CN109430056B - Method for inducing regeneration of adventitious buds of alum roots - Google Patents
Method for inducing regeneration of adventitious buds of alum roots Download PDFInfo
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- CN109430056B CN109430056B CN201811373025.0A CN201811373025A CN109430056B CN 109430056 B CN109430056 B CN 109430056B CN 201811373025 A CN201811373025 A CN 201811373025A CN 109430056 B CN109430056 B CN 109430056B
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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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- 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
Abstract
A method for inducing regeneration of adventitious bud of alum root comprises taking petioles or leaves of aseptic seedlings of alum root as explants, inoculating on an induction culture medium, and performing induction culture to obtain adventitious bud of alum root; wherein the induction culture medium takes MS as a basic culture medium, 2-4wt.% of sucrose, 0.5-0.7wt.% of agar powder, 0.05-0.15mg/L of naphthylacetic acid, 0.1-0.2mg/L of thidiazuron or 0.2-1.0mg/L of 6-benzylaminopurine is added, and the pH value is 5.5-6.0; the method simplifies the step of transferring the self-induced callus culture medium to the adventitious bud differentiation culture medium, effectively solves the vitrification problem of adventitious buds, is simple and convenient to operate, economic and effective, and can provide technical support for rapid propagation of alum root seedlings, cultivation of new varieties and improvement of alum root characters by using genetic engineering means.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing regeneration of adventitious buds of alum roots.
Background
Alum root (Heuchera spp.) is a plant of genus alum of family Saxifragaceae, and is a perennial evergreen herb flower, native to the central part of America. Because the flower leaves are beautiful, the color of the leaves is bright and the color is rich, the ornamental plant is a rare new excellent color leaf ornamental plant, can be used as materials of border decorations, cluster plants, ground cover, pot plants and the like, and is continuously heated in the flower market of China.
Most alum root varieties are subjected to seedling production by means of asexual propagation methods, and the plant division propagation and the cutting propagation have certain limitations due to the defects of long period, low propagation coefficient, great influence of seasons and the like.
At present, the tissue culture technology is a main method for breeding alum root seedlings in large scale. The existing alum root tissue culture rapid propagation technology takes buds as initial propagation materials, and has the problems of difficult material acquisition, small quantity, large damage to mother plants and the like.
In the prior art, alum root 'Paris' petiole is taken as an initial propagation material, induction culture and differentiation culture are carried out on an induction culture medium, the petiole generates callus, and the callus is further transferred to an adventitious bud differentiation culture medium for differentiation culture, so that adventitious buds are obtained. In the method, the differentiation rate of the adventitious bud is relatively low, the callus transfer process is required, the operation is complex, the cost is high, and the pollution probability in the sterile operation process is increased.
Disclosure of Invention
The invention aims to provide a method for inducing regeneration of adventitious buds of alum root, which simplifies the step of transferring a self-induced callus culture medium to an adventitious bud differentiation culture medium, has an adventitious bud regeneration rate of more than 62.5 percent and can reach 89.58 percent, is simple, convenient, economical and effective, and can provide technical support for rapid propagation of alum root seedlings, cultivation of new varieties and improvement of alum root characters by using a genetic engineering means.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for inducing adventitious buds of alum roots to regenerate comprises the following steps:
1) inoculation of
Taking petioles or leaves of the alum root aseptic seedlings as explants, and inoculating the explants to an induction culture medium;
2) induced culture
Culturing the explant inoculated into the induction culture medium in the dark for 14-28 days, and then culturing the explant in the light for 25-40 days to obtain alum root adventitious buds;
wherein the culture temperature is 24 +/-2 ℃, and the illumination time of illumination culture is 10-14 h/d;
wherein the induction culture medium takes MS as a basic culture medium, and 2-4wt.% of sucrose, 0.5-0.7wt.% of agar powder, 0.05-0.15mg/L of naphthylacetic acid, 0.1-0.2mg/L of thidiazuron or 0.2-1.0mg/L of 6-benzylaminopurine is added, and the pH value is 5.5-6.0.
Further, in the step 1), when the explant is a leaf blade, the leaf blade is cut into a leaf disc with the length of 0.5-0.8 cm multiplied by 0.5-0.8 cm, and inoculation is carried out.
And in the step 1), when the explant is a petiole, cutting the length of the petiole to 0.8-1 cm, and inoculating the petiole on an induction culture medium.
Preferably, in step 2), the dark culture time is 21 days, and the light culture time is 30 days.
In the step 2), the culture temperature is 25 ℃, and the illumination time of illumination culture is 12 h/d.
Further, when the explant is a petiole, the 6-benzylaminopurine is 0.2-0.5mg/L and the pH value is 5.8 in the induction medium.
Preferably, when the explant is leaf, the 6-benzylaminopurine is 0.5-1.0mg/L, pH5.8 in the induction medium.
In the step 1), the variety of the alum root is 'Paris' or 'flash autumn'.
According to the invention, only one culture medium is used, and through dark culture and illumination culture with the culture period of about 50 days, high-quality alum root adventitious buds can be regenerated from two explants, namely, a petiole and a leaf, so that the step of transferring the self-induced callus culture medium to an adventitious bud differentiation culture medium is simplified, the technical operation difficulty is reduced, meanwhile, the culture cost is greatly reduced, and the vitrification problem of the adventitious buds is effectively solved.
The light has an inducing effect on the formation of plant organs, in the invention, dark culture is firstly carried out, then light culture is carried out, the explant is dedifferentiated under dark culture, redifferentiation is carried out under light culture, and callus and adventitious buds are sequentially formed on the explant; in the culture process, the plant growth regulator with lower concentration in the induction culture medium is utilized to promote the formation of adventitious buds and callus of the explant, achieve higher adventitious bud regeneration rate and effectively solve the vitrification problem of the adventitious buds.
In the culture medium, thidiazuron or 6-benzylaminopurine is added to promote the bud differentiation of alum roots, the added naphthylacetic acid has the effects of promoting cell division and expansion, the tissue culture of alum roots can promote the differentiation of callus, meanwhile, the addition of cytokinin and auxin is also an important influence factor for regulating the formation of callus or adventitious buds of explants, and if the concentration is too high, the vitrification problem is easily caused.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, the alum root petiole and the leaf can be used as explants, only one culture medium is used in the culture process, and after a culture period of about 50 days, the culture medium does not need to be replaced, so that the operation is simple and convenient, the production cost is greatly reduced, the adventitious bud regeneration rate is over 62.50 percent, and the vitrification problem of the adventitious bud is effectively solved.
The method can save the step of transferring from a callus induction culture medium to an adventitious bud differentiation culture medium, is simple, convenient, economical and effective, has the adventitious bud regeneration rate of more than 62.50 percent and can reach 89.58 percent, and can provide better technical support for the rapid propagation of alum root seedlings, the cultivation of new varieties and the improvement of alum root characters by using genetic engineering means.
Drawings
FIG. 1 shows the status of petiole explants of Paris' in example 2 of the present invention at 30 days of culture.
FIG. 2 is a state of leaf explant of ` Paris ` in example 7 of the present invention at 30 days of culture.
FIG. 3 is the state of leaf explants of Paris' in example 7 of the present invention at 50 days of culture.
Detailed Description
The present invention is further illustrated by the following specific examples.
Examples 1 to 5
Taking the petiole of the alum root Paris aseptic seedling as an explant, cutting the length of the petiole to 0.8-1 cm, inoculating the petiole on an induction culture medium, carrying out induction culture, directly inducing and differentiating to obtain an adventitious bud, wherein the culture process comprises the following steps: culturing in the dark for 21 days, and then culturing in the light for 30 days at the culture temperature of 24 +/-2 ℃.
Wherein, the induction culture medium takes MS as a basic culture medium, 0.1mg/L of Naphthylacetic acid (NAA), 3 wt.% of cane sugar and 0.6 wt.% of agar powder are added, Thidiazuron (TDZ) or 6-benzylaminopurine (6-BA) with different contents are added, a comparative example 1 and a comparative example 2 are arranged, the pH of the induction culture medium is 5.8, and the induction culture medium is sterilized at the high temperature and the high pressure of 121 ℃ and 0.11 MPa.
The amounts of TDZ and 6-BA added to the induction medium and the culture results of examples 1-5 and comparative examples 1-2 are shown in Table 1 and FIG. 1, wherein the number of adventitious buds is the number of effective adventitious buds obtained by total differentiation of 12 explants per dish; the regeneration rate of adventitious buds is the number of explants differentiated to obtain effective adventitious buds/the number of explants inoculated x 100%.
TABLE 1
As can be seen from table 1, when the TDZ concentration added to the culture medium is 0.1, 0.2mg/L, the regeneration rate of adventitious buds induced by the leaf stalks of alum root paris is above 65%, the regeneration rate of adventitious buds is high, and no vitrification state exists; when the TDZ concentration is 0.5mg/L, the regeneration rate of adventitious buds generated by induction of the petioles of the alum root 'Paris' is 2.08%, and the regeneration rate of the adventitious buds is very low.
When the concentration of 6-BA added into the culture medium is 0.2mg/L, 0.5mg/L and 1.0mg/L, the regeneration rate of adventitious buds generated by induction of the leaf stalks of the alum root Paris' is more than 85 percent, the regeneration rate of the adventitious buds is higher, and as can be seen from figure 1, a plurality of green adventitious buds are generated at the two ends of the leaf stalks when the culture medium is cultured for 30 days; when the concentration of 6-BA is 2.0mg/L, the regeneration rate of adventitious buds generated by induction of the alum root 'Paris' petioles is 62.50%, the regeneration rate of the adventitious buds is low, and part of the adventitious buds are in a vitrified state.
In addition, when the concentration of 6-BA added to the medium was 0.2mg/L, 0.5mg/L and 1.0mg/L, the regeneration rate of adventitious buds induced by the ` flash autumn ` petiole was 77.08%, 62.50% and 72.9%, and there was no vitrification.
Examples 6 to 8
Taking alum root 'Paris' and 'flash autumn' aseptic seedling leaves as explants, shearing the leaves into leaf disks of 0.5-0.8 cm multiplied by 0.5-0.8 cm, inoculating the leaf disks on an induction culture medium, and carrying out induction culture.
The induction culture medium takes MS as a basic culture medium, 0.1mg/L of NAA0.1mg/L of naphthylacetic acid, 3 wt.% of cane sugar and 0.6 wt.% of agar powder are added, 6-BA with different contents is added, a comparative example 3 is set, the pH of the induction culture medium is 5.8, and the induction culture medium is sterilized at the high temperature and the high pressure of 121 ℃ and 0.11 MPa.
Examples 6-8 and comparative example 3 the amount of 6-BA added to the induction medium and the culture results are shown in Table 2 and FIGS. 2-3.
TABLE 2
As can be seen from Table 2, when the concentrations of 6-BA added to the medium were 0.2mg/L, 0.5mg/L and 1.0mg/L, the regeneration rate of adventitious buds induced by alum root 'Paris' leaf was 60% or more, the regeneration rate of adventitious buds induced by alum root 'Paris' leaf was 66% or more, the regeneration rates of adventitious buds were both high, and when the concentration of 6-BA was 2.0mg/L, the regeneration rates of adventitious buds induced by alum root 'Paris' and 'autumn' petiole were 58.33% and 58.33%, respectively, but some of the adventitious buds were in a vitrified state.
As can be seen from FIGS. 2 to 3, when the leaves were cultured for 30 days, many dark green adventitious buds had been produced around the leaves; after 50 days of culture, adventitious buds induced around the leaves already form obvious regeneration plants with leaves.
Claims (8)
1. A method for inducing adventitious buds of alum roots to regenerate comprises the following steps:
1) inoculation of
Taking a petiole or a leaf blade of an aseptic alum root seedling as an explant, and inoculating the petiole or the leaf blade into an induction culture medium, wherein the variety of the alum root is 'Paris' or 'flash autumn';
2) induced culture
Culturing the explant inoculated into the induction culture medium in the dark for 14-28 days, and then culturing the explant in the light for 25-40 days to obtain alum root adventitious buds;
wherein the culture temperature is 24 +/-2 ℃, and the illumination time of illumination culture is 10-14 h/d;
wherein the induction culture medium takes MS as a basic culture medium, and 2-4wt.% of sucrose, 0.5-0.7wt.% of agar powder, 0.10mg/L of naphthylacetic acid, 0.2-1.0mg/L of 6-benzylaminopurine and pH5.5-6.0 are added.
2. A method for inducing adventitious buds of alum roots to regenerate comprises the following steps:
1) inoculation of
Taking the petiole of the alum root aseptic seedling as an explant, and inoculating the petiole into an induction culture medium, wherein the variety of the alum root is Paris or flash autumn;
2) induced culture
Culturing the explant inoculated into the induction culture medium in the dark for 14-28 days, and then culturing the explant in the light for 25-40 days to obtain alum root adventitious buds;
wherein the culture temperature is 24 +/-2 ℃, and the illumination time of illumination culture is 10-14 h/d;
wherein the induction culture medium takes MS as a basic culture medium, and is added with 2-4wt.% of sucrose, 0.5-0.7wt.% of agar powder, 0.10mg/L of naphthylacetic acid, 0.1-0.2mg/L of thidiazuron and pH 5.5-6.0.
3. The method for inducing regeneration of adventitious buds of a alum root according to claim 1 or 2, wherein in the step 1), when the explant is a leaf blade, the leaf blade is cut into a leaf disc with the length of 0.5-0.8 cm x 0.5-0.8 cm and inoculated.
4. The method for inducing regeneration of adventitious buds of a alum root according to claim 1 or 2, wherein in the step 1), when the explant is a petiole, the length of the petiole is cut to 0.8-1 cm and inoculated on an induction culture medium.
5. The method for inducing regeneration of adventitious buds of alum root according to claim 1 or 2, wherein in the step 2), the dark culture time is 21 days, and the light culture time is 30 days.
6. The method for inducing regeneration of adventitious buds of alum root according to claim 1 or 2, wherein in the step 2), the culture temperature is 25 ℃, and the illumination time for illumination culture is 12 h/d.
7. The method for inducing regeneration of adventitious bud of alum root according to claim 1 or 2, wherein when the explant is petiole, 6-benzylaminopurine is 0.2-0.5mg/L and pH is 5.8 in the induction medium.
8. The method for inducing regeneration of adventitious bud of alum root according to claim 1 or 2, wherein when the explant is leaf, 6-benzylaminopurine is 0.5-1.0mg/L, pH5.8 in the induction medium.
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| CN110199885A (en) * | 2019-07-16 | 2019-09-06 | 上海市农业科学院 | A method of separation alum root secondary color leaf obtains pure color aseptic seedling |
| CN111034615B (en) * | 2019-12-21 | 2021-05-18 | 北京花乡花卉科技研究所有限公司 | Tissue culture method for rapid propagation of alum root "tiramisu |
| CN111513061B (en) * | 2020-05-22 | 2022-05-03 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
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