CN113179951A - Rapid breeding method for tissue culture seedlings of cymbidium sinense - Google Patents
Rapid breeding method for tissue culture seedlings of cymbidium sinense 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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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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Abstract
The application provides a method for quickly breeding tissue culture seedlings of cymbidium sinense, and relates to the field of plant tissue and cell engineering. The specific method comprises selecting the top end of a newly germinated tender stem of herba Patriniae, only retaining the leaf sheath attached to the bud point part, and sterilizing as explant; cutting off the base part of the tender stem, cutting the top end until the terminal bud is exposed, cutting the tender stem into stem sections with buds, and putting the stem sections into a callus culture medium; after axillary buds bud and the cut grows granular callus, placing the axillary buds into an adventitious bud induction culture medium, further proliferating the callus when the axillary buds form new adventitious buds, growing green bud points on the surface and growing tender buds, transferring the adventitious bud proliferation culture medium into an adventitious bud elongation culture medium after the adventitious bud proliferation culture medium is proliferated to enable the adventitious buds to elongate to more than 1.5cm, adopting a two-step rooting method, sequentially placing two adventitious bud rooting culture media, and finally growing 5-10 roots of the adventitious buds. The technical scheme of the application enriches the rapid propagation method of the tissue culture seedling of the cymbidium maculatum, and improves the induction rate of adventitious buds and the rooting rate of the seedling.
Description
Technical Field
The invention belongs to the field of plant tissue and cell engineering, and particularly relates to a method for quickly breeding tissue-cultured seedlings of cymbidium sinense. The invention also provides a rapid propagation method suitable for the large-scale industrialized production of the spotted orchid seedlings.
Background
The Indian Kalimeris herb, known by the scientific name of Pandanus amarylius, Roxb, also called Vanilla leaf, Isatis leaf, gorgeous leaf, is a perennial evergreen spice plant of the genus Pandanus of the family Pandanaceae (Pandanaceae). The origin place is Indonesia gulu islands which are mainly distributed in tropical and subtropical regions of eastern hemisphere and planted in local counties in the southern part of Hainan China. The family Aristolochiaceae is 3 plants of about 800, wherein herba Patriniae is a plant with unique "rice dumpling fragrance" mainly from leaf blades, and the fragrance component of "brown fragrance" is 2-acetyl-1-pyrroline (2 AP). In addition, the leaf of the variegated orchid is rich in active ingredients such as squalene and linoleic acid, and has the effects of enhancing cell activity, improving human immunity and the like. The southeast Asia countries add the leaf juice as food flavor to cakes, ice cream and beverages to improve the quality, wherein the Xinjiapo cake-green cake is popular all over the world. Hainan popularizes various snacks such as local cakes and the like by using the leaves.
Since the spotted orchid can be planted in the forest, the leaves have higher economic value, and the demand of seedlings is increased dramatically in recent years. The spotted orchid in China is mainly introduced from southeast Asia countries, and the planting area is smaller at present. And the flower and fruit of the spotted orchid are not seen, so that the traditional breeding mode mainly adopts tillering breeding. The method requires that the stock plant is planted for at least more than two years, each stock plant can be tillered by about 10 tilleres per year, and the existing tillering can not meet the requirement of industrial development on large-scale seedlings. Therefore, the research and development of a rapid seedling breeding method are urgently needed, and sufficient and high-quality seedlings are provided for the industry. The tissue culture technology is an effective method for solving the problem, and can quickly proliferate the seedlings in a short time and realize the large-scale production of the seedlings. The scholars at home and abroad research the situation.
In the prior art, CN111226792B Ji train et al (2020) disclose a high-throughput breeding method of a cymbidium species seedling, which takes a stem segment base part which is close to a root end, semi-lignified and 3-5mm long as an explant, induces compact callus at the base part, and obtains a tissue culture seedling through the processes of callus induction, cluster bud induction, bud elongation, rooting and the like. However, in practical application, the selection of the stem base part which is close to the root end, is semi-lignified and has the length of 3-5mm has the defects that the callus induction and propagation needs a long time and the root system is not developed enough. Therefore, a new high-flux method for rapidly propagating the tissue culture seedlings of the spotted orchid is urgently needed.
Disclosure of Invention
The invention aims to provide a novel method for obtaining a cymbidium tissue culture seedling, which comprises the steps of taking newly-germinated and tender overground stem sections as explants, inducing granular callus through a section, germinating green bud points from the callus, developing adventitious buds from the green bud points, extending axillary buds during the callus section induction callus period, inducing the adventitious buds to proliferate during the callus section induction adventitious buds, inducing the adventitious buds to proliferate and inducing the adventitious buds to proliferate with the axillary buds, and realizing the simultaneous proceeding of axillary bud proliferation and callus bud growth and proliferation; after the adventitious buds are multiplied, the two-step rooting method takes root, the root system is developed and robust, and the tissue culture seedlings of the spotted orchid can be obtained in batches.
The invention also aims to provide a high-flux method for obtaining the cymbidium maculatum tissue culture seedling, which adopts a new explant sampling part and a new cutting mode to ensure that the same explant induces the axillary bud to proliferate and simultaneously induces the callus to develop into the adventitious bud through the section, thereby improving the adventitious bud induction rate, the seedling rooting rate and the transplanting survival rate.
The invention is realized by the following technical scheme, which comprises the following steps:
(1) explant collection: selecting a stock plant which grows strongly and has no plant diseases and insect pests, collecting 4-18cm of the top part of a faint yellow and newly germinated tender stem of the cymbidium, cutting off other leaf sheaths, and only keeping the leaf sheath attached to a bud point part as an explant;
(2) explant disinfection: cleaning, disinfecting and sterilizing the top end of the variegated orchid;
(3) explant preparation and callus induction: stripping leaf sheaths attached with buds, cutting off the base parts of tender stems by 1-5mm, cutting the top ends of the tender stems to expose terminal buds, cutting the terminal buds into stem sections with buds with the length of 0.5-1cm, vertically placing callus induction culture media according to the biological polarity direction for culturing for 20-30 days, inserting an incision at one end into the culture media, upwards cutting the other end, forming callus on an upward section at the stage, and extending axillary buds;
(4) adventitious bud induction: subculturing the cut stem segment with callus in adventitious bud induction culture medium for 2-4 times, each time for 20-30 days, further proliferating the cut granular callus, growing green bud points on the surface and further developing into tender buds, and further proliferating axillary buds at this stage;
(5) adventitious bud proliferation: cutting off leaves with more than 0.5cm from stem segments with tender shoots, cutting into 2-4 segments, transferring to adventitious bud proliferation culture medium for adventitious bud proliferation, and proliferating for 20-30 days each time, wherein the steps are circulated until enough adventitious buds are obtained;
(6) adventitious bud elongation: transferring the adventitious bud to an adventitious bud elongation culture medium, and culturing by scattering light at 25-30 deg.C to elongate the adventitious bud to more than 1.5 cm;
(7) adventitious bud rooting: transferring the elongated adventitious bud into a No. 1 rooting culture medium, culturing for 1-3 days, transferring into a No. 2 rooting culture medium, culturing for 20-30 days, and culturing under illumination at 25-30 deg.C to obtain 5-10 adventitious buds.
In a preferred embodiment, in the explant collecting step, the length of the explant is 4-18cm of newly germinated young stems.
In a preferred embodiment, in the step of explant sterilization, the young stems are washed by washing powder, washed by running water, transferred to a super clean bench, gently shaken by 0.05% carbendazim solution for 10 minutes, sterilized on the surface of 75% alcohol for 30 seconds, sterilized by 0.1% mercuric chloride for 3-5 minutes, and washed by sterile water for 3-5 times, each time for 2-3 minutes.
In a preferred embodiment, in the callus induction step, the callus is cultured under scattered light for 20 to 30 days at a temperature of 25 ℃ to 30 ℃ under conditions in which white granular callus grows from an upward section.
In a preferred embodiment, in the callus induction step, the callus induction medium comprises: MS culture medium, 1-5 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder and 30-40g/L sucrose, and adjusting pH to 5.8.
In a preferred embodiment, in the adventitious bud induction step, the induction conditions are scattered light culture at a temperature of 25 ℃ to 30 ℃; in the step of proliferating the adventitious bud, the proliferation condition is culture by scattering light, and the culture temperature is 25-30 ℃.
In a preferred embodiment, in the adventitious bud induction step, the adventitious bud induction medium includes: MS culture medium, 2-4 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder, 30-40g/L sucrose and 0.3-1g/L active carbon, and the pH value is adjusted to 5.8.
In a preferred embodiment, in the adventitious bud propagation step, the adventitious bud propagation medium includes: MS culture medium, 0.5-2 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder and 30-40g/L sucrose, and adjusting pH to 5.8.
In a preferred embodiment, in the adventitious bud elongation step, the adventitious bud elongation medium includes: MS culture medium, 5g/L agar powder and 30-40g/L cane sugar, and the pH value is adjusted to 5.8.
In a preferred embodiment, in the adventitious bud rooting step, the adventitious bud number 1 rooting medium: MS culture medium, 2-3mg/L indolebutyric acid, 5g/L agar powder and 10-30g/L sucrose, and adjusting the pH value to 5.8; the No. 2 rooting culture medium is MS culture medium, 7g/L agar powder and 10-30g/L cane sugar, and the pH value is adjusted to 5.8.
Compared with the prior art, the novel method for obtaining the cymbidium sinense tissue culture seedlings has the following advantages:
(1) the invention finds that the spotted-blue is extremely easy to brown in the experimental process, so that the bud points and stem sections are effectively protected by keeping the leaf sheaths attached to the buds when the tender stems are cleaned and disinfected, the damage to the bud points and stem sections caused by direct cleaning and disinfection after the leaf sheaths are completely stripped is avoided, and the treated buds are strong and easy to grow.
(2) In addition, the invention keeps the leaf sheaths attached with the buds to clean and disinfect the buds and the stem sections, the whole section is grown with callus, and after all the leaf sheaths are directly cleaned and disinfected, the callus is only grown on the section of the medulla, thereby effectively improving the axillary bud germination rate and the section callus induction rate and accelerating the axillary bud germination time.
(3) According to the invention, the carbendazim solution with 0.05% of effective components is added before the conventional disinfection for 10 minutes, compared with the conventional disinfection method, the pollution rate is reduced by about 10%, and the failure of dedifferentiation of explants in the subsequent treatment process is avoided.
(4) The invention obtains the tissue culture seedling of the spotted orchid by inducing the callus on the section of the stem section and then inducing the adventitious bud, rooting and other processes, thereby enriching the rapid propagation method of the tissue culture seedling of the spotted orchid.
(5) The invention obtains the tissue culture seedling of the spotted orchid by inducing the callus on the section of the stem, so that the same explant can be proliferated through axillary buds and the section, thereby greatly improving the breeding efficiency and accelerating the development of related fields.
(6) The invention adopts a two-step rooting method, the induced root is strong and healthy, the number of the root is reasonable, and the transplanting survival rate reaches 100 percent.
(7) The tissue culture seedling of the spotted orchid obtained by the stem section has vigorous growth, and keeps the original fragrance of the rice dumpling.
(8) The method has simple process flow, and the adopted culture medium components are easy to obtain, thereby having great application value.
Drawings
FIG. 1 is a process for explant collection and preparation;
from left to right, the drawings are as follows: the leaf sheaths attached to the buds of the spotted-orchid growing outdoors and the top ends of the stems collected just now are reserved and torn off.
FIG. 2 shows the callus induction and proliferation process of the example;
wherein, the left side picture is: cutting into stem segments with buds, wherein the right picture is as follows: germinating bud and cutting surface for healing.
FIG. 3 is the adventitious bud induction and proliferation process of the example;
wherein, the upper left diagram is: the section of the callus is cut to grow bud points, and the left lower graph is as follows: shoots further developed and the right panels are: the bud elongates.
FIG. 4 is the tissue culture seedling of Banana prepared by the example;
wherein, the left side picture is: rooting tissue culture seedling of herba Sphaeranthi Indici; the right side is as follows: strong, extended and developed root system.
FIG. 5 shows callus induced in example and comparative example 1;
wherein, the left side picture is: example induced callus: the stem segments are green, the germinated buds are strong, and the callus is quickly formed; the right side is as follows: comparative example 1 induced callus: stem sections become brown, do not germinate or germinate with weak buds, and calluses are difficult to form.
FIG. 6 is a group of prepared Zealand tissue culture seedlings of comparative example 2;
wherein, the left side picture is: tissue culture seedlings of the non-rooted cymbidium; the right side is as follows: expanding and healing, and the stem base without growing root.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
The culture medium components used in the method for quickly breeding the tissue culture seedlings of the cymbidium sinense provided by the invention can be purchased from the market;
the biological polarity direction refers to: in the culture sheaths, shoots, young leaves and young roots, auxin is transported from the morphologically upper end to the morphologically lower end; in this context, callus induction is carried out by placing callus induction medium vertically from the sheath, shoot, young leaf to young root in the direction of biological polarity.
Examples
Explant collection: selecting a stock plant which grows strongly and has no plant diseases and insect pests in the morning of sunny days for more than 2 continuous days, collecting faint yellow and newly-germinated tender stems of herba Zealanae 4-18cm, and cutting off leaf sheaths attached to bud parts to serve as explants; the collection and preparation process of the specific explants is shown in the left-to-right and the first 3 groups of the figure 1.
Cleaning and disinfecting explants: washing tender stems one by one under running water, paying attention to poking leaf sheaths, washing dust in the leaf sheaths clean, then putting the tender stems into a beaker containing detergent, gently rubbing the surfaces of the tender stems, gently rubbing the tender stems under running water, washing off foams on the surfaces of stem segments, then transferring the tender stems into a super clean workbench, gently shaking a 0.05 percent carbendazim solution for 10 minutes, disinfecting the surfaces of 75 percent alcohol for 30 seconds, disinfecting the surfaces of 0.1 percent mercuric chloride for 5 minutes, washing the tender stems with sterile water for 3 to 5 times, and draining the water after 2 to 3 minutes each time.
Explant preparation and callus induction: stripping leaf sheaths attached to buds, cutting off the base parts of tender stems by 1-5mm, cutting the top ends of the tender stems until the top buds are exposed, cutting the tender stems into 2-4 stem segments with buds, vertically placing the stem segments with the buds into a callus induction culture medium according to the biological polarity direction for culture, inserting an incision at one end into the culture medium, upwards cutting an incision at the other end, and culturing by scattering light, wherein the culture temperature is 25-30 ℃; the callus induction culture medium comprises: MS culture medium, 1 mg/L6-benzylaminopurine, 0.1mg/L alpha-naphthylacetic acid, 30g/L cane sugar and 5g/L agar powder, and adjusting pH to 5.8. The specific observation is shown in fig. 2.
Adventitious bud induction: subculturing the stem segments with the callus in adventitious bud induction culture medium for 1-2 times, each time for 25 days, and culturing at 25-30 deg.C under scattered light; further proliferating the cut granular callus, and further growing green bud points on the surface and further growing into tender buds; the adventitious bud induction culture medium comprises: MS culture medium, 3 mg/L6-benzylaminopurine, 0.3mg/L alpha-naphthylacetic acid, 0.5g/L active carbon, 30g/L cane sugar and 5g/L agar powder, and adjusting the pH value to 5.8.
Adventitious bud proliferation: cutting off leaves with a length of more than 0.5cm from stem segments with tender shoots, cutting into 2-4 pieces, transferring to adventitious bud proliferation culture medium to induce more adventitious buds, each time for 20 days; the culture conditions and the culture medium are different for inducing adventitious buds. The specific observations of adventitious bud induction and proliferation are shown in FIG. 3.
Adventitious bud elongation: transferring the adventitious bud to an adventitious bud elongation culture medium, and culturing by scattering light at 25-30 deg.C to elongate the adventitious bud to more than 1.5 cm; the adventitious bud elongation culture medium comprises: MS culture medium, 30g/L sucrose and 5g/L agar powder, and adjusting the pH value to 5.8.
Adventitious bud rooting: transferring the elongated adventitious bud into a No. 1 rooting culture medium for culturing for 1 day, then transferring into a No. 2 rooting culture medium for culturing for 20-30 days, and performing illumination culture at the culture temperature of 25-30 ℃ in the adventitious bud No. 1 rooting culture medium: MS culture medium, 3mg/L indolebutyric acid, 10g/L sucrose and 5g/L agar powder, and adjusting the pH value to 5.8; the No. 2 rooting culture medium is an MS culture medium, 30g/L of sucrose and 7g/L of agar powder, and the pH value is adjusted to 5.8. The final herba Epilobii tissue culture seedling is shown in FIG. 4, and the adventitious bud has 5-10 roots with length of 3-5cm, strong and white root tip.
Comparative example 1
Explant collection: selecting a stock plant which grows strongly and has no plant diseases and insect pests in the morning of sunny days for more than 2 continuous days, collecting faint yellow and newly-germinated tender stems of herba Zealanae 4-18cm, and cutting off leaf sheaths attached to bud parts to serve as explants; specific explants are shown in the right-most panel of FIG. 1.
Cleaning and disinfecting explants: peeling off leaf sheaths, placing the leaf sheaths under running water to gently scrub the surfaces of tender stems, then placing the tender stems into a beaker containing detergent, gently scrubbing the tender stems under the running water to flush out foams on the surfaces of stem segments, then transferring the stem segments to a super clean workbench, gently shaking a carbendazim solution with 0.05 percent of effective components for 10 minutes, disinfecting the surfaces of 75 percent of alcohol for 30 seconds, disinfecting the surfaces of the stem segments for 5 minutes by 0.1 percent of mercuric chloride, and then washing the stem segments for 3 to 5 times by using sterile water, wherein each time lasts for 2 to 3 minutes. And (5) draining the water.
The rest of the steps are the same as the example, and the specific callus conditions are shown on the right side of FIG. 5.
Comparative example 2
Adventitious bud rooting: directly transferring the elongated adventitious bud into a rooting culture medium for culturing for 20-30 days, and culturing under illumination at the culture temperature of 25-30 ℃ in the adventitious bud rooting culture medium: MS culture medium, 3mg/L indolebutyric acid, 10g/L sucrose and 5g/L agar powder, and adjusting the pH value to 5.8. The finally prepared herba Epilobii tissue culture seedling is shown in FIG. 6, and the adventitious bud base turns yellow, expands and does not sprout adventitious roots.
The other steps are the same as the embodiment, and the specific rooting condition is shown in figure 6.
Test examples
The method of the embodiment and the comparative examples 1-2 is used for carrying out the rapid propagation of the cymbidium tissue culture seedlings, and the specific data indexes are shown in the table 1:
TABLE 1 comparison of browning rate, axillary bud germination rate, callus induction rate and callus differentiation rate for different culture methods
| Examples | Comparative example 1 | Comparative example 2 | |
| Browning rate | 0％ | 100％ | 0％ |
| Rate of axillary bud sprouting | 100％ | 21.43％ | 100％ |
| Callus induction rate | 100％ | 40.91％ | 100％ |
| Differentiation rate of callus | 37.5％ | 5％ | 30％ |
| Rooting rate | 100％ | 100％ | 0％ |
As shown in the above table, in comparison with the examples, comparative example 1 was such that the leaf sheaths were completely peeled off when the explants were washed and sterilized. Because the protective action of the leaf sheath is not provided, the delicate bud points and stem segments are easily damaged in the disinfection process, thereby browning the spotted orchid. The browning process can inhibit the activity of enzyme and seriously affect the dedifferentiation of the explant. Moreover, leaf sheaths are completely stripped, direct cleaning and disinfection are carried out, and the callus grows only on the section of the medulla part, so that the adventitious bud and the axillary bud germination rate are greatly reduced.
Compared with the embodiment, in the rooting step of the comparative example 2, the traditional rooting culture medium is adopted for one-time completion, and the tender adventitious buds are not rooted, but in the scheme of the application, the traditional rooting culture medium is adopted for treatment firstly, and then the rooting culture medium is transferred into the hormone-free culture medium for rooting culture for two times, so that the rooting rate is greatly improved.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A method for quickly breeding tissue culture seedlings of spotted orchid is characterized by comprising the following steps:
(1) explant collection: selecting a stock plant which grows strongly and has no plant diseases and insect pests, collecting 4-18cm of the top part of a faint yellow and newly germinated tender stem of the cymbidium, cutting off other leaf sheaths, and only keeping the leaf sheath attached to a bud point part as an explant;
(2) explant disinfection: cleaning, disinfecting and sterilizing the top end of the variegated orchid;
(3) explant preparation and callus induction: stripping leaf sheaths attached with buds, cutting off the base parts of tender stems by 1-5mm, cutting the top ends of the tender stems to expose terminal buds, cutting the terminal buds into stem sections with buds, vertically placing the stem sections into a callus induction culture medium according to the biological polarity direction for culturing for 20-30 days, inserting a cut at one end into the culture medium, enabling a cut at the other end to be upward, forming callus on an upward section at the stage, and extending axillary buds;
(4) adventitious bud induction: subculturing the cut stem segment with callus in adventitious bud induction culture medium for 2-4 times, each time for 20-30 days, further proliferating the cut granular callus, growing green bud points on the surface and further developing into tender buds, and further proliferating axillary buds at this stage;
(5) adventitious bud proliferation: cutting off leaves with a length of more than 0.5cm from stem segments with tender shoots, cutting into 2-4 segments, transferring to adventitious bud proliferation culture medium, and performing adventitious bud proliferation for 20-30 days each time;
(6) adventitious bud elongation: transferring the adventitious bud to an adventitious bud elongation culture medium, and culturing by scattering light at 25-30 deg.C to elongate the adventitious bud to more than 1.5 cm;
(7) adventitious bud rooting: transferring the elongated adventitious bud into a No. 1 rooting culture medium, culturing for 1-3 days, transferring into a No. 2 rooting culture medium, culturing for 20-30 days, and culturing under illumination at 25-30 deg.C to obtain 5-10 adventitious buds.
2. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the explant collecting step, the length of the explant is 4-18cm of a newly germinated tender stem.
3. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the step of explant disinfection, the tender stem is washed by washing powder, washed by running water and transferred to a super clean workbench, a carbendazim solution with 0.05% of active ingredients is gently shaken for 10 minutes, the surface of 75% alcohol is disinfected for 30 seconds, 0.1% mercuric chloride is disinfected for 3-5 minutes, and then the tender stem is washed by sterile water for 3-5 times, and each time lasts for 2-3 minutes.
4. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the callus induction step, the induction conditions are that scattered light culture is carried out for 20-30 days, the culture temperature is 25-30 ℃, and white granular callus grows from an upward section at the moment.
5. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the callus induction step, the callus induction medium comprises: MS culture medium, 1-5 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder and 30-40g/L sucrose, and adjusting pH to 5.8.
6. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the step of inducing the adventitious bud, the inducing condition is that scattered light is used for culturing, and the culturing temperature is 25-30 ℃;
in the step of proliferating the adventitious bud, the proliferation condition is culture by scattering light, and the culture temperature is 25-30 ℃.
7. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the adventitious bud induction step, the adventitious bud induction medium includes: MS culture medium, 2-4 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder, 30-40g/L sucrose and 0.3-1g/L active carbon, and the pH value is adjusted to 5.8.
8. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the adventitious bud propagation step, the adventitious bud propagation medium includes: MS culture medium, 0.5-2 mg/L6-benzylaminopurine, 0.1-0.5mg/L alpha-naphthylacetic acid or indoleacetic acid, 5g/L agar powder and 30-40g/L sucrose, and adjusting pH to 5.8.
9. The method for rapid propagation of tissue culture seedlings of spotted orchid of claim 1,
in the adventitious bud elongation step, the adventitious bud elongation medium includes: MS culture medium, 5g/L agar powder and 30-40g/L cane sugar, and the pH value is adjusted to 5.8.
10. The method for tissue culture seedling rapid propagation of the cymbidium sinense as claimed in claim 1, wherein in the step of rooting the adventitious bud, the rooting culture medium of the adventitious bud No. 1: MS culture medium, 2-3mg/L indolebutyric acid, 5g/L agar powder and 10-30g/L sucrose, and adjusting the pH value to 5.8; the No. 2 rooting culture medium is MS culture medium, 7g/L agar powder and 10-30g/L cane sugar, and the pH value is adjusted to 5.8.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN114847161A (en) * | 2022-05-12 | 2022-08-05 | 中国热带农业科学院香料饮料研究所 | Method for improving transplanting survival rate and fragrance of leaf tissue culture seedlings of cymbidium sinense |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114097611A (en) * | 2021-10-29 | 2022-03-01 | 中国热带农业科学院橡胶研究所 | Low-cost method for breeding cymbidium maculatum tissue culture seedlings |
| CN115119747A (en) * | 2022-04-30 | 2022-09-30 | 中国热带农业科学院海口实验站 | Leaf tissue culture medium of cymbidium maculatum and tissue culture rapid propagation method |
| CN114847161A (en) * | 2022-05-12 | 2022-08-05 | 中国热带农业科学院香料饮料研究所 | Method for improving transplanting survival rate and fragrance of leaf tissue culture seedlings of cymbidium sinense |
| CN116267618A (en) * | 2023-03-30 | 2023-06-23 | 中国热带农业科学院橡胶研究所 | Tissue culture and rapid propagation method by using leaves of cymbidium sinense |
| CN116267618B (en) * | 2023-03-30 | 2024-03-08 | 中国热带农业科学院橡胶研究所 | Tissue culture and rapid propagation method by using leaves of cymbidium sinense |
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