Chap 2: Propagation of Cannabis, Part 1

Chapter 2 - Propagation of Cannabis

"Make the most of the Indian Hemp Seed and sow it every where."
- George Washington

Sexual versus Asexual Propagation

Cannabis can be propagated either sexually or asexually. Seeds are the result of sexual propagation. Because sexual propagation involves the recombination of genetic material from two parents we expect to observe variation among seedlings and offspring with characteristics differing from those of the parents. Vegetative methods of propagation (cloning) such as cuttage, layerage, or division of roots are asexual and allow exact replication of the parental plant without genetic variation. Asexual propagation, in theory, allows strains to be preserved unchanged through many seasons and hundreds of individuals.

When the difference between sexual and asexual propagation is well understood then the proper method can be chosen for each situation. The unique characteristics of a plant result from the combination of genes in chromosomes present in each cell, collectively known as the genotype of that individual. The expression of a genotype, as influenced by the environment, creates a set of visible characteristics that we collectively term the phenotype. The function of propagation is to preserve special genotypes by choosing the proper technique to ensure replication of the desired characteristics.

If two clones from a pistillate Cannabis plant are placed in differing environments, shade and sun for in stance, their genotypes will remain identical. However, the clone grown in the shade will grow tall and slender and mature late, while the clone grown in full sun will remain short and bushy and mature much earlier.

Sexual Propagation

Sexual propagation requires the union of staminate pollen and pistillate ovule, the formation of viable seed, and the creation of individuals with newly recombinant genotypes. Pollen and ovules are formed by reduction divisions (meiosis) in which the 10 chromosome pairs fail to replicate, so that each of the two daughter-cells contains one-half of the chromosomes from the mother cell. This is known as the haploid (in) condition where in = 10 chromosomes. The diploid condition is restored upon fertilization resulting in diploid (2n) individuals with a haploid set of chromosomes from each parent. Offspring may resemble the staminate, pistillate, both, or neither parent and considerable variation in offspring is to be expected. Traits may be controlled by a single gene or a combination of genes, resulting in further potential diversity.

The terms homozygous and heterozygous are useful in describing the genotype of a particular plant. If the genes controlling a trait are the same on one chromosome as those on the opposite member of the chromosome pair (homologous chromosomes), the plant is homozygous and will "breed true" for that trait if self-pollinated or crossed with an individual of identical genotype for that trait. The traits possessed by the homozygous parent will be transmitted to the offspring, which will resemble each other and the parent. If the genes on one chromosome differ from the genes on its homologous chromosome then the plant is termed heterozygous; the resultant offspring may not possess the parental traits and will most probably differ from each other. Imported Cannabis strains usually exhibit great seedling diversity for most traits and many types will be discovered.

To minimize variation in seedlings and ensure preservation of desirable parental traits in offspring, certain careful procedures are followed as illustrated in Chapter III. The actual mechanisms of sexual propagation and seed production will be thoroughly explained here.

The Life Cycle and Sinsemilla Cultivation

A wild Cannabis plant grows from seed to a seedling, to a prefloral juvenile, to either pollen- or seed-bearing adult, following the usual pattern of development and sexual reproduction. Fiber and drug production both interfere with the natural cycle and block the pathways of inheritance. Fiber crops are usually harvested in the juvenile or prefloral stage, before viable seed is produced, while sinsemilla or seedless marijuana cultivation eliminates pollination and subsequent seed production. In the case of cultivated Cannabis crops, special techniques must be used to produce viable seed for the following year without jeopardizing the quality of the final product.

Modern fiber or hemp farmers use commercially produced high fiber content strains of even maturation. Monoecious strains are often used because they mature more evenly than dioecious strains. The hemp breeder sets up test plots where phenotypes can be recorded and controlled crosses can be made. A farmer may leave a portion of his crop to develop mature seeds which he collects for the following year. If a hybrid variety is grown, the offspring will not ail resemble the parent crop and desirable characteristics may be lost.

Growers of seeded marijuana for smoking or hashish production collect vast quantities of seeds that fall from the flowers during harvesting, drying, and processing. A mature pistillate plant can produce tens of thousands of seeds if freely pollinated. Sinsemilla marijuana is grown by removing all the staminate plants from a patch, eliminating every pollen source, and allowing the pistillate plants to produce massive clusters of unfertilized flowers.

Various theories have arisen to explain the unusually potent psychoactive properties of unfertilized Cannabis. In general these theories have as their central theme the extraordinarily long, frustrated struggle of the pistillate plant to reproduce, and many theories are both twisted and romantic. What actually happens when a pistillate plant remains unfertilized for its entire life and how this ultimately affects the cannabinoid (class of molecules found only in Cannabis) and terpene (a class of aromatic organic compounds) levels remains a mystery. It is assumed, how ever, that seeding cuts the life of the plant short and THC (tetrahydrocannabinol the major psychoactive compound in Cannabis) does not have enough time to accumulate. Hormonal changes associated with seeding definitely affect all metabolic processes within the plant including cannabinoid biosynthesis. The exact nature of these changes is unknown but probably involves imbalance in the enzymatic systems controlling cannabinoid production. Upon fertilization the plantâ??s energies are channeled into seed production instead of increased resin production. Sinsemilla plants continue to produce new floral clusters until late fail, while seeded plants cease floral production. It is also suspected that capitate-stalked trichome production might cease when the calyx is fertilized. If this is the case, then sinsemilla may be higher in THC because of uninterrupted floral growth, trichome formation and cannabinoid production. What is important with respect to propagation is that once again the farmer has interfered with the life cycle and no naturally fertilized seeds have been produced.

The careful propagator, however, can produce as many seeds of pure types as needed for future research without risk of pollinating the precious crop. Staminate parents exhibiting favorable characteristics are reproductively isolated while pollen is carefully collected and applied to only selected flowers of the pistillate parents.

Many cultivators overlook the staminate plant, considering it useless if not detrimental. But the staminate plant contributes half of the genotype expressed in the offspring. Not only are staminate plants preserved for breeding, but they must be allowed to mature, uninhibited, until their phenotypes can be determined and the most favorable individuals selected. Pollen may also be stored for short periods of time for later breeding.

Biology of Pollination

Pollination is the event of pollen landing on a stigmatic surface such as the pistil, and fertilization is the union of the staminate chromosomes from the pollen with the pistillate chromosomes from the ovule.

Pollination begins with dehiscence (release of pollen) from staminate flowers. Millions of pollen grains float through the air on light breezes, and many land on the stigmatic surfaces of nearby pistillate plants. If the pistil is ripe, the pollen grain will germinate and send out a long pollen tube much as a seed pushes out a root. The tube contains a haploid (in) generative nucleus and grows downward toward the ovule at the base of the pistils. When the pollen tube reaches the ovule, the staminate haploid nucleus fuses with the pistillate haploid nucleus and the diploid condition is restored. Germination of the pollen grain occurs 15 to 20 minutes after contact with the stigmatic surface (pistil); fertilization may take up to two days in cooler temperatures. Soon after fertilization, the pistils wither away as the ovule and surrounding calyx begin to swell. If the plant is properly watered, seed will form and sexual reproduction is complete. It is crucial that no part of the cycle be interrupted or viable seed will not form. If the pollen is subjected to extremes of temperature, humidity, or moisture, it will fail to germinate, the pollen tube will die prior to fertilization, or the embryo will be unable to develop into a mature seed. Techniques for successful pollination have been designed with all these criteria in mind.

Controlled versus Random Pollinations

The seeds with which most cultivators begin represent varied genotypes even when they originate from the same floral cluster of marijuana, and not all of these genotypes will prove favorable. Seeds collected from imported shipments are the result of totally random pollinations among many genotypes. If elimination of pollination was at tempted and only a few seeds appear, the likelihood is very high that these pollinations were caused by a late flowering staminate plant or a hermaphrodite, adversely affecting the genotype of the offspring. Once the offspring of imported strains are in the hands of a competent breeder, selection and replication of favorable phenotypes by controlled breeding may begin. Only one or two individuals out of many may prove acceptable as parents. If the cultivator allows random pollination to occur again, the population not only fails to improve, it may even degenerate through natural and accidental selection of unfavorable traits. We must therefore turn to techniques of controlled pollination by which the breeder attempts to take control and deter mine the genotype of future offspring.

Data Collection

Keeping accurate notes and records is a key to successful plant-breeding. Crosses among ten pure strains (ten staminate and ten pistillate parents) result in ten pure and ninety hybrid crosses. It is an endless and inefficient task to attempt to remember the significance of each little number and colored tag associated with each cross. The well organized breeder will free himself from this mental burden and possible confusion by entering vital data about crosses, phenotypes, and growth conditions in a system with one number corresponding to each member of the population.

The single most important task in the proper collection of data is to establish undeniable credibility. Memory fails, and remembering the steps that might possibly have led to the production of a favorable strain does not constitute the data needed to reproduce that strain. Data is always written down; memory is not a reliable record. A record book contains a numbered page for each plant, and each separate cross is tagged on the pistillate parent and recorded as follows: "seed of pistillate parent X pollen or staminate parent." Also the date of pollination is included and room is left for the date of seed harvest. Samples of the parental plants are saved as voucher specimens for later characterization and analysis.

Pollination Techniques

Controlled hand pollination consists of two basic steps: collecting pollen from the anthers of the staminate parent and applying pollen to the receptive stigmatic surfaces of the pistillate parent. Both steps are carefully con trolled so that no pollen escapes to cause random pollinations. Since Cannabis is a wind-pollinated species, enclosures are employed which isolate the ripe flowers from wind, eliminating pollination, yet allowing enough light penetration and air circulation for the pollen and seeds to develop without suffocating. Paper and very tightly woven cloth seem to be the most suitable materials. Coarse cloth allows pollen to escape and plastic materials tend to collect transpired water and rot the flowers. Light-colored opaque or translucent reflective materials remain cooler in the sun than dark or transparent materials, which either absorb solar heat directly or create a greenhouse effect, heating the flowers inside and killing the pollen. Pollination bags are easily constructed by gluing together vegetable parchment (a strong breathable paper for steaming vegetables) and clear nylon oven bags (for observation windows) with silicon glue. Breathable synthetic fabrics such as Gore-Tex are used with great success. Seed production requires both successful pollination and fertilization, so the conditions inside the enclosures must remain suitable for pollen-tube growth and fertilization. It is most convenient and effective to use the same enclosure to collect pollen and apply it, reducing contamination during pollen transfer. Controlled "free" pollinations may also be made if only one pollen parent is allowed to remain in an isolated area of the field and no pollinations are caused by hermaphrodites or late-maturing staminate plants. If the selected staminate parent drops pollen when there are only a few primordial flowers on the pistillate seed parent, then only a few seeds will form in the basal flowers and the rest of the flower cluster will be seedless. Early fertilization might also help fix the sex of the pistillate plant, helping to prevent hermaphrodism. Later, hand pollinations can be performed on the same pistillate parent by removing the early seeds from each limb to be re-pollinated, so avoiding confusion. Hermaphrodite or monoecious plants may be isolated from the remainder of the population and allowed to freely self-pollinate if pure-breeding offspring are desired to preserve a selected trait. Selfed hermaphrodites usually give rise to hermaphrodite offspring.

Pollen may be collected in several ways. If the propagator has an isolated area where staminate plants can grow separate from each other to avoid mutual contamination and can be allowed to shed pollen without endangering the remainder of the population, then direct collection may be used. A small vial, glass plate, or mirror is held beneath a recently-opened staminate flower which appears to be releasing pollen, and the pollen is dislodged by tap ping the anthers. Pollen may also be collected by placing whole limbs or clusters of staminate flowers on a piece of paper or glass and allowing them to dry in a cool, still place. Pollen will drop from some of the anthers as they dry, and this may be scraped up and stored for a short time in a cool, dark, dry spot. A simple method is to place the open pollen vial or folded paper in a larger sealable container with a dozen or more fresh, dry soda crackers or a cup of dry white rice. The sealed container is stored in the refrigerator and the dry crackers or rice act as a desiccant, absorbing moisture from the pollen.

Any breeze may interfere with collection and cause contamination with pollen from neighboring plants. Early morning is the best time to collect pollen, as it has not been exposed to the heat of the day. All equipment used for collection, including hands, must be cleaned before continuing to the next pollen source. This ensures protection of each pollen sample from contamination with pollen from different plants.

Staminate flowers will often open several hours before the onset of pollen release. If flowers are collected at this time they can be placed in a covered bottle where they will open and release pollen within two days. A carefully sealed paper cover allows air circulation, facilitates the release of pollen, and prevents mold.

Both of the previously described methods of pollen collection are susceptible to gusts of wind, which may cause contamination problems if the staminate pollen plants grow at all close to the remaining pistillate plants. There fore, a method has been designed so that controlled pollen collection and application can be performed in the same area without the need to move staminate plants from their original location. Besides the advantages of convenience, the pollen parents mature under the same conditions as the seed parents, thus more accurately expressing their phenotypes.

The first step in collecting pollen is, of course, the selection of a staminate or pollen parent. Healthy individuals with well-developed clusters of flowers are chosen. The appearance of the first staminate primordia or male sex signs often brings a feeling of panic ("stamenoia") to the cultivator of seedless Cannabis, and potential pollen parents are prematurely removed. Staminate primordia need to develop from one to five weeks before the flowers open and pollen is released. During this period the selected pollen plants are carefully watched, daily or hourly if necessary, for developmental rates vary greatly and pollen may be released quite early in some strains. The remaining staminate plants that are unsuitable for breeding are destroyed and the pollen plants specially labeled to avoid confusion and extra work.

As the first flowers begin to swell, they are removed prior to pollen release and destroyed. Tossing them on the ground is ineffective because they may release pollen as they dry. When the staminate plant enters its full floral condition and more ripe flowers appear than can be easily controlled, limbs with the most ripe flowers are chosen. It is usually safest to collect pollen from two limbs for each intended cross, in case one fails to develop. If there are ten prospective seed parents, pollen from twenty limbs on the pollen parent is collected. In this case, the twenty most flowered limb tips are selected and all the remaining flowering clusters on the plant are removed to prevent stray pollinations. Large leaves are left on the remainder of the plant but are removed at the limb tips to minimize condensation of water vapor released inside the enclosure. The portions removed from the pollen parent are saved for later analysis and phenotype characterization.

The pollination enclosures are secured and the plant is checked for any shoots where flowers might develop outside the enclosure. The completely open enclosure is slipped over the limb tip and secured with a tight but stretchable seal such as a rubber band, elastic, or plastic plant tie-tape to ensure a tight seal and prevent crushing of the vascular tissues of the stem. String and wire are avoided. If enclosures are tied to weak limbs they may be supported; the bags will also remain cooler if they are shaded. Hands are always washed before and after handling each pollen sample to prevent accidental pollen transfer and contamination.

Enclosures for collecting and applying pollen and preventing stray pollination are simple in design and construction. Paper bags make convenient enclosures. Long narrow bags such as light-gauge quart-bottle bags, giant popcorn bags or bakery bags provide a convenient shape for covering the limb tip. The thinner the paper used the more air circulation is allowed, and the better the flowers will develop. Very thick paper or plastic bags are never used. Most available bags are made with water soluble glue and may come apart after rain or watering. All seams are sealed with waterproof tape or silicon glue and the bags should not be handled when wet since they tear easily. Bags of Gore-Tex cloth or vegetable parchment will not tear when wet. Paper bags make labeling easy and each bag is marked in waterproof ink with the number of the individual pollen parent, the date and time the enclosure was secured, and any useful notes. Room is left to add the date of pollen collection and necessary information about the future seed parent it will pollinate.

Pollen release is fairly rapid inside the bags, and after two days to a week the limbs may be removed and dried in a cool dark place, unless the bags are placed too early or the pollen parent develops very slowly. To inspect the progress of pollen release, a flashlight is held behind the bag at night and the silhouettes of the opening flowers are easily seen. In some cases, clear nylon windows are in stalled with silicon glue for greater visibility. When flowering is at its peak and many flowers have just opened, collection is completed, and the limb, with its bag attached, is cut. If the limb is cut too early, the flowers will not have shed any pollen; if the bag remains on the plant too long, most of the pollen will be dropped inside the bag where heat and moisture will destroy it. When flowering is at its peak, millions of pollen grains are released and many more flowers will open after the limbs are collected. The bags are collected early in the morning before the sun has time to heat them up. The bags and their contents are dried in a cool dark place to avoid mold and pollen spoilage. If pollen becomes moist, it will germinate and spoil, therefore dry storage is imperative.

After the staminate limbs have dried and pollen re lease has stopped, the bags are shaken vigorously, allowed to settle, and carefully untied. The limbs and loose flowers are removed, since they are a source of moisture that could promote mold growth, and the pollen bags are re sealed. The bags may be stored as they are until the seed parent is ready for pollination, or the pollen may be re moved and stored in cool, dry, dark vials for later use and hand application. Before storing pollen, any other plant parts present are removed with a screen. A piece of fuel filter screening placed across the top of a mason jar works well, as does a fine-mesh tea strainer.

Now a pistillate plant is chosen as the seed parent. A pistillate flower cluster is ripe for fertilization so long as pale, slender pistils emerge from the calyxes. Withered, dark pistils protruding from swollen, resin encrusted calyxes are a sign that the reproductive peak has long passed. Cannabis plants can be successfully pollinated as soon as the first primordia show pistils and until just before harvest, but the largest yield of uniform, healthy seeds is achieved by pollinating in the peak floral stage. At this time, the seed plant is covered with thick clusters of white pistils. Few pistils are brown and withered, and resin production has just begun. This is the most receptive time for fertilization, still early in the seed plantâ??s life, with plenty of time remaining for the seeds to mature. Healthy, well flowered lower limbs on the shaded side of the plant are selected. Shaded buds will not heat up in the bags as much as buds in the hot sun, and this will help protect the sensitive pistils. When possible, two terminal clusters of pistillate flowers are chosen for each pollen bag. In this way, with two pollen bags for each seed parent and two clusters of pistillate flowers for each bag, there are four opportunities to perform the cross successfully. Remember that production of viable seed requires successful pollination, fertilization and embryo development. Since interfering with any part of this cycle precludes seed development, fertilization failure is guarded against by duplicating all steps.

Before the pollen bags are used, the seed parent information is added to the pollen parent data. Included is the number of the seed parent, the date of pollination, and any comments about the phenotypes of both parents. Also, for each of the selected pistillate clusters, a tag containing the same information is made and secured to the limb below the closure of the bag. A warm, windless evening is chosen for pollination so the pollen tube has time to grow before sunrise. After removing most of the shade leaves from the tips of the limbs to be pollinated, the pollen is tapped away from the mouth of the bag. The bag is then carefully opened and slipped over two inverted limb tips, taking care not to release any pollen, and tied securely with an expandable band. The bag is shaken vigorously, so the pollen will be evenly dispersed throughout the bag, facilitating complete pollination. Fresh bags are sometimes used, either charged with pollen prior to being placed over the limb tip, or injected with pollen, using a large syringe or atomizer, after the bag is placed. However, the risk of accidental pollination with injection is higher.

If only a small quantity of pollen is available it may be used more sparingly by diluting with a neutral powder such as flour before it is used. When pure pollen is used, many pollen grains may land on each pistil when only one is needed for fertilization. Diluted pollen will go further and still produce high fertilization rates. Diluting 1 part pollen with 10 to 100 parts flour is common. Powdered fungicides can also be used since this helps retard the growth of molds in the maturing, seeded, floral clusters.

The bags may remain on the seed parent for sometime; seeds usually begin to develop within a few days, buttheir development will be retarded by the bags. The propagator waits three full sunny days, then carefully removes and sterilizes or destroys the bags. This way there is little chance of stray pollination. Any viable pollen that failed to pollinate the seed parent will germinate in the warm moist bag and die within three days, along with many of the unpollinated pistils. In particularly cool or overcast conditions a week may be necessary, but the bag is removed at the earliest safe time to ensure proper seed development without stray pollinations. As soon as the bag is removed, the calyxes begin to swell with seed, indicating successful fertilization. Seed parents then need good irrigation or development will be retarded, resulting in small, immature, and nonviable seeds. Seeds develop fastest in

warm weather and take usually from two to four weeks to mature completely. In cold weather seeds may take up to two months to mature. If seeds get wet in fall rains, they may sprout. Seeds are removed when the calyx begins to dry up and the dark shiny perianth (seed coat) can be seen protruding from the drying calyx. Seeds are labeled and stored in a cool, dark, dry place, This is the method employed by breeders to create seeds of known parentage used to study and improve Cannabis genetics.

Seed Selection

Nearly every cultivated Cannabis plant, no matter what its future, began as a germinating seed; and nearly all Cannabis cultivators, no matter what their intention, start with seeds that are gifts from a fellow cultivator or extracted from imported shipments of marijuana. Very little true control can be exercised in seed selection unless the cultivator travels to select growing plants with favorable characteristics and personally pollinate them. This is not possible for most cultivators or researchers and they usually rely on imported seeds. These seeds are of unknown parentage, the product of natural selection or of breeding by the original farmer, Certain basic problems affect the genetic purity and predictability of collected seed.

1 - If a Cannabis sample is heavily seeded, then the majority of the male plants were allowed to mature and release pollen, Since Cannabis is wind-pollinated, many pollen parents (including early and late maturing staminate and hermaphrodite plants) will contribute to the seeds in any batch of pistillate flowers. If the seeds are all taken from one flower cluster with favorable characteristics, then at least the pistillate or seed parent is the same for all those seeds, though the pollen may have come from many different parents. This creates great diversity in offspring.

2 - In very lightly seeded or nearly sinsemilla Cannabis, pollination has largely been prevented by the removal of staminate parents prior to the release of pollen. The few seeds that do form often result from pollen from hermaphrodite plants that went undetected by the farmer, or by random wind-borne pollen from wild plants or a nearby field. Hermaphrodite parents often produce hermaphrodite offspring and this may not be desirable.

3 - Most domestic Cannabis strains are random hybrids. This is the result of limited selection of pollen parents, impure breeding conditions, and lack of adequate space to isolate pollen parents from the remainder of the crop.

When selecting seeds, the propagator will frequently look for seed plants that have been carefully bred locally by another propagator. Even if they are hybrids there is a better chance of success than with imported seeds, pro vided certain guidelines are followed:

1 - The dried seeded flower clusters are free of staminate flowers that might have caused hermaphrodite pollinations.

2 - The flowering clusters are tested for desirable traits and seeds selected from the best.

3 - Healthy, robust seeds are selected. Large, dark seeds are best; smaller, paler seeds are avoided since these are usually less mature and less viable.

4 - If accurate information is not available about the pollen parent, then selection proceeds on common sense and luck. Mature seeds with dried calyxes in the basal portions of the floral clusters along the main stems occur in the earliest pistillate flowers to appear and must have been pollinated by early-maturing pollen parents. These seeds have a high chance of producing early-maturing offspring. By contrast, mature seeds selected from the tips of floral clusters, often surrounded by immature seeds, are formed in later-appearing pistillate flowers. These flowers were likely pollinated by later-maturing staminate or hermaphrodite pollen parents, and their seeds should mature later and have a greater chance of producing hermaphrodite off spring. The pollen parent also exerts some influence on the appearance of the resulting seed. If seeds are collected from the same part of a flower cluster and selected for similar size, shape, color, and perianth patterns, then it is more likely that the pollinations represent fewer different gene pools and will produce more uniform offspring.
5 - Seeds are collected from strains that best suit the locality; these usually come from similar climates and latitudes. Seed selection for specific traits is discussed in detail in Chapter III.

6 - Pure strain seeds are selected from crosses between parents of the same origin.

7 - Hybrid seeds are selected from crosses between pure strain parents of different origins.

8 - Seeds from hybrid plants, or seeds resulting from pollination by hybrid plants, are avoided, since these will not reliably reproduce the phenotype of either parent.

Seed stocks are graded by the amount of control exerted by the collector in selecting the parents. Grade #1 - Seed parent and pollen parent are known and there is absolutely no possibility that the seeds resulted from pollen contamination.

Grade #2 - Seed parent is known but several known staminate or hermaphrodite pollen parents are involved. Grade #3 - Pistillate parent is known and pollen parents are unknown.

Grade #4 - Neither parent is known, but the seeds are collected from one floral cluster, so the pistillate seed parent age traits may be characterized.

Grade #5 - Parentage is unknown but origin is certain, such as seeds collected from the bottom of a bag of imported Cannabis.

Grade #6 - Parentage and origin are unknown.

Asexual Propagation

Asexual propagation (cloning) allows the preservation of genotype because only normal cell division (mitosis) occurs during growth and regeneration. The vegetative (non-reproductive) tissue of Cannabis has 10 pairs of chromosomes in the nucleus of each cell. This is known as the diploid (2n) condition where 2n = 20 chromosomes. During mitosis every chromosome pair replicates and one of the two identical sets of chromosome pairs migrates to each daughter cell, which now has a genotype identical to the mother cell. Consequently, every vegetative cell in a Cannabis plant has the same genotype and a plant resulting from asexual propagation will have the same genotype as the mother plant and will, for all practical purposes, develop identically under the same environmental conditions.

In Cannabis, mitosis takes place in the shoot apex (meristem), root tip meristems, and the meristematic cambium layer of the stalk. A propagator makes use of these meristematic areas to produce clones that will grow and be multiplied. Asexual propagation techniques such as cuttage, layerage, and division of roots can ensure identical populations as large as the growth and development of the parental material will permit. Clones can be produced from even a single cell, because every cell of the plant possesses the genetic information necessary to regenerate a complete plant.

Asexual propagation produces clones which perpetuate the unique characteristics of the parent plant. Because of the heterozygous nature of Cannabis, valuable traits may be lost by sexual propagation that can be preserved and multiplied by cloning. Propagation of nearly identical populations of all-pistillate, fast growing, evenly maturing Cannabis is made possible through cloning. Any agricultural or environmental influences will affect all the members of that clone equally.

The concept of clone does not mean that all members of the clone will necessarily appear identical in all characteristics. The phenotype that we observe in an individual is influenced by its surroundings. Therefore, members of the clone will develop differently under varying environmental conditions. These influences do not affect genotype and therefore are not permanent. Cloning theoretically can pre serve a genotype forever. Vigor may slowly decline due to poor selection of clone material or the constant pressure of disease or environmental stress, but this trend will re verse if the pressures are removed. Shifts in genetic composition occasionally occur during selection for vigorous growth. However, if parental strains are maintained by in frequent cloning this is less likely. Only mutation of a gene in a vegetative cell that then divides and passes on the mutated gene will permanently affect the genotype of the clone. If this mutated portion is cloned or reproduced sexually, the mutant genotype will be further replicated. Mutations in clones usually affect dominance relations and are therefore noticed immediately. Mutations may be induced artificially (but without much predictability) by treating meristematic regions with X-rays, colchicine, or other mutagens.

The genetic uniformity provided by clones offers a control for experiments designed to quantify the subtle effects of environment and cultural techniques. These subtleties are usually obscured by the extreme diversity resulting from sexual propagation. However, clonal uniformity can also invite serious problems. If a population of clones is subjected to sudden environmental stress, pests, or disease for which it has no defense, every member of the clone is sure to be affected and the entire population may be lost. Since no genetic diversity is found within the clone, no adaptation to new stresses can occur through recombination of genes as in a sexually propagated population.

In propagation by cuttage or layerage it is only necessary for a new root system to form, since the meristematic shoot apex comes directly from the parental plant. Many stem cells, even in mature plants, have the capability of producing adventitious roots. In fact, every vegetative cell in the plant contains the genetic information needed for an entire plant. Adventitious roots appear spontaneously from stems and old roots as opposed to systemic roots which appear along the developing root system originating in the embryo. In humid conditions (as in the tropics or a green house) adventitious roots occur naturally along the main stalk near the ground and along limbs where they droop and touch the ground.

Rooting

A knowledge of the internal structure of the stem is helpful in understanding the origin of adventitious roots.

The development of adventitious roots can be broken down into three stages: (1) the initiation of meristematic cells located just outside and between the vascular bundles (the root initials), (2) the differentiation of these meristematic cells into root primordia, and (3) the emergence and growth of new roots by rupturing old stem tissue and establishing vascular connections with the shoot.

As the root initials divide, the groups of cells take on the appearance of a small root tip. A vascular system forms with the adjacent vascular bundles and the root continues to grow outward through the cortex until the tip emerges from the epidermis of the stem. Initiation of root growth usually begins within a week and young roots appear within four weeks. Often an irregular mass of white cells, termed callus tissue, will form on the surface of the stem adjacent to the areas of root initiation. This tissue has no influence on root formation. However, it is a form of regenerative tissue and is a sign that conditions are favorable for root initiation.

The physiological basis for root initiation is well understood and allows many advantageous modifications of rooting systems. Natural plant growth substances such as auxins, cytokinins, and gibberellins are certainly responsible for the control of root initiation and the rate of root formation. Auxins are considered the most influential. Auxins and other growth substances are involved in the control of virtually all plant processes: stem growth, root formation, lateral bud inhibition, floral maturation, fruit development, and determination of sex. Great care is exercised in application of artificial growth substances so that detrimental conflicting reactions in addition to rooting do not occur. Auxins seem to affect most related plant species in the same way, but the mechanism of this action is not yet fully understood.

Many synthetic compounds have been shown to have auxin activity and are commercially available, such as napthaleneacetic acid (NAA), indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4 DPA), but only indoleacetic acid has been isolated from plants. Naturally occurring auxin is formed mainly in the apical shoot men stem and young leaves. It moves downward after its formation at the growing shoot tip, but massive concentrations of auxins in rooting solutions will force travel up the vascular tissue. Knowledge of the physiology of auxins has led to practical applications in rooting cuttings. It was shown originally by Went and later by Thimann and Went that auxins promote adventitious root formation in stem cuttings. Since application of natural or synthetic auxin seems to stimulate adventitious root formation in many plants, it is assumed that auxin levels are associated with the formation of root initials. Further research by Warmke and Warmke (1950) suggested that the levels of auxin may determine whether adventitious roots or shoots are formed, with high auxin levels promoting root growth and low levels favoring shoots.

Cytokinins are chemical compounds that stimulate cell growth. In stem cuttings, cytokinins suppress root growth and stimulate bud growth. This is the opposite of the reaction caused by auxins, suggesting that a natural balance of the two may be responsible for regulating nor mal plant growth. Skoog discusses the use of solutions of equal concentrations of auxins and cytokinins to pro mote the growth of undifferentiated callus tissues. This may provide a handy source of undifferentiated material for cellular cloning.

Although Cannabis cuttings and layers root easily, variations in rootability exist and old stems may resist rooting. Selection of rooting material is highly important. Young, firm, vegetative shoots, 3 to 7 millimeters (1/8 to Â¼ inch) in diameter, root most easily. Weak, unhealthy plants are avoided, along with large woody branches and reproductive tissues, since these are slower to root. Stems of high carbohydrate content root most easily. Firmness is a sign of high carbohydrate levels in stems but may be con fused with older woody tissue. An accurate method of determining the carbohydrate content of cuttings is the iodine starch test. The freshly cut ends of a bundle of cuttings are immersed in a weak solution of iodine in potassium iodide. Cuttings containing the highest starch content stain the darkest; the samples are rinsed and sorted accordingly. High nitrogen content cuttings seem to root more poorly than cuttings with medium to low nitrogen content. Therefore, young, rapidly-growing stems of high nitrogen and low carbohydrate content root less well than slightly older cuttings. For rooting, sections are selected that have ceased elongating and are beginning radial growth. Staminate plants have higher average levels of carbohydrates than pistillate plants, while pistillate plants exhibit higher nitrogen levels. It is unknown whether sex influences rooting, but cuttings from vegetative tissue are taken just after sex determination while stems are still young. For rooting cloning stock or parental plants, the favorable balance (low nitrogen-to-high carbohydrate) is achieved in several ways:

1 - Reduction of the nitrogen supply will slow shoot growth and allow time for carbohydrates to accumulate. This can be accomplished by leaching (rinsing the soil with large amounts of fresh water), withholding nitrogenous fertilizer, and allowing stock plants to grow in full sun light. Crowding of roots reduces excessive vegetative growth and allows for carbohydrate accumulation.

2 - Portions of the plant that are most likely to root are selected. Lower branches that have ceased lateral growth and begun to accumulate starch are the best. The carbohydrate-to-nitrogen ratio rises as you move away from the tip of the limb, so cuttings are not made too short.

3 - Etiolation is the growth of stem tissue in total darkness to increase the possibility of root initiation. Starch levels drop, strengthening tissues and fibers begin to soften, cell wall thickness decreases, vascular tissue is diminished, auxin levels rise, and undifferentiated tissue begins to form. These conditions are very conducive to the initiation of root growth. If the light cycle can be con trolled, whole plants can be subjected to etiolation, but usually single limbs are selected for cloning and wrapped for several inches just above the area where the cutting will be taken. This is done two weeks prior to rooting. The etiolated end may then be unwrapped and inserted into the rooting medium. Various methods of layers and cuttings rooted below soil level rely in part on the effects of etiolation.

4 - Girdling a stem by cutting the phloem with a knife or crushing it with a twisted wire may block the downward mobility of carbohydrates and auxin and rooting cofactors, raising the concentration of these valuable components of root initiation above the girdle.