CN106069748B - The commercial tissue culture of sprout mating system of American red-maple splendidness in October - Google Patents

Abstract

The invention provides a commercial tissue culture seedling breeding method of American red maple "October Brilliant", comprising the following steps: (1) selecting young shoots of individual seedlings, removing leaves and excessively long petioles, disinfecting, and cutting off dead parts; 2) inoculate and cultivate in the starting medium; (3) cut the germinated axillary buds into individual buds longitudinally along the stem section, inoculate them in the solid proliferation medium and cultivate them; (4) divide and transfer the proliferated cluster buds Carry out strong seedling culture in the strong seedling medium; (5) the tissue culture seedling that will pass through the strong seedling culture, excise the bottom stem segment, stem segment is inserted in the rooting medium to induce rooting culture; (6) will carry out the tissue culture of rooting culture The seedlings were domesticated and transplanted in the greenhouse to obtain commercial seedlings. The invention is economically feasible, stable in character, neat in seedlings, and can be applied to large-scale commercial production of woody plant tissue culture methods, which can meet the needs of commercial seedling production.

Description

Propagation Method of Commercial Tissue Culture Seedlings of American Red Maple October Brilliant

technical field

The invention relates to a seedling breeding method for commercial large-scale tissue culture of American red maple "October Brilliant".

Background technique

American red maple "October Glory", Acer rubrum 'October Glory' is a woody plant of the family Aceraceae, Acer genus, and is one of the improved horticultural species of American red maple. It is a large deciduous tree with the fastest growth, strong adaptability, cold resistance, drought resistance and humidity resistance. Acidic to neutral soils enhance fall colors. Strong resistance to harmful gases, especially strong absorption of chlorine. It is widely used in parks, residential areas, and streets for planting. It can be used for landscaping and street trees. It is deeply loved by people. It is one of the ideal and rare tree species introduced in recent years for beautifying and greening urban gardens. It is also the only color-leaved tree species that can be used as a street tree. At present, American red maple "October Brilliant" seedlings can be obtained through cutting propagation and seeding propagation. With the development of landscaping and urban landscape in recent years, its role as a dust-reducing plant in urban landscape has become increasingly prominent. The use of seedlings The amount is getting bigger and bigger.

The earliest introduction of plant tissue culture technology was introduced from abroad by the scientific research institutions of various universities in my country. After nearly 50 years of scientific research, especially in the past 20 years, with the improvement of the quality requirements of seeds and seedlings, especially in the field of ornamental herbal plants It has been vigorously promoted and rapidly commercialized. At present, my country's annual output of tissue culture seedlings (bulbs) exceeds 3 billion plants (grains), but it is mainly concentrated in herbaceous ornamental horticultural plants, potatoes, strawberries and other herbaceous economic crops.

Due to factors such as the longer physiological life of woody plants, more chromosome numbers, high cortex and xylem differentiation, and scientific research and education systems, a large number of research results carried out by many research institutes in my country are separated from the economic feasibility of commercial production. Various indicators such as stability and uniformity of seedlings cannot be applied to large-scale commercial production. The commercial activity of using woody plant tissue culture technology for seedling breeding has only just begun in recent years as the market's demand for commercial seedlings with neat batches and consistent traits has continued to increase.

Contents of the invention

The purpose of the present invention is to provide a tissue culture seedling breeding method of American Red Maple October Brilliant, so as to overcome the defects in the prior art and meet the needs of the development of commercial seedling breeding field.

The method of the present invention comprises the steps of:

(1) Explant selection: select the non-lignified young shoots that germinate naturally in the spring of individual seedlings with a growth period of 3 to 4 years. The young shoots are 10 to 15 cm in length and have 3 to 4 internodes;

Remove leaves and excessively long petioles, and disinfect;

Preferably, the disinfection method comprises the steps of:

Wipe the dust on the surface of the explant with alcohol cotton, and then rinse it in 1:2.5-3.5, preferably 1:3 bleach (commercial bleach:sterile water, volume ratio) for 10-20 minutes, preferably 15 minutes. Shake gently 3 to 5 times, then rinse in sterile water for 2 to 4 times, let stand for 1 to 3 minutes, then use sterile filter paper to blot the surface water, cut off the petiole and the dead parts of the upper and lower cuts of the stem, and keep Morphologically, the upper end is 0.8-1.2 cm, preferably 1 cm, and the lower end is 1.0-2.0 cm, preferably 1.5 cm;

Alternatively, the disinfection method comprises the steps of:

Use hydrogen peroxide or sodium hypochlorite for disinfection, the disinfection conditions are: the concentration of hydrogen peroxide is 5% to 15%, the disinfection time is 10 to 30 minutes, the weight concentration of sodium hypochlorite is 2% to 15%, the disinfection time is 5 to 30 minutes, and then use sterile filter paper blot dry surface water;

(2) Start culture: Inoculate the sterilized explants in the start medium, and transfer them to the culture room, at 20-30°C, preferably 25°C, and cultivate them in dark light for 40-60 hours, preferably 48 hours, and then give Illumination of 1200-1800LUX, preferably 1500LUX, photoperiod of 12-20/8h, preferably 16/8h, start-up culture for 2-4 weeks, preferably 3 weeks, dormant axillary buds germinate, and start-up culture is completed;

The starting medium, using water as a solvent, contains the following components:

PH value 5.50～6.00, preferably 5.80;

This formula can quickly break the dormancy of axillary buds, promote the germination of axillary buds, and effectively avoid the generation of callus.

The WPM mentioned above comes from commercially available prefabricated WPM finished products, which contain various chemical nutrients and trace elements, but do not contain sucrose and agar; 1/2WPM refers to the commercially available WPM finished products weighing 1/2 mass;

The TDZ is derived from commercially available imported subpackaged TDZ (Chinese name "thiadizuron" drug, analytically pure), a potent cytokinin substance;

The GA ₃ is derived from commercially available imported sub-packaged GA ₃ (Chinese name gibberellin), which is a kind of gibberellin substance and a kind of plant growth regulator, and is mainly used to break plant dormancy.

(3) Proliferation culture: the axillary buds that start the culture and germinate are cut into individual buds longitudinally along the stem section, inoculated in a solid proliferation medium, 20-30 ° C, preferably 25 ° C, dark culture for 40-60 hours, preferably 48 hours, Then give light, 1200～1800LUX, preferably 1500LUX, photoperiod 12～20/8h, preferably 16/8h, proliferate and cultivate for 3～5 weeks, preferably 4 weeks, and the multiplication rate reaches 3 times, and obtain the lump bud that proliferates, preferably , divide the multiplied buds into individual buds every 4 weeks, and re-inoculate them in the multiplication medium, and expand the multiplication;

The described solid proliferation medium uses water as a solvent and contains the following components:

PH value is 5.50～6.00, preferably 5.80;

The IAA is derived from commercially available imported sub-packaged IAA medicines, the Chinese name is indole acetic acid, and it is analytically pure;

The KT is derived from commercially available imported sub-packaged KT medicine, the Chinese name is kinetin, and it is analytically pure;

This formula can ensure that the proliferation of axillary buds is artificially controllable, the multiplication rate is 3-4 times, and the buds are strong, and the vitrification rate is lower than 0.1%;

(4) Cultivation of strong seedlings: the sprouts proliferated in the proliferation medium are divided and transferred to the medium for strong seedlings, illuminated at 2000-3000LUX, preferably 2500LUX, with a photoperiod of 12-20/8h, preferably 16/8h, 20-30°C, preferably 25°C, culture for 5-7 weeks, preferably 6 weeks, a single bud grows robustly, and the growth height reaches 3cm, and the tissue cultured seedlings cultivated by strong seedlings are obtained;

Described medium for strong seedlings, using water as a solvent, contains the following components:

WPM 1～2g/L, preferably 1.35g/L

Sucrose 20～30g/L

Agar 5.0～7.0g/L

PH value is 5.50～6.00, preferably 5.80;

(5) Rooting culture: With the tissue cultured seedlings cultivated by strong seedlings, cut off the stem section at the bottom of the stem section 3-5mm, insert the stem section into the rooting medium 8-10mm, insert vertically, light 2000-3000LUX, preferably 2500LUX, light The period is 12-20/8h, preferably 16/8h, 20-30°C, preferably 25°C, cultured for 2-5 weeks, preferably 3 weeks, each plant has 2-5 effective and strong roots, and the root length is 20-40mm. Lateral root, obtain rooting culture tissue culture seedling;

Described rooting culture medium, with water as solvent, comprises the component of following proportioning:

PH value is 5.50～6.00, preferably 5.80;

The IBA is derived from commercially available imported subpackaged IBA medicines, the Chinese name is indole butyric acid, and it is analytically pure;

AC is derived from commercially available imported subpackaged AC medicines, the Chinese name is activated carbon, ultrafine powder, bamboo charcoal;

The formula can promote the connection between the root of the plant and the structure of the plant body, avoid the generation of pseudoroots from the callus, and the grown roots are strong and have no lateral roots, which can reduce damage and survive quickly during transplanting.

(6) Acclimatization: the tissue cultured seedlings that have been rooted and cultivated for 3 weeks are transferred to the greenhouse, covered with 85% shading rate sunshade net, covered with two layers, kept for 5 to 10 days, preferably 7 days, and then covered with 1 layer of 85% shading rate 5-10 days with the sunshade net, preferably 7 days, can make the tissue-cultured seedlings of American red maple "October Brilliant" adapt to the variable temperature and natural light environment, and the seedlings can be obtained;

Preferably, also include step (7) transplanting:

2-4 days before transplanting, preferably 3 days, ventilate and keep the temperature in the greenhouse at 13-35°C, preferably 18--26°C;

Transplant the domesticated tissue cultured seedlings in step (6) into 72-hole tree trays, and the transplanting medium is configured as perlite:vermiculite with a volume ratio of 1:1;

Immediately after the transplanting is completed, 550-650 mesh, preferably 600-mesh nozzle, pour water thoroughly, cover plastic film and 95% sunshade net for 2 to 4 days, preferably 3 days, remove the plastic film, cover a single layer of 90% sunshade net and cultivate for 3 days ~ 7 days, preferably 5 days;

Then remove the sunshade net, and use 550-650 mesh, preferably 600-mesh nozzles to replenish fertilizer water for foliar fertilization. Use 20-20-20 quick-acting fertilizer for foliar fertilizer, EC value is 0.6ms/cm, spray once a day at 10 am on sunny days, and maintain normally after half a month.

The quick-acting fertilizer described in 20-20-20 quick-acting fertilizer is the US Peters brand 20-20-20 water-soluble balanced fertilizer

The beneficial effect of the present invention is: it is an economically feasible, stable character, neat seedling, and can be applied to large-scale commercial production woody plant tissue culture method, which can meet the demand of commercial seedling production.

Detailed ways

Example 1

(1) Explant is selected: select the unlignified young shoot that the growth period of greenhouse is 4 years old seedling individual spring natural germination, and the young shoot draws material length and is 15cm, tool 4 internodes; Remove blade and excessively long petiole, sterilize; The disinfection method is as follows:

Wipe the dust on the surface of the explants with alcohol cotton, then rinse in 1:3 bleach (commercial bleach: sterile water, volume ratio) for 15 minutes, shake gently 4 times during the period, and then rinse in sterile water 3 times, let it stand for 2 minutes, then blot the surface water with sterile filter paper, cut off the petiole and the dead parts of the upper and lower incisions of the stem section, and keep the top 1cm and bottom 1.5cm of morphology;

(2) Start-up culture: Inoculate the sterilized explants in the start-up medium, and transfer them to the culture room, culture in dark light at 25°C for 48 hours, then light 1500LUX with a photoperiod of 16/8h for start-up culture After 3 weeks, the dormant axillary buds germinated, and the start-up culture was completed.

The described priming medium, with water solvent, comprises the following components:

pH 5.80.

(3) Proliferation culture: Cut the axillary buds germinated from the start-up culture into individual buds longitudinally along the stem section, inoculate them in solid proliferation medium, culture in the dark at 25°C for 48 hours, then give light 1500LUX, photoperiod 16/8h, proliferate Cultivate for 4 weeks to obtain proliferated buds, or divide the proliferated buds into individual buds every 4 weeks, and re-inoculate them in the proliferation medium to expand and proliferate.

Described solid proliferation medium, with water solvent, comprises the component of following ratio:

The pH value is 5.80.

(4) Cultivation of strong seedlings: transfer the group buds proliferated in the proliferation medium to the medium for strong seedlings after division, light 2500LUX, photoperiod 16/8h, 25°C, and cultivate for 6 weeks. A single bud grows robustly. And the growth height reaches 3cm, and the tissue culture seedlings cultivated by strong seedlings are obtained.

The medium for strong seedlings, using water as a solvent, comprises the following components:

WPM 1.35g/L

Sucrose 30g/L

Agar 6.5g/L

The pH value is 5.80.

(5) Rooting culture: With the tissue cultured seedlings cultivated through strong seedlings, cut off the stem section at the bottom 3mm of the stem section, insert the stem section into the rooting medium 8--10mm, insert vertically, light 2500LUX, photoperiod 16/8h, 25°C, cultured for 6 weeks, each plant has 2 effective and robust roots, root length 10-30mm, no lateral roots, and rooted culture tissue culture seedlings are obtained.

Described rooting culture medium, with water as solvent, comprises the component of following proportioning:

The pH value is 5.80.

(6) domestication: the tissue culture seedling that will carry out rooting culture 6 weeks is transferred in the greenhouse, covers 85% shading rate sunshade net, covers two layers, keeps 7 days, then covers 1 deck 85% shading rate sunshade nets 7 days, namely It can promote the tissue culture seedlings of American red maple "October Brilliant" to adapt to variable temperature and natural light environment.

(7) Transplanting:

3 days before transplanting, ventilate and keep the temperature in the greenhouse at 18--26°C;

Transplant the domesticated tissue cultured seedlings in step (6) into 72-hole tree trays, and the transplanting medium is configured as perlite:vermiculite with a volume ratio of 1:1;

Immediately after the transplanting is completed, pour water with a 600-mesh nozzle, cover the plastic film and 95% sunshade net for 3 days, remove the plastic film, and cover the single layer of 90% sunshade net for another 4 days;

Then remove the sunshade net, 600 mesh sprinklers replenish fertilizer water for foliar fertilization. Use 20:20:20 quick-acting fertilizer for foliar fertilizer, EC value is 0.6ms/cm, spray once a day at 10 am on sunny days, and maintain normally after half a month.

Example 2

(1) explant is selected: the selection growth period is the non-lignified young shoot that germinates naturally in the spring of 3-year seedling individual, and the young shoot draws material length and is 10cm, tool 3 internodes;

Remove the leaves and excessively long petioles, and disinfect; the disinfection method is as follows:

Disinfect with hydrogen peroxide, the disinfection conditions are: the concentration of hydrogen peroxide is 10%, the disinfection time is 20 minutes, and then dry the surface water with sterile filter paper;

(2) Start-up culture: Inoculate the sterilized explants in the start-up medium, and transfer them to the culture room, culture in dark light at 25°C for 48 hours, then light 1500LUX with a photoperiod of 16/8h for start-up culture After 3 weeks, the dormant axillary buds germinated, and the start-up culture was completed.

The starting medium, using water as a solvent, includes the following components:

pH 5.80.

(3) Proliferation culture: Cut the axillary buds germinated from the start-up culture into individual buds longitudinally along the stem section, inoculate them in solid proliferation medium, culture in the dark at 25°C for 48 hours, then give light 1500LUX, photoperiod 16/8h, proliferate Cultivate for 4 weeks to obtain proliferated buds, or divide the proliferated buds into individual buds every 4 weeks, and re-inoculate them in the proliferation medium to expand and proliferate.

The solid proliferation medium, using water as a solvent, includes the following components:

The pH value is 5.80.

(4) Cultivation of strong seedlings: transfer the group buds proliferated in the proliferation medium to the medium for strong seedlings after division, light 2500LUX, photoperiod 16/8h, 25°C, and cultivate for 6 weeks. A single bud grows robustly. And the growth height reaches 3cm, and the tissue culture seedlings cultivated by strong seedlings are obtained.

The medium for strong seedlings, using water as a solvent, comprises the following components:

WPM 1.35g/L

Sucrose 30g/L

Agar 6.5g/L

The pH value is 5.80.

(5) Rooting culture: With the tissue cultured seedlings cultivated through strong seedlings, cut off the stem section at the bottom 3mm of the stem section, insert the stem section into the rooting medium 8--10mm, insert vertically, light 2500LUX, photoperiod 16/8h, 25°C, cultured for 6 weeks, each plant has 2 effective and robust roots, root length 10-30mm, no lateral roots, and rooted culture tissue culture seedlings are obtained.

Described rooting culture medium, with water as solvent, comprises the component of following proportioning:

The pH value is 5.80.

(6) domestication: the tissue culture seedling that will carry out rooting culture 6 weeks is transferred in the greenhouse, covers 85% shading rate sunshade net, covers two layers, keeps 7 days, then covers 1 deck 85% shading rate sunshade nets 7 days, namely It can promote the tissue culture seedlings of American red maple "October Brilliant" to adapt to variable temperature and natural light environment.

(7) Transplanting:

3 days before transplanting, ventilate and keep the temperature in the greenhouse at 18--26°C;

Transplant the domesticated tissue cultured seedlings in step (6) into 72-hole tree trays, and the transplanting medium is configured as perlite:vermiculite with a volume ratio of 1:1;

Immediately after the transplanting is completed, the 600-mesh nozzle is poured with water, covered with plastic film and 95% sunshade net for 3 days, removed the plastic film, and covered with a single layer of 90% sunshade net for another 4 days;

Then remove the sunshade net, 600 mesh sprinklers replenish fertilizer water for foliar fertilization. Use 20-20-20 quick-acting fertilizer for foliar fertilizer, EC value is 0.6ms/cm, spray once a day at 10 am on sunny days, and maintain normally after half a month.

Claims (3)

1. The commercial tissue culture seedling breeding method of American Red Maple "October Brilliant", is characterized in that, comprises the following steps: (1) select growth period to be the unlignified seedling individual tender shoot of spring natural germination of 3～4 seedling individual, tender shoot draws material length and is 10～15cm, tool 3～4 internode; Remove blade and excessively long petiole, After disinfection, cut off the petiole and the dead parts of the upper and lower incisions of the stem section, and keep the morphological upper end of 0.8-1.2 cm and the lower end of 1.0-2.0 cm; (2) Inoculate the sterilized explants in the starting medium and cultivate them; The cultivation method is as follows: culture in dark light at 20-30°C for 40-60 hours, then light 1200-1800LUX, photoperiod 12-20/8h, culture for 2-4 weeks, dormant axillary buds germinate; The starting medium, using water as a solvent, contains the following components: PH value is 5.50~6.00; (3) the axillary buds that start the culture and germinate are cut into individual buds longitudinally along the stem section, inoculated in the solid proliferation medium and cultivated to obtain the blob buds that have multiplied; The cultivation method is as follows: culture in dark at 20-30°C for 40-60 hours, then light, 1200-1800LUX, photoperiod 12-20/8h, culture for 3-5 weeks, and obtain proliferated buds; Divide the multiplied buds into individual buds every 4 weeks, and re-inoculate them in the multiplication medium to proliferate in large quantities; The solid proliferation medium, using water as a solvent, includes the following components: PH value is 5.50~6.00; (4) transfer the group buds proliferated in the proliferation medium to the strong seedling medium after segmentation, and cultivate the tissue cultured seedlings that obtain the strong seedling culture; The cultivation method is as follows: light of 2000-3000LUX, photoperiod of 12-20/8h, culture at 20-30°C for 5-7 weeks, and obtain tissue-cultured seedlings cultivated by strong seedlings; The medium for strong seedlings, using water as a solvent, comprises the following components: 1/2WPM 1～2g/L Sucrose 20～30g/L Agar 5.0～7.0g/L PH value is 5.50~6.00; (5) For the tissue cultured seedlings that have been cultivated through strong seedlings, cut off the stem section at the bottom of the stem section 3-5mm, insert the stem section vertically into the rooting medium for 8-10mm culture, light 2000-3000LUX, and photoperiod 12-20/8h, 20-30°C, cultivate for 2-5 weeks, and obtain rooted cultured tissue culture seedlings; Described rooting culture medium, with water as solvent, comprises the component of following proportioning: PH value is 5.50~6.00; (6) domesticate the tissue cultured seedlings that will be rooted and cultivated in the greenhouse to obtain commercial seedlings; The acclimatization method is as follows, cover with 85% shading rate sunshade net, cover two layers, keep for 5-10 days, then cover one layer of 85% shading rate sunshade net for 5-10 days. 2. The method according to claim 1, characterized in that in step (1), the disinfection method includes the following steps: wipe the dust on the surface of the explant with alcohol cotton, and then place it at 1:2.5-3.5, Rinse in water for 10-20 minutes, then rinse in sterile water for 2-4 times, let stand for 1-3 minutes, and then blot the surface water; Alternatively, the disinfection method comprises the steps of: Use hydrogen peroxide or sodium hypochlorite for disinfection. The disinfection conditions are: the weight concentration of hydrogen peroxide is 5% to 15%, the disinfection time is 10 to 30 minutes, the weight concentration of sodium hypochlorite is 2% to 15%, and the disinfection time is 5 to 30 minutes. water. 3. the method according to claim 1 or 2, is characterized in that, also comprises transplanting step (7): 2-4 days before transplanting, ventilate and keep the temperature in the greenhouse at 13-35°C; Transplanting the domesticated tissue culture seedlings in step (6) in the tree tray, the transplanting medium is configured as perlite: vermiculite is a volume ratio of 1:1; Immediately after the transplanting is completed, 550-650 meshes, water the nozzles thoroughly, cover plastic film and 95% sunshade net for 2-4 days, remove the plastic film, cover a single layer of 90% sunshade net and cultivate for 3-7 days; Then remove the sunshade net, use 550-650 mesh, apply fertilizer water to the nozzle for foliar fertilization, use 20-20-20 quick-acting fertilizer for foliar fertilizer, EC value is 0.6ms/cm, spray once a day at 10 am on sunny days, half Normal maintenance after one month.