using a temporary-immersion technique, the RITA system. Shoot cultures from existing tissue culture material (maintained in semi-solid medium) at FRIM’s Tissue Culture Laboratory were used as starting material for this study. Subsequent shoots obtained from this study were successfully rooted and acclimatised using our proprietary weaning chamber in our greenhouse.

Key words: Shorea leprosula, Meranti, Micropropagation, Tissue-Culture, Dipterocarp.

Introduction

The use of plant tissue culture (or in vitro) techniques is extremely valuable for the production of high quality uniform planting material within a short time frame, in a specified, usually limited space. Although, the technique itself may be simple, but developing an efficient propagation system for a targeted plant species is relatively complex. An efficient propagation system should include a defined culturing procedure for each target plant species, including specific medium requirements for the various developmental stage (multiplication, elongation and rooting, weaning or hardening), sub-culture interval and growth conditions.

Shorea leprosula belongs to the family Dipterocarpaceae, and its synonyms are Hopea maranti Miq., Shorea maranti Burck, Shorea astrostricta Scort. Ex. Foxw, and the common name (in Malaysia) is meranti tembaga. This Dipterocarp species has been propagated mostly through seeds and cuttings; however the seed production is erratic and infrequent. Furthermore, the seeds deteriorate rapidly in storage (a behavior associated with recalcitrant seeds). In addition, production of cuttings from mature trees is difficult and plagiotropic growth of shoots from cuttings is common. In recent years, many researchers have been reporting on the use of tissue culture techniques to address these problems (Scott & Rao1988, 1989, Vaario & Soda 1995, Roy 1997, Roy et. al. 1997, Nakamura et. al. 1994, 1999), however most used embryo as starting material (explant), while Nakamura’s and Roy’s groups used axillary buds as starting material. Nevertheless, there is an increasing interest in clonal propagation of dipterocarp species by tissue culture due to the rapid disappearance of the Dipterocarpaceae forest. Thus, the development of an efficient culture method for mass production of dipterocarp is important.

Method and materials

Explant preparation

Selection of suitable explants (or starting materials in an in vitro system) and its subsequent decontamination procedure is the key towards successful culture establishment. For Shorea leprosula the best explant material seems to be the mature seeds, followed by shoot-tip and nodal cuttings of young seedlings.

Seed explant: Seeds collected were carefully de-winged, washed in running tap water and soaked in fungicide solution (e.g. 0.1% Benlate or Furan) containing a drop of Tween-20 (or any domestic detergent) for an hour. Seeds were then washed in sterilised distilled water (SDW) and soaked in 70% ethanol for not more than 30 sec, and agitated in 10% bleach solution (e.g. any domestic bleach) containing a drop of Tween-20 (or any domestic detergent) for 30 min successively. Seeds were again washed in SDW (at least five times), and agitated in 50% bleach solution containing a drop of Tween-20, for 20 min. Finally, seeds were washed in SDW (again, at least five times or until all traces of detergent were gone), blot-dried (on sterile tissue towels) and were ready to be either germinated directly on to culture medium, or dissected to expose its zygotic embryo (to initiate embryo culture).

Shoot-tip and Nodal explants: Explants were harvested from 2-3 feet tall seedlings (wildings, established under greenhouse conditions can also used), early morning (preferably before 9.00am), washed in running tap water and soaked in fungicide solution (containing a drop of Tween-20) for an hour. Explants were then washed in SDW, and soaked in 70% ethanol for not more than 60 sec and agitated in 5% bleach solution (domestic bleach) containing a drop of Tween-20 for 30 min successively. Explants were washed in SDW (at least five times) and again, agitated in 20% bleach solution containing a drop of Tween-20 for 30 min. Following a final wash in SDW (again, at least five times or until all traces of detergent were gone), explants are now ready to be dissected to remove all bleached tissue and inoculated onto suitable culture medium.

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