Rhododendron sexual polyploidy seedling production method

Abstract

The invention relates to the field of plant planting, in particular to a rhododendron sexual polyploidy seedling production method which comprises parent selection and is characterized by further comprising parent planting: planting containers: the parent planting containers are suggested to be planted in degradable non-woven fabric bags; planting soil: planting parent plants in the soil, wherein peat, perlite and vermiculite are proportioned according to a ratio of 3: 1: 1, and carbendazim is used for sterilization and disinfection before use.

Description

Technical Field

[0001] The invention relates to the field of plant planting, and in particular to a method for producing rhododendron seedlings. Background Art

[0002] Currently, the rhododendrons that can be used in gardening have problems such as few varieties, single flower colors, and poor environmental adaptability. Summary of the invention

[0003] The purpose of the present invention is to provide a method for producing sexual polyploid rhododendron seedlings with low energy consumption, so as to increase the variety and color of rhododendrons and improve the performance of rhododendrons through polyploid seedlings.

[0004] The technical problem solved by the present invention can be achieved by adopting the following technical solutions:

[0005] A method for producing sexual polyploid rhododendron seedlings, comprising parent selection, characterized in that it also comprises parent planting:

[0006] Planting container: It is recommended that the parent planting container be planted in a biodegradable non-woven bag;

[0007] Planting soil: The parent plant is planted in soil with peat, perlite and vermiculite in a ratio of 3:1:1. It should be sterilized with carbendazim before use.

[0008] Preferably, the gamete induction period is also selected:

[0009] Selection of 2n female gamete induction period: flower buds of different growth stages were collected in the morning of a sunny day, and placed in FAA fixative according to different shapes and sizes. They were filtered with a vacuum filter for about 5 minutes, fixed in the fixative for more than 24 hours, and then transferred to 70% alcohol and stored in a refrigerator at 4°C for later use; conventional paraffin section technology was used to grade the young flower buds of rhododendron for cytological observation, and the inflorescence size when most of the megaspore mother cells of the flower buds were in the early stage of meiosis I was taken as the effective period of mutagen induction;

[0010] Selection of 2n male gamete induction period: Take flower buds of different growth stages in the morning of a sunny day, and fix them in fresh Carnoy's fixative for more than 2 hours according to different shapes and sizes; then wash them with 95% and 80% ethanol in a gradient manner until there is no acetic acid smell, and then immerse them in 70% ethanol and store them in a refrigerator at 4℃ for later use. Take out the flower buds, place them on filter paper from small to large, absorb the excess preservation solution on the flower buds, pick out the anthers and place them on a clean slide, crush them with tweezers to remove excess tissue, add 2 to 3 drops of fuchsin staining solution, cover with a coverslip, and stain for 15 minutes; gently pick out the stained anthers and place them on a slide, add a coverslip, add a filter paper on the coverslip, and gently squeeze to disperse the cells; observe the progress of microspore mother cell meiosis with an optical microscope, and record the external morphological characteristics of inflorescences and stamens at different periods; the inflorescence when most microspore mother cells are in prophase I of meiosis is taken as the induction period of 2n microspores.

[0011] Preferably, the method further comprises colchicine-induced treatment: 0.1% colchicine is mixed with 1% dimethyl sulfoxide, and the flower buds are continuously treated by cotton embedding method, excess leaves around the flower buds are removed, the flower buds are wrapped with cotton, and the wrapped flower buds are dripped with medicine every day. The absorbent cotton is kept moist during the treatment. The treatment is carried out for 4 days. After the treatment, the cotton is peeled off and the flower buds are repeatedly rinsed with clean water to prevent the flower buds from being affected by the residual drug solution.

[0012] Preferably, the 2n gamete ratio estimation is also included: when most of the flower buds are in the meiotic tetrad stage, the treated flower buds are sampled in the morning of a sunny day, and immediately placed in fresh Carnoy's solution for fixation for 24 hours. The flower buds are taken out and washed with 95%, 80%, and 70% ethanol solutions respectively until there is no acetic acid smell, and then transferred to 70% ethanol and stored in a 4°C refrigerator for later use. The flower buds are placed in a 60°C 1mol / L Soften in HCl for 8-10 minutes, then rinse with distilled water, remove the anther and place it on a clean slide, crush it with tweezers to remove excess tissue, add 2-3 drops of fuchsin staining solution, stain for 15 minutes, cover with a coverslip, add a filter paper on the coverslip, gently squeeze to disperse the cells, observe under a microscope, select 700-1000 sporophytes in the meiotic tetrad stage, and estimate the 2n gamete ratio = 2×number of dyads+number of tripartites / 2×number of dyads+3×number of tripartites+4×number of tetrads.

[0013] Preferably, it also includes artificial hybridization pollination:

[0014] Before the female parent blooms, it is emasculated and bagged. When its stigma secretes mucus, the male parent's pollen is gently smeared on the surface of the stigma until pollen grains are visible on the stigma to the naked eye. Then it is re-bagged for isolation and marked with a tag.

[0015] Preferably, the method further comprises ovule culture: based on the pollination day of the female parent, capsules 2, 3, and 4 months after pollination are selected, tissue culture is performed on the immature seeds, the villi on the surface of the harvested ovary are removed, the ovary is placed in a sterile bottle containing 70% ethanol and shaken for 30 seconds, and then sterilized in a 10% sodium hypochlorite solution containing 0.01% Tween 20 for 15 minutes, the ovary is washed 3 times with sterile distilled water, and then the ovary is moved into another sterile bottle and soaked in an appropriate amount of sterile water, after rinsing with sterile water, the ovary is cut into 5 parts longitudinally on sterile paper, the seeds are peeled from the ovary, and then inoculated into the prepared culture medium, sealed, and cultured at 25°C, 12h light conditions, using Anderson medium as the basal medium, adding 30g / L sucrose, 50mg / L GA3, pH=5.0, and after inoculation and germination, it is transferred to the rooting medium after 120d of culture, and the rooting medium adopts Anderson medium without any hormones.

[0016] Preferably, the method further comprises seed collection: fully mature seeds are collected around October and placed in a sealed glass container filled with color-changing silica gel for later use.

[0017] Preferably, it also includes seedling management: the transplanted seedlings are placed in an incubator and the culture conditions are set to two stages: the first stage is a temperature of 28°C, a light of 6000LX, and a time of 16h; the second stage is a temperature of 18°C, a light of 0LX, and a time of 8h. The soil should always be kept moist during the culture period. After a period of time, 1g of Huaduoduo is diluted with 2L of water and sprayed on the leaves of the hybrid seedlings or the root soil. Spray once every 20 days or so to promote the growth of the hybrid seedlings.

[0018] Preferably, flow cytometry detection is also included: when the seedlings grow 7-8 true leaves, the ploidy of the seedlings is determined by flow cytometry, and about 1 cm 2 The young leaves were placed in a 60 mm nucleic acid culture dish containing 400 μL nucleic acid extract, quickly chopped with a double-sided blade, and then 1600 μL of staining solution was added, filtered through a 50 μm nylon mesh, and the filtrate was placed in a centrifuge, centrifuged for 5 minutes, the supernatant was discarded, 1 ml of sheath fluid was added, mixed, centrifuged for 5 minutes, the supernatant was discarded, 5 ml of DAPI staining solution was added and mixed, and after dark staining for 5 minutes, the samples were loaded for analysis. At least 2000 cell nuclei were detected in each analysis, and 3 samples were collected from each plant body for analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0020] Figure 1 is a schematic diagram of the present invention;

[0021] Figure 2 It is a partial structural diagram of a non-woven bag.

[0022] Explanation of symbols:

[0023] 1. Greenhouse; 2. Sandwich wall; 3. Water source heat pump system; 4. Air heat exchanger; 5. Water pump; 6. Backfill well; 7. Underground aquifer; 8. Composting chamber; 9. Pull rope; 10. Water pipe. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings.

[0025] Method for producing sexual polyploid rhododendron seedlings, including parent selection:

[0026] Varieties: The parents should be selected from varieties that have strong potting adaptability, good ornamental value, large number of flowers, long flowering period, and similar or similar flowering periods. Preferably, the flowering period of the parents overlaps by 3-10 days.

[0027] Male parent: Take a small amount of pollen on a glass slide, add 1-2 drops of 1% acetic acid magenta dye solution, dye for 1 minute, and observe under a fluorescence microscope. The dark red pollen grains are active, the light red parts are inactive, and the inactive or sterile pollen is colorless. The dark red pollen grains that account for more than 95% of the field of view are selected as the male parent.

[0028] Female parent: collect flowers in the flowering period, cut off the stigma, place them in a benzidine-hydrogen peroxide reaction solution (1% benzidine: 3% hydrogen peroxide: water = 4: 11: 22), observe the bubbles generated by the stigma, judge the relative strength of the stigma receptivity according to the speed and amount of bubble generation, and select the one that generates more bubbles and lasts for a longer period of time as the female parent. Preferably, take the average value of bubble generation as a reference value, and select the one that generates more bubbles than the reference value and lasts for more than 2 days as the female parent.

[0029] The method for producing sexual polyploid rhododendron seedlings also includes planting the parents:

[0030] Planting container: It is recommended that the parent planting container be planted in a biodegradable non-woven bag, and the specifications are determined by the size of the parent plants. The volume of the non-woven bag is preferably equal to 2 to 3 times the total volume of the plant roots.

[0031] Planting soil: The parent plant is planted in an acidic or acidic soil (PH value 5.0-6.5) matrix. It is recommended to use peat, perlite and vermiculite in a ratio of 3:1:1. Before use, use carbendazim to sterilize and disinfect according to the instructions.

[0032] Preferably, the non-woven bag preferably has two layers, including an inner non-woven bag located in the inner layer and an outer non-woven bag located in the outer layer. At least three drawstrings 9 are provided between the inner non-woven bag and the outer non-woven bag, and one of the at least three drawstrings is located at the bottom of the non-woven bag, one is located at the mouth of the non-woven bag, and one is located in the middle of the non-woven bag. Thus, the volume of the non-woven bag can be changed by using the drawstring 9 to adapt to plants of different sizes. The density of the inner non-woven bag is less than that of the outer non-woven bag. A water pipe 10 is also provided between the inner non-woven bag and the outer non-woven bag. The water pipe 10 has a through hole facing the inner non-woven bag, and a water distribution pipe is connected to the through hole, and the water distribution pipe is inserted into the inner non-woven bag. Thus, the water pipe and the water distribution pipe can be used to introduce water into the non-woven fabric. The planting soil is filled in the inner non-woven bag. The inner non-woven bag and the outer non-woven bag are filled with foam particles. The foam particles are used to shape, permeable and breathable. The water pipe is located between two adjacent drawstrings. The water pipe is arranged in a corrugated shape. When the drawstring changes the shape of the non-woven bag, the water pipe can follow the deformation. Preferably, the drawstring and the water pipe surround the non-woven bag in the horizontal direction, and the water pipe is located below the non-woven bag.

[0033] The method for producing sexual polyploid rhododendron seedlings also includes gamete induction:

[0034] Selection of the induction period of 2n female gametes: flower buds of different growth stages were collected in the morning of a sunny day, and placed in FAA fixative (70% alcohol: glacial acetic acid: formalin = 90:5:5) according to different shapes and sizes. They were filtered with a vacuum filter for about 5 minutes, fixed in the fixative for more than 24 hours, and then transferred to 70% alcohol and stored in a refrigerator at 4°C for later use. Conventional paraffin section technology was used to grade the young flower buds of azalea for cytological observation, and the size of the inflorescence when most of the megaspore mother cells of the flower buds were in the early stage of meiosis I was used as the effective period of mutagen induction. 2n female gametes refer to 2n gametes produced without reduction during the meiosis of megaspore mother cells.

[0035] Selection of 2n male gamete induction period: Take flower buds of different growth stages in the morning of a sunny day, and fix them in fresh Carnoy's fixative (95% ethanol: glacial acetic acid = 3:1) for more than 2 hours according to different shapes and sizes. Then wash them with 95% and 80% ethanol in a gradient until there is no acetic acid smell, then immerse them in 70% ethanol and store them in a refrigerator at 4°C for later use. Take out the flower buds, place them on filter paper from small to large, absorb the excess preservation solution on the flower buds, pick out the anthers and place them on a clean slide, crush them with tweezers to remove excess tissue, add 2 to 3 drops of fuchsin staining solution, cover with a coverslip, and stain for 15 minutes. Gently pick out the stained anthers and place them on a slide, add a coverslip, add a filter paper on the coverslip, and gently squeeze to disperse the cells. Observe the progress of meiosis of its microspore mother cells with an optical microscope, and record the external morphological characteristics of inflorescences and stamens at different stages. The inflorescence when most microspore mother cells are in prophase I of meiosis is taken as the induction period of 2n microspores. 2n male gametes refer to the 2n gametes produced without reduction during meiosis of microspore mother cells.

[0036] Colchicine induced treatment: 0.1% colchicine was mixed with 1% dimethyl sulfoxide, which is a sulfur-containing organic compound that can enhance the penetration capacity in plant tissues. The flower buds were continuously treated by cotton embedding method, and the excess leaves around the flower buds were removed and wrapped with cotton. The wrapped flower buds were dripped with medicine every day, and the absorbent cotton was kept moist during the treatment. After 4 days of treatment, the cotton was peeled off and repeatedly rinsed with clean water to prevent the flower buds from being affected by the residual liquid. Colchicine is an alkaloid, which is highly toxic and can induce gene mutations. Colchicine can inhibit the formation of spindles during cell division, so that the movement of chromosomes to the two poles is blocked and stays in the metaphase, so that the cells cannot continue to divide, thereby producing nuclei with doubled chromosome numbers.

[0037] Estimation of 2n gamete ratio: When most flower buds are in the meiotic tetrad stage, sample the treated flower buds on a sunny morning and immediately place them in fresh Carnoy's solution (ethanol: acetic acid = 3:1) for 24 hours. Take out the flower buds and wash them with 95%, 80%, and 70% ethanol solutions in a gradient manner until there is no acetic acid smell, then transfer them to 70% ethanol and store them in a 4℃ refrigerator for later use. Take the flower buds and place them in 60℃ 1mol / L HCl to soften for 8-10min, then rinse them with distilled water, take out the anthers and place them on a clean slide, crush them with tweezers to remove excess tissue, add 2-3 drops of fuchsin staining solution, stain for 15min, cover with a coverslip, add a filter paper on the coverslip, and gently squeeze to disperse the cells. Observe under a microscope, select 700-1000 sporophytes in the meiotic tetrad stage, and estimate the 2n gamete ratio (2n gamete ratio = 2×number of dyads + number of triplets / 2×number of dyads + 3×number of triplets + 4×number of tetrads). This process can be used to preliminarily estimate the number of polyploid seedlings produced.

[0038] The method for producing sexual polyploid rhododendron seedlings also includes artificial hybridization and pollination:

[0039] Selection of flowers for pollination: When azalea flowers bloom, observe whether their external morphology has changed, and select flower buds whose external morphology has changed significantly compared with the control as the objects of artificial hybridization pollination.

[0040] Artificial hybridization: The female parent is emasculated and bagged before flowering. When the stigma secretes mucus, the male parent's pollen is gently applied to the surface of the stigma until pollen grains are visible on the stigma to the naked eye. Then the plant is re-bagged for isolation and marked with a label.

[0041] Pollen in situ germination observation: Take flowers 2h, 4h, 6h, 8h, 12h, 16h, 24h, 36h, 48h, and 72h after pollination, remove the sepals, fix the style and ovary in Carnoy's fixative (ethanol: glacial acetic acid = 3:1) for 24h, rinse with 95% and 80% ethanol in a gradient, place in 70% ethanol, and store in a refrigerator at 4℃ for later use. Soften with 2mol / LNaOH for 1h, rinse with distilled water, and then stain with 1% water-soluble aniline blue for 2h. Use a dissecting needle to gently dissect the style and ovary, place on a slide, add an appropriate amount of distilled water, cover the slide and gently flatten, observe with a fluorescence microscope, and observe and count the pollen germination rate. This step is used to observe whether the two selected varieties are hybrid compatible.

[0042] The method for producing sexual polyploid rhododendron seedlings also includes seedling cultivation:

[0043] Ovule culture: Calculated by the pollination date of the mother parent, capsules 2, 3, and 4 months after pollination were selected, and tissue culture was performed on the immature seeds. Remove the villi on the surface of the harvested ovary, place the ovary in a sterile bottle containing 70% ethanol, shake for 30 seconds, and then place it in a 10% sodium hypochlorite solution containing 0.01% Tween 20 for sterilization for 15 minutes. After washing with sterile distilled water three times, move the ovary into another sterile bottle and pour an appropriate amount of sterile water to soak. After rinsing with sterile water, cut the ovary into 5 parts longitudinally on sterile paper. Peel the seeds from the ovary, then inoculate them into the prepared culture medium, seal them, and culture them at 25°C and 12h light conditions. Anderson medium was used as the basic culture medium, 30g / L sucrose, 50mg / L GA3, pH=5.0, and inoculated after germination. After 120d of culture, transfer to the rooting medium, which uses Anderson medium without any hormones.

[0044] Seed collection: Harvest fully mature seeds around October and place them in a sealed glass container filled with color-changing silica gel (silica gel oxide) for later use.

[0045] Sowing: After harvesting mature seeds, rinse them with clean water and soak them in water for 12 hours before sowing. Sprinkle the seeds obtained after hybridization evenly on the surface of the charcoal soil, add water to the tray so that the seedling tray is completely immersed in water, and place them in the incubator for germination. Set the incubator temperature to 28°C, spray water on the seed surface every day, and keep water in the tray so that the plug tray is always immersed in water until the seeds germinate. Transplant when the seedlings grow 2-3 true leaves, at this time the transplant survival rate is the highest.

[0046] Seedling stage substrate: The seedling transplanting substrate should be evenly mixed in the ratio of peat: perlite: vermiculite = 3:1:1.

[0047] Seedling management: Place the transplanted seedlings in an incubator. Set the culture conditions to two stages: the first stage is 28°C, 6000LX of light, and 16h; the second stage is 18°C, 0LX of light, and 8h. The soil should always be kept moist during the culture period. After a period of time, add 1g of Huaduoduo to 2L of water and spray it on the leaves of the hybrid seedlings or the soil for root irrigation. Spray it once every 20 days or so to promote the growth of the hybrid seedlings.

[0048] The method for producing sexual polyploid seedlings of azalea also includes ploidy detection: flow cytometry detection: when the seedlings grow 7-8 true leaves, the ploidy of the seedlings is determined by flow cytometry. The leaf samples of diploid varieties are used as controls. About 1 cm2 of young leaves are placed in a 60 mm nucleic acid culture dish containing 400 μL of nucleic acid extract, quickly chopped with a double-sided blade, and then 1600 μL of staining solution is added and filtered through a 50 μm nylon mesh. The filtrate is placed in a centrifuge, centrifuged for 5 minutes (room temperature, 1500 rpm), the supernatant is poured out, 1 ml of sheath liquid is added, mixed, centrifuged for 5 minutes (room temperature, 1500 rpm), the supernatant is poured out, 5 ml of DAPI staining solution is added and mixed, and after dark staining for 5 minutes, the sample is loaded for analysis. At least 2000 nuclei are detected in each analysis, and 3 samples are collected from each plant body for analysis.

[0049] Chromosome detection: polyploid seedlings detected by flow cytometry were selected, and young leaves were selected as test materials between 8:30 and 10:00 in the morning. They were pretreated with a mixture of 0.1% colchicine and 0.002 mol / L 8-hydroxyquinoline at room temperature for 3 hours, fixed with a fixative (anhydrous alcohol: chloroform: glacial acetic acid = 6:3:1), dissociated with 1 mol / L hydrochloric acid at 60°C for 8 minutes, and then stained with modified phenol fuchsin to press the slides. The chromosome number was observed under a microscope, and the polyploid cells accounting for more than 85% of the field of view were regarded as polyploid homozygous seedlings.

[0050] Gradual management: Polyploid seedlings that have passed ploidy identification are picked out and managed separately. As the seedlings grow, morphological observations are carried out at regular intervals, including leaf thickness, leaf length, leaf width, leaf area, distribution of leaf surface hairs, petiole length and other morphological indicators and ornamental traits. This work is repeated until sexual polyploid rhododendron germplasm with high ornamental value and strong environmental adaptability is bred.

[0051] Both parent planting and seedling cultivation are preferably carried out in a low-energy-consuming azalea seedling greenhouse. Figure 1 As shown, the low-energy consumption azalea seedling greenhouse includes a greenhouse 1 and a temperature control system;

[0052] The temperature control system includes a water source heat pump system 3, a water source pipeline system, and a refrigerant pipeline system for circulating a refrigerant. The water source heat pump system 3 has two heat exchangers: one heat exchanger is a heat exchanger for exchanging heat with groundwater, called a water source heat exchanger, and the water source heat exchanger includes a water source inlet and a water source outlet; the other heat exchanger is a heat exchanger for exchanging heat with a refrigerant, called a refrigerant heat exchanger, and the refrigerant heat exchanger includes a refrigerant inlet and a refrigerant outlet.

[0053] The water source pipeline system includes a water pump 5 and a backfill well 6. The water inlet of the water pump 5 is connected to a pipeline reaching the underground aquifer 7. The water outlet of the water pump 5 is connected to the water source inlet of the water source heat exchanger. The water source outlet of the water source heat exchanger is connected to the backfill well 6.

[0054] The refrigerant piping system includes a fluid pump and an air heat exchanger 4. The air heat exchanger 4 is arranged in the greenhouse 1. The refrigerant outlet of the refrigerant heat exchanger is connected to the pump inlet of the fluid pump. The pump outlet of the fluid pump is connected to the pipe inlet of the air heat exchanger 4. The pipe outlet of the air heat exchanger 4 is connected to the refrigerant inlet of the refrigerant heat exchanger. At least two air heat exchangers 4 are provided. The refrigerant is one of water and air.

[0055] The low-energy azalea seedling greenhouse also includes a ventilation system, which includes an air duct arranged along the edge of the greenhouse 1, an air outlet facing the greenhouse 1, and fans are respectively arranged at the ends of the air ducts. The air outlet of the fan faces the inside of the air duct, and an air heat exchanger 4 is arranged in front of the air outlet of the fan; at least two air ducts are arranged symmetrically. The water source heat pump system 3 is used to exchange heat with groundwater in the underground aquifer 7, and heat is extracted from the groundwater in winter and released to the groundwater in summer. Then, the exchanged heat is exchanged with the coolant again, so that the temperature of the coolant is suitable for the temperature of the greenhouse 1. The coolant exchanges heat with the air in the greenhouse 1 again through the air heat exchanger 4, and further transfers heat to the greenhouse 1, so as to control the temperature of the seedling greenhouse 1 at 25-28°C. Its beneficial effect is that it reduces the energy consumption of maintaining the temperature of the greenhouse 1 and saves the operating cost of the greenhouse 1.

[0056] The air exchange system uses duct technology to push the air after heat exchange around the air heat exchanger 4 to a place far away from the air heat exchanger 4. There are two symmetrically arranged ducts, and each duct is provided with an air heat exchanger 4. Its beneficial effect is to maintain the uniform temperature in the greenhouse 1 and make the temperature control effect of the heat pump system better.

[0057] The air heat exchanger 4 adopts one of a plate-fin heat exchanger and a tube heat exchanger. The plate-fin heat exchanger and the tube heat exchanger have a larger contact area with the air, so the heat exchange effect is higher. The refrigerant passes through the pipes of the heat exchanger, and the air surrounds the outer periphery of the pipes to achieve heat exchange. The air heat exchanger 4 adopts one of a plate-fin heat exchanger and a tube heat exchanger. The beneficial effect is that the plate-fin heat exchanger and the tube heat exchanger have a simple structure and can reduce the manufacturing cost.

[0058] The low-energy azalea seedling greenhouse also includes a photovoltaic power generation system as a supplementary energy output system. The photovoltaic power generation system has a power transmission interface, and the power supply of the water source heat pump system 3 is connected to the power transmission interface of the photovoltaic power generation system. The photovoltaic power generation system, as a supplementary energy output system, can supply power to the water source heat pump system 3, and its beneficial effect is to reduce the energy consumption cost of maintaining the temperature of the greenhouse 1.

[0059] The low-energy azalea seedling greenhouse also includes a wind turbine system as a supplementary energy output system. The wind turbine system has a power transmission interface, and the water source heat pump system 3 is connected to the power transmission interface of the wind turbine system. The wind turbine system, as a supplementary energy output system, can supply power to the water source heat pump system 3, which has the beneficial effect of reducing the energy consumption cost of maintaining the temperature of the greenhouse 1.

[0060] A pressure spray device is provided in the greenhouse 1, and a sprinkler device is provided on the roof of the greenhouse 1. The low-energy azalea seedling greenhouse also includes a groundwater supply system, which includes a water pump 5; the water outlet of the water pump 5 is connected to the water inlet of the pressure spray device; the water outlet of the water pump 5 is connected to the water inlet of the sprinkler device. In summer, the surface temperature is higher than the temperature required for the seedlings, so it is necessary to cool down. The water supply system extracts groundwater from the underground aquifer 7, and the temperature of the groundwater is lower than the surface temperature. The groundwater is atomized by the pressure spray device in the greenhouse 1. When the groundwater evaporates, a large amount of heat is consumed, which reduces the indoor temperature; the sprinkler device on the roof of the greenhouse 1 sprays the groundwater onto the roof, and the temperature of the roof is reduced by the evaporation of water, which also indirectly reduces the indoor temperature. In this embodiment, the beneficial effect is that the greenhouse 1 is cooled by using groundwater, which saves the consumption of electric energy and reduces the operating cost of the greenhouse 1.

[0061] The wall of the greenhouse 1 is a wall with a sandwich structure; the outer wall of the sandwich structure wall is provided with a composting chamber 8 for composting straw fertilizer, and the composting chamber 8 includes a composting cavity, and the composting cavity and the sandwich layer of the greenhouse 1 are connected by a channel. When the straw fertilizer is composted, biological heat energy is released, and the biological heat energy is guided into the greenhouse 1 through the composting cavity connected to the sandwich layer of the sandwich wall 2 to provide the temperature in the greenhouse 1. Its beneficial effect is to save electric energy and reduce the operating cost of the greenhouse 1.

[0062] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for producing sexual polyploid rhododendron seedlings, comprising parent selection, characterized in that: Also includes parent planting: Planting container: It is recommended that the parent planting container be planted in a biodegradable non-woven bag; Planting soil: The parent plant is planted in soil with peat, perlite and vermiculite in a ratio of 3:1:

1. It should be sterilized with carbendazim before use.

2. The method for producing sexual polyploid rhododendron seedlings according to claim 1, characterized in that: Also includes gamete induction period selection: Selection of 2n female gamete induction period: flower buds of different growth stages were collected in the morning of a sunny day, and placed in FAA fixative according to different shapes and sizes. They were filtered with a vacuum filter for about 5 minutes, fixed in the fixative for more than 24 hours, and then transferred to 70% alcohol and stored in a refrigerator at 4°C for later use; conventional paraffin section technology was used to grade the young flower buds of rhododendron for cytological observation, and the inflorescence size when most of the megaspore mother cells of the flower buds were in the early stage of meiosis I was taken as the effective period of mutagen induction; 2n Selection of male gamete induction period: Take flower buds of different growth stages in the morning of a sunny day, and fix them in fresh Carnoy's fixative for more than 2 hours according to different shapes and sizes; then wash them with 95% and 80% ethanol in a gradient manner until there is no acetic acid smell, and then immerse them in 70% ethanol and store them in a refrigerator at 4°C for later use. Take out the flower buds, place them on filter paper from small to large, absorb the excess preservation solution on the flower buds, pick out the anthers and place them on a clean glass slide, crush them with tweezers to remove excess tissue, add 2 to 3 drops of fuchsin staining solution, cover with a coverslip, and stain for 15 minutes; gently pick out the stained anthers and place them on a glass slide, add a coverslip, add a filter paper on the coverslip, and gently squeeze to disperse the cells; observe the progress of meiosis of its microspore mother cells with an optical microscope, and record the external morphological characteristics of inflorescences and stamens at different stages; The inflorescence when most microspore mother cells were in prophase I of meiosis was regarded as the induction period of 2n microspores.

3. The method for producing sexual polyploid rhododendron seedlings according to claim 2, characterized in that: It also includes colchicine-induced treatment: 0.1% colchicine is mixed with 1% dimethyl sulfoxide, and the flower buds are continuously treated by cotton embedding method. The excess leaves around the flower buds are removed, and the flower buds are wrapped with cotton. The wrapped flower buds are dripped with medicine every day. The absorbent cotton is kept moist during the treatment. The treatment is carried out for 4 days. After the end, the cotton is peeled off and the flower buds are repeatedly rinsed with clean water to prevent the flower buds from being affected by the residual drug solution.

4. The method for producing sexual polyploid rhododendron seedlings according to claim 2, characterized in that: It also includes the estimation of the 2n gamete ratio: when most of the flower buds are in the meiotic tetrad stage, the treated flower buds are sampled on a sunny morning and immediately placed in fresh Carnoy's solution for fixation for 24 hours. The flower buds are taken out and washed with 95%, 80%, and 70% ethanol solutions in a gradient manner until there is no acetic acid smell, then transferred to 70% ethanol and stored in a 4°C refrigerator for later use. The flower buds are placed in a 60°C 1mol / L Soften in HCl for 8-10 minutes, then rinse with distilled water, remove the anther and place it on a clean slide, crush it with tweezers to remove excess tissue, add 2-3 drops of fuchsin staining solution, stain for 15 minutes, cover with a coverslip, add a filter paper on the coverslip, gently squeeze to disperse the cells, observe under a microscope, select 700-1000 sporophytes in the meiotic tetrad stage, and estimate the 2n gamete ratio = 2×number of dyads+number of tripartites / 2×number of dyads+3×number of tripartites+4×number of tetrads.

5. The method for producing sexual polyploid rhododendron seedlings according to claim 2, characterized in that: Also includes artificial cross pollination: Before the female parent blooms, it is emasculated and bagged. When its stigma secretes mucus, the male parent's pollen is gently smeared on the surface of the stigma until pollen grains are visible on the stigma to the naked eye. Then it is re-bagged for isolation and marked with a tag.

6. The method for producing sexual polyploid rhododendron seedlings according to claim 5, characterized in that: The method also includes ovule culture: based on the pollination day of the female parent, capsules 2, 3, and 4 months after pollination are selected, and tissue culture is performed on the immature seeds. The villi on the surface of the harvested ovary are removed, and the ovary is placed in a sterile bottle containing 70% ethanol and shaken for 30 seconds, and then sterilized in a 10% sodium hypochlorite solution containing 0.01% Tween 20 for 15 minutes. After washing with sterile distilled water for 3 times, the ovary is moved to another sterile bottle and soaked in an appropriate amount of sterile water. After rinsing with sterile water, the ovary is cut into 5 parts longitudinally on sterile paper, the seeds are peeled off from the ovary, and then inoculated into the prepared culture medium, sealed, and cultured at 25°C, 12h light conditions, using Anderson medium as the basal medium, adding 30g / L sucrose and 50mg / L GA3, pH=5.0, and after inoculation and germination, it is transferred to the rooting medium after 120d of culture. The rooting medium uses Anderson medium without any hormones.

7. The method for producing sexual polyploid rhododendron seedlings according to claim 1, characterized in that: The method also includes seed collection: fully mature seeds are collected around October and placed in a sealed glass container filled with color-changing silica gel for later use.

8. The method for producing sexual polyploid rhododendron seedlings according to claim 1, characterized in that: It also includes seedling management: place the transplanted seedlings in an incubator and set the culture conditions to two stages: the first stage is a temperature of 28°C, a light of 6000LX, and a time of 16h; the second stage is a temperature of 18°C, a light of 0LX, and a time of 8h. The soil should always be kept moist during the culture period. After a period of time, mix 1g of Huaduoduo with 2L of water and spray it on the leaves of the hybrid seedlings or the soil for root irrigation. Spray it once every 20 days or so to promote the growth of the hybrid seedlings.

9. The method for producing sexual polyploid rhododendron seedlings according to claim 1, characterized in that: It also includes flow cytometry testing: when the seedlings grow 7-8 true leaves, the ploidy of the seedlings is determined by flow cytometry. 2 The young leaves were placed in a 60 mm nucleic acid culture dish containing 400 μL nucleic acid extract, quickly chopped with a double-sided blade, and then 1600 μL of staining solution was added, filtered through a 50 μm nylon mesh, and the filtrate was placed in a centrifuge, centrifuged for 5 minutes, the supernatant was discarded, 1 ml of sheath fluid was added, mixed, centrifuged for 5 minutes, the supernatant was discarded, 5 ml of DAPI staining solution was added and mixed, and after dark staining for 5 minutes, the samples were loaded for analysis. At least 2000 cell nuclei were detected in each analysis, and 3 samples were collected from each plant body for analysis.

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