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    Thread: Pollination Techniques

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      Default Pollination Techniques

      Pollination Techniques

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

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

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

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

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

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

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

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

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

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

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

      Now a pistillate plant is chosen as the seed parent. A pistillate flower cluster is ripe for fertilization so long as pale, slender pistils emerge from the calyxes. Withered, dark pistils protruding from swollen, resin encrusted calyxes are a sign that the reproductive peak has long passed. Cannabis plants can be successfully pollinated as soon as the first primordia show pistils and until just before harvest, but the largest yield of uniform, healthy seeds is achieved by pollinating in the peak floral stage. At this time, the seed plant is covered with thick clusters of white pistils. Few pistils are brown and withered, and resin production has just begun. This is the most receptive time for fertilization, still early in the seed 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 Cannabis genetics.

      Last edited by pistils; 20th January 2006 at 06:34 PM.

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