CN115176704B - Method for tissue culture of poplar variety' Senhai No. 2 - Google Patents
Method for tissue culture of poplar variety' Senhai No. 2 Download PDFInfo
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- CN115176704B CN115176704B CN202210854816.5A CN202210854816A CN115176704B CN 115176704 B CN115176704 B CN 115176704B CN 202210854816 A CN202210854816 A CN 202210854816A CN 115176704 B CN115176704 B CN 115176704B
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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
- A01H4/002—Culture media for tissue culture
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Abstract
The invention relates to the field of plant tissue culture, and particularly discloses a method for tissue-culturing a poplar variety 'Senhai No. 2', which comprises the following steps: s1, dividing a sterile blade of 'Senhai No. 2' into two sections along the direction perpendicular to a main vein, taking the cut edge of a near-petiole section as an explant, and inoculating the explant into an induction culture medium to induce and culture callus, so as to obtain a blade with the callus; s2, transferring the leaf with the callus in the step S1 to a differentiation medium, and differentiating and culturing cluster seedlings; s3, transferring the cluster seedlings growing to 1.0-1.5cm in height to a rooting culture medium for rooting culture to obtain tissue culture seedlings of 'Senhai No. 2'. The invention mainly aims at the improved poplar seed 'Senhai No. 2', comprehensively considers each link in the tissue culture process, optimizes the tissue culture condition and establishes a set of efficient tissue culture system suitable for 'Senhai No. 2'.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for tissue-culturing a poplar variety 'Senhai No. 2'.
Background
The poplar improved variety 'Senhai No. 2' is obtained by artificial control pollination and breeding of the Chinese forest institute of forestry Hu Jianjun and the like, and the variety (number: national S-SV-PS-001-2021) with the improved variety passing approval is obtained in 2021. The male parent is populus tomentosa, and the female parent is populus tomentosa. The affinity of populus pie and populus pie is relatively close, the hybridization compatibility is high, and the populus pie and populus pie is a very good hybridization combination (Hu, fan Junfeng, high construction, etc. the artificial hybridization of populus americana and populus, chuan Yang Hebo, and the determination of hybrid seedling growth and disease resistance [ J ]. The university of Zhejiang, 2009, 26 (6): 778-783.). 'Senhai No. 2' has excellent rapidity and lumber property, but has poor saline-alkali resistance and insect resistance. The tissue culture regeneration system is the basis of the conservation and utilization of germplasm resources and the research of gene transformation, and the establishment of the efficient tissue culture regeneration system of poplar 'Senhai No. 2' can lay the foundation for introducing a saline-alkali resistant gene or an insect resistant gene into the 'Senhai No. 2' group by an agrobacterium tumefaciens-mediated method to obtain a good new variety.
Disclosure of Invention
The technical problem to be solved by the invention is how to efficiently tissue culture poplar improved variety 'Senhai No. 2'.
In order to solve the technical problems, the invention provides a method for tissue culture (tissue culture) of poplar variety 'Senhai No. 2', which comprises the following steps:
s1, dividing a sterile blade of 'Senhai No. 2' into two sections along the direction perpendicular to a main vein, taking the cut edge of a near-petiole section as an explant, and inoculating the explant into an induction culture medium to induce and culture callus, so as to obtain a blade with the callus;
s2, transferring the leaf with the callus in the step S1 to a differentiation medium, and differentiating and culturing cluster seedlings;
s3, transferring the cluster seedlings growing to 1.0-1.5cm in height to a rooting culture medium for rooting culture to obtain tissue culture seedlings of 'Senhai No. 2';
the induction medium is solid medium obtained by taking MS basic medium as basic medium and adding 6-BA, ZT, IBA and 2,4-D into the basic medium, wherein the concentration of 6-BA in the induction medium is 0.5mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 The concentration of 2,4-D is 1.0 mg.L -1 ；
The differentiation medium is a solid medium obtained by taking MS basic medium as basic medium, adding 6-BA, ZT and IBA into the basic medium, and the concentration of 6-BA in the differentiation medium is 0.5 mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 ；
The rooting culture medium is a solid culture medium obtained by taking 1/2MS basic culture medium as basic culture medium and adding IBA and NAA into the basic culture medium, wherein the concentration of IBA in the rooting culture medium is 0.05 mg.L -1 NAA concentration of 0.02 mg.L -1 。
In the above method, the size of the explant is preferably (0.8 to 1.2) cm× (0.8 to 1.2) cm.
In the method, the culture condition of the induction culture is that the temperature is 24-25 ℃ and the induction culture is dark.
In the above method, the induction culture is performed every 2 weeks.
In the above method, the induction culture is performed for 3 to 4 weeks.
In the method, the culture conditions of the differentiation culture and the rooting culture are that the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
In the above method, the differentiation culture is performed for 3 to 4 weeks.
In the above method, the rooting culture is performed for 15-21d.
In the method, the process for obtaining the aseptic blade of the Senhai No. 2 comprises the following steps:
a1, taking a new tender branch of 'Senhai No. 2', cleaning and sterilizing, shearing into a stem section containing 1-2 buds, and vertically placing the stem section on a rooting culture medium for culture until the buds of lateral buds grow to 1-2cm;
a2, taking off the lateral buds obtained by the culture in the step A1, and placing the lateral buds into a rooting culture medium for culture until seedlings are obtained;
a3, taking the terminal buds of the seedlings in the step A2, putting the terminal buds into a rooting medium for culturing for 25-30d, and taking the leaves on the terminal buds to obtain the aseptic leaves of 'Senhai No. 2'.
In the method, the culture conditions of the culture in the steps A1-A3 are 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
In the above method, the culture of A2 is carried out until the seedlings are obtained for 30 days.
The method also comprises the step of seedling hardening and transplanting.
The invention mainly aims at the improved poplar seed 'Senhai No. 2', comprehensively considers each link in the tissue culture process, optimizes the tissue culture condition and establishes a set of efficient tissue culture system suitable for 'Senhai No. 2'.
Drawings
FIG. 1 is a photograph of a cluster seedling differentiated from the improved poplar strain `Senhai No. 2` in example 1 of the present invention.
FIG. 2 is a photograph showing the cultivation of the improved poplar strain `Senhai2` of example 1 of the present invention on a rooting medium for 30 days.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The poplar improved variety 'Senhai No. 2' used in this example was a variety (No. S-SV-PS-001-2021) which was approved by the improved variety obtained in 2021.
The medium used in this example is as follows:
induction medium: MS+6-BA 0.5mg.L -1 +ZT 0.25mg·L -1 +IBA 0.25mg·L -1 +2,4-D 1.0mg·L -1 . The induction medium is a basic medium comprising MS basic medium, 6-BA (6-benzylaminopurine), ZT (zeatin), IBA (indolebutyric acid) and 2,4-D (2, 4-dichlorophenoxyacetic acid) are added into the basic medium, and the concentration of 6-BA in the induction medium is 0.5mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 The concentration of 2,4-D is 1.0 mg.L -1 。
Differentiation medium: MS+6-BA 0.5mg.L -1 +ZT 0.25mg·L -1 +IBA 0.25mg·L -1 . The differentiation medium is prepared by adding 6-BA (6-benzylaminopurine), ZT (zeatin) and IBA (indolebutyric acid) into MS basic culture medium as basic culture medium, wherein the concentration of 6-BA in the differentiation medium is 0.5mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 。
Rooting medium: 1/2MS+IBA 0.05 mg.L -1 +NAA 0.02mg·L -1 . The rooting medium is obtained by adding IBA (indolebutyric acid) and NAA (naphthylacetic acid) into 1/2MS basic medium as basic medium, wherein the concentration of IBA in rooting medium is 0.05mg.L -1 NAA concentration of 0.02 mg.L -1 。
1. Preparation of materials
A1, cutting new shoots from annual cutting seedlings of the improved poplar variety 'Senhai No. 2' after 5 months, and flowing and flushing the seedlings under tap water for 2 hours. Soaking in 75% ethanol for 30s in an ultra-clean workbench, washing with sterile water for 1 time, soaking in 10% sodium hypochlorite for 30min, washing with sterile water for 4-5 times, shearing into stem segments with length of about 2cm, each stem segment containing 1-2 bud points, vertically placing on rooting medium, and culturing until bud length of lateral bud (obtained by bud point growth) is 1-2cm.
A2, taking off the lateral buds obtained by culturing in the step A1, and culturing the lateral buds on a rooting medium for 30 days to grow strong seedlings.
A3, taking the terminal buds of the seedlings in the step A2, putting the terminal buds into a rooting medium, and culturing the seedlings for 25-30 days, wherein the seedlings are good in leaf status (fresh green, strong and good in leaf growth) and suitable for being used as explants.
The culture conditions of A1-A3 are as follows: the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h.
2. Induction and differentiation of callus
S1, dividing the vertical main vein of the leaf into two parts, leaving a clear upper section of the main vein, cutting a circle along the edge of the leaf to remove the edge of the leaf, placing the cut leaf serving as an explant (with the size of (0.8-1.2) cm multiplied by (0.8-1.2) cm) on an induction culture medium (repeated for 3 times, inoculating 30 leaves each time), culturing in darkness at the temperature of 24-25 ℃ for 3-4 weeks, repeating every 2 weeks, and counting the induction rate on the 28 th day of the induction culture.
Inductivity = leaf number of induced callus/total leaf number
The results showed that the induction rate reached 100%.
S2, transferring the explant inducing the callus to a differentiation culture medium (repeated for 3 times, inoculating 30 leaves each time), wherein the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, the illumination is 16 h/dark for 8h, and the cluster seedlings can be differentiated after 3-4 weeks of differentiation culture. Statistical differentiation rate at day 28 of differentiation culture:
differentiation rate = number of clumped seedlings differentiated/total number of leaves
The differentiation rate reaches more than 90 percent. Photographs of the differentiated seedlings are shown in FIG. 1.
3. Rooting culture
Transferring to rooting culture medium (repeating for 3 times, transferring about 27 cluster seedlings each time) when cluster seedlings grow to 1.0-1.5cm high, wherein the temperature is 24-25deg.C, the illumination intensity is 1600-2000lux, the illumination is 16 h/dark 8h, and the rooting rate is counted on 21 st day of rooting culture:
rooting rate = number of rooting plants/total number of plants
The total plant number is the plant number placed on the rooting culture medium.
The result shows that the rooting rate reaches more than 80%. The picture of rooting condition of culturing on rooting culture medium for 30 days is shown in figure 2.
4. Transplanting
Rooting culture for 15d, hardening off at normal temperature (or room temperature) for 2-3d after strong roots grow out: the rubber band is loosened firstly in the first day, the cover is opened in the shade in the second day, and sunlight can be directly radiated in the third day. And after hardening seedlings for 2-3 days, when the new roots grow to 1cm, the culture medium of the root seedlings is washed clean and transplanted into soil. And (5) covering a preservative film and performing on-duty watering after transplanting for 5-7 days.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (1)
1. A method for tissue culture of poplar variety 'Senhai No. 2', which is characterized by comprising the following steps:
s1, dividing a sterile blade of 'Senhai No. 2' into two sections along the direction perpendicular to a main vein, taking the cut edge of a near-petiole section as an explant, and inoculating the explant into an induction culture medium to induce and culture callus, so as to obtain a blade with the callus;
s2, transferring the leaf with the callus in the step S1 to a differentiation medium, and differentiating and culturing cluster seedlings;
s3, transferring the cluster seedlings growing to 1.0-1.5cm in height to a rooting culture medium for rooting culture to obtain tissue culture seedlings of 'Senhai No. 2';
the induction medium is solid medium obtained by taking MS basic medium as basic medium and adding 6-BA, ZT, IBA and 2,4-D into the basic medium, wherein the concentration of 6-BA in the induction medium is 0.5mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 The concentration of 2,4-D is 1.0 mg.L -1 ；
The differentiation medium is a solid medium obtained by taking MS basic medium as basic medium, adding 6-BA, ZT and IBA into the basic medium, and the concentration of 6-BA in the differentiation medium is 0.5 mg.L -1 The ZT concentration was 0.25 mg.L -1 IBA concentration of 0.25 mg.L -1 ；
The rooting culture medium is a solid culture medium obtained by taking 1/2MS basic culture medium as basic culture medium and adding IBA and NAA into the basic culture medium, wherein the concentration of IBA in the rooting culture medium is 0.05 mg.L -1 NAA concentration of 0.02 mg.L -1 ；
The culture condition of the induction culture is that the temperature is 24-25 ℃ and the induction culture is dark;
the induction culture is carried out once every 2 weeks;
the induction culture is carried out for 3-4 weeks;
the culture conditions of the differentiation culture and the rooting culture are that the temperature is 24-25 ℃, the illumination intensity is 1600-2000lux, and the illumination is 16 h/dark 8h;
the differentiation culture is carried out for 3-4 weeks;
the rooting culture is carried out for 15-21 days;
the process for obtaining the aseptic blade of the 'Senhai No. 2' comprises the following steps:
a1, taking a new tender branch of 'Senhai No. 2', cleaning and sterilizing, shearing into a stem section containing 1-2 buds, and vertically placing the stem section on a rooting culture medium for culture until the buds of lateral buds grow to 1-2cm;
a2, taking off the lateral buds obtained by the culture in the step A1, and placing the lateral buds into a rooting culture medium for culture until seedlings are obtained;
a3, taking terminal buds of the seedlings in the step A2, putting the terminal buds into a rooting medium for culturing for 25-30d, and taking the leaves on the terminal buds as sterile leaves of 'Senhai No. 2';
a2, culturing until the seedlings are obtained for 30 days;
the method also comprises the step of seedling hardening and transplanting.
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| US5354943A (en) * | 1988-05-04 | 1994-10-11 | The United States Of America As Represented By The Secretary Of The Agriculture | Methods of high frequency tissue regeneration, regeneration of herbicide-tolerant populus plants therewith, and the herbicide-tolerant plants made thereby |
| CN112335550A (en) * | 2020-12-23 | 2021-02-09 | 江苏省中国科学院植物研究所 | Method for establishing regeneration system of populus colourianus by taking petioles as explants |
| CN112715363A (en) * | 2021-01-26 | 2021-04-30 | 北京林业大学 | Optimization method of poplar callus budding regeneration system |
| CN113317200B (en) * | 2021-06-25 | 2022-04-12 | 四川大学 | Tissue culture medium for male populus diversifolia plants and application of tissue culture medium |
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