CN112369331A - Method for establishing regeneration system of populus variegatus by taking leaves as explants - Google Patents
Method for establishing regeneration system of populus variegatus by taking leaves as explants Download PDFInfo
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- CN112369331A CN112369331A CN202011511922.0A CN202011511922A CN112369331A CN 112369331 A CN112369331 A CN 112369331A CN 202011511922 A CN202011511922 A CN 202011511922A CN 112369331 A CN112369331 A CN 112369331A
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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
The invention belongs to the field of tissue culture propagation, and discloses a method for establishing a regeneration system of populus colourianus by taking leaves as explants, which comprises the following steps: explant selection was first performed, followed by 0.1% HgCl2Sterilizing for 12min, inoculating to an induction culture medium for adventitious bud induction, controlling the culture temperature at 24-26 ℃, then culturing for 25 days under the illumination time of 10-14 h/day and the illumination intensity of 1400 plus 1800lx, then cutting the obtained seedlings of the adventitious buds of the colored red poplar, transferring the seedlings into a subculture medium for culture, and finally inoculating the aseptic 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 leaves as explants, belonging to the technical field of woody ornamental plant seedling propagation.
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
In order to overcome the defects in the prior art, the invention provides a method for establishing a regeneration system of populus colourianus by taking leaves as explants.
The technical scheme is as follows:
a method for establishing a regeneration system of Populus variegatus by taking leaves as explants is characterized by comprising the following steps: A) explant selection and disinfection, B) adventitious bud induction, C) subculture and proliferation, D) root induction.
Further, the method comprises the steps of:
A) explant selection and disinfection: selecting explants, oscillating with a detergent for 10 minutes, washing with running water for 30 minutes, sterilizing with 75% ethanol for 1min, sterilizing with 0.1% mercuric chloride for 12min, and washing with sterile water for 6 times;
B) induction of adventitious buds: cutting edges of the sterilized leaf explants, and keeping the area of the cut edges to be about 3cm2Inoculating the explant into an induction culture medium for adventitious bud induction, controlling the culture temperature to be 24-26 ℃, and culturing for 1 day by red light irradiation; then continuing to culture for 24 days, wherein the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx;
C) subculture and proliferation: cutting the color red poplar seedlings obtained in the step B), and then inoculating the cut color red poplar seedlings into a subculture medium for subculture for 30 days, 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: and C) inoculating the color red poplar test-tube plantlet 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 the surface area of the color populus in the step A) to be about 5cm2The young and tender leaves of (1).
Preferably, the first and second electrodes are formed of a metal,
the induction medium used in step B) was: MS +6-BA 1mg/L + NAA0.2 mg/L.
Preferably, the first and second electrodes are formed of a metal,
the subculture medium used in step C) was: MS +6-BA 1mg/L + NAA0.2 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 wavelength range of the red light is 700-740nm, and the light intensity is 4000-5000 lx.
More preferably still, the first and second liquid crystal compositions are,
the wavelength range of the red light is 720-740nm, and the light intensity is 4800 lx.
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 50% by 8%, and the reason is probably that infrared light with certain intensity can activate an enzyme system required for inducing bud germination, thereby promoting the induction rate of the adventitious bud and also promoting the induction action of 6-BA and NAA on the adventitious bud. While the red light treatment in the secondary proliferation and root induction has no positive promotion effect. Furthermore, red light also had no substantial effect on adventitious bud induction in the absence of 6-BA and NAA.
The tissue culture method is adopted to breed the populus colourianus, a large number of aseptic seedlings can be effectively obtained, and the problem of rapid breeding of the populus colourianus can be solved after domestication and transplantation; through the selection of the optimal explant, the induction culture medium, the subculture medium and the rooting culture medium, the multiplication rate of 1 culture period reaches 160 times, and the aseptic populus diversifolia seedlings can be efficiently obtained.
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 variegatus by taking leaves as explants comprises the following steps:
1. explant selection and disinfection: selecting the surface area at 5cm at the bottom of 6 months2The left and right young leaves are shaken for 10 minutes by the detergent and washed for 30 minutes by running water. Shaking with 75% ethanol for 1min, then shaking with 0.1% mercuric chloride for 12min, washing with sterile water for 6 times to remove residual disinfectantAnd (3) preparing. Treating 100 explants in total;
2. induction of adventitious buds: cutting edges of the sterilized leaf explants, and keeping the area of the cut edges to be about 3cm2Inoculating the explant into an induction culture medium MS +6-BA 1mg/L + NAA0.2 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 1600 lx; after 25 days, the contamination rate of the explants is 50%, the induction rate is 42%, namely 2-5 adventitious buds with different numbers grow on 21 explants, and each explant is proliferated by 4.0 on average;
3. subculture and proliferation: cutting adventitious bud seedlings of the color red poplar, inoculating the cut adventitious bud seedlings into a subculture medium MS +6-BA 2mg/L + NAA0.2 mg/L, inoculating 1 seedling into each bottle, inoculating 82 bottles in total, carrying out subculture for 25 days at the culture temperature of 26 ℃, the illumination time of 14 h/day and the illumination intensity of 1600lx, carrying out subculture for 25 days at the proliferation multiple of 3.5 times to obtain 287 sterile seedlings, and carrying out subculture for 2 times (every interval is 25 days, and the average proliferation is 3.5 times) to obtain 3516 sterile seedlings;
4. root induction: and (3) inoculating the test-tube plantlets of the color red poplar obtained by the subculture into a rooting culture medium 1/2MS + IBA2.0 mg/L for rooting culture for 25 days, wherein the culture temperature is 26 ℃, the illumination time is 12 h/day, and the illumination intensity is 1600 lx. The rooting rate reaches 90 percent, so that 3164 strains are obtained.
3164 plants can be proliferated from 21 rachis tops in 1 culture period, and the proliferation rate is 150 times.
Example 2
A method for establishing a regeneration system of Populus variegatus by taking leaves as explants comprises the following steps:
1. explant selection and disinfection: selecting the surface area at 5cm at the bottom of 6 months2The left and right young leaves are shaken for 10 minutes by the detergent and washed 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 adventitious buds: cutting edges of the sterilized leaf explants, and keeping the area of the cut edges to be about 3cm2Inoculating the explant into an induction culture medium MS +6-BA 1mg/L + NAA0.2 mg/L for adventitious bud induction,controlling the culture temperature at 26 deg.C, culturing for 1 day under irradiation of red light with electromagnetic radiation of 720nm wavelength and light intensity of 4800 lx; then continuing to culture for 24 days, wherein the illumination time is 14 h/day, and the illumination intensity is 1600 lx; after 25 days, the contamination rate of the explants is 46 percent, the induction rate is 50 percent, namely 2 to 5 adventitious buds with different numbers grow on 27 explants, and each explant is proliferated by 4.1 on average;
3. subculture and proliferation: cutting adventitious bud seedlings of the color red poplar, inoculating the cut adventitious bud seedlings into a subculture medium MS +6-BA 2mg/L + NAA0.2 mg/L, inoculating 1 seedling into each bottle, inoculating 111 bottles together, carrying out subculture for 25 days at the culture temperature of 26 ℃, the illumination time of 14 h/day and the illumination intensity of 1600lx, carrying out subculture for 25 days at the proliferation multiple of 3.5 times to obtain 388 aseptic seedlings, and carrying out subculture for 2 times (every 25 days, the average proliferation is 3.5 times) to obtain 4759 aseptic seedlings;
4. root induction: and (3) inoculating the test-tube plantlets of the color red poplar obtained by the subculture into a rooting culture medium 1/2MS + IBA2.0 mg/L for rooting culture for 25 days, wherein the culture temperature is 26 ℃, the illumination time is 12 h/day, and the illumination intensity is 1600 lx. The rooting rate reaches 91 percent, so that 4330 strains are obtained.
4330 plants can be proliferated from the tops of 27 rachis in 1 culture period, and the proliferation rate is 160 times.
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 (8)
1. A method for establishing a regeneration system of Populus variegatus by taking leaves as explants is characterized by comprising the following steps: A) explant selection and disinfection, B) adventitious bud induction, C) subculture and proliferation, 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, sterilizing with 75% ethanol for 1min, sterilizing with 0.1% mercuric chloride for 12min, and washing with sterile water for 6 times;
B) induction of adventitious buds: cutting edges of the sterilized explants, and keeping the remaining area about 3cm2Inoculating the explant into an induction culture medium for adventitious bud induction, controlling the culture temperature to be 24-26 ℃, and culturing for 1 day by red light irradiation; then normal culture is continued for 24 days, the illumination time is 10-14 h/day, and the illumination intensity is 1400-1800 lx;
C) subculture and proliferation: cutting the color red poplar seedlings obtained in the step B), and then inoculating the cut color red poplar seedlings into a subculture medium for subculture for 30 days, 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: and C) inoculating the color red poplar test-tube plantlet 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 explant of step A) is selected from Populus variegatus with a surface area of about 5cm2The young and tender leaves of (1).
4. The method according to claim 2, wherein the induction medium used in step B) is: MS +6-BA 1mg/L + NAA0.2 mg/L.
5. The method according to claim 2, wherein the subculture medium used in step C) is: MS +6-BA 2mg/L + NAA0.2 mg/L.
6. The method according to claim 2, wherein the rooting medium used in step D) is: 1/2MS + IBA2 mg/L.
7. The method as claimed in claim 2, wherein the wavelength range of the red light is 700-740nm, and the light intensity is 4000-5000 lx.
8. The method as claimed in claim 7, wherein the wavelength range of the red light is 720-740nm, and the light intensity is 4800 lx.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113748989A (en) * | 2021-10-26 | 2021-12-07 | 山东省林业科学研究院 | Method for inducing somatic embryo of in vitro leaf of 107 populus deltoids |
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