Techniques of Layering
Almost all layering techniques rely on the principle of etiolation. Both soil layering and air layering involve depriving the rooting portion of the stem of light, promoting rooting. Root-promoting substances and fungicides prove beneficial, and they are usually applied as a spray or powder. Root formation on layers depends on constant moisture, good air circulation and moderate temperatures at the site of rooting.
Soil Layering
Soil layering may be performed in several ways. The most common is known as tip layering. A long, supple vegetative lower limb is selected for layering, carefully bent so it touches the ground, and stripped of leaves and small shoots where the rooting is to take place. A narrow trench, 6 inches to a foot long and 2 to 4 inches deep, is dug parallel to the limb, which is placed along the bottom of the trench, secured with wire or wooden stakes, and buried with a small mound of soil. The buried section of stem may be girdled by cutting, crushed with a loop of wire, or twisted to disrupt the phloem tissue and cause the accumulation of substances which promote rooting. It may also be treated with growth regulators at this time.
Serpentine layering may be used to create multiple layers along one long limb. Several stripped sections of the limb are buried in separate trenches, making sure that at least one node remains above ground between each set of roots to allow shoots to develop. The soil surrounding the stem is kept moist at all times and may require wetting several times a day. A small stone or stick is inserted under each exposed section of stem to prevent the lateral shoot buds rotting from constant contact with the moist soil surface. Tip layers and serpentine layers may be started in small containers placed near the parental plant. Rooting usually begins within two weeks, and layers may be re moved with a sharp razor or clippers after four to six weeks. If the roots have become well established, transplanting may be difficult without damaging the tender root system. Shoots on layers continue to grow under the same conditions as the parent, and less time is needed for the clone to acclimatize or harden-off and begin to grow on its own than with cuttings.
In air layering, roots form on the aerial portions of stems that have been girdled, treated with growth regulators, and wrapped with moist rooting media. Air layering is an ancient form of propagation, possibly invented by the Chinese. The ancient technique of goo tee uses a ball of clay or soil plastered around a girdled stem and held with a wrap of fibers. Above this is suspended a small container of water (such as a bamboo section) with a wick to the wrapped gootee; this way the gootee remains moist. The single most difficult problem with air layers is the tendency for them to dry out quickly. Relatively small amounts of rooting media are used, and the position on aerial parts of the plant exposes them to drying winds and sun. Many wraps have been tried, but the best seems to be clear polyethylene plastic sheeting which allows oxygen to enter and retains moisture well. Air layers are easiest to make in greenhouses where humidity is high, but they may also be used outside as long as they are kept moist and don’t freeze. Air layers are most useful to the amateur propagator and breeder because they take up little space and allow the efficient cloning of many individuals.
Making an Air Layer
A recently sexed young limb 3-10 mm (1/8 to 3/8 inch) in diameter is selected. The site of the layer is usually a spot 30 centimeters (12 inches) or more from the limb tip. Unless the stem is particularly strong and woody, it is splinted by positioning a 30 centimeter (12 inch) stick of approximately the same diameter as the stem to be layered along the bottom edge of the stem. This splint is tied in place at both ends with a piece of elastic plant-tie tape. This enables the propagator to handle the stem more confidently. An old, dry Cannabis stem works well as a splint. Next, the stem is girdled between the two ties with a twist of wire or a diagonal cut. After girdling, the stem is sprayed or dusted with a fungicide and growth regulator, surrounded with one or two handfuls of unmilled sphagnum moss, and wrapped tightly with a small sheet of clear polyethylene film (4-6 mil). The film is tied securely at each end, tightly enough to make a waterproof seal but not so tight that the phloem tissues are crushed. If the phloem is crushed, compounds necessary for rooting will accumulate outside of the medium and rooting will be slowed. Plastic florist’s tape or electrician’s tape works well for sealing air layers. Although polyethylene film retains moisture well, the moss will dry out eventually and must be remoistened periodically. Unwrapping each layer is impractical and would disturb the roots, so a hypodermic syringe is used to inject water, nutrients, fungicides, and growth regulators. If the layers become too wet the limb rots. Layers are checked regularly by injecting water until it squirts out and then very lightly squeezing the medium to remove any extra water. Heavy layers on thin limbs are supported by tying them to a large adjacent limb or a small stick anchored in the ground. Rooting begins within two weeks and roots will be visible through the clear plastic within four weeks. When the roots appear adequately developed, the layer is removed, carefully unwrapped, and transplanted with the moss and the splint intact. The layer is watered well and placed in a shady spot for a few days to allow the plant to harden-off and adjust to living on its own root system. It is then placed in the open. In hot weather, large leaves are removed from the shoot before removing the layer to prevent excessive transpiration and wilting.
Layers develop fastest just after sexual differentiation. Many layers may be made of staminate plants in order to save small samples of them for pollen collection and to conserve space. By the time the pollen parents begin to flower profusely, the layers will be rooted and may be cut and removed to an isolated area. Layers taken from pistil late plants are used for breeding, or saved and cloned for the following season. Layers often seem rejuvenated when they are re moved from the parent plant and begin to be supported by their own root systems. This could mean that a clone will continue to grow longer and mature later than its parent under the same conditions. Layers removed from old or seeded parents will continue to produce new calyxes and pistils instead of completing the life cycle along with the parents. Rejuvenated layers are useful for off-season seed production.
Grafting
Intergeneric grafts between Cannabis and Humulus (hops) have fascinated researchers and cultivators for decades. Warmke and Davidson (1943) claimed that Humbles tops grafted upon Cannabis roots produced ". . . as much drug as leaves from intact hemp plants, even though leaves from intact hop plants are completely nontoxic." According to this research, the active ingredient of Cannabis was being produced in the roots and transported across the graft to the Humulus tops. Later research by Crombie and Crombie (1975) entirely disproves this theory. Grafts were made between high and low THC strains of Cannabis as well as intergeneric grafts between Cannabis and Humulus, Detailed chromatographic analysis was performed on both donors for each graft and their control populations. The results showed ". . . no evidence of transport of inter mediates or factors critical to cannabinoid formation across the grafts." Grafting of Cannabis is very simple. Several seedlings can be grafted together into one to produce very interesting specimen plants. One procedure starts by planting one seed ling each of several separate strains close together in the same container, placing the stock (root plant) for the cross in the center of the rest. When the seedlings are four weeks old they are ready to be grafted. A diagonal cut is made approximately half-way through the stock stem and one of the scion (shoot) seedlings at the same level. The cut portions are slipped together such that the inner cut surfaces are touching. The joints are held with a fold of cellophane tape. A second scion from an adjacent seedling may be grafted to the stock higher up the stem. After two weeks, the unwanted portions of the grafts are cut away. Eight to twelve weeks are needed to complete the graft, and the plants are maintained in a mild environment at all times. As the graft takes, and the plant begins to grow, the tape falls off.
Pruning
Pruning techniques are commonly used by Cannabis cultivators to limit the size of their plants and promote branching. Several techniques are available, and each has its advantages and drawbacks. The most common method is meristem pruning or stem tip removal. In this case the growing tip of the main stalk or a limb is removed at approximately the final length desired for the stalk or limb. Below the point of removal, the next pair of axial growing tips begins to elongate and form two new limbs. The growth energy of one stem is now divided into two, and the diffusion of growth energy results in a shorter plant which spreads horizontally. Auxin produced in the tip meristem travels down the stem and inhibits branching. When the meristem is re moved, the auxin is no longer produced and branching may proceed uninhibited. Plants that are normally very tall and stringy can be kept short and bushy by meristem pruning. Removing meristems also removes the newly formed tissues near the meristem that react to changing environmental stimuli and induce flowering. Pruning during the early part of the growth cycle will have little effect on flowering, but plants that are pruned late in life, supposedly to promote branching and floral growth, will often flower late or fail to flower at all. This happens because the meristemic tissue responsible for sensing change has been removed and the plant does not measure that it is the time of the year to flower. Plants will usually mature fastest if they are allowed to grow and develop without interference from pruning. If late maturation of Cannabis is desired, then extensive pruning may work to delay flowering. This is particularly applicable if a staminate plant from an early maturing strain is needed to pollinate a late-maturing pistil late plant. The staminate plant is kept immature until the pistillate plant is mature and ready to be pollinated. When the pistillate plant is receptive, the staminate plant is allowed to develop flowers and release pollen.
Other techniques are available for limiting the size and shape of a developing Cannabis plant without removing meristematic tissues. Trellising is a common form of modification and is achieved in several ways. In many cases space is available only along a fence or garden row. Posts 1 to 2 meters (3 to 6 feet) long may be driven into the ground 1 to 3 meters (3 to 10 feet) apart and wires stretched between them at 30 to 45 centimeters (12 to 18 inches) intervals, much like a wire fence or grape trellis. Trellises are ideally oriented on an east-west axis for maxi mum sun exposure. Seedlings or pistillate clones are placed between the posts, and as they grow they are gradually bent and attached to the wire. The plant continues to grow upward at the stem tips, but the limbs are trained to grow horizontally. They are spaced evenly along the wires by hooking the upturned tips under the wire when they are 15 to 30 centimeters (6 to 12 inches) long. The plant grows and spreads for some distance, but it is never allowed to grow higher than the top row of wire. When the plant be gins to flower, the floral clusters are allowed to grow up ward in a row from the wire where they receive maximum sun exposure. The floral clusters are supported by the wire above them, and they are resistant to weather damage. Many cultivators feel that trellised plants, with increased sun exposure and meristems intact, produce a higher yield than freestanding unpruned or pruned plants. Other growers feel that any interference with natural growth patterns limits the ultimate size and yield of the plant.
Another method of trellising is used when light exposure is especially crucial, as with artificial lighting systems. Plants are placed under a horizontal or slightly slanted flat sheet of 2 to 5 centimeters (1 to 2 inches) poultry netting which is suspended on a frame 30 to 60 centimeters (12 to 24 inches) from the soil surface perpendicular to the direction of incoming light or to the lowest path of the sun. The seedlings or clones begin to grow through the netting al-‘ most immediately, and the meristems are pushed back down under the netting, forcing them to grow horizon tally outward. Limbs are trained so that the mature plant will cover the entire frame evenly. Once again, when the plant begins to flower, the floral clusters are allowed to grow upward through the wire as they reach for the light. This might prove to be a feasible commercial cultivation technique, since the flat beds of floral clusters could be mechanically harvested. Since no meristem tissues are re moved, growth and maturation should proceed on schedule. This system also provides maximum light exposure for all the floral clusters, since they are growing from a plane perpendicular to the direction of light. Sometimes limbs are also tied down, or crimped and bent to limit height and promote axial growth without meristem removal. This is a particularly useful technique for greenhouse cultivation, where plants often reach the roof or walls and burn or rot from the intense heat and condensation of water on the inside of the greenhouse. To prevent rotting and burning while leaving enough room for floral clusters to form, the limbs are bent at least 60 centimeters (24 inches) beneath the roof of the green house. Tying plants over allows more light to strike the plant, promoting axial growth. Crimping stems and bending them over results in more light exposure as well as inhibiting the flow of auxin down the stem from the tip. Once again, as with meristem removal, this promotes axial growth.
Limbing is another common method of pruning Cannabis plants. Many small limbs will usually grow from the bottom portions of the plant, and due to shading they re main small and fail to develop large floral clusters. If these atrophied lower limbs are removed, the plant can devote more of its floral energies to the top parts of the plant with the most sun exposure and the greatest chance of pollination. The question arises of whether removing entire limbs constitutes a shock to the growing plant, possibly limiting its ultimate size. It seems in this case that shock is minimized by removing entire limbs, including proportional amounts of stems, leaves, meristems, and flowers; this probably results in less metabolic imbalance than if only flowers, leaves, or meristems were removed. Also, the lower limbs are usually very small and seem of little significance in the metabolism of the total plant. In large plants, many limbs near the central stalk also become shaded and atrophied and these are also sometimes removed in an effort to increase the yield of large floral clusters on the sunny exterior margins. Leafing is one of the most misunderstood techniques of drug Cannabis cultivation. In the mind of the cultivator, several reasons exist for removing leaves. Many feel that large shade leaves draw energy from the flowering plant, and therefore the flowering clusters will be smaller. It is felt that by removing the leaves, surplus energy will be available, and large floral clusters will be formed. Also, some feel that inhibitors of flowering, synthesized in the leaves during the long noninductive days of summer, may be stored in the older leaves that were formed during the noninductive photoperiod. Possibly, if these inhibitor-laden leaves are removed, the plant will proceed to flower, and maturation will be accelerated. Large leaves shade the inner portions of the plant, and small atrophied floral clusters may begin to develop if they receive more light.
In actuality, few if any of the theories behind leafing give any indication of validity. Indeed, leafing possibly serves to defeat its original purpose. Large leaves have a definite function in the growth and development of Cannabis. Large leaves serve as photosynthetic factories for the production of sugars and other necessary growth sub stances. They also create shade, but at the same time they are collecting valuable solar energy and producing foods that will be used during the floral development of the plant. Premature removal of leaves may cause stunting, because the potential for photosynthesis is reduced. As these leaves age and lose their ability to carry on photo synthesis they turn chlorotie (yellow) and fall to the ground. In humid areas care is taken to remove the yellow or brown leaves, because they might invite attack by fungus. During chlorosis the plant breaks down substances, such as chlorophylls, and translocates the molecular components to a new growing part of the plant, such as the flowers. Most Cannabis plants begin to lose their larger leaves when they enter the flowering stage, and this trend continues until senescence. It is more efficient for the plant to reuse the energy and various molecular components of existing chlorophyll than to synthesize new chlorophyll at the time of flowering. During flowering this energy is needed to form floral clusters and ripen seeds. Removing large amounts of leaves may interfere with the metabolic balance of the plant. If this metabolic change occurs too late in the season it could interfere with floral development and delay maturation. If any floral inhibitors are removed, the intended effect of accelerating flowering will probably be counteracted by metabolic upset in the plant. Removal of shade leaves does facilitate more light reaching the center of the plant, but if there is not enough food energy produced in the leaves, the small internal floral clusters will probably not grow any larger. Leaf removal may also cause sex reversal resulting from a metabolic change.
If leaves must be removed, the petiole is cut so that at least an inch remains attached to the stalk. Weaknesses in the limb axis at the node result if the leaves are pulled off at the abscission layer while they are still green. Care is taken to see that the shriveling petiole does not invite fungus attack. It should be remembered that, regardless of strain or environmental conditions, the plant strives to reproduce, and reproduction is favored by early maturation. This produces a situation where plants are trying to mature and reproduce as fast as possible. Although the purpose of leafing is to speed maturation, disturbing the natural progressive growth of a plant probably interferes with its rapid development. Cannabis grows largest when provided with plentiful nutrients, sunlight, and water and left alone to grow and mature naturally. It must be remembered that any alteration of the natural life cycle of Cannabis will affect productivity. Imaginative combinations and adaptations of propagation techniques exist, based on specific situations of cultivation. Logical choices are made to direct the natural growth cycle of Cannabis to favor the timely maturation of those products sought by the cultivator, without sacrificing seed or clone production.
Chapter 3 - Genetics and Breeding of Cannabis
Genetics
Although it is possible to breed Cannabis with limited success without any knowledge of the laws of inheritance, the full potential of diligent breeding, and the line of action most likely to lead to success, is realized by breeders who have mastered a working knowledge of genetics. As we know already, all information transmitted from generation to generation must be contained in the pollen of the staminate parent and the ovule of the pistillate parent. Fertilization unites these two sets of genetic information, a seed forms, and a new generation is begun. Both pollen and ovules are known as gametes, and the transmitted units determining the expression of a character are known as genes. Individual plants have two identical sets of genes (2n) in every cell except the gametes, which through reduction division have only one set of genes (in). Upon fertilization one set from each parent combines to form a seed (2n). In Cannabis, the haploid (in) number of chromosomes is 10 and the diploid (2n) number of chromosomes is 20. Each chromosome contains hundreds of genes, influencing every phase of the growth and development of the plant. If cross-pollination of two plants with a shared genetic trait (or self-pollination of a hermaphrodite) results in off spring that all exhibit the same trait, and if all subsequent (inbred) generations also exhibit it, then we say that the strain (i.e., the line of offspring derived from common ancestors) is true-breeding, or breeds true, for that trait. A strain may breed true for one or more traits while varying in other characteristics. For example, the traits of sweet aroma and early maturation may breed true, while off spring vary in size and shape. For a strain to breed true for some trait, both of the gametes forming the offspring must have an identical complement of the genes that influence the expression of that trait. For example, in a strain that breeds true for webbed leaves, any gamete from any parent in that population will contain the gene for webbed leaves, which we will signify with the letter w.
Since each gamete carries one-half (in) of the genetic complement of the offspring, it follows that upon fertilization both "leaf shape" genes of the (2n) offspring will be w. That is, the offspring, like both parents, are ww. In turn, the offspring also breed true for webbed leaves because they have only w genes to pass on in their gametes.
On the other hand, when a cross produces offspring that do not breed true (i.e., the offspring do not all resemble their parents) we say the parents have genes that segregate or are hybrid. Just as a strain can breed true for one or more traits, it can also segregate for one or more traits; this is often seen. For example, consider a cross where some of the offspring have webbed leaves and some have normal compound-pinnate leaves. (To continue our system of notation we will refer to the gametes of plants with compound-pinnate leaves as W for that trait. Since these two genes both influence leaf shape, we assume that they are related genes, hence the lower-case w and upper-case W notation instead of w for webbed and possibly P for pinnate.) Since the gametes of a true-breeding strain must each have the same genes for the given trait, it seems logical that gametes which produce two types of offspring must have genetically different parents. Observation of many populations in which offspring differed in appearance from their parents led Mendel to his theory of genetics. If like only sometimes produces like, then what are the rules which govern the outcome of these crosses? Can we use these rules to predict the outcome of future crosses? Assume that we separate two true-breeding populations of Cannabis, one with webbed and one with compound-pinnate leaf shapes. We know that all the gametes produced by the webbed-leaf parents will contain genes for leaf-shape w and all gametes produced by the compound-pinnate individuals will have W genes for leaf shape. (The offspring may differ in other characteristics, of course.) If we make a cross with one parent from each of the true-breeding strains, we will find that 100% of the off spring are of the compound-pinnate leaf phenotype. (The expression of a trait in a plant or strain is known as the phenotype.) What happened to the genes for webbed leaves contained in the webbed leaf parent? Since we know that there were just as many w genes as W genes combined in the offspring, the W gene must mask the expression of the w gene. We term the W gene the dominant gene and say that the trait of compound-pinnate leaves is dominant over the recessive trait of webbed leaves.
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