Recurrent Selection

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Recurrent Selection

Overview
The goal of recurrent selection is to improve the mean performance of a population of plants; a secondary goal, but nevertheless very important, is to maintain the genetic variability present in the population to the extent possible.

Progress in selection is based on the heritability of the trait and the types of genetic variation controlling the trait in the particular population under selection and on the selection differential.

We will initially discuss various recurrent selection methods, and then discuss the genetic parameters that are associated with them, as a means to differentiate between them.

The generalized method consists of three parts: (1) Development of a base population with which to begin selection, (2) Evaluation of individuals from the population, and (3) Selection of superior individuals and intercrossing to form a new population.

Evaluation of individuals in the population

Individuals can be evaluated in one of two ways:

Phenotypic evaluation considers the individual plant per se, so that no evaluation of the actual breeding value of the individual is possible.

In contrast, genotypic evaluation is based on the performance of the progeny of the plant under evaluation, and therefore, provides information on the individual’s breeding value. Three types of progeny may be evaluated: (1) half-sib progeny, (2) full-sib progeny, and (3) selfed progeny. Half-sibs are formed by crossing the individuals to be evaluated to a common parent (which can be a population, an inbred line, or a specific genotype), called a tester; thus, all half-sib families have the tester in common.

To produced full-sib families, the individuals to be evaluated are crossed in pairwise combinations–thus, full-sib families do not have any parents in common, although all the parents come from the same population. Finally selfed progeny are produced by self-fertilizing the individuals to be evaluated for one or two generations. These partially inbred progeny are evaluated.


Methods of intrapopulation improvement

Recurrent phenotypic selection
Recurrent phenotypic selection (RPS), also termed mass selection (because this term is used in other situations, I prefer to not use it here), is the simplest form of recurrent selection. Typically, RPS has three stages: (1) a population of plants is planted in a nursery in a manner that allows notes to be taken on individual plants, (2) the population is evaluated for the trait/traits of interest and the best individuals are identified, and (3) seed is harvested from the selected individuals to constitute a new population from which the next cycle will begin.

A major determinant in rate of progress is parental control; that is, is the seed that is harvested from the selected plants the result of pollination from only those selections (i.e., both parents are controlled), or is it formed from pollen derived from all the plants in the nursery, both selected and unselected (i.e., only the female parent is controlled).

The former should theoretically double the rate of progress (i.e., the genetic gain from selection). However, in some instances, control of both parents is not possible. Consider selection for grain yield in maize. Evaluation can only occur after pollination, obviously, so when the best ears are selected from individual plants, clearly the pollen has derived from the entire population, and only the female parent is controlled. Perennial plants, in contrast, can be dug up from their location and all selections can be transplanted to a isolated crossing nursery (called a polycross nursery) where the plants are typically replicated to ensure random intercrossing among all the selections, or to the greenhouse to enable hand crossing among them.

Selection of plants spaced widely apart suffers from two major problems. First, the spaced planting may not reflect the density of commercial production, and if the correlation between performance in space planting and performance under commercial density is low, then selection will be ineffective and progress slow or non-existent.

Second, single plant performance can be significantly affected by variation in the field. Thus, if for some environmental reason plants perform better on one side of the nursery than the other, phenotype-based selection will be ineffective because the phenotype is undually affected by the environment, not the genotype. One way around this problem is gridding, in which the field is divided into a number of equally sized subdivisions. Selections are then made within each grid, such that an equal number of individuals are identified in each subdivision, for example, five plants within each 5 x 5 grid.

As suggested by the example of gridding, RPS can be improved by making small adjustments that enable better evaluation of phenotypes. Further improvements to the RPS system can be envisioned, and Burton {, 1982 #829} summarizes a number of modifications that he has adapted to selection of forage yield in bahiagrass. He terms his program, Recurrent Restricted Phenotypic Selection (RRPS), because he has placed restrictions on how he handles his material to ensure the greatest gain. Gridding is just one of many such restrictions. His method is a classic example of practical breeding–attention to the smallest detail and hard work, as his last sentence says, have resulted in the most effective forage breeding program in the world.

Selection will undoubtedly result in diminishing genetic variability, particularly if few genes are involved in the trait, because as they become fixed, genetic variability goes down. Selection is most effective when allele frequencies are near 0.5; if the desirable alleles are below 0.5, increasing their frequency will lead to increased genetic variation for the trait they control, until their frequency passes 0.5.

Recurrent half-sib selection
The general scheme for recurrent half-sib selection (RHSS) is to cross individuals being evaluated to a common tester, evaluate the half-sib progeny of each plant, select the best individuals, and intercross them. RHSS can be conducted for either general or specific combining ability. General combining ability (GCA) refers to the average performance of an individual’s progeny, which are developed by crossing that individual to a variety of other individuals. If the tester being used is a population of plants, then the progeny performance represents the parental plant’s GCA. If the tester were an inbred line, then the progeny performance would represent specific combining ability (SCA), because it would represent the ability of a particular plant to combine with a specific inbred rather than with a broad selection of genotypes.

The earliest RHSS program used the ear-to-row method (Hopkins, 1899). For this method, he produced half-sib seed by interpollinating a number of individuals from a population; thus, the population per se is the tester, and selection will be for GCA. The ears thus represent half-sib families, with the pollen coming from the entire population.

The ears were then planted into a single row in an unreplicated trial in one environment (hence, literally ear-to-row), and selection was based on this trial. (Note that this evaluation is not unlike that for RPS, except that here we’re evaluating progeny performance rather than single plants). The ears of selected plants, which received pollen from the population (although this was skewed toward more pollen from the neighboring half-sib families rather than a random sample of the population’s alleles), were then planted into rows next year. Note that if two seasons of maize could be grown in a single year, two cycles of selection could be completed per year. This was a simple, though not particularly effective, selection method.

The modified ear-to-row method (Lonnquist, 1964, and Fig 15.5 in Fehr, 1987) made a number of alterations to Hopkins’ method to avoid some of its shortcomings. First, testing the half-sib families is done at three locations with one replication per location. At two of these locations, data is collected and plants are harvested as normal done. At the third location, the half-sib families are planted in isolation. Between half-sib families, rows consisting of a bulk of all half-sib families are planted. The half-sib families are detasseled and the bulk rows are used as males to effect pollination.

Selections are based on the data from the first two locations. From the selected families in the isolation, the five best ears are selected and bulked to form the next cycle; these will be the half-sib family rows next season. These modifications still only control the female parent, but at least the pollination occurs with the entire population rather than just neighboring half-sib families. The within row selection is likely only moderately effective at best, but it may add a bit to the progress.

A better approach to those discussed above would be to control both parents (Jenkins, 1940 and Fig 15.6 in Fehr, 1987). In order to effect this, however, would require an extra season–that is, evaluations need to be conducted, and then, based on those evaluations, the parents would be intercrossed. Unless parents can be maintained in the field (as with perennial forage or turf species), the intercrossing needs to be done with remnant seed; if half-sib seed is to be used for evaluation, then some can be held in reserve to be used for crossing if that parent is selected. Intercrossing of selections is done as described for the modified ear-to-row method above, except that the male rows consist of a bulk of only the selected half-sib families. The intercrossing can be done in an off-season nursery, so that a cycle of selection can still be completed in one year.

Selection within half-sib families during the intercrossing phase cannot be done if an off-season nursery is used, simply because that nursery is in an environment outside the target region of the crop. (So, to be more specific, selection could be done, but most likely, it would not be advantageous to advancing the genetic potential of your populations.) However, if intercrossing is done in the target environment, either because two seasons per year can be grown there or because no winter nursery is used, then selection could be practiced within half-sib families. Of course, this is essentially RPS within families, and selection based on unreplicated single plants is not the most effective type of selection, but some additional gain might be possible, depending on the trait being selected. A further discussion of this topic with forage crops as the primary focus is Vogel and Pedersen {, 1993 #38}.

Now, even though intercrossing based on remnant half-sib seed of selected plants is better than only controlling the female parents, the half-sib seed being intercrossed only contains half of its alleles from the selected parent; the remainder comes from the population as a whole, which includes a preponderance of nonselected plants. Thus, an even more effective intercrossing method would be to use selfed seed rather than half-sib seed. (Again, if the plants themselves can be maintained indefinitely, or can be cloned, then using them is clearly the best strategy of all.)

The difficulty here is in getting both selfed and half-sib seed from the same plant. In some species, this would not be a problem. For maize it is. Two alternatives exist in maize. First, if the maize population reliably produces two ears, then one can be used for selfing and other for producing ½ sib seed. Many maize populations do not have this ability, so in this case, you can self pollinate the single ear on the individual to be tested and use pollen from that individual to pollinate several individuals of the tester. The ears on the tester, then, represent the half-sib family to be tested. Recombining selfed progeny will require three seasons: (1) selfing and crossing to the tester, (2) evaluation, and (3) intercrossing selfed progeny. This will lengthen a cycle of selection. The benefit gained from using selfed rather than half-sib seed may be offset by the longer selection cycle. More on this subject will be discussed in the next section.

Other types of testers besides the population per se can be used. If the population per se is the tester, then the methods discussed above can be completed in the shortest time if half-sib seed is used for recombination. If a different population or an inbred line is used as the tester, then following recombination, individuals need to be crossed to the tester, necessitating three seasons to complete a cycle, similar to using selfed seed for recombination. Thus, the tester chosen can influence gain per cycle. Note that using an inbred tester could pose a problem if the population developed does not combine well with a different tester.

Recurrent full-sib selection
Recurrent full-sib selection (RFSS) tests paired plant crosses rather than crosses of individual plants with a population (or inbred) tester. For this method, paired crosses are made between individuals in the population in season one. Evaluations are conducted in the field the next season and the best families are identified. The third season is used to recombine the best families using remnant seed from the first season. Thus, the second selection cycle begins in the fourth season, with paired crosses between individuals. To streamline procedures, paired crosses could be done during the third season, combining recombination of the best families with full-sib family development for the next evaluation cycle. Thus, paired crosses between individuals from the selected families can be made.

The disadvantage of this is that less recombination occurs between cycles of selection. The obvious advantage is that it allows a cycle to be completed each year, with the use of a single off-season nursery.

Recurrent selection among selfed families
a. Evaluation is based on self-pollinated progeny, usually formed by one generation of selfing, although two generations has also been used.

b. Season 1: S0 plants from the population are selfed to produces S0:1 lines.

c. Season 2: S0:1 lines evaluated, best lines selected

d. Season 3: Remnant S1 seed used to intercross selected lines, producing the Cycle 1 population with new S0 plants.

e. Season 4: Self S0 plants as in Season 1.....

f. Many modifications are possible. Could test S1:2 lines or S0:2 lines, depending on the desire of the breeder. The latter (S0:2) is useful if you cannot produce enough S0:1 seed for testing.

Methods of interpopulation improvement

Reciprocal recurrent selection
designed to improve both the general and specific combining ability of two populations. The general idea is to test and select in each of two populations, using the opposite population as a tester. Alleles do not migrate between populations, however!

2. Two starting populations should be genetically divergent (although selection will probably drive them apart anyway)–the point is that they should combine well together.

Reciprocal half-sib selection
a. Season 1: Individual plants (e.g., 100) from each population are (1) selfed and (2) outcrossed to several (e.g., 6) randomly chosen plants from the opposite population to generate ½ sib families

b. Season 2: 100 ½ sib families from each population are evaluated in replicated tests; top 10 are identified

c. Season 3: Go back to selfed seed of superior individuals and intercross to form Cycle 1 population within each population. DO NOT mix the selections of the two populations! Note that if the plants can be maintained clonally, you could go back to the original plants and intercross, which would mean you wouldn’t have to produce S1 seed in Season 1.

d. Season 4: Begin next cycle using Cycle 1 seed of each population.

e. Modifications:
i. Use inbred testers developed from the opposite population. The inbred tester would be replaced by superior ones as the program progressed.

ii. Produce half-sib seed of each population as in ear-to-row selection. Then plant half-sib families in alternate rows with the opposite tester population, detassel the half-sib lines and use the open pollinated seed for testing. The best lines are then intermated using the remnant half-sib seed of each parent. The disadvantage is that only the female parent is controlled.

Reciprocal full-sib selection
a. Used when the desired outcome is commercial hybrid seed.

b. Season 1: 200 phenotypically desirable S0 plants in Pop A paired with 200 from Pop B. Each plant is both selfed and crossed to produce S1 and full-sib seed. Selfed seed can be stored for intercrossing of desirable parents and also advanced to develop inbred lines.

c. Season 2: Evaluation and selection

d. Season 3: Intercrosss selfed seed of top 20 individuals from each population–>Cycle 1 populations for A and B.

e. Season 4: Begin again.

f. Alternative if can’t produce both selfed and crossed seed at the same time: use S0:1 lines to make crosses. Adds a year.

Importance of mode of reproduction
The mode of reproduction of a crop determines its genetic composition, which, in turn, is the deciding factor to develop suitable breeding and selection methods. Knowledge of mode of reproduction is also essential for its artificial manipulation to breed improved types. Only those breeding and selection methods are suitable for a crop which does not interfere with its natural state or ensure the maintenance of such a state. It is due to such reasons that imposition of self-fertilization on cross-pollinating crops leads to drastic reduction in their performance. Likewise, it is practically impossible to maintain permanent heterozygosity in self-fertilizing crops rendering the development of hybrids an unattractive breeding approach. Asexual propagation is another type of reproduction where any plant or part of it can be used for multiplication without even a slight genetic change from generation to generation. The methods of breeding and multiplication for such crop are thus entirely different than those of sexually reproducing crops.

Self fertilizing crops (autogamous crops)
Certain restrictions caused the mechanisms for self fertilization (partial and full self fertilization) to develop in a number of plant species.

Some of the reasons why a self fertilizing method of reproduction is so effective are the efficacy of reproduction, as well as decreasing genetic variation and thus the fixation of highly adapted genotypes. Most of the loci get fixed at a high rate; this can be ascribed to the fact that with each generation of self fertilization the rate of heterozygotes decreases by 50%. Homozygosity will thus be obtained in 5-8 generations. The 3rd reason for the efficacy of self fertilization is that in mixed stands of self and cross pollinating crops, the self fertilizing plants can donate pollen to both plant types, where the cross fertilizing plants are restricted concerning the contribution it can make to the population with regard to pollen donation.

Almost no inbreeding depression occurs in self fertilizing plants because the mode of reproduction allows natural selection to take place in wild populations of such plants. Thus, the genetically non-superior or unstable plants are removed from the population at an early evolutionary stage. Populations derived from self pollination are sometimes not as evolutionary adaptable as with other reproductive methods, but are known to utilize specific ecological niches more effectively.

Critical steps in the improvement of self fertilizing crops are the choice of parents and the identification of the best plants in segregating generations. The breeder should also have definite goals with the choice of parents. Self fertilizing cultivars are easier to maintain, but this could lead to misuse of seed.

Some of the agronomically important, self fertilizing crops include wheat, rice, barley, dry beans, soy beans, peanuts, cotton, tomatoes, etc. The basic methods by which new genetic variation can be established in self fertilizing crops are by means of introduction, selection and hybridization.

A few different selection methods are applied in self fertilizing crops, such as mass selection, single plant-, pedigree-, bulk population-, back cross-, recurrent-, F1 hybrid, as well as single seed descend (SSD) selection. In most breeding programs a combination of these methods are applied. The different selection methods can be summarized as follows:

Mass selection
This method of selection depends mainly on selection of plants according to their phenotype and performance. The seed from selected plants are bulked for the next generation. This method is used to improve the overall population by positive or negative mass selection. Mass selection is only applied to a limited degree in self fertilizing plants and is an effective method for the improvement of land races. This method of selection will only be effective for highly heritable traits. Some of the shortages of mass selection are that it is not possible to know whether selected plants are hetero- or homozygous.

Heterozygous plants will segregate in the next generation and it may thus necessitate repetition of phenotypic selection. Secondly, the influence that the environment has on growth of the plants includes development, phenotype and performance. It is not always clear whether the phenotypically superior plants are also genotypically superior and strong environmental differences may lead to low selection efficacy.

Single plant selection (pure line selection)
A variety developed by this method will be more uniform than those developed by mass selection because all of the plants in such a variety will have the same genotype. The seed from selected plants are not added together but are kept apart and used to perform offspring tests. This is done to study the breeding behaviour of the selected plants. The high uniformity in stand and performance has been stressed in the past, but the risk of highly specialized pathogens evolving is very high. More genetic variability could buffer the crop against such pathogens as well as stability of production under varied environmental conditions.

Selection Methods for the Development of Pure Breeding Cultivars from Crosses
Crosses between varieties, germplasm introduction and breeding lines are made to create new gene combinations. In the generations to follow, superior gene combinations are selected and fixed in the homozygous state by means of self fertilization and selection. These selections are tested extensively with the goal of releasing them for cultivation.

Pedigree selection

Parental lines are crossed and selection of plants with new gene combinations already takes place in the F2 generation (the generation of plants formed from crossing F1 hybrids). The offspring of selected populations in the generations to follow are repeatedly subjected to selection until genetic uniformity is reached. Records are kept of the origin of the selected individuals or lines. The amount of generations of single plant and line selections, as well as selection intensities, can be varied in practice according to the crop and availability of facilities.

It is usually traits with high heritability that are quick and easy to measure that are concentrated on when using this method of selection. One of the main objections against this method is that the genetic variation, available for selection of quantitative traits, are drastically decreased in later generations. Seed purification and multiplication is usually incorporated in one of the final generations of pedigree selection. This method is very labour intensive.

Breeding efficiency is one of the goals with early generation testing. This is done by early identification of superior heterogeneous populations. The early elimination of inferior populations and subsequent concentration of selection efforts within superior populations is assumed to result in increased efficiency. Accurate evaluation of heterogeneous populations is essential to the success of this method and assumes that transgressive segregants from inferior populations will not exceed selections from superior populations in performance.

Bulk population selection
With this method of selection the offspring from a crossing are planted at planting densities equal to commercial planting densities. During this period, which may include a number of generations, the level of homozygosity in the bulk population increases.

This method is simple and cheap and involves less work than pedigree selection in the earlier generations. It is necessary to plant large populations to ensure that the best segregates are selected when selection starts. Segregating generations are subjected to another single plant selection step. Fewer records are kept during earlier generations than with pedigree selection. This type of selection is especially carried out with crops which are usually planted at high planting densities, e.g. small grain crops.

Single seed selection
This method was introduced to keep as many F2 plants as possible intact and thus prevent the loss of variation of traits with low heritability in earlier generations. This method is also used to decrease the time that is needed to grow segregating generations. Because only one seed is harvested per plant, it is not necessary for optimal plant development and conditions can be manipulated so that 2-3 generations can be harvested per year. This can cause the process from the starting to finishing time to decrease by 1-3 years.

This method does not eliminate weak plants such as in other methods and there is also no provision for selection of superior plants in the F2 generation. Modification of this method is possible and record keeping is not necessary in early generations.

Doubled haploid method
Haploid plants may be produced by chromosome elimination in wide crosses, ovule culture or by anther culture. Anther culture, however, is mostly used because of its ability to produce haploid plants with much larger quantity compare with the other two methods. Stresses are usually necessary to alter the development pathways of microspores from producing pollen to forming haploid plants.

The chromosome numbers of the haploid plants are then doubled with the use of colchicine. Spontaneously doubled haploid plants, however, can also be produced directly from the three methods. Embryo rescue methods can be used to ensure that seed from these wide crosses or stigma culture plants survive and doesn’t get aborted. This method has the potential of shortening genetic improvement cycles in comparison to pedigree or bulk methods. Like the single seed selection method, early generations are not subject to selection, but most of the lines are eliminated during the land evaluation trials.

This method is very labor intensive and the most expensive of the procedures that increases the amount of generations per year. For this method to be successful, the plants must be genetically stable.

Back crossing: a selection method for the upgrading of genotypes
This is a type of repeated selection where a specific gene can be incorporated into otherwise superior cultivars. One of the parental varieties is highly productive and commercially successful but lacks a specific gene (e.g. disease resistance). This trait is usually present in the other parental variety. After each back cross, hybrid plants are identified with the gene under consideration and are back crossed again with the repeating parent.

This technique is easy when traits are added which are easily inherited, dominant, and easily identified in hybrid plants. If closely linked, unwanted genes are present with the useful genes, the unwanted genes could be transferred together and the offspring may actually be less productive. An advantage of the back crossing method is that extensive testing is not necessary. This method is used to create hybrid varieties in self fertilizing crops and to establish male sterility in parental lines.

Marker-assisted backcrossing is routinely applied in breeding programs for gene introgression.

Traditional selection methods
A Relatively small gene pool is created with traditional selection methods, there is not a lot of opportunity for gene recombination and few opportunities for the breakage of linkage blocks. Breeders are looking to overcome these shortcomings by increasing the amount of crosses made, as well as to start testing for performance in earlier generations and to introduce systems for improvement of populations (e.g. recurrent selection).

Other breeding strategies include multilines (a composite of genetically identical lines which have different genes for example stem rust resistance). Lines that are basically genetically identical, except for a single gene, are called isogenic lines. Mixed varieties (composites) are also used and are less uniform than pure varieties should be revised in regular intervals to prevent shifts in the proportion of components. Recurrent selection is most often used in cross fertilizing crops, but can be successfully used in self fertilizers only when a male sterility gene is present. Hybrid varieties are a very important method in self fertilizing crops and have the following advantages:

Heterosis
Easier to create varieties with multiple resistance genes
Attractive method for private breeders because of built-in mechanisms for protection of varieties.

Raising hybrid seed has been one of the major goals of horticultural and agricultural practice, because hybrid plants are more productive (due to hybrid vigour) and more uniform in quality than plants derived from self-pollination or random pollination. To raise hybrid seed, self-pollination and sib-pollination (pollination by a plant of the same hybrid) must be circumvented. One method is hand emasculation of the line used as female parent, which is then naturally cross-pollinated by pollen from the line serving as male parent and planted in an adjacent row. However, this process is very labour intensive and invariably expensive. If the crop plants can be made self-incompatible by the introduction of the genes controlling self-incompatibility, then all seeds produced will be hybrids resulting from cross-pollination between two different lines. This would facilitate the production and increase the yield of hybrid seed and, at the same time, reduce the labour costs.

Selection of cross-pollinated crops
The natural state of self-fertilizing crops is homozygosity and genetic uniformity, whereas cross-fertilizing crops are characterized by a high degree of heterozygosity. Plant species where normal mode of seed set is through a high degree of cross-pollination have characteristic reproductive features and population structure.Existence of self-sterility, self-incompatibility, imperfect flowers, and mechanical obstructions make the plant dependent upon foreign pollen for normal seed set. Each plant receives a blend of pollen from a large number of individuals each having different genotypes. Such populations are characterized by a high degree of heterozygosity with tremendous free and potential genetic variation, which is maintained in a steady state by free gene flow among individuals within the populations. It is inappropriate, and could be rather hazardous, to take one or a few individuals to investigate or improve these populations. The enhanced fitness of heterozygotes over homozygotes of cross-pollinated crops has been manipulated in the form of two different breeding approaches namely, population improvement and hybrid breeding in such crops. In the development of hybrid varieties, the aim is to identify the most productive heterozygote from the population, which then is produced with the exclusion of other members of the population. In contrast, the population improvement envisages a stepwise elimination of deleterious and less productive alleles through repeated cycles of selective mating of genotypes that are more productive. Population improvement is slow, steady and a long-term program, whereas the production of hybrids is aimed to maximize the genetic gains in much less time. Both of these breeding approaches are complementary rather than mutually exclusive and are based on sound genetic theory. The different selection methods can be summarized as follows:

Mass selection
It is the simplest, easiest and oldest method of selection where individual plants are selected based on their phenotypic performance, and bulk seed is used to produce the next generation. Mass selection proved to be quite effective in maize improvement at the initial stages but its efficacy especially for improvement of yield, soon came under severe criticism that culminated in the refinement of the method of mass selection. The selection after pollination does not provide any control over the pollen parent as result of which effective selection is limited only to female parents.

Recurrent selection
This type of selection is a refined version of the mass selection procedure and differs as follows:
Visually selected individuals out of the base population undergo progeny testing
Individuals selected on basis of the progeny test data are crossed with each other in every possible way to produce seed to form the new base population.

Half-sib selection with progeny testing
Selections are made based on progeny test performance instead of phenotypic appearance of the parental plants. Seed from selected half-sibs, which have been pollinated by random pollen from the population, is grown in unreplicated progeny rows for the purpose of selection. A part of the seed is planted to determine the yielding ability, or breeding value, for any character of each plant. The seed from the most productive rows or remnant seed from the outstanding half-sibs is bulked to complete one cycle of selection.

Full-sib selection with progeny testing
A number of full-sib families, each produced by making crosses between the two plants from the base population are evaluated in replicated trials. A part of each full-sib family is saved for recombination. Based on evaluation the remnant seed of selected full-sib families is used to recombine the best families.

Selections with test cross performance
The purpose of this type of selection is a slight deviation from the concept of intra-population improvement in the sense that the population is improved, not only for performance, but also with respect to combining ability with a specific reference population. It involves genetic modifications of the population with an aim on its better use for the exploitation of heterosis. It involves three steps:
Self-pollination and test crossing of individuals
Evaluation of test crosses in replicated trials
Recombination from selfed remnant seed of selected plants

Selfed family selection
The plants in the original base population are selfed to produce S1 progenies, which are evaluated in the next season in replicated multi-environmental trials to identify promising S1 families. The remnant S1 seed of such selected families is then recombined in the third season as a result of which one cycle is completed in three seasons. Hence, the units of selection and recombination are S1 progenies.

Breeding of Asexually Propagated Crops
Asexual reproduction covers all those modes of multiplication of plants where normal gamete formation and fertilization does not take place making these distinctly different from normal seed production crops. In the absence of sexual reproduction, the genetic composition of plant material being multiplied remains essentially the same as its source plant.

Clones of mother plants can be made with the exact genetic composition of the mother plant. Superior plants are selected and propagated vegetatively; the vegetative propagated offspring are used to develop stable varieties without any deterioration due to segregation of gene combinations. This unique characteristic of asexual reproduction helped to develop a number of cultivars of fruits and vegetables including grapes, apples, pears and peaches.

Improving asexual plant material through selection
The selection in these crops is restricted to the material introduced from other sources, such as field plantations. The promising selections are tested in large scale trials which, if successful, can be multiplied and released for commercial cultivation.The improvement of asexually propagated plants through induced mutations has distinct advantages and limitations. Any vegetative propagule can be treated with mutagens and even a single desirable mutant or a part of a mutated propagule (chimera) can be multiplied as an improved type of the original variety.

Selection of asexual plants
Selection, in the case of asexual plants, can be defined as the selection of the best performing plant and the vegetative propagation thereof. Because plants are not totally genetically stable, it can be expected that deviations would occur through the years. Selection is thus an ongoing process where deviants are selected or removed from the selection program. The main purpose of selection is to better the quality and yield of forthcoming plantations. Such as any breeder, the breeder has to have a good knowledge of the characteristics of the cultivar under consideration. Different approaches can be followed in the selection process of asexual plants, such as mass selection and clone selection from clone blocks.

In mass selection there are some factors that must be considered when selecting plants in a mother block, e.g. vineyard. Time of selection is a big factor, because you have to select when most of the characteristics of the plant are clearly showing. With asexual perennials the best time is just before harvest. For the best results the selected plant must be evaluated during the next season, when growth-abnormalities, leave disfigurations and virus symptoms are best visualized. Mass selection is done annually on the same plant for a minimum of three years. A plant that does not conform to the requirements in any given year of the selection cycle is discarded from the program.

Older plantations which were exposed to harsh growth conditions are seen as a preferred selection sources. The plants that grew under these circumstances and performed well are seen to have good genetic properties. In these older plantations natural selection took care of most poor performing plants.

New clone development
The development and registration of new clones take place by means of local clone selection in old plantations, as well as the importation of high quality clones from abroad, for local evaluation.

A clone is the vegetative offspring of one specific mother plant; it does not show any genetic, morphologic or physiologic deviations from the mother plant. Evaluation takes place with the different selected clones after selection. The dissimilar clones are compared to each other to determine their quality and resistance capabilities. Breeding is not involved in clone selection; the clone cannot be bred for resistance of certain types of viruses, emphasis must be put on making sure that the clone material leaving the nursery are virus free. Techniques are developed to test the clones for any harmful viruses.
Harmful viruses sometimes do not show in the preliminary evaluations. Phytosanitary development (virus detection and virus eradication) is thus performed in laboratories and greenhouses, parallel with field- and quality evaluation in field clone trials.

When a clone complies with the minimum quality and phytosanitary standards prescribed by the Plant Improvement Association (PIA), it is officially registered for certification and commercialization. The PIA is an association that complies for all plant improvement, including grapes, apple, pear and peaches.

Clones are made of cuttings from a field-grown mother plant. Due to bad management and infection from neighbouring plantations there is only a few virus free mother plants in selecting plantations. The clone developers had to incorporate techniques such as tissue cultures and in vitro propagation to develop virus free clones from the limited mother material. The apical meristem is free of any harmful viruses. By using the apical meristem for tissue culture a virus free clone can be developed.

Multiplication
During the first phase of multiplication, a nucleus cutting from each candidate and/or registered clone is kept in a PIA approved insect free nucleus block green house. From here all future multiplication and evaluation will be done. During the second phase of multiplication, rootstocks and grafted cuttings are established in foundation blocks in insect free facilities and open field isolated areas, from where further evaluation are done.

The scion and rootstock material from the second phase source are grafted and callused. The grafted plants are then planted in isolated areas for the establishment of mother blocks. These blocks are used for multiplication purposes. The third phase is the establishment of scion mother blocks from the above mentioned source on farms of contracted collaborator producers in pre-selected virgin soil of which about 4 km² are maintained.

Summary of Essential Points of Breeding

1 - The genotypes of plants are controlled by genes which are passed on unchanged from generation to generation.

2 - Genes occur in pairs, one from the gamete of the staminate parent and one from the gamete of the pistillate parent.

3 - When the members of a gene pair differ in their effect upon phenotype, the plant is termed hybrid or heterozygous.

4 - When the members of a pair of genes are equal in their effect upon phenotype, then they are termed true-breeding or homozygous.

5 - Pairs of genes controlling different phenotypic traits are (usually) inherited independently.

6 - Dominance relations and gene interaction can alter the phenotypic ratios of the F1, F2, and subsequent generations.

Polyploidy

Polyploidy is the condition of multiple sets of chromosomes within one cell. Cannabis has 20 chromosomes in the vegetative diploid (2n) condition. Triploid (3n) and tetraploid (4n) individuals have three or four sets of chromosomes and are termed polyploids. It is believed that the haploid condition of 10 chromosomes was likely derived by reduction from a higher (polyploid) ancestral number (Lewis, W. H. 1980). Polyploidy has not been shown to occur naturally in Cannabis; however, it may be induced artificially with colchicine treatments. Colchicine is a poisonous compound extracted from the roots of certain Colchicum species; it inhibits chromosome segregation to daughter cells and cell wall formation, resulting in larger than average daughter cells with multiple chromosome sets. The studies of H. E. Warmke et al. (1942-1944) seem to indicate that colchicine raised drug levels in Cannabis. It is unfortunate that Warmke was unaware of the actual psychoactive ingredients of Cannabis and was therefore unable to extract THC. His crude acetone extract and archaic techniques of bioassay using killifish and small freshwater crustaceans are far from conclusive. He was, however, able to produce both triploid and tetraploid strains of Cannabis with up to twice the potency of dip bid strains (in their ability to kill small aquatic organisms). The aim of his research was to "produce a strain of hemp with materially reduced marijuana content" and his results indicated that polyploidy raised the potency of Cannabis without any apparent increase in fiber quality or yield.

Warmke's work with polyploids shed light on the nature of sexual determination in Cannabis. He also illustrated that potency is genetically determined by creating a lower potency strain of hemp through selective breeding with low potency parents.

More recent research by A. I. Zhatov (1979) with fiber Cannabis showed that some economically valuable traits such as fiber quantity may be improved through polyploidy. Polyploids require more water and are usually more sensitive to changes in environment. Vegetative growth cycles are extended by up to 30-40% in polyploids. An extended vegetative period could delay the flowering of polyploid drug strains and interfere with the formation of floral clusters. It would be difficult to determine if cannabinoid levels had been raised by polyploidy if polyploid plants were not able to mature fully in the favorable part of the season when cannabinoid production is promoted by plentiful light and warm temperatures. Greenhouses and artificial lighting can be used to extend the season and test polyploid strains.

The height of tetraploid (4n) Cannabis in these experiments often exceeded the height of the original diploid plants by 25-30%. Tetraploids were intensely colored, with dark green leaves and stems and a well developed gross phenotype. Increased height and vigorous growth, as a rule, vanish in subsequent generations. Tetraploid plants often revert back to the diploid condition, making it difficult to support tetraploid populations. Frequent tests are performed to determine if ploidy is changing.

Triploid (3n) strains were formed with great difficulty by crossing artificially created tetraploids (4n) with dip bids (2n). Triploids proved to be inferior to both diploids and tetraploids in many cases.

De Pasquale et al. (1979) conducted experiments with Cannabis which was treated with 0.25% and 0.50% solutions of colchicine at the primary meristem seven days after generation. Treated plants were slightly taller and possessed slightly larger leaves than the controls, Anomalies in leaf growth occurred in 20% and 39%, respectively, of the surviving treated plants. In the first group (0.25%) cannabinoid levels were highest in the plants without anomalies, and in the second group (0.50%) cannabinoid levels were highest in plants with anomalies, Overall, treated plants showed a 166-250% increase in THC with respect to controls and a decrease of CBD (30-33%) and CBN (39-65%). CBD (cannabidiol) and CBN (cannabinol) are cannabinoids involved in the biosynthesis and degradation of THC. THC levels in the control plants were very low (less than 1%). Possibly colchicine or the resulting polyploidy interferes with cannabinoid biogenesis to favor THC. In treated plants with deformed leaf lamina, 90% of the cells are tetraploid (4n 40) and 10% diploid (2n 20). In treated plants without deformed lamina a few cells are tetraploid and the remainder are triploid or diploid.

The transformation of diploid plants to the tetraploid level inevitably results in the formation of a few plants with an unbalanced set of chromosomes (2n + 1, 2n - 1, etc.). These plants are called aneuploids. Aneuploids are inferior to polyploids in every economic respect. Aneuploid Cannabis is characterized by extremely small seeds. The weight of 1,000 seeds ranges from 7 to 9 grams (1/4 to 1/3 ounce). Under natural conditions diploid plants do not have such small seeds and average 14-19 grams (1/2-2/3 ounce) per 1,000 (Zhatov 1979).

Once again, little emphasis has been placed on the relationship between flower or resin production and polyploidy. Further research to determine the effect of polyploidy on these and other economically valuable traits of Cannabis is needed.

Colchicine is sold by laboratory supply houses, and breeders have used it to induce polyploidy in Cannabis. However, colchicine is poisonous, so special care is exercised by the breeder in any use of it. Many clandestine cultivators have started polyploid strains with colchicine. Except for changes in leaf shape and phyllotaxy, no out standing characteristics have developed in these strains and potency seems unaffected. However, none of the strains have been examined to determine if they are actually polyploid or if they were merely treated with colchicine to no effect. Seed treatment is the most effective and safest way to apply colchicine. * In this way, the entire plant growing from a colchicine-treated seed could be polyploid and if any colchicine exists at the end of the growing season the amount would be infinitesimal. Colchicine is nearly always lethal to Cannabis seeds, and in the treatment there is a very fine line between polyploidy and death. In other words, if 100 viable seeds are treated with colchicine and 40 of them germinate it is unlikely that the treatment induced polyploidy in any of the survivors. On the other hand, if 1,000 viable treated seeds give rise to 3 seedlings, the chances are better that they are polyploid since the treatment killed all of the seeds but those three. It is still necessary to determine if the offspring are actually polyploid by microscopic examination.

The work of Menzel (1964) presents us with a crude map of the chromosomes of Cannabis, Chromosomes 2-6 and 9 are distinguished by the length of each arm. Chromosome 1 is distinguished by a large knob on one end and a dark chromomere 1 micron from the knob. Chromosome 7 is extremely short and dense, and chromosome 8 is assumed to be the sex chromosome. In the future, chromosome *The word "safest" is used here as a relative term. Coichicine has received recent media attention as a dangerous poison and while these accounts are probably a bit too lurid, the real dangers of exposure to coichicine have not been fully researched. The possibility of bodily harm exists and this is multiplied when breeders inexperienced in handling toxins use colchicine. Seed treatment might be safer than spraying a grown plant but the safest method of all is to not use colchicine. mapping will enable us to picture the location of the genes influencing the phenotype of Cannabis. This will enable geneticists to determine and manipulate the important characteristics contained in the gene pool. For each trait the number of genes in control will be known, which chromosomes carry them, and where they are located along those chromosomes.

Breeding
All of the Cannabis grown in North America today originated in foreign lands. The diligence of our ancestors in their collection and sowing of seeds from superior plants, together with the forces of natural selection, have worked to create native strains with localized characteristics of resistance to pests, diseases, and weather conditions. In other words, they are adapted to particular niches in the ecosystem. This genetic diversity is nature's way of protecting a species. There is hardly a plant more flexible than Cannabis.

As climate, diseases, and pests change, the strain evolves and selects new defenses, programmed into the genetic orders contained in each generation of seeds. Through the importation in recent times of fiber and drug Cannabis, a vast pool of genetic material has appeared in North America. Original fiber strains have escaped and become acclimatized (adapted to the environment), while domestic drug strains (from imported seeds) have, unfortunately, hybridized and acclimatized randomly, until many of the fine gene combinations of imported Cannabis have been lost.

Changes in agricultural techniques brought on by technological pressure, greed, and full-scale eradication programs have altered the selective pressures influencing Cannabis genetics. Large shipments of inferior Cannabis containing poorly selected seeds are appearing in North America and elsewhere, the result of attempts by growers and smugglers to supply an ever increasing market for marijuana. Older varieties of Cannabis, associated with long standing cultural patterns, may contain genes not found in the newer commercial varieties. As these older varieties and their corresponding cultures become extinct, this genetic information could be lost forever. The increasing popularity of Cannabis and the requirements of agricultural technology will call for uniform hybrid races that are likely to displace primitive populations worldwide.

Limitation of genetic diversity is certain to result from concerted inbreeding for uniformity. Should inbred Cannabis be attacked by some previously unknown pest or disease, this genetic uniformity could prove disastrous due to potentially resistant diverse genotypes having been dropped from the population. If this genetic complement of resistance cannot be reclaimed from primitive parental material, resistance cannot be introduced into the ravaged population. There may also be currently unrecognized favorable traits which could be irretrievably dropped from the Cannabis gene pool. Human intervention can create new phenotypes by selecting and recombining existing genetic variety, but only nature can create variety in the gene pool itself, through the slow process of random mutation.

This does not mean that importation of seed and selective hybridization are always detrimental. Indeed these principles are often the key to crop improvement, but only when applied knowledgeably and cautiously. The rapid search for improvements must not jeopardize the pool of original genetic information on which adaptation relies. At this time, the future of Cannabis lies in government and clandestine collections. These collections are often inadequate, poorly selected and badly maintained. Indeed, the United Nations Cannabis collection used as the primary seed stock for worldwide governmental research is depleted and spoiled.

Several steps must be taken to preserve our vanishing genetic resources, and action must be immediate:
Seeds and pollen should be collected directly from reliable and knowledgeable sources. Government seizures and smuggled shipments are seldom reliable seed sources. The characteristics of both parents must be known; consequently, mixed bales of randomly pollinated marijuana are not suitable seed sources, even if the exact origin of the sample is certain. Direct contact should be made with the farmer-breeder responsible for carrying on the breeding traditions that have produced the sample. Accurate records of every possible parameter of growth must be kept with carefully stored triplicate sets of seeds.

Since Cannabis seeds do not remain viable forever, even under the best storage conditions, seed samples should he replenished every third year. Collections should be planted in conditions as similar as possible to their original niche and allowed to reproduce freely to minimize natural and artificial selection of genes and ensure the preservation of the entire gene pool. Half of the original seed collection should be retained until the viability of further generations is confirmed, and to provide parental material for comparison and back-crossing. Phenotypic data about these subsequent generations should be carefully recorded to aid in understanding the genotypes contained in the collection. Favorable traits of each strain should be characterized and catalogued.

It is possible that in the future, Cannabis cultivation for resale, or even personal use, may be legal but only for approved, patented strains. Special caution would be needed to preserve variety in the gene pool should the patenting of Cannabis strains become a reality.

Favorable traits must be carefully integrated into existing strains.

The task outlined above is not an easy one, given the current legal restrictions on the collection of Cannabis seed. In spite of this, the conscientious cultivator is making a contribution toward preserving and improving the genetics of this interesting plant.

Even if a grower has no desire to attempt crop improvement, successful strains have to be protected so they do not degenerate and can be reproduced if lost. Left to the selective pressures of an introduced environment, most drug strains will degenerate and lose potency as they acclimatize to the new conditions. Let me cite an example of a typical grower with good intentions.

A grower in northern latitudes selected an ideal spot to grow a crop and prepared the soil well. Seeds were selected from the best floral clusters of several strains avail able over the past few years, both imported and domestic. Nearly all of the staminate plants were removed as they matured and a nearly seedless crop of beautiful plants resulted.

After careful consideration, the few seeds from accidental pollination of the best flowers were kept for the following season, These seeds produced even bigger and better plants than the year before and seed collection was performed as before. The third season, most of the plants were not as large or desirable as the second season, but there were many good individuals. Seed collection and cultivation the fourth season resulted in plants inferior even to the first crop, and this trend continued year after year. What went wrong? The grower collected seed from the best plants each year and grew them under the same conditions. The crop improved the first year. Why did the strain degenerate?

This example illustrates the unconscious selection for undesirable traits. The hypothetical cultivator began well by selecting the best seeds available and growing them properly. The seeds selected for the second season resulted from random hybrid pollinations by early-flowering or overlooked staminate plants and by hermaphrodite pistil late plants. Many of these random pollen-parents may be undesirable for breeding since they may pass on tendencies toward premature maturation, retarded maturation, or hermaphrodism. However, the collected hybrid seeds pro duce, on the average, larger and more desirable offspring than the first season. This condition is called hybrid vigor and results from the hybrid crossing of two diverse gene pools. The tendency is for many of the dominant characteristics from both parents to be transmitted to the F1 off spring, resulting in particularly large and vigorous plants. This increased vigor due to recombination of dominant genes often raises the cannabinoid level of the F1 offspring, but hybridization also opens up the possibility that undesirable (usually recessive) genes may form pairs and express their characteristics in the F2 offspring. Hybrid vigor may also mask inferior qualities due to abnormally rapid growth. During the second season, random pollinations again accounted for a few seeds and these were collected. This selection draws on a huge gene pool and the possible F2 combinations are tremendous. By the third season the gene pool is tending toward early-maturing plants that are acclimatized to their new conditions instead of the drug-producing conditions of their native environment. These acclimatized members of the third crop have a higher chance of maturing viable seeds than the parental types, and random pollinations will again increase the numbers of acclimatized individuals, and thereby increase the chance that undesirable characteristics associated with acclimatization will be transmitted to the next F2 generation. This effect is compounded from generation to generation and finally results in a fully acclimatized weed strain of little drug value.

With some care the breeder can avoid these hidden dangers of unconscious selection. Definite goals are vital to progress in breeding Cannabis. What qualities are desired in a strain that it does not already exhibit? What characteristics does a strain exhibit that are unfavorable and should be bred out? Answers to these questions suggest goals for breeding. In addition to a basic knowledge of Cannabis botany, propagation, and genetics, the successful breeder also becomes aware of the most minute differences and similarities in phenotype. A sensitive rapport is established between breeder and plants and at the same time strict guidelines are followed. A simplified explanation of the time-tested principles of plant breeding shows how this works in practice.

Selection is the first and most important step in the breeding of any plant. The work of the great breeder and plant wizard Luther Burbank stands as a beacon to breeders of exotic strains. His success in improving hundreds of flower, fruit, and vegetable crops was the result of his meticulous selection of parents from hundreds of thou sands of seedlings and adults from the world over.

Bear in mind that in the production of any new plant, selection plays the all-important part. First, one must get clearly in mind the kind of plant he wants, then breed and select to that end, always choosing through a series of years the plants which are approaching nearest the ideal, and rejecting all others.

Luther Burbank (in James, 1964)
Proper selection of prospective parents is only possible if the breeder is familiar with the variable characteristics of Cannabis that may be genetically controlled, has a way to accurately measure these variations, and has established goals for improving these characteristics by selective breeding. A detailed list of variable traits of Cannabis, including parameters of variation for each trait and comments pertaining to selective breeding for or against it, are found at the end of this chapter. By selecting against unfavorable traits while selecting for favorable ones, the unconscious breeding of poor strains is avoided.

The most important part of Burbank's message on selection tells breeders to choose the plants "which are approaching nearest the ideal," and REJECT ALL OTHERS! Random pollinations do not allow the control needed to reject the undesirable parents. Any staminate plant that survives detection and roguing (removal from the population), or any stray staminate branch on a pistillate her maphrodite may become a pollen parent for the next generation. Pollination must be controlled so that only the pollen- and seed-parents that have been carefully selected for favorable traits will give rise to the next generation.

Selection is greatly improved if one has a large sample to choose from! The best plant picked from a group of 10 has far less chance of being significantly different from its fellow seedlings than the best plant selected from a sample of 100,000. Burbank often made his initial selections of parents from samples of up to 500,000 seedlings. Difficulties arise for many breeders because they lack the space to keep enough examples of each strain to allow a significant selection. A Cannabis breeder's goals are restricted by the amount of space available. Formulating a well defined goal lowers the number of individuals needed to perform effective crosses. Another technique used by breeders since the time of Burbank is to make early selections. Seedling plants take up much less space than adults. Thousands of seeds can be germinated in a flat. A flat takes up the same space as a hundred 10-centimeter (4-inch) sprouts or six-teen 30-centimeter (12-inch) seedlings or one 60-centimeter (24-inch) juvenile. An adult plant can easily take up as much space as a hundred flats. Simple arithmetic shows that as many as 10,000 sprouts can be screened in the space required by each mature plant, provided enough seeds are available. Seeds of rare strains are quite valuable and exotic; however, careful selection applied to thousands of individuals, even of such common strains as those from Colombia or Mexico, may produce better offspring than plants from a rare strain where there is little or no opportunity for selection after germination. This does not mean that rare strains are not valuable, but careful selection is even more important to successful breeding. The random pollinations that produce the seeds in most imported marijuana assure a hybrid condition which results in great seed ling diversity. Distinctive plants are not hard to discover if the seedling sample is large enough.

Traits considered desirable when breeding Cannabis often involve the yield and quality of the final product, but these characteristics can only be accurately measured after the plant has been harvested and long after it is possible to select or breed it. Early seedling selection, therefore, only works for the most basic traits. These are selected first, and later selections focus on the most desirable characteristics exhibited by juvenile or adult plants. Early traits often give clues to mature phenotypic expression, and criteria for effective early seedling selection are easy to establish. As an example, particularly tall and thin seedlings might prove to be good parents for pulp or fiber production, while seed lings of short internode length and compound branching may be more suitable for flower production. However, many important traits to be selected for in Cannabis floral clusters cannot be judged until long after the parents are gone, so many crosses are made early and selection of seeds made at a later date.

Hybridization is the process of mixing differing gene pools to produce offspring of great genetic variation from which distinctive individuals can be selected. The wind performs random hybridization in nature. Under cultivation, breeders take over to produce specific, controlled hybrids. This process is also known as cross-pollination, cross-fertilization, or simply crossing. If seeds result, they will produce hybrid offspring exhibiting some characteristics from each parent.

Large amounts of hybrid seed are most easily produced by planting two strains side by side, removing the staininate plants of the seed strain, and allowing nature to take its course. Pollen- or seed-sterile strains could be developed for the production of large amounts of hybrid seed without the labor of thinning; however, genes for sterility are rare. It is important to remember that parental weak nesses are transmitted to offspring as well as strengths. Because of this, the most vigorous, healthy plants are al ways used for hybrid crosses.

Also, sports (plants or parts of plants carrying and expressing spontaneous mutations) most easily transmit mutant genes to the offspring if they are used as pollen parents. If the parents represent diverse gene pools, hybrid vigor results, because dominant genes tend to carry valuable traits and the differing dominant genes inherited from each parent mask recessive traits inherited from the other. This gives rise to particularly large, healthy individuals. To increase hybrid vigor in offspring, parents of different geo graphic origins are selected since they will probably represent more diverse gene pools.

Occasionally hybrid offspring will prove inferior to both parents, but the first generation may still contain recessive genes for a favorable characteristic seen in a parent if the parent was homozygous for that trait. First generation (F1) hybrids are therefore inbred to allow recessive genes to recombine and express the desired parental trait. Many breeders stop with the first cross and never realize the genetic potential of their strain.

They fail to produce an F2 generation by crossing or self-pollinating F1 offspring. Since most domestic Cannabis strains are F1 hybrids for many characteristics, great diversity and recessive recombination can result from inbreeding domestic hybrid strains. In this way the breeding of the F1 hybrids has already been accomplished, and a year is saved by going directly to F2 hybrids. These F2 hybrids are more likely to express recessive parental traits. From the F2 hybrid generation selections can be made for parents which are used to start new true-breeding strains. Indeed, F2 hybrids might appear with more extreme characteristics than either of the P~ parents. (For example, P1 high-THC X P1 low-THC yields F1 hybrids of intermediate THC content. Selfing the F1 yields F2 hybrids, of both P1 [high and low THC] phenotypes, inter mediate F1 phenotypes, and extra-high THC as well as extra-low THC phenotypes.)

Also, as a result of gene recombination, F1 hybrids are not true-breeding and must be reproduced from the original parental strains. When breeders create hybrids they try to produce enough seeds to last for several successive years of cultivation, After initial field tests, undesirable hybrid seeds are destroyed and desirable hybrid seeds stored for later use. If hybrids are to be reproduced, a clone is saved from each parental plant to preserve original parental genes.

Back-crossing is another technique used to produce offspring with reinforced parental characteristics. In this case, a cross is made between one of the F~ or subsequent offspring and either of the parents expressing the desired trait. Once again this provides a chance for recombination and possible expression of the selected parental trait. Back-crossing is a valuable way of producing new strains, but it is often difficult because Cannabis is an annual, so special care is taken to save parental stock for back-crossing the following year. Indoor lighting or greenhouses can be used to protect breeding stock from winter weather. In tropical areas plants may live outside all year. In addition to saving particular parents, a successful breeder always saves many seeds from the original P1 group that produced the valuable characteristic so that other P1 plants also exhibiting the characteristic can be grown and selected for back-crossing at a later time.

Several types of breeding are summarized as follows:

1 - Crossing two varieties having outstanding qualities (hybridization).

2 - Crossing individuals from the F1 generation or selfing F1 individuals to realize the possibilities of the original cross (differentiation).

3 - Back crossing to establish original parental types.

4 - Crossing two similar true-breeding (homozygous) varieties to preserve a mutual trait and restore vigor.

It should be noted that a hybrid plant is not usually hybrid for all characteristics nor does a true-breeding strain breed true for all characteristics. When discussing crosses, we are talking about the inheritance of one or a few traits only. The strain may be true-breeding for only a few traits, hybrid for the rest. Monohybrid crosses involve one trait, dihybrid crosses involve two traits, and so forth. Plants have certain limits of growth, and breeding can only pro duce a plant that is an expression of some gene already present in the total gene pool. Nothing is actually created by breeding; it is merely the recombination of existing genes into new genotypes. But the possibilities of recombination are nearly limitless.

The most common use of hybridization is to cross two outstanding varieties. Hybrids can be produced by crossing selected individuals from different high-potency strains of different origins, such as Thailand and Mexico. These two parents may share only the characteristic of high psycho activity and differ in nearly every other respect. From this great exchange of genes many phenotypes may appear in the F2 generation. From these offspring the breeder selects individuals that express the best characteristics of the parents. As an example, consider some of the offspring from the P1 (parental) cross: Mexico X Thailand. In this case, genes for high drug content are selected from both parents while other desirable characteristics can be selected from either one. Genes for large stature and early maturation are selected from the Mexican seed-parent, and genes for large calyx size and sweet floral aroma are selected from the Thai pollen parent. Many of the F1 offspring exhibit several of the desired characteristics. To further promote gene segregation, the plants most nearly approaching the ideal are crossed among themselves. The F2 generation is a great source of variation and recessive expression. In the F2 generation there are several individuals out of many that exhibit all five of the selected characteristics. Now the process of inbreeding begins, using the desirable F2 parents.

If possible, two or more separate lines are started, never allowing them to interbreed. In this case one accept able staminate plant is selected along with two pistillate plants (or vice versa). Crosses between the pollen parent and the two seed parents result in two lines of inheritance with slightly differing genetics, but each expressing the desired characteristics. Each generation will produce new, more acceptable combinations.

If two inbred strains are crossed, F1 hybrids will be less variable than if two hybrid strains are crossed. This comes from limiting the diversity of the gene pools in the two strains to be hybridized through previous inbreeding. Further independent selection and inbreeding of the best plants for several generations will establish two strains which are true-breeding for all the originally selected traits. This means that all the offspring from any parents in the strain will give rise to seedlings which all exhibit the selected traits. Successive inbreeding may by this time have resulted in steady decline in the vigor of the strain.

When lack of vigor interferes with selecting phenotypes for size and hardiness, the two separately selected strains can then be interbred to recombine nonselected genes and restore vigor. This will probably not interfere with breeding for the selected traits unless two different gene systems control the same trait in the two separate lines, and this is highly unlikely. Now the breeder has produced a hybrid strain that breeds true for large size, early maturation, large sweet-smelling calyxes, and high THC level. The goal has been reached!

Wind pollination and dioecious sexuality favor a heterozygous gene pool in Cannabis. Through Anbreeding, hybrids are adapted from a heterozygous gene pool to a homozygous gene pool, providing the genetic stability needed to create true-breeding strains. Establishing pure strains enables the breeder to make hybrid crosses with a better chance of predicting the outcome. Hybrids can be created that are not reproducible in the F2 generation. Commercial strains of seeds could be developed that would have to be purchased each year, because the F1 hybrids of two pure-bred lines do not breed true. Thus, a seed breeder can protect the investment in the results of breeding, since it would be nearly impossible to reproduce the parents from F2 seeds.
 

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