Botany

by R. C. Clarke

is a tall, erect, annual . Provided with an open sunny environment, light well-drained composted soil, and ample irrigation, can grow to a height of 6 meters (about 20 feet) in a 4-6 month growing season. Exposed river banks, meadows, and agricultural lands are ideal habitats for since all offer good sunlight. In this example an imported seed from Thailand is grown without pruning and becomes a large female plant. A cross with a cutting from a male plant of Mexican origin results in hybrid seed which is stored for later planting. This example is representative of the outdoor growth of in temperate climates.

Seeds are planted in the spring and usually germinate in 3 to 7 days. The seedling emerges from the ground by the straightening of the hypocotyl (embryonic stem). The cotyledons (seed leaves) are slightly unequal in size, narrowed to the base and rounded or blunt to the tip. The hypocotyl ranges from 1 to 10 centimeters (1A to 3 inches) in length. About 10 centimeters or less above the cotyledons, the first true leaves arise, a pair of oppositely oriented single leaflets each with a distinct petiole (leaf stem) rotated one-quarter turn from the cotyledons. Subsequent pairs of leaves arise in opposite formation and a variously shaped leaf sequence develops with the second pair of leaves having 3 leaflets, the third 5 and so on up to 11 leaflets. Occasionally the first pair of leaves will have 3 leaflets each rather than 1 and the second pair, 5 leaflets each.

If a plant is not crowded, limbs will grow from small buds (located at the intersection of petioles) along the main stem. Each sinsemilla (seedless drug ) plant is provided with plenty of room to grow long axial limbs and extensive fine roots to increase floral production. Under favorable conditions grows up to 7 centimeters (21A inches) a day in height during the long days of summer.

shows a dual response to daylength; during the first two to three months of growth it responds to increasing daylength with more vigorous growth, but in the same season the plant requires shorter days to flower and complete its life cycle.

LIFE CYCLE OF I Juvenile Stage

flowers when exposed to a critical daylength which varies with the strain. Critical daylength applies only to plants which fail to flower under continuous illumination, since those which flower under continuous illumination have no critical daylength. Most strains have an absolute requirement of inductive photoperiods (short days or long nights) to induce fertile flowering and less than this will result in the formation of undifferentiated primordia (unformed flowers) only.

The time taken to form primordia varies with the length of the inductive photoperiod. Given 10 hours per day of light a strain may only take 10 days to flower, whereas if given 16 hours per day it may take up to 90 days. Inductive photoperiods of less than 8 hours per day do not seem to accelerate primordia formation. Dark (night) cycles must be uninterrupted to induce flowering (see appendix).

is a dioecious plant, which means that the male and female flowers develop on separate plants, although monoecious examples with both sexes on one plant are found. The development of branches containing flowering organs varies greatly between males and females: the male flowers hang in long, loose, multi-branched, clustered limbs up to 30 centimeters (12 inches) long, while the female flowers are tightly crowded between small leaves.

Note: Female flowers and plants will be referred to as pistillate and male flowers and plants will be referred to as staminate in the remainder of this text. This convention is more accurate and makes examples of complex aberrant sexuality easier to understand.

The first sign of flowering in is the appearance of undifferentiated flower primordia along the main stem at the nodes (intersections) of the petiole, behind the stipule (leaf spur). In the prefloral phase, the sexes of are indistinguishable except for general trends in shape.

When the primordia first appear they are undifferentiated sexually, but soon the males can be identified by their curved claw shape, soon followed by the differentiation of round pointed flower buds having five radial segments. The females are recognized by the enlargement of a symmetrical tubular calyx (floral sheath). They are easier to recognize at a young age than male primordia. The first female calyxes tend to lack paired pistils (pollen-catching appendages) though initial male flowers often mature and shed viable pollen. In some individuals, especially hybrids, small non-flowering limbs will form at the nodes and are often confused with male primordia.

Cultivators wait until actual flowers form to positively determine the sex of

The female plants tend to be shorter and have more branches than the male. Female plants are leafy to the top with many leaves surrounding the flowers, while male plants have fewer leaves near the top with few if any leaves along the extended flowering limbs.

*The term pistil has developed a special meaning with respect to which differs slightly from the precise botanical definition. This has come about mainly from the large number of cultivators who have casual knowledge of plant anatomy but an intense interest in the reproduction of . The precise definition of pistil refers to the combination of ovary, style and stigma. In the more informal usage, pistil refers to the fused style and stigma. The informal sense is used throughout the book since it has become common practice among cultivators.

The female flowers appear as two long white, yellow, or pink pistils protruding from the fold of a very thin membranous calyx. The calyx is covered with resin exuding glandular trichomes (hairs). Pistillate flowers are borne in pairs at the nodes one on each side of the petiole behind the stipule of bracts (reduced leaves) which conceal the flowers. The calyx measures 2 to 6 millimeters in length and is closely applied to, and completely contains, the ovary.

In male flowers, five petals (approximately 5 millimeters, or 3/16 inch, long) make up the calyx and may be yellow, white, or green in color. They hang down, and five stamens (approximately 5 millimeters long) emerge, consisting of slender anthers (pollen sacs), splitting upwards from the tip and suspended on thin filaments. The exterior surface of the staminate calyx is covered with non-glandular trichomes. The pollen grains are nearly spherical slightly yellow, and 25 to 30 microns (p) in diameter. The surface is smooth and exhibits 2 to 4 germ pores.

Before the start of flowering, the phyllotaxy (leaf arrangement) reverses and the number of leaflets per leaf decreases until a small single leaflet appears below each pair of calyxes. The phyllotaxy also changes from decussate (opposite) to alternate (staggered) and usually remains alternate throughout the floral stages regardless of sexual type.

The differences in flowering patterns of male and female plants are expressed in many ways. Soon after dehiscence (pollen shedding) the staminate plant dies, while the pistillate plant may mature up to five months after viable flowers are formed if little or no fertilization occurs. Compared with pistillate plants, staminate plants show a more rapid increase in height and a more rapid decrease in leaf size to the bracts which accompany the flowers. Staminate plants tend to flower up to one month earlier than pistillate plants; however, pistillate plants often differentiate primordia one to two weeks before staminate plants.

Many factors contribute to determining the sexuality of a flowering plant. Under average conditions with a normal inductive photoperiod, will bloom and produce approximately equal numbers of pure staminate and pure pistillate plants with a few hermaphrodites (both sexes on the same plant). Under conditions of extreme stress, such as nutrient excess or deficiency, mutilation, and altered light cycles, populations have been shown to depart greatly from the expected one-to-one staminate to pistillate ratio.

Just prior to dehiscence, the pollen nucleus divides to produce a small reproductive cell accompanied by a large vegetative cell, both of which are contained within the mature pollen grain. Germination occurs 15 to 20 minutes after contact with a pistil. As the pollen tube grows the vegetative cell remains in the pollen grain while the generative cell enters the pollen tube and migrates toward the ovule. The generative cell divides into two gametes (sex cells) as it travels the length of the pollen tube.

Pollination of the pistillate flower results in the loss of the paired pistils and a swelling of the tubular calyx where the ovule is enlarging. The staminate plants die after shedding pollen. After approximately 14 to 35 days the seed is matured and drops from the plant, leaving the dry calyx attached to the stem. This completes the normally 4 to 6 month life cycle, which may take as little as 2 months or as long as 10 months. Fresh seeds approach 100% viability, but this decreases with age.

The hard mature seed is partially surrounded by the calyx and is variously patterned in grey, brown, or black. Elongated and slightly compressed, it measures 2 to 6 millimeters (1/16 to 3/16 inch) in length and 2 to 4 millimeters (1/16 to 1/8 inch) in maximum diameter.

Careful closed pollinations of a fewselected limbs yield hundreds of seeds of known parentage, which are removed after they are mature and beginning to fall from the calyxes. The remaining floral clusters are sinsemilla or seedless and continue to mature on the plant. As the unfertilized calyxes swell, the glandular trichomes on the surface grow and secrete aromatic THC-laden resins. The mature, pungent, sticky floral clusters are harvested, dried, and sampled. The preceding simplified life cycle of sinsemilla exemplifies the production of valuable seeds without compromising the production of seedless floral clusters.

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

- George Washington

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 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 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 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.

A wild 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 cultivation eliminates pollination and subsequent seed production. In the case of cultivated 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 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 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 . 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 ) 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 ) 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 plant’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.

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.

The seeds with which most cultivators begin represent varied genotypes even when they originate from the same floral cluster of , 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.

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.

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 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 , 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. 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 plant’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 genetics.

Nearly every cultivated plant, no matter what its future, began as a germinating seed; and nearly all cultivators, no matter what their intention, start with seeds that are gifts from a fellow cultivator or extracted from imported shipments of . 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.

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:

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 .

Grade #6 - Parentage and origin are unknown.

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 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 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 , 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 , 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 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.

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 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 ¼ 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:

Cuttings of relatively young vegetative limbs 10 to 45 centimeters (4 to 18 inches) are made with a sharp knife or razor blade and immediately placed in a container of clean, pure water so the cut ends are well covered. It is essential that the cuttings be placed in water as soon as they are removed or a bubble of air (embolism) may enter the cut end and block the transpiration stream in the cutting, causing it to wilt. Cuttings made under water avoid the possibility of an embolism. If cuttings are exposed to the air they are cut again before being inserted into the rooting medium.

The medium should be warm and moist before cut tings are removed from the parental plant. Rows of holes are made in the rooting medium with a tapered stick, slightly larger in diameter than the cutting, leaving at least 10 centimeters (4 inches) between each hole. The cuttings are removed from the water, the end to be rooted treated with growth regulators and fungicides (such as Rootone F or Hormex), and each cutting placed in its hole. The cut end of the shoot is kept at least 10 centimeters (4 inches) from the bottom of the medium. The rooting medium is lightly tamped around the cutting, taking care not to scrape off the growth regulators. During the first few days the cuttings are checked frequently to make sure every thing is working properly. The cuttings are then watered with a mild nutrient solution once a day.

The cuttings usually develop a good root system and will be ready to transplant in three to six weeks. At this time the hardening-off process begins, preparing the delicate cuttings for a life in bright sunshine. The cuttings are removed and transplanted to a sheltered spot such as a greenhouse until they begin to grow on their own. It is necessary to water them with a dilute nutrient solution or feed with finished compost as soon as the hardening-off process begins. Young roots are very tender and great care is necessary to avoid damage. When vegetative cuttings are placed outside under the prevailing photoperiod they will react accordingly. If it is not the proper time of the year for the cuttings to grow and mature properly (near harvest time, for example) or if it is too cold for them to be put out, then they may be kept in a vegetative condition by supplementing their light to increase daylength. Alternatively they may be induced to flower indoors under artificial conditions.

After shoots are selected and prepared for cloning, they are treated and placed in the rooting medium. Since the discovery in 1984 that auxins such as IAA stimulate the production of adventitious roots, and the subsequent discovery that the application of synthetic auxins such as NAA increase the rate of root production, many new techniques of treatment have appeared. It has been found that mixtures of growth regulators are often more effective than one alone. IAA and NAA a—e often combined with a small percentage of certain phenoxy compounds and fungicides in commercial preparations. Many growth regulators deteriorate rapidly, and fresh solutions are made up as needed. Treatments with vitamin B1 (thiamine) seem to help roots grow, but no inductive effect has been noticed. As soon as roots emerge, nutrients are necessary; the shoot cannot maintain growth for long on its own reserves. A complete complement of nutrients in the rooting medium certainly helps root growth; nitrogen is especially beneficial. Cuttings are extremely susceptible to fungus attack, and conditions conducive to rooting are also favorable to the growth of fungus. "Cap tan " is a long-lasting fungicide that is sometimes applied in powdered form along with growth regulators. This is done by rolling the basal end of the cutting in the powder before placing it in the rooting medium.

The initiation and growth of roots depends upon atmospheric oxygen. If oxygen levels are low, shoots may fail to produce roots and rooting will certainly be inhibited. It is very important to select a light, well-aerated rooting medium. In addition to natural aeration from the atmosphere, rooting media may be enriched with oxygen (02) gas; enriched rooting solutions have been shown to increase rooting in many plant species. No threshold for damage by excess oxygenation has been determined, although excessive oxygenation could displace carbon dioxide which is also vital for proper root initiation and growth. If oxygen levels are low, roots will form only near the surface of the medium, whereas with adequate oxygen levels, roots will tend to form along the entire length of the implanted shoot, especially at the cut end.

Oxygen enrichment of rooting media is fairly simple. Since shoot cuttings must be constantly wetted to ensure proper rooting, aeration of the rooting media may be facilitated by aerating the water used in irrigation. Mist systems achieve this automatically because they deliver a fine mist (high in dissolved oxygen) to the leaves, from where much of it runs off into the soil, aiding rooting. Oxygen enrichment of irrigation water is accomplished by installing an aerator in the main water line so that atmospheric oxygen can be absorbed by the water. An increase in dissolved oxygen of only 20 parts per million may have a great influence on rooting. Aeration is a convenient way to add oxygen to water as it also adds carbon dioxide from the atmosphere. Air from a small pump or bottled oxygen may also be supplied directly to the rooting media through tiny tubes with pin holes, or through a porous stone such as those used to aerate aquariums.

Water is a common medium for rooting. It is inexpensive, disperses nutrients evenly, and allows direct observation of root development. However, several problems arise. A water medium allows light to reach the submerged stem, delaying etiolation and slowing root growth. Water also promotes the growth of water molds and other fungi, sup ports the cutting poorly, and restricts air circulation to the young roots. In a well aerated solution, roots will appear in great profusion at the base of the stem, while in a poorly aerated or stagnant solution only a few roots will form at the surface, where direct oxygen exchange occurs. If rootings are made in pure water, the solution might be replaced regularly with tap water, which should contain sufficient oxygen for a short period. If nutrient solutions are used, a system is needed to oxygenate the solution. The nutrient solution does become concentrated by evaporation, and this is watched. Pure water is used to dilute rooting solutions and refill rooting containers.

Solid media provide anchors for cuttings, plenty of darkness to promote etiolation and root growth, and sufficient air circulation to the young roots. A high-quality soil with good drainage such as that used for seed germination is often used but the soil must be carefully sterilized to prevent the growth of harmful bacteria and fungus. A small amount of soil can easily be sterilized by spreading it out on a cookie sheet and heating it in an oven set at "low," approximately 820 C (180~ F), for thirty minutes. This kills most harmful bacteria and fungus as well as nematodes, in sects and most weed seeds. Overheating the soil will cause the breakdown of nutrients and organic complexes and the formation of toxic compounds. Large amounts of soil may be treated by chemical fumigants. Chemical fumigation avoids the breakdown of organic material by heat and may result in a better rooting mix. Formaldehyde is an excellent fungicide and kills some weed seeds, nematodes, and in sects. One gallon of commercial formalin (40% strength) is mixed with 50 gallons of water and slowly applied until each cubic foot of soil absorbs 2-4 quarts of solution. Small containers are sealed with plastic bags; large flats and plots are covered with polyethylene sheets. After 24 hours the seal is removed and the soil is allowed to dry for two weeks or until the odor of formaldehyde is no longer present. The treated soil is drenched with water prior to use. Fumigants such as formaldehyde, methyl bromide or other lethal gases are very dangerous and cultivators use them only outside with appropriate protection for themselves.

It is usually much simpler and safer to use an artificial sterile medium for rooting. Vermiculite and perlite are often used in propagation because of their excellent drain age and neutral pH (a balance between acidity and alkalinity). No sterilization is needed because both products are manufactured at high heat and contain no organic material. It has been found that a mixture of equal portions of medium and large grade vermiculite or perlite promotes the greatest root growth. This results from increased air circulation around the larger pieces. A weak nutrient solution, including micro-nutrients, is needed to wet the medium, because little or no nutrient material is supplied by these artificial media. Solutions are checked for pH and corrected to neutral with agricultural lime, dolomite lime, or oyster shell lime.

Layering is a process in which roots develop on a stem while it remains attached to, and nutritionally sup ported by the parent plant. The stem is then detached and the meristematic tip becomes a new individual, growing on its own roots, termed a layer. Layering differs from cutting because rooting occurs while the shoot is still attached to the parent. Rooting is initiated in layering by various stem treatments which interrupt the downward flow of photosynthates (products of photosynthesis) from the shoot tip. This causes the accumulation of auxins, carbohydrates and other growth factors. Rooting occurs in this treated area even though the layer remains attached to the parent. Water and mineral nutrients are supplied by the parent plant because only the phloem has been interrupted; the xylem tissues connecting the shoot to the parental roots remain intact (see illus. 1, page 29). In this manner, the propagator can overcome the problem of keeping a severed cutting alive while it roots, thus greatly in creasing the chances of success. Old woody reproductive stems that, as cuttings, would dry up and die, may be rooted by layering. Layering can be very time-consuming and is less practical for mass cloning of parental stock than removing and rooting dozens of cuttings. Layering, however, does give the small-scale propagator a high-success alternative which also requires less equipment than cuttings.

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 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.

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 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.

Intergeneric grafts between and Humulus (hops) have fascinated researchers and cultivators for decades. Warmke and Davidson (1943) claimed that Humbles tops grafted upon 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 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 as well as intergeneric grafts between 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 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 techniques are commonly used by 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 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 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 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 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 . 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 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.

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 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 to favor the timely maturation of those products sought by the cultivator, without sacrificing seed or clone production.

"The greatest service which can be rendered to any country is to add a useful plant to its culture."

- Thomas Jefferson

Although it is possible to breed 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 , 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 , 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. This seems logical since the normal phenotype in has compound-pinnate leaves. It must be remembered, however, that many useful traits that breed true are recessive. The true-breeding dominant or recessive condition, WW or ww, is termed the homozygous condition; the segregating hybrid condition wW or Ww is called heterozygous. When we cross two of the F1 (first filial generation) offspring resulting from the initial cross of the ~1 (parental generation) we observe two types of offspring. The F2 generation shows a ratio of approximately 3:1, three compound pinnate type-to-one webbed type. It should be remembered that phenotype ratios are theoretical. The real results may vary from the expected ratios, especially in small samples.

In this case, compound-pinnate leaf is dominant over webbed leaf, so whenever the genes w and W are combined, the dominant trait W will be expressed in the phenotype. In the F2 generation only 25% of the offspring are homozygous for W so only 25% are fixed for W. The w trait is only expressed in the F2 generation and only when two w genes are combined to form a double-recessive, fixing the recessive trait in 25% of the offspring. If compound-pinnate showed incomplete dominance over webbed, the genotypes in this example would remain the same, but the phenotypes in the F1 generation would all be intermediate types resembling both parents and the F2 phenotype ratio would be 1 compound-pinnate :2 intermediate :1 webbed.

The explanation for the predictable ratios of offspring is simple and brings us to Mendel's first law, the first of the basic rules of heredity:

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

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

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

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

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

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

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

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

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

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

All of the grown in North America today originated in foreign lands. The diligence of our ancestors in their collection and sowing of seeds from superior plants, together with the forces of natural selection, have worked to create native strains with localized characteristics of resistance to pests, diseases, and weather conditions. In other words, they are adapted to particular niches in the ecosystem. This genetic diversity is nature's way of protecting a species. There is hardly a plant more flexible than . As climate, diseases, and pests change, the strain evolves and selects new defenses, programmed into the genetic orders contained in each generation of seeds. Through the importation in recent times of fiber and drug , a vast pool of genetic material has appeared in North America. Original fiber strains have escaped and become acclimatized (adapted to the environment), while domestic drug strains (from imported seeds) have, unfortunately, hybridized and acclimatized randomly, until many of the fine gene combinations of imported have been lost.

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

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

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

Several steps must be taken to preserve our vanishing genetic resources, and action must be immediate:

• Seeds and pollen should be collected directly from reliable and knowledgeable sources. Government seizures and smuggled shipments are seldom reliable seed sources. The characteristics of both parents must be known; consequently, mixed bales of randomly pollinated are not suitable seed sources, even if the exact origin of the sample is certain. Direct contact should be made with the farmer-breeder responsible for carrying on the breeding traditions that have produced the sample. Accurate records of every possible parameter of growth must be kept with carefully stored triplicate sets of seeds.
• Since seeds do not remain viable forever, even under the best storage conditions, seed samples should he replenished every third year. Collections should be planted in conditions as similar as possible to their original niche and allowed to reproduce freely to minimize natural and artificial selection of genes and ensure the preservation of the entire gene pool. Half of the original seed collection should be retained until the viability of further generations is confirmed, and to provide parental material for comparison and back-crossing. Phenotypic data about these subsequent generations should be carefully recorded to aid in understanding the genotypes contained in the collection. Favorable traits of each strain should be characterized and catalogued.
• It is possible that in the future, cultivation for resale, or even personal use, may be legal but only for approved, patented strains. Special caution would be needed to preserve variety in the gene pool should the patenting of strains become a reality.
• Favorable traits must be carefully integrated into existing strains.

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

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

A grower in northern latitudes selected an ideal spot to grow a crop and prepared the soil well. Seeds were selected from the best floral clusters of several strains avail able over the past few years, both imported and domestic. Nearly all of the staminate plants were removed as they matured and a nearly seedless crop of beautiful plants resulted. After careful consideration, the few seeds from accidental pollination of the best flowers were kept for the following season, These seeds produced even bigger and better plants than the year before and seed collection was performed as before. The third season, most of the plants were not as large or desirable as the second season, but there were many good individuals. Seed collection and cultivation the fourth season resulted in plants inferior even to the first crop, and this trend continued year after year. What went wrong? The grower collected seed from the best plants each year and grew them under the same conditions. The crop improved the first year. Why did the strain degenerate?

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

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

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

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

• Luther Burbank (in James, 1964)

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

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

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

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

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

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

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

Occasionally hybrid offspring will prove inferior to both parents, but the first generation may still contain recessive genes for a favorable characteristic seen in a parent if the parent was homozygous for that trait. First generation (F1) hybrids are therefore inbred to allow recessive genes to recombine and express the desired parental trait. Many breeders stop with the first cross and never realize the genetic potential of their strain. They fail to produce an F2 generation by crossing or self-pollinating F1 offspring. Since most domestic strains are F1 hybrids for many characteristics, great diversity and recessive recombination can result from inbreeding domestic hybrid strains. In this way the breeding of the F1 hybrids has already been accomplished, and a year is saved by going directly to F2 hybrids. These F2 hybrids are more likely to express recessive parental traits. From the F2 hybrid generation selections can be made for parents which are used to start new true-breeding strains. Indeed, F2 hybrids might appear with more extreme characteristics than either of the P~ parents. (For example, P1 high-THC X P1 low-THC yields F1 hybrids of intermediate THC content. Selfing the F1 yields F2 hybrids, of both P1 [high and low THC] phenotypes, inter mediate F1 phenotypes, and extra-high THC as well as extra-low THC phenotypes.)

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

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

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

3 - Back crossing to establish original parental types.

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

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

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

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

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

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

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

At this time it seems unlikely that a plant patent would be awarded for a pure-breeding strain of drug . In the future, however, with the legalization of cultivation, it is a certainty that corporations with the time, space, and money to produce pure and hybrid strains of will apply for patents. It may be legal to grow only certain patented strains produced by large seed companies. Will this be how government and industry combine to control the quality and quantity of "drug" ?

Much of the breeding effort of North American cultivators is concerned with acclimatizing high-THC strains of equatorial origin to the climate of their growing area while preserving potency. Late-maturing, slow, and irregularly flowering strains like those of Thailand have difficulty maturing in many parts of North America. Even in a green house, it may not be possible to mature plants to their full native potential.

To develop an early-maturing and rapidly flowering 8train, a breeder may hybridize as in the previous example. However, if it is important to preserve unique imported genetics, hybridizing may be inadvisable. Alternatively, a pure cross is made between two or more Thai plants that most closely approach the ideal in blooming early. At this point the breeder may ignore many other traits and aim at breeding an earlier-maturing variety of a pure Thai strain. This strain may still mature considerably later than is ideal for the particular location unless selective pressure is exerted. If further crosses are made with several individuals that satisfy other criteria such as high THC content, these may be used to develop another pure Thai strain of high THC content. After these true-breeding lines have been established, a dihybrid pure cross can be made in an attempt to produce an F1 generation containing early-maturing, high-THC strains of pure Thai genetics, in other words, an acclimatized drug strain.

Crosses made without a clear goal in mind lead to strains that acclimatize while losing many favorable characteristics. A successful breeder is careful not to overlook a characteristic that may prove useful. It is imperative that original imported genetics be preserved intact to protect the species from loss of genetic variety through excessive hybridization. A currently unrecognized gene may be responsible for controlling resistance to a pest or disease, and it may only be possible to breed for this gene by back-crossing existing strains to original parental gene pools.

Fixing traits (producing homozygous offspring) in 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 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 . 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 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.

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 , 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.

breed. Wind pollination and dioecious sexuality, which

account for the great adaptability in , 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 plants as a whole while the remainder concern the qualities of seedlings, leaves, fibers, and flowers. Finally a list of various strains is provided along with specific characteristics. Following this order, basic and then specific selections of favorable characteristics can be made.