The sequence of steps is as follows: 1. Species and Provenance Trials 2. Mass Selection 3. Progeny Testing 4. Advance Generation Breeding 5. Genetic Engineering.
Step # 1. Species and Provenance Trials (S & P Trials):
The foremost step in any tree improvement programme is the right choice of species followed by selection of suitable provenance within a species. It is necessary to conduct species and provenance test before making right choice of species and provenance for specific site. Species and provenance trial should be conducted according to written plans, which will usually form part of research programme of national forest authority.
ADVERTISEMENTS:
Plan of this sort must state clearly precise objectives of the particular species or provenance trial project. These should be derived from the objectives of national afforestation schemes and constraints upon them. Species and provenance trials must be aimed at reducing a large number of entries to relatively few proven species or provenances suitable for production of desired forest products.
The Basic Features of Species and Provenance Trials:
i. Review of literature, correspondence and personal knowledge of distribution and variation of species
ii. Selection of parent stands of a species in natural forest or seed stand in plantation. Procurement of seed and storage
iii. Assessment and selection of site, planning and designs of experiments
iv. Design and conduct of nursery experiments, nursery assessment and early testing like juvenile mature correlations
ADVERTISEMENTS:
v. Techniques and assessment in forest stage. The main activities are the duration of the trials (short, medium and long), designing and conduct of experiments and assessment
Phasing if Species and Provenance Trials:
Before starting large scale plantation programme, it is essential that adequate information about the requirement of the species and characteristics of site are obtained. Often this information is lacking at initial stages. Therefore the best way to get this information on suitable species/ provenance is to lay trials of number of species in small plots on localities which have been carefully selected. Species or provenance should represent geographic variation of the species. Sites selected for experimentation should be done on ecological requirement of the species.
The best way is to obtain gradual information by phasing the S & P trials as given below:
ADVERTISEMENTS:
a. Species Trials:
Initially large number of species is tested. Ultimately, one or two suitable species are recommended for plantation. Species selection passes through number of stages. Species are selected on the basis of their suitability for plantation sites. Initially trials are preliminary and are conducted on small sized plots for short period followed by trials with large plots and larger period.
An idealized sequence for species trials will be:
i. The Arboretum Phase:
ADVERTISEMENTS:
It involves testing of large number of species. The number of individuals of species is very low. The species are kept for longer duration, probably for their full life.
ii. The Species Elimination Phase:
The objective is to compare performance of large number of different species on one or more sites and to select a smaller number for more intensive trials. The number of species to be included in the trial depends upon availability of funds, seed, infrastructure, suitability of species to site and desired end product.
iii. The Species Testing Phase:
The objective is to compare restricted number of promising species within a broad climatic region. Properly statistically designed layouts are important in this phase. Plots must be large enough to enable reliable assessment to be made up to at least first thinning stage.
Sites should be stratified in order to evaluate the interaction between site variation and species difference. Again, the number of species tested depends upon availability of funds, infrastructure and adequate supply of seed from natural forests or local seed stands and number of suitable species from previous phase.
iv. The Species Proving Phase:
The objective is to confirm under normal plantation conditions, the results shown by the small number of species that have shown themselves superior in early phase. Trials should large enough to provide data on growth and yield for full rotation. Surrounds should be large enough to minimize edge effects. The trials also include different types of nursery methods, different types of planting methods, spacing and thinning trials etc.
b. Provenance Trials:
After selection of suitable species it is important that the most suitable seed source for planting site is selected. This is accomplished by selecting suitable provenances. Provenance is the area on which any stand of tree is growing. The stand may be indigenous or non- indigenous.
For an indigenous stand of trees the origin is the place in which the trees are growing; for a non-indigenous stand the origin is the place from which the seeds or plants were originally introduced. Provenance trial is also conducted in phases.
i. Range wide Provenance Phase:
The objective is to determine the content and pattern of variation between provenances of promising species. The number of provenances included in this phase depends upon the geographical distribution and variation in the species. This phase is run concurrently with species elimination phase. It is desirable to sample the entire range of the species.
This provides insurance against the possibility that some overlooked region is capable of providing the best trees. Seeds should be collected from several trees per locality. Parent trees could be average or selected and then bulked. A range wide provenance test usually indicates total range of genetic variation within a species
ii. Restricted Provenance Phase:
The objective is to find sub regions and ultimately provenances most suited to site under test. It detects minor trends and gives location of specific stands or small areas having the best germplasm. The test provides information on comparative amount of geographic and individual tree variability to function ultimately as seed orchard.
iii. Provenance Proving Phase:
The objective is to confirm under normal plantation conditions, the results shown by small number of provenances that have shown themselves superior in earlier phase. Here also the plots should be large enough to provide data on growth and yield for full rotation and surrounds should be large enough to minimize edge effects. In addition to normal plantation conditions a range of management techniques need to be tested.
Step # 2. Mass Selection:
Once the species has been selected and promising provenance has been delineated, the next step in tree improvement is selection. Simple form of selection is mass selection (Seed Production area etc.) which is an extension of natural selection with choice of trees left to man instead of nature.
Seed Production Area (SPA):
It is a seed-producing stand which has been developed either from existing planted stand or from stand specially planted and generally upgraded and opened by removal of undesirable trees and then cultured for early and abundant seed production. SPA constitutes a reliable source of seed of certain genetic quality, until such time the seed orchards come into production.
They are a very effective way of making available seed supply of an inexpensive but somewhat improved genetic quality. Although the amount of genetic improvement from a seed production area is expected to be small, the seed obtained from seed production areas still has better genetic qualities than seed from commercial collections in routine plantations, especially in adaptability, form and pest resistance.
SPA is a form of mass selection in which gains can be obtained by selecting or characters showing high heritability’s. A gain of 5-10 per cent may be possible but real genetic value is not known since the trees in the stand are not subjected to progeny trials.
Development of Seed Production Areas from Existing Planted Stands:
The quickest way to make a seed production area is to convert existing mature stands of good quality trees for this purpose. A seed production area can also be developed from a progeny or provenance trial. There are no specific age limitations but the stand should be old enough to produce seed.
A planted stand suitable for conversion to a seed production area should have the following specifications:
i. Details of the seed source used to establish the stand should be known:
Knowledge of the geographic origin and genetic base, which ideally will be broad, is important in determining the suitability of the stand for development into a seed production area.
ii. The trees should be mature for seed production, but not too old:
There are no age limitations, other than that the stand must be old enough for reliable selection and be sexually mature to produce seed. Individual trees must have sufficient crown density to potentially produce large seed crops. Seeds from very young or very old trees often are of inferior quality compared to those from middle age trees. In addition, old trees tend not to respond to thinning by further crown development.
iii. The stand should be near full stocking and contain a large number of trees of good phenotype:
A suitable stand before conversion should be near full stocking to increase the selection intensity. The final stocking of a seed production is usually 150-200 stems per ha. An initial survey should reveal that the number of trees of good phenotypes is equivalent to that.
iv. Free from pests and diseases:
Evidence of pests or diseases may be an indication of inferior adaptability of the seed source. If the stand shows widespread symptoms of attack by insects or diseases it should not be used for conversion.
v. The trees should have proven capacity to produce flowers and seeds in the area:
To avoid failure, a detailed survey of the candidate species’ ability to flower and seed in a particular environment should be a prerequisite to establishing seed production area. This is especially important for exotic species where flowering may fail or seeds are not produced due to incompatibility with the site. Non-favourable environments, such as drought prone areas, may be unsuitable for seed production area. Availability of pollinators can also be important.
vi. The area should be easily accessible:
The conversion of a stand into a seed production area and subsequent management operations, such as harvest of seeds, requires that the area is accessible throughout the year. This implies that it should be relatively flat but well drained. Ideally it should not be in a very remote area or too far from available labour for ease of maintenance and management.
vii. The selected stand should not be subject to commercial harvesting:
Seed production areas will be maintained for many years. It is necessary, therefore, to ensure that the stand in question is safe from commercial harvesting operations. Good communication with the forest managers will help avoid the accidental loss of valuable seed production areas.
viii. Size of Seed Production Areas:
In general a minimum of 4 ha is recommended for practical management of seed production areas for most tree species. Managing small stands is inefficient and there is a great danger of contamination from outside pollen. However, for some species like eucalypts, an area as small as 0.5 ha may be sufficient due to prolific seed production. The area should be as close to square as possible, rather than a long linear block to facilitate more cross pollination among the trees.
ix. Isolation:
Poor isolation will be a common disadvantage of seed production area developed from existing stands. Usually such stands are selected from mature plantations of the same species. It is virtually impossible to eliminate completely, contamination by stray pollen.
However, an isolation zone or pollen dilution zone surrounding the seed production area can reduce it. The dilution zone may be an open area of some 200 m. If trees are to be grown in the dilution zone they must be of a species that does not hybridize with the species of interest for seed production.
Selection of Trees for a Seed Production Area:
Desired attributes of the trees left in a seed production area are similar to, but less rigorous than, the qualifications required for selecting tree to be used in an intensive tree improvement program. Only trees in the dominant and co-dominant crown classes are considered for retention because of their growth and seed-producing potential.
For timber species, fast growth, straight bole, good crown development, self-pruning etc. are the criteria for selection of trees. Inferior trees should not be kept even if there is a gap. It is essential that the crowns of the trees be exposed to full sunlight of at least three sides if good seed production is to be realized.
Selection of trees should be done in small groups for ease of comparison. By dividing the stand into small plots, a more even spread of seed trees will be retained after final thinning. Trees that do not meet the desirable standard are marked for roguing. Some species such as Casuarinas are dioecious, i.e. trees are unisexual with separate male and female trees. Care must be taken to ensure that there are sufficient male trees left for pollination. A final stocking of 150-200 trees per ha is considered suitable for cross pollination and subsequent seed production in a wide range of commercial tree species.
Plus trees are individual trees of outstanding merit, initially selected on the basis of superior phenotypic characters like height, diameter, clear bole etc. Phenotypic selection of superior individuals forms the basic part of the most tree improvement programmes. Selection of superior phenotypes in forestry is most effective, as even a small genetic gain can be of enormous economic benefit.
Selected individuals are used as a base population for future breeding programme. The selection of superior parents from stands having uniform environment is of great importance. Selection from plantations gives more gain than from the natural stands.
Two factors are of paramount importance in developing a grading scheme for selection of superior trees. First the trait under consideration should be under at least moderate to strong genetic control. Second, the trait must have considerable economic value. Growth rate is nearly always key characteristics in a selection programme.
Stand Selection:
Within a provenance/seed zone, selection starts with designation of suitable stands. Stands should be chosen on several site classes to avoid selection of trees adapted to narrow range of environmental conditions. On the contrary selection of stands on better sites is also justified because these are the sites that will have priority in regeneration programme.
Select middle aged stands, close to expected future rotation as selection in stands of present rotation age will not lead to identification of genotypes that perform best under management conditions. If choice is to be made between even aged and uneven aged stands, the former should be preferred because they allow more accurate tree comparison.
Number of Plus Trees:
The number of plus trees selected depends upon overall breeding plan for the selected species. A large number of plus trees provide a broad genetic and avoid inbreeding. The trees can be selected within few years. A breeding/ production population should be at least consisting of 20-30 clones/genotypes.
Choice of Traits:
The characters to be improved may vary from species to species and objectives of the programme. Selection should be restricted to few traits as too many traits will increase the cost. Traits with long term economic value should be chosen. Improve only those traits which cannot be improved through silviculture or improved industrial technology.
The general characteristics of tree for timber purpose should be:
(i) Rapid growth,
(ii) Straight bole,
(iii) No forking,
(iv) Branches: thin, small, slow growing,
(v) Narrow crown,
(vi) Self pruning habit,
(vii) Quality timber, and
(viii) Tolerant to diseases and pest.
Methods of Plus Tree Selection:
The procedure used for selection of plus tree in the selected stands will depend upon number of factors like type and number of traits considered, heritability’s, selection intensity, stand structure etc.
Based on Van Buijtenen (1969), following are the methods of selection of plus trees:
i. Ocular Selection:
Vigorous, healthy trees of good form are rapidly identified without measurement or rating of individual traits. This procedure is adopted in both the situations where heritability of character is either low or high. It is applicable when selection is restricted to very few traits.
ii. Comparison Tree:
The method is suitable in plantations and natural stands of uniform age. A candidate tree which is phenotypically superior in chosen traits is compared with its nearest neighbours, or average or best trees in the stand. The comparison trees should be selected from dominant or co-dominant crown with similar age and site conditions approximately with 100 meter range from the candidate tree. The superiority of candidate tree over the average of comparison trees is worked out for each trait. The candidate tree is designated as plus tree if it proves superior to comparison trees; otherwise it is rejected.
iii. Regression Method:
This method is applied for uneven aged or mixed species forests. This requires the development of tables relating the desired character with age. Regression line is developed for a stand or site, relating the expression of characters to tree age by sampling a number of trees.
The sampling requires the trees of different ages. Different regressions are developed for different sites. Once the regression curve has been developed, the candidate values are plotted against the regression line and the trees that meet the minimum selection standards or are above the regression line are selected as plus tree. This method cannot be used for those species whose age cannot be determined.
The regression system of selection is particularly suitable for all aged or mixed species stands. It consists of developing a curve of production (growth is illustrated) for different ages of trees on a given site. Candidate Tree A falls above the curve, therefore, it has the desired growth for its age.
Tree B is average, therefore, its use depends on other characteristics, whereas Tree C has inferior growth for its age and should not be used. The regression line should be based on at least 50 trees if the age spread is considerable.
The Mother Tree System:
It consists of locating good trees. Seed is obtained from these trees to lay out progeny trials. On the basis of progeny trials, either the best parent trees or best trees of best families can be used in a vegetative seed orchard. The progeny test may also be converted to seedling seed orchard. This type of selection is used in situation where comparison tree or regression method cannot be used.
The main problem with this method is that time is lost in progeny testing before establishment of seed orchard for commercial seed production. However, this method is highly suitable for hardwood where planting programmes are small and seed is not immediately needed.
Step # 3. Progeny Testing:
Once plus trees are selected on the basis of their physical appearance and planted in a seed orchard, their genetic worth can be known only on the basis of performance of their progeny. The relative contribution of genotype and environment towards phenotype of the selected tree can be determined through progeny test.
The objectives of progeny testing are:
i. To evaluate family lines for the purpose of rouging out seed orchards;
ii. To establish plantings from which to make second generation selections.
Progeny testing is advantageous in case where most of the variation is environmental. One can make substantial gains by separating the total variation in environmental and genetic components through progeny testing. Progeny trials are either half sibs or full sibs. In half sib progeny trials, open pollinated seeds are collected from selected parents to lay out trials for assessment of General Combining Ability (GCA). In full sib progeny trials, controlled cross pollinated seeds are collected to lay out trials for the assessment of Specific Combining Ability (SCA). A summary of advantages and disadvantages of different progeny testing method is given in Table 18.5.
Location, Establishment and Management of Progeny Test:
i. Location:
The location where progeny test is to be carried out should be chosen carefully. It should represent the area where commercial planting is to be carried out in future. It is important to consider both climatic and soil characteristics when selecting the site. Uniformity of site is very important. Even in the most uniform site, there are difference in soil composition, drainage and fertility which makes it necessary to repeat the planting several times. The progenies may be tested on more than one site.
ii. Establishment:
Site preparation activities must be more intensive than done for commercial plantings. The objective is to create as uniform as an environment as possible so that genetic differences can be detected at an early age. Land should be cleared from other vegetation. Spacing should be wide enough to allow for maintenance during initial years.
The seedlings in the nursery should be raised under uniform conditions so as to provide equal advantage to all the entries. Similarly utmost care need to be taken while transporting the planting material from nursery to planting site so that the seedlings are not put under any kind of stress.
iii. Experimental Designs:
As indicated above, the site conditions are not always uniform and the progenies have to be properly evaluated. It is necessary to minimize the environmental factors operating on the progenies. Many experimental designs have been developed to control the environmental effects in the experiments.
However, the most commonly used design is the randomized complete block design or incomplete block designs. Replication should be done in both space and time. A poorly replicated plot on a highly variable site will yield little reliable information. Progenies within a replication should be properly randomized. Border rows should be planted to remove the edge effects.
iv. Plot Size:
In the past, progeny tests were conducted using the plot size as in the provenance trials that are square plots containing 25-100 trees. This will often result in the large size of the plots thereby increasing variation among the plots. To overcome this problem, in many programmes, progeny tests are conducted using row plots.
Use of small plots ensures that the genetic differences among the progenies are accurately evaluated. Row plots are easier to plant and measure than traditional square or rectangular plots. It is often desirable to use 10 tree row plots and six replications at the beginning of the testing programme.
v. Management:
Usually progeny trials run for half or full rotation age. Therefore, it is imperative that proper labeling and documentation of the trials in the field as well as in the office is done. The maps of the site, layout of the trial should be properly maintained. The trial should be protected from fire by establishing fire lines around the test.
Diseases and insects should be controlled as per standard protocols. Thinning’s may or may not be done in the progeny test. If thinning is carried out, proper records should be maintained. The measurement of height, diameter etc. in the trial is done periodically. Data should be recorded accurately.
Step # 4. Advance Generation Breeding:
Mass selection (Seed production area/ plus tree selection) is carried out in the natural population or plantations which are the source populations. The selected individuals are used to establish seed orchards. The seed orchards are rouged on the basis of progeny test. Information from progeny test is used to selected individuals which are intercrossed using different mating designs to develop populations which are source of second and third generation seed orchards.
Advance generation populations also provide estimates of genetic parameters such as additive and non-additive genetic variation, heritability, genetic gain and production of commercial end product. In improvement strategy, for every generation, an interim seed orchard has been created, which is improved through grafting of selected material following inter-mating of good combiners.
Broader genetic base is maintained through infusion of new entries from the unimproved populations in later generations. Tree improvement programs consist of two main lines of endeavor, the operational phase and developmental phase, as indicated. The quality of the breeding clone bank will determine long-term success and gains from the production seed orchards determine immediate success.
Step # 5. Genetic Engineering:
Traditional approaches to tree improvement have involved the identification of mature trees with desirable phenotypes, followed by their incorporation into breeding programs. The length of time needed for trees to reach reproductive maturity, often in excess of 20 years, before controlled crosses can be made is the limiting factor for tree improvement. Furthermore, there is no guarantee that the desired progeny phenotypes will be identified. The application of biotechnology can overcome many of the drawbacks associated with conventional breeding strategies.
This may be achieved by the use of genetic modification technologies. A Genetically Modified (GM) tree (sometimes referred to as a genetically engineered tree or a transgenic tree), can be defined as a tree that through human intervention in a laboratory, has had its genome, or genetic code, deliberately altered through the mechanical insertion of a specific identified sequence of genetic coding material (generally DNA) that has been either manufactured or physically excised from the genome of another tree. The new genes are passed on to future generations. Genetic modification may be used to alter or introduce a wide range of traits, including insect and disease resistance, herbicide tolerance, tissue composition and growth rate.
The first GM tree trial was carried out in Belgium in 1988. Since that time many tree species, both for fruit and timber production, have been genetically modified and tested in field trials. Trials have been conducted in Europe, North and South America and New Zealand, principally for facilitating pulp production and for increasing productivity. In China the State Forestry Administration has approved GM poplar trees for commercial planting and one million insect resistant GM poplars have been planted. There is enormous potential for speeding up tree breeding cycles by the use of genetic modification.
Systems have been developed for gene transfer, selection of novel gene-containing shoots and stimulating regeneration for both broadleaved and coniferous trees. The systems use either the soil-based microorganism Agrobacterium or DNA-coated particle bombardment, known as biolistics. Two species from the genus Agrobacterium (A. tumefaciens and A. rhizogenes) have been used for gene transfer into trees.
A small section of the bacterial DNA, known as T-DNA, is transferred into and expressed within the plant nuclear genome. Agrobacterium tumefaciens, the causative agent for crown gall disease, is widely used after the functional deletion of the auxin and cytokinin biosynthetic genes from the Ti, or tumor-inducing, plasmid. A separately replicating plasmid, known as a binary vector, can be introduced and used to deliver desired genes into the plant cell, from which phenotypically normal transgenic trees can be regenerated.
Agrobacterium rhizogenes, responsible for hairy root disease, transfers genes from root- inducing, plasmid. Applications of genetic modifications to trees include altered wood properties, speeding up breeding cycles, forests as pharmaceutical factories, dendroremediation and improved resistance to pests and diseases, as well as the restoration of sensitive landscapes.
But the application of GM technologies to trees has raised a number of potential public concerns. Many of these concerns are the same as those raised for GM annual crop plants. This includes the potential for spread of antibiotic or herbicide resistance genes to other non-target species from GM trees; the potential for long-distance pollen spread over many years from long-lived trees; the potential for adverse effects on biodiversity from forests of GM trees etc. Although so far adverse effect of GM trees on environment has not been found, still the issue is debatable and needs much more research on safety of GM trees.
Molecular Techniques:
Traditionally genetic variation was assessed through morphological, growth and cytological characteristics. The problem with morphological and growth characteristics is that they are highly influenced by environment. They reflect only part of the genetic variability since only functional genes are taken into consideration.
With the advent of isozymes and other biochemical markers, the effectiveness in estimating genetic diversity increased tremendously. However isozymes are handicapped by low polymorphism and influence of isozyme variation by environment to certain extent. Now DNA based molecular techniques such as Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR) are being increasingly used to detect genetic variation in populations.
RAPD utilizes Polymerase Chain Reaction (PCR) to screen the DNA for detecting variability. Polymorphism i.e. difference between two genotypes with reference to particular location in the DNA results from changes that effect amplification of DNA located between two primers. These changes include most probably single base substitution as well as deletion or insertions that either changes the primer sequence or the size of the amplified DNA.