CN109362567B - Method for inducing somatic embryo and regenerating plant by using hippeastrum leaves - Google Patents
Method for inducing somatic embryo and regenerating plant by using hippeastrum leaves Download PDFInfo
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- CN109362567B CN109362567B CN201811462413.6A CN201811462413A CN109362567B CN 109362567 B CN109362567 B CN 109362567B CN 201811462413 A CN201811462413 A CN 201811462413A CN 109362567 B CN109362567 B CN 109362567B
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- 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
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Abstract
The invention belongs to the technical field of plant induction and regeneration, and particularly discloses a method for inducing a somatic embryo and regenerating a plant by using hippeastrum leaves. The method comprises the steps of subculturing test-tube plantlets of hippeastrum, transversely cutting the test-tube plantlets perpendicular to the longitudinal axis of leaves after new leaves grow out, and cutting the test-tube plantlets into segments with proper lengths to serve as explants; placing the explant on an induction culture medium, and culturing under the conditions of proper temperature and darkness to generate somatic embryos on the surfaces of the leaves; transferring the explant producing the somatic embryo to a regeneration culture medium, and culturing for a plurality of weeks under the environment of proper temperature and illumination intensity for a certain time every day, wherein the somatic embryo germinates to form a complete plant. The invention establishes a plant regeneration system of an efficient somatic embryogenesis way, and provides a good technical basis for the large-scale sustainable industrialized seedling production of hippeastrum rutilum.
Description
Technical Field
The invention belongs to the technical field of plant induction and regeneration, and particularly relates to a method for inducing a somatic embryo and regenerating a plant by using hippeastrum leaves.
Background
Hippeastrum hybridum Hort also named as Hippeastrum, lonanthus, and armandin, etc., is an important perennial herb plant of Hippeastrum of Amaryllidaceae, and is a world-known bulbous flower in native tropical America.
The research of the tissue culture of the hippeastrum starts in the 70 th 20 th century, and mostly adopts bulbs, bulb plates, young capsules, young scapes, young embryos or hypocotyls as starting materials, and obtains callus, adventitious buds or somatic embryos by induction culture and then obtains complete plants by culture. The above regeneration techniques are inefficient. In the prior art in recent years, people establish an effective plant regeneration system by taking petals as explants, but the acquisition of the petal explants is limited by factors such as seasons and the like, and the propagation efficiency is still low.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for inducing a somatic embryo and regenerating a plant by using a hippeastrum leaves. The invention adopts the hippeastrum test-tube plantlet leaves as explants, and establishes a plant regeneration system with a high-efficiency somatic embryogenesis way. The method can effectively solve the problem that the acquisition of the petal explants is limited by factors such as seasons and the like in the prior art, and can obviously improve the tissue culture propagation efficiency of hippeastrum. The plant regeneration rate can reach 98.6 percent at most, and each explant can generate 26.8 plantlets at most on average.
The invention is realized by the following technical scheme.
A method for inducing somatic embryos and regenerating plants by using hippeastrum leaves comprises the following steps:
s1, explant acquisition: subculturing the test-tube plantlet of hippeastrum at 24-26 ℃ for 12-14 h daily in an illumination environment, cutting leaves after new leaves grow out, (transversely cutting perpendicular to the longitudinal axis of the leaves) and cutting the leaves into segments with proper length as explants;
s2, inducing somatic embryos: horizontally placing the explant processed in the S1 on an induction culture medium, and culturing at 24-26 ℃ under a dark condition to generate somatic embryos on the outer surfaces of leaves;
s3, plant regeneration: and transferring the somatic embryos generated in the S2 to a regeneration culture medium, and culturing the somatic embryos under the illumination environment of 24-26 ℃ for 12-14 h every day to germinate into complete plants.
Preferably, the length of the new leaves growing out selected from S1 is 0.5-5.0 cm, and the leaves are cut into suitable length segments of 0.5-1.0 cm.
Preferably, the culture medium for the subculture of S1 is MS culture medium as a basic culture medium, and 0-1.0 mg/L6-benzylamino adenine (6-BA) and 20-40 g/L sucrose are added, and the pH value is 5.8-6.0.
Preferably, the induction medium of S2 uses MS medium as basic medium, and 0.1-1.0 mg/L Thidiazuron (TDZ) and 20-40 g/L sucrose are added, and the pH value is 5.8-6.0. More preferably, the addition amount of the Thidiazuron (TDZ) in the induction medium is 0.2-0.7 mg/L.
Preferably, the regeneration medium of S3 uses MS medium as basic medium, and 0-1.0 mg/L6-benzylamino adenine (6-BA) and 20-40 g/L sucrose, pH 5.8-6.0 are added. More preferably, the addition amount of the 6-benzylaminopurine (6-BA) in the regeneration medium is 0-0.5 mg/L.
Preferably, the intensity of the illumination of S1 and S3 is 1000-1400 lux.
Preferably, the culture time in S2 is 8-12 weeks, and the culture time in S3 is 4-8 weeks.
More preferably, the test-tube plantlet of hippeastrum rutilum is subjected to subculture at 24-26 ℃ in an illumination environment for 12-14 h every day in S1, when a new leaf grows out and then the length of the new leaf is 0.5-5.0 cm, the new leaf is cut, transverse cutting is performed perpendicular to the longitudinal axis of the leaf, and the new leaf is cut into a segment with the proper length of 0.5-1.0 cm and serves as an explant.
More preferably, in S2, the explant treated in S1 is horizontally placed on an induction culture medium and cultured for 8-12 weeks under the dark condition at 24-26 ℃, and somatic embryos are generated on the outer surface of the leaf blades; the induction culture medium is an MS culture medium as a basic culture medium, 0.5mg/L Thidiazuron (TDZ) and 20-40 g/L cane sugar are added, and the pH value is 5.8-6.0.
More preferably, in S3, transferring the somatic embryos generated in S2 to a regeneration culture medium, culturing for 4-8 weeks in an environment with illumination intensity of 1000-1400 lux for 12-14 h every day at 24-26 ℃, and germinating the somatic embryos to form complete plants; the regeneration culture medium is an MS culture medium which is used as a basic culture medium, 0.5mg/L of 6-benzylamino adenine (6-BA) and 20-40 g/L of cane sugar are added, and the pH value is 5.8-6.0.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the culture process of the hippeastrum test-tube plantlets, new leaf tissues can be continuously formed without being limited by seasons. The leaf explant is very easy to obtain and very low in cost. The invention uses leaves as explants to carry out tissue culture and rapid propagation, and effectively solves the problem that the acquisition of petal explants is limited by factors such as seasons and the like in the prior art.
(2) At present, a high-efficiency plant regeneration system is not established by taking hippeastrum leaves as explants. The invention adopts the test-tube seedling leaves as the explants, establishes a plant regeneration system with a high-efficiency somatic embryogenesis way, has the highest somatic embryo induction rate of 96.7 percent and the highest plant regeneration rate of 98.6 percent, and can generate at most 26.8 plantlets per explant on average. Compared with the prior art, the regeneration efficiency of the plants is obviously improved.
(3) The method takes the regenerated plant and the leaf of the plant progeny of the regeneration plant and the subculture as explants, and can repeatedly realize the processes of somatic embryo induction and plant regeneration, thereby realizing the large-scale sustainable industrialized seedling production of the hippeastrum.
Drawings
FIG. 1 is a pictorial representation of somatic embryos induced by hippeastrum leaf explants.
FIG. 2 is a picture of a regenerated plant of hippeastrum rutilum.
Fig. 3 is a specific flowchart of embodiment 1 in the present embodiment.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the examples are not intended to limit the invention in any way. It will be understood by those skilled in the art that the scope of the present invention is defined by the claims, and any equivalents fall within the claims.
FIG. 1 is a diagram of a species of somatic embryos induced by a hippeastrum leaf explant, and FIG. 2 is a diagram of a species of a hippeastrum plant after regeneration. As can be seen from fig. 1 and 2, the technical effect of the present invention can be achieved.
Example 1
As shown in FIG. 3, the specific steps of the method for inducing somatic embryos and regenerating plants by using the leaves of hippeastrum in example 1 are as follows:
step 1, subculturing test-tube plantlets of hippeastrum at 24-26 ℃ for 12-14 h every day under the environment that the illumination intensity is 1000-1400 lux, cutting leaves when the length of the test-tube plantlets is 0.5-5.0 cm after new leaves grow out, transversely cutting the test-tube plantlets perpendicular to the longitudinal axis of the leaves, and cutting the test-tube plantlets into segments with proper length of 0.5-1.0 cm as explants;
step 2, horizontally placing the explants processed in the step 1 on an induction culture medium, culturing for 8-12 weeks at 24-26 ℃ under a dark condition, and generating somatic embryos on the outer surfaces of leaves; the induction culture medium is an MS culture medium which is used as a basic culture medium, 0.1-0.5 mg/L Thidiazuron (TDZ) and 20-40 g/L cane sugar are added, and the pH value is 5.8-6.0;
step 3, transferring the somatic embryos generated in the step 2 to a regeneration culture medium, culturing for 4-8 weeks in an environment with illumination intensity of 1000-1400 lux for 12-14 h every day at 24-26 ℃, and germinating the somatic embryos to form complete plants; the regeneration culture medium is an MS culture medium as a basic culture medium, 0.5mg/L of 6-benzylamino adenine (6-BA) and 20-40 g/L of cane sugar are added, and the pH value is 5.8-6.0.
Table 1 is provided to illustrate the effect of TDZ at a concentration of 0.1-1.0 mg/L on the induction rate of somatic embryos of hippeastrum leaves. Under the condition that other conditions are stable, as can be seen from the table, the induction rate of the somatic embryos is gradually improved along with the increase of the TDZ concentration when the concentration of the Thidiazuron (TDZ) is 0.1-0.4 mg/L, the induction rate of the somatic embryos reaches the highest value at 0.5mg/L, and then the induction rate of the somatic embryos starts to obviously reduce when the concentration of the TDZ is continuously increased from 0.6mg/L to 1.0 mg/L. It is understood that 0.5mg/L TDZ is the highest concentration of somatic embryo induction rate, therefore, in the embodiment 1, the 0.5mg/L Thidiazuron (TDZ) is selected when the method is implemented, so as to achieve the ideal implementation effect and improve the success rate of somatic embryo induction regeneration of hippeastrum leaf.
TABLE 1 influence of TDZ on somatic embryo induction of hippeastrum leaves
Note: somatic embryo induction (%) - (number of induced somatic embryo explants ÷ number of inoculated total explants) × 100%.
Example 2
The method for inducing somatic embryos and regenerating plants by using hippeastrum leaves in the embodiment 2 comprises the following specific processes:
step 1, subculturing test-tube plantlets of hippeastrum at 24-26 ℃ for 12-14 h every day under the environment that the illumination intensity is 1000-1400 lux, cutting leaves when the length of the test-tube plantlets is 0.5-5.0 cm after new leaves grow out, transversely cutting the test-tube plantlets perpendicular to the longitudinal axis of the leaves, and cutting the test-tube plantlets into segments with proper length of 0.5-1.0 cm as explants;
step 2, horizontally placing the processed explants on an induction culture medium, culturing for 8-12 weeks at 24-26 ℃ under a dark condition, and generating somatic embryos on the outer surfaces of leaves; the induction culture medium is an MS culture medium which is used as a basic culture medium, 0.5mg/L Thidiazuron (TDZ) and 20-40 g/L cane sugar are added, and the pH value is 5.8-6.0;
step 3, transferring the somatic embryos generated in the step 2 to a regeneration culture medium, culturing for 4-8 weeks in an environment with illumination intensity of 1000-1400 lux for 12-14 h every day at 24-26 ℃, and germinating the somatic embryos to form complete plants; the regeneration medium is an MS medium which is used as a basic medium, 0-1.0 mg/L of 6-benzylamino adenine (6-BA) and 20-40 g/L of cane sugar are added, and the pH value is 5.8-6.0.
Table 2 shows the effect of 6-BA at a concentration of 0 to 1.0mg/L on plant regeneration. Under otherwise stable conditions, it can be seen from Table 2 that 6-benzylaminopurine (6-BA) at a concentration range of 0-1.0 mg/L achieves the best effect of plant regeneration at a concentration of 0.5 mg/L.
TABLE 26 Effect of BA on plant regeneration
Note: plant regeneration rate (%) - (number of explants regenerated from plant ÷ total number of explants inoculated with somatic embryos) × 100%.
Claims (5)
1. A method for inducing a somatic embryo and regenerating a plant by using hippeastrum leaves is characterized by comprising the following steps:
s1, explant acquisition: subculturing the test-tube plantlet of hippeastrum at 24-26 ℃ for 12-14 h daily in an illumination environment, cutting leaves after new leaves grow out, transversely cutting the leaves perpendicular to the longitudinal axis of the leaves, and cutting the leaves into segments with proper length as explants;
s2, inducing somatic embryos: horizontally placing the explant treated in the S1 on an induction culture medium, and culturing at 24-26 ℃ under a dark condition to generate somatic embryos on the outer surface of the leaves;
s3, plant regeneration: transferring the somatic embryos generated in the S2 to a regeneration culture medium, and culturing the somatic embryos in a light environment of 12-14 h every day at the temperature of 24-26 ℃ to enable the somatic embryos to germinate to form complete plants;
wherein the culture medium of the subculture of S1 is MS minimal medium, 0-1.0 mg/L6-benzylamino adenine, 20-40 g/L sucrose and pH5.8-6.0; s2, wherein the induction culture medium is an MS minimal medium, 0.2-0.7 mg/L thidiazuron, 20-40 g/L cane sugar and pH5.8-6.0; s3 the regeneration medium is MS minimal medium, 0-0.5 mg/L6-benzylamino adenine, 20-40 g/L sucrose, pH5.8-6.0.
2. The method for inducing somatic embryos and regenerating plants by using hippeastrum leaves according to claim 1, which comprises the following steps: the intensity of illumination in S1 and S3 is 1000-1400 lux.
3. The method for inducing somatic embryos and regenerating plants by using hippeastrum leaves according to claim 1, which comprises the following steps: the culture time in S2 is 8-12 weeks, and the culture time in S3 is 4-8 weeks.
4. The method for inducing somatic embryos and regenerating plants by using hippeastrum leaves according to any one of claims 1 to 3, wherein the method comprises the following steps:
in S2, horizontally placing the explant treated in S1 on an induction culture medium, and culturing for 8-12 weeks at 24-26 ℃ under a dark condition, wherein somatic embryos are generated on the outer surface of leaves; the induction culture medium is MS minimal medium, 0.5mg/L thidiazuron, 20-40 g/L cane sugar and pH5.8-6.0.
5. The method for inducing somatic embryos and regenerating plants by using hippeastrum leaves as claimed in claim 4, wherein the method comprises the following steps: in S3, transferring the somatic embryos generated in S2 to a regeneration culture medium, and culturing for 4-8 weeks in an environment with the temperature of 24-26 ℃, the daily time of 12-14 h and the illumination intensity of 1000-1400 lux, wherein the somatic embryos germinate to form complete plants; the regeneration culture medium is MS minimal medium, 0.5 mg/L6-benzylamino adenine, 20-40 g/L sucrose and pH 5.8-6.0.
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| CN102388805A (en) * | 2011-09-09 | 2012-03-28 | 广东省农业科学院花卉研究所 | Spathiphyllum somatic embryo inducing and plant regenerating method |
| CN104585034A (en) * | 2015-01-20 | 2015-05-06 | 广东省农业科学院环境园艺研究所 | Method for regenerating plants by hippeastrum hybridum hort. petal tissue induction |
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| CN102388805A (en) * | 2011-09-09 | 2012-03-28 | 广东省农业科学院花卉研究所 | Spathiphyllum somatic embryo inducing and plant regenerating method |
| CN104585034A (en) * | 2015-01-20 | 2015-05-06 | 广东省农业科学院环境园艺研究所 | Method for regenerating plants by hippeastrum hybridum hort. petal tissue induction |
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