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Controlled Pollination of Maize |
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Resources for researchers and their assistants who are new to the field of performing controlled pollinations of maize/corn (Zea mays L.). Digital video clips: There are several digital video clips which illustrate various aspects of the methodology of controlled pollination. These are presented in a table and throughout the accompanying document. These are the methodologies used by many of the researchers of maize/corn at the University of Missouri-Columbia, USA. Controlled pollinations require several essential steps. These are described in the topics below. Select a topic title to access the associated text. Topics: Summary table of digital video clips on various aspects of controlled pollinations
Sources of Information:
Controlled pollinations require several essential steps:1. Ear Shoot Bagging:Bagging is a fundamental requirement and yet, of all the pollinating operations, it is the most difficult to do properly. The ears shoots (lateral branches) must be covered before the silks (styles) emerge in order to protect the silks from being contaminated by pollen, until the desired pollen can be applied. The bag used (Lawson 217) measures 2 x 1 x 7 inches (5 x 25 x 18 cm) and is made of semi-transparent treated 36 lb wet strength paper with waterproof glue. The daily shoot bagging operation should begin when the first tassels appear. At that time the tip of the first ear shoot may be visible in the axil of the 6th or 7th leaf down from the top of the plant. This shoot may be covered by placing the bag over the tip of the shoot with the longer lip of the bag next to the stem (culm) and so that the shorter lip of the bag slides between the tip of the ear shoot and the adjacent leaf sheath. The edges of the bag should be pulled around to conform to the shape of the culm. The bag should be given a sharp downward pull to firmly attach it between the tissues mentioned. The plants should be examined every day to cover new ears as they emerge. At this time those bags already on the plants may be pulled down again to make them more secure. If you have vigorous plants with large ears or leafy tips, they can grow through the end of closely tucked bags, especially during rainy weather. The bags may need to be loosened, the leafy part trimmed or soaked bags replaced, under such conditions. Other less vigorous plants, under certain growing conditions, can produce silks while the ear is still completely hidden in the sheath. If the plant is shedding pollen and no ear shoot has been bagged, check for the telltale bulging of the leaf sheath beneath the leaf where an ear is expected. Carefully pull back the sheath slightly and bend the leaf down, or rip the leaf off at the collar (of the leaf), so the shoot bag will fit securely over the ear and the leaf sheath surrounding it. Bagging too early is not very satisfactory because the tissues will not hold the bag firmly in place. Bagging too late risks exposure of silks and resulting contamination. Ears should not be bagged after silks appear. Complete honesty is required of all workers in this regard otherwise contamination will get completely out of hand. Some workers use a larger bag 2 ½ x 1 x 8 ½ inches (6.4 x 2.5 x 22 cm) (Lawson 218), bend back the 7th leaf and cover the whole auricle and ligule area at the top of the leaf sheath. They then staple the bag around the culm even before the first shoot appears. This allows one-trip shoot bagging but sometimes the wrong leaf axil is taken and the shoot comes out elsewhere and is exposed. In any shoot bagging practice care must be taken in handling the plant so that the support of the leaf sheath to the culm of the next node is not weakened; otherwise the top of the plant will break out and the whole plant will be lost.
Under natural conditions receptive silks appear over a 4-5 day period. They are pollinated by the pollen shed during each of these days and a full set (fertilized) ear results. In the case of hand pollination the pollen is applied only once in a very short period of time. The result would be a partially filled ear unless additional steps are taken. The standard practice to overcome this is called cutting back. 2. Cutting Back an Ear: This consists of cutting off, with a knife, the tip of the husks and silks on an ear having the first day's silks visible and then re-covering the ear to prevent exposure. The cut should be made squarely and cleanly across the ear and as far down the husks as possible, without cutting off the tip of the cob inside. Contamination is avoided because the wet ends of freshly cut silks are not receptive to stray pollen grains, which may fall on them during the operation. The shoot bag covering the cut silks should be marked by folding the corner to indicate which ears were cut back. The next day the silks will have grown to form a thick brush all the same length and all will be ready for pollination. Pollen applied at this time will reach all silks and result in a full set (fertilized) ear. Pollinated silks stop growing in an hour or so and become darker in color. Under normal conditions silks are usually receptive for about 3 days. After that the chance of successful pollination diminishes rapidly. Some inbred lines have very tight husk tips that protect them from bird damage. These husks must be cut back before silks can be seen, since they will be past the receptive stage before the mass of silks is large enough to push up through the husks. Note that unpollinated silks will grow continually during this period to a length of 6-8 inches (15-20 cm) or more. Failure of unpollinated silks to re-grow or irregular growth indicates that the silks were already past the most receptive stage and that conditions will not be good for pollen germination. 3. Pollen Production: Pollen is shed from mature anthers beginning on the upper third of the main spike of the tassel, usually 1 or 2 days before silks appear on the ear shoots. The shedding is in daily increments that spread downward to the tassel branches toward the base and upward towards the tip of the main spike. The shedding moves along the tassel in two waves about 2 days apart. The first wave begins from the anthers of the upper floret of each spikelet. The second wave of pollen shedding is from the lower floret of each spikelet. A healthy tassel should shed pollen for about 1 week. On a warm sunny day fresh anthers begin to extrude from the florets via elongation of the filaments, at about 7 AM (07:00). These anthers will open up (dehisce) about 30 minutes later and pollen will fall out and be scattered by air currents. As the morning progresses additional anthers will appear until about 10 a.m. After that and under these conditions no new anthers appear until the next morning. If the weather is cool or cloudy and humid the extrusion of anthers will be delayed until later, sometimes until evening. Some workers report successful pollinations made in the evening. The author finds this undependable in most conditions, and impossible in others. As long as the humidity is near 100% the anthers will not open. If the weather is hot and dry the pollen-shedding process will proceed more rapidly. 4. Pollination:4.1 Collecting Pollen: To make controlled pollinations it is necessary to collect viable pollen. This can be done by covering shedding tassels, before the time the pollen is shed, with a brown paper tassel bag (Lawson No.402 - "Showerproof'd") and then collecting and carrying the pollen to the desired protected silks. The bag is also the best place for recording information about the pollen parent, any traits seen in the ear parent, pollination date etc. Information about the pollen parent from which a single pollination is to be made can be written immediately after the tassels have been covered. If more than one female is to be pollinated from a single tassel, a list can be kept of bags to write or additional bags can be written up as the bags are put on and kept for the next day's pollinations. If the plants to be used as male parents are sturdy and heavy winds are not in the forecast, the best time to cover the tassels is in the evening or after pollen has shed on the previous day. If the plants are weak or heavy winds are expected, the tassels may be bagged after the dew is off the tassels but before shedding has ended on the morning of the day of the desired pollinations. Moisture from dew trapped in the tassel bag will prevent the anthers from opening (the humidity within the bag is raised and this inhibits the shedding of pollen). 4.2 Tassel Bagging for Collecting Pollen and the Method of Controlled Pollination: Cover the tassel with the bag (brown paper tassel bag) keeping the tassel as flat (horizontal) as possible. Pull the bag down past the first flag leaf (which may be removed) then fold the base of the bag firmly around the sheath and stem of the tassel and finally secure it in place with a regular paperclip. The bag can be stapled in place, but the time saved is often wasted removing the staple the next day, when speed is essential. If tassels were bagged the day before, pollination can begin as soon as nearby unbagged tassels are seen to be shedding. Bees determine this proper time very well and can be a good indicator. If tassels are bagged in the morning sufficient time (at least one-half hour) should be allowed for all stray pollen grains to die and for anthers with fresh pollen to emerge inside the bag. The tassel bags commonly used are water resistant, but not waterproof. If the bags have been soaked by heavy rain during the night and they will not dry before pollen shed begins, they can be replaced with dry bags, if you can't afford to loose a day of pollen shed. To collect the pollen, carefully bend the plant so that the open end of the bag is higher than the closed end. Remove the paperclip and shake the bag and tassel sharply. Withdraw the tassel while being careful not to allow the open end of the bag to be low enough so that the pollen falls out of the bag. The pollen can be carried in this manner to make a self pollination or to cross on silks of one or two nearby plants. One cannot depend upon pollen remaining viable in the removed tassel bag more than 10-30 minutes depending on conditions. Though there are as many variations in this procedure as there are principle investigators (researchers), the objectives of controlled pollinations remain the same: to apply the chosen pollen to the chosen silks and minimize contamination. One standard procedure for pollination is to carry the tassel bag, with open end folded, to the plant with silks intended for pollination. Place the stalk and tassel of the female parent under the pollinator's arm thus bending the plant so that the tassel is at the pollinator's back and the ear is immediately in front (this helps to protect the ear from pollen falling from its own tassel). Then raise the shoot bag slightly, tear off the top, and squeeze it so that the shoot bag forms a chimney above the silks (this protects the silks from pollen carried by air currents from the side). Open the tassel bag in such a way that the pollen can be poured out in small amounts and dust pollen on the silks; quickly fold over the open torn end of the shoot bag to protect the silks. Then pull the tassel bag over the pollinated ear so that the long lip slides between the stalk (culm) and the ear and the short lip hangs loosely on the outside. Pull the two edges on the long lip side of the bag near the bottom around the stalk and staple them together on the side of the stalk opposite the ear, thus securing the bag to the stalk so that the ear is free to enlarge without disrupting the bag. If additional pollinations are to be made from the same male, fold the top of the bag of pollen closed and proceed to the next female. 4.3 Performing multiple pollinations using a glassine bag: If a large number of pollinations are to be made on a single day from one pollen source (as many as 100 pollinations may be made from one tassel) the best procedure is as follows: prepare a glassine bag (2" x 7 ½ " [5 x 19 cm] ) by making a "Z"-shaped fold in the bag about halfway up the length of the bag. Then pour in the contents of the tassel bag. Sift the pollen past the first fold into the bottom fold, taking care to keep the anthers in the top from where they may be discarded. Turn the bag on its side and tear off the upper of the two bottom corners of the bag. Carry the pollen in this bag to the waiting silks where it may be sifted sparingly through the torn corner of the glassine bag into the top of the torn shoot bag covering each ear. Then fold the shoot bag to protect the pollinated silks and move rapidly to the next ear. Speed is essential here because the pollen will remain viable only a short time. Fresh pollen appears light yellow and will flow freely from the glassine bag. If the pollen becomes deeper yellow or clumps together these are signs that it is no longer viable (collectively speaking even though there may be some viable grains mixed among the majority of unviable ones). A pre-written tassel bag can be left between the shoot bag and the stalk (culm) (this bag is secured later). The pre-written information on the tassel bag should comprise the relevant information (identity) of the host plant bearing the ear (sometimes referred to as the female plant) and the identity of the pollen -producing plant (sometimes referred to as the male plant). Notes about the pollination and the date may also be written on the bag. After all pollinations from one source are completed then there is time to go back and place the tassel bags over the ears and staple 5. Some Precautions:5.1 Contamination. During the shedding period pollen will be airborne on air currents. Pollen will also be on leaves, the pollinator's clothing and hands. Most of it will already be unviable, but enough will be viable to make contamination a serious problem. Precautions should be taken to keep receptive silks covered at all times. 5.2 Pollen Viability. The life of a pollen grain is usually less than 20 minutes after it leaves the anther. It is less than that in hot dry weather and much more than that in cool humid weather. (Ways to considerably extend pollen viability are known but the author has not found them to be reliable. They require special knowledge and handling.) There is little hope for fertilization if the temperature goes much above 95oF (35°C) during the shedding period or several hours thereafter. Conversely pollen may appear to be normal but will not grow on the silks unless the temperature during pollination and some hours thereafter goes above 55°F (13%deg;C). Pollen is especially sensitive to drying conditions. Any time pollen grains change in color or begin to collapse irreversible degeneration may have already occurred. High humidity has different but equally important consequences. Anthers will not open in high humidity thereby trapping the pollen within. While this does not kill the pollen immediately it does pose mechanical difficulties in spreading pollen, and causes premature loss of viability. Moisture on silks, in bags or other containers for carrying pollen will cause immediate loss of viability. Pollen is promptly killed when immersed in water and most other liquids. Known exceptions are paraffin oil and properly buffered aqueous solutions. See Poehlman & Sleper (1995) for additional information on pollination practices. 5.3. Dating of tassel bags: (Information provided by Susan Melia-Hancock, Maize Mapping Project, Department of Agronomy, University of Missouri-Columbia, Columbia, MO; origin of methodology: considered to be Dr. E.H. Coe Jr., University of Missouri-Columbia) For some projects it is convenient to have the date of pollination recorded on all tassel bags, but it is time consuming to write on each bag. With a little practice hundreds of tassel bags can be dated in a few minutes. With the closed end of a tassel bag as the top, starting at the left margin and finishing at the right margin, make a series of 5 evenly-spaced marks across the top of a tassel bag with a permanent marker (Sharpie) or heavy pencil. The 3rd mark will be in the center of the bag. Under the left most mark write "16", next "8", center "4", next "2" and under the right most mark write a "1", this is your key and template. All dates from the 1st to the 31st can be made by adding the numbers associated with the marked location. The first day of the month is the right most mark. The 31st of the month is all 5 marks (16+8+4+2+1). The 17th is the left most and right most (16+1) marks etc. Make a template on a tassel bag which reflects the desired date of pollination. Fan a stack of 25 to 50 tassel bags so the closed ends are laid out like stair steps. Place the template on top of the sloped stack of underlying bags and make the combination of marks that add up to the desired pollination date, down the entire stack of bags. To discriminate between different months different colors of marker can be used. 6. Supplies and equipment - suggestions on where to obtain them in the USA Disclaimer of Endorsement: Reference herein to any specific commercial products, process, or service by trade name, trademark, manufacturer, supplier or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the author of this document or the University of Missouri-Columbia.
Envelopes: for storage, seed separation and seed preparation
Field stakes: for planting and marking plants
Hand Held Seed Planter
Plant tags: for marking plants in field
Ear shoot bags: to cover ear shoots
Tassel bags: for covering tassel and pollinated ear
Pollinating bags: for collecting and disseminating pollen
Cut back knife: for cutting ear shoots
Stapler: for securing bags on plants
Paper clips: for securing bags on tassels
Pollinators Apron: for carrying pollinating equipment
Sharpie fine point permanent markers: for marking and writing on tassel bags
Harvesting bags: for harvesting ears 10, 20, and 50 pound onion sacks - misprints can often be purchased cheaply
Fasteners: for attaching tag to ear
Ear tag: for labeling ears
Storage boxes for ear storage
8. Useful References:
Dicke, F. F. 1977. The Most Important Corn Insects. Chapter 9: Corn
and Corn Improvement. G. F. Sprague, ed. American Society of Agronomy,
Madison, Wisconsin.
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