CN115644067B - Method for producing polygonatum kingianum dihaploid - Google Patents
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Abstract
The invention relates to the technical field of medicinal plant tissue culture breeding, and particularly discloses a method for producing polygonatum kingianum dihaploid, which comprises the following steps: pre-treating flower buds; sterilizing buds and stripping anthers; inoculating anther into a callus induction culture medium for culture; inoculating the induced callus into a callus differentiation culture medium for culture; carrying out chromosome doubling treatment on the buds differentiated from the callus; continuously inoculating the buds subjected to chromosome doubling treatment into a callus differentiation culture medium to grow a complete plant; inoculating the differentiated plant into a rooting culture medium for rooting culture; carrying out chromosome ploidy detection on the rooted seedlings, and screening to obtain polygonatum kingianum double haploid plants; the callus induction culture medium in the step (3) is improved N6+2, 4-D0.6-0.8 mg/L +2-IP 0.2-0.5 mg/L + GA 3.4-0.5 mg/L, the rest components are unchanged, the sugar addition amount is changed to 50g/L, and the pH value is changed to 6.4; the callus induction culture medium adopted by the invention can successfully culture the anther into callus, and the callus induction rate is up to more than 80%.
Description
Technical Field
The invention relates to the technical field of medicinal plant tissue culture breeding, in particular to a method for producing polygonatum kingianum dihaploid.
Background
Polygonatum kingianum coll.et Hemsl.) is a perennial herb of Polygonatum of Liliaceae, and has effects of invigorating qi, nourishing yin, invigorating spleen, moistening lung, and invigorating kidney.
Doubled Haploids (DH) are a technology that has been developed in recent years, and specifically refer to 100% homozygotes obtained by chromosome doubling of individuals, tissues or cells having gamete chromosome numbers in the cells.
The polygonatum kingianum of each production place and germplasm has great differences in effective component content, single plant biological yield, disease and pest resistance and the like, but at present, the polygonatum kingianum germplasm breeding is mainly concentrated on selection, namely, wild germplasm collection is adopted, then high-quality germplasm is manually selected for reproduction, and at present, only Zhangfang et al' primordial guidance for polyploid polygonatum kingianum [ J ], china breed, 2011 (1): 48 to 49, li national Tai et al, breeding research of Polygonatum odoratum polyploid [ J ], china Special side product, 2018 (4): 14 to 16, reports polyploid breeding of Polygonatum kingianum and Polygonatum odoratum which belong to the same genus as Polygonatum kingianum.
The method is to obtain haploid buds by in vitro culture of polygonatum kingianum anther, and obtain doubled haploid by chromosome doubling, thereby making up for the deficiency of the artificial induced breeding technology of polygonatum kingianum.
Disclosure of Invention
The invention mainly aims to provide a method for producing polygonatum kingianum double haploid, which solves the problem of the deficiency of the artificial induction breeding technology of polygonatum kingianum.
In order to realize the purpose, the invention provides the following technical scheme:
a method for producing polygonatum kingianum double haploid comprises the following steps:
(1) Selecting Polygonatum kingianum flower buds with anthers for pretreatment;
(2) Sterilizing the pretreated flower buds, and stripping off anthers after sterilization;
(3) Inoculating anther into a callus induction culture medium for culture;
(4) Inoculating the induced callus into a callus differentiation culture medium for culture;
(5) Carrying out chromosome doubling treatment on the buds differentiated from the callus;
(6) Continuously inoculating the buds subjected to chromosome doubling treatment into a callus differentiation culture medium to grow seedling bodies;
(7) Inoculating the differentiated seedling body into a rooting culture medium for rooting culture;
(8) Carrying out chromosome ploidy detection on the rooted seedlings, and screening to obtain polygonatum kingianum double haploid plants;
the callus induction culture medium in the step (3) is improved N6+2, 4-D0.6-0.8 mg/L +2-IP 0.2-0.5 mg/L + GA 3.4-0.5 mg/L, the rest components are unchanged, the addition amount of sugar is changed to 50g/L, and the pH is changed to 6.4.
In the step (1), the pretreatment method is to treat the mixture for 24 to 36 hours in a full-dark environment at the temperature of 4 +/-2 ℃.
In the step (2), the disinfection method comprises the steps of disinfection for 20-30 s by 75% alcohol, disinfection for 20-30min by saturated bleaching powder solution and disinfection for 3-4 min by 0.05% mercuric chloride and 2 drops of Tween-80 solution.
In the step (5), the chromosome doubling treatment method is to soak 0.02-0.04% of colchicine, 0.05-0.06% of benazolin, 6-BA, 3.0mg/L and 0.5% of DMSO at 25 +/-2 ℃ for 36-48 hours in the dark.
The callus differentiation culture medium in the step (4) and the step (6) is MS + NAA 0.02-0.05 mg/L + IBA 0.1-0.2 mg/L + BR 0.4-0.5 mg/L + TDZ 2.0-3.0 mg/L.
And (5) and (6) the buds are in a state that the calluses are obviously raised and just have buds.
The rooting medium in the step (7) is 1/2MS + NAA 0.5-0.8 mg/L + IBA 1.0-1.2 mg/L + activated carbon 0.5g/L.
The invention has the beneficial effects that
1. The improved N6+2, 4-D0.6-0.8 mg/L +2-IP 0.2-0.5 mg/L + GA 3.4-0.5 mg/L adopted by the invention is used as a callus induction culture medium, so that the anther can be successfully cultured into callus, and the callus induction rate is up to more than 80%.
2. The haploid obtained by anther culture is generally subjected to chromosome doubling in a callus stage, and the callus obtained by anther culture is close to chromosome doubling, so that cluster buds cannot be differentiated, and a complete plant cannot be obtained.
3. After the callus induced by the polygonatum kingianum anther is differentiated into small buds, the high-concentration colchicine can induce the chromosome doubling of the small buds to obtain a double haploid, but the small buds can die in large quantity, but the small buds which do not die can not grow continuously by continuous differentiation culture to obtain plants with stems and leaves, and simultaneously, tetraploid or even more ploidy chimeras can be generated, while the low-concentration colchicine and other chemical inducers such as pendimethalin and asulam can not effectively induce the chromosome doubling.
4. The double haploid induced by the invention not only can screen out high-quality germplasm with extremely excellent characters, but also can be used as a male parent to perform crossbreeding with female parents of other excellent lines, so that a better pure hybrid is obtained, and the double haploid can also be used as a pure material for other gene breeding such as gene editing and the like.
Drawings
FIG. 1 is a graph of the effect of differentiated shoots on chromosome doubling as described in step 5 of example 1;
FIG. 2: after chromosome doubling treatment in step 6 in example 1, no affected effect pattern was observed in the differentiated shoot;
FIG. 3: the normal growth effect of the buds continuously differentiated and cultured in the step 7 in the example 1 is shown;
FIG. 4 is a schematic view of: example 1 the rooted shoots obtained in step 8.
Detailed Description
Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1
1. Selection and cleaning of materials: selecting a flower bud without obvious plant diseases and insect pests from polygonatum kingianum, dipping a saturated soap water solution by using a soft brush to scrub the surface of the flower bud, and after dirt on the surface is cleaned, rinsing the flower bud by using tap water until no soap water remains.
2. Pretreatment of materials: the cleaned flower buds are placed in a culture dish with filter paper and are placed in a dark environment at the temperature of 4 +/-2 ℃ for standing treatment for 24 hours.
3. And (3) disinfection of materials: transferring the pretreated flower buds into an ultra-clean bench, disinfecting with 75% alcohol for 20s, rinsing with sterile water for 3 times, disinfecting with a saturated bleaching powder solution for 20min, rinsing with sterile water for 4 times, dropwise adding 2 drops of Tween-80 into a 0.05% mercuric chloride solution, disinfecting for 3min, rinsing with sterile water for 6 times, disinfecting for 7 days with a pollution rate of 6.9%, and disinfecting with a death rate of 17.8%.
4. Callus induction: absorbing surface water of sterilized flower buds by using sterile filter paper, taking out anthers from the flower buds by using tweezers, cutting off slippery threads, inoculating the anthers into a modified N6+2, 4-D0.6 mg/L +2-IP (isopentenyl adenine) 0.2mg/L + GA3 (gibberellin) 0.4mg/L callus induction culture medium, culturing for 15 days in a dark environment at the temperature of 25 +/-2 ℃, keeping the temperature unchanged, continuously culturing for 30 days under light-dark alternation with the light intensity of 2000-3000 lx after 12-hour single-day irradiation, wherein the callus induction rate can reach 83.6 percent, the modified N6 culture medium has the advantages that other components are unchanged, the sugar addition amount is changed to 50g/L, and the pH value is changed to 6.4.
5. Callus primary differentiation (fig. 1): the callus induced by anther is inoculated on a callus differentiation culture medium of MS + NAA 0.02mg/L + IBA 0.1mg/L + BR 0.4mg/L + TDZ 2.0mg/L, and the callus is cultured for 25 days under the condition of light intensity of 3000-4000 LX and light-dark alternation of daily illumination for 12 hours at the temperature of 25 +/-2 ℃, so that obvious bulges appear on the callus, the bulges are in a bud state, and the callus differentiation rate is 97.8 percent.
6. Chromosome doubling treatment (fig. 2): the buds induced by the callus differentiation medium were immersed in a mixed solution of 0.02% colchicine, 0.05% oryzalin, 6-BA 3.0mg/L, and 0.5% DMSO (dimethyl sulfoxide) at 25. + -. 2 ℃ for 36 hours in a full dark condition.
7. Continued differentiation (fig. 3): the surface water of the gemmules after chromosome doubling treatment is absorbed by sterile filter paper, and then the gemmules are inoculated into a callus differentiation culture medium of MS + NAA 0.02mg/L + IBA 0.1mg/L + BR 0.4mg/L + TDZ 2.0mg/L, and cultured for 30 days under the condition of illumination intensity of 3000 to 4000LX and illumination of 12 hours in light and dark alternately at the temperature of 25 +/-2 ℃.
8. Rooting culture (fig. 4): the plant differentiated into the stem and leaf is inoculated into a rooting culture medium of 1/2MS + NAA 0.5-0.8 mg/L + IBA 1.0-1.2 mg/L + activated carbon 0.5g/L, and the plant is cultured for 50 days under the environment of 25 +/-2 ℃ and with the illumination intensity of 3000-4000 LX and the light-dark alternation of 12 hours of sunlight, and the rooting rate is 97.0 percent.
9. And (3) ploidy detection: taking the root tip of the plant cultured by rooting, carrying out microscopic examination after chromosome staining, and screening out the plant with 2n chromosomes, wherein the doubled haploid yield is 32.9%.
Example 2
1. Selection and cleaning of materials: selecting a flower bud without obvious plant diseases and insect pests from polygonatum kingianum, dipping a saturated soap water solution by using a soft brush to scrub the surface of the flower bud, and after dirt on the surface is cleaned, rinsing the flower bud by using tap water until no soap water remains.
2. Pretreatment of materials: the cleaned flower buds are placed in a culture dish with filter paper and are placed in a totally dark environment at the temperature of 4 +/-2 ℃ for standing treatment for 36 hours.
3. And (3) disinfection of materials: transferring the pretreated flower buds into a super clean bench, disinfecting with 75% alcohol for 30s, rinsing with sterile water for 3 times, disinfecting with saturated bleaching powder solution for 30min, rinsing with sterile water for 4 times, dropwise adding 2 drops of Tween-80 into 0.05% mercuric chloride solution, disinfecting for 4min, and rinsing with sterile water for 7 times. The disinfection pollution rate is 6.2 percent and the disinfection death rate is 18.8 percent in 7 days.
4. Callus induction: absorbing surface water of sterilized flower buds by using sterile filter paper, taking out anthers from the flower buds by using tweezers, cutting off smooth filaments, inoculating the anthers into a modified N6+2, 4-D0.8 mg/L +2-IP 0.5mg/L + GA 3.5 mg/L callus induction culture medium, culturing for 15 days in a dark environment at the temperature of 25 +/-2 ℃, keeping the temperature unchanged, continuously culturing for 30 days under light-dark alternation of single-day irradiation for 12 hours with the light intensity of 2000-3000 lx, wherein the callus induction rate is 84.7%, the modified N6 culture medium has the rest components unchanged, the sugar addition amount is changed to 50g/L, and the pH value is changed to 6.4.
5. Primary callus differentiation: the callus induced by anther is inoculated on a callus differentiation culture medium of MS + NAA 0.05mg/L + IBA 0.2mg/L + BR 0.5mg/L + TDZ 3.0mg/L, and is cultured for 30 days under the environment of 25 +/-2 ℃ and with the illumination intensity of 3000-4000 LX and the light-dark alternation of the sunlight for 12 hours, so that obvious bulges appear on the callus, the bulges are in a bud state, and the callus differentiation rate is 98.5%.
6. Chromosome doubling treatment: inducing buds by using a callus differentiation medium, placing the buds in a mixed solution of 0.04 percent colchicine, 0.06 percent benazolin, 6-BA, 3.0mg/L and 0.5 percent DMSO (dimethyl sulfoxide), and soaking for 36-48 hours under the conditions of the temperature of 25 +/-2 ℃ and full darkness.
7. And (4) continuing differentiation: absorbing surface water of the buds subjected to chromosome doubling treatment by using sterile filter paper, inoculating the buds into a callus differentiation culture medium of MS + NAA 0.05mg/L + IBA 0.2mg/L + BR 0.5mg/L + TDZ 3.0mg/L, and culturing for 30 days under the condition of illumination intensity of 3000-4000 LX and illumination intensity of 12 hours in the sun and dark alternately at the temperature of 25 +/-2 ℃ until plants with stems and leaves grow.
8. Rooting culture: the plants differentiated into the plants with stems and leaves are inoculated in a rooting culture medium of 1/2MS + NAA 0.8mg/L + IBA 1.2mg/L + activated carbon 0.5g/L, and are cultured for 60 days under the environment of 25 +/-2 ℃ and light-dark alternation of illumination intensity of 3000-4000 LX and daily illumination light of 12h, and the rooting rate is 98.3 percent.
9. And (3) ploidy detection: taking the root tip of the plant cultured by rooting, carrying out microscopic examination after chromosome staining, and screening out a pair of chromosome plants to obtain the polygonatum kingianum double haploid, wherein the double haploid yield is 35.1%.
Experimental verification
1. Callus culture experiment
(1) Influence of hormone on anther culture of Polygonatum kingianum
(1) Test with auxin only: tests with three auxins of 2,4-D, NAA and IBA at different concentrations show that only 2,4-D induces anthers to form callus but the induction rate is extremely low no matter how the concentration is changed, and the highest induction rate can only reach 7% through repeated tests, while the other two auxins can not induce the callus well when used alone.
(2) Test for compounding other plant growth regulators
N6 is used as a basic culture medium, 2,4-D with 0.5mg/L is compounded with other plant growth regulators to carry out orthogonal tests, and the specific test design is as follows in the following table 1:
as can be seen from the above table 2, the YS7 induction rate is the highest, and the YS8 times, and the callus growth of YS8 is excellent. Meanwhile, 2-IP is relatively sensitive to the anther-induced callus of polygonatum kingianum, but CPPU and PPP333 have opposite effects. Can well induce callus when being combined with 6-BA, KT and ZT, but the whole inductivity is lower than that of 2-IP. After repeated experiments, when 2,4-D and 2-IP are independently combined, the induction rate is highest and can reach 89%, when 2,4-D and other plant growth regulators are combined or when 2,4-D and 2-IP are combined, other plant growth regulators are added, the independent effect can not be achieved, but when GA3 is added, the induction rate is not obviously reduced, and the GA3 has the effect of promoting the callus growth.
(2) When the anther callus induction culture is carried out by adopting the N6 culture medium, the callus is found to be loose and not compact, the vitrification is serious, on one hand, the experimental operation is not facilitated, and on the other hand, the differentiation clump budding is not facilitated.
The member guesses that the method is possibly related to an osmotic type, and then changes the concentration of inorganic salt, adjusts the concentration of sugar and the dosage of agar, finds that the increase of the concentration of inorganic salt, the increase of the concentration of sugar and the dosage of agar can reduce vitrification to a certain extent and enable the callus to be more compact, but the adjustment of the concentration of inorganic salt can promote browning, non-growth and even death, and after the dosage of agar is increased, a culture medium is too hard, the test operation is difficult, and the culture medium has a certain growth inhibition effect, when the concentration of sugar is increased, the vitrification of the callus completely disappears, the callus is more compact, when differentiation is carried out, the differentiation rate basically reaches 100%, and the callus growth vigor is better than the callus growth vigor without increasing the concentration of sugar.
Modified N6+2, 4-D0.6-0.8 mg/L +2-IP 0.2-0.5 mg/L + GA 3.4-0.5 mg/L as callus induction culture medium.
2. Callus chromosome doubling test
Cutting the callus into 1cm 2 The single-factor multi-level chromosome doubling test is carried out under the conditions that the temperature is 25 +/-2 ℃ and the whole dark is carried out under the conditions of different concentrations and types of chemical inducers (0.5 percent of DMSO is added) and the inducing time, but after the chromosome is doubled into double haploid after being treated by colchicine with certain concentration, the proportion of the double haploid can reach about 7 percent, but the callus can not be differentiated into buds, and the high-concentration colchicine soaks the callus, not only can the double haploid not be differentiated into buds, but also can cause the death of the callus. When low-concentration colchicine and other chemical inducers such as pendimethalin, oryzalin, phosphorus methylaminoate, trifluralin and the like with any concentration are adopted, the proportion of doubling the callus chromosomes into double haploids is lower than 1 percent, and when the low-concentration colchicine and the other chemical inducers are mixed for use,the low-concentration colchicine and the appropriate-concentration oryzalin are compounded for use, so that the doubling rate of the callus chromosomes can be instantly increased to more than 20%, but the callus can still be hardly differentiated into buds, and the specific test data is shown in the following table 3:
after the treatment, the callus is placed in a differentiation medium for differentiation culture, after 30 days of culture, newly grown callus is taken for chromosome staining microscopy, and the test results are shown in the following table 4:
as can be seen from the above table 4, the chromosome multiplying factor of R8 is optimal, but part of the callus is blackened, the callus can not grow after the continuous culture in the later period, the chromosome multiplying factors of R3, R5 and R7 are also obviously higher than those of other treatments, but the callus death can be caused by R3, the callus can not grow after the continuous culture of R7 and the callus can not grow, and a certain proportion of tetraploids can be caused by R7. In comprehensive comparison, R5 is the best treatment for callus chromosome doubling.
3. Chromosome doubling assay with shoot after callus differentiation
The callus differentiated buds were treated by soaking with colchicine and oryzalin at different concentrations for different times, and the specific experimental design is as follows in table 5:
after the treatment is finished, inoculating the strain in a differentiation culture medium for continuous culture, then carrying out rooting culture, taking the root tip for chromosome staining microscopy, and testing results are shown in the following table 6:
as can be seen from Table 6 above, both XY3 and XY8 have good chromosome doubling effects, and do not affect the growth of the bud, nor generate tetraploids.
4. Effect of plant growth regulators on shoot chromosome doubling
And in the later stage, the colchicine and the benazolin are compounded and finely adjusted on the basis of 0.02-0.05% and on the basis of 0.03-0.06%, and the effect of 0.02-0.04% of colchicine and 0.05-0.06% of benazolin in composite soaking is optimal for 36-48 hours, and on the basis, 2,4-D, IAA, 6-BA, KT and GA3 with different concentrations are continuously added, wherein 2,4-D, IAA, 6-BA and KT can increase the doubling rate of chromosomes to a certain degree and do not influence the growth and rooting in the later stage of the buds, and the effect of the 6-BA with 3.0mg/L is optimal, so that the chromosome doubling rate can be increased to more than 32% and tetraploids cannot appear.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Claims (4)
1. A method for producing polygonatum kingianum double haploid is characterized by comprising the following steps:
(1) Selecting polygonatum kingianum flower buds with anthers for pretreatment;
(2) Sterilizing the pretreated flower buds, and stripping off anthers after sterilization;
(3) Inoculating anther into a callus induction culture medium for culture;
(4) Inoculating the induced callus into a callus differentiation culture medium for culture;
(5) Carrying out chromosome doubling treatment on the buds differentiated from the callus;
(6) Continuously inoculating the buds subjected to chromosome doubling treatment into a callus differentiation culture medium to grow seedling bodies;
(7) Inoculating the differentiated seedling body into a rooting culture medium for rooting culture;
(8) Carrying out chromosome ploidy detection on the rooted seedlings, and screening to obtain polygonatum kingianum double haploid plants;
the callus induction culture medium in the step (3) is improved N6+2, 4-D0.6-0.8 mg/L +2-IP 0.2-0.5 mg/L + GA 3 0.4-0.5 mg/L, the modified N6 culture medium is that the other components are unchanged, the adding amount of sugar is changed to 50g/L, and the pH is changed to 6.4;
in the step (5), the chromosome doubling treatment method is to soak 0.02 to 0.04 percent of colchicine, 0.05 to 0.06 percent of benazolin, 6-BA, 3.0mg/L and 0.5 percent of DMSO in the dark condition for 36 to 48 hours at the temperature of 25 +/-2 ℃;
the callus differentiation culture medium in the step (4) and the step (6) is MS + NAA 0.02-0.05 mg/L + IBA 0.1-0.2 mg/L + BR 0.4-0.5 mg/L + TDZ 2.0-3.0 mg/L;
and (5) and (6) the buds are in a state that the calluses are obviously raised and just have buds.
2. The method for producing the dihaploid of polygonatum kingianum according to claim 1, wherein in the step (1), the pretreatment method is to treat the dihaploid of polygonatum kingianum in a full-dark environment at 4 +/-2 ℃ for 24-36 hours.
3. The method for producing the dihaploid of polygonatum kingianum according to claim 1, wherein in the step (2), the sterilization method comprises the steps of sterilizing with 75% alcohol for 20-30 s, sterilizing with saturated bleaching powder solution for 20-30min, and sterilizing with 0.05% mercuric chloride and 2 drops of tween-80 solution for 3-4 min.
4. The method for producing the polygonatum kingianum doubled haploid according to claim 1, wherein the rooting medium in the step (7) is 1/2MS + NAA 0.5-0.8 mg/L + IBA 1.0-1.2 mg/L + activated carbon 0.5g/L.
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