Oriental cymbidium pollination method

Abstract

According to the method, the pollination problem of an endangered plant, namely the cymbidium serotina, is researched, and in order to improve the pollination rate of the cymbidium serotina and optimally select the pollen collection time, the pollination tool and the pollination time, the invention provides a method for adopting cactus thorns as the pollination tool for the first time, so that the pollination of the cymbidium serotina is facilitated, and the pollination efficiency of the cymbidium serotina is improved. The pollen utilization rate is greatly improved, the breeding number of the precious relict plants is increased, and an efficient pollination method is provided for breeding of the rare and endangered plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant pollination, and particularly relates to a Welwitschia pollination method. Background Art

[0002] Welwitschia mirabilis, also known as 100-year-old leaf, 100-year-old orchid, and 100-year-old leaf, is the only plant in the gymnosperm class, Welwitschiales order, and Welwitschiaceae family.

[0003] The Welwitschia plant grows exclusively in the deserts of Namibia and Angola along the southwest coast of Africa. It was first discovered in the Namib Desert in 1860 by the Austrian explorer Friedrich Welwitsch. Its limited distribution makes it an extremely rare plant, listed under Appendix II of the Convention on International Trade in Endangered Species (CITES). Considered a relict plant and a living fossil, the Welwitschia plant is recognized worldwide as a rare and valuable plant species. As a link in plant evolution, the Welwitschia plant retains some of the primitive characteristics of spore plants while possessing the evolutionary characteristics of seed plants, thus occupying a crucial position in plant phylogeny.

[0004] Welwitschia is a dioecious plant. The female cones, also called megasporophylls, each contain an ovule in each bract. Upon fertilization, the ovule develops into a seed. The megasporophylls are arranged opposite each other to form the megasporophylls. The male cones are called microsporophylls. Each microsporophyll contains six microsporangia, which contain microspores, or pollen. Because it's impossible to distinguish between male and female Welwitschias before flowering, the ratio of male to female plants is uncertain. This is especially true during artificial cultivation, where the small number of plants makes it difficult to control the ratio of male to female flowers. This makes sexual reproduction difficult, and therefore artificial pollination is essential during Welwitschia cultivation. Summary of the Invention

[0005] The purpose of the present invention is to provide a Welwitschia pollination method that improves the pollination success rate of Welwitschia, increases the breeding quantity of this precious relic plant, and develops an efficient method for the breeding of rare and endangered plants.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A Welwitschia pollination method, comprising the following steps:

[0008] (1) During the flowering period, cover the male and female flowers of the Welwitschia plant with a net to prevent flies, ants, and hoverflies from eating them;

[0009] (2) Pollen collection: Collect Centella asiatica pollen between 8:30 and 11:30 in the morning and place the collected pollen in a 1.5 ml centrifuge tube. In actual work, in order to improve work efficiency, it is recommended to collect pollen at 11:30 and collect all the mature pollen of the day at one time for pollination.

[0010] (3) Pollination was carried out during the period of 10:30-16:30 when the pollination drops of the female cones of Welwitschia orchid appeared, and the preferred pollination period was 11:30-15:00; the thorns of the tortoise shell cactus were used as pollination tools; the pollen was directly pollinated into the pollination drops of the female cones after the thorns of the tortoise shell cactus were dipped in the pollen.

[0011] Preferably, when collecting pollen, the following methods can be used: open the lid of a sterile centrifuge tube and place it vertically just below the target pollen, 5-10 mm away from the pollen, to ensure that the pollen falls freely into the tube; or take a clean weighing paper and let the pollen fall on the weighing paper. After collecting all the pollen, fold the weighing paper longitudinally along the center line to form a V-shaped groove. Through the action of gravity, the pollen particles slide along the V-shaped groove to the bottom of the crease. Then align the crease end of the weighing paper with the mouth of the sterile centrifuge tube, and tap the back of the paper with your fingers to allow the pollen to flow into the centrifuge tube.

[0012] Preferably, the pollen is collected by gently picking the Welwitschia pollen with sterile tweezers, a toothpick or a cactus thorn.

[0013] In addition, if the female and male flowers do not bloom at the same time, the Welwitschia pollen needs to be stored and pollinated when the female flowers mature. Specifically, the collected Welwitschia pollen can be refrigerated. Welwitschia pollen can be stored in a 4°C refrigerator, a -20°C refrigerator, or a -80°C ultra-low temperature refrigerator for a short period of time. During the 3-month storage period, the pollen germination rate does not decrease significantly. Therefore, the Welwitschia pollen can be refrigerated and pollinated when the female flowers bloom.

[0014] Advantages of the present invention:

[0015] The present invention studies the pollination problem of the endangered plant Welwitschia, and in order to improve its pollination rate, optimizes the pollen collection time, pollination tools and pollination time. It is proposed for the first time to use the thorns of the tortoise shell cactus as a pollination tool, which is beneficial to the pollination of Welwitschia, greatly improves the pollen utilization rate, increases the breeding quantity of this precious relic plant, and provides an effective method for the reproduction of rare and endangered plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the hoverfly that feeds on the pollen of the centenarian orchid;

[0017] Figure 2 It is the ants that suck the pollen of the centenarian flowers;

[0018] Figure 3 It is the flies that feed on the pollen of the centenarian flowers;

[0019] Figure 4 The isolation net is used to protect the centenarian pollen plants;

[0020] Figure 5 It is a count of the number of male flowers that bloomed between 8:30 and 14:30 on June 4th;

[0021] Figure 6 It is a statistical analysis of the increase in the number of male flowers that bloomed between 8:30 and 14:30 on June 4th;

[0022] Figure 7 It is a direct pollen collection method;

[0023] Figure 8 It is a weighing paper assisted collection method;

[0024] Figure 9 The number of pollination drops observed on 18 female cones between 10:30 and 16:30 on April 25th.

[0025] Figure 10 This is a real picture of the Tortoise Shell Cactus;

[0026] Figure 11 This is an electron microscope photo of the spines of Tortoiseshell Cactus;

[0027] Figure 12 It is a comparison of several pollination tools;

[0028] Figure 13 It is a precise point-injection method using the thorns of the tortoise shell cactus. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0032] Example 1 Welwitschia pollination method

[0033] 1. Protection of male and female flowers

[0034] During the cultivation process in the production greenhouse of the National Botanical Garden (North Garden), we observed that the insects feeding on the pollen and pollination drops of the Welwitschia orchid were mainly flies, ants, and hoverflies (see Figure 1-3 ), they appear in large numbers in the morning when pollen and pollination droplets appear. If these insects are not driven away, they will eat the pollen and pollination droplets, resulting in a situation where there is no pollen and pollination droplets.

[0035] In order to protect the pollen, a frame was made of lead wire and a mesh was made of gauze (see Figure 4 ) to protect the male flowers, ensuring pollen collection. A net was also made using the same method to protect the female flowers. The net was used from the time the male and female flowers began to bloom until the end of the flowering period.

[0036] 2. Collection of Centennial Orchid Pollen

[0037] 1. Determination of the time for collecting Centella asiatica pollen

[0038] 1.1 Single-day flowering observation of Welwitschia male flowers

[0039] For an 11-year-old male plant grown in the production greenhouse of the National Botanical Garden (North Garden), the leaf blades were 15 cm wide, 60 cm long, and the stem diameter was 10 cm. Flowering was counted based on visible anther sacs with pollen; no pollen was observed.

[0040] Observations were conducted on June 4th. The temperature in the greenhouse was 19.3℃-32.9℃ that day. The main observation times were 8:30, 9:30, 10:30, 11:30, 12:30, 13:30, and 14:30 (Beijing time). The number of male flowers that opened every hour was recorded until no new male flowers opened.

[0041] The male flower bulbs were cleaned after pollen was collected the day before the observation to ensure that the pollen observed on the observation day was all pollen produced by new flowering on that day.

[0042] Figure 5 It shows that the male flowers of Welwitschia begin to open at 8:30 and continue to open until 11:30, after which no new male flowers open.

[0043] Figure 6 It shows the number of new male flowers every hour. The maximum number of male flowers blooming in Welwitschia is 30 from 8:30 to 9:30, 16 from 9:30 to 10:30, and 5 from 10:30 to 11:30. No new male flowers bloom after 11:30.

[0044] As you can see, the male flowers of Welwitschia orchid bloom between 8:30 AM and 11:30 AM. Pollen can be collected starting at 8:30 AM. In practice, for increased efficiency, it's recommended to collect pollen at 11:30 AM, gathering all the mature pollen for the day at once for pollination. Harvested pollen can be placed in a 1.5ml centrifuge tube for easy storage.

[0045] 2. Collection of Centella asiatica Pollen

[0046] 2.1 Direct Collection

[0047] like Figure 7 As shown, when collecting centennial flower pollen, you can open the lid of a sterile centrifuge tube (specification: 1.5ml) and place it vertically just below the target pollen, about 5-10mm away from the pollen, to ensure that the free-falling pollen can fall into the tube.

[0048] Use sterile tweezers, toothpicks or cactus thorns to gently poke the pollen to cause it to fall off naturally and fall directly into the centrifuge tube; after collection, cover the lid tightly and use it directly for pollination or refrigerate it and wait for the best time to pollinate.

[0049] 2.2 Weighing paper assisted collection

[0050] like Figure 8 As shown, place a clean piece of weighing paper under the pollen. Use sterile tweezers, a toothpick, or a cactus thorn to pry the pollen directly onto the weighing paper. Once all the pollen has been collected, fold the paper in half lengthwise along its centerline to form a V-shaped groove. Gravity will cause the pollen grains to slide along the V-shaped groove to the bottom of the fold. Align the folded end of the weighing paper with the opening of a sterile centrifuge tube and gently tap the back of the paper with your finger to allow the pollen to flow into the tube. Once collected, secure the lid tightly and use directly for pollination or refrigerate.

[0051] 3. Storage of Centella asiatica Pollen

[0052] During the cultivation process, it is often the case that the female and male flowers do not bloom in the same season. At this time, it is necessary to store the pollen of the Welwitschia and wait until the female flowers mature for pollination.

[0053] The collected Centella asiatica pollen was collected in 1.5 ml centrifuge tubes and stored in a 4°C refrigerator, a -20°C refrigerator, and a -80°C ultra-low temperature refrigerator, and taken out regularly for pollen germination.

[0054] Preparation of culture medium: Dissolve 2.5 g of sucrose in distilled water and add to a 25 ml volumetric flask.

[0055] Experimental process: drop 2 drops of culture solution in the groove of the slide, put the pollen into the culture solution, put the slide pad with moistened filter paper into the culture dish, cover it, wrap it with a plastic bag, and place it in a 35℃ artificial climate box. Take it out after 7 hours of culture, and repeat 3 times for each culture.

[0056] A Leica dm3000 led microscope was used to observe and photograph all pollen.

[0057] Germination rate = number of germinations / number of pollen * 100%. Repeat 3 times and take the average value. Perform germination experiment every 30 days and observe for 3 months. The results are shown in Table 1:

[0058] Table 1: Results of germination of Centella asiatica pollen

[0059] Group Initial germination rate 1 month germination rate 2-month germination rate 3-month germination rate 4℃ 41.27 40.57 39.27 38.76 -20℃ 41.59 41.71 40.26 43.62 -80℃ 42.18 41.53 40.22 41.88

[0060] As can be seen from Table 1, Welwitschia pollen can be stored in a 4°C refrigerator, a -20°C refrigerator, or a -80°C ultra-low temperature refrigerator for a short period of time. During the three-month storage period, there was no significant decrease in the pollen germination rate. Therefore, Welwitschia pollen can be refrigerated and wait for the female flowers to bloom for pollination.

[0061] 3. Pollination of Welwitschia female flowers

[0062] 1. Observation of the appearance of pollination drops in Welwitschia orchids on a single day

[0063] On April 25th, in the production greenhouse at the National Botanical Garden (North Garden), temperatures ranged from 14.7°C to 39°C. Pollination droplets were observed every half hour from 10:30 AM to 4:30 PM (Beijing Time). Eighteen female cones reached maturity that day. Individual cones were used as observation units, and pollination droplets were counted if visible to the naked eye. Those not visible were counted as absent. The number of pollination droplets present in each of the 18 female cones was recorded.

[0064] Five of the 18 female cones had no pollination droplets on that day, and no trend is shown in the graph. Figure 9 The figure shows the number of pollen droplets appearing in the remaining 13 cones between 10:30 and 16:30. The number of pollen droplets in the female Welwitschia cones gradually increased from 10:30, reached a peak at 14:00, and gradually decreased after 15:00.

[0065] In practice, to ensure effective pollination, we begin pollination when pollination droplets appear at 10:30 AM. The number of pollination droplets increases significantly between 11:30 AM and 3:00 PM, making artificial pollination more efficient during this time. Pollination must be completed before the pollination droplets disappear.

[0066] 2. Selection of Welwitschia pollination tools

[0067] Due to the scarcity of pollen in Centella asiatica, it is necessary to improve the utilization rate of pollen. We cannot use the general method of pollination by dipping pollen in a brush, including special pollination pens. These tools have high requirements for the amount of pollen. We choose the thorns of Tortoise Shell Cactus as pollination tools. Through electron microscope observation at 500 times magnification, we found that the thorn tips of Tortoise Shell Cactus have a scale structure, which is conducive to fixing pollen ( Figure 10 、 Figure 11 Compare the thorns of the Tortoiseshell Cactus, toothpicks, tweezers, and pollination sticks ( Figure 12 ), it can be seen that the surface of the toothpick is relatively smooth. It is difficult to pick up pollen with tweezers without using it to dip the pollen, and it is not conducive to pollination. The amount of pollen picked up by the brush is too large, which easily leads to waste. Therefore, in the actual operation process, since the use of conventional pollination tools for Welwitschia pollination is not very ideal, through observation of the thorns of the Tortoise Shell Cactus, it is preliminarily speculated that it is conducive to fixing pollen. Therefore, the experiment used the thorns of the Tortoise Shell Cactus as a pollination tool and studied its pollination effect:

[0068] Place two drops of distilled water in the groove of a glass slide. Dip the tip of a tortoise shell cactus into Welwitschia pollen. If the pollen is yellow, use it as the standard. Then wash the pollen in distilled water. If no pollen is visible on the tip of the thorn, use it as the standard. Observe the washed pollen under a microscope and record it as one group. Take a photo and count the pollen. Repeat 10 groups in total. The results are shown in Table 2.

[0069] Table 2 Pollen quantity of Tortoiseshell Cactus

[0070]

[0071] As can be seen from Table 2, the number of pollen picked up by the thorn tip of the tortoise shell cactus each time is between 431 and 880, which is sufficient for pollination and will not be wasted. The thorns of the tortoise shell cactus are chosen as pollination tools mainly because the thorn length can reach more than 7 cm, which is easy to hold and operate, the tip diameter of 100 μm is thin enough, the wood is not easy to bend, and the hardness is suitable. The thorn tip of the tortoise shell cactus picks up 431 to 880 grains of pollen each time, which fully meets the pollination needs. Compared with several other common toothpicks, tweezers, and pollination sticks, it has its own advantages.

[0072] 3. Welwitschia pollination techniques

[0073] In order to improve the efficiency of pollination, we choose the method of artificial pollination. We use the tip of the tortoise shell cactus to dip the pollen and then directly pollinate the pollen in the pollination drop to avoid waste. The pollen germinates in the pollination drop and enters the ovule ( Figure 13 ), this point pollination method saves pollen and improves pollen utilization.

[0074] Ultimately, we collected pollen during the optimal pollination period for male flowers and pollinated during the optimal pollination period for female flowers, ultimately harvesting 504 seeds. In 2023, we harvested 987 seeds. Continuous pollination increases the probability of Welwitschia seed production. If experimental conditions permit, increasing the number of pollinations can significantly increase the pollination success rate.

[0075] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A Welwitschia pollination method, characterized in that: The method comprises the following steps: (1) During the flowering period, cover the male and female flowers of the Welwitschia plant with a net to prevent flies, ants, and hoverflies from eating them; (2) Pollen collection: Collect pollen from Centella asiatica between 8:30 and 11:30 in the morning and place the collected pollen in a 1.5 ml centrifuge tube; (3) Pollination was carried out during the period of 10:30-16:30 when the pollination drops of the female cones of Welwitschia orchid appeared. The thorns of Tortoise Shell Cactus were used as pollination tools. The pollen was dipped into the tip of the thorns of Tortoise Shell Cactus and then directly pollinated into the pollination drops of the female cones.

2. The Welwitschia pollination method according to claim 1, characterized in that: Open the lid of a sterile centrifuge tube and place it vertically just below the target pollen, 5-10mm away from the pollen, to ensure that the pollen falls freely into the tube; or take a clean piece of weighing paper and let the pollen fall on it. After collecting all the pollen, fold the weighing paper longitudinally along the center line to form a V-shaped groove. Under the action of gravity, the pollen particles slide along the V-shaped groove to the bottom of the crease. Then align the crease end of the weighing paper with the mouth of the sterile centrifuge tube and tap the back of the paper with your finger to allow the pollen to flow into the centrifuge tube.

3. The Welwitschia pollination method according to claim 1, characterized in that: When collecting pollen, use sterile tweezers, toothpicks or cactus thorns to gently pick the pollen from the Welwitschia orchid.

4. The Welwitschia pollination method according to claim 1, characterized in that: If the flowering period of female and male flowers does not coincide, the collected Welwitschia pollen should be refrigerated first.

5. The Welwitschia pollination method according to claim 4, characterized in that: The refrigerator temperature is -80°C to 4°C.

6. The Welwitschia pollination method according to claim 1, characterized in that: The pollen collection time of step (1) is 11:30; the pollination time period of step (3) is 11:30-15:00.

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