CN112335550A - Method for establishing regeneration system of populus colourianus by taking petioles as explants - Google Patents
Method for establishing regeneration system of populus colourianus by taking petioles as explants Download PDFInfo
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- CN112335550A CN112335550A CN202011539529.2A CN202011539529A CN112335550A CN 112335550 A CN112335550 A CN 112335550A CN 202011539529 A CN202011539529 A CN 202011539529A CN 112335550 A CN112335550 A CN 112335550A
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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
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Abstract
The invention provides a method for obtaining a regeneration plant by using a colored red poplar leaf stalk as an explant to induce callus, which comprises the following steps: explant selection was first performed, followed by 0.1% HgCl2Sterilizing for 12min, inoculating into an induction culture medium for callus induction, controlling the culture temperature at 24-26 ℃, then culturing under the conditions of illumination time of 10-14 h/day and illumination intensity of 1400 plus 1800lx, then transferring the obtained calli of the colored red poplar into a differentiation culture medium for culturing for 30 days, and finally inoculating the sterile seedlings of the colored red poplar into a rooting culture medium for rooting culture for 25 days. The method can effectively solve the problem of in vitro rapid propagation of the color red poplar.
Description
Technical Field
The invention relates to a tissue culture propagation method of color populus, in particular to a method for establishing a color populus regeneration system by taking petioles as explants, belonging to the technical field of seedling propagation and breeding of woody ornamental plants.
Background
The populus colouris a new variety of populus tremuloides 2025 populus americanus induced by gene, belonging to the populus genus of the salicaceae family. The color of the leaves of the branches in the annual growth period is bright red, light yellow, orange yellow and yellow-green from top to bottom respectively. At present, the variety is mainly bred by two modes of cuttage and grafting. In the cutting seedling process, the attention points required by cutting of the seed, preparation of cutting slips, cutting time, cutting density and seedling management technology are basically the same as those of other poplar varieties. When grafting and seedling raising are carried out, the selection of the rootstocks is very important. During grafting propagation, the black poplar is highest in the survival rate and best in growth, the poplars are inferior in variety, the survival rate of the European and American poplars is low, and the poplars and the small-leaf poplars are not suitable for being used as grafting stocks. The grafted seedling management technology is basically the same as that of other poplar. Stock grafting and scion cuttage are best in spring.
The variety has strong disease and pest resistance and drought resistance, and can be widely applied to greening construction in gardens, suburbs, roads, tourist attractions and new rural areas. At present, the number of seedlings of the variety is very small in China, and investigation shows that cutting seedlings and grafting seedlings cannot meet market demands, so that the color red poplar resource is in short supply and the application of the color red poplar resource is limited.
The tissue culture of the color populus is rarely reported at home and abroad. The establishment of a primary sterile line is very difficult, and a regeneration system method of the color populus is not established yet, wherein the differentiation rate is high, and the pollution rate is low.
In conclusion, the tissue culture and propagation of the Populus variegatus is one of the important ways for solving the resource shortage, and has very important significance for the development, popularization and utilization of the Populus variegatus.
Disclosure of Invention
The invention mainly solves the technical problem of disclosing a method for establishing a regeneration system of populus colourianus by taking petioles as explants.
The technical scheme is as follows:
a method for establishing a regeneration system of Populus variegatus by taking petioles as explants is characterized by comprising the following steps: A) explant selection and disinfection, B) callus induction, C) differentiation culture, and D) root induction.
Specifically, the method comprises the following steps:
A) explant selection and disinfection: selecting explants, oscillating with a detergent for 10 minutes, washing with running water for 30 minutes, then shaking with 75% alcohol for 1 minute, then shaking with 0.1% mercuric chloride for 12 minutes, and washing with sterile water for 6 times;
B) induction of callus: taking the sterilized petiole explant, cutting off 0.5cm of each of two ends of the sterilized petiole explant, inoculating the petiole explant into an induction culture medium for adventitious bud induction, controlling the culture temperature to be 26 ℃, and carrying out red light irradiation culture for 1 day; then continuing to culture for 24 days, wherein the illumination time is 14 h/day, and the illumination intensity is 1800 lx; after 25 days, callus with different sizes grows on the explants;
C) differentiation culture: transferring the callus obtained in the step B) into a differentiation culture medium to culture for 25 days to obtain aseptic seedlings, wherein the culture temperature is 24-26 ℃, the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx;
D) root induction: inoculating the sterilized red poplar seedlings obtained in the step C) into a rooting culture medium for rooting culture for 25 days, wherein the culture temperature is 24-26 ℃, the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx.
Preferably, the first and second electrodes are formed of a metal,
selecting tender petioles of 2-3cm long color red poplars from the explants in the step A).
Preferably, the first and second electrodes are formed of a metal,
the induction medium used in step B) was: MS +6-BA 1mg/L + NAA0.4 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the differentiation medium used in step C) was: MS +6-BA 0.5mg/L + NAA0.1 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the rooting medium adopted in the step D) is as follows: 1/2MS + IBA2.0 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the intensity of the red light is 4000-.
Preferably, the first and second electrodes are formed of a metal,
the red light has a wavelength of 700-760 nm.
More preferably still, the first and second liquid crystal compositions are,
the intensity of the red light was 4800 lx.
More preferably still, the first and second liquid crystal compositions are,
the red light has a wavelength of 720 nm.
The advantages of the present invention over the prior art mainly include, but are not limited to, the following aspects:
the propagation of the color red poplar usually adopts cuttage and grafting propagation, and is influenced by the quantity of scions and the quality of rootstocks, and the rapid propagation speed and the propagation rate are very low.
In the process of inducing the adventitious bud, the invention adopts high-intensity red light treatment firstly, so that the induction rate of the adventitious bud is greatly improved from 42% to 59%, and is improved by 17%, probably because infrared light with certain intensity and wavelength can activate an enzyme system required for inducing the adventitious bud of the callus of the explant to sprout, thereby promoting the induction rate of the adventitious bud of the callus, and also promoting the induction effect of 6-BA and NAA on the explant. While the red light treatment in the secondary proliferation and root induction has no positive promotion effect. Red light also had no substantial effect on explant induction in the absence of 6-BA and NAA.
The method adopts a tissue culture method to breed the populus colourianus, can effectively obtain a large amount of aseptic seedlings, and can solve the problem of rapid propagation of the populus colourianus after domestication and transplantation; the selection of the optimal explant, the induction culture medium, the subculture medium and the rooting culture medium can enable the rooting rate to reach 90%, 4651 populus chromopterus aseptic seedlings can be efficiently obtained from 100 explants, and the establishment of a regeneration system has very important significance for the genetic engineering transformation of poplar varieties and the molecular biological research on new poplar varieties.
Drawings
FIG. 1: influence of red light irradiation time on induction rate;
FIG. 2: influence of red wavelength on the inductivity.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for establishing a regeneration system of populus tremuloides by taking petioles as explants comprises the following steps:
1. explant selection and disinfection: and at the bottom of 6 months, selecting tender petioles with the length of 2-3cm, oscillating the detergent for 10 minutes, and washing the petioles for 30 minutes by running water. The residual disinfectant was removed by shaking with 75% alcohol for 1 minute, then with 0.1% mercuric chloride for 12 minutes and washing with sterile water 6 times. Treating 100 explants in total;
2. induction of callus: taking the sterilized petiole explant, cutting off 0.5cm of each of two ends of the sterilized petiole explant, inoculating the petiole explant into an induction culture medium MS +6-BA 1mg/L + NAA0.4 mg/L for adventitious bud induction, controlling the culture temperature at 26 ℃, and culturing for 25 days under the culture conditions: the illumination time is 14 h/day, and the illumination intensity is 1800 lx; after 25 days, the induction rate of the explants is 42 percent, namely, callus with different sizes grows on 42 explants;
3. differentiation culture: transferring the calli of the colored red poplar obtained in the step B) into a differentiation culture medium MS +6-BA 0.5mg/L + NAA0.1 mg/L to be cultured for 25 days, wherein the culture temperature is 26 ℃, the illumination time is 14 h/day, and the illumination intensity is 1800 lx; the differentiation rate reaches 40% in 25 days, thus obtaining a sterile seedling 287 strain, and then carrying out subculture for 2 times (every 25 days) to obtain a sterile seedling 3516 strain;
4. root induction: and (3) inoculating the color red poplar test-tube plantlets obtained by subculture into a rooting culture medium 1/2MS + IBA 1.0 mg/L for rooting culture for 15 days, wherein the culture temperature is 26 ℃, the illumination time is 14 h/day, the illumination intensity is 1800lx, and the rooting rate reaches 90 percent, so that 3164 plants are obtained.
3164 plants can be differentiated from the callus in 1 culture period, and the establishment of a regeneration system has great significance for the genetic engineering transformation of poplar varieties and the molecular biological research on new poplar varieties.
Example 2
A method for establishing a regeneration system of populus tremuloides by taking petioles as explants comprises the following steps:
1. explant selection and disinfection: and at the bottom of 6 months, selecting tender petioles with the length of 2-3cm, oscillating the detergent for 10 minutes, and washing the petioles for 30 minutes by running water. The residual disinfectant was removed by shaking with 75% alcohol for 1 minute, then with 0.1% mercuric chloride for 12 minutes and washing with sterile water 6 times. Treating 100 explants in total;
2. induction of callus: taking a sterilized petiole explant, cutting off 0.5cm from each of two ends of the sterilized petiole explant, inoculating the petiole explant into an induction culture medium MS +6-BA 1mg/L + NAA0.4 mg/L for adventitious bud induction, controlling the culture temperature to be 26 ℃, and culturing for 1 day by red light irradiation, wherein the red light has electromagnetic radiation with the wavelength of 720nm, and the light intensity is 4800 lx; then, normal culture is continued for 24 days, the illumination time is 14 h/day, and the illumination intensity is 1800 lx; after 25 days, the induction rate of the explants is 59 percent, namely callus with different sizes grows on 59 explants;
3. differentiation culture: transferring the calli of the colored red poplar obtained in the step B) into a differentiation culture medium MS +6-BA 0.5mg/L + NAA0.1 mg/L to be cultured for 25 days, wherein the culture temperature is 26 ℃, the illumination time is 14 h/day, and the illumination intensity is 1800 lx; the differentiation rate reaches 40% in 25 days, 421 aseptic seedlings can be obtained, and 5206 aseptic seedlings can be obtained after 2 subcultures (every 25 days);
4. root induction: the color red poplar test-tube plantlets obtained by subculture are inoculated into a rooting culture medium 1/2MS + IBA 1.0 mg/L for rooting culture for 15 days, the culture temperature is 26 ℃, the illumination time is 14 h/day, the illumination intensity is 1800lx, and the rooting rate reaches 90 percent, so that 4651 strains are obtained altogether, and the establishment of a regeneration system has very important significance for modifying poplar varieties by genetic engineering and carrying out molecular biology research on new poplar varieties.
Example 3
As shown in figure 1, the induction rate of the explant is higher in the red light illumination time, the induction rate is increased along with the increase of the illumination time, when the induction rate is increased to 24 hours, the maximum value is 59%, the induction rate is increased by 17 percentage points compared with 42% of a control group without red light, the red light illumination time is continuously increased, and the induction rate is not obviously influenced. In the process of inducing the adventitious bud, the invention adopts high-intensity red light treatment firstly, so that the induction rate of the adventitious bud is greatly improved, probably because infrared light with certain intensity can activate an enzyme system required for inducing the adventitious bud of the callus of the explant to sprout, thereby promoting the induction rate of the adventitious bud of the callus, and also promoting the induction action of 6-BA and NAA on the explant. The differentiation culture and root induction processes adopt red light treatment, and the differentiation rate and the rooting rate are not positively promoted. The present invention also found that in the absence of 6-BA and NAA, the induction of explants by red light was not substantially affected. In addition, as shown in FIG. 2, the wavelength of red light also has an influence on the induction rate, the wavelength of 700nm or less has a smaller influence on the induction rate, and the promotion effect of red light with the wavelength between 700 and 760nm is the best.
Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made to the invention or the method can be practiced without the specific embodiments. Accordingly, it is intended that all such modifications, improvements and extensions that do not depart from the spirit of the invention, be considered within the scope of the invention as claimed.
Claims (10)
1. A method for establishing a regeneration system of Populus variegatus by taking petioles as explants is characterized by comprising the following steps: A) explant selection and disinfection, B) callus induction, C) differentiation culture, and D) root induction.
2. Method according to claim 1, characterized in that it comprises the following steps:
A) explant selection and disinfection: selecting explants, oscillating with a detergent for 10 minutes, washing with running water for 30 minutes, then shaking with 75% alcohol for 1 minute, then shaking with 0.1% mercuric chloride for 12 minutes, and washing with sterile water for 6 times;
B) induction of callus: taking the sterilized petiole explant, cutting off 0.5cm of each of two ends of the sterilized petiole explant, inoculating the petiole explant into an induction culture medium for adventitious bud induction, controlling the culture temperature to be 26 ℃, and carrying out red light irradiation culture for 1 day; then normally culturing for 24 days, wherein the illumination time is 14 h/day, and the illumination intensity is 1800 lx; after 25 days, callus with different sizes grows on the explants;
C) differentiation culture: transferring the callus obtained in the step B) into a differentiation culture medium to culture for 25 days to obtain aseptic seedlings, wherein the culture temperature is 24-26 ℃, the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx;
D) root induction: inoculating the sterilized red poplar seedlings obtained in the step C) into a rooting culture medium for rooting culture for 25 days, wherein the culture temperature is 24-26 ℃, the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx.
3. The method as claimed in claim 2, wherein the explants in step A) are selected from tender petioles of 2-3cm long colored red poplar.
4. The method according to claim 2, wherein the induction medium used in step B) is: MS +6-BA 1mg/L + NAA0.4 mg/L.
5. The method according to claim 2, wherein the differentiation medium used in step C) is: MS +6-BA 0.5mg/L + NAA0.1 mg/L.
6. The method according to claim 2, wherein the rooting medium used in step D) is: 1/2MS + IBA2.0 mg/L.
7. The method as claimed in claim 2, wherein the intensity of the red light is 4000-.
8. The method according to claim 2, characterized in that the red light has a wavelength of 700 and 760 nm.
9. A method according to claim 7, characterized in that the intensity of red light is 4800 lx.
10. A method according to claim 2, characterized in that said red light has a wavelength of 720 nm.
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Cited By (1)
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| CN115176704A (en) * | 2022-07-18 | 2022-10-14 | 北京市农林科学院 | Method for tissue culture of poplar variety' Senhai No. 2 |
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