TECHNICAL REPORT ITIO PD 106/01 REV. 1 (F)

TECHNICAL REPORT Ino PD 106/01 REV. 1 (F)

Conservation and Genetic Improvement of Indigenous Tree Species of Tropical Rain

Forest of Indonesia

Prepared by Anto Rimbawanto

© 2006 by Faculty of Forestry, GMU and International Tropical Timber Organization

This publication was made possible by a generous grant from the International Tropical

Timber Organization (ITIO), Yokohama, Japan.

Published by

ITIO PO 106/01 Rev. 1 (F)

Faculty of Forestry, GMU

Yogyakarta, Indonesia

Available from

ITIO Project, Faculty of Forestry, GMU

Phone/Fax. 62-274-545639

E-mail:itto-gmu@yogya.wasantara.net.id

ISBN: 979-99818-7-5

Printed in Indonesia

ii Technical Report PO 106/01 Rev.1 (F)

Contents

1. Introduction

2. Conservation and Utilization Strategy

3. Genetic Diversity

3.1. Isozyme diversity

3.2. Molecular diversity

4. Genetic Resources

4.1. Natural Stands

4.2. Genetic Conservation Stands

4.3. Genetic Trial Stands .....

5. Deployment Strategy

5.1. Clonal Propagation

5.2. Seed Propagation

6. Synthesis and Implications

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Preface

Massive deterioration of Indonesia's natural forest is evident from

the sharp decline of official timber production, which stood at over 30 million m3 annually between 1970 - 1990s, to a mere 6 million m3 in recentyears.lndoriesia's lowland tropical forests, the richest

in timber resources and biodiversity, are most at risk. Environmental implications of the degraded natural forests in Kalimantan and Sumatra have manifested in natural disasters such as drought, flood

and landslide.

The Government of Indonesia has identified conservation and sus­tainable development of biological resources as a national priority of prime importance. The focus ofthe policy is to preserve biodiversity

for ensuring the dynamic stability of ecosystems. The preservation and conservation policy also serves to provide a pool of genetic materials for subsequent research and development to meet future national and regional needs.

In orderto address the issue of genetiC conservation oftropical tree species, an InO-funded project on "Ex situ Conservation of Shorea

leprosula and Lophopetalum multinervium and Their Use in Future Breeding and Biotechnology" (PD 16/96 Rev.4 (F)) was implemented

in 1998. The Project was designed to address the issue of conserv­inggenetic resources oftwo model species. Follow on project namely PO 106/01 Rev.1 (F) "Increasing Genetic Diversity of Shorea leprosula

and Lophopetalum multinervium for Breeding and Genetic Improve­ment" was carried out from 2002 - 2005 to enrich the collection of genetic diversity of the species.

This Technical Report synthesizes the outcomes of those projects

and discuss the implications for genetic conservation and genetic improvement programs. The established conservation and genetic test plots are invaluable resources for future utilization and there­fore should be maintained in a sustainable manner.

iv Technical ReportPD 106/01 Rev. 1 (F)

TECHNICAL REPORT ITTO PD 106/01 REV. 1 (F)

Conservation and Genetic Improvement of In­digenous Tree Species of Tropical Rain Forest of

Indonesia

1. Introduction Indonesia's rich natural resources are fundamental to the country's growth and development. Terrestrial and ma­rine resources have provided suste­nance and livelihoods to millions of In­donesians for generations. Exploitation

offorest, mineral, gas and oil resources fueled rapid economic growth from the 1970s through the mid 1990s. Yet this natural resource base is under threat as natural resource management is no longer sustainable. Indonesia is expe­riencing the negative economic and

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ecological consequences of this as her forest and marine resources disappear while conflict over these limited re­

sources increases. Decentralized natu­ral resources management provides a unique opportunity for regaining sus­tainable natural resources manage­ment.

Forest degradation due to excessive logging and forest fire of tropical rain

forest in Indonesia has been taking place indiscriminately. Many experts believe that the natural forests in two

Technical ReportPD 106/01 Rev.l (F)

of Indonesia's major islands namely Sumatra and Kalimantan will be de­

funct by year 2015 and 2020 respec­tively. The Government of Indonesia has

identified conservation and sustain­able development of biological re­sources as a national priority of prime importance. The focus of the policy is

to preserve biodiversityforensuringthe dynamic stability of ecosystems. The preservation and conservation policy

also serves to provide a pool of genetiC materials for subsequent research and development to meet future national and regional needs.

In orderto address the issue of genetiC conservation of tropical tree species, an ITIO-funded project on "Ex situ Con­servation of Shorea leprosula and Lophopetalum multinervium and Their Use in Future Breedingand Biotechnol­ogy" (PO 16/96 Rev.4 (F)) was imple­mented in 1998. The Project was de­

signed to address the issue of conserv­ing genetic resources of two ecologi­cally distinct sites namely well-drained

and swampy sites that dominate the landscape of tropical rain forest of In­donesia. One model species for each site was selected for the conservation and breeding works, namely Shorea leprosula and Lophopetalum multinerviumwhich are under threats of serious genetic erosion due to de­forestation and excessive logging. If no measures were taken the plantation

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program (using these two species) will

be severely inferior due to low genetic diversity. The Development Objective of

the Project was to create a Centre of

Excellence for ex situ conservation in Indonesia that will not only be a ben­efit to Indonesia but also to the neigh­boring countries.

When the Project was completed in 2000 it had established 10 ex situ con­

servation plots of S. leprosula in East, West and South Kalimantan, Jambi Sumatra and West Java, consisting of 14 populations from Kalimantan and

Sumatra, and 8 ex situ conservation plots of L. multinervium. Similarly im-

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portant achievement ofthe Project was its impact on generating awareness amongst decision makers, forestry managers, research institutions and field workers of the importance of ge­netic conservation.

2. Conservation and Utili­zation Strategy

Subsequent to the completion of the PD 16/96 RevA (F) in 2001, two fol­low on projects were initiated, namely PD 41/00 Rev.3 (F,M) "Model Devel­opmentto Establish Commercial Plan­tation of Dipterocarps" and PD 106/ 01 Rev.1 (F) "Increasing Genetic Diver­sity of Shorea leprosula and Lophopetalum multinervium for Breed­ing and Genetic Improvement". Both projects were operational until 2005.

The former was designed to develop technology that will provide means to produce good quality seedlings of the suitable dipterocarps species. Such technology would be readily applied for large-scale commercial plantations. Study was also being done to examine the economic of establishing and man­aging dipterocarps plantations. Such information would be useful for private sectors tomake decision on investing in plantations. The latter project was to continue the conservation efforts by collecting ge­netic materials from more populations

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and evaluating the genetic trials that

were established in the previous iu project. The major outputs from these ' genetic trials would be information on the best genetically superior families of S.leprosula in growth traits. Planting materials of these families would be used for operational plantations.

Ideally, genetic conservation should be imbedded into the management of natural forest. However, when large part ofthe forests is destroyed and deterio­ration is continuing, such approach is no longer effective. Ex situ conserva­tion, for many of the dipterocarps spe­cies is the most effective measures to safe the remaining genetic resources.

3. Genetic Diversity

Natural population oftree species pro­vides a pool of gene diversity essential for the long-term survival of the spe­cies. In the context of conservation of genetic resources, determining the level of genetic diversity that exists in a species is fundamentally important. Quantitatively, genetiC diversity is com­monly expressed as: polymorphism to describe the proportion of available gene loci, average number of alleles. per locus, average heterozygosity to describe the proportion of all heterozy­gous gene loci, and level of among­population differentiation (Yeh 2000).

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The genetic diversity ofthe two species, namely S. /eprosu/a and L. mu/tinervium was examined using isozymes and DNA markers. Isozymes

(also known as isoenzymes) are en­zymes that differ in amino acid se­quence but catalyze the same chemi­

cal reaction. These enzymes usually display different kinetic parameters (Le. different KM values), or different regulatory properties.

Genetic diversity can also be assessed at molecular level. Whilst biochemical markers examine the products of genes, molecular markers examine di­

versity at DNA level in particular it re­fers to the unique position within the genome of the DNA segment. The DNA marker used for this study is called

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RAPDs (random amplified polymorphic

DNAs). RAPD marker (Williams et al

1990) is a PCR based in which a single, short primer of 10 bases long is used (rather than 2 flanking primers of around 20 bases each used in regular

PCR). RAPD primers are of arbitrary se­quence. By chance the sequences matching a given RAPD primer will oc­

cur in many places. The RAPD marker detects DNA polymorph isms due to substitution, deletion and insertion at primer binding site.

The technical ease of RAPD markers and their application to any species has led to their use in many genetic studies of tree species including genetic link­

age mapping and population genetic

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3.1. Isozyme diversity

Our study examined the diversity of S. leprosula from 5 populations namely Ragusa west Sumatra, Carita Banten west Java, Kotawaringin Timur central Kalimantan, Semaras Pulau Laut south Kalimantan and Batu Ampar east Kalimantan. Using polyacryla­mide vertical slab gel electrophore­sis 100 samples from each popu­lation were subjected to 7 enzyme systems, namely Acid Phospate/ ACP, Diaphoras8jDIA, GlycerateHydrogenase/ G2DH,Glutamate Oxaloacetat Tran­saminase/GOT, 6-Phosphogluconate Dehydroge­nase/6-PEG and Shikimate Dehy­drogenase/ShDH. However, only four enzymes (EST, GOT, 6-PG and SkDH) gave clear band patterns. A total of five loci were detected in these four enzyme systems. Analysis of the diversity ofthe spe­cies showed a high degree of ge­netic diversity evidenced by the high level of heterozygosity He = 0~338

for Sumatra and He = 0.334 for Kalimantan, up to 63% of the total diversity resides within populations. Similar results has also been re­ported by Lee et al. (2000) working on nine populations of S./eprosula based on eight polymorphic loci.

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3.2. Molecular diversity

DNA analysis of S. leprosula was carried out to evaluate the genetic diversity within and between popu­lations and the genetic relation­ships of population in Indonesia. Sample collected from forty-four trees from 3 populations, i.e. Jambi (Sumatera), Tanah Grogot(southern­East Kalimantan), and Mahakam River (northern-East Kalimantan). Samples from Jambi were collected from 2 sub-populations namely PT Dalek Hutani Esa and PT Sadarnila.

Forty-four wildlings were randomly selected and leaf samples were taken from the wildling and used for DNA analysis. Sixteen (16) prim­ers with high reproducibility and have clear bands were chosen for the population study.

Heterozygosity (H.,) value within population was high (0.310) in Jambi population (Sumatra) but low in both of the Kalimantan popula­tions (0.257 and 0.247 in Sungai Mahakam and Tanah Grogot popu­lations, respectively). The values within subpopulations were lower than that within Jambi population but higher than those within Kalimantan populations. The GST value was 0.152 among 3 popula­tions. The values between two re-

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gions (Sumatra and Kalimantan re­gion), between populationsin the

same region (Sungai Mahakam and

Tanah Grogot populations,

Kalimantan region), and between subpopulations (Dalek and Sadarnila subpopulation in Jambi population, Sumatra) were 0.088, 0.080, and 0.065 respectively.

The mean value of genetic dis­tances between individuals (d)

within populations was 0.341, lower than the mean of between

populations (0.393), and between regions was the highest (0.406) as expected. However, the mean value within subpopulation (0.355) was higher than that of between sub­population (0.353) or within popu­lation (0.341). The UPGMA

dendogram of each individual formed two major clusters. Cluster 1was consisted of only the individu­als in Kalimantan region, but clus­ter 2 was consisted of both

Kalimantan and Sumatra region.

Genetic conservation for breeding purpose aims at obtaining variable genetic materials rather than sim­ply preserving genetic composition

of a species. Our results indicated that the geographically distant popu­lation could possess the different genetic composition, and the ge­netic diversity within population is

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lower than species' total diversity. It

is therefore advisable to sample

trees from several populations in

the ex-situ conservation plots.

High level of genetic diversity in S. /eprosu/a can be due to a number of factors such as geographical range, high fecundates, outcrossing, long life span and occurrence in late succession phase (Loveless 1992).

However, since the genetic diversity obtained was relatively higher com­pared to other tropical species with similar life history traits, the species'

evolutionary history in which ances­tors ofs./eprosu/a had acquired very high genetiC diversity duringspecia­tion has also been considered as

another reason causingthe higher value of genetic diversity. Outcross­ing, insect pollinated and wide dis­tribution of the species are consid­ered as other reasons in having high level of diversity. From the conser­vation point of view, conservation

strategy for a widespread distribu­tion such as S. /eprosu/a, in which the species existence is not seri­ously under threat, is directed to safeguard the long-term evolution­ary fitness ofthe species.

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4. Genetic Resources

4.1.. Natural stands

S. leprosula occurs naturally from Penninsular Thailand, throughout Penninsular Malaysia, Sumatra and Kalimantan. It belongs to the red meranti group and has been exten­sively harvested because of its high economic value. The species had suffered massive population reduc­tion and Oh the IUCN Red List itis categorized as endangered. Natu­ral populations of S./eprosula were limited to small populations in few areas in Kalimantan and Sumatra.

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4.2. Genetic conservation stands

The objective of establishing ex situ conservation stand is to collect pools of genetic diversity that exist within the species and maintain them in a genetic conservation plot. In the longterm this plot could serve as important genetic resources, particularly when the natural popu­lation is so depleted.

Genetic materials of S. leprosula

were collected from nine natural stands across four provinces in Kalimantan, e.g., East Kalimantan (Malinau, Sangkulirang, Sambarata, Sentawar, Meratus­Birawa), South Kalimantan (Barabai), Central Kalimantan (Bukit Baka, Muarateweh), and West Kalimantan (Gunung Bunga). A total of 262,000 seeds were col­lected from 610 families but not all families were used, limited by the available number of seedlings re­quired for establishing the plot. The number of families from each popu­lation used forthe trial is: Sentawar 35, Meratus-Birawa 55, Samba rata 36, Barabai 97, Bukit Baka 125, Gunung Bunga 90 and Muarateweh 28.

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The ex situ conservation plots were

established in 6 locations namely Teras South Sumatra, Carita west

Java, Gunung Kencana west Java, Bukit Baka central Kalimantan,

Semaras South Kalimantan and Birawa East Kalimantan. In total 81.5 ha of conservation plot have been established.

Ex situ genetic conservation plots for i. multinervium have also been established. i. multinervium is a wet­land species valuable for light gen­

eral construction, interior finishing, paneling and furniture. The species is endemic to the northern part of East Kalimantan found along the

riverbanks. Its existence in their na­tive habitat is under threats be­cause of high demand of the tim­ber, encroachment and land conver­

sion.

Genetic materials were collected from Berau, Betayau, Seputuk and Pimping Rawa in East Kalimantan. Originally the plan was to establish the conservation plots in East

Kalimantan and West Kalimantan. However, because of land tenure issue the plan for East Kalimantan

was cancelled and the plot was eventually established at Mandor and Segedong near Pontianak West Kalimantan. The conservation plot covers an area of 55 ha.

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The choice for having ex situ con­servation plots for i. multinervium

was made as a mean to develop a

model of genetic conservation for wetland species. Our primary con­

cern is with the deterioration of eco­nomically and ecologically impor­tant wetland species such as ramin (Gonystylus spp). i. multinervium is relatively easy to propagate and

seeds are abundant. In contrast, propagation techniques of Gonystylus spp is virtually unknown.

The experience and knowledge in establishing ex situ conservation plots of L. multinervium will be used to develop similar approach for Gonystylus spp.

Regular measurement and mainte­nance have been carried out to en­

sure that the plots are in good con­ditions a nd that useful information on the performance of the trees dif­ferent populations can be obtained.

4.3. Genetic trials stands

The long-term objective of estab­

lishing genetic trials, in the context of the Project is to establish prog­eny trial, is to produce genetically

improved seeds for operational

plantation by converting the prog­eny test into seed orchard. In the short-term, progeny test can provide information on the adaptability and

growth of the trees.

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The progeny trial was established in February 2003 at Nanga Nuak

Central Kalimantan. It consists of 31 seed lots originated from Bukit Baka population in Central

Kalimantan and SO seedlots from Gunung Bunga population in West Kalimantan. The trials were laid out

in ndomized complete block design,

3- tree plots with S repl ications.

Evaluation of the progeny trials at

2.5 years indicated a significant dif­ference for height and stem diam­eterfor all seedlots. Mean value of height was 5.29 m of Bukit Baka

and 3.3 m of Gunung Bunga.

5. Deployment Strategy

5.1 Clonal propagation

Because of the inherent irregular flowering of dipterocarps, deploy­

ment of genetic materials is best done byclonal propagation. Conven­tional propagation techniques such as cuttihgs have been developed for many dipterocarps species

(Subiakto 200S). Large-scale pro­duction of dipterocarps by cuttings has been developed by FORDA and Komatsu Ltd. Thetechnique known as KOFCO (Komatsu-FORDA Fog Cooling) system is suitable to pro­duce dipterocarps planting stocks from shoot cuttings. The propaga-

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tion technology controls humidity,

temperature and light intensity to a

level suitable for transpiration and photosynthesis. The cutting tech­nique has been studied to 36 dipterocarps species including S. leprosula. Rooting percentage

ranges from 0% to 99% depending on species. The technique is suit­

able for producing planting stocks

of dipterocarps when seeds are not

available, and for mass propagation of superior clones generated through breeding program.

5.2. Seed Propagation

Propagation of dipterocarps by seeds is hindered the nature ofthe seed which loses its viability within weeks. The phenomenon is known

as recalcitrant, in which the seed embryo is sensitive to desiccation.

Unlike orthodox seed which can stand desiccation (it is viable at water content of 5%) and therefore can be stored for long period oftime under suitable storage conditions, recalcitrant seeds can not be stored for more than several weeks. Seed­lings of dipterocarps can be pro­

duced only when the seeds are brought to germination within days.

Further complication of propagation by seeds is the irregularity offlower­ing. When seeds are storable, un-

Technical Report PD 106/01 Rev.1 (F)

availability of seeds can be over­come. using seeds from previous seasons. Because dipterocarps seed is recalcitrant seed stock is never available and therefore this propagation method is not a reliable one for mass production of plant­ing materials.

6. Synthesis and Implica tions

ITIO Project PO 106/01 Rev.1 (F) fo­cused on enrichment of genetic diver­sity oftwo selected indigenous species (S. /eprosu/a and L. multinervium) to provide pools of genetic materials for future breeding and biotechnology. Major outputs of the project have been: a) conservation plots of wider popula­tions of the two species, and b) prog­eny plantation of S./eprosu/a.

Main characteristic ofthe project is its approach to use conserved genetic materials for breeding and genetic im­provement. The project is designed to provide answers on a) the most effec­tive means for maintaining genetic re­sources ofthe selected species and b) the utilization of the conserved genetic resources for establishing highly pro­ductive plantation incorporating breed­ingand genetic improvement.

ITIO strategy towards sustainable tropi­cal forest management and trade in

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tropical timber production stipulated that the sustainability of tropical forest is a major theme. Three assessment criteria have been set up to assess sustainability, namely productivity (quality and quantity), biodiversity, and capacity of soils to sustain forest growth.

The objective of establishing ex situ

conservation plots under this Project is to provide pools of genetic materials for maintaining genetiC diversity of the species while securing the remaining diversity from further depletion. There­fore it is important that a wide-range of populations is covered. The range of genetic materials collected from a wide range of population of S. /eprosu/a (a total 18 populations) should ensure that sufficient genetic diversity has been captured and conserved in genetiC conservation plots.

From the view point of conserving the genetic diversity of the species of inter­est, the outputs of this Project is a sig­nificant achievement. The plots of ge­netic conservation are an invaluable resource both for conservation and genetiC improvement. Another mile­stone of this Project is the establish­ment of progeny test of S. /eprosu/a.

This has set a direction for improving the genetic quality ofthe species using conventional breeding method.

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Coupled with the vegetative propaga- References tion method genetically improved plant-

ing materials should be available Loveless, M.D. 1992. Isozyme varia-

within foreseeable future. tion in tropical trees: pattern of geneti<;:

organization. In: Adam, W.T., Strauss,

S.H. and Copes, D.L. (eds). Population Genetic of Forest Trees. Kluwer Aca­

demic Publishers, Netherlands. Pp: 67-94.

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Subikato, A., Sakai, C, Purnomo, S., and Taufiqurahman. 2005. Cutting propa­gation as an alternative technique for mass production of dipterocarps plant­ing stocks in Indonesia. Paper pre­

sented at the 8th Round Table Confer­ence on Dlpterocarps, Ho Chi Minh City, 15-17 November 2005.

Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, JA and Tingey, S.V. (1990) DNA polymorph isms amplified byarbi­trary primers are useful as genetiC mark­ers. Nucleic Acids Res. 18: 6531-6535 Yeh, FC. 2000. Population genetics. In:

Young, A, Boshier, D and Boyle, T (eds.). Forest Conservation Genetics: Prin­ciples and Practices. CSIRO Publ. Aus-

tralia. Pp: 21-37.

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