CN114303946A - Efficient induction method of clerodendrum japonicum polyploid - Google Patents
Efficient induction method of clerodendrum japonicum polyploid Download PDFInfo
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Abstract
The invention discloses a high-efficiency induction method of clerodendrum japonicum polyploid. The method directly uses petioles with leaves in Clerodendron petiolum aseptic seedlings as an inducing material, and comprises the steps of preculture, polyploid induction, regeneration of adventitious buds of the petioles and polyploid flow detection. The method overcomes the problem of tissue culture browning of the clerodendrum petasitum caused by colchicine mutagenesis, can complete the callus induction and the regeneration process of petiole adventitious buds in one step, has the characteristics of simple operation, low browning rate, short regeneration period, high mutagenesis efficiency and the like, and can provide technical support for creating superior and new varieties of clerodendrum petasitum polyploids.
Description
Technical Field
The invention belongs to the technical field of plant polyploid breeding, and particularly relates to a high-efficiency induction method of clerodendrum japonicum polyploid.
Background
Clerodendron petiolatus (Clerod japonica (Thunb.) Sweet) is a plant of Clerodendron cyrtophyllum of Verbenaceae, also known as Zhuangyuanhong, and widely distributed in southern provinces of China. Clerodendrum petasitum is a traditional wild flower in China, has bright color like fire, has a full-year flowering period as long as 6 months, is called as "whooping red", and is popular with people in the south of the Linglory. Meanwhile, Clerodendron petiolatus is a traditional Chinese medicinal material in the south of Ling, is a whole herb medicine and is recorded in Zhonghua materia Medica for treating diseases such as palpitation and insomnia, carbuncle and swelling and sore, lung heat and cough and the like. In addition, Clerodendron petiolatus is a highly-resistant shrub plant, can grow in forests with a canopy density as high as 0.8, does not need to be nursed too much, and is very suitable for planting in forests in the south. Therefore, Clerodendron petiolatus has great industrial development potential as a multi-effect economic plant variety under welfare with both landscape and medicinal values.
At present, the wild cutting is mainly used for clerodendrum japonicum seedlings on the market, the artificial breeding is late, and the germplasm genetic basis is relatively narrow, so that the new clerodendrum japonicum variety is extremely deficient. Polyploid breeding has a broad prospect in the aspects of changing plant types, promoting the synthesis of pharmacodynamic ingredients, improving the stress resistance and the like, and is one of important approaches to the breeding of clerodendrum japonicum. However, no studies have been reported on induction of Clerodendron petalum polyploid, and related technologies only relate to a suspension cell propagation technology (CN 104255481A) and a tissue culture rapid propagation technology (CN 111202002A), etc. In addition, the traditional method of using seeds or stem buds as polyploid inducing materials cannot ensure the synchronous doubling of somatic chromosomes, so that the chimera rate is high, the later-stage separation operation is complicated, and the inducing efficiency is low.
Disclosure of Invention
The present invention aims to provide a method for inducing Clerodendron petorum polyploid, which is simple in operation, low in browning rate, short in regeneration cycle and high in mutagenesis efficiency, and which does not have the disadvantages of the conventional method for inducing Clerodendron petorum polyploid.
The present invention provides a high-efficiency induction method of Clerodendron petiolum, which is based on adventitious bud regeneration and uses petiole as an induction material, and the obtained polyploid Clerodendron petiolum has positive significance in landscape and drug effect utilization.
The invention relates to a high-efficiency induction method of Clerodendron slurtumnade polyploid, which comprises the following steps:
a. pre-culturing: selecting the 3 rd to 6 th completely unfolded leaves of the Clerodendron petiolus aseptic seedlings from top to bottom, cutting off petioles with leaves as an inducing material, inoculating the petioles with leaves into a pretreatment culture medium, and performing pre-culture;
b. polyploid induction: after the cut end of the petiole slightly expands, taking out the petiole with the leaf, immersing the petiole with the leaf in colchicine aqueous solution for polyploid induction culture, then taking out the petiole with the leaf, washing the petiole with sterile water for 3 to 5 times, and then sucking water by using filter paper;
c. regeneration of adventitious buds of petioles: b, inoculating the leafy petioles with leaves obtained in the step b into an induced differentiation integrated culture medium for culture, inducing callus at the cut of the petioles, continuing to culture, and differentiating adventitious buds;
d. polyploid screening: cutting off original leaves when the adventitious bud grows to a height of more than 1cm, transferring the adventitious bud into a multiplication culture medium for culture, and taking 2-3 leaves for ploidy detection after more than 3 leaves are grown and fully developed.
Preferably, the petiole with the leaf is 0.8-1.5cm long and is worn with 0.5-3cm2A blade of a size.
Preferably, the pretreatment medium: contains 2mg of 6-BA, 0.05mg of NAA, 30g of sucrose and 9g of agar per liter, and the balance is MS culture medium with pH of 5.8.
Preferably, the pre-culture in the step a is performed in a dark culture at a temperature of 23 ℃ for 5 d.
Preferably, the polyploid induction culture in the step b is a dark culture at 25 ℃ for 2-6h by immersing in colchicine aqueous solution with the concentration of 20 mg/L.
More preferably, the polyploid induction culture in the step b is submerged in colchicine water solution with the concentration of 20mg/L and is cultured for 4 hours in dark under the condition of the temperature of 25 ℃.
Preferably, the differentiation induction integrated culture medium: each liter contains 2mg of 6-BA, 0.05-0.1mg of NAA, 1g of PVP, 30g of cane sugar and 9g of agar, the rest is MS culture medium, and the pH value is 5.8.
Preferably, the culturing in step c is: culturing at 23 deg.C in dark for 5 days, and culturing under illumination intensity of 2000lx for 16 h/d.
Preferably, the proliferation medium: contains 2mg of 6-BA, 0.5mg of IBA, 30g of sucrose and 7g of agar per liter, and the balance is MS culture medium with pH of 5.8.
Preferably, the culture conditions in step d are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d.
The MS culture medium refers to a general culture medium with a formula known in the art, and the components and the configuration method are shown in Murashige T, SkoogF (1962) A recycled medium for rapid growth and bioassays with a microbacco tissue cultures, Physiologia plantarum 15: 473-.
The invention has the beneficial effects that:
the invention directly uses petioles with leaves in Clerodendron petersatumbunge aseptic seedlings as an inducing material, overcomes the problem of petiole tissue culture browning caused by colchicine mutagenesis, can complete the callus induction and petiole adventitious bud regeneration process in one step, has the characteristics of highest tetraploid induction rate of 43.3 percent, simple operation, low browning rate, short regeneration period, high mutagenesis efficiency and the like, and has important significance for clerodendrum petersatumbunge polyploid breeding.
Drawings
FIG. 1 shows the regeneration of adventitious buds of Clerodendron petiolum when cultured for 40d after the treatment with colchicine in example 1 (circled part).
FIG. 2 is a flow-type ploidy identification map (Clerodendron petioloides) of example 1.
FIG. 3 is a flow-type ploidy identification map (Clerodendron trichotomum tetraploid) of example 1.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
A method for efficiently inducing Clerodendron petiolatum polyploid, comprising the steps of:
a. pre-culturing: selecting healthy Clerodendron petioloides aseptic seedling as material, completely unfolding leaf from top to bottom, cutting leaf petiole with leaf as inducing material, inoculating into pretreatment culture medium, and pre-culturing. The leafy petiole with the leaf comprises: the leaf stalk is 0.8-1cm long and is provided withIs irradiated by 0.5-0.8cm2A blade of a size. The pretreatment culture medium: each liter contains 2mg of 6-BA, 0.05mg of NAA, 30g of sucrose and 9g of agar, the balance is MS culture medium, and the pH value is 5.8; the culture conditions are as follows: the temperature is 23 ℃, and the culture is carried out for 5d in dark.
b. Polyploid induction: when the cut end of the petiole slightly expands, the whole petiole with the petiole is taken out and immersed in colchicine aqueous solution with the concentration of 20mg/L for polyploid induction. Induction culture conditions: the temperature is 25 ℃, and dark culture is carried out for 4 h. Then taking out leafstalk with leaf, washing 3-5 times by sterile water, and then sucking water by sterile filter paper.
c. Regeneration of adventitious buds of petioles: and c, inoculating the leafy petioles obtained in the step b into an induction and differentiation integrated culture medium, and synchronously performing callus induction and adventitious bud differentiation. The induced differentiation integrated culture medium comprises: each liter contains 2mg of 6-BA, 0.05mg of NAA, 1g of PVP, 30g of cane sugar and 9g of agar, the balance is MS culture medium, and the pH value is 5.8; the culture conditions are as follows: culturing at 23 deg.C in dark for 5d, and culturing under illumination intensity of 2000lx for 16 h/d. When the culture is carried out for 20 days, green compact callus is induced at the cut of the petiole, and the induction rate is 86.7 percent. Culturing for 20d without changing culture medium and culture conditions, and differentiating strong green adventitious bud at callus position (FIG. 1), with differentiation rate of 76.7%.
d. Polyploid screening: and c, when the adventitious bud of the petiole grows to be 1cm high in the step c, shearing off the original leaf, and transferring the adventitious bud into a multiplication culture medium. The proliferation culture medium: each liter contains 2mg of 6-BA, 0.5mg of IBA, 30g of sucrose and 7g of agar, the balance is MS culture medium, and the pH value is 5.8; the culture conditions are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d. After 20 days of culture, 2-3 leaves are taken and completely unfolded, and the ploidy detection is carried out by a flow cytometer. FIG. 2 shows the results of flow ploidy identification of a Clerodendron petioloides diploid, and FIG. 3 shows the results of flow ploidy identification of a Clerodendron petioloides diploid whose induction has succeeded.
With respect to the inducing material:
example 2
The method steps of the embodiment and the embodiment 1 are the same except for the following differences; the difference lies in that: in this example, leaves were completely opened from the 5 th to the 6 th leaves of Clerodendron petiolatus Miq,cutting a petiole with a blade as an inducing material, wherein the petiole with the blade is characterized in that: the petiole is 1-1.5cm long and 2-3cm long2A blade of a size. The regeneration effect of the adventitious bud is shown in Table 1.
Comparative example 1
The procedure of this comparative example was the same as that of example 1 except for the following differences; the difference lies in that: in the comparative example, only in-vitro petioles of aseptic seedlings were used as an inducing material, both ends of the petioles were cut to obtain wounds, and the corresponding adventitious bud regeneration effects are shown in table 1.
Comparative example 2
The procedure of this comparative example was the same as that of example 1 except for the following differences; the difference lies in that: in the comparative example, only in-vitro leaves of aseptic seedlings were used as the inducing material, 2 to 3 wounds were scratched on the leaves, and the corresponding adventitious bud regeneration effect was as shown in table 1.
The parallel culture was performed according to the methods of example 1, example 2, comparative example 1 and comparative example 2, respectively, and the culture time after colchicine treatment was 40d, and the results are shown in Table 1 by statistics.
TABLE 1 Effect of different Induction materials on callus induction and adventitious bud regeneration of Clerodendron petiolatus after colchicine treatment
As can be seen from Table 1, when the leafstalk with leaves is used as an inducing material, the callus induction rate of the leafstalk base part exceeds 81% when the leafstalk base part is cultured for 20 days after colchicine treatment, the adventitious bud regeneration rate of the leafstalk base part exceeds 65% when the leafstalk base part is cultured for 40 days, and the adventitious bud regeneration effect is far better than that of the comparative example 1 and the comparative example 2. Wherein, the callus incidence rate of the inducing material of the embodiment 1 can reach 86.7 percent, and the adventitious bud regeneration rate can reach 76.7 percent.
The formula of the induction and differentiation integrated culture medium comprises the following components:
example 3
The method steps of the embodiment and the embodiment 1 are the same except for the following differences; the difference lies in that: the concentration of NAA in the induced differentiation integrated culture medium used in the step c is 1 mg/L; the corresponding callus induction rate and adventitious bud regeneration effect are shown in Table 2.
Comparative example 3
The procedure of this comparative example was the same as that of example 1 except for the following differences; the difference lies in that: the concentrations of 6-BA and NAA in the culture medium used in the step c are respectively 0.5mg/L and 0.05 mg/L; the corresponding callus induction rate and adventitious bud regeneration effect are shown in Table 2.
Comparative example 4
The procedure of this comparative example was the same as that of example 1 except for the following differences; the difference lies in that: the culture medium used in the step c contains 6-BA and 2,4-D, the concentrations of 6-BA and 2,4-D are respectively 5mg/L and 1 mg/L; the corresponding callus induction rate and adventitious bud regeneration effect are shown in Table 2.
Comparative example 5
The procedure of this comparative example was the same as that of example 1 except for the following differences; the difference lies in that: the culture medium used in the step c contains 6-BA and IBA, and the concentrations of the 6-BA and the IBA are respectively 2mg/L and 0.2 mg/L; the corresponding callus induction rate and adventitious bud regeneration effect are shown in Table 2.
The parallel culture was performed according to the methods of example 1, example 3, comparative example 4 and comparative example 5, respectively, and the culture time was 40d, and the results were shown in Table 2 by statistics.
TABLE 2 Effect of different Medium formulations on Clerodendron calluses Induction and adventitious bud regeneration after colchicine treatment
As can be seen from Table 2, using the differentiation-inducing integrative medium of examples 1 and 3, the callus induction rate of the petiole base was more than 83.3% at 20 days of culture after colchicine treatment, the adventitious bud regeneration rate of the petiole base was more than 70% at 40 days of culture, and the callus induction and adventitious bud regeneration effects were superior to those of comparative examples 3, 4 and 5.
With respect to induction concentration and time:
examples 4 to 5
Examples 4 to 5 the process steps of example 1 were the same except for the following differences; the difference lies in that: immersing in colchicine aqueous solution for a treatment time length, wherein example 4 is immersing in colchicine aqueous solution for dark culture for 2h, and example 5 is immersing in colchicine aqueous solution for dark culture for 6 h. The results of adventitious bud regeneration rate and tetraploid induction rate are shown in table 3.
Comparative examples 6 to 8
Comparative examples 6 to 8 the process steps of example 1 were the same except for the following differences; the difference lies in that: the concentration of colchicine in water and the length of treatment time used. Comparative examples 6-8 all used colchicine aqueous solution concentrations were 10mg/L, wherein comparative example 6 was submerged in colchicine aqueous solution for dark culture for 2h, comparative example 7 was submerged in colchicine aqueous solution for dark culture for 4h, and comparative example 8 was submerged in colchicine aqueous solution for dark culture for 6 h. The results of adventitious bud regeneration rate and tetraploid induction rate are shown in table 3.
Comparative examples 9 to 11
Comparative examples 9 to 11 have the same procedure as in example 1 except for the following differences; the difference lies in that: the concentration of colchicine in water and the length of treatment time used. Comparative examples 9-11 all used colchicine aqueous solution concentrations of 40mg/L, wherein comparative example 9 was submerged in colchicine aqueous solution for dark culture for 2h, comparative example 10 was submerged in colchicine aqueous solution for dark culture for 4h, and comparative example 11 was submerged in colchicine aqueous solution for dark culture for 6 h. The results of adventitious bud regeneration rate and tetraploid induction rate are shown in table 3.
Comparative examples 12 to 14
Comparative examples 12 to 14 the process steps of example 1 were the same except for the following differences; the difference lies in that: the concentration of colchicine in water and the length of treatment time used. Comparative examples 12-14 all used colchicine concentrations of 60mg/L, wherein comparative example 12 was submerged in colchicine aqueous solution for dark culture for 2h, comparative example 13 was submerged in colchicine aqueous solution for dark culture for 4h, and comparative example 14 was submerged in colchicine aqueous solution for dark culture for 6 h. The results of adventitious bud regeneration rate and tetraploid induction rate are shown in table 3.
The results of parallel culture according to the methods of example 1, example 4, example 5 and comparative examples 6 to 14, culture time 60d, adventitious bud regeneration rate and tetraploid induction rate were statistically shown in Table 3.
TABLE 3 influence of different colchicine induction times and concentrations on Clerodendron petiolatus polyploid Induction Rate
As can be seen from Table 3, using the colchicine induction concentrations and times of examples 1, 4, 5, the adventitious bud regeneration rate after 60 days of culture was higher than 70%, the tetraploid induction rate was higher than 36.7%, and the tetraploid induction efficiency was superior to that of comparative examples 6-14. Wherein, by using the colchicine induction concentration and time of the embodiment 1, the tetraploid induction rate can reach 43.3 percent.
Claims (10)
1. A method for efficiently inducing Clerodendron petioloides polyploid, comprising the steps of:
a. pre-culturing: selecting the 3 rd to 6 th completely unfolded leaves of the Clerodendron petiolus aseptic seedlings from top to bottom, cutting off petioles with leaves as an inducing material, inoculating the petioles with leaves into a pretreatment culture medium, and performing pre-culture;
b. polyploid induction: after the cut end of the petiole slightly expands, taking out the petiole with the leaf, immersing the petiole with the leaf in colchicine aqueous solution for polyploid induction culture, then taking out the petiole with the leaf, washing the petiole with sterile water for 3 to 5 times, and then sucking water by using filter paper;
c. regeneration of adventitious buds of petioles: b, inoculating the leafy petioles with leaves obtained in the step b into an induced differentiation integrated culture medium for culture, inducing callus at the cut of the petioles, continuing to culture, and differentiating adventitious buds;
d. polyploid screening: cutting off original leaves when the adventitious bud grows to a height of more than 1cm, transferring the adventitious bud into a multiplication culture medium for culture, and taking 2-3 leaves for ploidy detection after more than 3 leaves are grown and fully developed.
2. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein said petiole with leaf has a length of 0.8 to 1.5cm and is worn at a length of 0.5 to 3cm2A blade of a size.
3. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein said pretreatment medium: contains 2mg of 6-BA, 0.05mg of NAA, 30g of sucrose and 9g of agar per liter, and the balance is MS culture medium with pH of 5.8.
4. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein the preculture in the step a is carried out at 23 ℃ for 5 days in the dark.
5. The method of efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein said polyploid inducing culture in said step b is carried out by immersing the resulting polyploid in an aqueous solution of colchicine having a concentration of 20mg/L and carrying out dark culture at a temperature of 25 ℃ for 2 to 6 hours.
6. The method of efficiently inducing Clerodendron clerodendrum polyploid according to claim 5, wherein said polyploid induction culture in said step b is carried out by immersing the resulting product in an aqueous solution of colchicine having a concentration of 20mg/L and incubating the resulting product at 25 ℃ for 4 hours in the dark.
7. The method for efficiently inducing Clerodendron petersatumpolyploid according to claim 1, wherein said medium integrating induction and differentiation: each liter contains 2mg of 6-BA, 0.05-0.1mg of NAA, 1g of PVP, 30g of cane sugar and 9g of agar, the rest is MS culture medium, and the pH value is 5.8.
8. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein said culturing in the step c is carried out by: culturing at 23 deg.C in dark for 5 days, and culturing under illumination intensity of 2000lx for 16 h/d.
9. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein said propagation medium: contains 2mg of 6-BA, 0.5mg of IBA, 30g of sucrose and 7g of agar per liter, and the balance is MS culture medium with pH of 5.8.
10. The method for efficiently inducing Clerodendron clerodendrum polyploid according to claim 1, wherein the culture conditions in said step d are: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986527A (en) * | 2012-11-05 | 2013-03-27 | 湖北大学 | Method for inducing petiole callus to form multiploid London planes |
| CN111202002A (en) * | 2020-01-17 | 2020-05-29 | 广东省林业科学研究院 | Tissue culture and rapid propagation method of clerodendrum japonicum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986527A (en) * | 2012-11-05 | 2013-03-27 | 湖北大学 | Method for inducing petiole callus to form multiploid London planes |
| CN111202002A (en) * | 2020-01-17 | 2020-05-29 | 广东省林业科学研究院 | Tissue culture and rapid propagation method of clerodendrum japonicum |
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