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Training Report No. 2B

ITTO Project PO 16/96 Rev.4{F) EX SITU CONSERVATION OF SHOREA LEPROSULA AND LOPHOPETALUM MULTINERVIUM AND THEIR USE FOR FUTURE BREEDING AND BIOTECHNOLOGY

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TRAINING IN CONSERVATION AND MANAGEMENT OF

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GENETIC RESOURCES

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YOGYAKARTA, 19-23 MARCH 1999

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PART 11

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FACULTY OF FORESTRY GADJAH MADA UNIVERSITY YOGYAKARTA

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1999

Table of Contents

Part 11. Handouts and Reading Materials

1. Introduction to Conserving Forest Resource Prof. Bart A. Thielges 2. In situ Conservation Prof. Soekotjo 3.

Forest Tree Breeding Prof. Oemi Hani'in Suseno

4. The Application of isoenzyme analysis for Breeding and Conservation Dr. M. Na'iem 5. ITIO PO 16/96 Rev.4(F) Workplan (revised edition)

Introduction to Conserving Forest Resources

prof. Bart. A. Tbielges Oregon State Universit~

I. Need for Conservation of Forest Resources The world is losing large numbers of populations of plants and animals - including entire species - at an increasing rate. Much of that loss is attributable to clearing of land for agriculture and urbanization, but other man-caused factors, including forest practices that encourage high grading and over cutting, also play a significant role. And then there are natural factors at work as well, such as global warming and the various types of atmospheric pollution present in the developed and developing world. Just a few examples from North America are adequate to illustrate the extent of the losses that may be incurred. Introduced pests and pathogens - Many North American commercial tree species have been decimated by problem organisms brought in from other countries. Examples include American chestnut (Castanea dentata) which was completely eliminated from eastern North America by an introduced fungal disease. To a lesser degree, American elm (U/mus americana), and North American white pines (Pinus strobus, P. montico/a, P. lamberliana) have suffered the same fate from introduced pathogens. And the European gypsy moth and the Japanese beetle have also eliminated marginal populations of many broad-leaved trees. Native pests and pathogens - Not all of the North American problems with population and species demise from pests are exotic in origin. Several spp. of Populus in North America suffer from native leaf and twig beetles and a wide array of homegrown fungal and bacterial pests, as well. These domestic organisms are not generally as devastating as introduced ones, because the hosts generally have some degree of evolved resistance to the disease organisms and insect predators. But the results can be devastating, especially to those host populations growing under stressed conditions at the margins of the species range. Pollutants -_ In many urban areas, ozone and other air pollutants generated by internal combustion engines are eliminating previously viable populations of sensitive Pinus and other genera of forest trees. And often the pollutants act to weaken trees and make them more susceptible to insects and drought. Heavy metals and acidic runoff from mining operations are destroying important riparian populations in the US west and southeast and in central and western Canada. And high elevation populations of Picea, Abies, and other coniferous spp. in rural areas all over North America are being weakened and eliminated by acid deposition emanating from industrial sites far removed from the site of the damages. Climatic Change - If global climatic warming is real, then within the next century major changes could affect virtually every forested area on the globe. Many boreal and temperate species, especially at the southern edges of their ranges, might be all but eliminated from forests, especially in the Northern Hemisphere. In the tropical and subtropical regions, areas of rainforests, and the species therein, might be changed to more open, drier forests due to droughty cycles. Because forests exert such a major effect on local and regional climates, and upon all that lives beneath them, the loss of biological diversity in forests is especially significant. Changes in dominant forest cover may lead to major changes in species composition, and thus food chain dynamics, that might trigger much greater losses in

Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

biodiversity. Forests create vertically stratified habitats upon which virtually all other forest organisms - mammals, birds, insects, small plants, microorganisms - depend. And to the point of this workshop, the loss of biological diversity due to elimination of forest tree species may be far less of a danger than the more insidious and less dramatic losses of subspecific sources of variation which are occurring much more rapidly and over larger landscapes than species losses. Such things as clearing of lowlands, especially riparian areas, for crop production; conversion of uplands to pastures and homesites; and general urbanization/industrialization are causing the losses of genetic resources especially at the edges of species ranges or site distributions. And forestry - through clearing of natural forests and plantation as well as high-grading harvesting and other silvicultural practices - contributes to these losses in subspecific diversity or variation, as well. The conversion of the vast North American "heartland" to seemingly endless fields of wheat, maize, and soybeans and pasturelands for domestic animals caused incalculable loss of genetic material in "fringe" populations of dozens of native forest tree species which breeders may later have found useful for increasing drought, disease, or insect resistance of those species. Finally, these losses which have been occurring in temperate zones for centuries e.g., Scotland and Ireland were heavily forested before the conquering English decided they were better suited for grazing of sheep - are now being repeated in the tropics. And the reasons are the same - these countries are looking for more land upon which to house and feed increasing populations. But there's a big difference, I believe. It's only been about 150 years since Darwin, Mendel, and other pioneers of evolutionary biology and genetics began to lay much of the scientific groundwork for what we now can call "conservation biology". Europeans and the "new world" colonizers of the 15th through 19th Centuries didn't have the benefit of this way of thinking. The Industrial Revolution didn't have ecology or genetics textbooks to set its parameters and guide its progress. But we have this knowledge and this historical record now to guide development in the tropics , and the advancing technologies to implement conservation much more effectively as we proceed with development in the tropics. And we also now know now that the tropical landscapes with their productive climates and soils and accompanying tremendous resources of biological diversity, are vitally important not only to mankind, but to the entire balance of life on our planet. That's sort of a "big picture" summary of what we're about here for the next few days. This ex situ genetic conservation project for two endemic tropical hardwood species based here at GMU and sponsored by the Government of Indonesia and IITO, is a unique opportunity for all of us present to get in on the ground floor of genetic conservation of forest trees. We've done a great deal of talking about this issue, certainly since the late 1960's, but we haven't done much else. As we say in the USA, it's now time to "put our money where our mouth is" or, more recently, "if you're going to talk the talk, then you've got to walk the walk". Sure, some of the things that we have done have made contributions - nature reserves and "wilderness areas" are types of in situ conservation measures. And botanical gardens, arboreta, and various "plant germ plasm banks" recently developed are


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examples of ex situ techniques. Closer to home, research-wise, the provenance and progeny tests and the seed orchards and clone banks developed by tree breeders are definitely ex situ conservation efforts. But these are generally quite limited and ex post facto measures that are truly secondary to much more limited and focused programs for economically linked genetic gains. And there are even some exceptions to that many of the very early IUFRO provenance testing of the 1930's was aimed at a true sampling of ranges of important tree species and conserving that sampled variation in replicated and protected plantations by national agencies. And many of the NC-51/-99 tests set up in central and eastern North America by Jonathan Wright, Scott Pauley, Chris Heimberger and others in the 1950-80 era also were basically intended to serve those purposes. But this ITIO project is different in several important ways. Number one, it is quite large and comprehensive. The sampling is quite extensive in terms of truly representing the ranges of both species and also intensive in terms of numbers of· parent trees selected and progeny collected to try to encompass the extent of the genetic variation present. Protection of this major investment of scientific talent, time, and money is maximized by geographically separating plantations of each of the extensive and well-replicated ex situ collections. And, unlike many of the past efforts in this area the preservation of the forest tree germ plasm is the a priori reason for this study, and not merely an afterthought of normal procedures for tree improvement programs. But the primary and most important difference, and the reason that brings us all together here this week is that this ITIO project contains a significant educational element. We hope that the project will stand as a model of proper ex situ conservation of forest germplasm not only for Indonesia, but also for the Southeast Asia region and even the world. This workshop will be followed by others on more specific topics, and the collections will serve as field demonstration areas. We want to develop an efficient way of establishing and managing the data that this huge project will generate, and share those systems with colleagues elsewhere. And we are hopeful that establishment of an ITIO Project web-site will be a contribution to technology transfer as we proceed. We will return to more of the specifics of this ITIO Project later on in the workshop, but for now, we want to look at the principles and theories guiding genetic conservation for forest trees.

11. Overview of Genetic Conservation Objectives of Genetic Resources Conservation. There are many, depending on the goals. "to conserve the adaptive, evolutionary, commercial, and amenity potential (public forest of trees and the ecosystems in which they exist." management) "to ensure access to genetic resources" (forest tree breeder)

Training in Conservation and Management of Genetic Resources Yogyakarta. 19-23 March 1999

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"to maintain the evolutionary capacity of species to adapt to changing economic and ecological requirements, and the viability of the supporting ecosystems" (evolutionary biologist) "to preserve biological diversity" (Iayperson) The entire above are valid, for they define the perceptions and aims of the various persons or agencies. Definitions of Diversity. (taxonomic) based on descriptions of organisms present (species, genus, family, etc.) (ecology) based on numbers of plant/animal taxa, and their interactions within and between ecosystems. (evolutionary) based on number of taxonomic groups within habitats, species richness, relative abundance or rarity, trophic levels, etc. (genetics/breeding) Based upon amounts of intraspecific genetic variation both between and within populations of a species. This last definition is the one that is of interest to us in this course and the ITIO Project, as well. We will be concerned with the conservation and use of gene complexes, genes, alleles, DNA sequences for both current and future potential uses. This means that we also must consider natural breeding systems, including determination of population size and structure, pollination mechanisms, gene flow patterns, etc. In other words, we are dealing with the management of the population genetics of the species in question. We want to assure that rare or specialized alleles are conserved and protected from loss due to various factors in the natural and man-made environments in which the plants must live. It might be helpful at this point to use a very simple illustration that has special meaning here in Indonesia, especially on Java. Let's assume that a mainland population (the main population) of a species has evolved to contain two common alleles of a gene important for adaptation - say, alleles A & B. A much smaller island population of that same species has, however, evolved a third, much rarer allele - C, which confers some selective advantage to the species in this harsher island environment (e.g., salt tolerance or a root system resistant to strong winds). If that island population is somehow eliminated, allele C will be lost and may not again be available for centuries of evolution. Recolonization from the available seed source on the mainland will not replace allele C. We may postulate that this simple example is illustrative of the genetic loss(es) which occurred when Krakatoa erupted in 1883. Other disasters such as fires and floods might also be responsible for such losses, but some of you will remember from population genetics the theories of random drift and extinction, by which genes in very small and isolated populations may be lost simply by chance. So, dramatic events are not always required for losing genes and variation in nature! However, it is obvious to us all that environmental factors are a driving force in initiating losses in biodiversity, or, more specifically, genetic diversity. Among the many factors driving genetic change are -


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global and local climatic changes soil, air, and water pollution managerial interventions, such as selective management of natural forests enrichment of natural forests with exotic spp. forest fragmentation clearcutting and replacement with other spp. (or favored spp.) agricultural clearing, industrialization, urbanization The foregoing generalized comments apply to all species - even animals - but we can also make the case that forest trees are "special" organisms in their ecosystems. There are many valid reasons why society should take special steps to conserve forest ecosystems and the significant genetic resources that they represent. First, trees are ecologically important. They are the dominant component in many ecosystems and are responsible for ecosystem structure and function through their dominant positions. Globally, there are> 50,000 spp. of forest trees, so it is highly probable that all cannot be conserved. Decisions on which to conserve depend on economic, ecological, aesthetic, or other social values. Second, many tree species are economically important. In the USA, for example, of 684 native tree spp., about 270 (about 40%) can be used to produce wood products. Even though production may concentrate heavily on only a few spp., regionally (e.g. lobi oily pine in south, Douglas-fir, hemlock, w. red cedar in west) there may be good reasons to conserve other "reserve" spp. as well (ex. chestnutga k, Doug fir[fll d er). Third, non-wood products of trees and forests are now just beginning to be appreciated and exploited in many parts of the world, especially in western cultures. Major examples are Taxus (taxol) and the recent trend to over-the-counter "natural" drugs. Fourth, there is a global importance of forests. They exert tremendous influences on local and regional climates through C02 <->02 exchange in PS/RS, controlling floods and erosion, stabilizing influence on temperatures/humidity. Disturbing the forest also, of course, has effects on other living components of the ecosystem (both flora and fauna). Fifth, healthy, diverse forests are a source of recreational and esthetic enjoyment for society. No one would be willing to travel long distances to camp in a maize field or hike through rice paddies! Lastly, there are ethical issues to consider when managing natural forests. As foresters and scientists, we should have a "stewardship" outlook, and strive to conserve our important forest resources for the future generations. This is a large and significant responsibility of forestland management. And finally, we may also make a "special case" for genetic conservation of tropical forest trees. It is extremely important in tropical areas, where population pressures and industrialization are just now driving major land clearing. As an example, the International Union for Conservation of Nature and Natural Resources (IUCN) recently compiled some startling statistics -


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Of 250,000 vascular plant spp on earth, 170,000 (68%) are tropical or subtropical endemics. Of these 170,000 spp, 60,000 (30%) are at risk over the next 50 years as compared with only 8,000 at risk (10%) of the 80,000 temperate zone plants. Furthermore, of the 130,000 tropical plant spp not endemic to Amazonia or Equatorial Africa where change is slower and not so dramatic as in other tropical reasons) these 60,000 "at risk" spp represent almost 50% of the total flora. The IUCN proposes that Botanical Gardens should be a focus of the conservation effort. But, as the IUCN has indicated that 35,000 plants are "immediately endangered" and they also emphasize a further 15,000 spp of "known economic value" these 50,000 spp represent an enormous conservation target. And there are only about 250 major botanical gardens in the world that could be called upon to partiCipate in this major effort. So, each of these botanical gardens would have to take responsibility for conserving 200 species, each adequately represented as to range and genetic diversity! Obviously the logistical problems and costs of this effort would be enormous, especially where widespread species of forest trees are concerned. And, pragmatically speaking, while most of the T&E plant species are in the tropics, most of the major botanical gardens are in temperate countries. This situation further contributes to an unworkable imbalance. So, these are some of the major reasons why genetic conservation of the T & E forest tree resources are so important, as well as some of the significant obstacles to accomplishing effective conservation. And, the "action zone" is definitely here in the tropics so you all have a chance to play an important role in some of the initial major efforts in genetic conservation. Next, we'll take a look at how we go about assessing and sampling diversity in forest ecosystems in order to set priorities for gene conservation efforts.

Ill. Determining Needs/Candidacy for Genetic Conservation Programs There are a great many variables that must contribute to a decision on which species are candidates for genetic conservation. As I mentioned earlier, these involve economic, ecological, and even social value judgements. But some very broad generalities may be made, especially from the standpoint of ecological risk. On that basis, we may say that "prime candidates" for genetic conservation include (1) Forest trees in general - they are "big and immobile targets" often falling prey to fires, logging, land clearing operations, etc. (2) Medicinal plants. Proven or potential values as pharmacologicals, insecticides, etc. (These two categories are arrived at based mainly on economic and social values) (3) "Keystone Species" of an ecosystem. E. g., the food plant of a major T&E animal, important link in whole food chain, etc. (4) Rare and Endangered


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a). Historical Data. From herbarium, surveys, etc. The actual geographical range of some spp. may be seriously underrepresented (sampling errors). This is especially true with smaller, rarer spp. Sampling may not have been random, favoring overrepresentation of rare or "sought after" spp., underrepresenting more common spp. Biases toward more "valuable" spp., neglect of non-commercial. b) Stages of development. Developmental morphology (changes) Simply stated, it is easier to miss seedling and other understory representatives than mature. At other stages, may be hard to recognize mature (can't see leaves in canopy, e.g.) c) Annual and longer cyclic differences. Examples Lack of current fruiting and thus no forest floor reproduction for several years may bias results based on surveys of reproduction. These cycles may be influenced/caused by drought, fire, insects, disease, etc, d) Seral Stage differences of communities. When sampling landscapes, need to take successional variation into account and compensate in survey design. 2. Locational Scale a) Physical Position. These may be site differences - wet, dry, low, high, substrate, etc. b) Geographic Scale. Basically, we must be aware of the pattern of distribution of the spp. of interest. For example, searching at the extreme of a species range may greatly bias survey results towards "scarcity". So, now that we have designed our survey with the above potential sources of error in mind, what are some of the indicators of biodiversity we must look for and include in our decision-making? I n addition to direct or sampled counts of spp or individuals in a given area, and an indication of population dynamics, there are five sets of information that facilitate or add to biodiversity indicators in support of decisions on genetic conservation. These are(1) Species-area relationships. This helps to estimate the minimum population size needed in reserves or other conservation areas. (2) Keystone Species Status. These spp are those which play a major role in maintaining ecosystem structure and integrity; e.g., an old-growth "climax" spp, or one with fruit important to ecosystem food chain, or N-fixer.


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(3) Taxic Groups. Basically, this is relative "related ness' - a spp with relatively few nearby close relatives is higher priority for conservation. (4) Functional Groups. Groups of spp filling same "niche" or having same function because of similar morphological structure - more "unique" is relatively better candidate. (5) Known or potential economic value of spp. With those biodiversity indicator criteria in mind, we are now ready to design and implement an assessment strategy in the field. There are several methods available. Assessment Methods (1) Traditional Forest Inventory. Or, "Vegetation Analysis". Well-suited to mature trees, but not to many other organisms because of limitations on data recording. (2) Molecular Methods. Looking at genetic diversity present and population structure by sampling at protein or DNA levels. Some techniques are - Isozyme analysis, restriction fragment length polymorphism (RFLP), randomly-amplified polymorphic DNA (RAP D), DNA fingerprinting, microsatellite analysis, etc. (3) Remote Sensing. May be valuable to identify unique or rare communities with characteristic foliage, flowers, etc. but currently limited. (4) Databases and GIS. This is an area that is currently developing and may have great applicability for future surveys of biodiversity. However, must remember that the GIS is only as good as the data that went into creating it. A poorly created database will yield a poor GIS. IV. Determination of Method of Conservation Once a decision has been made on the species, it is then necessary to select the most appropriate method for genetic conservation. This selection will vary with the species as well as the objectives of the genetic conservation program. There are two basic strategies or techniques - in situ or "in place" , and ex situ or "off site" conservation. But these are not mutually exclusive and, in fact, they may be combined into an optimal genetic conservation strategy for some spp.

In situ conservation basically depends upon establishing some sort of "protected reserves" of the species in locations appropriate to its range and pattern of distribution. In theory, those areas should then be managed to ensure survival and reproduction of the species. But in practice, these reserves are not managed well and often become areas of "benign neglect". (Ledig) Prof. Soekotjo will later be discussing in more detail the theories and practices of in situ conservation and I will then concentrate on ex situ practices. But it might be productive, at this point, to generally compare and contrast these two conservation techniques. In theory, in situ is the preferable long-term genetiC conservation solution for most species. This is because by "dedicating" the site(s) containing the populations to be conserved, you are also preserving,. in effect, the set of ecosystems in which the


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selected species populations are growing. This then allows for the continuation of G X E interactions, adaptations, and evolution of the selected populations. So, the in situ program provides for a long-term "dynamic" situation wherein the populations or collections continue to evolve in nature. But severe drawbacks to the in situ system are obvious. One, it is a very landintensive method. This is especially true of tropical hardwoods where there may be only one or a few specimens of a spp per hectare. It goes without saying then that two, they are expensive. Third, it is difficult to adequately protect these reserves, and they may be liable to damage from fire and other environmental agents. Four, the very best reserves are the primeval "old growth" stands and these are not only high value, but rare. Five, because of their natural" primeval state, the reserves may be difficult to access and to work in. These are only the very pragmatic problems with in situ systems. Other drawbacks include Problems surrounding the durability and long-term significance of the conservation effort. If administrations and priorities change, will the reserves be maintained and protected? The a priori choosing of priorities for conservation, e.g., wildlife habitat vs. timber. Ethical questions surrounding the wisdom of "human intervention" in natural systems. Despite the above problems and obstacles to implementation, most evolutionary biologists adhere to the basic theoretical utility of the in situ technique, considering the value of ex situ techniques generally only as a "backup" system for the in situ reserves. V. Ex situ Conservation Techniques On the other hand, many conservation biologists and evolutionists are quick to realize the value of ex situ methods, especially where resources are limiting. Returning to the tropical hardwood issue, Frankel et al. observed that to ensure a long term viable population of 500 individuals in an in situ reserve, a species with a distribution of 10 trees/km2 would need an initial reserve of 1000 trees or 100 km2 of reserve. Obviously, there are few organizations that could afford this investment in land, timber resources, and management and protection services. We have already disposed of the possibilities for Botanical Gardens to make much of a contribution to ex situ genetic conservation of trees, especially tropical trees. So, let's take a look at some of the options that are open for ex situ techniques for tropical trees. Some considerations necessary for selection of ex situ gene conservation strategies include The purposes and priorities of the conservation effort. Selection of specific conservation targets among R & E species. The scope and purpose of the genetic representation desired in the target spp. Is remediation (redistribution of spp.) of natural environments a goal? The role of databases in the program.


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We'll look at each of these in a bit more detail later as we proceed to describe the ITIO Project. For now, there are a few rules of thumb to determine the efficacy of ex situ techniques. It would seem logical that species that are at once useful and also R & E should have top priority for ex situ genetic conservation. Since preserving genetic diver-sity is paramount (or essential) to ex situ methods, as opposed to in situ which may also be species/type/system preservation focused, we need to give careful consideration to (a) the number of sites (sample number) selected for collections, and (b) the number of individuals (sample size) per collection site. The implicit purpose in preserving the genetic diversity of R & E or .JillY species is to keep open as many options as possible - for research, for utilization, for conservation, and if necessary, for remediation. Frankel et al., note that "ex situ conservation not only complements in situ conservation, it makes a specific contribution through being readily available and under direct controL" The retention of "genetic variation" richness".

= maximizing #

of alleles per locus, i.e .. , "allelic

When conserving natural R & E species, as with crop plants, the attention should be on common allleles - those with a frequency of >0.05 to 0.10 (as is common in preservation of crop spp.) This because the aim of the conservation program is to be able to recover at least one copy of such alleles with a probability of 0.90 to 0.95, which requires a sampling of about 50-100 randomly-selected plants per collection site. And this purpose must be stated clearly - the initial emphasis must be on species survival - the rarer alleles for breeding are a later consideration, from within the ex situ collections. As we shall see, this is a strong argument for the very strategy that we employed in our ITIO Project. But in recognizing the many restrictions and obstacles to the above strategy such as size, logistics, difficulty of collection, etc. Brown and Briggs (1991) have developed a fall-back to a "minimum sampling target". They have employed the logarithmic view of genetic diversity - that is that "useful variation" increases in proportion to the logarithm rather than the absolute size of the sample, so that their reasoning drops the requirement to a minimum of 50 individuals/species, optimally 5 populations of 10 individuals each. Having seized upon ex situ conservation as "the" best means of preserving genetic diversity in tropical tree species, it is wise to heed the costs of doing so. As Frankel, et al., have observed "the preservation of genetic diversity even on a modest scale, involves substantial costs in scientific effort, organization, and resources. As a rule some form of selection (of species) takes place. It appears that, more often than not, selection is motivated by actual or potential interest, concern, or use. But in their absence, can the burden of preserving genetic diversity be justified?" This is a very interesting observation. First, it certainly recognizes that it is only human nature to take an action requiring a large investment if there is some sort of future "payoff'. And secondly, it is a perfect bit of logic explaining why agencies and private


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industry need to invest and participate in genetic conservation programs, while at the same time providing a strong answer to those critics skeptical of why industry participates in this type of conservation effort! It goes without saying that even the minimal investment in ex situ conservation is a significant one, and that initial investment must be protected by adequate recordkeeping. Establishment and maintenance of a good database is an absolute must for this type of program, and especially if the collections are to be used now or at some future date for breeding and/or genetic transformation purposes. Returning now to a linkage between in situ and ex situ techniques it is worth observing that even the best-designed in situ reserve needs to be sampled periodically. If seed and/or pollen is difficult to store (as it is for almost all tropical tree species) then the ex situ planting is truly the "conserving adjunct" of the reserve. And this returns us to the issue of sampling strategy. Frankel et al., take a conservative approach to this kind of "backup" collection. They suggest collections of propagules from the number of trees one order of magnitude less than the population size in the in situ reserve; i.e., 100 from a reserve population of 1000 trees. Sampling should be random, and if seed is to be used, more than one fruit per individual should be collected to reduce chances for correlated paternity and inbreeding. Since most trees are outcrossing, sampling several fruits from each tree will amplify the pollen sources and increase the effective size of the sample, in terms of preserving genetic variability. To finish this discussion of the underlying principles of ex situ techniques, we can assume that ex situ conservation may offer a refuge for a greater diversity of spp and with somewhat better security than that available in the in situ "nature preserves". Yet even here there are severe limitations imposed by costs, space availability, staff, facilities, competing demands for all these, and most importantly in the long term, shifts in organizational policies due to staff changes, or to shifts in political control, or to shifts in public concern. Because of these latter risk factors for change (which also apply to in situ programs, of course) it is possible that such programs may be viable for only 1-3 (human) generations. But, I think that the significant question to ask at this point might be "what is the cost of not implementing these genetic conservation measures?" That's a good thought to hold as we now go into a discussion of the more pragmatic" nuts-and-bolts" of an ex situ program in preparation for reviewing the ITIO Project. VI. A "Generic" Strategy for Ex situ Gene Conservation of Forest Trees (From Ledig, 1992)

A. Sampling the Population(s) to Be Conserved In general, the sampling procedure should be based upon the genetic structure - the level and distribution of genetic diversity and how it is partitioned within and among individuals and populations - within the species. Some species, such as the North American Pinus resinosa have a very simple genetic structure that is reflected in lack of phenotypic variation throughout its range. Other species (and probably many tropical tree spp) have high levels of genetic diversity - an example from my personal research is the great range of variability in Populus deltoides especially in regard to its reactions to various native and introduced disease organisms.


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In addition to the level of genetic diversity, another important factor to consider is the pattern of that diversity. In simple terms, the diversity or variation can follow some (usually environmental) gradient and is then termed "clinal", or be irregular in its distribution that is termed "ecotypic". Unfortunately, these patterns of variation are not known for many tree species, even the most widely-studied European ones. So when information is incomplete, as it usually is, (location in temperate zone or tropics notwithstanding), it is always wise to sample systematically. No major "gaps" should be left. In western North America, "seed zone" boundaries may serve as a In that situation, the collection grid is approximately 80 to 160 km guide. latitudinally/longitudinally and 300m elevationally. But that system might be completely unsuitable in this region of the world, as it is based primarily on growing seasons or "frost-free" days in the Northern Hemisphere. At any rate, it illustrates that the sampling guidelines might be best-based on the prevalent agents of selection for the species. It goes without saying that unique or isolated populations should also be sampled, even if they violate the systematic grid established. Often, these populations contain alleles that are important to survival of the spp on the "fringes" of its range. In nature, such populations were usually associated with some sort of edaphic or elevational feature now they may be areas where the spp is persisting under selection by air or soil pollutants. Then there is the "numbers game" - how many sample points and how many individuals per point, etc. The number of points depends on spp range and the physiography - the larger and more complex, the more populations sampled. Trees per site depends on population structure and breeding system. The former is often completely unknown so it is best to err on the side of conservatism - perhaps 30-50 trees per site and 1000 seed per tree is the "safety zone" to strive for. Finally, locations and identities must be carefully maintained. If collections are made on a per tree basis, then the identity system must be based on parent trees. All information - location, elevation, soil and climatic data, parent tree conditions, seed yields, etc. must be put into a comprehensive, well-organized and well-managed data base. B. Field Plantations - Genebanks for Tropical Tree Species. Seed of tropical forest species does not store well, thus precluding the "seedbank" concept popular with temperate and boreal plant breeders and conservationists. So in the tropics, the ex situ conservation areas must do "double duty" as the dynamic gene banks for the species to be conserved. Living, growing trees must be maintained over time in carefully selected and well-managed plantations, preferably well-replicated and located in several appropriate sites for safety. Selection of planting sites should be done using the best possible local and regional knowledge of soils and climates that is available. Needless to say, the planting sites need also to be maintained and protected, especially if they are to serve as resources for current or future breeding/transformation programs. It is also advisable to involve local officials and even the local populace in these plantings. As we'll see later, the ex situ collections have tremendous potential for educational and training sites.


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C.Collection Renewals Once ex situ collections are "in place" they come to represent a valuable genetic resource. It is important that their genetic integrity be maintained. While breeding programs may utilize these areas for studies involving inbreeding, wide-crossing, and even interspecific controlled hybridization, when it comes time to renew the trees in the collection, such recombinations must be avoided at all costs. So, the very best way to regenerate these collections is via vegetative propagation. This insures that the genetic diversity so painstakingly collected in the original accessions is not lost. If effective vegetative techniques are not currently available for the species to be conserved, a parallel research program to develop these would be prudent. D. Use in Forest Regeneration There is absolutely nothing wrong, and close to "everything right" about using these ex situ collections for genetics and breeding research and development. Again, vegetative propagation will be the most efficient and environmentally "safe" means for providing propagules adapted to reforestation of specific areas, but there are absolutely no reasons why the collections cannot serve as a "breeding population" or as a source of materials for genetic transformation studies. The only rules would be to maximize "non-destructive" use of the plantings and to preserve genetic identities .. E. Research, Education, and Training As long as the integrity of the collections is maintained, the possibilities here are almost endless. Probably number one on the list is research on how to vegetatively propagate the ex situ collection! Further ex situ work would benefit from expanded studies of geographic variation, genetic structure of the species, basic reproductive biology, mating patterns, general population genetics. Universities, agencies, industries could benefit from the use of these sites as demonstration/training areas. In the not-toodistant future, industries and public forest management agencies may find it useful perhaps even necessary - to maintain their own germ plasm collections, as do major agricultural seed companies at present. And of course, as we evolve a system of germplasm collections, we'll need highly-trained and competent people to assemble and manage these on scales that might surprise us 20 or so years from now! F. Need for Cooperation All of the above represents a sort of "brave new world"'approach to forestry, of course! It's obvious that it won't be possible to accomplish this without a great deal of cooperation among the various sectors of the forestry community. In Indonesia, the Ministry of Forestry and the Forest Service are the landowners and the policy-makers so they play a crucial role in making germ plasm available, and maintaining it in wellmanaged and protected sites. Forest industry has the manpower and, to some degree, the resources necessary to finance regional and national programs. And, of course, the universities and government have the technical expertise to provide the research necessary to maintain the programs. Finally, universities can play a major facilitating role in education, training, and transfer of technology by working closely with the management and utilization sectors to sustain viable, effective, and efficient genetic conservation programs.


14

G. Funding of Genetic Conservation Programs for Forest Tree Species Because the dangers to forest germ plasm are increasing at an alarming rate, these conservation programs must be sponsored by the government - the stewards of this invaluable resource - and by the industries which profit from the maintenance of productive populations of trees - their raw material. Research and development in this area should not have to compete with existing, limited funds - there should be a "new initiative" to conserve these genetic resources. And this new initiative is needed now!

VII. The IITO Project for Ex Situ Conservation of Shorea leprosula and Lophopetalum multinervium. Ledig (1992) outlined a "generic" genetic conservation program for forest trees which has met with wide acceptance during this decade. The genetic conservation program managed by Gadjah Mada University's faculty of Forestry and funded by IITO measures-up closely to all of the aspects Ledig mentioned. Let's take a quick, summary look at that program and where it's at in 1999 by organizing it along the lines of Ledig's idealized program. According to Ledig (1992) an effective program would include the following eight functions. We have inserted a summary of the IITO Project's goals, methodology, and progress to date under each of those eight major points. Following that summary, we wish to have an open discussion of the IITO Project. ELEMENTS OF AN EFFECTIVE GENETIC CONSERVATION PROGRAM FOR A FOREST TREE SPECIES (As proposed by Ledig, 1992) AND THE ITTO PROJECT'S APPROACH TO EACH OF THOSE EIGHT PROGRAM ELEMENTS An effective genetic conservation program for a forest tree species would include1) Collection of Forest germ plasm. The IITO Project is collecting propagation materials from both S. leprosula and L.. multinervium. This is Output 2.1 of the Project. At least five naturally distributed populations of S. leprosula and one of L. multiinervium will be collected each year for three years within each species range. These will be very comprehensive collections with at least 100 parent trees per collection site. 2) Storage of Germplasm in Seed Banks or as living Trees in Field Genebanks. The IITO Project will establish nurseries in cooperation with industry and government, and also in the establishment of 11 ex situ conservation plantations throughout the ranges of the two species and elsewhere. These plantation shall be prepared, managed, and protected by the cooperators.


15

3) Evaluation of Germplasm in test Sites to Measure Its Potential Range of Use The IITO plantation sites shall also serve as replicated provenance and progeny tests for further evaluation of each species. Again, cooperators shall assist in measurements and record keeping at each site. Faculty of GMU shall be responsible for coordinating data collection, analyses, and interpretation. 4) Inventory, Monitoring, and Management of Conservation Reserves. The IITO Project plans, with its cooperators, to maintain the original collection sites and trees, at least in the short run. The parent trees will be able to be evaluated via the provenance and half-sib progeny tests and may serve as important interim seed collection areas. In addition, maintaining parental selections will allow for replacement of materials if needed. 5) Regeneration of Seed or Clones for Second and Advanced Cycles and Multiplication for Planting. The IITO Project shall maintain cooperator nursery facilities for propagation studies as well as for pilot-scale production of clonal materials for replacements in ex situ collections. Cooperators may choose to use materials in tests via broad-scale reproduction of collection materials, via seed an/or vegetative techniques. 6) Supporting research and Training. This Workshop is only the first in a series that the IITO Project plans to conduct throughout the study period. Also, the nurseries and the ex situ plantation shall be available for demonstrations and for training of forestry students and government and industry personnel. Comprehensive research programs of Gadjah Mada University's faculty of Forestry will concentrate on population genetics, seed technology, vegetative propagation, breeding and genetic transformation using the materials in plantations and parent stands. This research shall be coordinated with Project cooperators and others. 7) Provision for Exchange of Germplasm Among Nations It is the intention of the IITO Project to make collection materials, research information, and educational and training opportunities generated by the Project available to ASEAN partners and others, as appropriate. 8) Development of Data Systems on Germplasm Materials. With the assistance of Project Consultants, the IITO Project intends to develop and maintain a series of comprehensive and unique databases and communication systems - possibly via Internet - which will not only assure the integrity of Project records but will also serve as educational materials for cooperators and colleagues in other countries.


16

A PARTIAL REFERENCE LIST FOR GENETIC CONSERVATION OF FOREST TREES

I.

References Used in the ITIO Project Workshop, Yogyakarta, 19-23 March, 1999

Brown, AH.D. and J.D. Briggs. 1991. Sampling strategies for genetic variation in ex situ collections of endangered plant species. Chapter 7, Pp. 99119 hGenetics and conservation of rare plants, D.A Falk and K.E. Holsinger, Eds. Oxford Univ. Press, New York. Burley, J. and I. Gauld. 1994. Measuring and monitoring forest biodiversity; a commentary. Chapter 3 Pp.19-46 In Measuring and monitoring biodiversity in tropical and temperate forests, T.J.B. Boyle and B. Boontawee, Eds. Proceedings of a IUFRO Symposium, Chiang Mai, Thailand, Aug. 27-Sept. 2, 1994. Frankel, O.H., AH.D. Brown and J.J. Burdon. 1995. The conservation of plant biodiversity (Chapters 6 and 7, Pp. 148-192). Cambridge University Press. ITIO Project Executing Team. 1999. Ex situ conservation of Shorea leprosula and Lophopetalum multinervium and their use for future breeding and biotechnology. Work Plan (Revised) August 1998 to July 2001. ITIO Project PD 16/96 Rev.4 (F) Faculty of Forestry, Gadjah Mada Univ., Yogyakarta, Indonesia. Ledig, F.T. 1992. A comprehensive strategy for the conservation of forest genetic resources. Pp. 325-344 In The development of natural resources and environmental preservation, Proceedings of an INRE Symposium, Korea University, Seoul, October 13-18, 1992. Namkoong, G. 1984. A control concept for gene conservation. Silvae Genetica 33:160-163. Rogers, D. L. 1996. Status of temperate forest tree genetic resources in North America. Pp. 13-27 In The status of temperate North American forest genetic resources. Report No. 16 .University of California Genetic Resources Conservation Program, Davis, CA

Training in Conservation and Management Genetic Resources Yogyakarta, 19-23 March 1999

17

11.

Additional references

Brown, AH.D. 1992. Human impact on plant gene pools and sampling for their conservation. Oikos 63: 109-118. Brown, AH.D. 1995. The core collection at the crossroads. Pp. 3-19 In Core collections of plant genetic resources, T. Hodgkin, et al., Eds. IPGRI. Brown, AH.D. and D.R. Marshall. A basic sampling strategy: theory and practice. Pp. 75-91 In Collecting plant genetic diversity: technical guidelines. Eds. L. Guarino, et al.. CAB International. Kitzm ill er, J.H. 1990. Managing genetic diversity in a tree improvement program. Forest Ecology and Management 35: 131-149.


18

In situ Conservation

Prof. Dr. soekotjo Gaojab Maoa Ul1iversit;'9

In situ Conservation Soekotjo Date: 19-3-1999 General information: balance between conservation and wise use of resources; reason for conservation and priority; the advantages; selection of target species; strategies for genetic conservation

Date: 22-3 ... 1999 Technical information: design, size, monitoring, management, practical model and future challenges and opportunity

Balance between conservation and wise use of resources

Genetic resource conservation

Commercial plantations: - productive - efficient - competitive - sustainable

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1. Reason for Conservation and Priority a. Tropical Rain Forest (1) The inventory of plant species is far from complete - Tropical rain forests cover 70/0 of the planet, yet it harbors 500/0 of the species .. Indonesia covers only 1.3 % of the land surface, but it includes .. 100/0 of the world's flowering plant species .. 120/0 of the world's mammal • species .. 160/0 of all reptile and amphibian species .. 17% of the world's bird . species - 250/0 or more of the world's fish species (2) Areas of high species endemism

(3) Many of tropical species are threatened, endangered and are being extinct ... deforestation .. over exploitation .. encroachment .. forest fire (4) Human dependence on flora and fauna ... bioactive compounds .. wood .. food ... chemical .. environment (5) Ethical .. Moral right of all species to exist (6) Esthetic ... Recreation (7) Improvement of our plantations (8) Ecosystem functioning stably

The Advantages of in situ Conservation

1. Tropical species is still poorly studied on the other sides it is a treasure house of potentially useful species 2. For evolutionary development of a . species 3. An individual species is never its own isolated ecosystem, it interacts with others in may ways 4. Ethical and political concern

Selection of Target Species 1. Reason - Limited technical and financial resources available 2. Factors that influence selection .. Potential economic use - Economic importance to human kind .. Increase yield; increased disease resistance; improvement of growth rate; wider adaptability to environmental conditions; improve nutritional quality .. Threat of genetic erosion - Based on scoring individual parameter such as taxon rarity, local ecological condition local agricultural condition and development. The higher the score, the greater is the risk of genetic . erosion

.. IUCN Red list Individual number and changes in number, location of individuals and analysis of the probability of extinction ... Biologically important species - Keystone species to provide the appropriate habitat for ensuring target species survival. Example: wild potato -- Pinus ponderosa, Jumiperus deppeana and Eupressus arizonica .. The target taxon is likely to have a mutualistic relationship with animal pollinators and seed dispersers - Culturally important species Local or national cultural significance Ba

- Genetic distinction

Convention on Biological Diversity Rio, June 1992 Strategies: Ex situ conservation .. Conservation of components of biological diversity outside their natural habitats .. Involves the sampling, transfer and storage of target species from target area

In situ conservation .. Conservation of ecosystems and natural habitats and the maintenance and recovery of variable populations of species in their natural environments .. Involves the designation, management and monitoring of target species where they are encountered

Strategy: in situ conservation Technique: genetic reserve Advantages: - Dynamic conservation in relation to environmental changes, pests and diseases ... Provide easy access for evolutionary and genetic studies - Appropriate method for recalcitrant' . species .. Allows easy conservation of a diverse range of wild relative .. Possibility of multiple target species reserve Disadvantages: .. Material not easily available for utilization .. Vulnerable to natural and man directed disasters (fire) .. Requires high level of active supervision and monitoring

Strategy: ex situ conservation Technique: Field gene bank Advantages: - Easy access for utilization - Easy access for characterization and evaluation .. Material can be evaluated while being conserved Disadvantages: .. Involves large areas of land - High maintenance cost once materials is conserved

In Situ Conservation Monday March 22, 1999 1. Planning and establishment of in situ Conservation * ecogeographic and site evaluation - effective size to ensure the continued presence of viable population of target species with minimal genetic erosion - number of small conservation area vs single large conservation area * socio economic and socio factors costs and benefit of in situ conservation * Conservation design - optimal conservation design - conservation size & population

2. Operational plan 2.1 objective to ensure that the target species remain sufficiently abundant at the site 2.2 .. Describe the physical and biological environment - Describe the management practices required to achieve the objectives - Monitor community dynamics within the conservation area to assess management effectiveness - Organize human and financial resources - Ensure consistency between the reserve and national and regional conservation plan - Facilitate communication and collaboration among genetic reserve

3. Monitoring * Status, change and trends of conservation * Develop - Assessment question - Indicator of performance * Indicator - measurable + hard numbers + percentages - simple quantification - Index-period stability - Regional responsiveness * Monitoring regime - where to sample - How to sample - Frequency - Data accumulation

4. Institutional Development - Forestry department and institution - Forest company University - National herbaria and botanical garden - National gene bank

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Scientific and technical development - New research methods - New conservation technique - Practical application

6. National Conservation Plant

Forest Tree Breeding ~

~--------------------------------~

prof. Dr. Oemi Han]']n Suseno Gaojab Maoa Un]versit;9

FOREST TREE BREEDING by Oemi Hani'in Suseno

What is tree improvement? Tree improvement is currently included as an essential part of forest management operations. No longer is tree improvement considered an impractical academic exercise that require special treatment and financing but contributes little to the income from forest land. Most scientists differentiate between Forest tree breeding, forest genetics, forest tree improvement, although some authors, such as TODA equate those three terms. Forest genetics Activities that are restricted to genetic studies of forest trees. The objective is to determine the genetic relationship among trees and species. An example of a forest genetic activity is the attempt to determine crossability pattern among species within a genus.The crooses are made to determine relationship . They have no special breeding objective. Forest tree breeding Activities that geared to solve some specific problem or to produce a specially desired product. An example of such directed breeding is the development of pest-resistant strains of trees or breeding trees that possess specially desired wood. Forest tree improvement Forest tree improvement is applied when control of parentage is combined with other forest management activities such as site preparation or fertilization, to improve the overall yields and quality of product from forestland. Forest tree improvement merupakan kegiatan yang bertujuan untuk meningkatkan produktivitas baik kuantitas maupun kualitas dengan pengendalian asal-usul pohon diperpadukan dengan kegiatan pengelolaan hutan. Di Indonesia hanya dikenal satu sebutan yaitu pemuliaan pohon hutan. Dan dulu juga disebut seleksi. Secara sederhana dapat dikatakan bahwa "forest tree improvement" sebagai alat tambahan silvikultur yang menyangkut susunan genetik pada pohon-pohon di dalam hutan. Di kalangan kehutanan, pemuliaan pohon hutan diartikan sebagai aplikasi ilmu genetik hutan pada praktek silvikulur dalam upaya untuk meningkatkan produk yang lebih tinggi nilainya.


1

Peran pemuliaan pohon hutan contoh: 1. USA Populus deltoides daur dapat diperpendek dari 8 jadi 4 tahun 2. Australia dan New Zealand pad a Pinus radiata (tinggi, diameter batang, volume pohon, percabangan ) 3. Brazil - ekaliptus riap tahunan meningkat 50 m3/ha 4. Congo - ekaliptus riap tahunan bisa berkisar antara 30-50 m3/ha .. 5. Brazil & Congo menghasilkan bastar ekaliptus dapat meningkatkan dari 36 menjadi 70-100 m3/ha. 6. PT Arara abadi menghasilkan HTI Acacia mangium dengan riap 20-30 m3/ha/tahun pad a daur 7-8 tahun serta riap tahunan 40 - 60 m31 ha dengan daur 5-6 tahun. All beginning for improvement programs rely on and consist of the following: 1. A determination of the species or geographic sources within a species that should be used in a given area 2. A determination of the amount, kind and causes of variability within the species. 3. A packaging of the desired qualities into improved individual, such as to develop trees with combinations of desired characteristics. 4. Mass producing improved individuals for reforestation purposes. 5. Developing and maintaining a genetic base population broad enough for needs in advance generation.

Pemuliaan Pohon dan Hukum klebs

genetik

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Proses fisiologis Etu m buh IP r oduktivitas

lingkungan / - menyangkut site - menyangkut pengelolaan

Keterangan : Faktor genetik dan faktor lingkungan menentukan pertumbuhan pohon lewat proses fisiologis dan pertumbuhan pohon menentukan produktivitas tegakan. Produktivitas tinggi dicapai apabila bibit yang ditanam genetik baik dengan lingkungan tumbuh pohon yang baik. Bibit genetik baik adalah hasil pekerjaan pemuliaan pohon (breeding) Bagaimanakah ciri-ciri hutan yang produktif? homogenitas tinggi (variabilitas rendah) --- genotype terbatas pertumbuhan cepat bentuk batang lurus, silindris percabangan baik (?) tajuk ram ping Karena genotype terbatas, harus ada cadangan yang disimpan . Karena itu , breeding tidak dapat dipisahkan dari konservasi. Training in Conservation and Management Genetic Resources Yogyakarta, 19-23 March 1999

2

Kedudukan Pemuliaan Pohon Hutan? Konservasi in-situ 0 Konservasi ex-situ 0 Breeding & Bioteknologi

EH

U TAN KOMERSIAL Konservasi ex-situ jembatan antara konservasi in-situ dan breeding & bioteknologi

Model Pemuliaan Pohon Pemuliaan pohon seringkali diilustrasikan dalam bentuk model yang menggambarkan interaksi, umpan balik, dinamika dari setiap langkah dari program pemuliaan. Contoh : Rangkaian Pemuliaan Pohon Eksplorasi

~

Evaluasi

~

Pemuliaan

~

perbiyakan Distribusi Konservasi gen •

Eksplorasi Eksplorasi dilakukan untuk kepentingan spesies yang akan dimuliakan. Sasarannya adalah lokasi sebaran alam dimana species itu tumbuh. Apabila diketemukan pohonl spesies yang diiginkan, tempat tumbuh didiskripsi; fenotipe dipelajari; koleksi benih dilakukan. Pekerjaan ini dilakukan meliputi seluruh sebaran alami untuk kemudian dipetakan

•

Evaluasi Benih dari hasil eksplorasi kemudian dipergunakan untuk evaluasi, misalnya dalam bentuk uji provenans untuk evaluasi antar provenans dan uji keturunan atau uji progeni untuk evaluasi variasi antar keturunan dalam suatu provenans. Bisa juga dalam bentuk uji kombinasi antar provenans dan keturunan

•

Pemuliaan Langkah pemuliaan umumnya terdiri dari seleksi pohon plus di Areal Produksi Benih, seleksi pohon plus untuk pembuatan kebun benih dan uji keturunan untuk evaluasi pohon induk.


3

•

Perbanyakan Penyilangan (terbuka maupun terkendali) dan perbanyakan perbanyakan massal dari pohon-pohon terseleksi dalam kebun benih (ada kebun benih semai dan kebun benih klon) atau kebun pangkas. APB merupakan langkah sementara sebelum kebun benih ada atau menghasilkan benih.

•

Distribusi Distribusi benih atau bahan vegetatif yang bermutu baik ke persemaian dan tapak penanaman, pemapanan dan pemeliharaan yang baik merupakan langkah akhir untuk menjamin suplai material tanaman yang bermutu.

•

Konservasi gen Konservasi gen merupakan aktivitas yang inheren dalam pemuliaan pohon untuk menjamin tersedianya material genetik yan diinginkan dalam program pemuliaan di masa yang akan datang yang sulit diramalkan. Konservasi yang dilakukan mencakup konservasi in-situ dan konservasi ex-situ.

•

Siklus Pemuliaan Variasi genetik

D

Seleksi Rekombinasi

penUjian Benih Unggul

D

Hutan Produksi Siklus pemuliaan pohon dimulai dengan seleksi, kemudian dilanjutkan dengan pengujian material yang telah diseleksi. Dalam hat ini adanya variasi genetik dalam populasi sangat penting. Material genetik yang telah dimuliakan (unggul) diperbanyak untuk pembuatan hutan produksi. Dinamika dari pemuliaan pohon digambarkan oleh keperluan untuk membuat rekombinasi guna mendapatkan material baru untuk seleksi dan perbaikan yang berkelanjutan. •

Komponen Program Pemuliaan Pohon. Komponen umum dari suatu program pemuliaan dan bagaimana material genetik mengalami peningkatan dari generasi kegenerasi melalui proses seleksi dan penyilangan dapat digambarkan sebagai berikut:

Training in Conservation and Management Genetic Resources Yogyakarta. 19-23 March 1999

4

l. .· · _· · ·

POPULASI DASAR Seleksi

POPULASI PEMULlAAN GENERASII

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..............................~

~

Seleksi intensif

POP.PERBANYAKAN (K. BENIH GEN. I) ....................~ POP.PRODUKSI (T.KOMERSIAL)

Seleksi dan Penyilangan

~

POPULASI PEMULlAAN GENERASIII

~ Populasi Dasar: berupa hutan alam atau tanaman dimana seleksi (misalnya pemilihan pohon plus) dilakukan. Untuk suatu program pemuliaan pohon, populasi dasar hendaknya berbasis genetik luas. Populasi Pemuliaan: Pohon-pohon terpilih dan keturunannya dalam suatu seri uji keturunan dan bank klon dimana siklus seleksi dan penyilangan akan dilakukan berulangulang dari generasi ke generasi. Pada generasi lanjut popuasi dasar ini berupa tanaman uji genetik. Populasi Perbanyakan: Pohon-pohon terpilih secara intensif (umumnya kurang dari 100) dalam kebun benih atau area perbanyakan dimana kombinasi gen yang terpilih dalam populasi pemuliaan diproduksi secara massal sebagai bibit unggul. Populasi Produksi: merupakan hutan tanaman untuk produksi yang berasal dari populasi produksi. Populasi pemuliaan dan populasi perbanyakan biasanya dibangun secara terpisah. Tetapi kadang-kadang fungsi pemuliaan dan perbanyakan ditangani oleh populasi yang sama, misalnya kebun benih uji keturunan tusam. Metode Pemuliaan Pohon Metode pemuliaan yang umumnya diterapkan pada tumbuh-tumbuhan ialah pemuliaan mutasi, "inbreeding", hibridisasi, pemuliaan silang balik dan seleksi. Seleksi merupakan metode yang paling umum diterapkan di pohon-pohon hutan. Metode ini sesuai bagi kebanyakan jenis-jenis penyerbukan silang dan yang variabilitasnya besar seperti jenisjenis yang masih liar. Seleksi memberi harapan untuk memperoleh hasil-hasil genetik


5

yang agak besar pada generasi pertama serta untuk memenuhi kebutuhan benih unggul dalam jumlah yang besar. Seleksi dapat dilakukan dengan: 1. Seleksi massa a. Seleksi massa pad a seed production area b. Seleksi massa pada program pemuliaan. 2. Seleksi famili a. Seleksi famili-half sib (saudara tiri) - Hasil penyerbukan bebas. - Hasil penyerbukan terkendali. b. Seleksi famili full-sib (saudara kandung) Penjelasan : Seleksi massa atau seleksi individu mendasarkan pemilihan individu pohon hanya pad a fenotipenya, tanpa memperhatikan informasi tentang penampilan induk, keturunan atau kerabatnya. Seleksi massa paling berguna untuk sifat2 yang nilai heritabilitasnya tinggi, dimana fenotipe mencerminkan sifat genetiknya. Seleksi famili dilakukan dengan memilih famili dengan dasar nilai re rata fenotipenya. Seleksi individu di dalam famili tidak dilakukan dan nilai individu digunakan untuk menghitung rerata. Seleksi tipe ini berguna bagi sifat-sifat dengan heritabilitas rendah .. Pertanaman Uji Genetik ("TEST PLANTATION") Salah satu segi penting dalam teknik pemuliaan pohon adalah pengujian tanaman baik di pesemaian maupun di lapangan. Uji keturunan adalah salahsatu contoh pertanaman uji gentik. Di dalam pertanaman uji genetik, seringkali digunakan istilah-istilah khusus, antara lain: Seedlot

Plot Replikasi

Slok

Pertanaman ("Plantation") Eksperimen

sekelompok pohon-pohon yang berkerabat (misalnya suatu klon atau suatu fa miIi) diberi nomor dan diidentifikasi sebagai suatu unit eksperimen sekelompok pohon yang jumlahnya 1 - 100 dari suatu seed lot yang ditanam berdampingan satu sama lain di pesemaian atau di lapangan praktek penanaman plot-plot yang berbeda dari seedlot yang sama di lokasi yang berbeda dalam suatu pertanaman, atau pertanaman yang berbeda atau tahun penanaman yang berbeda sebagian dari pertanaman yang berisi satu plot dari masing-masing seedlotlklon dan dalam blok terdapat beberapa seedlot. Slok yang lengkap ialah apabila blok berisi seluruh seed lot yang ada dalam eksperimen yang untuk masing-masing seedlot ditanam satu plot. Slok dapat berbentuk teratur dan dapat juga tidak teratur sekelompok blok yang ditanam berdekatan satu sama lain yang seringkali berdampingan terdiri dari uji pesemaian dan satu atau lebihpertanaman dimaksudkan untuk pengujian sekelompok seed lot tertentu. Eksperimen yang modern menyangkut beberapa pertanaman yang terpencar letaknya, yang berbeda negara


6

Famili

Genotip

Fenotipe

keturunan dari satu pohon dengan penyerbukan terbuka atau keturunan dari sepasang pohon hasil penyerbukan terkendali (pada penyerbukan terbuka tidak dikenal tetua pejantan sedang pad a penyerbukan terkendali kedua tetua dikenal) seluruh susunan genetik dari suatu organisme yang nampak atau tersembunyi juga dapat dikatakan untuk suatu klompok organisme yang nampak atau tersembunyi sifat - sifat yang nampak hasil interaksi suatu genotipe dan lingkungan


7

General Concepts of Tree improvement Research and development For long term breeding

Production line seed for operational planting Wild stands and plantation

Wild stands and plantation

I

Pick the best (300+ trees~

Pick the very best (30 to 40 trees)

Special test Such as wide Cross test

•

First-generation seed orchard

~ Selection

Progeny test

Rogued firstGeneration orchard

Intensil selection

Hybridization

j Breeding Clone Bank

Cross among clone bank trees - progeny testselect the best trees from the best families

Improved first-generation seed orchard

Progeny test

~

Second - generation seed orchard

Specialty orchard

Continue crossing and Preserving genotypes Advanced generation orchard


8

Dalam program pemuliaan harus ada dua aspek yang diperhatikan, yaitu aspek yang menyangkut perolehan langsung produk yang diinginkan serta aspek penelitian dan pengembangan jangka panjang seperti nampak jelas pada gambar diatas . STRATEGI PEMULlAAN Tujuan umum dari suatu pemuliaan pohon adalah : 1. Memuliakan secara progresif populasi dasar (base population) dan populasi pemuliaan (breeding population) 2. Membiakkan material yang dimuliakan untuk membua populasi perbanyakan (propagation population) yang unggul. 3. Menjaga variabilitas dan ukuran poplasi pada populasi dasar dan populasi pemuliaan; dan 4. Mencapai semuanya ini secara ekonomis. Perolehan yang terbesar dalam jangka panjang dicapai melalui seleksi yang efektif, populasi yang besar dan variabel , dengan pengendalian kekerabatan dalam generasi mendatang. Strategi pemuliaan Menentukan sistem yang paling efisien dalam mengelola berbagai bidang dalam suatu program pemuliaan dalam kendala waktu dan sumberdaya yang tersedia merupakan masalah yang kompleks dan diperlukan penyelesaian yang memperhatikan berbagai kepentingan. Skema yang dibuat untuk mencapai tujuan yang diinginkan dalam program dengan memperhatikan semua kondisi yang ada dinamakan strategi pemuliaan atau "breeding strategy" Metode pemuliaan Proses untuk mengimplementasikan strategi pemuliaan disebut metode pemuliaan atau "breeding method". Teknik pemuliaan Prosedur biologi seperti pembiakan vegetatif, penyerbukan terkendali, penanaman dsb merupakan teknil pemuliaan Unsur strategi pemuliaan: 1. Tujuan 2. Populasi dasar, populasi pemuliaan 3. Seleksi Dalam memilih karakteristik yang akan dimuliakan sebaiknya memilih sifat-sifat yang tidak dapat dimanipulasi dengan teknik silvikultur atau teknologi lain dengan biaya murah. Pertimbangan untuk memuliakan sifat dengan ciri-ciri : Nilai ekonomi tinggi sifat yang di masa yang akan datang tetap penting. variabilitas dan heritabilitas tinggi, memberikan potensi perbaikan genetik yang tinggi Beberapa sifat sesungguhnya memenuhi persyaratan unuk semua keperluan dan sifat ini tetap diperlukan di masa mendatang seperti, kecepatan tumbuh, batang lurus, percabangan


9

4. Uji genetik - tingkatkan gain melalui pengendalian lingkungan, sehingga dapat meningkatkan akurasi seleksi 5. Managemen kekerabatan - hindarkan depresi inbreeding pad a populasi produksi dan hindarkan kehilangan aiel yang mungkin berguna di masa yang akan datang Kunci Program pemuliaan : 1. Perencanaan 2. Populasi dasar berbasis luas, kalau perlu diinfus 3. Seleksi yang akurat 4. Penelitian terapan dan pengembangan. Kendala umum: mendapatkan material genetik yang baik, biologi reproduktif, pilihan kriteria seleksi yang tepat, uji keturunan dan sumberdaya 5. Managemen interaksi G - L 6. Seleksi tenaga, pelatihan, motivasi dan menjaga tenaga yang ada agar betah. Rencana pemuliaan yang baik memiliki unsur: 1. Tujuan yang spesifik 2. Mempertimbangkan semua sumberdaya 3. Mengembangkan pilihan dan memilih yang terbaik 4. Mendapatkan material genetik dan informasi biologi 5. Penaksiran parameter 6. Kriteria seleksi 7. Pengendalian inbreeding dan menjaga variabilitas 8. Mengembangkan teknologi 9. Menentukan tanggung jawab 10. Menentukan jadwal kegiatan 11. Menjaga fleksibilitas

Training in Conservation and Management Genetic Resources Yogyakarta. 19-23 March 1999

10

An alis is Iso zim dan Pem anf aat ann ya di Bid ang Ke hut ana n

Dr. Ir. Mobal11l11tlo Na'iem Gaojab Maoa Universit;g

ANALlSIS ISOZIM DAN PEMANFAATANYA DI BIDANG KEHUTANAN *)

Oleh: Dr. Ir. Mohammad Na'iem**)

PEtlDAHULUAN

Prinsip pengembangan

prinsip terhadap

genetik

tanaman,

pada

dasarnya

hukum Mendel dan genetika

adalah

populasi.

Di

bidang Pertanian dan Holtikultura pengembangan terhadap

prinsip

ini,

tentang

seperti estimasi besarnya variasi genetik,

aliran dalam gen,

studi

gen (gene flow), efektifitas penyilangan, efesiensi kebun benih, identifikasi klon dan telah

kultivar,

lama dilakukan. Sehingga hasilnya

banyak

dirasakan

adalah

telah

oleh masyarakat. Hasil

banyak

ditemukan

saat

tersebut

galur-galur

murni

gen

konservasi ini

telah

diantaranya (terutama

tanaman·semusim), yang arahnYB menuju kepada pemanfaatan variasi genetik yang sempit dan terbatas. Didalam

*)

**)

melakukan

studi

genetika

tanaman

baik di bidang

Disarnpaikan pad a Training in Conservation and Management of Genetic Resources, tanggal18-23 Maret 1999, Radisson Hotel Staf Pengajar Fakultas kehutanan UGM, Yogyakarta


Pertanian maupun Holtikultura, diperlukan adanya penanda genetik (marker

gene).

sifat-sifat individu penanda

Penanda

genetik ini berupa

tersebut

biasanya

suatu

terekspresi

sifat

dalam

fenotipe

yang bersangkutan. Penanda genetik ini dikenal

dengan

morfologis (morphological marker). Tetapi karena

sifat

morfologis tersebut sangat terpengaruh oleh kondisi maka hasil yang diperoleh ada kalanya

(biochemical marker) yang isoenzim

lingkungan,

kurang cermat.

Sejak dekade terakhir diperkenalkan

Analisis

yang

adanya penanda biokimia

dikenal dengan isoenzyme atau isozim.

~

(isozim) telah digunakan secara

luas

sejak

beberapa dekade yang lalu, untuk melakukan investigasi

genetik

bagi

tanaman

sejumlah besar organisme, mulai dari lalat

buah,

liar, tanaman budidaya hingga manusia. Kendala biasanya

yang

terbentur

dihadapi untuk pada

bahan

mengaplikasikan kimia

yang

teknik

relatif

ini

mahal,

peralatan loratorium yang khusus dan juga tersedianya sumberdaya manusia memiliki

yang

memadai.

Namun

demikian

mengingat

manfaat yang cukup banyak untuk keperluan

teknik

ini

investigasi

genetik, maka ada baiknya teknik ini diperkenalkan.

BEBERAPA PENGERTIAN

a). Apakah Enzim ? Protein peranan

adalah

penting

molekul-molekul

dalam

organik

berbagai proses


yang

biologi.

memainkan Peranan 2

ini

pengangkutan

meliputi menyediakan reaksi

dukungan

kimia

dan

penyipanan

molekul

mekanis, dan mengendalikan

dalam organ tubuh. Protein yang

lainnya,

hampir

semua

memiliki

fungsi

dalam pengendalian seluruh reaksi kimia ini disebut Enzim. Enzim ini

juga

mempunyai sifat mampu

meningkatkan

kecepatan

suatu

reaksi kimia, tanpa ikut tereaksi. Protein

disusun

oleh sub-unit sub-unit yang

disebut

asam

amino. Asam amino adalah molekul organik yang memiliki satu basa (NH2) grup dan satu asam karboksil (COOH) grup.

amino asam

amino

grup

grup

memiliki grup grup ion yang lain

sebagai

tambahan

amino dan karboksil yang membentuk

ikatan

peptide.

grup

Apabila

Beberapa

grup tersebut terletak pada permukaan

luar

dari

molekul protein, ion ion ini akan memiliki suatu jaringan aliran listrik (net electric charge) dan karenanya akan bergerak

dalam

suatu medan listrik. Dasar

pewarisan genetik terletak didalam molekul

DNA

yang

ada dalam Chromosom yang terdapat pada inti sel semua sel hidup. Molekul

terikat

oleh

ikatan hidrogen antara molekul molekul basa organik. Ada 4

basa

organik karena

DNA

terdiri dari 2 rantai molekul

(Adenine, ukuran

ini

hidrogen dalam 2 cara, yaitu Adenine hanya dapat terikat

dengan

basa

dapat

Basa

ikatan

dan

strukturnya hanya

Thymine).

membentuk

Thymine

dan

Cytosine, Guanine dan

gula

Guanine dengan Cytosine. Rangkaian

tertentu

dari

genetik

yang

ini dalam molekul DNA ternyata membawa kode

mengandung informasi untuk diteruskan pada generasi

berikutnya.

Rangkaian basa yang membentuk bagian molekul DNA tersebut, Training in Conservation and Management of Genetic Resources Yogyakarta. 19-23 March 1999

juga 3

mengendalikan spesifik asam amino. Oleh karena itu suatu rangkaian

molekul

DNA

juga

mengendalikan

segmen protein

molekul

mengingat protein merupakan asam asam amino yang secara

bersama

membentuk ikatan. Oleh

karena protein secara langsung merupakan

translasi

produk

DNA, maka hasil analisis protein mestinya akan

dari mirip

dengan hasil yang diperoleh dari analisis DNA itu sendiri.

b). Mengapa electroforesis ? Electroforesis yang

telah

listrik. pada

adalah

teraliri

suatu proses dimana

listrik

bergerak

Kecepatan bergerak molekul enzim

besarnya

muatan

listrik. Pemisahan

molekul

enzyme

suatu

medan

melalui

tersebut molekul

tergantung enzim

oleh

proses elektroforesis terjadi karena 2 proses, yaitu - Besar kecilnya muatan listrik - Besar kecilnya ukuran dan bentuk dari partikel

c). Mengapa enzim electroforesis ? Ada

beribu enzim yang berbeda, yang masing masing

fungsi

yang

tubuh.

Beberapa

identik, yang

spesifik pada reaksi kimia

tertentu

dari enzim ini ada yang memiliki

berbedanya hanya pada lokasi dan

memiliki

dalam fungsi

aktifitasnya.

memiliki bentuk yang berbeda tetapi memiliki

organ yang Enzim

fungsi

yang

identik disebut Isoenzim. Variasi mungkin

juga

genetik dapat

muncul karena menyebabkan

terjadinya terjadinya


mutasi.

Mutasi

perobahan

dalam 4

rangkaian

molekul

DNA.

Berhubung rangkaian

molekul

DNA

mengendalikan asam amino tertentu, maka perobahan ini juga menyebabkan

perobahan

pada rangkaian asam

amino

pada

ini akan

rantai

polypeptide.

Different form of gene (-allele)

Segment of chromosome (- gene)

codes for specific enzyme

Chain of mfno acids linked by ,tide bonds

IVM. •••

) AL •••

~

~

~

LEU

~

•••

Gambar :1. Diagram

PHE

Enzyme now

1- G~~ I

/'.

A1lozymes

representatif

Amino acid substitution

negatively charged

LEU.

••

suatu

enzim

dan asal adanya

variasi genetik yang menyebabkan munculnya allozim. Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

5

Perbedaan

bentuk

dari suatu gen yang

memproduksi

diistilahkan

protein

dimana

strukturnya sedikit berobah

dengan

Adapun

enzim yang secara struktur berbeda yang dihasilkan

aIel yang berbeda adalah tipe khusus daru isoenzim yang

AIel. oleh

disebut

Alozim. Alozim disebabkan molekul

ini

memiliki

oleh

adanya

muatan

listrik

substansi asam

yang

yang

berbeda

amino

pada

enzim. Alozim ini akan bergerak dengan

permukaan

kecepatan

yang

berbeda pada medan listrik. Oleh karena itu elektroforesis

pada

enzim

dari

dapat

mutasi

menunjukkan perbedaan genetik

gen. Dengan cara ini

yang

berasal

perbedaan genetik antara

individu

atau populasi dapat diteliti dengan mudah dan cepat.

PENGGUNAAN PENANDA GENETIK (GENETIC MARKER)

Langkah populasi genetik

awal

sebelum

melakukan

analisis

diperlukan adanya penanda genetik. Dua yang

dapat dipergunakan yaitu

penanda

genetik macam

suatu penanda

morfologi

dan

penanda biokimia.


6

1. Penanda morfologi . morfologi

Penanda biasanya

terekspresi

sifat-sifat

menggunakan

dalam fenotipe

suatu

yang

jenis

untuk

penanda genetik. Misalnya bentuk. letak, ukuran dan

warna

dari bagian vegetatif maupun generatif tanaman. Di samping 1tu produk sampingan seperti flavopoid dan terpenoid . juga dapat digunakan sebagai marker untuk studi genetik. sulit

untuk mencari hubungan antara genotip

dengan

marker morfologi. Hal ini

morfologi faktor

pada

resesif

disebabkan

umumnya dikontrol oleh

lingkungan pada

yang

kompleks.

individu

dan

D1

yang

Namun fenotip

sifat-sifat

gen

majemuk

samping

itu

dan gen tidak

heterozigot

diekspresikan. Penentuan

pola

pewarisan

penyilangan

memerlukan

terkendali dan analisis individu hasil uji keturunan

yang

biasanya

yang

memerlukan waktu yang cukup lama. biasanya

diperoleh

dipengaruhi

Fenotip

sehingga

dominasi,

menyu11tkan interpretasi genetik.

2. Penanda biokimia Penanda biokimia menggunakan hasil analisis pada

bagian

tanaman

melalui

ana11sis

pertama

kali

untuk

isozim.

penanda Istilah

nllsalnya

genet1k, 1soz1m

oleh Markert dan Holler

tahun

biok1mia

d1perkenalkan 1959

Na"iem dan Soeseno (1993). Isozim (isozyme) atau

1soenzlm

(isoenzyme) disebutkan sebagai variasi yang terdapat Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

dalam

pada 7

enzim

yang

sama,

yang

memiliki

kemiripan

fungsi

dan

terdapat pad a individu yang sama. Hartl (1980) menyebutkan bahwa

apabila variasi enzim tersebut dikontrol oleh

alel

atau gen yang berbeda pada suatu lokus dan dapat dideteksi dengan elektroforesis maka disebut alozim (allozyme). Menurut (1993),

Bailey

jika

menyebabkan molekul

(1983)

menghasilkan

Na·iem

dan

komposisi asam amino di atas perbedaan

dan

dalam

pula

dalam

berbeda

muatan

ion,

konfigurasinya. Perbedaan isozim kecepatan

gerak

yang

Soeseno

tidak

akan ukuran

juga sama

akan bila

dikondislkan dalam medan listrik dan medium gel yang

semi

porus, sehingga setiap enzim yang berbeda akan menyebabkan pola berkas (banding pattern) yang berbeda pula. Na·iem

dan

Soeseno

(1993)

menguraikan

keunggulan

penggunaan isozim sebagai penanda genetik sebagai berikut: 1. Di

alam,

aIel

kodominan,

pada kebanyakan

sehingga

mempermudah

lokus

isozim

pemisahan

adalah antara

heterozlgot dan homozlgot. 2.· Isozim tidak terpengaruh oleh lingkungan.

3. Isozim

tidak terpengaruh oleh resesifitas suatu

sifat

seperti yang terjadi pada fenotip tanaman. 4. Dengan analisis isozim dapat dilakukan pengujian banyak sampel,

sehingga

dapat diteliti

banyak

lokus

dalam

waktu yang relatif singkat. 5. Isozim dapat diidentifikasi dengan materi dari berbagai bagian jaringan tanaman. Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

8

PROSEDUR KERJA D1 LABORATOR1UK

Terdapat sejumlah prosedur kerja untuk analisis isozim, yang pada umumnya tergantung pada macam gel yang dipergunakan (Starch gel atau polyacrylamide gel) dan bahan penelitian yang digunakan (daun,

akar, kambium, tepungsari, jaringan megagametofit

Namun

secara

garis besar urutan kerja

sama.

Kalaupun

yang

berbeda biasanya hanya pada

dll).

dilakukan komposisi

hampir ekstrak

bufer, terutama untuk bahan penelitian yang berasal dari vegetatif banyak

tanaman. Bahan vegetatif tanaman biasanya

tanin

dan

gambaran

pola

ekstraksi

bufer

memberikan

phenol, sehingga

bercak

(banding

pattern)

perlu ditambahkan

gambaran

yang

lebih

untuk

dapat

jelas,

pada

mengandung

menghasilkan jelas,

pada

tertentu.

Untuk

yang

kemikalia

bagian

kesempatan

ini

diberikan contoh prosedur kerja untuk penelitian analisis isozim pada Pinus merkusii, dengan menggunakan 8 sistim enzim. Delapan

sistim

enzim

yang

dimaksud

adalah

Phosphate;

E.C.

3.13.2), D1A (Diaphorase; E.C.

(Esterase;

E.C.

3.1.1.1), GDH (Glutamate

1.4.1.2), (Glutamate (Leucine

G2DH

Oxaloacetate Amino

(Acid

2.6.4.3),

Dehydrogenase:

DehydrogA~ase;E.C.

(Glycerate

ACP

E.C.

1.1.1.29),

E.C.2.6.1.1.),

Transaminase;

Peptidase; E.C. 3.4.11.1)

dan

SHD

EST

GOT LAP

(Shikimate

Dehydrogenase ;E.C.l.l.l.25). Secara

terperinci

prosedur

kerja

dapat

dipelajari

pada

uraian berikut : Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

9

1. Persiapan bahan (materi yang akan diuJi). Bahan darl

yang dlgunakan adalah

benih

jaringan

P. meu'kusii dari kebun

megagametofit

benih

Pinus

Jember.

Untuk

masing-masing nomor pohon diambil 20 benih.

Jumlah

sampel

1nl dlanggap cukup valid dalam menentukan

genotlp

suatu 100

indivldu, karena kebenaran genotip

akan

% jika jumlah benih yang digunakan adalah

sesua!

dengan

rumus yang dlkemukakan

ulah

mendekatl benlh,

8

Chellak

dan

Pltel (1984) yaitu :

=

Persentase kebenaran genotip 1_(~)n-1, dengan n jumlah megagametoflt

=

Gambar 3. Perendaman benih dalam Hidrogen Peroksida (H202)

Benih Hidl'ogen

diperlakukan Pel'oksida

dengan perendalllan

(H 2 0 2 )

selama

24

jam

pada

(Gambar

Tujuannya

adalah untlllt skarifilmsi dan ::t.erillsasl

sehingga

dapat.

membant.u

menghi langltan ..i amul'. Agar masing-masing

nomor

mehmakkan t

b.llit

larutan 3),.

benlh

benih

dan

idak t.er'c'ampul· maka benih untuk

pohon


inciuk

djpisahkan

dengan

10

dalam

membungkus respirasi

maka

aerator.

kain

udara

Selanjutnya

kassa.

Untuk

dipompa ke benih

dalam

kertas

dalam cawan petri dan diberi label

nomor

masing-masing.

dijaga.

Pengecambahan

diletakkan

larutan

dikecambahkan

Kelembaban

proses

membantu

dengan

dengan

media

sesuai

media

dengan

kertas

dilakukan selama ±

2

dalam almari es dengan tujuan

selalu

minggu

agar

dan

kecepatan

berkecambah dapat diperlambat.

2. Ekstraksi sampel. dan

Kulit

embrio

digunakan

hanya

berupa

bersifat

haploid

(n).

dihancurkan

sehingga

yang

megagametofit

yang

dibuang,

jaringan Selanjutnya

megagametofit

dengan menggunakan mOl'tal' dan

larutan

diberi

benih

extraot

buffel'

0,5

pestle

ml.

Adapun

ini dengan cara

pembuatan e4y:traot buffer dapat dilihat dalam tabel berikut Tabel 5. Extraot buffer yang dipergunakan dalam penelitian Nama Bahan

Jumlah

Penggunaan

1.EXT

1 M Tris - Hel, pH 7,5 Air suling

8,45 g 50 ml

2 g

2.EXT

G1Y081'ol Air suling

75,6 40

g ml

6,67 g

3.EXT

Tween 80 Air suling

7,85 g 42,5 ml

1 g

4.EXT :

Di tiotlu'ei tol Air suling

mg ml

1 g

Pol.vvin.v l-polYPY1'olldone Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

330 10,5 1,2

g

1,2 g 11

Setelah

telah

centl'lfuge ependorf

dan homogen

dingin

maka

dimasukkan

disiapkan

Centrlfuge

ependOl'f.

tabung

15.000

hancur

±

(suhu

0

clan

°C),

apabila

maka

dapat dimasukkan untuk diputar dengan

rpm

selama 20 menit.

selesai

maka

larutan

bagian.

Bagian

Setelah

sampel

akan

proses terpisah

atas berwarna bening

berupa

bahan

padat

tabung

kecepatan centrlfuge

menjadi

dibuang.

(pellet)

2

yang

(supel'natallt)

akan digunakan dalam proses elektroforesis, sedang bawah

dalam

bagian Proses

ekstraksi sampel dapat dilihat dalam Gambar 4 berikut :

Gambar 4. Proses ekstraksi sampel mAgagametofit

3. Persiapan pembuatan gel polyacrylamide. Gel running

polyacrylamide gel

konsentrasi atas

running

yang

terdiri

terletak

dari dlbagian

7,5 % dan spacer gel yang gel

2

dengan kepekatan

bagian, bawah

te~letak

3,75

%.

yaitu dengan

dibagian Bahan

dan

komposisi yang digunakan untuk pembuatan gel adalah : Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

12

Tabel 6. Larutan utama untuk menyiapkan gel polyacrylamide

A.

TriSJ11a base

91,5

120 ml TEMED 0,575 ml Dilarutkan dalam 500 ml air suling 150

Acrylamlde

BIS

D.

g

(pH 8,9) IN HCl

B.

4

ml TEHED 1,15 ml Dilarutkan dalam 500 ml air 5uling

E.

g

14,95 g

Trisma base

(pH 6,7) IN Hel

g

Dilarutkan dalam 500 ml air sul1ng

C.

Bahan

Larutan

Bahan

Larutan

AmonilllJ1 persulfate 140 mg F.

Dilarutkan dalam 100 ml air 5ullng

120

37,5 g 6,25 g Dllarutkan dalam 500 ml air 5uling Acrylamide

BIE

Riboflavin 20 mg Dllarutkan dalam 1000 ml air 5uling

Running gel

Untuk membuat running gel diperlukan larutan A, B, dengan

perbandingan 1

diperlukan larutan

larutan

C.

Ketiga

1 : 2. Untuk mengisi 6 plat

A & B masing-masing 25 ml dan larutan

tersebut

dicampur

menggunakan magnetic stiz'rer dan divaltum selama 20 Setelah homogen campuran ini dapat dimasukkan dalam

C

kaca ml

50

dengan menit. glass

electz'ophoresls, yaitu alat berupa sepasang kaca setebal 5

mm yang khusus dirancang untuk elektroforesis. Pada bagian tepi kiri, kanan, dan bawah dipasang sekat (shield Sekat

inl

harus dipasang dengan

cermat

sehingga

tube).

dapat

membentuk rongga antar lapisan lapisan kaca dengan tebal ± 1

mm

dan

harus dijaga

agar

jangan

sampal

larutannya

merembes keluar. Untuk satu buah plat kar.a memerlukan ± 15 ml

larutan.

menjadi

rata

Selanjutnya

untuk

membuat~

permukaan

dapat ditambahkan alkohol atau


air

gel dengan l3

ketinggian ± 1 mm. Agar·gel menJadi padat diperlukan waktu ± 1 Jam dengan menggunakan lampu untuk mempercepat

proses

pemadatan.

SpaceI' Gel Spaoer dengan

gel dibuat dengan menoampur larutan D,

perbandingan 1 : 2 : 1. Untuk mengisi 6 plat

d1perlukan

magnetlc

stlI'I'eI' dan d1vakum selama campuran

homogen,

electI'ophoI'esls

Setelah

dalam

glass

d1 atas I'unnlng gel. dan

menggunakan

men1t.

dimasukkan

pada spacel' gel

waktu

selama ± 30

men1t

ml

10

Sample

dilepas

dengan

comb

setelah

gel memadat. B1asanya untuk memadatkan spaceI'

diperlukan lampu

tepat

d1pasang

kemud1an

1n1

20

F

kaca

ml larutan D, 20 ml larutan E, dan

10

larutan F. Ket1ga larutan ini d10ampur dengan

spaceI'

E,

gel

menggunakan

untuk memperoepat proses pemadatan. Berikut

adalah

gambar proses pemadatan gel dengan bantuan lampu :

Gambar 5. Proses pemadatan gel Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

14

Sdmple yang

comb merupakan alat yang

dirancang

sedemikian

rupa

sisir

m~enyerupai

sehingga

saat

dilepas

meninggalkan lubang sedalam 2 cm dan antar lubang berjarak 5

mm. Dalam penelitian ini jumlah lubang

yang

digunakan

adalah 20 buah. Sebelum

lubang

dlisi

dengan

larucan

superndtdnt,

lebih dulu dibilas dengan larutan Bromophenol Blue (20

Bromophenol Blue dilarutkan 1 Lair sullng) sampai 3 den~an

cara menuang dan mengisapnya kembali. Pada

terakhir

larutan

ditinggalkan

setengahnya

mg kali

bilasan

karena

akan

digunakan sebagal indikator. Lubang-lubang ini selanjutnya digunakan sebagai tempat sampel. Cara pencucian gel dengan larutan Bromophenol Blue dapat dilihat pada Gambar 6 :

~"r'.~ . ..

..

~~

Gambar 6. Pencucian gel dengan Bromopllellol Blue

4. Proses elektroforesis. Elektroforesis elektroforesis, alat

ini

dilakukan

lengkap

dengan

dengan power

menggunakan 6 set gel, maka


menggunakan supplynya. se~iap

unit

alat Karena dapat 15

digunakan

maksimal

dipelajari

setiap

Setiap

satu

pengisian

untuk kali

6

macam

proses

enzim

elektroforesis

gel dapat digunakan untuk larutan

superna tan t

20

dapat

yang

selesai.

sampel. lubang

dalam

Car a sampel

disajlkan dalam Gambar 7 berikut

Gambar 7. Cara pengislan larutan supernatant

Sebelum pemasangan plat kaca pada bak dlpastikan

elektroforesis

dulu slrkulator menunjukkan Buhu

tidak

lebih

darl 4 °C. Penutup bak elektroforesis dibuka dan bak diisi larutan

elektroda rUnniJlg buffel' yang telah

dipersiapkan

sebelumnya hingga setinggi 2 cm. Selanjutnya klip penjeplt dan

shield

plat

kaca

tube darl plat kaca dllepas

dipasang

pad a

bak

dan

selanjutnya

elektroforesis

secara

berhadap-hadapan, densan plat kaca yang bertakik berada d1 sebelah

dalam.

gelembung holdel'

udara

Pada di

d lkencangltan.

saat

pemasangan

antara plat Selanjutnya


kaca,

tidak

boleh

kemudian

dl tambahkan

ada

palang larutan 16

l~unning

telah

buffer (Tabel 7) ke bagian dalam plat

kaca

dipasang berhadapan tersebut hingga tepat di

yang bawah

takik. Tabel 7. Running buffer Pembuatan

Larutan

No. 1.

Stock buffer

60 g 288 g Air suling hingga larutan 5.000 ml

2.

Running buffer

Stock buffer 250 ml Air suling hingga larutan 5.000 ml

gel

Setelah larutan

Trizma base Glycine

dipasang

superllstant

sebanyak

diisikan

pada ke

alat

elektroforesis,

dalam

lubang

sampel

alat

inJeksi

10, mikroliter dengan menggunakan

yang disebut stepper. SelanJutnya rwmi11g buffer

diisikan

lagi hingga penuh dan bak penutup dipasang kembali. Power

suply

elektroforesis Ampere.

gel

dan

dengan

Proses

Bromophenol

dihidupkan

ini

arus

untuk listrik

dihentikan

menJalankan sebesar

100

setelah

proses mili

kedudukan

Blue ± 0,5 - 1 cm di atas dari dasar

running

memerlukan waktu ± 180

menit.

biasanya

200

Berikut disaJikan gambar proses elektroiol'esis


17

Gambar 8. Proses elektroforesis

5. Proses staining. Staining

melalui

adalah

proses

Pewarnaan

pewarnaan

elektroforesis

dllakukan

dikeluarkan

proses

dari

dengan

glass

gel

yang

sistem

dengan

meletakkan

electrophoresis

enzim.

gel

yang

telah

ke

dalam

baki

plastik berukuran 20 cm x 15 cm, kemudian dituang staining

yang telah dipersiapkan sebelumnya.

larutan

Selanjutnya

dibiarkan selama beberapa saat dalam keadaan gelap dlgoyang-goyang. larutan dilakukan

Lama

staining ±

30

yang menit

perendaman

1nl

dlgunakan. sebelum

dengan

tergantung

Pembuatan

proses

telah

pada

staining

elektroforesis

berakhir. Nama bahan kimia dan komposisi pembuatan larutan staining dapat dilihat pada Tabel 8, 9, 10 dan 11.


18

Tabel 8. Komposisi larutan staining Enzim

Staining Btlffer

ACP

ACP.BUF 50 ml

DIA

EST

GDH

C.BUF 50 ml

Substrat, Koenzim dan Komponen Lain

Fast Garnei; GBe salt

0,5 ml 25 mg

Q-Naphf:yl Acid PJJospJJate

100 mg

S.DIA

1 m! 0,2 m! 2 m!

MG

NADH MTI'

EST BUF 50 ml

A. O,lM a-Naphtyle propionate

EST BUF 50 ml

B.

A.BUF 50 ml

G2DH

A.BUF 50 ml

GOT

GOT BUF 50 m!

1 200 10 2 190 10 100

ml mg m! ml mg m! mg

NAD NBT PMS

1 1 1 1

g m! ml ml

S.G2DH NAD NBT R1S

4 1 1 1

m! m! m! m!

100 1 500 5 5 33,3 10 100

mg m! mg m! m! mg ml mg

1 100 10 30

ml mg ml mg

1 1 1 1 0,2

ml ml ml ml ml

[ a-Naplltyle propiollate Etanol O,lM a-Naphtyl aoetate [ a-Naphtyle aoetate Eta/l0l Fast Blue BR salt

L-Glutd/11io aoid

L-Aspartio aoid

10 % a-Ketoglutaric aoid [ (l-Ketoglutario acid

Air suling 0.33 % Piridoxall-5~-phosphate [ Piridoxall-5"-phosphate

Air sttling Fast Blue BB sal t

LAP

LAP.BUF 50 ml

1 % L-Leucyl-O-llaphtylamide [ L-Leucyl-Ll-l1aphtylaalide

Air suling Fast Black K salt

SHD

C.BUF 50 ml

S.SHD MG NADP t1TI'

PHS


19

Tabel 9. Komposisi stalnl11g buffalo Buffer

pH

A.BUF

7,0

g 3,88 Air suling sampai larutan 500 ml

ACP.BUF

5,0

g 5,74 1,8 ml Air suling sampai larutan 500 ml

C.BUF

8.0

g 2,22 g 1,33 Air suling sampai larutan 500 ml

EST.BUF

5,6

g 7,80 NaH2P04·2H20 g 1,42 NaH2P01 Air su ing sampai larutan 500 ml

GOT.BUF

7,0

g 1,18 NaOH Air suling sampai larutan 200 ml g 6,80 KH2P04·2H20 Air suling sampai larutan 500 ml

LAP.BUF

6,0

g 9,80 g 12,10 Air suling sampai larutan 300 ml g 0,6 NaOH Air suling sampai larutan 500 ml

Larutan Tl'isma pH 7,0 crystal

Na ace ta te (al'1hydl'i de ) Acetic acid" (glacial) Tl'izma Hel Tl'i sma Base

Ha 1 e i c al'1hydl'a te Trisma Base

Tabel 10. Komposisi substrat Substrat S.DIA

•

Komposisi

2. is Dichlol'ophemol-I1'1<:iophaJ10l 2.6 DichloropheJl01-!Jldophenol sodium salt 25 mg

Air suling

25 ml

S.G2DH

0,2 M DL-r..71ycel'ic acid Air suling

25 mg 1 ml

S.SHD

2% Shikimic acid Shikimic acid

Air suling Training in Conservation and Management of Genetic Resources Yogyakarta. 19-23 March 1999

500 mg 25 ml 20

Tabel 11. Komposisi Koenzim dan Bahan Lain Komposisi

Koenzim dan Bahan Lain

10,165 g 100 ml

MG

MgCl2·6H20 Air suling

MTT

MTT Air Buling

125 25

mg ml

NAD

NAD Air suling

500 20

mg ml

NADH

NADH Air Buling

500 5

mg ml

NADP

NADP Air suling

133 20

mg ml

NBT

NBT Air suling

200 20

mg ml

PMS

PMS Air suling

125 25

mg ml

Staining dilakukan

setelah

proses

elektroforesis

berakhir. Gel dar1 bak elektroforesis d1ambil dengan

cara

mematikan powel' supply, membuang larutan elektroda melalui saluran

pembuangan sampai habis, membuka bak penutup

plang 1101 del',

mengendorkan

sehingga

plat

kaca

dan

dapat

diambil. Pelepasan menggunakan kaca

gel

plat

kaca

dilakukan

pipih dan pisau cutter.

plat

diletakkan di atas meja yang telah diberi alas

ka1n

posisi

kaca

yang

bertakik

di

sebelah

Pemisahan

kaca

mencongkel

tepi kaca dengan hati-hati dengan

sendok

dengan

Setiap

dengan

sendok

dari

pipih.

bertakik

Lembar

gel

dari

akan


kaca

bersekat

tertinggal

atas. dengan

menggunakan pad a

kaca 21

bersekat. Gel yang akan direndam hanya bagian l'uJwing gel, sahingga antara bagian spacer gel dan runJliJlg gel dipotong dengan

pisau cutter. Sisi-sisi gel disayat

cuttez'

dan

pada

plastik

baki

Selanjutnya perendaman'

perlahan-lahan gel diangkat.

baki

dan

larutan

plastik

tersebut

dengan Gel

pisau

diletakkan

staiJling ditutup.

dituang. Temperatur

adalah 37 ·C, sedangkan lama perendaman

untuk

masing-masing enzim dapat dilihat pada Tabel 12 berikut :

Tabel 12. Waktu perendaman masing-masing sistem enzim Enzim

Waktu

ACP DIA EST A.

20 - 180 menit 30 - 60 menit 10 menit 60 menit 30 - 60 menit

GDH

B.

Adapun

cara

Waktu

Enzim G2DH GOT LAP SHD

30 15 15 15

melepas gel dan proses

-

60 30 30 30

menit menit menit menit

staining

dapat

dilihat pada Gambar 9 dan 10 berikut :

Gambar 9. Proses pelepasan gel Training in Conservation and Management of Genetic Resources Yogyakarta, 19-23 March 1999

22

Gambar 10. Proses staiJling

6. Pengamatan gel Pengamatan

pola

berkas (baJldlng pattern)

pada

gel

harus dilakukan segera setelah proses stalning berlangsung karena

apabila

kondisi sulit

pengamatan dilakukan

setelah

over stainlng interpretasi terhadap

gel

pola

dalam berkas

dilakukan. Pola berkas harus disalin dengan benar dalam

blangko

data (lihat Lampiran 3). Posisi berkas pada gel ditetapkan berdasarkan masing masing nilai Rf. Rf ialah

perbandingan

jarak antara permukaan gel dengan posisi sua tu berkas permukaan

gel

dengan

penanda

Bromophenol

Blue.

dan Untuk

menetapkan posisi alel, suatu famili yang genotipnya telah dicek perlu diikutkan pada elektroforesis sebagai kontrol.

7. Fiksasi gel. Gel

yang

telah diwarnai segera


difiksasi.

Larutan 23

stalnlng dibuang, dan larutan fiksasi dituangkan pada

sebanyak sistem 13.

50 ml. Larutan fiksasi yang

dipaka.i

tergantung

enzim yang digunakan dan dapat d1l1hat pada

Selanjutnya

gel d1simpan dalam suhu

gel

dingin

Tabel (4

·C)

selama 24 jam, dan d1tutup agar larutan t1dak menguap.

Tabel 13. Larutan f1ksas1 untuk mas1ng-mas1ng s1stem enz1m Enz1m

Larutan F1ksas1 F1ksas1 F1ksas1 F1ksas1 F1ksasi

ACP DIA EST GDH

Keterangan : F1ksas1 A - Fiksas1 B

Enz1m

B A B A

Larutan F1ksas1 Fiksasi F1ksas1 F1ksas1 Fiksasi

G2DH GOT LAP SHD

= 50 % Alkohol = 50 % Alkohol

A B B A

+ 5 % Aceton

8. Pengeringan gel. Supaya dengan

gel

dapat dis1mpan, maka ,

menggunakan

kertas kaca

setelah

Penger1ngan "dllakukan

selesai.

perlu

dengan

d1keringkan

proses

fiksasi

cara

sebaga1

berikut : Kertas kaca yang telah d1rendam dalam a1r su11ng d1bentangkan d1basah1

di

kaca yang

sebelumnya

juga

air. Ukuran kertas kaca leb1h besar ±

masing-mas1ng menggunakan merata

pada

disiram

air

dengan

at as

tep1

kaca. Kertas

kaca

2cm

diratakan

penyapu gelembung hingga kertas kaca

kertas

kaca. Gel diletakkan su11ng dan kaca

di

atasnya,

perlahan-lahan

basah.

Air


disapu

telah

ditutup kembali

dar1 dengan

melekat kemudian kembali dengan 24

penyapu

gelembung

bergelembung.

hingga kertas kaca

merata

dan

tidak

Maaing-maaing tepi kertaa kaca dilipat

dan

dijepit dengan klip penjepit. Gel dibiarkan kering

aecara

alami

dengan diletakkan pad a rak aelama ± 24 jam.

Setiap

gel diberi label aeauai dengan aiatem enzim, nomor

famili

dan

tanggal

dilakukan

proaea

elektroforeaia.

Cara

pengeringan dapat dilihat pada Gambar 11 dan 12 berikut :

Gambar 11. Pelapiaan gel dengan kertas kaca

Gambar 12. Pengeringan dengan dianginkan Training in Conservation and Management of Genetic Resources Yogyakarta. 19-23 March 1999

25

9. Penyimpanan gel. Gel

yang telah kering diambil.

mengenai yang

keterangan

tanggal pengamatan, nama famili dan

nama

enzim

diletakkan

dalam

foto untuk penyimpanan. Gel kering ini tahan

untuk

disUnakan

album

Berbagai

perlu dicatat

sebelum

disimpan, sehingga masih dapat dimanfaatkan untuk

diamati

pada waktu-waktu mendatang • • 3.4. Analisis Hasil Pengamatan Pengamatan sampel

dilakukan terhadap seluruh nomor

untuk setiap sistem enzim

pada

famili.

masing-masing

gel kering dilakukan dengan perhitungan

alel

dan

nilai Rf (Relative value from the Bromophenol Blue front). yang

Parameter

dihitung

berdasarkan

data

yang

diperoleh dari hasil interpretasi pola berkas ialah 1. Jumlah untuk

lokus

dan jumlah

mengetahui

banding pattez'ns)

alel

pewarisan pola

masing-masing berkas

seedlot

(inheritance

dari enzim polimorfik (frekuensi alel

umum yang muncul < 99 %).

2. Perhitungan

Chi-kuadrat

/ Chi-squal'e

(X2)

alel-alel

dalam setiap genotip.


26

Keterangan : X2 Chi-kuadrat

= o = Frekuensi E

=Frekuensi

3. Frekuensi

aIel yang muncul aIel yang diharapkan

aIel,

yaitu rata-rata

lokus

CA) dan Heterozigositas /

untuk

mengetahui

besarnya

jumlah

aIel

aetiap

He terazygosJ. ty

variasi

genetik

CH) ,

dalam

populasi.

Keterangan :

H

=Heterosigositas

Pi

= Frekuensi

aIel ke-i


27

KEGUNAAN ANALISIS ISOZIK UNTUK BUDIDAYA TANAKAN

Apabila isozim

materi

dapat

atau enzim yang

cocok

dimanfaatkan untuk membantu

tersedia,

analisis

memecahkan

beberapa

masalah yang erat kaitannya dengan identifikasi variasi

genetik

atau menentukan level variasi genetik. Beberapa contoh pemanfaatan analisis isozim diantaranya adalah a. Identifikasi pohon induk dengan klon, digunakan pad a kebun

benih

identiflkasl

klon

(clonal

seed

orchard),

seed lot dengan pohon induk

serta

balk

pada

pengujian keturunan maupun pengujian provenans. Mlsalnya dilakukan oleh Hamrlck et al. (1981)

dalam

Adams

jenls

(1983)

Pseudotsuga

dengan

25

menziesii,

klon

pohon

ternyata darl 9

plus blok

yang

dlgunakan terdapat kesalahan pelabelan genetlk

2-13

% pada 6 bIok, sehingga perlu aegera dldeteksl

dan

dlkoreksl. b. Stud1

eflalena1

orchard), hasl1

kebun benih semai

(seedling

untuk mencegah penggunaan produksi

allang

dalam

(inbreeding)

dan

seed benih

kontaminasi

serbuk sari darl luar kebun benlh. Mlsalnya klon

dl1akukan Adams (1983) dengan kebun

benih

P. mellzlesi1 berumur 20 tahun, diperoleh

produkai

benih merupakan hasil penyerbukan

8

%

sendiri

(selfing) .


28

c. Identifikasi

jenis-jenis bastar,

baik untuk bastar alam

maupun pembastaran buatan. Sebagaimana

yang dilakukan oleh Adam

bahwa

pembastaran

dari

sebanyak

43

terkendali

(1983),

diketahui

P.

menziesii

pada

pasang, diperoleh hasil bahwa diduga

39

%

benih hasil persilangan terkena kontaminasi/tidak berasal dari kedua induk yang ingin disilangkan. Kenyataan

diatas

secara mudah dapat

dijelaskan

dengan

bagan berikut : Genotipe induk betina

Keturunan 1

2

3

4

5

Locus 1

AA

AA

AA

~

AA

AA

Locus 2

Bb

BB

bb

Bb

BB

Bb

Locus 3

CC

CC

§]

CC

Locus 4

dd

dd

dd

Dd

[;J

dd

Amb

OutX

~

CC

OutX

OutX

Amb

Tipe keturunan

Catatan : Amb : penyerbukan sendiri OutX: penyerbukan silang d. Studi

taxonomi

jenis jenis

yang sulit dideteksi dengan

karakter morphologi. Crawford (1989) membedakan 2 species dari genus nia

yang berhabitat rawa. Kedua species ini secara

phologis sulit dibedakan. Oari analisis isozim hasil

Salicor-

bahwa

antara

keduanya

tidak

diperoleh

terdapa~

variasi.

Keduanya menghasilkan pola bercak yang monomorfik, tidak menunjukkan gejala persilangan antar jenis. demikian

tidak terdapat bukti untuk

membedakan

mor-

serta Oengan species

yang dimaksud menjadi 2 jenis yang berbeda. Training in Conservation and Management of Genetic Resources Yogyakarta. 19-23 March 1999

29

e. Pendugaan tingkat variasi genetik dalam populasi alami. Tabel berikut

mer~ngkum

beberapa hasil penelitian tentang

besarnya variasi genetik untuk beberapa species. Jenis

Heterozigositas

P. densiflora P. kesiya P. pungens

0,314 0,169 0,204

P. thunbergii C. obtusa P. glauca

0,279 0,305 0,290

Jenis konifer Jenis daun lebar

0,207 0,211

Semua jenis tanaman

0,141

Sumber

Na'iem (1991) Boyle et.al (1991) Gibson dan Hamrick (1990) Shiraishi (1988) Shiraishi et.ai.(1987) Purnier dan Clyde(1990)

Hamrick et.al.(1981) Hamrick dan Loveless (1989) Hamrick (1979)

f. Menyediakan informasi untuk konservasi genetic Strategi konservasi suatu biodiversitas in situ

memerlu-

kan data informasi tentang besarnya variasi genetik dalam maupun antar populasi species yang akan

baik

dilindungi

(Adam, 1983).


30

ITTO PROJECT PO 16/96 REV. 4(F}

EX SITU CONSERVATION OF Shorea leprosula AND Lophopetalum multinervium AND THEIR USE FOR FUTURE BREEDING AND BIOTECHNOLOGY WORK PLAN AUGUST 1998 TO JULY 2001

(REVISED EDITION)

PREPARED BY

PROJECT EXECUTING TEAM PO 16/96 REV. 4(F) TECHNICAL EDITOR: BART A. THIELGES

FACULTY OF FORESTRY GADJAH MADA UNIVERSITY YOGYAKARTA, INDONESIA . 1999

Chapter I

Introduction Ex situ conservation refers to the conservation of components of biological diversity outside their natural habitats. Ex situ genetic conservation is achieved by preservation of collections of various types of plant genetic resources such as seeds, pollen, seedlings or vegetative propagules in field collections and/or gene banks. These activities are essential to accommodate and assist the users of the plant germ plasm who need ready access for their genetic improvement programs. Ex situ conservation may also serve to conserve natural genetic diversity that might otherwise be lost in to natural disasters or to land-clearing operations. ITTO Project PD 16/96 Rev. 4 (F) focuses on the ex situ genetic conservation of two selected tropical hardwood species and on their use for future breeding and biotechnology programs. The Project will initiate fundamental research activities that will: (1) avert declines in natural genetic variability within Shorea leprosula and Lophopetalum multinervium and (2) design and establish these ex situ genetic collections so that they may also contribute to more effective breeding and biotechnology programs in the future. The expected outputs of this project could be used to meet national policy goals for forest conservation and also to develop ex situ conservation action plans with sound scientific foundations. Additionally, this project will be utilized as: (1) A model to demonstrate the design and establishment of ex situ conservation plantations; (2) The plant materials collection pOints for subsequent breeding and biotechnology programs to improve health, productivity, and quality of plantations of Shorea leprosula and Lophopetalum multinervium; (3) Training and educational facilities for students and industry and government personnel; and (4) Research and demonstration sites for networking, cooperation and partnership among national and international institutions engaged in ex situ conservation.

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The project will be undertaken and managed by the Faculty of Forestry, Gadjah Mada University, in cooperation with the Directorate General of Forest Management (GOI), the Directorate General of Forest Protection and Nature Conservation (GOI), P.T. INHUTANII to V, PERUM PERHUTANI, and Oregon State University (USA). This work plan elaborates specific outputs to be achieved within the three -year project time frame, and provides a list of the specific activities and resources needed to accomplish these results. This project relies on concerted team efforts involving various collaborating institutions, and will be facilitated by a series of well-planned meetings and technical training sessions.

Chapter 11

Project Objectives 2.1 Development Objective At present, ex situ conservation activities for forest trees in Indonesia are limited to collecting and preserving genetic resources in Botanical Gardens, Arboreta, and Taman Hutan Raya. In all of these very initial efforts, the sample size is small and severely limited both in number of specimens and in their geographic distribution. Furthermore, in general, the samples are poorly characterized and documented, so it will not be possible to use these collections for breeding purposes. To meet the needs of future breeding and biotechnology programs aimed at improving productivity and quality of tropical forest products, IITO is supporting project PO 16/96 Rev. 4 (F): on ex situ conservation of native Indonesian hardwoods. The Development Objective of IITO project PO 16/96 Rev. 4 (F): on ex situ conservation of Shorea /eprosu/a and Lophopeta/um multinervium and their use for future breeding and biotechnology is to create a Center of Excellence for ex situ conservation which will serve Indonesia and neighboring countries through research, technology development, training and education in the genetic conservation and improvement of selected tropical tree species. This project will focus on determining appropriate methods of conserving two selected indigenous species of the tropical rain forest and also to provide a reservoir of genetic materials for future breeding and biotechnology efforts to genetically improve those species.

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The design and methodologies developed during this project will be well documented through publication.

They will also be demonstrated through on-site

training of scientists and foresters engaged in genetically improving productivity and quality of products of tropical rain forests while also conserving genetic resources of target species. The methodologies developed will also contribute to the conservation of biological diversity in tropical production forests. And technologies developed and transferred during this project will also provide the bases for refining and adapting these

ex situ methods for gene conservation and improvement of additional tropical tree species. 2.2 Specific Objectives 2.2.1. To create effective programs of education and technology transfer that may be used to train appropriate personnel in methods for ex situ conservation of tropical forest trees. Scientific publications often address the importance of ex situ genetic conservation in the context of global efforts to conserve biological resources. The critical relationship of these conservation efforts to the future availability and use of these genetic resources to effectively respond to changing environmental and economic conditions cannot be overstated. Conscious of the intrinsic value of ex situ conservation and aware of the general lack of knowledge regarding the combination of values of conservation with those of wise utilization, project personnel recognize that the availability of technology to establish ex situ conservation is of paramount importance. Furthermore, the establishment of genetic collections at many sites of PT INHUTANII to V and PERUM PERHUTANI is of critical importance as a medium of technical training and public education. In this regard, the project shall: (1) Design, establish, and maintain programs for scientific and technical education and training in ex situ conservation methodology, and link these with in situ conservation and with breeding and biotechnology programs; (2) Promote and encourage research that contributes to the conservation and utilization of genetic resources for improving productivity, quality of products and maintaining biological diversity.

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Project personnel also realize that knowledge, rather than the endowment of natural resources, is becoming a primary determinant of the economic performance of nations. Countries that have invested heavily in science and technology are observed to make more rapid economic progress than those that have not done so. However, science and technology are advancing at a much faster pace in industrialized countries than in most of the developing countries. Urgent attention is required to improve the capabilities and potentials of scientists in developing countries. In this regard, the transfer of technology by creating strong partnerships with industrialized countries is one way to narrow the knowledge gap. Accordingly, the Faculty of Forestry, Gadjah Mada University has developed a partnership with Oregon State University (OSU), in the USA. The role of OSU is basically to provide project support in education and transfer of technology. This includes consultation and collaboration during both the pre-project phase and the implementation phase. More specifically, OSU will provide scientific back up and expertise in planning and implementing research, education and training to transfer current ex situ genetic conservation theory and technology to Indonesian scientists and practitioners. As such, the project will promote and facilitate the cooperative use of scientific advances in biological diversity research by developing methods for the conservation and sustainable use of tropical forest resources.

2.2.2. To establish ex situ conservation methodologies of Shorea leprosula and

Lophopetalum multinervium and to develop these as models for general ex situ conservation techniques for tropical forest trees. Genetic resources of two selected species will be collected and established at several sites of PT INHUTANI I to V and PERUM PERHUTANI. The establishment of these ex situ conservation areas will also be designed to meet future requirements for more efficient breeding and biotechnology programs. As the genetic conservation model for these two indigenous Indonesian species is designed and tested in this project's activities, development of ex situ conservation techniques for other indigenous tropical tree species will be facilitated, as well. The project's ex situ conservation collections will be utilized as a model for the demonstration of general deSign and establishment techniques of ex situ conservation plantations. In addition, the ex situ collections will serve to provide fundamental genetic information, as well as plant materials needed for further research to establish OPAGE 040

valuable, high-yielding forest plantations. Therefore, the ex situ conservation plantations developed in this project will serve as a unique model for future programs designed to address both genetic conservation and utilization of tropical hardwood resources. By establishing collections combining both genetic conservation and the potential for further genetic improvement in their basic design, the project is taking innovative approaches to providing a scientific basis for sound forest management. The reasons for selecting Shorea leprosula and Lophopetalum multinervium relate to characteristics of these species contributing to their use as general models for ex situ conservation techniques. Basically, we wish to represent two common and widely

distributed Indonesian hardwood forest habitats: (1) well-drained sites, and (2) swampy sites. In this context, Shorea leprosula serves as a model species for the former site, and Lophopetalum multinervium for the latter. And it should also be noted that these two model species are of considerable commercial value in the Indonesian economy.

Chapter

III

The Outputs 3.1.

Specific Objective I: To create effective programs of education and technology transfer that

may be used to train appropriate personnel in methods for

ex situ conservation of

tropical forest trees.

This specific objective has three expected outputs: Output: 1.1 A report on technology transferred by US experts to Indonesian scientists through various workshops and seminars. Technology transfer activities encompass planning, design, conduct, and evaluation of ex situ conservation methodologies. US experts, together with key Indonesian scientists, will plan, design and organize in-country training for Indonesian field technicians. This training will enable the Government of Indonesia to later establish ex situ conservation plantations for other important target species.

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Output: 1.2 Well-trained personnel in ex situ genetic conservation, biotechnology and tree improvement. Two persons of the Project Executing Agency and Project Executing Team will explore the possibility of obtaining support for research on ex situ conservation and linkages with in situ conservation, biotechnology, and tree improvement. Output: 1.3 Dissemination of research results. Four types of information will be disseminated; technical, educational, executive, and informational/public reporting to partners and to other interested institutions /agencies. In addition, prior to the termination of this project, project personnel will organize an international conference on genetic conservation methods for tropical tree species. 3.2.

Specific Objective 11: To establish ex situ conservation of Shorea leprosula and Lophopetalum

mUltinervium as a model for general ex situ conservation of tropical forest tree species. Specific Objective 11 has two main outputs, these are: Output 2.1: Collections representing genetic variability in each of the two selected species established as ex situ conservation plantations. In cooperation with PT INHUTANI I to V and PERUM PERHUTANI project personnel will explore natural stands of each species and design appropriate sampling schemes to select representative trees and collect materials for propagation. At least five natural populations of Shorea leprosula and one population of Lophopetalum will be collected in each species range each year. Shorea leprosula will be collected from January to March and Lophopetalum multinervium will be collected from June to September, each year for three years. Nurseries for raising seedlings of the two species will be established at appropriate sites in INHUTANII to V and PERUM PERHUTANI, and these seedlings will be used to establish the ex situ conservation plantations at or near each nursery site. The ex situ conservation plantations established in this project will represent a subset of the Indonesian ex situ conservation efforts for Shorea leprosula and

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Lophopetalum multinervium. Breeding and biotechnology strategies for these two

species will be developed upon the completion of ex situ conservation activities. The combined conservation and genetic improvement programs are intended to serve as a model to demonstrate the general establishment of similar conservation and improvement programs for other species. It is hoped that project activities will assist the Government of Indonesia to develop policies, regulations, and action plans for in situ and ex situ conservation, and to also develop comprehensive genetic improvement programs aimed at achieving productive and sustainable forests as quickly as possible.

Output 2.2: A report on the results of isozyme and other molecular analyses of genetic structure of geographic populations of each species. Project scientists will employ polyacrylamide vertical slab gel electrophoresis techniques using several enzyme systems to elucidate patterns of genetic variation. The subsequent isozyme analyses will estimate genetic variation within and between natural populations of the two selected species. Data generated by isozyme analyses will be used to further confirm levels of genetic variation by calculating polymorphic loci, number of alleles per locus, expected heterozygosity, genetic identity, and genetic distance. Molecular information on genetiC structure of geographic populations and on centers of distribution of Shorea leprosula and Lophopetalum multinervium will be available in 2001. This information will greatly assist the further development of efficient breeding strategies for both species.

Chapter IV

Organization and Management 4.1.

Management Structure The project management will consist of a Project Steering Committee and a

Project Executing Agency. The Project Executing Team and Secretariat will support project implementation by the Faculty of Forestry, Gadjah Mada University, acting as the Project Executing Agency. The Project Executing Team shall be composed of

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representatives from the staffs of the Faculty of Forestry, PT INHUTANI I to V, and PERUM PERHUTANI. The Project Executing Team shall be responsible for implementing the Project according to the procedures specified in the Project Document and the detailed Work Plan approved by ITIO. During Project implementation, appropriate coordination and monitoring shall be maintained among all levels of management. The main responsibilities of the Project Executing Team/Secretariat are (1) to prepare and implement the work plan and budget, (2) to coordinate national and international experts and field technicians collecting genetic materials and establishing those collections at the ex situ conservation plantation sites, (3) to organize PSC meetings and other related meetings, (4) to organize the international workshop on ex situ conservation

The Project Executing Team shall do its utmost to ensure that the Project is implemented on schedule, remains within budgetary limits, and generally operates to achieve project objectives. The Chairperson of the Project Executing Team, as the team leader, has the power to manage with full support from all members of the Project Executing Team in accordance with their job descriptions, to achieve the stated objectives. The Project Executing Team will hire national experts in ex situ conservation, tree improvement, and biotechnology in accordance with the Project Document and the ITTO Guidelines on the Selection and Employment of Consultants. The Terms of Reference of the national experts are attached as Appendix 1. The Project Executing Team will also invite international experts on ex situ conservation, tree improvement, biotechnology and data management to share their expertise and experience with ex situ conservation techniques and uses of these genetic collections

for further

genetic

improvement through

breeding

and

biotechnology. Each of these experts will spend one month in Indonesia in years one and three of the project. The invitation of international experts will be accomplished in accordance with the Project Document and the ITTO Guidelines on the Selection and Employment of Consultants. The Terms of Reference of the international experts are attached as Appendix 2.

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The project Executing Team I Secretariat is comprised of the following personnel in compliance with the Letter of Decision of the Dean of the Faculty of Forestry Gadjah Mada University No UGM/KT/1166/UM/01/39: Chairperson ViceChairperson

Prof. Dr. Ir. Oemi Hani'in Soeseno (Faculty of Forestry GMU Prof. Dr. Ir. Soekotjo (Faculty of Forestry GMU)

Secretary I

Dr. Ir. Mohammad Naiem (Faculty of Forestry GMU)

Secretary "

Drs. Yogi Setiadi Halim (Ministry of Forestry and Estate Crops)

Member

1. Ir. Sadhardjo Sm, M.Sc. (Perum Perhutani) 2. Dr. Irsyal Yasman (P.T.lnhutani I) 3. Ir. Sunarno (P.T.lnhutani 11) 4. Ir. Kurdi Nuryanto (P.T. Inhutani Ill) 5. Ir. Sabaris Wantono (P.T. Inhutani IV) 6. Ir. Sutomo (P.T. Inhutani V)

The Chairperson of the project Executing Team shall recruit the following project personnel (see Appendix 3, letter of Appointment)

(1). Administrative and Financial Officer to assist the project in complying with administrative arrangements during project implementation. She shall be responsible for preparing a financial statement that will be submitted to IITO and tabled for discussion during the Project Steering Committee Meetings. (2). Technicians to assist national experts. (3). Other labor.

The Project Steering Committee shall be composed of the representatives of the Ministry of Forestry and Plantation Crops, the Faculty of Forestry of Gadjah Mada University, IITO, the donors, PT INHUTANI I to V and

PERUM PERHUTANI, in

accordance with the criteria established by the Steering Committee for membership. The Ministry of Forestry and Estate Crops will appoint the Project Steering Committee. The duties of the Project Steering Committee are (1) guiding the technical conduct of the Project to achieve its objectives within the approved budget, work plan, and time

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frame as outlined in the Project Document and in any supplementary arrangement agreed upon by IITO, GOI, and The Project Executing Agency; (2) approve programs and budgets of the various sub-projects within the framework of the Project as approved by IITO; (3) approve progress reports before submittal to IITO and GOI. The Director of Foreign Cooperation and Investment Bureau, Ministry of Forestry and Plantation Crops, GOI, will chair the Project Steering Committee. The Project Steering Committee shall meet at least twice a year. However, the first meeting shall take place shortly after the start of the Project. The Project Steering Committee is comprised of the following members, in compliance with the S.K. Menhutbun No 495/Kpts-II'98 : Chairman

: Dr. Ir. Untung Iskandar

Vice chairman

: Dr. Sambas Sabarnurdin

Secretary I

: Dr. Benni H. Sormin

Secretary 11

: Dr. Ir. Suhardi, M.Sc.

Members

: 1. Prof. Dr. Ir. H. Oemi Hani'in Soeseno 2. Ir. Oesman Yoesoep 3. Ir. Yaman Mulyana 4. Ir. Dwiatmo Siswomartono 5. Ir. Hendi Suhaendi 6. Ir. Hardjono, M.Eng. 7. Dr. Efransjah 8. Director of Perum Perhutani 9. Director of P.T. Inhutani I 10. Director of P.T. Inhutani 11 11. Director of P.T. Inhutani III 12. Director of P.T. Inhutani IV 13. Director of P.T. Inhutani V 14. Attache of Agriculture, Embassy of Japan 15. Mr. Osamo Tando

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Chapter V

Project Activities 5.1. Specific Objective I : Output 5.1.1: Technology transfer by US experts to Indonesian Scientists. Activities: 5.1.1.1. US experts assist Indonesian Project scientists in planning, conducting and evaluating ex situ conservation • Ex situ conservation expert • Biotechnology expert • Tree improvement expert • Database management expert 5.1.1.2. US experts, together with Indonesian scientists design, conduct, and evaluate short course(s) in ex situ conservation (theory and techniques) for Indonesian technicians • Ex situ conservation expert • Biotechnology expert • Tree improvement expert • Database management expert Output 5.1.2. Well trained Indonesian personnel in ex situ conservation, biotechnology (sterility, plant resistant to diseases), and tree improvement. Activities : 5.1.2.1 Two persons of The Project Executing Agency I Team will explore the possibility of getting support for research on ex situ conservation and linkages with in situ conservation, biotechnology and tree improvement. 5.1.2.2 Organize in-country training for Indonesian technicians and young scientists.

Output 5.1.3.: Dissemination of results Activities: 5.1.3.1 Organize an international workshop to discuss ex situ conservation strategy and linking ex situ conservation with tree improvement, biotechnology, in situ conservation and plantation programs. 5.1.3.2 Publish research results and other information.

5.2.

Specific Objective 11 :

Output 5.2.1.: Collections representing existing genetic variability in each of two selected species established as ex ~itu conservation plantations.

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Activities: 5.2.1.1 Explore genetic resources of the selected species in their natural geographic distributions and design an appropriate sampling scheme to collect materials for propagation. 5.2.1.2 A minimum of five natural populations or provenances of Shorea JeprosuJa per year is the target for collections. From each natural population 100 to 200 trees will be selected as parents. For LophopetaJum multinervium the project will collect from one natural population per year. From each natural population of LophopetaJum

multinervium 100 to 150 parent trees will be selected. 5.2.1.3 Collect and store by parent tree the fruits of Shorea JeprosuJa and the seeds of

LophopetaJum muJtinervium. Some of the fruit and seed collections will be bulked and used as materials for ex situ conservation plantations, while others will be maintained by parent tree identity as families and will be used for progeny tests. 5.2.1.4 Establish local nurseries for raising seedlings from collected fruits or seeds at P.T. INHUTANII to Vand PERUM PERHUTANI's plantation sites. 5.2.1.5 Establish the ex situ conservation plantations at appropriate local sites as identified on the maps of Appendices 4,5,6,7,8 and 9 and also establish progeny test plantations. 5.2.1.6 Protect and maintain all ex situ conservation and progeny test plantations.

Output 5.2.2

Report the results of isozyme

and other molecular analyses of

genetic structures of geographic populations of each species. Activities: 5.2.2.1. Employ polyacrylamide vertical slab gel electrophoresis techniques using several enzyme system to elucidate patterns of genetic variation. 5.2.2.2. Use isozyme analyses to estimate the genetic variation within and between the provenances of each species. 5.2.2.3. Using data generated by activities 5.2.2.2., confirm levels of genetic variation by calculating polymorphic loci, number of alleles per locus, expected heterozygosity, genetic identity and genetic distance.

A summary of these activities is presented in Table V.1.

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Chapter VI

Monitoring, Reporting and Evaluation The Project Executing Team must develop efficient and effective physical and financial record systems so that project monitoring will be easily accomplished. Representatives of ITIO will twice monitor the project. The first monitoring mission will visit the project one year after signing the agreement, and the final monitoring mission will visit the project during the time for organization of the International Workshop on Ex situ Conservation.

The Chairperson of the Project Executing Team shall prepare a progress report semi-annually to be reviewed by the PSC before it is submitted to ITIO and the Government of Indonesia. The major reports will consist of The Project Progress Report and The Project Completion Report. In addition to those reports, the Project will also prepare and distribute published information of four types: technical, educational, executive, and informational I public reporting. This information will be disseminated to partners and other interested institutions. Other formats for dissemination of information will be research results in scientific Journals; published proceedings of conferences, seminars, and workshops; training materials and reports; press releases and other media outlets. The Project will be subject to evaluation in accordance with ITIO procedures. A final evaluation will be conducted based on project outputs, impact on National Policy, and possible future actions.

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Chapter VII

Project Cost and Financing 7.1.

Budget Plan 1999 to 2001 The overall budget plan for 1999 to 2001 follows the Project Document and

Agreement Document as shown in Table VII.1.

7.2.

Project Financing Procedure ITIO will disburse funds for all approved activities to the Project Executing

Teaml Faculty of Forestry, Gadjah Mada University by means of direct payment upon

official request of the Chairman of the Project Steering Committee with reference to the endorsement made at the Steering Committee meeting(s). ITIO will make payments to the bank account of The Project Executing Team at PT. Bank Rakyat Indonesia, Yogyakarta Cik Ditiro Branch, JI. Cik Ditiro 3 Yogyakarta as follows, unless otherwise advised by the Project Steering Committee: 1. An initial installment of US $ 70,000.00 (Seventy thousand United States dollars) after signing of the agreement by all parties, and upon submission of a detailed work plan for the execution of the Project, and upon notification to the Executive Director by the Project Executing Team that the Project is about to begin. 2. A second installment of US $ 130,000.00 (One hundred thirty thousand United States dollars) upon receipt of the Report of the Meeting of the Steering Committee that reviewed and approved the detailed work plan of the project. 3. A third Installment of US $ 160,000.00 (One hundred fifty thousand United States dollars) upon receipt of the Report of the Meeting of the Project Steering Committee that reviewed the first year's activities in accordance with the procedures of ITIO and the Project Executing Team's request for the third payment. 4. A Fourth installment of US $ 196,866.00 (One hundred ninety five thousand

eight hundred sixty six United States dollars) upon receipt of a midterm report submitted by the Project Executing Team and the Report of the Meeting of the Steering Committee that reviewed the second year's activities in accordance with the

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procedures of IITO and the Project Executing Team's request for the fourth payment. The amount of US $ 54,134.00 ( fifty four thousand three hundred United States dollars ) will be retained by IITO from the total financing approved by IITC to meet IITO's other costs . The Financial and Administrative officer will be recruited to assist the Project Executing Team to comply with necessary administrative

arrangements during the

project implementation. She shall be responsible for preparing a financial statement that will be submitted to IITO and tabled for discussion during the Project Steering Committee meeting. An independent auditor will review all financial statements at the end of the project and certify the balance.

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Glossary of Technical Terms Biological diversity means the variability among living organisms from all sources including diversity within species, between species and of ecosystems. Biotechnology means any technological application that uses biological systems, living organisms or derivative thereof to make or modify products or process for specific use Breeding means the science or art of changing the £enetic constitution of a population of plants or animals. Ex situ conservation means the conservation of components of biological diversity outside their natural habitats Ex situ genetic conservation means the conservation of genetic material of plant outside their natural habitat Family means the offspring of a single tree after open pollination or of single pair of trees after controlled pollination Genetic variability means a measure of genetic characteristic of differing from the average value. This terms are usually used in general sense and qualified by such words as low, moderate; and high. For quantitative ccmparisons, the concept of variance is used Plant genetic resources means genetic materials of plant of actual or potential value. Population means a group of individuals related by common descent and treated as a unit for convenience. Reservoir of genetic materials mean~ collection of any material ')f plant containing functional units of heredity User germplasm means breeder or biotechnologist who need ready access of the sum total of the genes and cytoplasmic factors governing inheritance

96 Rev.4{F)

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