Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
  • A01H1/027—Apparatus for pollination

Definitions

• the present invention relates generally to a plant breeding techniques and,
• Horticultural plants whose flowering and particularly their pollen production may be manipulated for plant development and breeding purposes include lettuce, endive and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums. Double haploid pollen production limits or enables plant development and breeding processes which are that often characterized as successful or not based on the amount of seed that is set on the plants. Seed set is greatly influenced by the amount of viable pollen from the haploid or partial doubled haploid or the doubled haploid that actually reach the silks and ultimately the egg.
• tassels and other pollen producing structures on haploid and chimeric (diploid/haploid) doubled haploid corn, wheat, barley, rice, etc. and other haploid plants produce very small amounts of viable pollen.
• the conventional method of pollen collection which involves putting a bag over the tassel, is usually an inadequate method for pollen collection, particularly when small amounts of viable pollen have been produced, as is often the case in haploid and doubled or partially doubled haploid and some doubled haploid plants.
• the present invention provides a means to collect viable pollen from fertile anthers of haploid and doubled haploid, or other corn plants producing small amounts of viable pollen, and use the collected pollen for direct placement onto the receptive stigmas (silk) to produce seed.
• the present invention represents a novel apparatus and method to collect small amounts of viable corn pollen from tassels of corn plants, particularly pollen producing anthers on plant structures which may include the tassels of haploid and doubled haploid plants.
• the present invention includes a device consisting of (a) a nozzle to direct the collection of pollen, (b) a vacuum source, (c) a cylinder capable of generating a cyclonic air movement, and (d) a disposable receptacle in which to collect viable corn pollen.
• the pollen is captured via cyclonic air movement wherein the viable pollen grains settle out of the air stream and are collected in a receptacle.
• the viable corn pollen is then placed directly onto the receptive stigmatic tissues (silks).
• the present inventions uses the method for collecting pollen from anthers of a plant, comprising the steps of providing a cyclonic chamber having a vacuum port, a pollen collection port, and a pollen deposition port, providing a source of a vacuum connected to the vacuum collection port, passing the pollen collection port adjacent the anthers of the plant to draw into the cyclonic chamber pollen from the anthers that would not be shed naturally by the anthers; and depositing the collected pollen in a reservoir connected to the deposition port of the cyclonic device where it is viable for the pollination of the same or another plant.
• Another method of the present invention is useful for breeding monocot plants comprising barley, rye, triticale, wheat, rice and maize.
• the method of breeding in particular maize or corn plants comprises the steps of: growing a corn plant to a stage of pollen viability in the anthers; providing a cyclonic chamber having a vacuum port, a pollen collection port, and a pollen deposition port; providing a source of a vacuum connected to the vacuum collection port; passing the pollen collection port adjacent the anthers of the plant to draw into the cyclonic chamber pollen from the anthers; depositing the collected pollen in a reservoir connected to the deposition port of the cyclonic device; and pollinating the same or another corn plant with the collected pollen.
• a novel feature of this apparatus is that it provides a means to collect very small amounts of viable pollen generated on haploid and doubled haploids plants, each of which may generate a limited number of functional anthers.
• the collected viable corn pollen grains can be placed directly onto the silks.
• Fig. 1 is photograph of an embodiment of the present invention.
• FIG. 2 is a photograph of an alternative embodiment of the present invention.
• Fig. 3 is a composite photograph showing a vacuum pump for use with the present invention, a cyclone vacuum pollen collection apparatus, collected urediosposres, and a photomicrograph of an anther which is producing only a small amount of pollen.
• FIG. 4 is a photograph of two ears that were produced using pollen collected by the present invention.
• Fig. 5 is a photograph of a device of the present invention showing pollen collected from a pictured diploid corn plant wherein the anthers did not dehisce well.
• haploid and doubled haploid corn plants have inadequate amounts of viable pollen, Some haploid and doubled plants may have spontaneous sectors of the tassel bearing fertile flowers, but often these regions are infrequent and may produce limited amounts of viable pollen. As such, self pollination and seed production is often not possible with the above described haploid and doubled haploid plants.
• the conventional method of pollen collection in maize for example is to use a tassel bag.
• a tassel bag may be problematic due to any or all of (a) the plant not being large enough to support the weight of the bag, (b) the plant not being large enough to tolerate the increased wind-based movement of the plant due to the sail-like effect of the bag, or (c) the inability of the operator to remove a suitable amount of pollen from the bag.
• removal of limited amounts of viable pollen from the bag onto the receptive silk tissue can be problematic in a field environment due to inadvertent release of pollen from the pollen bag due to having to manipulate a large tassel bag amongst the leaves of adjacent plants.
• the tassel bag is immediately adjacent to the ear silks, wind movement can blow the pollen away from the silks. In each of these examples, reduced seed production can result.
• the present invention provides a means to easily collect and distribute significant amounts viable pollen from haploid or doubled haploid plants directly onto the ear silks and, thus, results in more haploid and doubled haploid plants producing homozygous seed and the production of significantly greater amounts of seed per ear.
• FIG. 1 is a drawing of an embodiment of the Cyclonic rust spore collector which is used in the method of the present invention which shows a nozzle 20 which is used not for spores but to direct the collection of pollen 22.
• a vacuum line 30 which is connected to the cyclonic cylinder 40 vacuum source 32 ( shown in Figure 3) which is not shown in Figure 1.
• the nozzle 20 is connected to a cyclonic cylinder 40 which is capable of generating a cyclonic air movement.
• This cyclonic air movement acts to deposit the pollen 22 in the disposable receptacle 50.
• the disposable receptacle is a h lf of a pill or a single gel cap 52 similar to the gel capsules 54 used for providing medicine to large farm animals.
• the pollen 22 is captured via cyclonic air movement 42 (shown in Fig 3) wherein the viable pollen grains 22 settle out of the air stream and are collected in a receptacle 50.
• the use of a disposable receptacle 50 significantly decreases any issues of cross contamination of pollen between pollen collections.
• the vacuum system is cleansed of extraneous pollen by running the vacuum system for short periods without a receptacle attached between collecting pollen from different plant genotypes.
• the viable corn pollen is then placed directly onto the receptive stigmatic tissues (silks) (not shown).
• FIG 2 shows a alternative embodiment of the present invention, which is based on the ease of use of a automated pipetting device 300.
• the figure is an automated battery operated pipetting system that releases set amounts of material.
• the design of the hand shaped gun system would work well for pollen collection in both green houses but also in fields.
• the vacuum power could be turned off and on by with the grip of the hand, leaving the other hand free to collect and replace receptacles from the device.
• the receptacle replacement could be automated in a cartridge system where each receptacle has a second gel cap pushed Onto it to form an enclosed capsule once the receptacle is filled.
• the enclosed gel capsule is shown in Figure 3a.
• Figure 3 shows four different figures 3a, 3b, 3c, and 3d.
• Figure 3b shows a vacuum pump 32 for use in the method of the present invention.
• This vacuum source can be transported or carried in a back pack, strapped to a forearm, slung around the shoulder or neck with an attached strap.
• the vacuum line 30 in Figure 3B attaches to the improved cyclone column 101 with the cyclonic cylinder 140 below.
• the vacuum pollen collection apparatus shown in Figure 3a has the vacuum line 30 attached on one end to the vacuum source 32 and the other end is attached to the improved cyclone column 101 with the nozzle 122 located approximate the cyclonic cylinder 140.
• the cyclonic cylinder 140 optionally has dual detachable collectors 150 and 160.
• This improved vacuum system 100 allows for less clogging and decreases any associated damage to the pollen from the prior art device of figure 1.
• the device 100 is approximately 23 cm in size. The size can be altered but it must be capable of free pollen movement and reduced pollen damage.
• the present invention has a larger area for pollen flow in the nozzle 122 and cyclonic air movement shown by two arrows 41 in Fig 3a 5 with dual detachable collectors 150 and 160 (160 shown in Figure 5) to decrease pollen contamination.
• the dual detachable collectors fulfill two important goals. The first goal is to allow for larger amounts of pollen collection than can be achieved by the disposal collector 150, and the second goal is to allow for additional cleaning of the pollen collection area by removing the larger collector portion 160.
• Figure 3D in comparison shows that the urediospores cyclone collector is not well adapted for larger amounts of pollen which may need to be collected.
• Figure 3c shows the relative size of the tassel and the anthers that are on a maize tassel, similar anthers are on wheat, oat, barley, triticale and rye spikes and rice panicles.
• Figure 4 shows a photograph of the two ears that were produced from pollen collected using the method of this invention and the device of Figure 3a. This device collected substantially undamaged viable pollen. The adapted device permits anthers to be collected even from plant anthers that did not dehisce well.
• the present inventions uses the method for collecting pollen from anthers of a plant, comprising the steps of providing a cyclonic chamber having a vacuum port, a pollen collection port, and a pollen deposition port, providing a source of a vacuum connected to the vacuum collection port, passing the pollen collection port adjacent the anthers of the plant to draw into the cyclonic chamber pollen from the anthers that would not be shed naturally by the anthers; and depositing the collected pollen in a reservoir connected to the deposition port of the cyclonic device where it is viable for the pollination of the same or another plant.
• the pollen was placed on the surface of a piece of glassine weigh paper.
• a rust spore collection device was hooked to the lab vacuum system and the input tube of the cyclone collector was placed near the pollen.
• a small amount of the pollen was sucked into the vacuum system and via cyclonic air movement, deposited in the gel cap.
• the pollen was allowed to move in a circular motion within the gel cap for approx. 30 seconds.
• the gel cap containing the pollen was then transferred directly back to the greenhouse and used to pollinate an ear.
• Vacuum collection of pollen significantly improves seed production on doubled haploid plants because current methods of self pollination are not particularly well suited for situations where limited amounts of viable pollen are available.
• some partially fertile anthers on doubled haploid tassels do not dehisce well, and thus, self pollination becomes problematic due to the lack of pollen shed.
• a cyclonic system in which the anthers spin around in the collection receptacle for a period of time provides an opportunity to break open the anthers in such a way that the viable pollen grains may exit the anther and subsequently be collected (along with other pollen grains) in the pollen collecting receptacle.
• seed production should be possible on haplid, partially doubled haploids and doubled haploid plants that typically would be problematic if not impossible to self-pollinate.
• Doubled haploid maize plants may extrude limited amounts of
• pollen from doubled haploid plants is collected from the tassel in a bag. To produce doubled haploid seed, the pollen then is distributed over the top of brush of silks on that same plant.
• the process of collecting pollen in a bag and distributing that pollen on a brush of silks can be problematic.
• the purpose of this experiment was to evaluate the cyclonic pollen collection device (Fig. 3) in terms of its ability to collect pollen from corn tassels, and test how viable and functional the pollen is post collection by using the assembled pollen to pollinate ears of corn, as measured by seed production.
• the pollen collection device consists of a vacuum source, a cyclonic tube shaped container to separate the pollen from the vacuum airstream, a nozzle used to direct a vacuum to regions of the tassel in order to remove the pollen and anthers from the tassel, and a disposable gel cap which is used to collect the pollen.
• the pollen collection device worked quite well to collect and deposit pollen in the gel cap container.
• the device was light weight and easily handled amongst the plants. Pollen and anthers were easily sucked into the nozzle of the device as it was moved up and down along the length of each tassel branch.
• the vacuum air stream from the nozzle as well as rubbing the nozzle against the tassel branch helped to dislodge anthers and shake loose pollen, both of which resulted in very good pollen collection
• FIG. 2 is a picture of the hand-operated vacuum collector. In this case, a
• cyclonic action is incorporated into the device in order to collect pollen, possibly into a disposable collection receptacle.
• the device is economically modeled to look something like existing hand-held auto-pipetters. This device could be used to both collect and transfer pollen from the tassel to the silk.
• the belt or backpack vacuum pump and energy source would be connected to this hand-held pollen collection unit via tubing.
• the pollen collection device readily collected pollen and the collected pollen was viable and able to produce seed on pollinated ears at a level reasonably comparable to hand pollination.

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• Life Sciences & Earth Sciences (AREA)
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• General Health & Medical Sciences (AREA)
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• Developmental Biology & Embryology (AREA)
• Environmental Sciences (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

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• 2012
  • 2012-03-12 WO PCT/US2012/028833 patent/WO2012125593A2/en active Application Filing

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