CN113475396B - Hybrid orchid high-efficiency seedling method of spring sword and cymbidium - Google Patents
Hybrid orchid high-efficiency seedling method of spring sword and cymbidium Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention discloses a hybrid orchid high-efficiency seedling method of spring Sword and cymbidium, which comprises the following steps: selecting strong new buds from hybrid orchid, and processing to obtain sterilized small lateral buds and sterilized stem tips; inducing and culturing the sterilized stem tip on an original bulb induction culture medium, inoculating sterilized small lateral buds into a growth culture medium for re-greening and growing culture, and then inoculating the small lateral buds onto the original bulb induction culture medium for inducing and culturing; dividing the obtained protocorm, and inoculating to a secondary proliferation/differentiation culture medium for culture; cutting off adventitious buds obtained by culture, inoculating the adventitious buds serving as buds to a rooting and seedling strengthening culture medium for culture, and obtaining transplanting seedlings. The high-efficiency seedling method for tissue culture of the hybrid orchid is suitable for tissue culture and rapid propagation of hybrid orchid varieties formed by taking spring sword and cymbidium as parents, and solves the problem of slow propagation process of traditional parting.
Description
Technical Field
The invention discloses a high-efficiency seedling method for hybrid orchid of cymbidium kanran, which relates to the field of plant tissue culture.
Background
Spring sword (Cymbidium longibracteatum) and Cymbidium (Cymbidium hybridum) both belong to perennial herbs of the genus Cymbidium (Cymbidium) of the orchid family (Orchidaceae). Spring sword is one of the representative varieties of the national blue, mainly produced in southwest regions of China, is as green as spring sword in four seasons, has elegant flower color She Tingzhi, and is clear and pleasant in flower fragrance based on white, yellow, green and red; cymbidium is a kind of orchid variety obtained by hybridization and breeding of some large-flower-type epiphytic orchids in orchid, and has the advantages of large plant type and flower type, gorgeous and colorful flower color, long flower and peppers, large flowering quantity, long flowering period and no flower fragrance. The hybrid orchid obtained by hybrid breeding of the spring sword and the cymbidium faberi has the excellent quality of parents, inherits the elegant gesture and faint aroma of the spring sword, has moderate plant type size, is suitable for household placement, has the characteristics of large and bright color, regular and plump flower type and the like of the cymbidium faberi, and has higher economic and ecological values.
In actual production, the realization of rapid propagation of seedlings is an important aspect of commercial mass production of plants and creation of economic benefits, and the hybrid orchid formed by spring sword and cymbidium can be propagated and propagated in a tissue culture mode, so that the propagation efficiency is greatly improved.
At present, the culture modes of the hybrid orchid comprise:
1. method and Medium for rapid propagation of hybrid orchid Using root (application number: 201310299505.8) inform:
step one, selecting root segments of hybrid orchid, inoculating the root segments on an induction culture medium for dark culture, and inducing and proliferating protocorms; step two, transferring the protocorms on the induction culture medium into light culture, and developing the protocorms into clustered seedlings; and thirdly, cutting the cluster seedlings into single seedlings, transferring the single seedlings to a strong seedling rooting culture medium, and culturing to obtain rooting plants.
The induction culture medium in the first step and the culture medium used for transferring the culture medium into the light culture in the second step are the same induction culture medium, and the induction culture medium is MS+6-BA0.5mg/L+PIC0.6mg/L+200 mg/L of hydrolyzed casein+30000 mg/L of sucrose+5000 mg/L of agar powder.
The rooting culture medium for strong seedlings is MS+AC100 mg/L+500 mg/L of hydrolyzed casein+30000 mg/L of sucrose+5000 mg/L of agar powder.
2. The method for tissue culture and rapid propagation of hybrid orchid (application number 201310339643.4) is reported as follows:
the induction culture medium contains 1/2MS+6-BA (6-benzylaminoadenine) 1.0-3.0 mg/L+NAA (naphthalene acetic acid) 0.05-0.10 mg/L+10.0-20.0% coconut juice+3.0-5.0 g/L active carbon; the culture medium contains 30g/L sugar, 0.7% agar and pH 5.5-5.8.
The cluster bud proliferation culture medium contains 1/2MS+6-BA (6-benzylaminoadenine) 1.0-3.0 mg/L+AD (adenine) 1.0-3.0 mg/L+10.0-20.0% coconut juice+3.0-5.0 g/L active carbon.
The secondary culture medium contains 1/2MS+6-BA (6-benzylaminoadenine) 1.0-3.0 mg/L+AD (adenine) 1.0-3.0 mg/L+10.0-20.0% coconut juice+3.0-5.0 g/L active carbon.
The rooting culture medium contains 1/2MS+NAA (naphthalene acetic acid) 0.5-1.0 mg/L+10.0-20.0% coconut juice+3.0-5.0 g/L active carbon.
3. Method for tissue culture of stem tip of hybrid Langeiger (application number: 2018113331689):
the sterilized stem tip was inoculated into a starter medium: no. 1 American flower, 5000-15000mg/L of potato juice, 5000-15000mg/L of banana juice and 1g/L of active carbon.
In the cluster bud culture stage, leaves and roots of the small buds induced by the stem tips are cut off, and then the small buds are transferred to a culture medium: murashige and Skoog 1962+ naphthalene acetic acid 0.1-5mg/L + 6-benzylaminoadenine 1-5mg/L + kinetin 1-5mg/L + banana juice 5000-15000mg/L.
And (3) strong seedling culture and selection stage: the formula of the seedling strengthening culture medium is that Huabao No. 1 in the United states is mixed with 5000-15000mg/L of potato juice, 5000-15000mg/L of banana juice and 1g/L of active carbon.
At the rooting and seedling stage, inoculating 3cm or more plantlets into a culture medium of American Huabao No. 1, 5000-15000mg/L of potato juice, 5000-15000mg/L of banana juice and 1g/L of activated carbon.
4. A method for obtaining sterile materials of hybrid orchid efficiently by using new lateral buds (2018108302258) comprises inoculating small lateral buds and stem tips which have been removed respectively into a protocorm induction medium: 1/2MS+1.0mg/L NAA+1.0mg/L6-BA+100 ml/L Sucus Cocois+30 g/L sucrose+5 g/L agar+1 g/L activated carbon.
Disclosure of Invention
The technical problem solved by the invention is to provide a tissue culture method for high-efficiency seedling formation of hybrid orchid of spring Sword and cymbidium, and a large number of hybrid orchid tissue culture seedlings can be obtained by adopting the method.
In order to solve the technical problems, the invention provides a hybrid orchid high-efficiency seedling method (tissue culture method) of cymbidium hybridum, which comprises the following steps:
1) Selection (preferred) and sterilization of explants:
selecting a new bud with strong growth (3-6 cm long and no disease spots) from the hybrid orchid, and cleaning, sterilizing and stripping the new bud to obtain a small lateral bud and a stem tip which are used as explants, wherein the small lateral bud and the stem tip are respectively sterilized and cleaned to correspondingly obtain the sterilized small lateral bud and the sterilized stem tip;
2) Protocorm induction:
performing induction culture on the sterilized stem tip on an protocorm induction culture medium until protocorms with diameters more than or equal to 0.5cm grow at the positions of stem tip growing points; the induction culture conditions are as follows: illuminating for 8-10 hours every day, wherein the illumination culture temperature is 25+/-1 ℃, and the illumination intensity is 800+/-100 lux; the dark culture temperature is 20+/-1 ℃;
inoculating the sterilized small lateral buds into a growth medium for re-greening and growing up for 1.5-2 months, then stripping off the stem tip, washing with sterile water, and inoculating onto an original bulb induction medium for induction culture until an original bulb with the diameter of more than or equal to 0.5cm grows up at the position of a stem tip growth point; the induction culture conditions are the same as above;
description: the culture medium is not required to be replaced in the induction culture and the growth and cultivation process of the re-greening;
a large number of propagation is completed through the continuous propagation of the protocorms;
3) Protocorm proliferation and adventitious bud differentiation
The protocorm was divided (into small pieces of about (0.4 to 0.45) mm× (0.4 to 0.45)) and inoculated into a secondary proliferation/differentiation medium for culture (to increase the volume of the protocorm), and the method was divided into the following two cases:
case 1, need subculture:
performing multiplication culture for 50-60 days according to multiplication culture conditions (the volume of protocorm can be increased to about 1.3cm multiplied by 1.3 cm), and performing primary subculture (the culture medium needs to be replaced once for each subculture);
case 2, no subculture was required:
firstly, carrying out proliferation culture for 50-60 days according to proliferation culture conditions, and then directly changing into differentiation culture conditions to carry out differentiation culture until adventitious buds with the length of 3-5 cm are obtained;
the proliferation culture conditions are as follows: illuminating for 8-10 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 500-1000 lux (preferably 800 lux); the culture temperature is 20+/-1 ℃ during dark culture,
the differentiation culture conditions are as follows: illuminating for 10-14 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 1000-1500 lux (preferably 1200 lux); the culture temperature is 20+/-1 ℃ in dark culture;
note that: in the invention, the culture medium is not required to be replaced in the middle of the culture medium, and the culture medium is subjected to subculture once every 50-60 days; and continuously repeating the steps for a plurality of times to obtain a large number of proliferated protocorms, thereby achieving the purpose of seedling proliferation. After the required number of protocorms are obtained, the adventitious buds are continuously differentiated on the culture medium, namely, the adventitious buds are not transferred after the target propagation amount is reached for a plurality of times by continuous subculture, and the adventitious buds are cultivated directly according to the differentiation culture condition; thus, the last subculture, starting from the protocorm pieces until the adventitious buds are obtained, generally lasts for 4 to 6 months, without the need to change the culture medium.
4) Strengthening seedlings and rooting
Cutting off the adventitious bud with the length of 3-5 cm obtained in the step 3), inoculating the adventitious bud as a young bud into a rooting and seedling strengthening culture medium for culture until the number of the obtained roots with the length of more than or equal to 3cm is at least 3, and the seedling height of more than or equal to 8cm; obtaining transplantable seedlings;
the culture conditions are as follows: illuminating for 12-14 hours every day, wherein the light culture temperature is 26+/-1 ℃, and the illumination intensity is 1500-2000 lux; the dark culture temperature was 20.+ -. 1 ℃.
As an improvement of the tissue culture method for high-efficiency seedling formation of hybrid orchid of spring Sword-cymbidium, the invention further comprises the following step 5):
5) And seedling out-bottle transplanting (namely, transplanting after hardening seedlings of the bottle):
firstly, seedling (bottle seedling) is subjected to greenhouse seedling hardening: the illumination intensity in the daytime is 4000-5000 lux, the temperature is 25-30 ℃, and the temperature at night is 15-20 ℃;
after hardening seedlings for 11-13 days, opening a bottle mouth, placing for 2-3 days, taking out and cleaning the seedlings (aiming at cleaning a culture medium at the root of the seedlings), and soaking the seedlings in a carbendazim diluent for 15-20 minutes; air-drying, transplanting into a matrix with the ratio of bark, deer-marsh soil to Xian soil being 1:1:1, and culturing in a conventional manner.
As a further improvement of the tissue culture method for efficient seedling formation of hybrid orchid of spring Sword X cymbidium,
in the step 2):
the protocorm induction medium is: 1/2MS+0.5-1.0 mg/L NAA+0.5-1.0 mg/L6-BA+20-30 g/L sucrose+5 g/L agar+1 g/L active carbon, pH 5.6-5.8,
the growth medium is: MS+0.5-1.0 mg/L IBA+0.5-1.0 mg/L6-BA+50 g/L banana puree+20-30 g/L sucrose+6 g/L agar+0.8 g/L active carbon, and the pH is 5.6-5.8;
in the step 3):
the secondary proliferation/differentiation medium was: hyponex 1 (Huabao No. 1) +0.5 mg/L6-BA+0.2-0.5 mg/L NAA+10% -15% coconut juice+30 g/L sucrose+5-7 g/L agar+2 g/L hydrolyzed casein+0.5-1 g/L activated carbon, pH 5.6-5.8;
in the step 4):
the rooting culture medium for strong seedlings comprises the following components: MS+50-100 g/L banana puree+30 g/L sucrose+2 g/L hydrolyzed casein+7 g/L agar+1 g/L active carbon, and pH is 5.6-5.8.
As a further improvement of the tissue culture method for high-efficiency seedling formation of hybrid orchid of spring Sword and cymbidium, the culture conditions of the step 2) double-green and long-growing culture are as follows:
inoculating sterilized lateral buds into a growth medium for 1 month, and illuminating for 8-10 hours every day, wherein the illumination culture temperature is 25+/-1 ℃, and the illumination intensity is 500-1000 lux (preferably 800 lux); inoculating for 1 month until the set culture time (1.5-2 months) is reached, and illuminating for 10-14 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 2000lux; the dark culture temperature was 20.+ -. 1 ℃.
Further improvement of the tissue culture method for efficient seedling formation of hybrid orchid of spring Sword X cymbidium hybridum of the present invention: the induction culture of step 2) was inoculated with 1 shoot tip (as explant) per 20ml of the protocorm induction medium.
As a further improvement of the tissue culture method for efficient seedling of hybrid orchid of spring Sword X cymbidium, the step 1) is as follows:
selecting new buds with strong growth vigor (3-6 cm long and no disease spots) from hybrid orchid, soaking the new buds in washing powder water with the concentration of 10% -15% (W/V) for cleaning after removing surface impurities and dirt, washing with running water, and then placing the new buds on an ultra-clean workbench for sterilization treatment;
the sterilization treatment is as follows: immersing the new buds into 75% alcohol for 1-1.5 minutes, taking out and cleaning with sterile water; removing 1-2 bracts, immersing in 10% (V/V) sodium hypochlorite for sterilization for 10-15 minutes, and cleaning with sterile water; immersing in mercuric chloride disinfectant for disinfection for 12-18 minutes, and cleaning with sterile water; peeling off the leaves layer by layer, peeling off small lateral buds of each layer and stem tips exposing growth points of leaf bases as explants, soaking for 10-20 seconds by 0.05% (W/V) mercuric chloride, and cleaning with sterile water to obtain sterilized small lateral buds and sterilized stem tips respectively;
the mercuric chloride disinfectant is as follows: each 500ml of 0.1% (W/V) mercuric chloride was added with 50. Mu.l Tween-80 before use.
As a further improvement of the tissue culture method for efficient seedling formation of hybrid orchid of spring Sword X cymbidium, the preferred culture medium is:
the protocorm induction medium is: 1/2MS+0.5mg/L NAA+1.0 mg/L6-BA+30 g/L sucrose+5 g/L agar+1 g/L active carbon, pH 5.6-5.8;
the growth medium is: MS+1.0mg/L IBA+0.5 mg/L6-BA+50 g/L banana puree+30 g/L sucrose+6 g/L agar+0.8 g/L active carbon, pH 5.6-5.8.
The invention discloses a high-efficiency seedling method for hybrid orchid of spring sword (female parent) x cymbidium (male parent), which utilizes protocorms with vigorous cell division to carry out mass proliferation, so that the proliferation multiple is higher and the proliferation speed is high. The invention combines the proliferation and differentiation steps into one, accelerates the proliferation and differentiation speed, shortens the seedling time, simultaneously reduces the material and labor cost, and has the advantage of high seedling efficiency.
The invention has the following technical advantages:
1. the conditions are controllable: the tissue culture conditions can be regulated and controlled manually according to the optimal growth environment or specific production requirements of plants, and are not limited by external climatic environments and seasonal factors;
2. the seedling efficiency is high: the tissue culture propagation is based on somatic cell proliferation, and the formed offspring has high uniformity and can obtain a large number of seedlings with uniform specification and high quality. In order to improve the seedling efficiency, the invention combines the proliferation and differentiation steps into one, shortens the seedling time, and reduces the material and labor cost;
3. the propagation speed is high: under the condition of providing the optimal growth conditions and nutrition requirements of plants, the plant growth speed is increased, the propagation coefficient is higher, and compared with the traditional plant dividing mode, the quantity of seedlings produced in the same time is larger.
Therefore, the tissue culture method is utilized to realize high-efficiency seedling formation, so that the propagation speed of the hybrid orchid is greatly improved, the quality of seedlings is easy to accurately control, the consistency and commodity performance of products are improved, and the seedlings are tidy and the quality of flowers is better; is beneficial to accelerating popularization and application of hybrid orchid, reducing seedling cost and meeting market demands.
In conclusion, the invention is based on the principle of plant cell totipotency, uses the bud growth point of the parent new bud to induce protocorm as a nutrition propagation carrier, and can form a large number of seedlings with consistent genetic background and unified specification, thereby achieving the purpose of rapid propagation of seedlings.
Drawings
FIG. 1 shows the protocorms induced in example 1.
FIG. 2 shows the differentiation of the protocorm into adventitious buds in example 1.
FIG. 3 shows the state in which adventitious buds continue to grow in example 1.
FIG. 4 shows the development of seedlings into whole plants to form seedlings in bottles in example 1.
FIG. 5 shows the growth state of seedlings transplanted in example 1 after 1 month.
Detailed Description
In the culture process of each stage, the need of changing the culture medium is not explicitly informed, and the culture medium does not need to be changed.
Example 1, tissue culture method for efficient seedling formation of hybrid orchid of spring Sword 'Tonghai Sword' ×cymbidium faberi 'KK560', the following steps are sequentially carried out:
1. treatment of explants:
selecting new buds with strong growth vigor (3-6 cm long and no disease spots) from hybrid orchid plants, removing surface impurities and dirt, taking the new buds back to a laboratory, soaking the new buds in washing powder water with the concentration of 10% -15% (W/V) for cleaning, soaking for about 1 hour, washing for 1 hour by running water, and placing the new buds on an ultra-clean workbench for sterilization: immersing the new buds in 75% (vol%) alcohol for 1 minute, and washing with sterile water 3 times (1 minute each time); removing 1-2 bracts, immersing in 10% (V/V) sodium hypochlorite for sterilization for 10 minutes, and cleaning with sterile water for 3 times (1 minute each time); immersing in mercuric chloride disinfectant for sterilization for 16 minutes, and washing with sterile water for 5 times (1 minute each time); the leaves are peeled layer by layer, small lateral buds of each layer and 1 stem tip (each layer has small lateral buds and only 1 stem tip) exposing growth points of the basal parts of the leaves are peeled as explants, and the small lateral buds and the stem tips after the sterilization treatment are respectively obtained by soaking the explants in 0.05% (W/V) mercuric chloride for 20 seconds and cleaning the explants with sterile water for 6 times (1 minute each time).
The mercuric chloride disinfectant is as follows: each 500ml of 0.1% (W/V) mercuric chloride was added with 50. Mu.l Tween-80 before use.
Description: 10-15% (W/V), namely 10-15 g/100ml; the rest and so on.
2. Protocorm induction:
performing induction culture on the sterilized stem tip on an original bulb induction culture medium, wherein the culture medium does not need to be replaced until an original bulb with the diameter of more than or equal to 0.5cm grows at the position of a stem tip growing point; that is, when the protocorm grows to a diameter of about 0.5cm, the culture in this step may be ended, and the induction culture time in this step may be about 1.5 to 2 months. The induction culture conditions are as follows: 1 stem tip as explant was inoculated per bottle (containing 20ml of protocorm induction medium) and irradiated for 10 hours daily at 25.+ -. 1 ℃ under irradiation with an intensity of 800.+ -. 100lux; the dark culture temperature is 20+/-1 ℃;
inoculating the sterilized small lateral buds into a growth culture medium for re-greening and growing culture for 1.5-2 months, wherein the re-greening and growing culture conditions comprise: within 1 month of inoculation, the light is irradiated for 10 hours every day, the light culture temperature is 25+/-1 ℃, and the light intensity is 800lux; inoculating for 1 month until the set culture time (1.5-2 months) is reached, and illuminating for 14 hours every day, wherein the light culture temperature is 26+/-1 ℃, and the illumination intensity is 2000lux; the dark culture temperature was 20.+ -. 1 ℃. At the moment, the small lateral buds are greened again, the stem tips are stripped off, and the small lateral buds are inoculated on an original bulb induction culture medium for induction culture after being washed by sterile water until the original bulbs with the diameters more than or equal to 0.5cm grow at the positions of the stem tip growing points; the induction culture time is about 1.5-2 months, and the induction culture conditions are the same.
The components of the protocorm induction culture medium are as follows: 1/2MS+0.5mg/L NAA+1.0 mg/L6-BA+30 g/L sucrose+5 g/L agar+1 g/L active carbon, pH 5.6-5.8;
the growth medium comprises the following components: MS+1.0mg/L IBA+0.5 mg/L6-BA+50 g/L banana puree+30 g/L sucrose+6 g/L agar+0.8 g/L active carbon, pH 5.6-5.8.
The protocorm induction rate was 58.06%.
Protocorm induction = protocorm induction/total inoculated stem tip x 100%.
3. Protocorm proliferation and adventitious bud differentiation
The protocorm is gently broken off, divided into small blocks with the diameter of about (0.4-0.45) mm, inoculated on a secondary proliferation/differentiation culture medium for proliferation culture, wherein the proliferation culture conditions are as follows: illuminating for 10 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 800lux; the temperature is 20+/-1 ℃ in dark culture; the medium is not required to be replaced in the middle, the proliferation culture time is about 50-60 days, and the volume of the protocorm is increased to about 1.3cm multiplied by 1.3cm and the protocorm does not bud yet; then, the proliferation culture is repeated once, and the culture medium is replaced once for each time of the subculture.
When the continuous subculture for many times reaches the target propagation amount, the transfer is not carried out any more, and the culture is continued on the primary subculture propagation/differentiation culture medium according to the differentiation culture condition until the adventitious bud with the length of 3-5 cm grows; the differentiation culture conditions are as follows: illuminating for 12 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 1200lux; the culture temperature in dark culture is 20+/-1 ℃.
Thus, the last subculture, starting from the small pieces of the protocorm until the adventitious buds of 3-5 cm are obtained, is generally continued for 4-6 months, and the culture medium does not need to be replaced.
The secondary proliferation/differentiation medium was: hyponex 1 (Huabao No. 1) +0.5 mg/L6-BA+0.5 mg/L NAA+10% coconut juice+30 g/L sucrose+6 g/L agar+2 g/L hydrolyzed casein+0.5 g/L activated carbon, pH 5.6-5.8.
4. Strengthening seedling and rooting
Cutting off 3-5 cm adventitious buds obtained in the step 3 to serve as buds, cutting off the buds, inoculating the buds to a rooting and seedling strengthening culture medium, and performing rooting culture until root systems are strong and developed, namely, the root systems are required to meet the condition that the length is more than or equal to 3cm, the root thickness is more than or equal to 2mm, the rooting number is at least 3 (namely more than or equal to 3), and the seedling height is more than or equal to 8cm, so that transplanting seedlings are obtained;
the culture time of the rooting and seedling strengthening culture medium is about 50-70 d, the culture medium is not required to be replaced in the middle, and the rooting and seedling strengthening culture medium can generally achieve the following steps: the seedling height can be 8-12 cm, the root length is 3-5, and the root length is 3-5 cm; and transferring the bottle seedlings to outdoor domestication and transplanting.
The strong seedling rooting culture medium comprises the following components: MS+100g/L banana puree+30 g/L sucrose+2 g/L hydrolyzed casein+7 g/L agar+1 g/L active carbon, and the pH is 5.6-5.8.
The culture conditions are as follows: illuminating for 14 hours every day, wherein the light culture temperature is 26+/-1 ℃, and the illumination intensity is 2000lux; the dark culture temperature was 20.+ -. 1 ℃.
5. Seedling bottle-out transplanting
Seedling hardening of bottle seedlings (seedlings) in a greenhouse under illumination for 12 days, wherein the specific conditions are as follows:
the strong scattered light at 4000-5000 lux in the daytime (14 hours) is controlled at 25-30 ℃ and at the night (10 hours) is not lower than 15 ℃ (generally 15-20 ℃).
Then opening the bottle mouth and standing for 3 days, taking out and cleaning the seedlings (aiming at cleaning the culture medium at the root of the seedlings), and soaking the seedlings in the 1000-fold carbendazim diluent for 15min; air-drying, transplanting into a matrix with the mass ratio of bark, deer-marsh soil to Xian soil being 1:1:1 (the prepared matrix is sterilized by high-temperature steam in advance for 20-30 min and used after cooling), and then culturing in a conventional manner.
The management measures (conventional manner) after the tissue culture seedlings of the hybrid orchid can be, for example, as shown in Table 1.
The 1000-fold carbendazim diluent is obtained by diluting 1 part by mass of carbendazim with water to 1000 parts by mass.
TABLE 1 management measures after the tissue culture seedlings of hybrid orchid
The invention adopts the same culture medium formula in the proliferation and differentiation stages of the hybrid orchid, and simultaneously utilizes the characteristic that the hybrid orchid is sensitive to hormone, and can produce the seedlings with vigorous growth and developed root systems without adding any hormone in the strong seedling rooting stage, thereby being beneficial to improving the transplanting survival rate, simplifying the culture medium preparation procedure and reducing the production cost.
The method can obtain a large number of hybrid orchid test-tube seedlings for a long time, the multiplication factor of the protocorm can reach 4.9, the transplanting survival rate is more than or equal to 80 percent (table 2), the propagation speed is far higher than that of the traditional plant division mode, a large amount of time cost is saved, and huge economic benefits can be generated.
Multiplication factor = [ weight of protocorm after multiplication culture (g) -weight of protocorm initiation (g) ]/weight of protocorm initiation (g);
survival rate (%) = (number of surviving plants/total number of transplanted plants after 1 month of transplantation) ×100
TABLE 2 transplanting survival of seedlings
| Batch of | Initial number of transplants (plant) | Survival number after 1 month (strain) | Survival rate (%) |
| 1 | 236 | 212 | 89.83 |
| 2 | 206 | 187 | 90.78 |
| 3 | 174 | 154 | 88.50 |
Comparative example 1, "Hyponex 1 (flower bud No. 1)" in proliferation/differentiation medium was changed to "MS medium", and the rest was identical to example 1.
The comparison of the results with example 1 is:
by variance analysis, the multiplication factor of the protocorm of the 'Hyponex 1 (Huabao 1)' group of the example 1 is as high as 4.90, while the multiplication factor of the protocorm of the 'MS' group of the comparative example 1 is 4.03, which is remarkably lower than that of the 'Hyponex 1 (Huabao 1)' group of the example 1, and the defect of premature differentiation and budding, namely, differentiation and budding during multiplication culture, is caused, and the defect can lead to slow multiplication speed and increase the transfer times, thereby reducing the production efficiency and improving the labor cost.
Comparative example 2-1 the remainder of the primordial bulb induction medium was identical to example 1 except that "0.5mg/L NAA, 1.0 mg/L6-BA" was changed to "0.1mg/L NAA, 1.0 mg/L6-BA".
The protocorm induction rate of example 1 was about 58.06% (the rest was about 35.48% of direct seedling formation, the others were rigidified or dead), while the protocorm induction rate of comparative example 2-1 was only 33.33%, and about 53.33% of the stem tip growth points of comparative example 2-1 were directly sprouted to seedlings, and the seedlings were re-induced to obtain protocorms, resulting in an increase in the operation steps, an increase in the induction time, and a decrease in the production efficiency.
Comparative example 2-2, "0.5mg/L NAA, 1.0 mg/L6-BA" in the protocorm-inducing medium was changed to "1.0mg/L NAA, 1.0 mg/L6-BA", and the remainder was identical to example 1.
The protocorm induction rate of comparative example 2-2 was 34.38%; and 62.50% of stem tip growing points are directly sprouted to form seedlings, the seedlings are required to be re-induced to obtain protocorms, and the defects of increasing operation steps and prolonging induction time are overcome.
Comparative example 3 the balance was identical to example 1 except that the "100g/L banana puree" in the rooting medium for strong seedlings was changed to "50g/L banana puree".
Compared with the group of 100g/L banana puree, the group of 50g/L banana puree has shorter root system, fewer roots and slow seedling growth (Table 3), thereby prolonging the seedling emergence time and being unfavorable for large-scale, batch and high-efficiency seedling production.
TABLE 3 influence of different banana puree addition on the rooting stage of strong seedlings
| Banana mud addition amount | Root length | Number of roots | High growth of seedlings |
| 50g/L | 3.79 | 6.76 | 1.82 |
| 100g/L | 5.03 | 7.20 | 2.90 |
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (4)
1. The high-efficiency seedling method for the hybrid orchid of spring Sword and cymbidium hybridum is characterized by comprising the following steps of:
1) Selection and sterilization of explants:
selecting a strong new bud from the hybrid orchid, and cleaning, sterilizing and stripping the new bud to obtain a small lateral bud and a stem tip which are used as explants, wherein the small lateral bud and the stem tip are respectively sterilized and cleaned to correspondingly obtain a sterilized small lateral bud and a sterilized stem tip;
the method comprises the following steps:
selecting strong new buds from hybrid orchid, removing surface impurities and dirt from the new buds, soaking the new buds in washing powder water with the concentration of 10% -15% (W/V), washing the new buds with flowing water, and then placing the new buds on an ultra-clean workbench for sterilization treatment;
the sterilization treatment is as follows: immersing the new buds into 75% alcohol for 1-1.5 minutes, taking out and cleaning with sterile water; removing 1-2 bracts, immersing in 10% (V/V) sodium hypochlorite for sterilization for 10-15 minutes, and cleaning with sterile water; immersing in mercuric chloride disinfectant for disinfection for 12-18 minutes, and cleaning with sterile water; peeling off the leaves layer by layer, peeling off small lateral buds of each layer and stem tips exposing growth points of leaf bases as explants, soaking for 10-20 seconds by 0.05% (W/V) mercuric chloride, and cleaning with sterile water to obtain sterilized small lateral buds and sterilized stem tips respectively;
the mercuric chloride disinfectant is as follows: 0.1% (W/V) of mercury per 500ml, 50 μl Tween-80 was added before use;
2) Protocorm induction:
performing induction culture on the sterilized stem tip on an protocorm induction culture medium until protocorms with diameters more than or equal to 0.5cm grow at the positions of stem tip growing points; the induction culture conditions are as follows: illuminating for 8-10 hours every day, wherein the illumination culture temperature is 25+/-1 ℃, and the illumination intensity is 800+/-100 lux; the dark culture temperature is 20+/-1 ℃;
inoculating the sterilized small lateral buds into a growth medium for re-greening and growing up for 1.5-2 months, then stripping off the stem tip, washing with sterile water, and inoculating onto an original bulb induction medium for induction culture until an original bulb with the diameter of more than or equal to 0.5cm grows up at the position of a stem tip growth point; the induction culture conditions are the same as above;
the protocorm induction medium is: 1/2MS+0.5mg/L NAA+1.0 mg/L6-BA+30 g/L sucrose+5 g/L agar+1 g/L active carbon, pH 5.6-5.8;
the growth medium is: MS+1.0mg/LIBA+0.5 mg/L6-BA+50 g/L banana puree+30 g/L sucrose+6 g/L agar+0.8 g/L active carbon, pH 5.6-5.8;
3) Protocorm proliferation and adventitious bud differentiation
The protocorm is divided and inoculated into a secondary proliferation/differentiation medium for culture, and the following two conditions are adopted:
case 1, need subculture:
performing proliferation culture for 50-60 days according to proliferation culture conditions, and performing primary subculture;
case 2, no subculture was required:
firstly, carrying out proliferation culture for 50-60 days according to proliferation culture conditions, and then directly changing into differentiation culture conditions to carry out differentiation culture until adventitious buds with the length of 3-5 cm are obtained;
the proliferation culture conditions are as follows: illuminating for 8-10 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 500-1000 lux; the culture temperature is 20+/-1 ℃ during dark culture,
the differentiation culture conditions are as follows: illuminating for 10-14 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 1000-1500 lux; the culture temperature is 20+/-1 ℃ in dark culture;
the secondary proliferation/differentiation medium was: hyponex 1+0.5 mg/L6-BA+0.5 mg/L NAA+10% coconut juice+30 g/L sucrose+6 g/L agar+2 g/L hydrolyzed casein+0.5 g/L activated carbon, pH 5.6-5.8;
4) Strengthening seedlings and rooting
Cutting off the adventitious bud with the length of 3-5 cm obtained in the step 3), inoculating the adventitious bud as a young bud into a rooting and seedling strengthening culture medium for culture until the number of the obtained roots with the length of more than or equal to 3cm is at least 3, and the seedling height of more than or equal to 8cm; obtaining transplantable seedlings;
the culture conditions are as follows: illuminating for 12-14 hours every day, wherein the light culture temperature is 26+/-1 ℃, and the illumination intensity is 1500-2000 lux; the dark culture temperature is 20+/-1 ℃;
the rooting culture medium for strong seedlings comprises the following components: MS+100g/L banana puree+30 g/L sucrose+2 g/L hydrolyzed casein+7 g/L agar+1 g/L active carbon, and the pH is 5.6-5.8.
2. The method for efficiently growing hybrid orchids of cymbidium kanran x cymbidium in spring sword x according to claim 1, further comprising the following step 5):
5) And (5) seedling bottle-out transplanting:
firstly, seedling hardening in a greenhouse: the illumination intensity in the daytime is 4000-5000 lux, the temperature is 25-30 ℃, and the temperature at night is 15-20 ℃;
after hardening seedlings for 11-13 days, opening a bottle mouth, placing for 2-3 days, taking out and cleaning the seedlings, and soaking the seedlings in the carbendazim diluent for 15-20 minutes; air-drying, transplanting into a matrix with the ratio of bark, deer-marsh soil to Xian soil being 1:1:1, and culturing.
3. The method for efficiently forming hybrid orchid seedlings of cymbidium kanran by spring sword x cymbidium hybridum according to claim 1 or 2, wherein the method comprises the steps of:
the culture conditions of the step 2) of the compound green growth culture are as follows:
inoculating sterilized small lateral buds into a growth medium for 1 month, and illuminating for 8-10 hours every day, wherein the illumination culture temperature is 25+/-1 ℃, and the illumination intensity is 500-1000 lux; inoculating for 1 month until the set culture time is reached, and illuminating for 10-14 hours every day, wherein the illumination culture temperature is 26+/-1 ℃, and the illumination intensity is 2000lux; the dark culture temperature was 20.+ -. 1 ℃.
4. The method for efficiently growing hybrid orchids of cymbidium kanran x cymbidium in spring sword x according to claim 3, wherein: the induction culture of the step 2) is carried out, and 1 stem tip is inoculated to each 20ml of protocorm induction culture medium.
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