Controlled_Pollination_in_Western_White_PineEXCERPT.pdf

Controlled Pollination in Western White Pine Pollen Collection and Extraction

Pollen carries the male generative cell which fertilizes the ovule in the female flower. Tree pollen is a fine, yellow dust borne on the male strobilus or pollen catkin. Most coniferous trees are monecious with both the male and female reproductive organs on the same tree.

Pollen may be collected for the purpose of making controlled pollinations between specific trees or it may be used to mass pollinate seed orchards.

Time of Collection. Pollen catkins usually ripen at the same time as the female flowers. The catkins are green or red, but turn yellow upon ripening. Pollen should be collected just prior to shedding. A test for determining ripeness is to squeeze a catkin. Pollen catkin ripeness proceeds as a thick yellow juice first, then a milky juice, followed by a clear juice. Just before pollen is shed, there is little or no juice. Pollen catkins should be collected during the clear juice phase.

At Hog Meadows, pollen collection has been taking place in the first two weeks of June.

Canyon Creek pollen collection is usually one week behind Hog Meadows (second to third week of June).

Picking Pollen. Pollen catkins are collected by using hand clippers (pruning shears). Clippers should be sterilized in rubbing alcohol, to avoid contamination of pollen collections, before proceeding to a new tree. The catkins are usually found lower in the crown than the female flowers and can sometimes be collected from the ground. If not, a cone hook may be used to pull a branch down to collect pollen; otherwise, the tree must be climbed.

Drying and cleaning the pollen is expedited by cutting away vegetative material such as needles and stems. Catkins should be gathered in a paper bag for transit to the laboratory. The bag should only be filled 2/3 to capacity to allow air circulation and to prevent molding. The identity of the pollen parent should be clearly noted on each bag, date collected, and for what purpose

(e.g., WP Vertical Resistance Study). See below:

WP Vertical Resistance Study

Species Code _________________ (2 alpha, WP)

Plantation Code _______________ (2 alpha, CC=Canyon Creek, HM=Hog Meadows)

Tree Number________ Resistance Type________ (NS, SS, BR,)

Collection Date _____/_____/_____

Collected by______________________________

Do not collect pollen on rainy days as it molds.

Protect the bags from direct sunlight or excess heat (keep bags cool and dry). The seams of each bag should be sealed with clear tape to avoid pollen contamination during transport and drying.

Double-fold the top of each bag and staple with 3-4 staples to keep pollen catkins inside bag.

When the pollen arrives back at the district or station, each individual bag should be hung on a clothes line strung inside a warm, dry building to facilitate pollen ripening. Pollen bags should be individually shook (gently) twice a day, while on the clothes line, to facilitate air circulation and drying (to prevent molding), until pollen is shipped to the CDA Nursery for further processing. Ship bags no later than 48 hours after collection.

Forcing Pollen. If pollen is to be used immediately, it is sometimes desirable to collect the catkins in advance of female flower development so that it will be dried and ready for use when the flowers are receptive. Pollen catkins that are not fully ripe can be forced by snipping off the catkin bearing branches, bringing them indoors and standing the branches, base down, in a container of water. This will speed up the ripening of the catkins. Separation procedures are the same as for field collected catkins. Some viability loss may occur depending on the degree of development of the catkins at the time the branched are cut. This procedure is not recommended for the vertical resistance study—pollen should be shipped to the nursery for processing.

Processing Pollen. Pollen extraction takes place at the Coeur d'Alene Nursery, which has the necessary equipment. When it is not important to prevent pollen contamination, and the pollen is required quickly, small quantities of pollen can be separated following these rather simple procedures:

In the laboratory or other indoor facility spread the freshly picked pollen catkins in homemade paper "boats" to dry. As the catkins dry, pollen will be shed in the bottom of the paper boat.

Occasional slight agitation will assure that all of the pollen is out of the catkins. When the catkins are empty they are disposed of and the dried pollen in the boat is poured through a fine mesh screen to remove foreign particles. Pollen should be stored in tightly capped, tinted bottles.

Pollen dried in boats is unacceptable for the WP Vertical Resistance Study, due to purity standards.

Properly dried pollen flows like water. The pollen moisture content has to be at 6% or less. If the pollen sticks to the side of the bottle, it should be dried further, preferably at Coeur d'Alene.

In localities where humidity is high, it may be necessary to heat the room where the pollen is being dried.

If the pollen is not to be used immediately, it should be stored in a freezer with a temperature at -

12 to -15 degrees Celsius. Pine pollens so dried and stored have retained their viability for 3 or more years. These same conditions can be used for pollen that is to be stored for several years.

Pollen used for controlled pollinations should be stored at 2 degrees Celsius during the pollination period, then dried down to 6% moisture content and put back in the freezer.

Pollen Testing. If pollen has been stored for any length of time, its viability should be tested before use. A simple test for pine pollen is prepare a 0.5% wt./vol. agar medium using Difco

Bacto agar. The agar is prepared as follows:

Ingredients: 0.625 grams of agar

125 ml distilled water

Thoroughly mix ingredients and heat until agar is dissolved being sure to stir the mixture regularly to prevent the agar from burning. The agar will appear clear when it is ready. To ensure sterility of your agar mixture, put it in the autoclave at 15 lbs pressure for 20 minutes. Let the agar cool slightly before pouring it into the petri dishes. Fill the dishes 1/4 full and let them sit undisturbed for at least 10 minutes. Minimize possible contamination by reducing exposure of agar to the air. When the agar has hardened, use a wire wand to lightly dust the surface with a small quantity or the pollen to be tested. Sterilize the wire after you dust each agar dish.

Incubate dishes around 29 degrees Celsius for 48 hours, viable pollen will develop elongated pollen tubes. These tubes should be at least as long as the pollen grain is wide at its widest dimension. Pollen grains are easily observed under a lower power dissecting microscope. This mixture makes at least 10 petri dishes 1/4 full.

The percentage of grains germinated, length of the pollen tube and length of time on the agar are all measures of the vigor and viability of the pollen. Dose rates may have to be increased for pollens with low viability. Pollens that produce few or no pollen tubes should be discarded.

Pollens with reduced viability should be replaced as soon as possible.

The ingredients used in the testing procedure are available from biological supply houses. This pollen testing procedure will not work for all species and the literature should be checked to determine procedures when a species other than white pine is being worked with.

Controlled Pollination

Controlled pollination is the transfer of pollen from a known source to a receptive female of a specific tree. Successful controlled pollination requires the exclusion of pollen from surrounding trees through the use of a special pollination bag. This technique is usually used only in high intensity tree improvement programs where full-sib or other specific crosses are desirable.

Controlled pollination has also been used to produce highly selected seed for reforestation.

Because of the high cost of the seed produced, this is seldom practical.

The five steps in controlled pollination in western white pine are:

First Season:

(1) Applying the pollination bag and flagging for monumentation

(2) Pollination (two to three times)

(3) Removing the pollination bag by end of June

Second Season:

(4) Applying a protective cone bag in May/early June

(5) Collecting the mature cone

All of these steps must be performed in the top of the tree and climbing is usually necessary.

Applying the Pollination Bag. In the late spring, after flower buds develop and before the flowers have opened, the bud is covered with a special pollination bag. In some species, the flower buds are formed the previous year. In pine, the flower buds form after the shoots begin to grow in the spring.

White paper bags ordered in bulk from Lawson Bags, 318 Happ Road, P.O. Box 8577, Northfield, IL 60093 (847) 446-8812, have proved to be the most economical and effective pollination bag. These bags have a plastic panel which permits viewing the developing cones, while the paper bag permits the foliage to "breathe" while pollen is excluded. Plastic bags do not breathe and therefore allow water to collect in the bag. Sausage skins, obtained from meat packing supply houses, have been used in the past but high temperatures tend to occur inside the bag when they are exposed to direct sunlight; this causes cone abortion.

Bag temperatures get high on sunny spring days and this is a frequent cause of flower mortality in the pollination bags. The effect of temperature can be minimized by turning the plastic panel towards the ground, to keep sunshine from touching the conelet.

Bags are prepared in advance so that enough will be ready when the time for applying them arrives. Each bag should be prepared with a strip of cotton wadding about 3" wide and 8" long.

The application of the pollination bag is the most time consuming of the five steps in controlled pollination. The period of bud development to flower development may only be two to three weeks in pine. Pollination bagging is usually the major factor limiting production in controlled pollinations.

When applying the bags, the sides should be "fluffed out" as much as possible. This keeps the bag expanded and allows more room for the shoot and flower to develop and results in less flower abortion.

The bags are placed over the flower bearing branch in a manner to encase the flowers themselves. Flowers may occur singly or in clusters of 2, 3, or more. Two or more branches ends may be encased in one bag but it is not desirable in pine at least, to have more than 6 flowers in one bag. When the flower-bearing branch is bagged, the flowers should not touch the sides or end of the bag to allow the conelet to develop.

A spot on the branch should be selected where the ends of the bag will be tied. The best spot is typically right below a whorl, where the twigs coming off the branch will keep the bag from slipping forward and off the branch. The needles are removed for about 3" on the branch, and the cotton is then wrapped around the branch where the needles have been removed. The bag is then slipped over the conelets, "fluffed out," crimped shut around the cotton at the bottom, and tied with a 6-8" plastic cable tie or several wraps of string. Brightly-colored flagging is then tied around the branch just below the pollination bag.

Pollination. Within the pollination bag, the flowers continue to develop as normal. When the flower emerges from the bud and the flower scales are open, the flower is ready to be pollinated.

There are five stages to flower development. Optimal receptivity is at stage five when the scales are perpendicular to the central axis of flower. The flower has the appearance of little umbrellas or awnings stacked one on top of the other (see below):

Dave Bramlett

There is also color variation in western white pine flowers ranging from the typical hot pink to yellow:

Aram Eramian

Pollen is carried to the field in a cooler, ice chest, or small battery operated refrigerator.

Recommend that pollen be preloaded in syringes at CDA Nursery. Use vinyl tape to write male identity, test location origin (if WP VR Study--CC=Canyon Creek and HM=Hog Meadows), and color coordinate vinyl tape with tree flagging (e.g., use yellow-green vinyl tape and flagging for selfs and orange vinyl tape and flagging for trait crosses). Pollen should be kept cool (with blue ice packs wrapped in newspaper to absorb moisture), dry (crumpled up newspaper), and out of the sun, while being used. Pollen is applied to be bagged flower by the use of a hypodermic syringe. The syringe is positioned about 1-2 inches away from the flower. The needle is pointed at the conelet as the bulb is compressed, enveloping the flower in a cloud of pollen. One or two light squirts for each flower is enough. Pouring pollen directly on the flower or squirting too much pollen on the flower, should be avoided since this may cause the flower to abort and it wastes pollen.

After the pollen has been applied, grab the branch at the base of the pollination bag and give it a few shakes. This will help to distribute the pollen evenly over the flower.

If contamination cannot be tolerated then it is necessary to seal the needle holes in the pollination bag with water-proof tape.

Seed yields have been improved, and the likelihood of complete pollination by the desired male parent can be enhanced, by giving the flower a second and/or third application of pollen two to three days after the initial application pollen., following the same procedures above.

If there is reason to believe that all ovules did not receive pollen or if the controlled pollination is being done in a species in which the action of micropyle is unknown,then it is necessary to leave the bags on the flowers until scales are visibly closed. This may be as long as one or two weeks after pollination.

If there are not enough atomizer/syringe setups for all crosses, the bulb, 2-way valves, syringe, and needles will have to be sterilized between tree applications, with alcohol. Make sure all supplies are thorougly dry before filling syringe with new pollen lot.

Disbagging. When the flowers are no longer receptive, the pollination bag is removed and the conelet is allowed to develop normally.

In white pine and other pines, the micropyle closes within 48 hours after ovule has been successfully pollinated and it is no longer receptive even though the scales may be open. Thus, the bags can be removed two days after the last pollination, but no later than July 1. Flower survival is improved if time in the bag is kept to a minimum. It is best however, to wait at least one week after the last controlled-pollination to minimize pollen contamination and floral survival.

A system must be used to identify those flowers that were control-pollinated once the bags have been removed. This is usually done by tying colored ribbon around the branch at the base of the pollination bag. All of the flowers on the branch between the flag and the branch tip will have been control pollinated. For the WP Vertical Resistance Study, the tree climber should mark the female and male parent numbers on the flagging and note whether the cross is a "self" or "trait" cross. The female number is always listed first, e.g., 234 x 445 self.

Cone Bagging. Most species develop from flower to mature cones the same year. Pine cones require two years to mature. Regardless of the phenology of cone development, it may be desirable to protect the cones from loss by applying protective cone bags. The advantage of cone bagging are:

1. The developing cones are protected from damaging insects.

2. The cones are protected from squirrels.

3. The cones collection period is extended. If the cones cannot be collected before they open, the seed is shed in the bag.

In western white pine, cone bags are applied at the beginning of the second growing season to protect the cones from seed bugs (Leptoglossus occidentalis)

A.F. Hedlin and the cone beetle (Conophthorus sp.), which can destroy up to 90% of the crop in a bad year.

The importance of cone bagging in late May cannot be overemphasized. Insects bags also seem to deter squirrels from clipping the cones in August. This technique may also be useful when collecting open-pollinated cones.

The cone bags currently being used in the white pine program are a white, reinforced, porous, 17-inch nylon laundry bag, NSN 3510-00-273-9738 (old item number GSA #8105-664-3725).

They are light weight and easy to see in the tops of trees. They weather rapidly and will not last more than one season. Regular cloth lunch sacks may also be used.

Cone Collection. If the cones are in cone bags (aka insect or laundry bags), they can be collected by cutting the branch at the base of the bag. Do not drop the bag to the ground as the cones may be damaged. The bags on an individual tree are then collected and the cones are consolidated into one bag.

Another system sometimes used, is to pinch the cone from the stem (with the cone bag still attached), then untie the cone bag with the cone and any shed seed inside. The cones from one cross are later consolidated into a large bag for shipment to the nursery. The advantage of this system is that it is less damaging to the tree and branches, and needles are eliminated from the bags in the process.

If the cones are not already bagged, then they are simply gathered and consolidated for shipment.

The cone bags themselves may be used to ship the cones but they are not big enough to hold large collections. Paper bags such as double weight grocery sacks have been used. They are cheap and easy to write on. Bag staplers are used to seal the top. The cones tend to mold rapidly in paper bags. Nylon laundry bags discussed above, make good bags to ship small seed lots.

They are expensive ($24.43 per dozen as of May 2000), but may be reused. They are porous and tend to minimize the mold problem.

Each bag of seed lots must be carefully and clearly identified with both male and female parent number. Use the following tags when collecting cones for shipment to CDA Nursery: R1-2470-

11 and R1-FS-2470-26. Place one set of tags inside the bag and another set of tags on the outside of the bag, i.e., there are four tags per bag. Numbers are easily transposed and sloppy writing increases the chance of error. Requirements vary, but the following minimum information should be on each seedlot (as required):

(1) Species

(2) Purpose (WP Vertical Resistance Study), Breeding Orchard

(3) Female parent

(4) Male Parent

(5) Subline Name and Number if for Breeding Orchard Purposes

(6) Forest or Field Test Name

(7) Type of cross (self, trait, full-sib, etc.)

(8) Resistance Mechanism (for WP Vertical Resistance Study)

(9) Date collected

(10) Collected by (full name)

If both open-pollinated and control-pollinated cones are being collected for the same species, then type of collection should also be marked on the bag (e.g., ancestral or woodsrun checklot cone collection).

Determining Cone Ripeness. For most species, cone ripeness can be determined by the color of the cone. For white pine, collection can begin when the cones turn from green to brown, red-brown, or yellow. In white pine, cone maturation seldom varies by more that a week from one year to the next. The cone crop should be monitored very closely since the color change may occur abruptly.

It may be possible to start collecting a few days even a week earlier if the seed cutting test is made. To make this test:

Cut the seed in half longitudinally with a sharp razor blade.

Use a 10X hand lens to examine the embryo. As the seeds ripen, the embryo elongated and is firm and yellow.

Examine 20 to 30 seeds from 5 to 6 trees in the area.

Determine the average embryo elongation. If the embryos occupy 75% or more of the cavity in the endosperm, the cones may be collected.

Cone color, seed coat and wing development may serve to indicate maturity. Another good test is cone flexibility. Grab a cone and twist in a diagonal motion. If the cone scales pop open when twisted the cone is ripe. If it is impossible to open the cone scales or to bend the cone at all, the cone is still immature.

Equipment Required For Controlled-Pollination

Safety

Radios for emergency calls (notify adjacent district each day you have tree climbers so

EMTs can be on stand by).

List of emergency phone numbers (work, home) for each person in field.

Hard Hats for ground crews

First Aid Kit

Bee Sting Kit

Follow saftey general safety procedures for operating a vehicle, field work, and tree climbing.

Only certified tree climbers climb trees, and must have one other person on site.

No climbing in the rain or if bark is wet.

Planning

Field Test Map

Field Sheets showing monitoring and crossing procedures

Pollen collection

Hand clippers (pruning shears) to both snip off branches from the tree and trim pollen cones from branches.

Small brown paper bags to hold pollen cones.

Clear tape to seal seams on paper bags.

Stapler and staples to seal paper bags.

WD-40 to clean and lubricate pruning shears.

Ethanol or rubbing alcohol to sterilize pruning shears.

Climbing harness and helmet if trees will be climbed for pollen.

Gloves to protect hands from pitch (optional).

"Cone hook" (broom handle with hook in the end) to bring branches within reach.

Daisy Chain for trees with long internodes.

Clothesline to hang pollen bags.

Clothespins for use on clothesline.

Small glass jars with screw tops to hold dry pollen.

Metal tags if monumentation poor, cow tags for vertical resistance study.

Stage 1: Applying the pollen bag

Binoculars to evaluate female readiness from the ground

Swedish climbing ladders to climb spindly trees (optional)

Pollination bags

Cotton strips 3-4" wide and about 8" long

Plastic cable ties

Brightly colored flagging

Gloves to protect hands when climbing trees

Stage 2: Pollination

18-gage disposable needles; bring spares

Plastic syringes

One-way rubber bulbs

Ethanol or rubbing alcohol for sterilization

Plastic tape for sealing syringe holes

Cooler with blue ice to keep pollen cool to and from the field

Newspaper

Climbing harness and helmet

Gloves to protect hands when climbing trees

Cone hooks

Swedish climbing ladder

Daisy Chain

Sharpie pen to note parents and type of cross on flagging

Stage 3: Removing the pollination bag

Climbing harness and helmet

Gloves to protect hands when climbing trees

Cone hooks

Swedish climbing ladder

Daisy Chain

Pruning shears to snip cable ties

Brightly colored flagging to replace any that has come loose

Garbage bags to collect used pollination bags

Stage 4: Install insect bags

Insect (17-inch nylong laundry) bags

Replacement Flagging, if needed

Sharpie pens

Stage 5: Cone collection

Two types of tags

Sharpie pens

Wire ties for cone tags on outside of bag

Pruning shears to snip cone bearing branches

Gloves

Go-Jo creme to clean pitch off of equipment and personnel

Garbage bags to remove residual flagging