CN110663555B - Rapid propagation method of odorous jasminum grandiflorum - Google Patents
Rapid propagation method of odorous jasminum grandiflorum Download PDFInfo
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Abstract
The invention discloses a rapid propagation method of Clerodendron dubia, belonging to the technical field of plant tissue culture and comprising the following steps: (1) material acquisition and processing: selecting tender stem tips and stem segments extracted from double-petal osmidium amadori pot plants as explants; (2) inducing cluster buds; (3) the method disclosed by the invention has the advantages that the average number of cluster buds induced by each explant reaches 19.30, the average number of roots induced by rooting reaches 12.33, the rooting rate reaches 100%, the propagation efficiency is high, meanwhile, the method provides theoretical support for deep research of the Clerodendron tonkinensis, and provides technical reference for large-scale and standardized production of the Clerodendron tonkinensis.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation method of double-petal osmidium odoratum.
Background
Flos Clerodendri Japonici（Clerodendrum philippinum）Is a shrub plant of Clerodendrum of Verbenaceae, also called as Clerodendron odoratum, Clerodendron trichotomum, Mirabilis jalapa, etc. China mainly has distribution in Guizhou, Yunnan, Fujian, Taiwan, Guangxi and Guangdong, Laos, Thailand, Cambodia and other Asian tropical and subtropical regions, and has normalized growth in Maoliyi, Hawaii and other places; the whole plant height of the plant is 50-120 cm. The strong-petal smelly jasmine is naturally grown and mostly distributed beside a stream or under a forest, and is also artificially cultivated to be used as an ornamental plant.
The double-petal smelly jasmine has fragrance because the corolla is red, light red or white, has ornamental value and can be used as an ornamental plant. The medicine can be used as root and leaf medicine, has mild and bitter taste, and is used for treating various human diseases; or extracting volatile oil from stem and leaf, and making into daily spice. The whole plant contains flavonoid glycoside, phenols, saponin and tannin, and has effects of dispelling pathogenic wind, promoting diuresis, eliminating phlegm, relieving cough, promoting blood circulation and detumescence. Can be used for treating bronchitis, eczema, skin pruritus, rheumatic arthritis, tinea pedis, edema, and leucorrhea.
At present, researches on the odorous double-petal jasmine are few, the research direction mainly comprises extraction and separation of effective components in a plant body and content detection, and no report is found on researches on the tissue culture aspect of the odorous double-petal jasmine. Chinese patent application No. CN201711084711.1, entitled tissue culture and propagation method of clerodendrum bungei, discloses a tissue culture and rapid propagation method of clerodendrum bungei of Clerodendrum of Verbenaceae, although the species and Clerodendron odoriferum Thunb of Clerodendrum of Verbenaceae are all plants of Clerodendron of Verbenaceae, but the species and Clerodendron odoriferum Thunb belong to completely different species, the physiological characteristics of the species and the Clerodendron odoriferum Thunb are different, moreover, the number of cluster buds induced by the method is only 10 at most, the bud induction rate is only 90% at most, furthermore, the inventor of the application proves through test results that the method is used for rapid propagation of Clerodendron odoriferum Thunb, the number and the induction rate of the cluster buds are lower, and the propagation efficiency is to be improved.
As mentioned above, the double-petal smelly jasmine has great ornamental value and medicinal value, but the wild resource is limited, and the natural growth cycle of the whole plant is longer. In order to expand the production, shorten the breeding cycle of the osmidium odoratum plant and meet the market demand, it is necessary to research the rapid breeding method, improve the breeding efficiency and reduce the rapid breeding cost.
Disclosure of Invention
The invention aims to provide a rapid propagation method of double-petal osmidium brevifolium to solve the problems.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a rapid propagation method of double-petal osmidium brevifolium comprises the following steps in sequence:
(1) material acquisition and processing: selecting tender stem tips and stem segments extracted from a double-petal osmidium odoratum pot plant as explants, carrying out aseptic treatment to obtain aseptic explants, then inoculating the explants to a culture medium, and placing the culture medium in a constant-temperature culture room to culture a large number of aseptic explants for inducing cluster buds;
(2) inducing cluster buds: under the aseptic condition, cutting terminal buds and stem segments of Clerodendron odoriferum Duck, inoculating in a culture medium for inducing cluster buds, wherein the adopted culture medium is MS culture medium + ZT1.0-3.0mg/L or MS culture medium + KT1.0-3.0 mg/L;
(3) rooting culture: shearing the strong multiple shoots obtained in the step (2) to perform rooting culture to obtain tissue culture seedlings; the adopted culture medium is 1/2MS culture medium + NAA0.1-0.3 mg/L;
(4) hardening and transplanting seedlings: and (4) hardening and transplanting the tissue culture seedlings obtained in the step (3) when the tissue culture seedlings grow to 3-5 cm.
As a preferred technical scheme: in the step (1), the aseptic processing method comprises the following steps: removing impurities on the surface of an explant, washing under running water, then disinfecting for 1min by 75% alcohol, washing for 3 times by sterile water, disinfecting for 6min by 0.1% mercuric chloride solution, washing for 5 times by sterile water, sucking water by filter paper, and shearing off black necrotic tissues by sterile scissors.
As a preferred technical scheme: in the step (1), the culture medium is MS + NAA0.1mg/L + 6-BA2.0mg/L. In the MS + NAA0.1mg/L +6-BA2.0mg/L culture medium, the bud growth vigor is fast, and sterile materials such as terminal buds and stem segments can be cultured fast, and the sterile terminal buds and stem segment explants can be obtained through repeated transfer to prepare a large number of sterile terminal bud and stem segment explants for the multiple bud induction experiment.
As a preferred technical scheme: in the step (2), the adopted culture medium is MS culture medium + ZT2.0 mg/L. On the culture medium, the number of cluster buds induced by each explant can reach 19.30 on average, the induction rate reaches 96.50%, and the generated cluster buds are strong, have large leaf amount, and are dark green and thick.
As a preferred technical scheme: in the step (2), the induction time is 21 d.
As a preferred technical scheme: in the step (3), the adopted culture medium is 1/2MS culture medium + NAA0.1mg/L. In the culture medium, the average root length of rooting induction is 2.66cm, the average number of roots is 12.33, the rooting rate is 100%, and the roots are strong and divergent.
As a preferred technical scheme: in the step (3), the culture time is 21 d.
As a preferable technical scheme: in the step (4), the seedling hardening method comprises the steps of placing the tissue culture seedlings in a light-transmitting chamber, opening a culture tank, allowing the tissue culture seedlings to receive natural light, keeping the humidity at 65-75%, and injecting a small amount of sterile water to ensure that the surface of the culture medium is just covered.
As a preferable technical scheme: in the step (4), the seedling exercising time is 2-3 d.
As a preferred technical scheme: in the step (4), the transplanting matrix is humus soil, vermiculite and river sand =1:1:1 in mass ratio.
Compared with the prior art, the invention has the advantages that: the invention explores a rapid propagation system of the Clerodendron bivalvifolia by using a modern plant tissue culture technology, and by adopting the method, the average number of cluster buds induced by each explant reaches 19.30, the average number of roots induced by the explants is 12.33, the rooting rate reaches 100%, the propagation efficiency is high, meanwhile, the invention provides theoretical support for the deep research of the Clerodendron bivalvifolia, and also provides technical reference for the large-scale and standardized production of the Clerodendron bivalvifolia.
Drawings
FIG. 1 is a diagram of multiple shoots of Choerospondias axillaris obtained in example 1 of the present invention;
FIG. 2 is a diagram showing the rooting of Mirabilis jalapa Thunb obtained in example 1 of the present invention;
FIG. 3 is a diagram showing the transplanted survival rate of Ottelia polyphylla in example 1 of the present invention.
Detailed Description
The invention will be further explained with reference to the drawings.
In this example
Test materials:
the double-petal smelly jasmine is obtained from Guangxi Baichou city. Planting the strong odorous jasmine plant in a constant-temperature culture chamber for culture, and taking the tender bud and the stem section with strong growth as explants for experiments after the plant shoots out new buds.
The culture conditions are as follows:
the axillary bud and the cluster bud are induced, proliferated and strong seedlings by using MS as a basic culture medium. 1/2MS culture medium is used in the rooting culture process, 1/2MS culture medium is MS minimal medium with half of macroelements and unchanged other components. In the minimal medium, plant growth regulators with different concentrations and different types are added according to the purpose of the experiment. The addition amounts of sucrose and agar in the culture medium are respectively 30.00g/L and 10.20g/L, the pH is adjusted to 5.85, the sterilization condition of the culture medium is 121 ℃, 101KPa, and the sterilization is carried out for 20 min.
The culture conditions in the thermostatic chamber are as follows: the illumination period is 12h/d, the illumination intensity is 1500-.
Test equipment:
SW-CJ-2FD double single-side ultra-clean operating platform, Sanyon full-automatic autoclave, MilliQbiool ultra-pure water system, culture shelf, tissue culture pot, pH meter, electronic balance, weighing paper, tweezers, scissors, filter paper, alcohol lamp, medicine spoon, beaker, glass rod, volumetric flask, culture dish, liquid-transferring gun, measuring cylinder and oven.
Test reagents:
culture medium mother liquor composition:
macroelements: KNO 3 、NH 4 NO 3 、MgSO 4 ·7H 2 O、KH 2 PO 4 、CaCl 2 ·2H 2 O；
Trace elements: h 3 BO 3 、MnSO 4 ·4H 2 O、ZnSO 4 ·7H 2 O、KI、Na 2 MoO 4 ·2H 2 O、CuSO 4 ·5H 2 O、CoCl 2 ·6H 2 O；
Iron salt: FeSO 4 ·7H 2 O、Na 2 EDTA·2H 2 O；
Organic matter: inositol, thiamine hydrochloride (VB) 1 ) Nicotinic acid (VB) 5 ) Pyridoxic acid hydrochloride and glycine.
Preparing mother liquor of 20, 100 and 200 times of macroelements, microelements, iron salts and organic matters, and storing at 4 ℃ for later use. The contents of the components of the culture medium and the manufacturers are shown in Table 1;
TABLE 1 MS culture Medium composition, content and manufacturer's Table
The 1/2MS culture medium of the invention means that macroelements in the MS minimal medium are reduced by half, and other components are unchanged.
Plant growth regulator:
naphthylacetic acid (NAA), Zeatin (ZT), 6-benzylamino adenine (6-BA), Kinetin (KT). Pre-dissolving NAA with absolute ethyl alcohol; ZT, 6-BA and KT were pre-dissolved in 0.1ml/LNaOH under heating, and prepared into 1mg/L mother liquor.
Storing NAA, 6-BA and KT mother liquor at a low temperature of 4 ℃; and storing the ZT mother liquor at a low temperature of-18 ℃ for later use.
The experimental plant growth regulators were all from sigma, germany.
Disinfectant:
75% ethanol solution, 0.1% mercuric chloride solution.
The analysis method comprises the following steps:
after the inoculation culture, the growth conditions such as culture medium pollution, culture death and the like are regularly observed. And (3) counting indexes such as the pollution rate, the death rate and the like of the material every 7 days, carrying out one-factor variance analysis on the experiment result by adopting SPSS17.0, and establishing a rapid propagation system of the jasminum grandiflorum according to the analysis result.
Contamination rate (%) = (number of contamination/number of inoculation) × 100%;
cluster bud induction (%) = (number of explants producing cluster buds/number of explants inoculated) × 100%;
rooting rate (%) = (number of rooted plants/number of inoculated plants) × 100%;
mortality (%) = (number of inoculated deaths/total number of inoculated) × 100%.
The invention adopts a direct organ generation mode, namely, the top bud and stem segment of the odorous double-petal jasmine are directly induced to generate secondary buds. Compared with the indirect generation mode, the direct organ generation mode has the advantages of reducing the probability of generating somatic cell variation, shortening the period of culturing the complete plant of the double-petal osmidium majus, and ensuring the genetic stability of the cultured regeneration plant.
Example 1:
a rapid propagation method of double-petal osmidium brevifolium is characterized by sequentially comprising the following steps:
(1) material acquisition and processing: dipping a small amount of washing powder by a brush, slightly brushing impurities on the surface of an exophyte, and washing for 2 hours under running water; sterilizing with 75% alcohol in an ultra-clean workbench for 1min, cleaning with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 6min, cleaning with sterile water for 5 times, drying with filter paper, and shearing off black necrotic tissue with sterile scissors to obtain sterile explant; inoculating the explant to MS + NAA0.1mg/L +6-BA2.0mg/L, and placing the explant in a constant-temperature culture chamber;
in the MS + NAA0.1mg/L +6-BA2.0mg/L culture medium, the bud growth vigor is fast, and sterile materials such as terminal buds and stem segments can be cultured fast, and the sterile terminal buds and stem segment explants can be obtained by repeated transfer to prepare a large amount of sterile terminal bud and stem segment explants for a bud induction experiment;
(2) inducing cluster buds: cutting the terminal bud and stem segment of Clerodendron odoriferum Thunb, inoculating into MS culture medium with different hormone concentration ratios, and inducing cluster bud. The cytokinins used are 6-BA, KT and ZT, the hormone proportion of an induction culture medium is shown in table 2, 20 explants are inoculated in each treatment, the culture temperature is 24 +/-1 ℃, observation is carried out once every 7d, the explants which are browned or infected with bacteria are treated, the number of cluster buds, the differentiation rate and the growth condition are counted after 21d, and the results are shown in table 3.
TABLE 2 different hormone concentration ratios for inducing cluster buds
The result shows that the multiple-petal smelly jasmine has good inducing effect and the whole inducing rate is more than 40 percent. The number of cluster buds induced by each explant is more than 8 on average in each experimental group, but the cluster buds induced by each culture medium and the growth conditions thereof are different: 1. the number of cluster buds induced by each explant in the culture medium 2, 3 or 6 is less than 12, the induction rate is less than 60 percent, and the induction effect is poor; 4. the number of cluster buds induced by each explant in the culture medium 7 or 8 is about 13.5, the induction rate is about 70%, and the induction effect is general; the number of cluster buds induced by each explant in the culture medium 9 is 17.57 on average, the induction rate is 87.85 percent, but the growth condition of the cluster buds is poor, so the comprehensive induction effect is poor. The average number of cluster buds induced by each explant in the No. 5 medium (MS + ZT2.0 mg/L) is the largest, 19.30, the induction rate is 96.50%, and the generated cluster buds grow robustly and have large leaf amount and strong green color. And the induction effect of the No. 5 culture medium is remarkably different from that of other culture media, and the induction effect is the best. Considering comprehensively, the optimal culture medium for inducing the cluster buds is as follows: MS medium + ZT2.0 mg/L. On the medium, the number of cluster buds induced by each explant can reach 19.30 on average, the induction rate reaches 96.50%, the generated cluster buds are strong, the leaf amount is large, the cluster buds are dark green and thick, and the photo is shown in figure 1.
TABLE 3 Effect of different hormone combinations on Cluster bud Induction
Note: a. the numbers are followed by one or several identical letters, all indicating no significant difference between the data (p < 0.05).
The number of + indicates the integrated growth of the cluster buds: the more symbols, the better the cluster buds grow.
(3) Rooting culture
Cutting strong single bud from the bud, and performing rooting culture. Rooting medium is shown in table 4 below. Each pot was inoculated with 1-2 single shoots, 10 flasks for each treatment, and the experiment was repeated 3 times. Culturing at 24 +/-1 ℃ under the condition of illumination intensity of 1000-1500 lx, observing once every 7d, and counting the average root number, rooting rate and seedling growth condition after 21d, wherein the results are shown in Table 5.
TABLE 4 concentration ratios of different hormones for root induction
The results show that: in the culture medium I and II, the average root number of the induced aseptic seedlings is small, the rooting rate is about 70 percent, the induced roots are thin and weak, brittle and tender and easy to break, and the induction effect is the worst. Culture medium No. 4 has the induced average root length of 2.88cm, the average number of roots of 8.20, the rooting rate of 100%, the induced root is thin and weak, the root diameter is small, and the induction effect is general. ③ rooting culture medium: the average root number of rooting induction is 12.33, and the induction result is obviously different from that of culture mediums of the first, second and fourth; the induced rooting rate is 100 percent, and is the same as the culture medium No. IV; the average root length of induction is 2.66cm, and the average root length has no obvious difference with the induction result of the culture medium No. IV; the roots obtained by the induction of the culture medium III are strong and divergent, and the induction effect is obviously different from that of the culture medium I, culture medium II and culture medium IV. The rooting induction result of the culture medium is shown in figure 2;
it can be seen that: the most suitable culture medium for rooting induction of the jasmine aseptic seedlings is No. three culture medium: 1/2MS culture medium + NAA0.1mg/L, in the culture medium, the average root length of rooting induction is 2.66cm, the average number of roots is maximum, 12.33, the rooting rate is 100%, the roots are strong and divergent.
TABLE 5 Effect of different hormone combinations on rooting Induction
Note: the inclusion of one or several identical letters after the number indicates no significant difference between the data (p < 0.05).
(4) Hardening and transplanting seedlings: and (4) hardening and transplanting the tissue culture seedlings obtained in the step (3) when the tissue culture seedlings grow to about 4 cm. Hardening seedlings before transplanting: placing the tissue culture seedling in a light-transmitting chamber, opening a culture tank, allowing the tissue culture seedling to receive natural light, keeping the humidity at about 70%, and injecting a small amount of sterile water to cover the surface of the culture medium. After hardening off the seedlings for 2-3d, gently washing off the culture medium adhered to the tissue culture seedling roots by using tap water, and then planting the tissue culture seedling roots into a thoroughly watered transplanting matrix (humus soil: vermiculite: river sand =1:1: 1). After transplanting, watering with thin water, and culturing the tissue culture seedling outdoors. The growth condition is observed regularly every two days and watered, and the sun is shaded and ventilated. After 21d, counting the transplanting survival rate and the growth condition of the tissue culture seedlings;
as a result: the transplanting survival rate is 100%, the seedlings grow to about 15cm, and the seedlings grow robustly as shown in figure 3.
Comparative example 1
In the step (2), the culture medium adopts MS basic culture with the addition of 1.0mg/L, NAA 0.05.05 mg/L of 6-BA, 0.2g/L of acid hydrolyzed casein and 200mg/L of yeast extract in the Chinese patent application No. CN201711084711.1 and the invention name of 'a tissue culture and propagation method of clerodendrum bungei', and the rest is unchanged, so that the following results are obtained: the average induction rate of regeneration bud is 7.9, the induction rate is only 39.5%, and the growth condition of the bud is "+", which indicates that the culture medium disclosed by the patent application is not suitable for inducing cluster buds of the jasminum grandiflorum at all.
Comparative example 2
In the step (3), the culture medium adopts the improved '1/2 MS culture medium + NAA0.05mg/L + acid hydrolyzed casein 0.2 g/L' in the Chinese patent application No. CN201711084711.1 and the invention name 'a tissue culture and propagation method of clerodendrum bungei', and the rest is unchanged, so that the results are as follows: the average number of the induced roots is 7.90, the rooting rate is 83 percent, and the average root length is 2.59 cm. It is explained that the culture medium disclosed in this patent application is not suitable for rooting culture of Onchidium brevicornum Hance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A rapid propagation method of double-petal osmidium brevifolium is characterized by sequentially comprising the following steps:
(1) material acquisition and processing: selecting tender stem tips and stem segments extracted from a double-petal osmidium amadori pot plant as explants, performing aseptic treatment to obtain aseptic explants, then inoculating the explants to a culture medium, and placing the culture medium in a constant-temperature culture room to culture a large amount of aseptic explants for inducing cluster buds, wherein the culture medium is MS + NAA0.1mg/L +6-BA2.0 mg/L;
(2) inducing cluster buds: under the aseptic condition, cutting terminal buds and stem segments of Clerodendron odoriferum Duck, inoculating in a culture medium for inducing cluster buds, wherein the adopted culture medium is MS culture medium + ZT1.0-3.0mg/L or MS culture medium + KT1.0-3.0 mg/L;
(3) rooting culture: shearing the strong multiple buds obtained in the step (2) to perform rooting culture to obtain tissue culture seedlings; the adopted culture medium is 1/2MS culture medium + NAA0.1-0.3 mg/L;
(4) hardening and transplanting seedlings: and (4) hardening and transplanting the tissue culture seedlings obtained in the step (3) when the tissue culture seedlings grow to 3-5 cm.
2. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (1), the aseptic processing method comprises the following steps: removing impurities on the surface of the explant, washing with running water, sterilizing with 75% alcohol for 1min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 6min, washing with sterile water for 5 times, draining water with filter paper, and shearing off black necrotic tissue with sterile scissors.
3. The rapid propagation method of origanum polyphylla according to claim 1, wherein the method comprises the following steps: in the step (2), the adopted culture medium is MS culture medium + ZT2.0 mg/L.
4. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (2), the induction time is 21 d.
5. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (3), the adopted culture medium is 1/2MS culture medium + NAA0.1mg/L.
6. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (3), the cultivation time was 21 days.
7. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (4), the seedling hardening method comprises the steps of placing the tissue culture seedlings in a light-transmitting chamber, opening a culture tank, allowing the tissue culture seedlings to receive natural light, keeping the humidity at 65-75%, and injecting a small amount of sterile water to ensure that the surface of the culture medium is just covered.
8. The rapid propagation method of Clerodendron bivalvis according to claim 1, which is characterized in that: in the step (4), the seedling exercising time is 2-3 d.
9. The rapid propagation method of origanum polyphylla according to claim 1, wherein the method comprises the following steps: in the step (4), the transplanting substrate is humus soil in mass ratio: vermiculite: river sand =1:1: 1.
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| CN102232359A (en) * | 2011-04-15 | 2011-11-09 | 江苏省中国科学院植物研究所 | In-vitro rapid propagation method of double-petal Jasminum sambac |
| CN105943889A (en) * | 2016-06-24 | 2016-09-21 | 郭兴芦 | Traditional Chinese medicine decoction for treating hypertension |
| CN107646690A (en) * | 2017-11-07 | 2018-02-02 | 中国计量大学 | The tissue cultures and propagation method of a kind of clerodendrum bungei |
| WO2019171374A1 (en) * | 2018-03-05 | 2019-09-12 | Bio-Bee Sde Eliyahu Ltd. | Novel methods for rearing and controlled release of biological control agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102232359A (en) * | 2011-04-15 | 2011-11-09 | 江苏省中国科学院植物研究所 | In-vitro rapid propagation method of double-petal Jasminum sambac |
| CN105943889A (en) * | 2016-06-24 | 2016-09-21 | 郭兴芦 | Traditional Chinese medicine decoction for treating hypertension |
| CN107646690A (en) * | 2017-11-07 | 2018-02-02 | 中国计量大学 | The tissue cultures and propagation method of a kind of clerodendrum bungei |
| WO2019171374A1 (en) * | 2018-03-05 | 2019-09-12 | Bio-Bee Sde Eliyahu Ltd. | Novel methods for rearing and controlled release of biological control agents |
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