CN108901849B - Tissue culture method of aseptic seedlings of hydrilla verticillata - Google Patents
Tissue culture method of aseptic seedlings of hydrilla verticillata Download PDFInfo
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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
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Abstract
The invention relates to a tissue culture method of a sterile seedling of leaf spring algae, which comprises the following steps: (1) preparing a primary culture medium and a proliferation culture medium, and sterilizing for later use; (2) cleaning and disinfecting explants; (3) absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into a primary culture medium, pouring 30-60ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in an incubator for induced culture for 4 weeks; (4) separating the explant and the generated new bud in the primary culture medium, transferring into a proliferation culture medium, pouring 30-60ml of cooled sterile water to submerge the new explant and the new bud, and performing proliferation culture for 4 weeks; (5) taking out the leaf spring algae plant with complete root system, rinsing with clear water, and transplanting to simulated natural water body for rooting culture. The method can obtain a large number of new plants without being affected by season, temperature, and region, the rooting rate of the new plants reaches 100%, the survival rate is high, and the method can quickly establish an aseptic propagation system for the secondary leaf moss.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of a sterile seedling of leaf spring algae.
Background
The leaf spring algae, also called water sifter, is a common ornamental aquatic weed. The leaf spring algae is a submerged herbaceous plant belonging to the family Amyda sinensis and monocotyledonous plants of the same genus as hydrilla verticillata. The leaf of the secondary leaf algae is beautiful, is an important arrangement material for submerged plants in the aquarium and is also a good greening material in landscape. The leaf spring algae has thin white petals and is suitable for planting in the foreground region of a flat aquarium, but the leaf spring algae is a positive grass and is responsible for light and CO2The water quality and the water depth have strict requirements, and the phenomena of plant withering, leaf falling and the like can be caused by carelessness, so the planting difficulty is extremely high, strong illumination and extremely soft water environment are required, and the plants cannot normally grow in water with too high water hardness and alkaline water; the phenomenon of leaf falling can also occur when the light is insufficient. Therefore, the propagation of the leaf spring algae in the aquarium field is difficult, is one of the aquatic weed species with higher cultivation difficulty, and the attribute of difficult propagation is also forbidden by a plurality of enthusiasts.
There is currently a lack of mature techniques for the large-scale propagation of engineered seedlings of secondary leaf algae of high quality while overcoming the problems of high environmental requirements and difficulty in propagating the species.
Disclosure of Invention
In order to overcome the above-identified deficiencies of the prior art, the present invention provides a tissue culture method of a sterile seedling of algae of leaf spring comprising leaf spring algae by inducing new seedlings of the algae of leaf spring in a suitable culture environment and with substantial illumination of light under sterile conditions to progressively form a sterile seedling system that provides a plurality of sterile seedlings in a later stage. The method is easy to implement, convenient to operate and high in yield, and solves the problems that the aquatic animals are difficult to propagate in the aquarium and the plant seedlings are difficult to obtain.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for culturing tissue of a sterile seedling of algae leaf spring comprising the steps of:
(1) preparing a primary culture medium and a proliferation culture medium, and sterilizing for later use;
(2) cleaning and disinfecting explants: taking a healthy plant without diseases and insect pests of the leaf spring algae as an explant, cleaning, then sequentially disinfecting with alcohol and 0.1% mercuric chloride solution, and then cleaning with sterile water for later use;
(3) absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into a primary culture medium, pouring 30-60ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in an incubator for induced culture for 4 weeks;
(4) separating the explant and the generated new bud in the primary culture medium, transferring into a proliferation culture medium, pouring 30-60ml of cooled sterile water to submerge the new explant and the new bud, and performing proliferation culture for 4 weeks;
(5) taking out the leaf spring algae plant with complete root system, rinsing with clear water, and transplanting to simulated natural water body for culturing.
Further, the primary medium in the step (1) is any one of MS medium, B5 medium, White medium and N6 medium.
Furthermore, the primary culture hormone added into the primary culture medium is 6 aminopurine and naphthylacetic acid, the adding concentration of the 6 aminopurine is 0.5-1.5mg/L, and the adding concentration of the naphthylacetic acid is 0.1-0.5 mg/L.
Further, the proliferation medium is any one of an MS medium, a B5 medium, a White medium and an N6 medium.
Furthermore, proliferation culture hormones are added into the proliferation culture medium and are kinetin and naphthylacetic acid, the addition concentration of the kinetin is 0.5-2mg/L, and the addition concentration of the naphthylacetic acid is 0.1-0.5 mg/L.
Further, the explants were subjected to leaf removal treatment prior to washing and sterilization, leaving 8-12 leaves near the bud center.
Further, the specific method for cleaning and disinfecting the explants in the step (2) comprises the following steps: washing explant with running water for 20-40min, sterilizing with 75% alcohol for 15-40s, and washing for 3 times; sterilizing with 0.1% mercuric chloride solution for 5-20min, and washing with sterile water for 5 times.
Further, the primary culture conditions in the step (3) are as follows: in a sterile environment, the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-27 ℃.
Further, the proliferation culture conditions in the step (4) are as follows: in a sterile environment, the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-27 ℃.
Compared with the prior art, the invention has the beneficial effects that:
(1) the tissue culture method of aseptic seedlings of leaf spring algae provided in the invention is characterized in that under the aseptic condition, a suitable culture environment is adopted, fixed illumination is provided, the growth of the sprouts of leaf spring algae is directly induced, the influence of season, temperature and region can be avoided, a large number of new plants can be obtained, a sterile propagation system of leaf spring algae can be quickly established, the technical support is provided for maintaining the excellent seed properties of the leaf spring algae, and the continuous aseptic seedlings are provided for vast enthusiasts. Experiments prove that through one-month culture, the tissue culture method can enable one explant to quickly obtain 6-8 new progeny plants.
(2) The leaf spring algae seedling aseptically cultured by the tissue culture method of the invention has 100 percent of rooting rate in natural water and high plant survival rate.
(3) The tissue culture method of the aseptic seedlings of the cladium SCHIZOMA of the invention is simple and easy to operate, has convenient operation and high yield, and overcomes the problems of difficult propagation in an aquarium and difficult acquisition of plant seedlings.
Detailed Description
The following examples are shown to illustrate certain embodiments of the invention in detail and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Specifically, the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
Example 1:
step 1: preparing primary culture medium and multiplication culture medium
1. The primary culture medium and the multiplication culture medium prepared in the example are MS culture media, and the formula is as follows: mg/L
2. Preparing primary culture medium
Adding agar 7g, sucrose 30g, 6-aminopurine (6BA)0.5mg, and naphthylacetic acid (NAA)0.1mg into 1L of the MS culture medium to prepare a solid culture medium, and sterilizing at the temperature of 98kPa, 115 ℃ and 125 ℃ for 20min for later use; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
3. Preparing proliferation culture medium
Adding agar 7g, sucrose 30g, Kinetin (KT)0.5mg, and naphthylacetic acid (NAA)0.1mg into the MS culture medium 1L, sterilizing at 125 deg.C under 98kPa for 20 min; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
Step 2: cleaning and disinfecting explants
Purchasing a leaf spring algae plant from the flower and bird market, returning the plant to a laboratory for cleaning away surface stains and dust with a detergent, and washing for half an hour under tap water; selecting healthy plants without diseases and insect pests as explants on a superclean workbench, removing leaves of the plants to a certain extent, leaving about 10 leaves away from the bud center, disinfecting for 15s by 75% alcohol, washing for three times, then disinfecting for 5min by 0.1% mercuric chloride solution, and washing for 5 times by using sterile water for standby.
And step 3: primary culture
Absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into the primary culture medium prepared in the step 1, pouring 30ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in a sterile culture box for induced culture for 4 weeks under the following induced culture conditions: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-24 ℃.
And 4, step 4: proliferation culture
Separating the explant and the generated new bud from the primary culture medium, transferring the explant and the generated new bud into the multiplication culture medium prepared in the step 1, pouring 30ml of cooled sterile water to submerge the new explant and the new bud, and carrying out multiplication culture for 4 weeks under the following conditions: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-24 ℃. The plant can root in the proliferation culture medium, so that no independent rooting culture is required.
And 5:
after 4 weeks of culture, the leaf spring algae plant with an intact root system is removed, rinsed with fresh water, and transplanted into a simulated natural water body, wherein the plant survives completely and the rooting rate reaches 100%.
The method can directly induce a new plant from an explant by culturing successive seedlings of a leaf spring algae supplied by a continuous separation and propagation process, and can induce 6 new progeny plants of the leaf spring algae having a survival rate greater than 90% after being transformed into an aquarium for 30 days.
Example 2:
step 1: preparing primary culture medium and multiplication culture medium
1. The primary culture medium and the proliferation culture medium prepared in this example were MS culture media, and the formulation was the same as that shown in example 1.
2. Preparing primary culture medium
Adding agar 7g, sucrose 30g, 6-aminopurine (6BA)1mg, and naphthylacetic acid (NAA)0.3mg into the MS culture medium 1L, sterilizing at 125 deg.C under 98kPa for 20 min; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
3. Preparing proliferation culture medium
Adding agar 7g, sucrose 30g, Kinetin (KT)1.25mg, and naphthylacetic acid (NAA)0.3mg into the MS culture medium 1L, sterilizing at 125 deg.C under 98kPa for 20 min; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
Step 2: cleaning and disinfecting explants
Purchasing a leaf spring algae plant from the flower and bird market, returning the plant to a laboratory for cleaning away surface stains and dust with a detergent, and washing for half an hour under tap water; selecting healthy plants without diseases and insect pests as explants on a superclean workbench, removing leaves of the plants to a certain extent, leaving about 10 leaves away from the bud center, disinfecting for 30s by 75% alcohol, washing for three times, then disinfecting for 8min by 0.1% mercuric chloride solution, and washing for 5 times by using sterile water for standby.
And step 3: primary culture
Absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into the primary culture medium prepared in the step 1, pouring 50ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in a sterile culture box for induced culture for 4 weeks, wherein the induced culture conditions are as follows: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 24-25 ℃.
And 4, step 4: proliferation culture
Separating the explant and the generated new bud from the primary culture medium, transferring the explant and the generated new bud into the multiplication culture medium prepared in the step 1, pouring 50ml of cooled sterile water to submerge the new explant and the new bud, and carrying out multiplication culture for 4 weeks under the following conditions: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 24-25 ℃. The plant can root in the proliferation culture medium, so that no independent rooting culture is required.
And 5:
after 4 weeks of culture, the leaf spring algae plant with an intact root system is removed, rinsed with fresh water, and transplanted into a simulated natural water body, wherein the plant survives completely and the rooting rate reaches 100%.
The method can directly induce a new plant from an explant by culturing successive seedlings of a leaf spring algae supplied by a continuous separation and propagation process, and can induce 8 new progeny plants of the leaf spring algae having a survival rate greater than 90% after being transformed into an aquarium for 30 days.
Example 3:
step 1: preparing primary culture medium and multiplication culture medium
1. The primary culture medium and the proliferation culture medium prepared in this example were MS culture media, and the formulation was the same as that shown in example 1.
2. Preparing primary culture medium
Adding agar 7g, sucrose 30g, 6-aminopurine (6BA)1.5mg, and naphthylacetic acid (NAA)0.5mg into the MS culture medium 1L, sterilizing at 98kPa, 115-; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
3. Preparing proliferation culture medium
Adding agar 7g, sucrose 30g, Kinetin (KT)2mg, and naphthylacetic acid (NAA)0.5mg into the MS culture medium 1L, sterilizing at 125 deg.C under 98kPa for 20 min; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
Step 2: cleaning and disinfecting explants
Purchasing a leaf spring algae plant from the flower and bird market, returning the plant to a laboratory for cleaning away surface stains and dust with a detergent, and washing for half an hour under tap water; selecting healthy plants without diseases and insect pests as explants on a superclean workbench, removing leaves of the plants to a certain extent, leaving about 10 leaves away from the bud center, disinfecting for 40s by 75% alcohol, washing for three times, then disinfecting for 20min by 0.1% mercuric chloride solution, and washing for 5 times by using sterile water for standby.
And step 3: primary culture
Absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into the primary culture medium prepared in the step 1, pouring 60ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in a sterile culture box for induced culture for 4 weeks, wherein the induced culture conditions are as follows: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 26-27 ℃.
And 4, step 4: proliferation culture
Separating the explant and the generated new bud from the primary culture medium, transferring the explant and the generated new bud into the proliferation culture medium prepared in the step 1, pouring 60ml of cooled sterile water to submerge the new explant and the new bud, and performing proliferation culture for 4 weeks under the conditions that: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 26-27 ℃. The plant can root in the proliferation culture medium, so that no independent rooting culture is required.
And 5:
after 4 weeks of culture, the leaf spring algae plant with an intact root system is removed, rinsed with fresh water, and transplanted into a simulated natural water body, wherein the plant survives completely and the rooting rate reaches 100%.
The method can directly induce a new plant from an explant by culturing successive cladium leaf cells seedlings by continuous separation and propagation, and can induce 7 new progeny plants from a leaf of secondary leaf spring algae for 30 days with a survival rate of greater than 85% after transformation into an aquarium.
Example 4:
1. the primary culture medium and the proliferation culture medium prepared in the example are white culture media, and the formula is as follows: mg/L
2. Preparing primary culture medium
Adding agar 7g, sucrose 30g, 6-aminopurine (6BA)1mg, and naphthylacetic acid (NAA)0.3mg into 1L of the white culture medium to prepare a solid culture medium, and sterilizing at the temperature of 98kPa, 115 ℃ and 125 ℃ for 20min for later use; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
3. Preparing proliferation culture medium
Adding agar 7g, sucrose 30g, Kinetin (KT)1.25mg, and naphthylacetic acid (NAA)0.3mg into 1L of the white culture medium to prepare a solid culture medium, and sterilizing at 98kPa, 115 ℃ and 125 ℃ for 20min for later use; the culture medium is divided into 150ml tissue culture bottles, and each bottle contains 30ml of the culture medium.
Step 2: cleaning and disinfecting explants
Purchasing a leaf spring algae plant from the flower and bird market, returning the plant to a laboratory for cleaning away surface stains and dust with a detergent, and washing for half an hour under tap water; selecting healthy plants without diseases and insect pests as explants on a superclean workbench, removing leaves of the plants to a certain extent, leaving leaves about 10 leaves away from the bud center, disinfecting for 30s by 75% alcohol, washing for three times, disinfecting for 8min by 0.1% mercuric chloride solution, and washing for 5 times by using sterile water for standby.
And step 3: primary culture
Absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into the primary culture medium prepared in the step 1, pouring 50ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in a sterile culture box for induced culture for 4 weeks, wherein the induced culture conditions are as follows: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 24-25 ℃.
And 4, step 4: proliferation culture
Separating the explant and the generated new bud from the primary culture medium, transferring the explant and the generated new bud into the multiplication culture medium prepared in the step 1, pouring 50ml of cooled sterile water to submerge the new explant and the new bud, and carrying out multiplication culture for 4 weeks under the following conditions: the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 24-25 ℃. The plant can root in the proliferation culture medium, so that no independent rooting culture is required.
And 5:
after 4 weeks of culture, the leaf spring algae plant with an intact root system is removed, rinsed with fresh water, and transplanted into a simulated natural water body, wherein the plant survives completely and the rooting rate reaches 100%.
The method can directly induce a new plant from an explant by culturing successive seedlings of a leaf spring algae supplied by a continuous separation and propagation process, and can induce 6 new progeny plants of the leaf spring algae having a survival rate greater than 88% after being transformed into an aquarium for 30 days.
In this embodiment, the medium is white medium, the concentration of 6 aminopurine added to the primary medium includes, but is not limited to, 1mg/L, and may also be 0.5mg/L or 1.5mg/L, and the concentration of 6 aminopurine added to the primary medium is 0.5-1.5 mg/L; the concentration of naphthylacetic acid added in the primary culture medium includes but is not limited to 0.3mg/L, and can also be 0.1mg/L or 0.5mg/L, and the concentration of naphthylacetic acid added in the primary culture medium is 0.1-0.5 mg/L. The added concentration of KT in the proliferation culture medium includes but is not limited to 1.25mg/L, and can also be 0.5mg/L or 2mg/L, and the added concentration of KT in the proliferation culture medium is 0.5-2 mg/L; the concentration of naphthylacetic acid added in the proliferation culture medium includes but is not limited to 0.3mg/L, and can also be 0.1mg/L or 0.5mg/L, and the concentration of naphthylacetic acid added in the proliferation culture medium is 0.1-0.5 mg/L. The explants are induced and cultured in the primary culture medium under the illumination of 2500Lux and the illumination period of 12h/d and the temperature of 23-27 ℃, then the explants are subjected to proliferation and culture in the proliferation culture medium under the illumination of 2500Lux and the illumination period of 12h/d and the temperature of 23-27 ℃ to obtain algae plants with complete root systems, and the algae plants are transplanted into a simulated natural water body, wherein the plants are all alive, and the rooting rate reaches 100%.
The medium used in the present invention includes, but is not limited to, MS medium and white medium in the examples, and further includes any one of B5 medium, N6 medium. A primary culture medium was prepared by adding 30g of agar, 7g of sucrose, 0.5-1.5mg/L of 6-aminopurine and 0.1-0.5mg/L of naphthylacetic acid to 1L of the above-mentioned culture medium, and a growth medium was prepared by adding 30g of agar, 7g of sucrose, 0.5-2mg/L of KT and 0.1-0.5mg/L of naphthylacetic acid to the above-mentioned culture medium. The tissue culture process of the present invention in these media also enables large-scale propagation of engineered seedlings of high quality algae leaf pass through the invention, the leaf of leaf spring algae can induce 6-8 new progeny plants over 30 days, and the engineered seedlings of leaf spring algae are transplanted into simulated natural water, the plants are fully viable, the rooting rate reaches 100%, and the survival rate after transfer into an aquarium is greater than 85%.
Claims (9)
1. A method for culturing tissue of a sterile seedling of algae leaf spring comprising the steps of:
(1) preparing a primary culture medium and a proliferation culture medium, and sterilizing for later use;
(2) cleaning and disinfecting explants: taking a healthy plant without diseases and insect pests of the leaf spring algae as an explant, cleaning, then sequentially disinfecting with alcohol and 0.1% mercuric chloride solution, and then cleaning with sterile water for later use;
(3) absorbing surface moisture of the sterilized explant by using a sterilization paper sheet, transferring the explant into a primary culture medium, pouring 30-60ml of cooled sterile water into a tissue culture bottle to enable the explant to sink in the sterile water, and placing the explant in an incubator for induced culture for 4 weeks;
(4) separating the explant and the generated new bud in the primary culture medium, transferring into a proliferation culture medium, pouring 30-60ml of cooled sterile water to submerge the new explant and the new bud, and performing proliferation culture for 4 weeks;
(5) taking out the leaf spring algae plant with complete root system, rinsing with clear water, and transplanting to simulated natural water body for culturing.
2. The method of tissue culture of a sterile sprout of algae of the type i (i) according to claim 1, wherein the primary culture medium of step (1) is any one of MS medium, B5 medium, White medium, N6 medium.
3. The method of tissue culture of a sterile sprout of algae of the type i (i) as recited in claim 2, wherein the primary culture medium supplemented with a primary culture hormone comprises 6BA and naphthaleneacetic acid, wherein the concentration of 6BA is 0.5-1.5mg/L and the concentration of naphthaleneacetic acid is 0.1-0.5 mg/L.
4. The method of tissue culture of a sterile seedling of algae of the type i t claim 1, wherein the propagation medium is any one of MS medium, B5 medium, White medium, N6 medium.
5. The method of tissue culture of a sterile sprout of algae according to claim 4, wherein the culture medium supplemented with culture hormones comprises kinetin and naphthylacetic acid, the concentration of kinetin is 0.5-2mg/L and the concentration of naphthylacetic acid is 0.1-0.5 mg/L.
6. The method of tissue culture of a sterile seedling of algae according to claim 1, wherein the explant is subjected to a leaf removal process prior to cleaning and sterilizing the explant to leave between 8 and 12 leaves adjacent the bud center.
7. A method for tissue culture of a sterile sprout of algae according to claim 1, wherein the specific method for cleaning and disinfecting the explant of step (2) is: washing explant with running water for 20-40min, sterilizing with 75% alcohol for 15-40s, and washing for 3 times; sterilizing with 0.1% mercuric chloride solution for 5-20min, and washing with sterile water for 5 times.
8. A method for culturing tissue of a sterile sprout of algae according to claim 1, wherein the primary culture conditions in step (3) are: in a sterile environment, the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-27 ℃.
9. A method for culturing tissue of a sterile sprout of algae according to claim 1, wherein the propagation conditions in step (4) are: in a sterile environment, the illumination is 2500Lux, the illumination period is 12h/d, and the temperature is 23-27 ℃.
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