Chap 3: Genetics and Breeding of Cannabis Part 2/3
Chapter 3 - Genetics and Breeding of Cannabis
Fixing Traits
Fixing traits (producing homozygous offspring) in Cannabis strains is more difficult than it is in many other flowering plants. With monoecious strains or hermaphrodites it is possible to fix traits by self-pollinating an individual exhibiting favorable traits. In this case one plant acts as both mother and father. However, most strains of Cannabis are dioecious, and unless hermaphroditic reactions can be induced, another parent exhibiting the trait is required to fix the trait. If this is not possible, the unique individual may be crossed with a plant not exhibiting the trait, inbred in the F1 generation, and selections of parents exhibiting the favorable trait made from the F2 generation, but this is very difficult.
If a trait is needed for development of a dioecious strain it might first be discovered in a monoecious strain and then fixed through selfing and selecting homozygous offspring. Dioecious individuals can then be selected from the monoecious population and these individuals crossed to breed out monoecism in subsequent generations.
Galoch (1978) indicated that gibberellic acid (GA3) promoted stamen production while indoleacetic acid (IAA), ethrel, and kinetin promoted pistil production in prefloral dioecious Cannabis. Sex alteration has several useful applications. Most importantly, if only one parent expressing a desirable trait can be found, it is difficult to perform a cross unless it happens to be a hermaphrodite plant. Hormones might be used to change the sex of a cutting from the desirable plant, and this cutting used to mate with it. This is most easily accomplished by changing a pistillate cutting to a staminate (pollen) parent, using a spray of 100 ppm gibberellic acid in water each day for five consecutive days. Within two weeks staminate flowers may appear. Pollen can then be collected for selfing with the original pistillate parent. Offspring from the cross should also be mostly pistillate since the breeder is selfing for pistillate sexuality. Staminate parents reversed to pistillate floral production make inferior seed-parents since few pistillate flowers and seeds are formed.
If entire crops could be manipulated early in life to produce all pistillate or staminate plants, seed production and seedless drug Cannabis production would be greatly facilitated.
Sex reversal for breeding can also be accomplished by mutilation and by photoperiod alteration. A well-rooted, flourishing cutting from the parent plant is pruned back to 25% of its original size and stripped of all its remaining flowers. New growth will appear within a few days, and several flowers of reversed sexual type often appear. Flowers of the unwanted sex are removed until the cutting is needed for fertilization. Extremely short light cycles (6-8 hour photoperiod) can also cause sex reversal. How ever, this process takes longer and is much more difficult to perform in the field.
Genotype and Phenotype Ratios
It must be remembered, in attempting to fix favorable characteristics, that a monohybrid cross gives rise to four possible recombinant genotypes, a dihybrid cross gives rise to 16 possible recombinant genotypes, and so forth.
Phenotype and genotype ratios are probabilistic. If recessive genes are desired for three traits it is not effective to raise only 64 offspring and count on getting one homozygous recessive individual. To increase the probability of success it is better to raise hundreds of offspring, choosing only the best homozygous recessive individuals as future parents. All laws of inheritance are based on chance and offspring may not approach predicted ratios until many more have been phenotypically characterized and grouped than the theoretical minimums.
The genotype of each individual is expressed by a mosaic of thousands of subtle overlapping traits. It is the sum total of these traits that determines the general phenotype of an individual. It is often difficult to determine if the characteristic being selected is one trait or the blending of several traits and whether these traits are controlled by one or several pairs of genes. It often makes little difference that a breeder does not have plants that are proven to breed true. Breeding goals can still be established. The selfing of F1 hybrids will often give rise to the variation needed in the F2 generation for selecting parents for subsequent generations, even if the characteristics of the original parents of the F1 hybrid are not known. It is in the following generations that fixed characteristics appear and the breeding of pure strains can begin. By selecting and crossing individuals that most nearly approach the ideal described by the breeding goals, the variety can be continuously improved even if the exact patterns of inheritance are never deter mined. Complementary traits are eventually combined into one line whose seeds reproduce the favorable parental traits. Inbreeding strains also allows weak recessive traits to express themselves and these abnormalities must be diligently removed from the breeding population. After five or six generations, strains become amazingly uniform. Vigor is occasionally restored by crossing with other lines or by backcrossing.
Parental plants are selected which most nearly approach the ideal. If a desirable trait is not expressed by the parent, it is much less likely to appear in the offspring. It is imperative that desirable characteristics be hereditary and not primarily the result of environment and cultivation. Acquired traits are not hereditary and cannot be made hereditary. Breeding for as few traits as possible at one time greatly increases the chance of success. In addition to the specific traits chosen as the aims of breeding, parents are selected which possess other generally desirable traits such as vigor and size. Determinations of dominance and recessiveness can only be made by observing the outcome of many crosses, although wild traits often tend to be dominant. This is one of the keys to adaptive survival. However, all the possible combinations will appear in the F2 generation if it is large enough, regardless of dominance.
Now, after further simplifying this wonderful system of inheritance, there are additional exceptions to the rules which must be explored. In some cases, a pair of genes may control a trait but a second or third pair of genes is needed to express this trait. This is known as gene inter action. No particular genetic attribute in which we may be interested is totally isolated from other genes and the effects of environment. Genes are occasionally transferred in groups instead of assorting independently. This is known as gene linkage, These genes are spaced along the same chromosome and may or may not control the same trait. The result of linkage might be that one trait cannot be inherited without another. At times, traits are associated with the X and Y sex chromosomes and they may be limited to expression in only one sex (sex linkage). Crossing over also interferes with the analysis of crosses. Crossing over is the exchanging of entire pieces of genetic material between two chromosomes. This can result in two genes that are normally linked appearing on separate chromosomes where they will be independently inherited. All of these processes can cause crosses to deviate from the expected Mendelian outcome. Chance is a major factor in breeding Cannabis, or any introduced plant, and the more crosses a breeder attempts the higher are the chances of success.
Variate, isolate, intermate, evaluate, multiplicate, and disseminate are the key words in plant improvement. A plant breeder begins by producing or collecting various prospective parents from which the most desirable ones are selected and isolated. Intermating of the select parents results in offspring which must be evaluated for favorable characteristics. If evaluation indicates that the offspring are not improved, then the process is repeated. Improved off spring are multiplied and disseminated for commercial use. Further evaluation in the field is necessary to check for uniformity and to choose parents for further intermating. This cyclic approach provides a balanced system of plant improvement.
The basic nature of Cannabis makes it challenging to breed. Wind pollination and dioecious sexuality, which account for the great adaptability in Cannabis, cause many problems in breeding, but none of these are insurmountable. Developing a knowledge and feel for the plant is more important than memorizing Mendelian ratios. The words of the great Luther Burbank say it well,
"Heredity is indelibly fixed by repetition."
The first set of traits concerns Cannabis plants as a whole while the remainder concern the qualities of seedlings, leaves, fibers, and flowers. Finally a list of various Cannabis strains is provided along with specific characteristics. Following this order, basic and then specific selections of favorable characteristics can be made.
List of Favorable Traits of Cannabis
in Which Variation Occurs
1. General Traits
a) Size and Yield
b) Vigor
c) Adaptability
d) Hardiness
e) Disease and Pest Resistance
f) Maturation
g) Root Production
h) Branching
i) Sex
2. Seedling Traits
3. Leaf Traits
4. Fiber Traits
5. Floral Traits
a) Shape
b) Form
c) Calyx Size
d) Color
e) Cannabinoid Level
f) Taste and Aroma
g) Persistence of Aromatic Principles and Cannabinoids
h) Trichome Type
i) Resin Quantity and Quality
j) Resin Tenacity
k) Drying and Curing Rate
I) Ease of Manicuring
m) Seed Characteristics
n) Maturation
o) Flowering
p) Ripening
q) Cannabinoid Profile
6. Gross Phenotypes of Cannabis Strains
1. General Traits
a) Size and Yield - The size of an individual Cannabis plant is determined by environmental factors such as room for root and shoot growth, adequate light and nutrients, and proper irrigation. These environmental factors influence the phenotypic image of genotype, but the genotype of the individual is responsible for overall variations in gross morphology, including size. Grown under the same conditions, particularly large and small individuals are easily spotted and selected. Many dwarf Cannabis plants have been re ported and dwarfism may be subject to genetic control, as it is in many higher plants, such as dwarf corn and citrus. Cannabis parents selected for large size tend to produce offspring of a larger average size each year. Hybrid crosses between tall (Cannabis sativa-Mexico) strains and short (Cannabis ruderalis-Russia) strains yield F1 offspring of intermediate height (Beutler and der Marderosian 1978). Hybrid vigor, however, will influence the size of offspring more than any other genetic factor. The increased size of hybrid offspring is often amazing and accounts for much of the success of Cannabis cultivators in raising large plants. It is not known whether there is a set of genes for "gigantism" in Cannabis or whether polyploid individuals really yield more than diploid due to increased chromosome count. Tetraploids tend to be taller and their water re quirements are often higher than diploids. Yield is determined by the overall production of fiber, seed, or resin and selective breeding can be used to increase the yield of any one of these products. However, several of these traits may be closely related, and it may be impossible to breed for one without the other (gene linkage). Inbreeding of a pure strain increases yield only if high yield parents are selected. High yield plants, staminate or pistillate, are not finally selected until the plants are dried and manicured. Because of this, many of the most vigorous plants are crossed and seeds selected after harvest when the yield can be measured.
b) Vigor - Large size is often also a sign of healthy vigorous growth. A plant that begins to grow immediately will usually reach a larger size and produce a higher yield in a short growing season than a sluggish, slow-growing plant. Parents are always selected for rich green foliage and rapid, responsive growth. This will ensure that genes for certain weaknesses in overall growth and development are bred out of the population while genes for strength and vigor remain.
c) Adaptability - It is important for a plant with a wide distribution such as Cannabis to be adaptable to many different environmental conditions. Indeed, Cannabis is one of the most genotypically diverse and phenotypically plastic plants on earth; as a result it has adapted to environ mental conditions ranging from equatorial to temperate climates. Domestic agricultural circumstances also dictate that Cannabis must be grown under a great variety of conditions,
Plants to be selected for adaptability are cloned and grown in several locations. The parental stocks with the highest survival percentages can be selected as prospective parents for an adaptable strain. Adaptability is really just another term for hardiness under varying growth conditions.
d) Hardiness - The hardiness of a plant is its overall resistance to heat and frost, drought and overwatering, and so on. Plants with a particular resistance appear when adverse conditions lead to the death of the rest of a large population. The surviving few members of the population might carry inheritable resistance to the environmental factor that destroyed the majority of the population. Breeding these survivors, subjecting the offspring to continuing stress conditions, and selecting carefully for several generations should result in a pure-breeding strain with increased resistance to drought, frost, or excessive heat.
e) Disease and Pest Resistance - In much the same way as for hardiness a strain may be bred for resistance to a certain disease, such as damping-off fungus. If flats of seedlings are infected by damping-off disease and nearly all of them die, the remaining few will have some resistance to damping-off fungus. If this resistance is inheritable, it can be passed on to subsequent generations by crossing these surviving plants. Subsequent crossing, tested by inoculating flats of seedling offspring with damping-off fungus, should yield a more resistant strain.
Resistance to pest attack works in much the same way. It is common to find stands of Cannabis where one or a few plants are infested with insects while adjacent plants are untouched. Cannabinoid and terpenoid resins are most probably responsible for repelling insect attack, and levels of these vary from plant to plant. Cannabis has evolved defenses against insect attack in the form of resin-secreting glandular trichomes, which cover the reproductive and associated vegetative structures of mature plants. Insects, finding the resin disagreeable, rarely attack mature Cannabis flowers. However, they may strip the outer leaves of the same plant because these develop fewer glandular tri chomes and protective resins than the flowers. Non-glandular cannabinoids and other compounds produced within leaf and stem tissues which possibly inhibit insect attack, may account for the varying resistance of seedlings and vegetative juvenile plants to pest infestation. With the popularity of greenhouse Cannabis cultivation, a strain is needed with increased resistance to mold, mite, aphid,- or white fly infestation. These problems are often so severe that greenhouse cultivators destroy any plants which are attacked. Molds usually reproduce by wind-borne spores, so negligence can rapidly lead to epidemic disaster. Selection and breeding of the least infected plants should result in strains with increased resistance.
f) Maturation - Control of the maturation of Cannabis is very important no matter what the reason for growing it. If Cannabis is to be grown for fiber it is important that the maximum fiber content of the crop be reached early and that all of the individuals in the crop mature at the same time to facilitate commercial harvesting. Seed production requires the even maturation of both pollen and seed parents to ensure even setting and maturation of seeds. An uneven maturation of seeds would mean that some seeds would drop and be lost while others are still ripening. An understanding of floral maturation is the key to the production of high quality drug Cannabis. Changes in gross morphology are accompanied by changes in cannabinoid and terpenoid production and serve as visual keys to deter mining the ripeness of Cannabis flowers.
A Cannabis plant may mature either early or late, be fast or slow to flower, and ripen either evenly or sequentially.
Breeding for early or late maturation is certainly a reality; it is also possible to breed for fast or slow flowering and even or sequential ripening. In general, crosses between early-maturing plants give rise to early-maturing offspring, crosses between late-maturing plants give rise to late-maturing offspring, and crosses between late- and early-maturing plants give rise to offspring of intermediate maturation. This seems to indicate that maturation of Cannabis is not controlled by the simple dominance and recessiveness of one gene but probably results from incomplete dominance and a combination of genes for separate aspects of maturation. For instance, Sorghum maturation is controlled by four separate genes. The sum of these genes produces a certain phenotype for maturation. Al though breeders do not know the action of each specific gene, they still can breed for the total of these traits and achieve results more nearly approaching the goal of timely maturation than the parental strains.
g) Root Production - The size and shape of Cannabis root systems vary greatly. Although every embryo sends out a taproot from which lateral roots grow, the individual growth pattern and final size and shape of the roots vary considerably. Some plants send out a deep taproot, up to 1 meter (39 inches) long, which helps support the plant against winds and rain. Most Cannabis plants, however, produce a poor taproot which rarely extends more than 30 centimeters (1 foot). Lateral growth is responsible for most of the roots in Cannabis plants. These fine lateral roots offer the plant additional support but their primary function is to absorb water and nutrients from the soil. A large root system will be able to feed and support a large plant. Most lateral roots grow near the surface of the soil where there is more water, more oxygen, and more avail able nutrients. Breeding for root size and shape may prove beneficial for the production of large rain- and wind-resistant strains. Often Cannabis plants, even very large ones, have very small and sensitive root systems. Recently, certain alkaloids have been discovered in the roots of Cannabis that might have some medical value. If this proves the case, Cannabis may be cultivated and bred for high alkaloid levels in the roots to be used in the commercial production of pharmaceuticals.
As with many traits, it is difficult to make selections for root types until the parents are harvested. Because of this many crosses are made early and seeds selected later.
h) Branching - The branching pattern of a Cannabis plant is determined by the frequency of nodes along each branch and the extent of branching at each node. For examples, consider a tall, thin plant with slender limbs made up of long internodes and nodes with little branching (Oaxaca, Mexico strain). Compare this with a stout, densely branched plant with limbs of short internodes and highly branched nodes (Hindu Kush hashish strains). Different branching patterns are preferred for the different agricultural applications of fiber, flower, or resin production. Tall, thin plants with long internodes and no branching are best adapted to fiber production; a short, broad plant with short inter nodes and well developed branching is best adapted to floral production. Branching structure is selected that will tolerate heavy rains and high winds without breaking. This is quite advantageous to outdoor growers in temperate zones with short seasons. Some breeders select tall, limber plants (Mexico) which bend in the wind; others select short, stiff plants (Hindu Kush) which resist the weight of water without bending.
i) Sex - Attempts to breed offspring of only one sexual type have led to more misunderstanding than any other facet of Cannabis genetics. The discoveries of McPhee (1925) and Schaffner (1928) showed that pure sexual type and hermaphrodite conditions are inherited and that the percentage of sexual types could be altered by crossing with certain hermaphrodites. Since then it has generally been assumed by researchers and breeders that a cross between ANY unselected hermaphrodite plant and a pistillate seed-parent should result in a population of all pistillate offspring. This is not the case. In most cases, the offspring of hermaphrodite parents tend toward hermaphrodism, which is largely unfavorable for the production of Cannabis other than fiber hemp. This is not to say that there is no tendency for hermaphrodite crosses to alter sex ratios in the offspring. The accidental release of some pollen from predominantly pistillate hermaphrodites, along with the complete eradication of nearly every staminate and staminate hermaphrodite plant may have led to a shift in sexual ratio in domestic populations of sinsemilla drug Cannabis. It is commonly observed that these strains tend toward 60% to 80% pistillate plants and a few pistillate hermaphrodites are not uncommon in these populations.
However, a cross can be made which will produce nearly all pistillate or staminate individuals. If the proper pistillate hermaphrodite plant is selected as the pollen-parent and a pure pistillate plant is selected as the seed-parent it is possible to produce an F1, and subsequent generations, of nearly all pistillate offspring. The proper pistillate hermaphrodite pollen-parent is one which has grown as a pure pistillate plant and at the end of the sea son, or under artificial environmental stress, begins to develop a very few staminate flowers. If pollen from these few staminate flowers forming on a pistillate plant is applied to a pure pistillate seed parent, the resulting F1 generation should be almost all pistillate with only a few pistillate hermaphrodites. This will also be the case if the selected pistillate hermaphrodite pollen source is selfed and bears its own seeds. Remember that a selfed hermaphrodite gives rise to more hermaphrodites, but a selfed pistillate plant that has given rise to a limited number of staminate flowers in response to environmental stresses should give rise to nearly all pistillate offspring. The F1 offspring may have a slight tendency to produce a few staminate flowers under further environmental stress and these are used to produce F2 seed. A monoecious strain produces 95+% plants with many pistillate and staminate flowers, but a dioecious strain produces 95+% pure pistillate or staminate plants. A plant from a dioecious strain with a few inter sexual flowers is a pistillate or staminate hermaphrodite. Therefore, the difference between monoecism and her maphrodism is one of degree, determined by genetics and environment.
Crosses may also be performed to produce nearly all staminate offspring. This is accomplished by crossing a pure staminate plant with a staminate plant that has produced a few pistillate flowers due to environmental stress, or selfing the latter plant. It is readily apparent that in the wild this is not a likely possibility. Very few staminate plants live long enough to produce pistillate flowers, and when this does happen the number of seeds produced is limited to the few pistillate flowers that occur. In the case of a pistillate hermaphrodite, it may produce only a few staminate flowers, but each of these may produce thou sands of pollen grains, any one of which may fertilize one of the plentiful pistillate flowers, producing a seed. This is another reason that natural Cannabis populations tend toward predominantly pistillate and pistillate hermaphrodite plants. Artificial hermaphrodites can be produced by hormone sprays, mutilation, and altered light cycles. These should prove most useful for fixing traits and sexual type.
Drug strains are selected for strong dioecious tendencies. Some breeders select strains with a sex ratio more nearly approaching one than a strain with a high pistillate sex ratio. They believe this reduces the chances of pistillate plants turning hermaphrodite later in the season.
2. Seedling Traits
Seedling traits can be very useful in the efficient and purposeful selection of future parental stock. If accurate selection can be exercised on small seedlings, much larger populations can be grown for initial selection, as less space is required to raise small seedlings than mature plants. Whorled phyllotaxy and resistance to damping-off are two traits that may be selected just after emergence of the embryo from the soil. Early selection for vigor, hardiness, resistance, and general growth form may be made when the seedlings are from 30 to 90 centimeters (1 to 3 feet) tall. Leaf type, height, and branching are other criteria for early selection. These early-selected plants cannot be bred until they mature, but selection is the primary and most important step in plant improvement.
Whorled phyllotaxy is associated with subsequent anomalies in the growth cycle (i.e., multiple leaflets and flattened or clubbed stems). Also, most whorled plants are staminate and whorled phyllotaxy may be sex-linked.
3. Leaf Traits
Leaf traits vary greatly from strain to strain. In addition to these regularly occurring variations in leaves, there are a number of mutations and possible traits in leaf shape. It may turn out that leaf shape is correlated with other traits in Cannabis. Broad leaflets might be associated with a low calyx-to-leaf ratio and narrow leaflets might be associated with a high calyx-to-leaf ratio. If this is the case, early selection of seedlings by leaflet shape could determine the character of the flowering clusters at harvest. Both compound and webbed leaf variations seem to be hereditary, as are general leaf characteristics. A breeder may wish to develop a unique leaf shape for an ornamental strain or increase leaf yield for pulp production.
A peculiar leaf mutation was reported from an F1-Colombian plant in which two leaves on the plant, at the time of flowering, developed floral clusters of 5-10 pistil late calyxes at the intersection of the leaflet array and the petiole attachment, on the adaxial (top) side of the leaf. One of these clusters developed a partial staminate flower but fertilization was unsuccessful. It is unknown if this mutation is hereditary.
From Afghanistan, another example has been observed with several small floral clusters along the petioles of many of the large primary leaves.
4. Fiber Traits
More advanced breeding has occurred in fiber strains than any other type of Cannabis. Over the years many strains have been developed with improved maturation, in creased fiber content, and improved fiber quality as regards length, strength, and suppleness. Extensive breeding programs have been carried on in France, Italy, Russia, and the United States to develop better varieties of fiber Cannabis. Tall limbless strains that are monoecious are most desirable. Monoeciousness is favored, because in dioecious populations the staminate plants will mature first and the fibers will become brittle before the pistillate plants are ready for harvest. The fiber strains of Europe are divided into northern and southern varieties. The latter require higher temperatures and a longer vegetative period and as a result grow taller and yield more fiber.
5. Floral Traits
Many individual traits determine the floral characteristics of Cannabis This section will focus on the individual traits of pistillate floral clusters with occasional comments about similar traits in staminate floral clusters. Pistillate flowering clusters are the seed-producing organs of Cannabis; they remain on the plant and go through many changes that cannot be compared to staminate plants.
a) Shape - The basic shape of a floral cluster is determined by the internode lengths along the main floral axis and within individual floral clusters. Dense, long clusters result when internodes are short along a long floral axis and there are short internodes within the individual compact floral clusters (Hindu Kush). Airy clusters result when a plant forms a stretched floral axis with long internodes between well-branched individual floral clusters (Thailand).
The shape of a floral cluster is also determined by the general growth habit of the plant. Among domestic Cannabis phenotypes, for instance, it is obvious that floral clusters from a creeper phenotype plant will curve upwards at the end, and floral clusters from the huge upright phenotype will have long, straight floral clusters of various shapes. Early in the winter, many strains begin to stretch and cease calyx production in preparation for rejuvenation and sub sequent vegetative growth in the spring. Staminate plants also exhibit variation in floral clusters. Some plants have tight clusters of staminate calyxes resembling inverted grapes (Hindu Kush) and others have long, hanging groups of flowers on long, exposed, leafless branches (Thailand).
b) Form - The form of a floral cluster is determined by the numbers and relative proportions of calyxes and flowers. A leafy floral cluster might be 70% leaves and have a calyx-to-leaf ratio of 1-to-4. It is obvious that strains with a high calyx-to-leaf ratio are more adapted to calyx production, and therefore, to resin production. This factor could be advantageous in characterizing plants as future parents of drug strains. At this point it must be noted that pistillate floral clusters are made up of a number of distinct parts. They include stems, occasional seeds, calyxes, inner leaves subtending calyx pairs (small, resinous, 1-3 leaflets), and outer leaves subtending entire floral clusters (larger, little resin, 3-11 leaflets). The ratios (by dry weight) of these various portions vary by strain, degree of pollination, and maturity of the floral clusters. Maturation is a reaction to environmental change, and the degree of maturity reached is subject to climatic limits as well as breeder's preference. Because of this interplay between environment and genetics in the control of floral form it is often difficult to breed Cannabis for floral characteristics. A thorough knowledge of the way a strain matures is important in separating possible inherited traits of floral clusters from acquired traits. Chapter IV, Maturation and Harvesting of Cannabis, delves into the secrets and theories of maturation. For now, we will assume that the following traits are described from fully mature floral clusters (peak floral stage) before any decline.
c) Calyx Size - Mature calyxes range in size from 2 to 12 millimeters (1/16 to 3/8 inch) in length. Calyx size is largely dependent upon age and maturity. Calyx size of a floral cluster is best expressed as the average length of the mature viable calyxes. Calyxes are still considered viable if both pistils appear fresh and have not begun to curl or change colors. At this time, the calyx is relatively straight and has not begun to swell with resin and change shape as it will when the pistils die. It is generally agreed that the production of large calyxes is often as important in deter mining the psychoactivity of a strain as the quantity of calyxes produced. Hindu Kush, Thai, and Mexican strains are some of the most psychoactive strains, and they are often characterized by large calyxes and seeds.
Calyx size appears to be an inherited trait in Cannabis. Completely acclimatized hybrid strains usually have many rather small calyxes, while imported strains with large calyxes retain that size when inbred.
Initial selection of large seeds increases the chance that offspring will be of the large-calyx variety. Aberrant calyx development occasionally results in double or fused calyxes, both of which may set seed. This phenomenon is most pronounced in strains from Thailand and India.
d) Color - The perception and interpretation of color in Cannabis floral clusters is heavily influenced by the imagination of the cultivator or breeder. A gold strain does not appear metallic any more than a red strain resembles a fire engine. Cannabis floral clusters are basically green, but changes may take place later in the season which alter the color to include various shades. The intense green of chlorophyll usually masks the color of accessory pigments, Chlorophyll tends to break down late in the season and anthocyanin pigments also contained in the tissues are unmasked and allowed to show through. Purple, resulting from anthocyanin accumulation, is the most common color in living Cannabis, other than green. This color modification is usually triggered by seasonal change, much as the leaves of many deciduous trees change color in the fall. This does not mean, however, that expression of color is controlled by environment alone and is not an inheritable trait. For purple color to develop upon maturation, a strain must have the genetically controlled metabolic potential to pro duce anthocyanin pigments coupled with a responsiveness to environmental change such that anthocyanin pigments are unmasked and become visible. This also means that a strain could have the genes for expression of purple color but the color might never be expressed if the environmental conditions did not trigger anthocyanin pigmentation or chlorophyll breakdown. Colombian and Hindu Kush strains often develop purple coloration year after year when subjected to low night temperatures during maturation. Color changes will be discussed in more detail in Chapter IV-Maturation and Harvesting of Cannabis.
Carotenoid pigments are largely responsible for the yellow, orange, red, and brown colors of Cannabis. They also begin to show in the leaves and calyxes of certain strains as the masking green chlorophyll color fades upon maturation. Gold strains are those which tend to reveal underlying yellow and orange pigments as they mature. Red strains are usually closer to reddish brown in color, although certain carotenoid and anthocyanin pigments are nearly red and localized streaks of these colors occasionally appear in the petioles of very old floral clusters. Red color in pressed, imported tops is often a result of masses of reddish brown dried pistils.
Several different portions of floral cluster anatomy may change colors, and it is possible that different genes may control the coloring of these various parts.
The petioles, adaxial (top) surfaces, and abaxial (bot tom) surfaces of leaves, as well as the stems, calyxes, and pistils color differently in various strains. Since most of the outer leaves are removed during manicuring, the color ex pressed by the calyxes and inner leaves during the late flowering stages will be all that remains in the final product. This is why strains are only considered to be truly purple or gold if the calyxes maintain those colors when dried. Anthocyanin accumulation in the stems is sometimes considered a sign of phosphorus deficiency but in most situations results from unharmful excesses of phosphorus or it is a genetic trait. Also, cold temperatures might interfere with phosphorus uptake resulting in a deficiency. Pistils in Hindu Kush strains are quite often magenta or pink in color when they first appear. They are viable at this time and turn reddish brown when they wither, as in most strains. Purple coloration usually indicates that pistillate plants are over-mature and cannabinoid biosynthesis is slowing down during cold autumn weather.
e) Cannabinoid Level - Breeding Cannabis for cannabinoid level has been accomplished by both licensed legitimate and clandestine researchers. Warmke (1942) and Warmke and Davidson (1943-44) showed that they could significantly raise or lower the cannabinoid level by selective breeding. Small (1975a) has divided genus Cannabis into four distinct chemotypes based on the relative amounts of THC and CBD. Recent research has shown that crosses between high THC: low CBD strains and low THC: high CBD strains yield offspring of cannabinoid content intermediate between the two parents. Beutler and der Marderosian (1978) analyzed the F1 offspring of the controlled cross C. Sativa (Mexico-high THC) X C. ruderalis (Russia-low THC) and found that they fell into two groups intermediate between the parents in THC level. This indicates that THC production is most likely controlled by more than one gene. Also the F1 hybrids of lower THC (resembling the staminate parent) were twice as frequent as the higher THC hybrids (resembling the pistillate parent). More re search is needed to learn if THC production in Cannabis is associated with the sexual type of the high THC parent or if high THC characteristics are recessive. According to Small (1979) the cannabinoid ratios of strains grown in northern climates are a reflection of the cannabinoid ratio of the pure, imported, parental strain. This indicates that cannabinoid phenotype is genetically controlled, and the levels of the total cannabinoids are determined by environment. Complex highs produced by various strains of drug Cannabis may be blended by careful breeding to produce hybrids of varying psychoactivity, but the level of total psychoactivity is dependent on environment. This is also the telltale indication that unconscious breeding with undesirable low-THC parents could rapidly lead to the degeneration rather than improvement of a drug strain. It is obvious that individuals of fiber strains are of little if any use in breeding drug strains.
Breeding for cannabinoid content and the eventual characterization of varying highs produced by Cannabis is totally subjective guesswork without the aid of modern analysis techniques. A chromatographic analysis system would allow the selection of specific cannabinoid types, especially staminate pollen parents. Selection of staminate parents always presents a problem when breeding for cannabinoid content. Staminate plants usually express the same ratios of cannabinoids as their pistiliate counterparts but in much lower quantities, and they are rarely allowed to reach full maturity for fear of seeding the pistillate portion of the crop. A simple bioassay for THC content of staminate plants is performed by leaving a series of from three to five numbered bags of leaves and tops of various prospective pollen parents along with some rolling papers in several locations frequented by a steady repeating crowd of marijuana smokers. The bag completely consumed first can be considered the most desirable to smoke and possibly the most psychoactive. It would be impossible for one per son to objectively select the most psychoactive staminate plant since variation in the cannabinoid profile is subtle. The bioassay reported here is in effect an unstructured panel evaluation which averages the opinions of unbiased testers who are exposed to only a few choices at a time. Such bioassay results can enter into selecting the staminate parent.
It is difficult to say how many genes might control THC-acid synthesis. Genetic control of the biosynthetic pathway could occur at many points through the action of enzymes controlling each individual reaction. It is generally accepted that drug strains have an enzyme system which quickly converts CBD-acid to THC-acid, favoring THC-acid accumulation. Fiber strains lack this enzyme activity, so CBD-acid accumulalion is favored since there is little con version to THC-acid. These same enzyme systems are probably also sensitive to changes in heat and light.
It is supposed that variations in the type of high associated with different strains of Cannabis result from varying levels of cannabinoids. THC is the primary psycho active ingredient which is acted upon synergistically by small amounts of CBN, CBD, and other accessory cannabinoids. Terpenes and other aromatic constituents of Cannabis might also potentiate or suppress the effect of THC. We know that cannabinoid levels may be used to establish cannabinoid phenotypes and that these phenotypes are passed on from parent to offspring. Therefore, cannabinoid levels are in part determined by genes. To accurately characterize highs from various individuals and establish criteria for breeding strains with particular cannabinoid contents, an accurate and easy method is needed for measuring cannabinoid levels in prospective parents. Inheritance and expression of cannabinoid chemotype is certainly complex.
f) Taste and Aroma - Taste and aroma are closely linked.
As our senses for differentiating taste and aroma are connected, so are the sources of taste and aroma in Cannabis. Aroma is produced primarily by aromatic terpenes produced as components of the resin secreted by glandular trichomes on the surface of the calyxes and subtending leaflets. When a floral cluster is squeezed, the resinous heads of glandular trichomes rupture and the aromatic terpenes are exposed to the air. There is often a large difference between the aroma of fresh and dry floral clusters. This is explained by the polymerization (joining together in a chain) of many of the smaller molecules of aromatic terpenes to form different aromatic and nonaromatic terpene polymers. This happens as Cannabis resins age and mature, both while the plant is growing and while curing after harvest. Additional aromas may interfere with the primary terpenoid components, such as ammonia gas and other gaseous products given off by the curing, fermentation or spoilage of the tissue (non-resin) portion of the floral clusters.
A combination of at least twenty aromatic terpenes (103 are known to occur in Cannabis) and other aromatic compounds control the aroma of each plant. The production of each aromatic compound may be influenced by many genes; therefore, it is a complex matter to breed Cannabis for aroma. Breeders of perfume roses often are amazed at the complexity of the genetic control of aroma, Each strain, however, has several characteristic aromas, and these are occasionally transmitted to hybrid offspring such that they resemble one or both parents in aroma. Many times breeders complain that their strain has lost the de sired aromatic characteristics of the parental strains. Fixed hybrid strains will develop a characteristic aroma that is hereditary and often true-breeding. The cultivator with preservation of a particular aroma as a goal can clone the individual with a desired aroma in addition to breeding it. This is good insurance in case the aroma is lost in the off spring by segregation and recombination of genes.
The aromas of fresh or dried clusters are sampled and compared in such a way that they are separated to avoid confusion. Each sample is placed in the corner of a twice-folded, labeled piece of unscented writing paper at room temperature (above 650). A light squeeze will release the aromatic principles contained within the resin exuded by the ruptured glandular trichome head. When sampling, never squeeze a floral cluster directly, as the resins will ad here to the fingers and bias further sampling. The folded paper conveniently holds the floral cluster, avoids confusion during sampling, and contains the aromas as a glass does in wine tasting.
Taste is easily sampled by loosely rolling dried floral clusters in a cigarette paper and inhaling to draw a taste across the tongue. Samples should be approximately the same size.
Taste in Cannabis is divided into three categories according to usage: the taste of the aromatic components carried by air that passes over the Cannabis when it is in haled without being lighted; the taste of the smoke from burning Cannabis; and the taste of Cannabis when it is consumed orally. These three are separate entities.
The terpenes contained in a taste of unlighted Cannabis are the same as those sensed in the aroma, but perceived through the sense of taste instead of smell. Orally ingested Cannabis generally tastes bitter due to the vegetative plant tissues, but the resin is characteristically spicy and hot, somewhat like cinnamon or pepper. The taste of Cannabis smoke is determined by the burning tissues and vaporizing terpenes. These terpenes may not be detected in the aroma and unlighted taste.
Biosynthetic relationships between terpenes and cannabinoids have been firmly established. Indeed, cannabinoids are synthesized within the plant from terpene precursors. It is suspected that changes in aromatic terpene levels parallel changes in cannabinoid levels during maturation. As connections between aroma and psycho activity are uncovered, the breeder will be better able to make field selections of prospective high-THC parents without complicated analysis.
g) Persistence of Aromatic Principles and Cannabinoids - Cannabis resins deteriorate as they age, and the aromatic principles and cannabinoids break down slowly until they are hardly noticeable. Since fresh Cannabis is only available once a year in temperate regions, an important breeding goal has been a strain that keeps well when packaged. Packageability and shelf life are important considerations in the breeding of fresh fruit species and will prove equally important if trade in Cannabis develops after legalization.
h) Trichome Type - Several types of trichomes are present on the epidermal surfaces of Cannabis. Several of these trichomes are glandular and secretory in nature and are divided into bulbous, capitate sessile, and capitate stalked types. Of these, the capitate stalked glandular trichomes are apparently responsible for the intense secretion of cannabinoid laden resins. Plants with a high density of capitate stalked trichomes are a logical goal for breeders of drug Cannabis. The number and type of trichomes is easily characterized by observation with a small hand lens (lOX to 50X). Recent research by V. P. Soroka (1979) concludes that a positive correlation exists between the number of glandular trichomes on leaves and calyxes and the various cannabinoid contents of the floral clusters. In other words, many capitate stalked trichomes means higher THC levels.
i) Resin Quantity and Quality - Resin production by the glandular trichomes varies. A strain may have many glandular trichomes but they may not secrete very much resin. Resin color also varies from strain to strain. Resin heads may darken and become more opaque as they mature, as suggested by several authors. Some strains, however, pro duce fresh resins that are transparent amber instead of clear and colorless, and these are often some of the most psycho active strains. Transparent resins, regardless of color, are a sign that the plant is actively carrying out resin biosynthesis. When biosynthesis ceases, resins turn opaque as cannabinoid and aromatic levels decline. Resin color is certainly an indication of the conditions inside the resin head, and this may prove to be another important criterion for breeding.
j) Resin Tenacity - For years strains have been bred for hashish production. Hashish is formed from detached resin heads. In modern times it might be feasible to breed a strain with high resin production that gives up its precious covering of resin heads with only moderate shaking, rather than the customary flailing that also breaks up the plant. This would facilitate hashish production. Strains that are bred for use as marijuana would benefit from extremely tenacious resin heads that would not fall off during packaging and shipment.
k) Drying and Curing Rate - The rate and extent to which Cannabis dries is generally determined by the way it is dried, but, all conditions being the same, some strains dry much more rapidly and completely than others. It is assumed that resin has a role in preventing desiccation and high resin content might retard drying. However, it is a misconception that resin is secreted to coat and seal the surface of the calyxes and leaves. Resin is secreted by glandular trichomes, but they are trapped under a cuticle layer surrounding the head cells of the trichome holding the resin away from the surface of the leaves. There it would rarely if ever have a chance to seal the surface of the epidermal layer and prevent the transpiration of water. It seems that an alternate reason must be found for the great variations in rate and extent of drying. Strains may be bred that dry and cure rapidly to save valuable time.
1) Ease of Manicuring - One of the most time-consuming aspects of commercial drug Cannabis production is the seemingly endless chore of manicuring, or removing the larger leaves from the floral clusters. These larger outer leaves are not nearly as psychoactive as the inner leaves and calyxes, so they are usually removed before selling as marijuana. Strains with fewer leaves obviously require less time to manicure. Long petioles on the leaves facilitate removal by hand with a small pair of scissors. If there is a marked size difference between very large outer leaves and tiny, resinous inner leaves it is easier to manicure quickly because it is easier to see which leaves to remove.
m) Seed Characteristics - Seeds may be bred for many characteristics including size, oil content, and protein con tent. Cannabis seed is a valuable source of drying oils, and Cannabis-seed cake is a fine feed for ranch animals. Higher-protein varieties may be developed for food. Also, seeds are selected for rapid germination rate.
n) Maturation - Cannabis strains differ greatly as to when they mature and how they respond to changing environment. Some strains, such as Mexican and Hindu Kush, are famous for early maturation, and others, such as Colombian and Thai, are stubborn in maturing and nearly always finish late, if at all. Imported strains are usually characterized as either early, average, or late in maturing; however, a particular strain may produce some individuals which mature early and others which mature late. Through selection, breeders have, on the one hand, developed strains that mature in four weeks, outdoors under temperate conditions; and on the other hand, they have developed green house strains that mature in up to four months in their protected environment. Early maturation is extremely advantageous to growers who live in areas of late spring and early fall freezes. Consequently, especially early-maturing plants are selected as parents for future early-maturing strains.
o) Flowering - Once a plant matures and begins to bear flowers it may reach peak floral production in a few weeks, or the floral clusters may continue to grow and develop for several months. The rate at which a strain flowers is independent of the rate at which it matures, so a plant may wait until late in the season to flower and then grow extensive, mature floral clusters in only a few weeks.
p) Ripening - Ripening of Cannabis flowers is the final step in their maturation process Floral clusters will usually mature and ripen in rapid succession, but sometimes large floral clusters will form and only after a period of apparent hesitation will the flowers begin to produce resin and ripen. Once ripening starts it usually spreads over the entire plant, but some strains, such as those from Thailand, are known to ripen a few floral clusters at a time over several months. Some fruit trees are similarly everbearing with a yearlong season of production. Possibly Cannabis strains could be bred that are true everbearing perennials that continue to flower and mature consistently all year long.
q) Cannabinoid Profile - It is supposed that variations in the type of high associated with different strains of Cannabis result from varying levels of cannabinoids. THC is the primary psychoactive ingredient which is acted upon synergistically by small amounts of CBN, CBD, and other accessory cannabinoids. We know that cannabinoid levels may be used to establish cannabinoid phenotypes and that these phenotypes are passed on from parent to offspring. Therefore, cannabinoid levels are in part determined by genes. To accurately characterize highs from various individuals and establish criteria for breeding strains with particular cannabinoid contents, an accurate and easy method is necessary for measuring cannabinoid levels in prospective parents.
Various combinations of these traits are possible and inevitable. The traits that we most often see are most likely dominant and any effort to alter genetics and improve Cannabis strains are most easily accomplished by concentrating on the major phenotypes for the most important traits. The best breeders set high goals of a limited scope and adhere to their ideals.
