CN115250921B - Efficient broussonetia papyrifera polyploid induction method with leaves as explants - Google Patents
Efficient broussonetia papyrifera polyploid induction method with leaves as explants Download PDFInfo
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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
- A01H4/002—Culture media for tissue culture
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Abstract
The invention discloses a high-efficiency paper mulberry polyploid induction method taking leaves as explants, which comprises the following steps: s1, taking aseptic seedling leaves of diploid paper mulberry as explants, and inoculating the explants on a preculture medium for preculture; s2, transferring the pre-cultured explant to a liquid colchicine culture medium for dark culture for 1-4 days; the liquid colchicine culture medium contains: MS+6-BA+IBA+sucrose+colchicine, wherein the pH value is 5.8-6.0, and the colchicine concentration is 250 mg/L-550 mg/L; s3, transferring the explant subjected to dark culture to an adventitious bud induction culture medium, and inducing to form an adventitious bud; cutting the formed adventitious buds, transferring the adventitious buds into a rooting culture medium, and inducing the adventitious buds to form re-rooting to obtain regenerated seedlings; s4, identifying the multiple of the root tip chromosome. The invention establishes the broussonetia papyrifera tetraploid induction system for the first time, so as to cultivate high-quality broussonetia papyrifera tetraploid varieties and meet the requirements of broussonetia papyrifera germplasm resources.
Description
Technical Field
The invention relates to the technical field of polyploid genetic breeding, in particular to a high-efficiency paper mulberry polyploid induction method taking leaves as explants.
Background
The Broussonetia papyrifera (Broussonetia papyrifera) is a perennial deciduous arbor of the genus Broussonetia (Moraceae), has the characteristics of high stress resistance and pollution resistance, quick growth, strong adaptability, wide distribution range, easiness in propagation and short rotation period, and can play an important role in ecological restoration. Specifically, the broussonetia papyrifera leaves contain higher crude protein content, belong to high-quality feed resources, and can reduce the feed cost; paper mulberry bast fiber is slender, is an advanced raw material for papermaking, and is mainly paper mulberry bark used in Cai Lun papermaking; because of thicker leaves, rough surface and soft hair on the back, the paper mulberry leaves have extremely strong stress resistance, harmful gas (sulfur dioxide and chlorine) absorption capacity and dust absorption capacity, paper mulberry is often planted as greening and anti-pollution tree species.
Polyploid plant has the advantages of large organ, high content of effective components, high stress resistance, high disease resistance, etc. and may be used widely in forestry, agriculture, gardening, etc. Therefore, the technology for efficiently inducing polyploid paper mulberry is researched, and the high-quality paper mulberry polyploid variety is cultivated, so that the paper mulberry quality can be improved, the requirement of paper mulberry germplasm resources is met, and the technology has important significance in aspects of feed, medicine, ecological restoration and the like. However, no technology for cultivating the paper mulberry polyploid is yet seen.
Disclosure of Invention
In order to make up for the blank of the prior art, the invention aims to provide a high-efficiency paper mulberry polyploid induction method taking leaves as explants.
In order to achieve the above object, the technical scheme of the present invention is as follows:
a high-efficiency paper mulberry polyploid induction method taking leaves as explants comprises the following steps:
s1, pre-culturing a leaf explant: taking diploid paper mulberry aseptic seedling leaves as explants, and inoculating the explants on a preculture medium for preculture;
s2, colchicine induction culture: transferring the pre-cultured explant to a liquid colchicine culture medium for dark culture for 1-4 days; the liquid colchicine culture medium contains: MS+6-BA+IBA+sucrose+colchicine, wherein the pH value is 5.8-6.0, and the colchicine concentration is 250 mg/L-550 mg/L;
s3, tissue culture seedling: transferring the dark cultured explant to an adventitious bud induction culture medium, and inducing to form an adventitious bud; cutting the formed adventitious buds, transferring the adventitious buds into a rooting culture medium, and inducing the adventitious buds to form re-rooting to obtain regenerated seedlings;
s4, multiple identification: and cutting root tips of the regenerated roots, counting chromosomes, and identifying multiples of the root tip chromosomes.
Further, the preculture medium in step S1 comprises MS+30 g/L sucrose+6.0 g/L agar and has a pH of 5.8-6.0.
Further, the adventitious bud induction culture medium comprises MS+1.0-2.0 mg/L6-BA+0-0.2 mg/L IBA+30 g/L sucrose+6.0 g/L agar, and the pH value is 5.8-6.0.
Further, the rooting induction base is 1/2 MS+0-0.5 mg/L NAA+30 g/L sucrose+5.0 g/L agar, and the pH value is 5.8-6.0.
Further, the conditions of the dark culture in step S2 are: culturing is carried out on a shaking table at the constant temperature of 26+/-2 ℃, and the rotating speed of the shaking table is 90-150 rpm/min.
In step S1, the pre-culture time is 1-5 d.
Further, the conditions of preculture in step S1, induction of adventitious bud formation in step S3 and induction of rooting are as follows: the temperature is 26+/-2 ℃, the illumination intensity is 2000lx, and the illumination time is 12 hours.
Further, the step S4 further includes the following steps: the leaves of the regenerated plantlets identified as tetraploids were then subjected to flow cytometry to determine final ploidy.
In the step S3, the cutting standard of the adventitious buds is that the length of the formed adventitious buds is 2-5 cm; in the step S4, the cutting standard of the regenerated root is that the regenerated root grows to 1-3 cm.
The cutting standard of the adventitious bud can ensure the survival rate of the regenerated seedling, because the adventitious bud is too tender when the adventitious bud is lower than 2cm, and the leaf area is large when the adventitious bud is higher than 5cm, the leaf area is easy to lose water and die.
When the regenerated root grows to 1-3 cm, the root tip of the regenerated root is vigorous in division, and more cells exist in the metaphase of mitosis, and the root tip of the regenerated root is cut at this time and is more than the chromosome number of the root tip.
Further, the blades are 3 rd to 5 th blades with the tops of the regenerated seedlings downward.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for inducing paper mulberry polyploid by taking leaves as explants for the first time.
According to the invention, researches show that when the colchicine concentration in the liquid colchicine culture medium is 250 mg/L-550 mg/L, the culture time is 1-4 d, the induction efficiency of the paper mulberry tetraploid is high, and the paper mulberry tetraploid has remarkable advantages.
The invention establishes a broussonetia papyrifera tetraploid induction system by using colchicine to induce polyploid, so as to improve broussonetia papyrifera quality, cultivate high-quality broussonetia papyrifera tetraploid varieties and meet the requirements of broussonetia papyrifera germplasm resources.
Drawings
FIG. 1 shows a root tip chromosome map (A) of a diploid Broussonetia papyrifera obtained in the comparative example and a root tip chromosome map (B) of a tetraploid Broussonetia papyrifera obtained in the present invention.
FIG. 2 is a flow cytometry graph (A) of diploid paper mulberry leaves obtained in the comparative example and a flow cytometry graph (B) of tetraploid paper mulberry regenerated seedling leaves obtained in the present invention.
FIG. 3 shows the pore patterns (A) of the diploid broussonetia papyrifera leaves obtained in the comparative example and the pore patterns (B) of the tetraploid broussonetia papyrifera regenerated seedling leaves obtained in the present invention.
FIG. 4 shows a diploid Broussonetia papyrifera seedling (A) obtained in the control example and a tetraploid Broussonetia papyrifera regenerated seedling (B) obtained in the invention.
Description of the embodiments
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. In the present specification, "part" and "%" respectively mean "part by mass" and "% by mass" unless otherwise specified.
Examples
S1, selecting healthy leaves of 3 rd to 5 th pieces of diploid paper mulberry aseptic seedlings from the top to the bottom, and cutting the healthy leaves into 1 x 1 cm pieces on an ultra-clean workbench 2 Taking the square leaves as explants, and placing the explants into a preculture medium for preculture of 3 d, wherein the preculture medium has a formula of MS+30 g/L sucrose+6.0 g/L agar and a pH value of 5.8-6.0;
s2, an induction method comprises the following steps: placing the pre-cultured leaves into a colchicine-containing liquid culture medium, soaking the leaves in a shaking table at the temperature of 26+/-2 ℃ and the rotating speed of 90-150 rpm/min for 3 d, and performing dark culture induction, wherein the colchicine-containing liquid culture medium has a formula of MS+2.0 mg/L6-BA+0.1 mg/L IBA+30 g/L sucrose+250 mg/L colchicine and a pH value of 5.8-6.0;
s3, culturing the colchicine-treated explant, namely taking the colchicine-treated explant out of a colchicine-containing liquid culture medium in an ultra-clean workbench, washing the colchicine-treated explant with sterile water for 2-3 times, and transferring the colchicine-treated explant to a solid adventitious bud induction culture medium without colchicine for continuous culture to form adventitious buds; the adventitious bud induction culture medium has a formula of MS+2.0 mg/L6-BA+0.1 mg/L IBA+30 g/L sucrose+6.0 g/L agar and a pH value of 5.8-6.0;
s4, adventitious buds root: cutting off the adventitious bud with the length of 2-5 cm in the step S3 on an ultra-clean workbench, putting the adventitious bud into a rooting culture medium, and inducing to form a rooting again, wherein the rooting culture medium has a formula of 1/2 MS+0.1 mg/L NAA+30 g/L sucrose+5.0 g/L agar and a pH value of 5.8-6.0;
s5, root tip chromosome counting: when the regenerated roots grow to about 1-3 cm, taking off root tips of the regenerated roots at 9:30-10:30 am, putting the root tips into a centrifuge tube filled with distilled water in advance, and putting the centrifuge tube into ice water for mixing for treatment 12-h; transferring the pretreated root tip into Carnot's fixative (absolute ethanol: glacial acetic acid=3:1) for fixation of 24-h; then taking out the fixed root tip, flushing the root tip for 15-20min by running water, transferring the root tip into HC1 with the concentration of L mol/L for dissociation, and dissociating for 6-9min at 60 ℃; washing the dissociated root tip with running water for 15-20min, staining with Kabao fuchsin dye liquor for 15min, tabletting, observing chromosome morphology and number under a microscope, and photographing;
s6, detecting ploidy by a flow cytometer:
leaves of the paper mulberry regenerated plantlet identified as tetraploid by root tip chromosome count were selected from 3 rd to 5 th leaves from the top down, and the leaves were subjected to flow cytometry to determine final ploidy.
Examples
The present embodiment differs from embodiment 1 in that: the colchicine-containing liquid medium in this example was formulated as MS+2.0 mg/L6-BA+0.1 mg/L IBA+30 g/L sucrose+350 mg/L colchicine.
Examples
The present embodiment differs from embodiment 1 in that: the colchicine-containing liquid medium in this example was formulated as MS+2.0 mg/L6-BA+0.1 mg/L IBA+30 g/L sucrose+450 mg/L colchicine.
Examples
The present embodiment differs from embodiment 1 in that: the colchicine-containing liquid medium in this example was formulated as MS+2.0 mg/L6-BA+0.1 mg/L IBA+30 g/L sucrose+550 mg/L colchicine and the colchicine treatment time was 2d.
Examples
The present embodiment differs from embodiment 2 in that: the dark culture induction time in step S2 in this embodiment is 1d.
Examples
The present embodiment differs from embodiment 2 in that: the dark culture induction time in step S2 in this example was 4d.
Comparative example
The other steps were performed in the same manner as in example 1, except that the precultured leaf was placed in a liquid medium containing no autumn-water for induction of the dark culture.
The induction culture of the paper mulberry tetraploid was performed according to the induction method of the above-described example 1-example 4, and the chimeric rate and the tetraploid rate were counted, respectively, to obtain the results shown in Table 1.
TABLE 1
| Colchicine concentration (mg/L) | Treatment time (d) | Chimeric rate (%) | Tetraploid rate (%) | |
| Example 1 | 250 | 3 | 4.65±4.09 | 2.56±4.44 |
| Example 2 | 350 | 3 | 6.73±5.92 | 3.70±6.42 |
| Example 3 | 450 | 3 | 19.25±8.34 | 18.65±5.63 |
| Example 4 | 550 | 2 | 6.36±5.53 | 3.33±5.25 |
| Example 5 | 450 | 1 | 7.98±4.03 | 4.31±3.73 |
| Example 6 | 450 | 4 | 11.11±19.25 | 8.33±14.43 |
| Comparative example | 0 | 0 | 0 | 0 |
The calculation formula in table 1 is as follows:
tetraploid induction = (tetraploid bud number/total bud number identified) ×100%.
As can be seen from Table 1, colchicine concentration and treatment time have a large effect on the chimera rate and tetraploid induction rate of explants.
The results of randomly picking up one strain of the tetraploid paper mulberry regenerated seedlings obtained by the induction method of the above example 1-example 4, respectively performing root tip chromosome counting, flow cytometry detection, paper mulberry regenerated seedling leaf pore scanning, and the like according to the same method in the control group are shown in fig. 1-3.
As can be seen from fig. 1 to 4, the regenerated plantlet obtained in the present invention is a tetraploid paper mulberry regenerated plantlet, and the regenerated plantlet obtained in the comparative example is a diploid paper mulberry regenerated plantlet. Compared with the diploid paper mulberry regenerated seedling of the comparative example, the tetraploid paper mulberry regenerated seedling of the invention has larger organs and more active ingredient content.
The invention establishes a broussonetia papyrifera tetraploid induction system by using colchicine induced polyploid, has high broussonetia papyrifera tetraploid induction efficiency and remarkable advantages, can improve broussonetia papyrifera quality, cultivates high-quality broussonetia papyrifera tetraploid varieties, and meets the requirements of broussonetia papyrifera germplasm resources.
The above embodiments are preferred embodiments of the present invention, and besides, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.
Claims (7)
1. The efficient paper mulberry polyploid induction method taking leaves as explants is characterized by comprising the following steps of:
s1, pre-culturing a leaf explant: taking diploid paper mulberry aseptic seedling leaves as explants, and inoculating the explants on a preculture medium for preculture;
s2, colchicine induction culture: transferring the pre-cultured explant to a liquid colchicine culture medium for dark culture for 1-4 days; the liquid colchicine culture medium is as follows: MS+6-BA+IBA+30 g/L sucrose+colchicine, wherein the pH value is 5.8-6.0, and the colchicine concentration is 250 mg/L-450 mg/L;
s3, tissue culture seedling: transferring the dark cultured explant to an adventitious bud induction culture medium, and inducing to form an adventitious bud; cutting the formed adventitious buds, transferring the adventitious buds into a rooting culture medium, and inducing the adventitious buds to form re-rooting to obtain regenerated seedlings;
s4, multiple identification: cutting root tips of the regenerated roots, counting chromosomes, and identifying multiples of the root tip chromosomes;
wherein, the preculture medium in step S1 is: MS+30 g/L sucrose+6.0 g/L agar, pH value is 5.8-6.0;
the adventitious bud induction culture medium is as follows: MS+1.0-2.0 mg/L6-BA+0.1-0.2 mg/L IBA+30 g/L sucrose+6.0 g/L agar, and the pH value is 5.8-6.0;
the rooting induction base is 1/2 MS+0.1-0.5 mg/L NAA+30 g/L sucrose+5.0 g/L agar, and the pH value is 5.8-6.0.
2. The method for inducing polyploidy of paper mulberry according to claim 1, wherein the conditions of dark culture in step S2 are: culturing is carried out on a shaking table at the constant temperature of 26+/-2 ℃, and the rotating speed of the shaking table is 90-150 rpm/min.
3. The method of claim 1, wherein the preculture time is 3 d.
4. The method for inducing polyploidy of paper mulberry according to claim 1, wherein the conditions of preculture in step S1, induction of adventitious bud formation in step S3, and induction of rooting are: the temperature is 26+/-2 ℃, the illumination intensity is 2000lx, and the illumination time is 12 hours.
5. The method of claim 1, wherein step S4 further comprises the steps of: the leaves of the regenerated plantlets identified as tetraploids were then subjected to flow cytometry to determine final ploidy.
6. The efficient paper mulberry polyploid induction method of claim 1, wherein in step S3, the cutting standard of the adventitious bud is that the formed adventitious bud is 2-5 cm long; in the step S4, the cutting standard of the regenerated root is that the regenerated root grows to 1-3 cm.
7. The method for inducing polyploidy in paper mulberry according to claim 5, wherein said leaves are 3 rd to 5 th leaves with the top of said regenerated seedling downward.
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| CN103168676B (en) * | 2013-03-08 | 2014-11-26 | 湖北大学 | New Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method |
| CN108633738A (en) * | 2018-05-15 | 2018-10-12 | 贵州务川科华生物科技有限公司 | A method of producing paper mulberry tissue-cultured seedling |
| CN109156343A (en) * | 2018-08-23 | 2019-01-08 | 云南兴构农业产业发展有限公司 | The culture medium and breeding method of paper mulberry are cultivated for blade |
| CN114097613B (en) * | 2021-11-12 | 2023-02-28 | 长江大学 | Method for establishing paper mulberry leaf tissue culture regeneration system |
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