CN108377910B - Tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins - Google Patents
Tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins Download PDFInfo
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- CN108377910B CN108377910B CN201810376851.4A CN201810376851A CN108377910B CN 108377910 B CN108377910 B CN 108377910B CN 201810376851 A CN201810376851 A CN 201810376851A CN 108377910 B CN108377910 B CN 108377910B
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins, which comprises the following steps: (1) selecting a female parent material; (2) explant treatment: taking the leaf vein of the young leaf of the female parent as a tissue culture explant, cutting the leaf from the female parent, and washing with tap water; soaking in alcohol, washing with sterilized water, soaking in hydrogen peroxide, and washing with sterilized water in sequence; cutting the vein along its edge; (3) starting culture: horizontally inoculating the cut leaf veins into a starting culture medium, and putting the starting culture medium into a culture room for culture; (4) differentiation culture of callus: after the start culture is finished, forming callus at the edge of the vein, and transferring the callus into a callus differentiation culture medium; (5) adventitious bud induction culture: after the differentiation culture of the callus is finished, the callus is enlarged and is transferred into a culture medium for adventitious bud induction culture, and adventitious buds are induced and differentiated on the callus. The method is easy to implement and simple and convenient to operate, reduces the browning rate of the explant, improves the induction rate of the callus, and improves the anthurium tissue culture technology.
Description
Technical Field
The invention belongs to the technical field of tissue culture of horticultural plants, and particularly relates to a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins, which is suitable for inducing and differentiating the adventitious buds in the tissue culture process of anthurium andraeanum.
Technical Field
Anthurium andraeanum (also called Anthurium andraeanum) is a tropical flower plant with high economic benefit. The flower has unique and beautiful shape, bright color and long flowering phase, and is a good flower decoration product in various places. With the continuous improvement of the living standard of people, the consumption of anthurium in the fields of home life, ceremony communication, event celebration and the like is continuously increased, so that the demand of the market for the anthurium is continuously expanded.
The excellent characters of the female parent are maintained by the plant progeny bred by the tissue culture technology, and meanwhile, the progeny can be efficiently bred in a large quantity and rapidly, and the seedling breeding production can be carried out annually.
The adventitious bud formation type is one of the important types of the tissue culture organ formation modes of plants, and is also the main mode of tissue culture and rapid propagation organ formation of anthurium andraeanum. Through the organ forming mode, the explant is dedifferentiated to form callus, and then the callus is redifferentiated to form adventitious buds. Therefore, callus induction is the primary link in the tissue culture process, and the mass formation of adventitious buds can be ensured only by reducing the occurrence of explant browning in the tissue culture process, improving the callus induction rate and obtaining more callus.
At present, tissue culture technology is mainly adopted in commercial anthurium andraeanum seedling breeding production, a large number of researches in the technical field of anthurium andraeanum tissue culture are carried out at home and abroad, but the problems of serious explant browning phenomenon and low callus induction rate exist, and a tissue culture method using the anthurium andraeanum leaf vein as the explant is not searched yet. In addition, in the tissue culture production of the commercialized anthurium andraeanum, if the leaf is used as the explant, the leaf veins are usually removed, and the waste of resources is also caused.
Disclosure of Invention
The invention aims to provide a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum leaf veins, which solves the problems that an explant is easy to brown and the callus induction rate is low in the anthurium andraeanum callus induction culture process, the browning rate of the explant is greatly reduced in the culture process by using the anthurium andraeanum leaf veins as the explant, the browning rate is reduced by 23.5-41.4%, the callus induction rate is greatly increased, and the induction rate is as high as 97.6%, so that the proliferation number of later generations is further increased, the method is easy to implement, the operation is simple and convenient, the browning rate of the explant is reduced, the callus induction rate is increased, and the tissue culture technology of anthurium andraeanum is improved.
In order to achieve the purpose, the invention provides the following technical scheme:
a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins comprises the following steps:
(1) selecting a female parent material: selecting a strong and beautiful anthurium plant as a female parent material.
(2) Explant treatment: taking the leaf vein of the female parent young leaf as the explant for tissue culture. And (3) cutting off the leaves from the female parent, washing the leaves for 20-30 min by using tap water, and gently rubbing the surfaces of the leaves by hands in the period. And then, sequentially soaking the substrate on an ultra-clean workbench for 30-60 seconds by using 70-75% (volume ratio) alcohol, washing the substrate with sterilized water for 2-3 times, soaking the substrate in 10-12% (volume ratio) hydrogen peroxide solution for 10-20 min, and washing the substrate with sterilized water for 3-5 times. The veins were cut along their edges and cut into 1cm long sections for use.
(3) Starting culture: and horizontally inoculating the cut leaf veins into a No. 1 culture medium, and putting the culture medium into a culture room for culture.
(4) Differentiation culture of callus: after the culture is started for 30-60 days, the edges of the veins form callus, and the callus is transferred into a No. 2 culture medium.
(5) Adventitious bud induction culture: after the callus is subjected to differentiation culture for 30-60 days, the callus is enlarged, punctate protrusions are visible on the surface, and the callus is transferred into a No. 3 culture medium. After the adventitious buds are subjected to induction culture for 30-60 days, adventitious buds with the height of 1-2 cm can be induced and differentiated on the callus.
The No. 1 culture medium is 1/2MS + TDZ 0.01-0.05 mg/L +6-BA 0.5-2.0 mg/L + NAA 0.1-1.0 mg/L, and the No. 1 culture medium mainly has the functions of explant starting culture and callus formation induction;
the No. 2 culture medium is 1/2MS +6-BA 0.5-2.0 mg/L + NAA 0.1-1.0 mg/L +2, 4-D0.01-0.5 mg/L, and the No. 2 culture medium mainly plays a role in promoting the callus to further dedifferentiate;
the No. 3 culture medium is MS +6-BA 0.2-1.5 mg/L + NAA 0.01-0.8 mg/L, and the No. 3 culture medium mainly plays a role in promoting adventitious bud induction differentiation;
the pH value of the culture medium No. 1, the culture medium No. 2 and the culture medium No. 3 is adjusted to be 5.8-6.0.
The culture conditions of the culture chamber are as follows: culturing at 23-27 deg.C in dark.
In the adventitious bud induction culture stage, the culture conditions of a culture room are 23-27 ℃, the illumination intensity is 1500-2000 lx and the illumination period is 12-14 h/d.
Furthermore, the diameter of the vein is more than 1.5mm, and if the diameter of the vein is too small, the inoculation operation is not facilitated, so the vein with the diameter more than 1.5mm is selected.
As shown in Table 1, the scheme solves the problems that the explant is easy to brown and the callus induction rate is low in the anthurium andraeanum callus induction culture process. By using the anthurium andraeanum vein as the explant, the browning rate of the explant is reduced by 23.5-41.4% in the culture process, and the callus induction rate is as high as 97.6%, so that the proliferation number of offspring is further increased. The invention is mainly different from the prior art in that the invention provides a tissue culture method for inducing differentiation adventitious buds by using the anthurium andraeanum vein as an explant, and the prior art reports do not search a case of using the anthurium andraeanum vein as the explant.
TABLE 1 comparison of tissue culture results of different explants
Compared with the prior art, the invention has the advantages and beneficial effects that:
(1) compared with other explants, the anthurium andraeanum leaf vein is not easy to lose water in the process of culturing the explant, and the risk of browning of the explant is reduced.
(2) The vein is used as an explant, the wound callus area of the cut edge of the vein is large after cutting, the vein is fully contacted with a culture medium, and the callus induction rate is greatly improved.
(3) After the explant veins are cut into small sections, the volume is small, the inoculation operation is convenient and quick, the inoculation operation time is reduced, and the risk of pollution caused by the inoculation operation is reduced.
(4) In the conventional anthurium andraeanum tissue culture method, if leaves are selected as explants, veins are usually cut off and discarded. The invention selects the vein as the explant, can be used as a supplementary form of the conventional tissue culture, and fully utilizes the leaf tissue.
Drawings
FIG. 1 shows an adventitious bud formed by a tissue culture method for inducing differentiation of adventitious buds by using the vein of anthurium,
wherein 1a is vein with diameter larger than 1.5 mm; 1b is the leaf vein margin of the explant forming callus; 1c is a punctate protuberance on the callus, and 1d is an adventitious bud induced and differentiated on the callus.
FIG. 2 shows an adventitious bud formed by a tissue culture method for inducing differentiation of adventitious buds by using the vein of anthurium,
wherein 1a is vein with diameter larger than 1.5 mm; 1b is the leaf vein margin of the explant forming callus; 1c is a punctate protuberance on the callus, and 1d is an adventitious bud induced and differentiated on the callus.
Detailed Description
The following describes specific embodiments of the present invention.
Example 1:
a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins comprises the following steps:
(1) selecting a female parent material: a potted anthurium andraeanum variety '17-04' with strong growth and beautiful plant type is selected as a female parent material from the base of Wuhan Weierfu biotechnology limited company.
(2) Explant treatment: young and tender leaves are selected and cut off from the female parent plant, and then are washed for 20min by tap water, and the surfaces of the leaves are gently kneaded by hands in the period. Soaking the mixture on a clean bench for 60s by using 75 percent (volume ratio) alcohol solution, washing the mixture by using sterile water for 2 times, soaking the mixture by using 10 percent (volume ratio) hydrogen peroxide solution for 15min, and washing the mixture by using sterile water for 5 times. The veins are cut from the lamina along their edges and veins greater than 1.5mm in diameter are cut into 1cm long sections for use (fig. 1 a).
(3) Starting culture: the cut veins are horizontally inoculated into a No. 1 culture medium 1/2MS, TDZ 0.01mg/L, 6-BA1.8mg/L and NAA 0.8mg/L, the pH value of the culture medium is adjusted to 5.95, and the culture medium is placed into a culture chamber for culture for 45 d. The culture environment condition of the culture room is controlled to be 25 +/-2 ℃ and the culture is carried out in dark.
(4) Differentiation culture of callus: after the start culture is finished, only 1.3 percent of explants are browned, callus is formed at the leaf vein edges of the other explants (figure 1b), the callus induction rate is 96.6 percent, and the callus is transferred to No. 2 culture medium 1/2MS +6-BA 1.5mg/L + NAA 0.2mg/L +2, 4-D0.2 mg/L. The pH of the culture medium is adjusted to 5.95, and the culture medium is placed into a culture room for culture for 45 d. The culture environment condition of the culture room is controlled to be 25 +/-2 ℃ and the culture is carried out in dark.
(5) Adventitious bud induction culture: after callus differentiation culture for 45 days, punctate protrusions were observed on the callus (FIG. 1c), which was transferred to medium No. 3 MS +6-BA 1.2mg/L + NAA 0.05mg/L, the pH of the medium was adjusted to 5.95, and the medium was placed in a culture room for culture. The culture environment conditions of the culture chamber are that the temperature is 25 +/-2 ℃, the illumination intensity is 1700-. After culturing for 40 days, the adventitious bud is induced and differentiated on the callus (figure 1d), and the adventitious bud induction rate is 97.8%.
Example 2:
a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins comprises the following steps:
(1) selecting a female parent material: selecting Anthurium '17-07' with no pest damage and beautiful plant type as female parent material from Wuhan Weierfu biotechnology limited base.
(2) Explant treatment: after young and tender leaves were selected and cut from the female parent, the leaves were rinsed with tap water for 30min, during which the surface of the leaves was gently kneaded with hands. Soaking the mixture on a clean bench for 50s by using 70 percent (volume ratio) of alcohol solution, washing the mixture for 3 times by using sterile water, soaking the mixture for 13min by using 12 percent (volume ratio) of hydrogen peroxide solution, and washing the mixture for 4 times by using the sterile water. The veins are cut along their edges and veins greater than 1.5mm in diameter are cut into 1cm pieces for use (fig. 2 a).
(3) Starting culture: the cut veins are horizontally inoculated into No. 1 culture medium 1/2MS + TDZ 0.04mg/L +6-BA0.5mg/L + NAA 0.2mg/L, the pH value of the culture medium is adjusted to 5.85, and the culture medium is placed into a culture room for culture for 60 days. The culture environment condition of the culture room is 25 +/-2 ℃ and dark culture.
(4) Differentiation culture of callus: after the start culture is finished, only 2.0% of explants are browned, callus is formed at the leaf vein edges of the other explants (figure 2b), the induction rate of the callus is 97.3%, the explant is transferred to No. 2 culture medium 1/2MS +6-BA 1.0mg/L + NAA 0.1mg/L +2, 4-D0.1 mg/L, the pH value of the culture medium is adjusted to 5.85, and the explant is placed in a culture room for culture for 30D. The culture environment condition of the culture room is 25 +/-2 ℃ and dark culture.
(5) Adventitious bud induction culture: after the callus differentiation culture was completed, punctate protrusions were observed on the callus (FIG. 2c), and the callus was transferred to medium No. 3 MS +6-BA 1.0mg/L + NAA 0.04mg/L, the pH of the medium was adjusted to 5.85, and the medium was placed in a culture room for culture. The culture environment conditions of the culture room are that the temperature is 25 +/-2 ℃, the illumination intensity is 1800-2000 lx, and the illumination period is 12 h/d. After the adventitious bud induction culture is carried out for 60 days, the adventitious bud can be induced and differentiated on the callus (figure 2d), and the adventitious bud induction rate is 98.2%.
Example 3:
a tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins comprises the following steps:
(1) selecting a female parent material: a potted anthurium andraeanum variety '17-04' or '17-07' which grows robustly and has beautiful plant types is selected as a female parent material from the base of Wuhan Weierfu biotechnology limited.
(2) Explant treatment: young leaves are selected and cut off from the female parent plant, and then are washed with tap water for 20 or 25 or 30min, and the surfaces of the leaves are gently rubbed by hands in the process. Soaking in 70 or 75% (volume ratio) alcohol solution for 30 or 40 or 50 or 60s, washing with sterile water for 2 or 3 times, soaking in 10 or 12% (volume ratio) hydrogen peroxide solution for 10 or 15 or 20min, and washing with sterile water for 3 or 4 or 5 times. The veins are cut from the lamina along their edges and veins greater than 1.5mm in diameter are cut into 1cm long sections for use (fig. 1 a).
(3) Starting culture: the cut veins are horizontally inoculated into a No. 1 culture medium 1/2MS + TDZ 0.01 or 0.02 or 0.04 or 0.05mg/L +6-BA0.5 or 1.0 or 1.5 or 1.8 or 2.0mg/L + NAA 0.1 or 0.2 or 0.4 or 0.8 or 1.0mg/L, the pH value of the culture medium is adjusted to 5.8 or 5.9 or 5.95 or 6.0, and the mixture is placed into a culture room for culture for 30 or 45 or 60 days. The culture room is controlled at 23 or 24 or 25 or 26 or 27 deg.C, and cultured in dark.
(4) Differentiation culture of callus: after the start culture is finished, only 0.9 to 1.4 percent of explants generate browning, the leaf vein edges of the rest explants form callus, the callus induction rate is 95.9 to 98.1 percent, and the callus is transferred into No. 2 culture medium 1/2MS +6-BA0.5 or 1.0 or 1.5 or 2.0mg/L + NAA 0.1 or 0.2 or 0.4 or 0.8 or 1.0mg/L +2, 4-D0.01 or 0.05 or 0.1 or 0.2 or 0.4 or 0.5 mg/L. Adjusting the pH value of the culture medium to 5.8 or 5.9 or 5.95 or 6.0, and placing the culture medium into a culture chamber for culture for 30 or 45 or 60 days. The culture room is controlled at 23 or 24 or 25 or 26 or 27 deg.C, and cultured in dark.
(5) Adventitious bud induction culture: after callus differentiation culture for 30 or 45 or 60 days, punctate protrusions are visible on the callus, and the callus is transferred into a No. 3 culture medium MS +6-BA 0.2 or 0.5 or 1.0 or 1.2 or 1.5mg/L + NAA 0.01 or 0.05 or 0.1 or 0.2 or 0.4 or 0.8mg/L, the pH of the culture medium is adjusted to 5.8 or 5.9 or 5.95 or 6.0, and the callus is placed into a culture chamber for culture. The culture room has culture environment conditions of 23 or 24 or 25 or 26 or 27 deg.c, 1700 or 1800 or 1900lx light intensity and 12 or 13 or 14h/d light period. After culturing for 30 or 40 or 50 or 60 days, adventitious buds are induced and differentiated on the callus, and the adventitious bud induction rate is 97.4-98.2%.
Claims (2)
1. A tissue culture method for inducing and differentiating adventitious buds by using anthurium andraeanum veins comprises the following steps:
(1) selecting a female parent material: selecting anthurium plants as a female parent material;
(2) the explant treatment, namely taking the vein of the young leaf of the female parent as the explant for tissue culture, cutting the leaf from the female parent, washing the cut leaf with tap water for 20 ~ 30min, then sequentially soaking the cut leaf in 70 ~ 75% alcohol for 30 ~ 60s, washing the cut leaf with sterilized water for 2 ~ 3 times, soaking the cut leaf in 10 ~ 12% hydrogen peroxide solution for 10 ~ 20min, washing the cut leaf with sterilized water for 3 ~ 5 times, cutting the cut leaf into 1cm for later use;
(3) starting culture: horizontally inoculating the cut leaf veins into a No. 1 culture medium, and putting the culture medium into a culture room for culture;
(4) callus differentiation culture, namely starting culture for 30 ~ 60d, forming callus at the edge of the vein, and transferring the callus into No. 2 culture medium;
(5) adventitious bud induction culture, namely, after the differentiation culture of the callus for 30 ~ 60d, the callus is enlarged, the surface of the callus has punctate protrusions, the callus is transferred into a No. 3 culture medium, and after the differentiation culture of the adventitious bud for 30 ~ 60d, the adventitious bud with the height of 1 ~ 2cm is induced and differentiated on the callus;
the No. 1 culture medium is 1/2MS + TDZ 0.01 ~ 0.05.05 mg/L +6-BA0.5 ~ 2.0.0 mg/L + NAA 0.1 ~ 1.0.0 mg/L;
the No. 2 culture medium is 1/2MS +6-BA0.5 ~ 2.0.0 mg/L + NAA 0.1 ~ 1.0.0 mg/L +2, 4-D0.01 ~ 0.5.5 mg/L;
the No. 3 culture medium is MS +6-BA 0.2 ~ 1.5.5 mg/L + NAA 0.01 ~ 0.8.8 mg/L;
the pH value of the culture medium No. 1, 2 or 3 is 5.8 ~ 6.0.0;
the culture conditions of the culture chamber are as follows: culturing at 23-27 deg.C in dark;
in the adventitious bud induction culture stage, the culture conditions of the culture chamber are 23-27 ℃, the illumination intensity is 1500 ~ 2000lx and the illumination period is 12 ~ 14 h/d.
2. The tissue culture method for inducing differentiation adventitious bud by using anthurium andraeanum vein according to claim 1, characterized in that: the diameter of the vein is more than 1.5 mm.
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| CN106386486A (en) * | 2016-09-06 | 2017-02-15 | 广州市名卉景观科技发展有限公司 | Anthurium tissue culture and fast propagation culture medium and anthurium tissue culture and fast propagation seed production method |
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| CN106165647A (en) * | 2016-08-04 | 2016-11-30 | 武汉市农业科学技术研究院作物科学研究所 | Red palm tissue-culturing rapid propagation inoculation method |
| CN106386486A (en) * | 2016-09-06 | 2017-02-15 | 广州市名卉景观科技发展有限公司 | Anthurium tissue culture and fast propagation culture medium and anthurium tissue culture and fast propagation seed production method |
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