Micropropagation of Eucalyptus hybrid

Micro propagation of  Eucalyptus hybrid (WK 15 Eh) Using Nodal Explants

Freddy Pangaribuan, SP

Plants Tissue Culture Laboratory – Tree Improvement Section

Research and Development PT Wirakarya Sakti –S Tapa - Jambi

Abstract: Large scale micro propagation of Eucalyptus species has been achieved from nodal explants.  Several media with different hormone and condition were tries in standardization of this plant.  Surface sterilization technique using Mercury chloride (HgCl₂) can be used as alternative for easy and efficient protocol.  Explants induce can produce new buds within 7-10 days.  Additional of silver nitrate (AgNO₃) have a promotory effect on shoot production and bacterial suppression.  Micro propagation by this protocol is rapid, reproducible and true to type plant can be achieved.

Introduction

Eucalyptus has been a major fiber source and the demand for pulp production is ever increasing.  The objective of this study is the establishment of standard protocols micro propagation of large scale Eucalyptus hybrid clones free of pest and disease.

Material and Methods

Plant material of Eucalyptus hybrid Wk 015 EH  was taken from PT Wirakarya Sakti R&D Nursery Centre in November 2008.  Nodal segment of 3-5 cm was harvested from newly sprouted branch of the stool plant (mother plant).  Leaves were trimmed and washed with detergent for 10 minutes.  Explants were surface sterilized with 1% of Mercury chloride for 1-3 minutes and then rinsed with sterile water.  Damage cut end were removed, trimmed to a size of 1-1.5 cm each explants containing at least one node one  node.  All the aseptic manipulation was carried out in laminar flow.  Selection of medium for inoculation using E6’ (Base E5, Vit E5, IAA 0.2 + BAP 0.4, Sugar 30 gr/l and as gelling agent using agar Jia Feng from China.  After inoculation culture were incubated in cool white fluorescent light (1600-2000lux) with 12-16 hours photoperiod at temperature 25-32oC.  The explants were oriented in a vertical position.  The response of the explants were estimated on contamination rate and bud initiation

Result and Discussion

The success of the micro propagation through nodal cultures involves number of factors which affect directly or indirectly the proper establishment of explants in the medium.

Of all treatment using HgCl₂ as a sterilant the respond of the explants varies.  Treatment with 1 minute of submerging and continuous agitation gives 100% contaminated while the best treatment is 5 minutes submerging and twice rinsing in sterile water.  The result may be different since the explants are not uniform but it is better to use second and third nodes i.e. with older tissue.  Young tissue use in this experiment shows the effect of toxicity and explants dies.

Table 1. Responds of explants with sterilization using HgCl2 in 1-5 minutes duration after 40 days.

HgCl2	Duration (mnt)	No Explants	Contamination (%)
	1	20	100
	2	20	40
	3	20	55
	4	20	70
	5	20	65

The nodal explants produce new buds within 7-10 days inE6 media and subsequent sub culturing were using this medium and added with 1 ppm of silver nitrate.  Silver nitrate have a promotory effect on shoot initiation and also anti microbial activity.  Problem of hyperhydricity occurs on some explants and the symptom is red, glassy and watery tissue.  The vitreous explants are separated from healthy tissue because vitreous tissue produces more than normal ethylene gas that can deform healthy tissue. 

Fig 1.  Culture of  WK 015 EH after 6 series of subculture

Healthy tissues can be multiplied and a multiplication rate of 2 can be achieved.  Elongated shoots were rooted on B2RE and rooting rate will be assessed.  Several sub culturing may contribute to high rooting rate, since young tissue can not developed root.

Fig 2. One day old rooting from above culture on B2RE (MSm NAA 1 + BAP 0.5).  Note that the stem still less lignified

 Fig 3.  Symptoms of Hyperhidricity on culture appear as glassy, watery and brittle tissues.

Fig 4.  Normal tissue with green leaves and high efficiency multiplication on ME 15 (MS  , IAA 1 + BA 0.5).

Reference

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