A tissue culture and propagation method for Fritillaria eyrei

Abstract

The invention discloses a tissue culture and propagation method of eye-spotted Fritillaria orchid, and relates to the field of eye-spotted Fritillaria orchid tissue culture technology. The method includes selecting a robust eye-spotted Fritillaria orchid plant, performing artificial cross-pollination on it during flowering; cutting off the debris remaining in the fruit pod, washing and disinfecting it and placing it in a sterile dish on a clean bench for standby; dissecting it along the fruit ridge with a cooling scalpel and tweezers, taking out the seeds and sprinkling them on the primary culture medium for dark culture, and light culture after germination; the germinated eye-spotted Fritillaria orchid continues to grow for 45-60 days, and when the leaves begin to differentiate, the seedlings are transferred to the secondary culture medium for culture for 100-120 days; the sterile seedlings are transferred to the rooting culture medium for rooting culture for 180-210 days; hardening and taming transplanting. The present invention overcomes the problems of slow traditional division propagation of eye-spotted Fritillaria orchid, incomplete seed embryo development, and it is difficult for seeds to independently develop into complete plants under natural conditions, while meeting a large number of breeding needs, reducing the excavation of wild resources, and protecting wild resources.

Description

Technical Field

[0001] The invention belongs to the technical field of eye-spotted Fritillaria orchid tissue culture, and particularly relates to a tissue culture and propagation method for eye-spotted Fritillaria orchid. Background Art

[0002] Coelogyne corymbosa belongs to the genus Coelogyne of the Orchidaceae family. It is native to the northwest to southeast of Yunnan and southern Tibet, and grows on tree trunks at the edge of forests or on moist rock walls. Currently, Coelogyne corymbosa is included in the "China Biodiversity Red List - Higher Plants Volume" and is a national second-level protected plant. It is named because of the four yellow eyespots surrounded by orange-red on its lip. It is loved by many viewers for its unique ornamental characteristics.

[0003] However, due to the problem that the seeds of the eye-spotted Fritillaria are difficult to develop into complete plants independently under natural conditions, the traditional method of propagation by division cannot meet market demand. For this reason, we provide a tissue culture and propagation method for the eye-spotted Fritillaria, which is used to solve the problem of rapid propagation of the eye-spotted Fritillaria seedlings, reduce the collection of wild resources, and protect wild resources. Summary of the invention

[0004] The purpose of the present invention is to provide a tissue culture propagation method for eye-spot Fritillaria orchid, which can effectively improve the speed and quantity of eye-spot Fritillaria orchid seedling propagation, control the stability of germplasm from the source, ensure the quality of seedlings, realize the factory production of high-quality eye-spot Fritillaria seedlings, meet the ornamental needs, and reduce the situation of rampant excavation of wild resources.

[0005] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0006] The present invention discloses a tissue culture and propagation method of Ocellated Fritillaria ovata, comprising the following steps:

[0007] Step 1: Explant selection

[0008] Select robust plants of the eye-spotted Fritillaria, perform artificial cross-pollination on them during the flowering period, and use them as explants when the fruit pods are ripe;

[0009] Step 2: Disinfection of explants

[0010] Cut off the debris left on the pods, clean the surface of the pods with tap water, disinfect them with medical alcohol and mercuric chloride on a sterile operating table, and finally put them in a sterile tray on a clean workbench for later use;

[0011] Step 3: Seeding and primary culture

[0012] Dissect the sterilized pods along the ridge with a cooled scalpel and forceps, take out the seeds and spread them on the primary culture medium for dark culture, and then culture them in the light after the seeds germinate;

[0013] Step 4: Subculture

[0014] The germinated eye-spotted Fritillaria orchid continues to grow for about 45-60 days. When the leaves begin to differentiate, the seedlings are transferred to the subculture medium on a sterile operating table and cultured for 100-120 days.

[0015] Step 5: Rooting and seedling cultivation

[0016] Transfer the sterile seedlings cultured for 100-120 days to rooting medium for rooting culture for 180-210 days;

[0017] Step 6: Hardening, acclimatization and transplanting

[0018] Select tissue culture seedlings with good rooting and robustness, move them to an outdoor environment and place them for 15-20 days, avoiding direct sunlight to allow them to adapt to the external environment; when transplanting, pour out the seedlings and wash the culture medium, soak the seedlings in 800-1000 times diluted carbendazim, wash and dry them in the shade, and then transplant them into the substrate for domestication.

[0019] Preferably, the specific method of explant selection in step 1 includes:

[0020] (1) Select healthy plants, artificially cross-pollinate them during the flowering period of the eye-spotted Fritillaria orchid in mid-April, and carefully cultivate them for 6-7 months;

[0021] (2) When the pods are ripe, pick the yellow-green pods with intact shells and place them in a bottle filled with sponge and store them at 4°C for 24 hours.

[0022] Preferably, the specific method for sterilizing the explant in step 2 includes:

[0023] (1) Remove the fruit pods, cut off the dried petals remaining in the fruit pods, and use a brush dipped in a small amount of detergent water to clean the surface of the seed shell. The cleaning action should be gentle to avoid scratching the fruit pods. Then rinse under tap water for 8-10 minutes, and rinse with sterile water 2-3 times;

[0024] (2) Place the fruit pods rinsed with sterile water into a clean beaker, move to the inoculation room and irradiate under ultraviolet light for 30 minutes, then soak them in 75% medical alcohol for 3 minutes on a sterile operating table, rinse them once with sterile water, then sterilize them in 0.1% mercuric chloride for 10 minutes, and then rinse them with sterile water 3-5 times;

[0025] (3) Finally, use sterile filter paper to absorb the surface moisture of the pods and place them in a sterile plate on the clean bench for later use.

[0026] Preferably, the specific method of sowing and primary culture in step 3 includes:

[0027] (1) Use a cooled scalpel to cut off the head and tail of the sterilized pods, and then cut open 3-4 petals along the ridge to reveal the white, thread-like seeds;

[0028] (2) Sowing the seeds on the primary culture medium, and then putting the bottle cap on the primary culture medium after flame sterilization. The primary culture medium is moved to the culture room for dark culture at a temperature of 25° C. for 30-45 days until the embryo swells and turns green.

[0029] (3) Then enter the light culture stage, the culture temperature is 22-25℃, the light intensity is 1500x, the lighting time is 8 hours / day, the primary culture medium is: MS+1mg / LNAA+0.25mg / L6-BA+1g / L acid hydrolyzed casein+1g / L activated carbon+30g / L white sugar+3g / L agar powder, and the pH is adjusted to 5.8-6.0.

[0030] Preferably, the specific method of the subculture and value-added culture in step 4 includes:

[0031] The germinated eye-spotted Fritillaria seedlings grow for 45-60 days, and the leaves begin to differentiate. The seedlings are transferred to the subculture medium on a sterile operating table and cultured for 100-120 days. The culture temperature is 22-25°C, the light intensity is 15001x, the lighting time is 8h / day, and the subculture growth medium is: 1 / 2MS+0.25mg / LNAA+1.0mg / L6-BA+1g / L acid hydrolyzed casein, the culture medium agar content is 4g / L, and the pH is adjusted to 5.8-6.0.

[0032] Preferably, the culture medium used for rooting and seedling cultivation in step 5 is: 1 / 2MS+1mg / LNAA+1.5mg / LIBA+1g / L acid hydrolyzed casein+50g / L banana+50g / L potato+1g / L acid hydrolyzed casein+1g / L activated carbon+30g / L white sugar, and the amount of agar in the culture medium is 4g / L;

[0033] The culture conditions are: culture temperature is 25-28℃, light time is 10-12h / d, light intensity is 1500lx, and multiple seedling transfer and culture can be carried out during the culture stage to avoid the growth of seedlings being affected by the small growth space.

[0034] Preferably, the specific methods of hardening the seedlings and acclimatizing and transplanting in step 6 include:

[0035] (1) Select tissue culture seedlings with good rooting and robustness, move them to an outdoor environment, seal them and place them for 15-20 days, avoid direct sunlight to allow them to adapt to the external environment;

[0036] (2) When transplanting, pour out the seedlings and wash the culture medium, soak the seedlings in 800-1000 times diluted carbendazim for 3 minutes, wash and dry them in the shade, and then transplant them into the substrate for acclimatization;

[0037] The cultivation medium is made of crushed humus and pine bark mixed evenly in a volume ratio of 3:4. 50g of organic fertilizer is added to each liter of cultivation medium. During the seedling hardening stage, avoid direct sunlight, maintain a shading rate of 60-70%, and the medium humidity is 40%.

[0038] The present invention has the following beneficial effects:

[0039] 1. The present invention can effectively improve the speed and quantity of breeding of eye-spotted Fritillaria seedlings, control the stability of germplasm from the source, ensure the quality of seedlings, realize the factory production of high-quality eye-spotted Fritillaria seedlings, meet the ornamental needs, and reduce the situation of rampant excavation of wild resources.

[0040] 2. The method for selecting explants and disinfecting explants of the present invention can effectively reduce the contamination rate caused by the materials themselves. During the implementation process, the contamination rate is controlled at 8%, which avoids material waste to a certain extent and saves the production cost of tissue culture and propagation.

[0041] 3. The primary culture medium used in the present invention has a germination rate of 55%-60% in 30 days during the implementation process, which meets the demand for the number of seedlings to be expanded by tissue culture; the secondary proliferation culture medium used has an average value-added rate of 2.5 times, and the time for starting secondary proliferation is selected when the leaf buds begin to differentiate, which is convenient for promoting seedling proliferation.

[0042] 4. The rooting and seedling strengthening medium adopted by the present invention has a rooting rate of 100% after 180 days, an average root diameter of 1.3mm, a root length of 5-7cm, a plant height of 4-6cm, a pseudobulb length of 2-3cm, a diameter of 5-6mm, a leaf length of 3-5cm, and a width of 1.5cm. The seedlings are strong, the leaves are dark green and shiny, and the survival rate of the seedlings is improved.

[0043] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. DETAILED DESCRIPTION

[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The present invention discloses a tissue culture and propagation method of Ocellated Fritillaria ovata, comprising the following steps:

[0046] Step 1: Selection of explants. Select healthy plants and artificially cross-pollinate them during the flowering period of the eye-spotted Fritillaria orchid in mid-April. After 6-7 months of careful cultivation, when the fruit pods are ripe, that is, the outer seed shell is yellow-green and intact, they are picked and placed in a bottle filled with sponge and stored at 4°C for 24 hours;

[0047] Step 2: Disinfection of explants. Take out the fruit pods, cut off the dry petals and other debris left in the fruit pods, use a brush dipped in a small amount of detergent water to brush the surface of the seed shell, and be gentle to avoid scratching the fruit pods. Then rinse under tap water for 8-10 minutes, and rinse with sterile water 2-3 times; put it in a clean beaker, move it to the inoculation room, irradiate it under ultraviolet light for 30 minutes, then soak it in 75% medical alcohol for 3 minutes on a sterile operating table, wash it once with sterile water, then put the fruit pods in 0.1% mercuric chloride for 10 minutes for disinfection, wash it with sterile water 3-5 times, and finally use sterile filter paper to absorb the surface moisture of the fruit pods, and put them in a sterile plate on the ultra-clean workbench for use;

[0048] Step 3: Sowing and primary culture. Cut off the head and tail of the sterilized pods with a cooled scalpel, and dissect 3-4 petals along the ridge to reveal the white filamentous endosperm, i.e., the seeds; sprinkle the seeds on the primary culture medium, seal the bottle cap after flame sterilization, and move it to the culture room for dark culture at a temperature of about 25°C. After 30-45 days of culture, the embryo swells and turns green; then enter the light culture stage, the culture temperature is 22-25°C, the light intensity is 1500x, and the light time is 8 hours / day. The primary culture medium is: MS+1mg / LNAA+0.25mg / L6-BA+1g / L acid hydrolyzed casein+1g / L activated carbon+30g / L white sugar+3g / L agar powder, and the pH is adjusted to 5.8-6.0.

[0049] Step 4: Subculture and proliferation. When the seedlings of the eye-spotted Fritillaria orchid grow for about 45-60 days and the leaves begin to differentiate, the seedlings are transferred to the subculture medium on a sterile operating table and cultured for 100-120 days. The subculture and proliferation medium is: 1 / 2MS+0.25mg / LNAA+1.0mg / L6-BA+1g / L acid hydrolyzed casein, the amount of agar in the culture medium is 4g / L, and the pH is adjusted to 5.8-6.0. The culture conditions are: temperature 22-25℃, light time 8h / day, light intensity 15001x.

[0050] Step 5: Rooting and seedling cultivation. The culture medium in the rooting and seedling cultivation step is: 1 / 2MS + 1mg / LNAA + 1.5mg / LIBA + 1g / L acid hydrolyzed casein + 50g / L banana + 50g / L potato + 1g / L acid hydrolyzed casein + 1g / L activated carbon + 30g / L white sugar, and the amount of agar in the culture medium is 4g / L. The preparation process of banana juice is as follows: select a ripe banana with a bright yellow peel and an aromatic odor, remove the peel, weigh 50g of the pulp, add 1L of sterile water and crush it with a wall breaker to obtain banana juice; the preparation process of potato juice is as follows: first steam the potatoes, weigh 50g of peeled potatoes, add 1L of sterile water and crush it with a wall breaker to obtain potato juice. The culture conditions are: temperature 25-28℃, light time 10-12h / d, light intensity 15001x. During the rooting and seedling cultivation stage, multiple seedling divisions and transfers can be carried out according to actual conditions to avoid affecting the growth of the seedlings due to the small growth space.

[0051] Step 6: Hardening, acclimatization and transplanting. Select tissue culture seedlings with good rooting and strong seedlings, move them to an outdoor environment, seal them and place them for 15-20 days, avoid direct sunlight, and let them adapt to the external environment; when transplanting, pour out the seedlings and wash the culture medium, soak the seedlings in 800-1000 times diluted carbendazim for 3 minutes, wash and dry them in the shade, and transplant them into the substrate for acclimatization. The cultivation substrate is made of crushed humus soil and crushed pine bark mixed in a volume ratio of 3:4, and 50g of organic fertilizer is added to each liter of substrate. Avoid direct sunlight during the seedling hardening stage, maintain a 60-70% shading rate, and keep the substrate humidity at around 40%. Pay special attention to the seedling hardening stage: the light should not be too strong to avoid burning the seedlings; pay attention to ventilation, especially when just after watering, ventilation should be strengthened to avoid excessive substrate humidity, otherwise it will easily cause plant rot.

[0052] By using the invention, the germination rate of the eye-spotted Fritillaria orchid can reach 55%-60%, the value-added rate can reach 2.5 times on average, and after multiple seedling divisions and transfers in the rooting and strong seedling cultivation stage, tissue culture seedlings with thick roots, strong seedlings, dark green and shiny leaves can be obtained after 6-7 months of rooting and cultivation.

[0053] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0054] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A tissue culture and propagation method for Fritillaria eyrei, characterized in that: The steps include: Step 1: Explant selection Select robust plants of the eye-spotted Fritillaria, perform artificial cross-pollination on them during the flowering period, and use them as explants when the fruit pods are ripe; Step 2: Disinfection of explants Cut off the debris left on the pods, clean the surface of the pods with tap water, disinfect them with medical alcohol and mercuric chloride on a sterile operating table, and finally put them in a sterile tray on a clean workbench for later use; Step 3: Seeding and primary culture Dissect the sterilized pods along the ridge with a cooled scalpel and forceps, take out the seeds and spread them on the primary culture medium for dark culture, and then culture them in the light after the seeds germinate; Step 4: Subculture The germinated eye-spotted Fritillaria orchids continue to grow for 45-60 days. When the leaves begin to differentiate, the seedlings are transferred to the subculture medium on a sterile operating table and cultured for 100-120 days. Step 5: Rooting and seedling cultivation Transfer the sterile seedlings cultured for 100-120 days to rooting medium for rooting culture for 180-210 days; Step 6: Hardening, acclimatization and transplanting Select tissue culture seedlings with good rooting and robustness, move them to an outdoor environment for 15-20 days, avoid direct sunlight to adapt them to the external environment; when transplanting, pour out the seedlings and wash the culture medium, soak the seedlings with 800-1000 times diluted carbendazim, wash and dry them in the shade, and then transplant them into the substrate for domestication; The specific method of explant selection in step 1 includes: Select healthy plants, artificially cross-pollinate them during the flowering period of the eye-spotted Fritillaria orchid in mid-April, and carefully cultivate them for 6-7 months; When the pods are ripe, pick the yellow-green pods with intact shells and place them in a bottle filled with sponge and store them at 4 ℃ for 24 h. The specific method of sowing and primary culture in step 3 includes: (1) Use a cooled scalpel to cut off the head and tail of the sterilized pods, and then cut open 3-4 petals along the ridge to reveal the white, thread-like seeds; (2) Sow the seeds on the primary culture medium, sterilize the bottle cap with flame and then cover it on the primary culture medium, move the primary culture medium to the culture room for dark culture at 25°C for 30-45 days, and the embryo will swell and turn green; Then it entered the light culture stage, the culture temperature was 22-25℃, the light intensity was 1500lx, the light time was 8 hours / day, the primary culture medium was: MS+1 mg / L NAA+0.25 mg / L 6-BA+1 g / L acid hydrolyzed casein+1 g / L activated carbon+30 g / L white sugar+3 g / L agar powder, and the pH was adjusted to 5.8-6.0; The specific method of the subculture proliferation culture in step 4 includes: The germinated eye-spotted Fritillaria seedlings grow for 45-60 days, and the leaves begin to differentiate. The seedlings are transferred to the subculture medium on a sterile operating table and cultured for 100-120 days. The culture temperature is 22-25 ° C, the light intensity is 1500lx, the light time is 8 h / day, and the subculture proliferation medium is: 1 / 2 MS+0.25 mg / L NAA+1.0 mg / L 6-BA+1 g / L acid hydrolyzed casein, the agar content of the culture medium is 4 g / L, and the pH is adjusted to 5.8-6.0; The culture medium used for rooting and seedling cultivation in step 5 is: 1 / 2 MS + 1 mg / L NAA + 1.5 mg / L IBA + 1 g / L acid hydrolyzed casein + 50 g / L banana + 50 g / L potato + 1 g / L acid hydrolyzed casein + 1 g / L activated carbon + 30 g / L white sugar, and the amount of agar in the culture medium is 4 g / L; The culture conditions are: culture temperature is 25-28 ℃, light time is 10-12 h / d, light intensity is 1500 lx, and multiple seedling transfer and culture can be carried out during the culture stage to avoid the growth of seedlings being affected by the small growth space.

2. The tissue culture propagation method of the eye-spotted Fritillaria orchid according to claim 1, characterized in that: The specific method for explant disinfection in step 2 includes: Take out the pods, cut off the dried petals remaining in the pods, and use a brush dipped in a small amount of detergent to clean the surface of the seed shell. Be gentle in the cleaning action to avoid scratching the pods, then rinse under tap water for 8-10 minutes, and rinse with sterile water 2-3 times; Place the fruit pods rinsed with sterile water in a clean beaker, move them to the inoculation room and irradiate them under ultraviolet light for 30 minutes, then soak them in 75% medical alcohol for 3 minutes on a sterile operating table, wash them once with sterile water, then sterilize them in 0.1% mercuric chloride for 10 minutes, and then wash them with sterile water 3-5 times; Finally, use sterile filter paper to absorb the surface moisture of the pods and place them in a sterile tray on the clean bench for later use.

3. The tissue culture propagation method of the eye-spotted Fritillaria orchid according to claim 1, characterized in that: The specific method of hardening the seedlings and acclimatizing and transplanting in step 6 includes: Select tissue culture seedlings with good rooting and robustness, move them to an outdoor environment, seal them and place them for 15-20 days, avoid direct sunlight to allow them to adapt to the external environment; When transplanting, pour out the seedlings and wash the culture medium, soak the seedlings in 800-1000 times diluted carbendazim for 3 minutes, wash and dry them in the shade, and then transplant them into the substrate for acclimatization; The cultivation medium is made of crushed humus and pine bark mixed evenly in a volume ratio of 3:

4. 50 g of organic fertilizer is added to each liter of cultivation medium. During the seedling hardening stage, direct sunlight should be avoided, a shading rate of 60-70% should be maintained, and the medium humidity should be 40%.