CN110192527B - Tissue culture method of aromatic plant Linglingqing - Google Patents
Tissue culture method of aromatic plant Linglingqing 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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention provides a tissue culture method of aromatic plant bellflower azure, which comprises the following steps (a 1): (a1) taking the adventitious bud of the Bell-glorybower, and culturing on a rooting culture medium to obtain a regenerated seedling; the rooting culture medium contains 0.1-2.0mg/L IAA or 0.1-2.0mg/L IBA or 0.1-2.0mg/L LNAA. According to the invention, the plant growth regulator with specific types and concentration is added into the culture medium used in each stage of tissue culture of the chimerican green, so that the propagation coefficient of the chimerican green is high, the speed is high, a large number of regenerated seedlings can be obtained in a short time, the propagation of the chimerican green is facilitated, and the large-scale production of the chimerican green is promoted.
Description
Technical Field
The invention relates to a tissue culture method of aromatic plant bellflower.
Background
Lindley bell (Anaphhalis hancockii Maxim) is a plant of the genus Anaphhalis (Compositae), the whole plant has an aromatic odor, and is one of important perfume plants. The application value of the bellflower green is high, and the whole plant can extract aromatic oil; can also be used as ornamental plant, is a natural dried flower material, and has aromatic odor for years; the medicine can be used for medicine, and the whole herb is used as the medicine, and has the efficacy of clearing heat and removing toxicity. In addition, it also has insecticidal effect.
People's research on the azure plants focuses on the extraction and analysis of the chemical components of plants such as the yellow-gland azure, the pearl azure, the milky-white azure, the azure and the like, and no research report on the aspect of tissue culture is found.
Disclosure of Invention
The invention aims to provide a tissue culture method of aromatic plant bellflower.
The tissue culture method of the aromatic plant bellflower azure provided by the invention comprises the following steps (a 1):
(a1) taking the adventitious bud of the Bell-glorybower, and culturing on a rooting culture medium to obtain a regenerated seedling;
in step (a1) of the above method, the rooting medium contains 0.1-2.0mg/L IAA, 0.1-2.0mg/L IBA, or 0.1-2.0mg/L LNAA, and the rooting medium may specifically contain 0.1mg/L IAA, 0.1-0.5mg/L IBA, or 0.1mg/L NAA;
(a1) the time of the culturing may be 45 d;
the rooting medium also contains an MS solid medium.
In the above tissue culture method, the adventitious bud may be an adventitious bud prepared by (b1) or (b2) below, or a multiple bud prepared by (b3) or (b4) below:
(b1) taking petiole explants of the fragrant bell green, and culturing on an adventitious bud induction culture medium (adventitious bud induction culture medium A) to obtain adventitious buds;
(b2) taking a leaf explant of the fragrant bell green, and culturing on an adventitious bud induction culture medium (adventitious bud induction culture medium B) to obtain an adventitious bud;
(b3) taking a petiole explant of the Bell-lily-of-the-valley, and culturing on a petiole cluster bud induction culture medium to obtain cluster buds;
(b4) taking a leaf explant of the Bell-lily buds, and culturing on a leaf cluster bud induction culture medium to obtain cluster buds;
in the step (b1), the adventitious bud induction medium A contains 1.0-4.0mg/L TDZ, and the adventitious bud induction medium A may specifically contain 2.0mg/L TDZ;
or the adventitious bud induction culture medium A contains 1.0-4.0mg/L KT, and the adventitious bud induction culture medium A can specifically contain 3.0mg/L KT;
the adventitious bud induction medium A also contains an MS solid medium.
In the (b1), the culturing time may be 45 d.
In the step (B2), the adventitious bud induction medium B contains 1.0-4.0mg/L TDZ, and the adventitious bud induction medium B may specifically contain 4.0mg/L TDZ;
or the adventitious bud induction culture medium B contains 1.0-4.0mg/L KT, and the adventitious bud induction culture medium B can specifically contain 3.0mg/L KT;
the adventitious bud induction medium B also contains an MS solid medium.
In the (b2), the culturing time may be 45 d.
In the above (b3), the petiole cluster shoot induction medium may contain 1.0-4.0mg/L of 6-BA, and the petiole cluster shoot induction medium may contain 2.0mg/L of 6-BA;
or the petiole clump bud induction culture medium contains 1.0-4.0mg/L of 2,4-D and 1.0-4.0mg/L of KT, and the petiole clump bud induction culture medium can specifically contain 1.0mg/L of 2,4-D and 4.0mg/L of KT;
the petiole clump bud induction culture medium also contains an MS solid culture medium.
In the (b3), the culturing time may be 45 d.
In the above (b4), the leaf cluster shoot induction medium may contain 1.0-4.0mg/L of 6-BA, and the leaf cluster shoot induction medium may specifically contain 2.0mg/L of 6-BA; or
The leaf cluster bud induction culture medium contains 1.0-4.0mg/L of 2,4-D and 1.0-4.0mg/L of KT, and specifically can contain 1.0mg/L of 2,4-D and 4.0mg/L of KT;
the leaf clumpy bud induction culture medium also contains an MS solid culture medium.
In the (b4), the culturing time may be 45 d.
In the above tissue culture, before the adventitious bud is subjected to rooting culture, the adventitious bud prepared in (b1) or (b2) or the multiple bud prepared in (b3) or (b4) may be subjected to propagation culture;
the proliferation culture is carried out on a proliferation culture medium;
the proliferation culture medium contains 2.0-4.0 mg/L6-BA and 1.0-4.0mg/L NAA, and the proliferation culture medium can specifically contain 3.0 mg/L6-BA and 1.0mg/L NAA;
the multiplication culture medium also contains an MS solid culture medium;
the time for the propagation culture may be 45 d.
When the multiple shoots obtained in (b3) and (b4) are subjected to propagation culture, the multiple shoots need to be cut into individual shoots and then subjected to propagation culture.
In the above (b1), (b2), (b3) and (b4), the method for preparing the petiole and leaf explant is as follows:
taking seeds of the fragrant bell green, culturing the seeds on a seed germination culture medium to obtain seedlings, and taking leaves and petioles of the fragrant bell green seedlings as explants;
the seed germination culture medium is an MS solid culture medium.
The culture time may be 20-30 days, and specifically may be 30 days.
The tissue culture method of the Bell jar caraway can further comprise the operation of hardening and transplanting the obtained regenerated seedlings.
The invention can further comprise the operation of preparing callus from petioles and leaf explants of the Bell-lily buds, wherein the callus can be further cultured into adventitious buds or used for extraction of aromatic substances and the like.
The operation may specifically be:
(c1) taking a petiole explant of the fragrant bell green, and culturing on a callus induction culture medium A to obtain a callus;
(c2) taking a leaf explant of the fragrant bell green, and culturing on a callus induction culture medium B to obtain a callus;
in the step (c1), the callus induction medium A contains 1.0-4.0mg/L2,4-D, and the callus induction medium A may specifically contain 1.0mg/L2, 4-D;
or callus induction culture medium A contains 1.0-4.0mg/L2,4-D and 1.0-4.0mg/LKT, and the callus induction culture medium A specifically contains 4.0mg/L2,4-D and 2.0mg/L KT;
the callus induction medium A also comprises an MS solid medium.
The time for culturing on callus induction medium A may be 45 d.
In the step (c2), the callus induction medium B contains 1.0-4.0mg/L2,4-D, and the callus induction medium B may specifically contain 1.0mg/L2, 4-D; or
Callus induction culture medium B contains 1.0-4.0mg/LKT and 1.0-4.0mg/L2,4-D, and the callus induction culture medium B specifically contains 4.0mg/L2,4-D and 2.0mg/L KT;
the callus induction medium B also comprises an MS solid medium.
The time for culturing on callus induction medium B may be 45 d.
The culture conditions are that the culture temperature is 25-26 ℃, the illumination time is 12h/d, and the illumination intensity is 2000-3000 lx.
The invention also provides a kit for tissue culture of the chimerica bell caraway, which comprises any one of the following components (e1) - (e 9):
(e1) the rooting medium;
(e2) the rooting culture medium and the adventitious bud induction culture medium A;
(e3) the rooting culture medium and the adventitious bud induction culture medium B;
(e4) the rooting culture medium and the petiole cluster bud induction culture medium;
(e5) the rooting culture medium and the leaf cluster bud induction culture medium;
(e6) the rooting medium, the adventitious bud induction medium A and the proliferation medium;
(e7) the rooting culture medium, the adventitious bud induction culture medium B and the proliferation culture medium;
(e8) the rooting medium, the petiole cluster bud induction medium and the proliferation medium;
(e9) the rooting culture medium, the leaf cluster bud induction culture medium and the proliferation culture medium;
the application of the tissue culture method or the kit in the propagation of the fragrant plant of the fragrant bell green also belongs to the protection scope of the invention.
According to the invention, the plant growth regulator with specific types and concentration is added into the culture medium used in each stage of tissue culture of the chimerican green, so that the propagation coefficient of the chimerican green is high, the speed is high, a large number of regenerated seedlings can be obtained in a short time, and the transplanting survival rate of the regenerated seedlings reaches 100%, thereby being beneficial to propagation of the chimerican green and promoting large-scale production of the chimerican green.
Drawings
FIG. 1 shows a seedless seedling, wherein A is a seedless seedling cultured for 4 days, and B is a seedless seedling cultured for 20 days;
FIG. 2 shows the effect of MS +6-BA2.0mg/L medium on adventitious bud induction, where A is leaf explant after 10 days of culture and B is multiple shoots generated when leaf explant is induced for 45 days of culture.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The composition and content (mg/L) of MS medium (pH 5.8) used in the following examples were:
examples
1. Material acquisition
The material used in the experiment is the seeds of the Bell-jar caraway, which is collected from the mountain of Beijing east Lingshan (40 degrees 1 '51' north latitude, 115 degrees 27 '19' east longitude) in Hippocampus at the altitude of 2100-.
2. Seed disinfection treatment and sterile seedling culture
The optimal disinfection scheme is as follows: alcohol sterilization for 30s, 10% hydrogen peroxide sterilization for 15min, and the pollution rate is 0.
Inoculating the disinfected Tulipa-Georgi seeds in an MS culture medium, and beginning to germinate about 4 days; the germination rate reaches 98.0% after about 15 days, and 2 cotyledons grow out of each seed; when the culture time is up to 20 days, 1-2 true leaves are generated (figure 1).
3. Induction of callus
3.1 Induction Effect of 2,4-D
The leaves and petioles of the seedlings of the bellflower turfgrass cultured for 30 days are taken as explants. And respectively inoculating the explants into MS culture medium, inoculating 5 explants in each bottle, inoculating 50 explants for each treatment, repeating the steps for 3 times, counting the induction conditions of different explants after 45 days, and calculating the induction rate.
Inoculating the petiole explant of the fragrant bell green to 2,4-D (1.0, 2.0, 3.0 and 4.0mg/L) MS culture media with different concentrations, observing the change and expansion of the explant after 5 days, obviously expanding the petiole when the petiole is cultured for 10 days, and generating granular yellow callus at the two ends of the petiole when the petiole is cultured for 20 days. The callus is continuously increased by continuous culture, mostly in a loose state and in a yellow-green color. The induction rate is 23.33% -75.33% at 45 d. Wherein when the concentration of the 2,4-D is 1.0mg/L, the induction rate of the petiole callus reaches 75.33%.
Inoculating the leaves of the Bell-jar Clerodendrum Bunge as explants in MS culture medium added with 2,4-D (1.0, 2.0, 3.0, 4.0mg/L) with different concentrations, and culturing for 20 days to obtain granular yellow callus around the leaves. The inductivity is 32.67-98.00% at 45 d. When the concentration of the 2,4-D is 1.0mg/L, the induction rate of the callus is as high as 98.00 percent.
Combined induction of Effect of 3.2, 2,4-D and KT
Inoculating the Linglorybower leaf explants to MS culture media added with 2,4-D and KT with different concentrations, analyzing statistical results to obtain that the 2,4-D and KT with different concentrations can induce callus at different degrees, and the induction rate of the callus is higher. When the concentration of 2,4-D was 2.0mg/L and the concentration of 2.0mg/L, KT was 2.0mg/L, the induction rate of 45D calli was the highest, 94.67% (Table 1). The callus is in green and loose state.
When the petiole explants of bellflower azure were inoculated into MS +2,4-D (1.0, 2.0, 3.0, 4.0mg/L) and KT (1.0, 2.0, 3.0, 4.0mg/L) media for culture, it was found that the induction rate of 45D petiole callus reached up to 85.33% at a concentration of 2.0mg/L, where 2,4-D was 4.0mg/L, KT (Table 1).
TABLE 1 Induction Effect of different concentrations of 2,4-D in combination with KT on callus
Note: data to
Meaning, non-identical letters indicate that the difference is significant. P is less than 0.05.
4. Induction of adventitious buds
4.1 Induction Effect of TDZ on adventitious buds
Inoculating the petiole explants into MS culture media added with TDZ (1.0, 2.0, 3.0 and 4.0mg/L) with different concentrations, and continuously increasing emerald green granular calluses when the seedlings are cultured for 20 days, so that green bud points appear, and the bud points gradually form adventitious buds. The induction rate is 28.20-46.67% at 45 d. When the concentration of TDZ reaches 2.0mg/L, the induction rate of the adventitious bud reaches the highest value, namely 46.67%.
The leaf explant of the fragrant bell green is inoculated in MS culture medium added with TDZ (1.0, 2.0, 3.0 and 4.0mg/L) with different concentrations for culture, the induction rate of the adventitious bud is increased along with the increase of the concentration of the TDZ, and the induction rate is 66.02-92.65% at 45 days. When the TDZ concentration is 4.0mg/L, the adventitious bud induction rate is as high as 92.65%.
4.2 Induction Effect of KT on adventitious buds
The petiole explant of the Bellamyda chinensis is inoculated into an MS culture medium of MS + KT (1.0, 2.0, 3.0 and 4.0mg/L), and the induction rate at 45d is 32.33 to 52.34 percent. When KT reaches 3.0mg/L, the highest induction rate of adventitious buds of the petioles is 52.34%.
The leaf explants of the Bellamydomonas L are inoculated into an MS culture medium of MS + KT (1.0, 2.0, 3.0 and 4.0mg/L), the induction rate of the adventitious buds tends to increase and then decrease along with the increase of the concentration of the KT, and the induction rate at 45d is 34.00-91.96%. When the concentration of KT is 3.0mg/L, the induction rate of the adventitious bud is the highest and is 91.96%.
4.3 Induction Effect of 6-BA on Cluster buds
Inoculating the petiole explant of the fragrant bell green into MS culture media added with 6-BA (1.0, 2.0, 3.0 and 4.0mg/L) with different concentrations, when the petiole explant is cultured for 10 days, the petiole explant is obviously expanded, and when the petiole explant is cultured for 20 days, granular yellow callus and green bud points appear at the edge of the explant, so that green cluster buds are formed. The cluster bud inductivity at 45d is 13.33-58.00%. When the concentration of 6-BA is 2.0mg/L, the inductivity of the petiole cluster buds is 58.00 percent respectively.
The leaf explant of the Bellamy leaf is inoculated into MS culture media added with 6-BA (1.0, 2.0, 3.0 and 4.0mg/L) with different concentrations, and the 6-BA is found to induce the Bellamy leaf to generate cluster buds, and the induction rate of the cluster buds is higher. The cluster bud inductivity at 45 days is 28.00% -78.00%. When the concentration of 6-BA is 2.0mg/L, the induction rate of the leaf explant clumpy buds is 78.00% (FIG. 2).
4.4 Induction Effect of combination of 2,4-D and KT on Cluster buds
When the leaves of Bellamydomonas L were inoculated into MS +2,4-D (1.0, 2.0, 3.0, 4.0mg/L) + KT (1.0, 2.0, 3.0, 4.0mg/L) MS medium, it was found that clumpy buds were induced more easily at a low concentration of 2,4-D than at a high concentration, and when the concentration of 2,4-D was 4.0mg/L at a concentration of 1.0mg/L, KT, clumpy buds were cultured for 45 days with an induction rate of 87.25% (Table 2).
After 2,4-D and KT with different concentrations are added into the MS culture medium, the effect of inducing the petioles to generate the cluster buds is different, and when the concentration of the 2,4-D is 1.0mg/L, the induction rate of the cluster buds is obviously higher than that of other concentrations. When 2,4-D is 1.0mg/L, KT 4.0.0 mg/L, the induction rate of the cluster buds is 59.33 percent at the highest after 45 days of culture.
TABLE 2 Induction Effect of different concentrations of 2,4-D and KT on Cluster buds
Note: data to
Meaning, non-identical letters indicate that the difference is significant. P is less than 0.05.
5. Multiplication culture of adventitious bud
The single adventitious bud (the cluster bud prepared in step 4.3 is cut into single adventitious buds) is inoculated into MS +6-BA (2.0, 3.0, 4.0mg/L) + NAA (1.0, 2.0, 3.0, 4.0mg/L) culture medium, and the amplification coefficient of the single adventitious bud is observed and counted. After 45d culture, the result shows that cluster buds can be induced to generate in different degrees in each culture medium. The MS +6-BA3.0mg/L + NAA 1.0mg/L culture medium has the best proliferation effect, the proliferation rate is 98.67%, and each single bud can averagely amplify 6 buds. Therefore, MS +6-BA3.0mg/L + NAA 1.0mg/L is the most suitable culture medium for adventitious bud proliferation.
TABLE 3 Effect of different concentrations of 6-BA and NAA combination on adventitious bud proliferation
Note: data are expressed in x ± SD, with non-identical letters indicating significant differences. P is less than 0.05.
5. Rooting culture
Selecting about 3cm adventitious buds (cluster buds prepared in step 4.3) with normal morphology, inoculating 50 adventitious buds in the MS culture medium, repeating for 3 times, culturing for 45 days, observing rooting conditions, and counting rooting rate, rooting number and root length.
TABLE 4 Effect of different concentrations of IAA, IBA on adventitious root Induction
Note: data to
Meaning, non-identical letters indicate that the difference is significant. P is less than 0.05.
When IAA and IBA are added into the culture medium, the rooting rate is high. In the MS and IBA0.1 mg/L culture medium, the rooting rate is up to 98.67%, the average number of roots is 22.33, and the length of adventitious roots is about 3.23cm (Table 4). When 0.1mg/L NAA was added to the medium, 82.00% rooting rate could be obtained, but the rooting rate decreased significantly with the increase in the concentration.
5. Hardening and transplanting seedlings
Opening a sealing film of a triangular flask, hardening the regenerated seedlings for about 7 days, carefully flushing the culture medium at the root with tap water, and transplanting the seedlings to peat soil: perlite: and (3) putting the sand into a mixed matrix with the relative humidity of 80-90% and the temperature of about 25 ℃ for culture, wherein the leaf length is large after 30 days, the leaf stalk is elongated, and the survival rate reaches 100%.
Claims (4)
1. A tissue culture method of aromatic plant bellflower comprises the following steps (a 1):
(a1) taking the adventitious bud of the Bell-glorybower, and culturing on a rooting culture medium to obtain a regenerated seedling;
the adventitious bud is an adventitious bud prepared by the following (b1) or (b2), or a cluster bud prepared by the following (b3) or (b 4):
(b1) taking a petiole explant of the fragrant bell green, and culturing on an adventitious bud induction culture medium A to obtain an adventitious bud;
(b2) taking a leaf explant of the fragrant bell green, and culturing on an adventitious bud induction culture medium B to obtain an adventitious bud;
(b3) taking a petiole explant of the Bell-lily-of-the-valley, and culturing on a petiole cluster bud induction culture medium to obtain cluster buds;
(b4) taking a leaf explant of the Bell-lily buds, and culturing on a leaf cluster bud induction culture medium to obtain cluster buds;
(b1) (b2), (b3) and (b4), the method for preparing the petiole and leaf explant is as follows:
taking seeds of the fragrant bell green, culturing the seeds on a seed germination culture medium to obtain seedlings, and taking leaves and petioles of the fragrant bell green seedlings as explants;
(b1) in the culture medium, the adventitious bud induction culture medium A contains 1.0-4.0mg/L TDZ; or the adventitious bud induction culture medium A contains 1.0-4.0mg/L KT;
(b2) in the culture medium, the adventitious bud induction culture medium B contains 1.0-4.0mg/L TDZ; or the adventitious bud induction culture medium B contains 1.0-4.0mg/L KT;
(b3) in the medium, the petiole cluster bud induction culture medium contains 1.0-4.0mg/L of 6-BA; or, the petiole cluster bud induction culture medium contains 1.0-4.0mg/L of 2,4-D and 1.0-4.0mg/L of KT;
(b4) in the medium, the leaf cluster bud induction medium contains 1.0-4.0mg/L of 6-BA; or the leaf clumpy bud induction culture medium contains 1.0-4.0mg/L of 2,4-D and 1.0-4.0mg/L of KT;
in step (a1), the rooting medium contains 0.1mg/L IAA or 0.1-0.5mg/L IBA or 0.1mg/L NAA;
the basic culture media of the rooting culture medium, the adventitious bud induction culture medium A, the adventitious bud induction culture medium B, the petiole cluster bud induction culture medium and the leaf cluster bud induction culture medium are MS culture media;
the seed germination culture medium is an MS solid culture medium.
2. The method of claim 1, wherein: the tissue culture method further comprises the operation of performing proliferation culture on the adventitious bud or the multiple bud before (a1) performing rooting culture on the adventitious bud;
the proliferation culture is carried out on a proliferation culture medium;
the proliferation culture medium contains 2.0-4.0 mg/L6-BA and 1.0-4.0mg/L NAA;
the basic culture medium of the proliferation culture medium is an MS culture medium.
3. The method of claim 1, wherein: the tissue culture method also comprises the operation of preparing the callus by the petiole and leaf explant of the Bell-glorybower,
the operation is as follows:
(c1) taking a petiole explant of the fragrant bell green, and culturing on a callus induction culture medium A to obtain a callus;
(c2) taking a leaf explant of the fragrant bell green, and culturing on a callus induction culture medium B to obtain a callus;
the basic culture media of the callus induction culture medium A and the callus induction culture medium B are MS culture media;
in the step (c1), the callus induction medium A contains 1.0-4.0mg/L2, 4-D; or callus induction culture medium A contains 1.0-4.0mg/L2,4-D and 1.0-4.0 mg/LKT;
(c2) in the callus induction culture medium B, 1.0-4.0mg/L2,4-D is contained; or callus induction medium B contains 1.0-4.0mg/LKT and 1.0-4.0mg/L2, 4-D.
4. Use of the tissue culture method of any one of claims 1-3 for the propagation of the aromatic plant Strobilanthes cusia.
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| CN106386484A (en) * | 2016-08-30 | 2017-02-15 | 云南金碧制药有限公司 | Anaphalis bulleyana cultivation and breeding method |
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| CN106386484A (en) * | 2016-08-30 | 2017-02-15 | 云南金碧制药有限公司 | Anaphalis bulleyana cultivation and breeding method |
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| Conservation of an endemic medicinal plant, Anaphalis eliptica DC. by employing plant tissue culture technique;P.Senthilkumar等;《Journal of Applied and Natural Science》;20101231;第2卷(第1期);第17-21页，尤其是摘要、第19页左栏第19行、表1-3、第19页右栏第10段、第19页左栏第11行、第17页右栏第1行、第19页左栏第3行 * |
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