1 CONSERVATION OF FREEZER POLLEN BY FREEZING In 1955, as part of her thesis work in Wageningen, Tijs Visser was interested in Storage and in vitro germination of pear and apple pollen. On this occasion, He finds that the pollen germinates better when the water content has been lowered and when it has been Stored at very low temperatures. Viability is not affected after two years of storage at Minus 180 ° C. It is therefore on the water content and the different conservation methods that Focus research for two to three decades. It was not only Viability of the pollen is retained, but also that the integrity of the envelope glycoproteins Stigma recognition allows for the germination of Pollen (Charrier 1990). This research was first conducted on woody plants Fruit and forestry, followed by cereals. Despite differences in conservation suitability by species, On the lowering of the water content to the threshold of 5% to 2% by vacuum desiccation to Ambient temperature and storage at negative temperature. It is on the basis of a Freeze-drying and freezing preservation of the Pollens of the National Museum of Natural History of Paris from 1983 (Cerceau - Larrival, 1995). Among the 250 species included a board cactus: Harrisia nashii. Perhaps there - She still? In 1998 (a, b) and 2000, Julia Buitink explains the maintenance of pollen viability by Physical and molecular mechanisms of intracellular vitrification (detailed In Derouet 2010, page 18). It shows the interactions between the temperature of Storage, water content and their influence on the aging of pollen. It was in April 2000 that an Israeli team (Metz, Nerd and Mizrahi) published a Dehydration of the pollen in order to keep it for several months by freezing to achieve the cross-fertilization of two cactus (Hylocereus undatus and H. polyrhizus). These cloned plants Intended to produce fruit for consumption are self-sterile and have peaks of Offset flowers in the season. The technique developed and the means adopted seem to be sufficiently simple to Work by the cactus lover. THE "HYLOCEREUS" TECHNIQUE Pollens of Hylocereus undatus and H. polyrhizus Have a water content of 18% to 22% When harvested in the morning and 45% to 50% evening. It is known that water-rich pollen is not Viable for a long time. A few hours at most. They must therefore be dehydrated. Fruit of Hylocereus undatus Photo by Maja Dumat - flickr.com
2 The dehydration It was carried out in a partial vacuum desiccator (half a atmosphere or - 50 kPa Or -38 cm of mercury relative to the mean atmospheric pressure). The recipient Contains crystals of silica gel to adsorb the water. The pollens stay there for two hours And half before reaching a water content of about 2%, regardless of The time of harvest. The conversation In this experiment, pollens were Stored at several temperatures: +4 to Refrigerator, -18 ° C and -70 ° C in Freezers and -196 ° C in liquid nitrogen For 3 months and 9 months. Only the Results are reported here. Fertilization The rehydration of pollen is Naturally by contact with the stigma. The pollinated flowers were fertilized at 100% for frozen pollen and Only 60% with pollen Stored at + 4 ° C. Fruiting For temperatures of -18 ° C and -70 ° C, the weight of fruit produced (pitayas) was the same as orchards those harvested in (410 g to 330 g H. undatus and H. polyrhizus) after fertilization With fresh pollen of any kind. Fruits from pollen stored at -196 ° C Weight about 10%. Pollen stored at + 4 ° C produced fruit from 20 to 30% lighter. Seeds The number of seeds Harvested is included in the Table opposite. These Are the average Of six samples per batch. The in vitro tests of Seed germination Were conducted with 400 Seeds per lot. Balance sheet Finally, we see that these conditions successfully tested in the laboratory and then in the orchard are Transposable within the framework of a domestic practice. In fact, it is simply a matter of Dehydrate the pollen under a partial vacuum and store it in the freezer.
3 THE VULGARIZED TECHNIQUE For cactus lovers, until now, practices for the conservation of fresh pollen In the refrigerator allow fertilization for only a few days. This New long-term conservation technique will allow fertilization or hybridization Of species whose flowering gap extends over a whole season. Manufacture of vacuum desiccator We are trying to achieve a residual pressure of 50 kilopascals, that is half a bar. To do this, we must empty the container half of its air. Technical Requirements - Vacuum gauge purchased at ExpertByNet: 19,90 € + port http://www.expertbynet.fr/fr/manometre--et--vacuometre-- oil / 8682 - vacuum gauge - round - 1 - a - 0 - bars-- diameter - 63mm - m1--4-- 3283159024227.html - Brass nipple: F 8x13 - M 12x17: € 1.45 (DIY plumbing department) - O-rings made of rubber: two from Internal diameter 6 mm, two diameter Inside 11 mm - Metal washers: one with an inside diameter of 13 mm for the vacuum gauge and two of 6 mm For the valves. containers Take jars called "TO" (Twist Off). I chose with a lid of 104 mm compared To a jar with an outer diameter of only 83 mm. It may be a bit Too large because the lid becomes very concave when it is under vacuum, with a click Characteristic to -0.2 bar. An intermediate option exists in the Pickle with Russian (Kühne) with a Diameter of 93 mm. - Desiccator version pollen: a jar of Gizzards of duck confit and sliced to 6.00 €. The same container exists with fat Duck or goose in the two euros ... - Desiccator version pollen and seeds: one Jar of cassoulet at 5,70 €
4 Equipment -- A drill - Wood drills with centering point of 6 mm and 13 mm Drilling - First drill a 3 mm hole at the future location of the vacuum gauge to accommodate the Center of the wick without deforming the lid. - Then start drilling the 13 mm hole for the vacuum gauge using the Centering through the outside of the lid without crossing, only so that the finish is clean. Turn the lid upside down and finish the hole from the inside of the lid without slowly. Finally, using the 6 mm drill bit, drill according to the same principle the hole of each Two valves. Mounting Remove the rubber hull around the vacuum gauge. It adds an unnecessary volume. To put An O-ring of 11 on the vacuum gauge and thread the assembly into the hole of the cover, To Inside the lid, add the washer of 13 then the nipple and tighten. If you notice a lack of waterproofing, you can add a flat rubber gasket Which will make a wider seating for the O-ring on the cover. In the picture below, the Attached to the inside of the lid, under the washer, is not essential. For the valves: put the O-ring on the valve, insert into the hole of the cover then Put a washer on the other side of the cover and the clamping screw. This is Valid for both internal and external valves.
5 To make the vacuum, a refrigerator group recovered on an old refrigerator or a freezer Constitutes an excellent vacuum pump. A few seconds is enough to reach a Depression of - 38 cm of mercury. If you can not retrieve one, there is also a manual option.
6 Dryer version caviste A vacuum pump in the opened wine bottles may well be enough to create the Vacuum required for pollen dehydration. The pump, and its reusable cap, Sell between 15 and 20 €. The vacuum at -50 kPa is quickly reached. Vacuometer: Same way to do that for the model previously described. Manual vacuum: - An Aqua Flow D1630 mounting junction Use only the central part (gray): 1,25 € http://www.leroymerlin.fr/v3/p/produits/jonction--de-- mounting - aqua - flow - d1630-- e144630 # & XT ™ = & junction xtcr = 2 - A reduced nipple. The female part will receive the part Center of the junction and the male part (diameter 16.5 mm Approximately) will pass through the lid. - Seals of the required dimensions: one to be between the Lid and nipple, two (flat and / or toric) superimposed For sealing between the plastic joint and the part Female from the nipple. - Any element to come screw on the nipple to Inside the jar. - Same containers as for the version "group refrigerator", Lid diameter 104 mm or slightly lower (93 mm). - Wine Pump (Vacu Pump): 15,90 € http://www.boutique--bar.com/pompe--vin--vacu--pump-- xml - 244_408--1340.html Equipment required Drill and two bits of wood with centering point: - One of 13 mm - One of 16 mm Mounting In this version, there are only two holes in the
7 Lid: one for the vacuum gauge and one for the vacuum pump since the system used Also allows the air to flow in.
8 Pollen collection The funnel The pollen is collected in a bag of crystal paper. For large flowers (Epiphyllum, Selenicereus and some Echinopsis), collection can be done by shaking the flower - above a Funnel connected to the crystal paper pouch. The scissors For the other flowers, one can use a pair of scissors and cut just below The dehiscent anther. It is then sufficient to pour into the sachet the anthers and the pollen left On the blades of the scissors. The cotton-tube The scissors technique The inconvenience of loading the bags Pollen and the remains of the anther. This set is then found On the brush during fertilization and could compete Occupation of the surface of Stigma by pollen. To separate Pollen pollen bags, it is necessary to So that this is done at the collection By a technique of suction of the pollen. Cottons are often Hollow plastic. The solution is simple: a cotton rod = two cotton tubes. Then, the cotton - tube must be connected suction device. It is then enough to walk the cotton Tube from the stamens to Aspirate the pollen. Before stopping Suction, the tube must be straightened And then emptying it in a Bag and patting it on, or Possibly by blowing into the Cotton tube with delicacy. Dehydration of pollen The sachet containing the pollen is folded and closed with a scotch and placed in the jar whose Was previously filled with a moisture-absorbing product. The partial vacuum is made in the container. The system is very hermetic because it can The same vacuum for a very long time ... as much as a bike can remain inflated. After 3 or 4 Hours, the pollen is considered to have reached a water content consistent with freezing. The valve is unscrewed and there is heard the whistling of the air entering the container. It is also a means of verifying that the vacuum was indeed made.
9 Freezing pollen The pollen bags are then stored in a freezer in a sealed jar with Crystals of silica gel to maintain dehydration. RESULTS Influence of the pollen collection technique on the quality of fertilization In the first stage, pollen was collected by cutting with the scissors the part Superior of the stamens with their pollen sac. The hypothesis of a competition of debris Pollen bags with pollen on stigma triggered comparison "aspiration" and "Scissors" on the number of seeds per fruit. The pollen was therefore, either sucked on a flower, or collected with scissors on another flower Of the same plant, the same day. The fertilization also took place obviously on two different flowers of the same Plant on the same day. The harvest of the fruits of each plant takes place on the same day. Of course, it is on a small number but it is found that the starting hypothesis was not Because in the present case, consisting solely of Echinopsis, fertilization by Pollen collected with scissors generate on average a little more seeds (400 vs 340). Both techniques therefore remain conceivable. Influence of a greater vacuum on the number of seeds produced. In their publication, Metz et al. Recommend a vacuum of -0.50 kPa. It was interesting to See if the amateur could dispense with a vacuum gauge to appreciate the depression in his Dehydration container. Pollen was collected, bagged and dehydrated under a high vacuum between -0.88 and -0.92 KPa for 3 to 4 hours, then frozen. Twelve flowers were fertilized with these pollen and the Half of them have not been fruitful.
10 These results are to be compared with the results of the 17 fertilizations obtained on Echinopsis With the 2011 pollen that was dehydrated under -0.50 kPa. The average number of seeds Obtained by fruit was 417. It is found that Echinopsis sp. "Yellow flower" seems very sensitive, which is not the case for Harrisia sp. Sensitivity to high vacuum may not be the same for all species ... It is certainly no coincidence that the Metz team set the vacuum value at -0.5 kPa. he There seems to be a deleterious effect on the viability of pollen and it is therefore essential to To be equipped with a vacuum gauge to stabilize the vacuum at about -0.50 kPa. Influence of the longevity of pollen storage on the number of seeds produced After 3 years of storage in the freezer, pollen remains viable even after several Brief exits at room temperature for fertilization, as below for 3 pollens: CONCLUSION After three years of testing, the demonstration is made that the technique is reliable and Simple enough to be implemented by the collector. It should Previously unrecognizable crossings because of the shifting of flowering. There is thus a technique of future and new perspectives for the amateurs and the breeders.
11 BIBLIOGRAPHY Buitink J. C. Walters, J. Hoekstra FA & Crane 1998a, Storage behavior of Typha latifolia pollen at low Water content: interpretation on the basis of water activity and glass concepts Physiologia. Plantarum, 103: 145-153. Buitink J., Claessens MAE, Hemminga MA, Hoekstra FA, 1998b, Influence of water content and Temperature on molecular mobility and intracellular glasses in seed and pollen, Plant Physiol. 118: 531--541 Buitink J., Leprince O., Hemminga M. A., Hoekstra F. A., 2000, The effects of moisture and temperature On the aging of kinetics of pollen: interpretation based on cytoplasmic mobility, Plant, Cell & Environment, 23, 9: 967-974 Cerceau - Larrival M .-- Th. , Delange Y., Youmbi E., Derouet L., Verhille A. M. And Carbonnier - Jarreau M .-- C., 1995, Contribution to the Preservation of the Male Genetic Heritage of Plant Collections live National Museum of Natural History, Paris, Grana, 34, 6: 371 - 407 Charrier A., 1990, Pollen and genetic resources, Bull. Soc. Bot. Fr., 2: 101--104. Derouet M., 2010, Seeds of cacti, from the flower to the seedling, page 18. http://www.cactuspro.com/articles/graines_de_cactees_de_la_fleur_a_la_plantule C. Metz, Nerd A., Y. Mizrahi, 2000. Viability of pollen of two fruit crop cacti of the genus Hylocereus is By HortScience, 35, 2: 199--201 Visser T., 1955, Germination and storage of pollen, Thesis, Mededelingen van de Landbouwhogeschool 55 (1): 68p, NL Summary: Long - term storage of pollen is interesting for enthusiasts seeking cacti butts. This paper has popularize home - made experiment based on the study developped by Metz et al. (2000). To obtain an efficient preservation of viable pollen, two processes are required: first Dehydration in a vacuum (about - 50kPa) (About 3 hours) and second, storage at subfreezing temperature. in Practice vacuum, vacuum wine, refrigerating unit And the storage in a household freezer. For pollination, the pollen is applied to mature stigmas Able to rehydrate the dried pollen. Under these conditions, we get fruits and seeds after Three years of storage. Key words: cactus, pollen, seed, dehydration, drying, storage, freezing I thank Georges Marchand (†) and Jean - Didier Hary for their friendly collaboration. Michel Derouet firstname.lastname@example.org Last updated May 14, 2014
Original French text:
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