CN107750946B - Method suitable for cultivating medicinal plants sweet wormwood herb and Chinese yam polyploid variety - Google Patents
Method suitable for cultivating medicinal plants sweet wormwood herb and Chinese yam polyploid variety Download PDFInfo
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Abstract
The invention discloses a method suitable for cultivating medicinal plants of sweet wormwood herb and a yam polyploid variety. The method comprises the steps of selecting single plant callus of sweet wormwood, dioscorea zingiberensis and dioscorea composita as a material, carrying out low-temperature co-culture after short-time soaking treatment by using a calcium ion solution and a colchicine solution, then inoculating the callus into a culture medium, adding colchicine with different concentrations for further mutagenesis treatment, doubling the plant, and then identifying by using a ploidy identification technology to obtain polyploid sweet wormwood, dioscorea zingiberensis and dioscorea composita plant. The method provides optimal colchicine concentration, treatment time, calcium ion concentration, low temperature co-culture time, etc. for inducing sweet wormwood herb and yam polyploidy. The method can remarkably improve the inductivity of the polyploidy of the sweet wormwood and the yam plants, and has the advantages of short time, high efficiency and low cost.
Description
Technical Field
The invention belongs to the technical field of polyploid induction cultivation of plants, and particularly relates to a method suitable for innovatively cultivating medicinal plants of sweet wormwood herb and a yam polyploid variety.
Background
Artemisia annua L (also called artemisia annua L.) is a cross-pollinated plant, and due to the complex heterogeneity of the population, the offspring always has character separation and shows diversity, and the excellent characters are difficult to be stably inherited, which is the root cause that the variety of the artemisia annua in China is easy to mix and degenerate. In the conventional breeding of the sweet wormwood, multiple selections must be performed under the condition of properly controlling pollination in order to obtain stable homozygous progeny and ensure the selection effect, and the cross-pollinated plant is not resistant to selfing, so that the viability is remarkably reduced due to selfing, and the breeding difficulty is high, the time is long, and the germplasm character is unstable. The method is widely applied internationally at present, is suitable for a breeding method of medicinal plants with functional active ingredients, and simultaneously, in order to obtain more stable homozygous progeny and ensure the selection effect, the most direct and rapid method at present is to directly carry out asexual propagation on the selected polyploid plant by using a tissue culture rapid propagation technology, avoid germplasm variation of seeds and cutting propagation seedlings, thereby obtaining a large amount of polyploid artemisia apiacea high-quality seedlings.
Dioscorea (Dioscorea) has important medicinal value, and is called medicinal gold by the medical community. It is documented that there are about 650 dioscoreaceae plants and 62 plants in our country worldwide. Peltate yam is a unique species in China, and is cold-resistant and drought-resistant. Dioscorea composita Hemsl, which is drought-resistant and cold-resistant, is planted in Yunnan and Hainan areas of China, and the Dioscorea composita grows continuously all the year round. Therefore, the industrial cultivation of the dioscorea zingiberensis and the dioscorea composita in the dioscorea zingiberensis has better economic prospect, and is worthy of being popularized and planted with non-grain energy and medicine dual-purpose crops. The dioscorea zingiberensis and the dioscorea composita are mainly planted artificially, and the variety is degraded, the quality and the yield of medicinal materials are reduced and chemical components are unstable due to the fact that only seeds are not selected for a long time. Through the isolated culture and chemical mutagenesis technology, the chromosome number of the homologous tetraploid obtained by artificial induction is doubled, and the volumes of cell nucleuses and cells are correspondingly increased, so that the organs of the trophosome of the polyploid plant are enlarged, the yield is improved, and the contents of some effective components in the body can be increased.
Polyploid materials have breeding resources with potential application values, and it is believed that with the deep research, new germplasm with high yield and high diosgenin and high artemisinin content is expected to be bred from polyploid plants. Therefore, developing the breeding of the sweet wormwood, the dioscorea zingiberensis and the dioscorea composita polyploid varieties and obtaining polyploid innovative varieties have important significance for solving the problems in production.
Generally, chemical reagents are adopted for polyploidy induction, and the combination effect of the selected reagents on plant tubulin is utilized to effectively prevent microtubule polymerization, so that the formation of spindles is inhibited, and the effect of inducing chromosome doubling is achieved. Colchicine (Colchicine) is a common polyploid mutagen, and when a living body induction treatment method is utilized, the exophyte has high death rate, low mutagenesis rate and high mosaic rate, the fertility of the obtained tetraploid plant is low, and a large amount of tetraploids are not easy to obtain; although treated under ex vivo culture conditionsThe frequency of obtaining polyploids is somewhat high, but few plants are capable of ideal polyploidy induction rates. This may be related to the different characteristics of various plants, as well as numerous factors that affect the rate of polyploid induction. The invention integrates the concentration of different mutagens, the treatment time and different Ca2+The method is characterized in that factors such as concentration, co-culture time and the like are designed, corresponding experimental schemes are designed to carry out different treatments on the callus of the sweet wormwood herb, the dioscorea zingiberensis and the dioscorea composita, the effects of the factors in the process of inducing the polyploidy of the sweet wormwood herb, the dioscorea zingiberensis and the dioscorea composita are discussed, and an optimal treatment combination scheme suitable for inducing the polyploidy of various medicinal plants such as the sweet wormwood herb, the dioscorea zingiberensis and the dioscorea composita is expected.
Disclosure of Invention
The invention aims to solve the problems and provides a rapid propagation cultivation method of sweet wormwood herb and Chinese yam polyploidy. The method can remarkably improve the inductivity of the polyploidy of the sweet wormwood and the yam plants, and has the advantages of short time, high efficiency and low cost. The purpose of the invention is realized by the following modes:
a method for cultivating medicinal plants Artemisia annua and rhizoma Dioscoreae polyploid breed comprises the following steps:
(1) explant selection
Selecting green bud formed embryonic callus of Artemisia annua or Dioscorea opposita, taking out and cutting into small pieces;
(2) mutagenesis treatment
1.0-3.0mmol/LCa of the callus processed in the step (1)2+Soaking the solution; then soaking in 0.1% colchicine solution for 30-90 min; then directly transferring the cells into a solid culture medium without cleaning, co-culturing at 4 ℃, taking out and cleaning, and drying moisture on an ultra-clean workbench; inoculating on solid culture medium containing colchicine at concentration of 1.0-5.0mg/L, and treating for 20-60 d;
(3) morphological and chromosomal characterization
When the plant in the step (2) grows to 3-4cm and the root grows to 0.5-1cm, comparing and analyzing the plant with a diploid control plant, preliminarily screening the plant with obvious phenotypic variation for rooting according to the difference of the width, the thickness and the stem thickness of the leaves in appearance morphology and the size and the density of stomatal guard cells of the plant and the diploid control plant, and then taking a part of the root tip of the variation seedling, which comprises a meristem part, for karyotype identification; the polyploidy is obtained by multiplying the chromosome number by taking a chromosome set as a unit;
(4) transplantation management
Opening a bottle cap of the plant identified as tetraploid, hardening off seedlings, planting, ventilating and carrying out normal management on the seedlings.
The method is suitable for cultivating medicinal plants Artemisia annua and rhizoma Dioscoreae polyploid varieties, and the rhizoma Dioscoreae comprises Dioscorea zingiberensis or Dioscorea composita.
The method for cultivating the medicinal plant sweet wormwood herb and the yam polyploid variety preferably comprises the following steps of (1) selecting embryonic callus of sweet wormwood herb or yam, transferring the embryonic callus into a differentiation culture medium for culture, and taking out and cutting the embryonic callus into small blocks of 1.0-3.0cm when the callus begins to form green bud points. Further preferably, the embryogenic callus of the high-quality single plant sweet wormwood herb, dioscorea zingiberensis C.H.Wright or dioscorea composita L is selected in the step (1), and is transferred into a differentiation culture medium of MS +6-BA1.0mg/L + IAA0.5mg/L for culture for 3-10 d; when the callus begins to form green bud, it is cut into 1.0-3.0cm, placed in a petri dish with 4-5 layers of sterile filter paper, and blown on a clean bench for 1-5h, preferably 2 h.
The method suitable for cultivating medicinal plants of sweet wormwood herb and yam polyploid varieties is preferably as follows:
step (2) using 1.0-3.0mmol/LCa of the callus processed in the step (1)2+Soaking in the solution for 20-40min, preferably 3.0mmol/LCa2+The solution is preferably soaked for 30 min.
Ca in step (2)2+Soaking the solution treated callus in 0.1% colchicine solution for 60 min.
The callus after the colchicine soaking in the step (2) is directly transferred into MS without being washed0Co-culturing in culture medium at 4 deg.C for 72 hr, taking out, cleaning for 5-6 times, placing on sterile filter paper, draining water, and blowing on ultra-clean bench for 10-60min, preferably 30 min.
In step (2), the callus is inoculated on a solid culture medium MS + BA2.0mg/L + NAA0.2mg/L + paclobutrazol with the concentration of 1.0-5.0mg/L colchicine for treatment for 20-60 days, and preferably cultured in a solid culture medium containing 1.0mg/L colchicine for 40 days.
Culturing the callus in the step (2) in a solid culture medium containing colchicine at 25 ℃, with the illumination intensity of 2000LX for 10-12 h.
The method suitable for cultivating medicinal plants of sweet wormwood herb and yam polyploid varieties is preferably as follows:
and (4) opening a bottle cap of the plant identified as the tetraploid, hardening the seedling, taking out and cleaning a root culture medium, planting the plant in an incubator filled with the culture medium after autoclaving, watering thoroughly after planting, covering the plant, starting ventilation for 1-2 times/7 d after 20d of transplantation, gradually increasing the ventilation time from 10min to the whole day, not watering before uncovering, spraying a small amount of clear water on the leaf surface during ventilation, and gradually removing the cover after 30-35d of ventilation, thereby carrying out normal seedling management. The transplanting survival rate is more than 95%.
By implementing the specific invention content of the invention, the following effects can be achieved:
the invention relates to a method suitable for polyploid cultivation of medicinal plants Artemisia annua, Dioscorea zingiberensis C.H.Wright and Dioscorea composita, which is simple in operation, can quickly realize new species breeding of Artemisia annua, Dioscorea zingiberensis C.H.Wright and Dioscorea composita, shortens breeding period, further effectively improves cultivation speed of high-quality seedlings of Artemisia annua, Dioscorea zingiberensis C.H.Wright and overcomes the problems that the characteristics are easy to separate in conventional breeding, the excellent characteristics are difficult to stably inherit, and the cultivated new species is.
Compared with the prior art, the invention has the beneficial effects that:
1. the method of the invention is not only suitable for artemisia plant artemisia apiacea, but also suitable for polyploid innovative breeding of dioscorea zingiberensis and dioscorea composita.
2. Ca for use in the invention2+Soaking callus to induce depolymerization of microtubule skeleton, soaking with colchicine for a short time to make the solution easily penetrate into callus, and transferring into MS0Basic cultureThe callus is not soaked in colchicine solution for a long time, the toxic action is small, and the culture medium can provide sufficient nutrition and is beneficial to the recovery and growth of explants.
3. After the colchicine is co-cultured at low temperature, the colchicine is air-dried on a super clean workbench, the water content in the callus is obviously reduced, the toxic action of the explant is reduced, and the recovery and the growth of the explant are facilitated.
4. Due to the influence of multiple advantages, the death rate of the explant is low, the chimeric rate is low, the mutagenesis efficiency is greatly improved, and the disadvantage of long-time soaking treatment of colchicine is thoroughly solved.
Description of the drawings:
FIG. 1 analysis of growth vigor in diploid and tetraploid regenerated seedling bottles
1. The growth condition of the sweet wormwood mutation seedling is shown;
2. the growth condition of the dioscorea zingiberensis mutant seedlings is shown;
3. the growth condition of the dioscorea composita mutant seedlings is shown;
4. the growth condition of the sweet wormwood diploid regenerated seedling is shown;
5. the growth condition of the diploid regenerated seedlings of the dioscorea zingiberensis is shown;
6. the growth condition of the diploid regenerated seedlings of the dioscorea composita of the invention;
7. the growth condition of the southernwood tetraploid regenerated seedlings is shown;
8. the growth condition of the tetraploid regenerated seedlings of dioscorea zingiberensis is shown in the specification;
9. the growth condition of the tetraploid regenerated seedlings of the dioscorea composita of the invention is shown;
FIG. 2 field growth analysis of diploid and tetraploid regenerated seedlings
1. Is the sweet wormwood diploid plant;
2. is the southernwood tetraploid plant of the invention;
3. comparing the sizes of the sweet wormwood leaves (diploid on the left and tetraploid on the right);
4. is the diploid plant of dioscorea zingiberensis of the invention;
5. is the tetraploid plant of dioscorea zingiberensis C.H.Wright of the invention;
6. comparing the sizes of the peltate yam leaves (diploid on the left and tetraploid on the right);
7. is the diploid plant of Dioscorea composita of the invention;
8. is the tetraploid plant of Dioscorea composita of the invention;
9. comparing the sizes of the dioscorea composita leaves (diploid on the left and tetraploid on the right);
FIG. 3 chromosome karyotype analysis of diploid and tetraploid regenerated seedlings
1. Artemisia apiacea diploid chromosome morphology and karyotype plot, x 1000(2n ═ 2x ═ 18);
2. dioscorea zingiberensis diploid chromosome morphology and karyotype plot, × 1000(2n ═ 2x ═ 20);
3. dioscorea composita diploid chromosome morphology and karyotype plot, x 1000(2n ═ 2x ═ 20);
4. southernwood tetraploid chromosome morphology and karyotype plot, x 1000(2n ═ 4x ═ 36);
5. dioscorea zingiberensis tetraploid chromosome morphology and karyotype plot, x 1000(2 n-4 x-40);
6. the tetraploid chromosome morphology and karyotype of Dioscorea composita, x 1000(2 n-4 x-40).
Detailed Description
The invention is further described with reference to specific embodiments, but the scope of the methods and protection described herein is not limited thereto:
materials and methods
(1) Explant selection
Selecting embryonic callus of herba Artemisiae Annuae, rhizoma Dioscoreae Zingiberensis, and rhizoma Dioscoreae Jerusalem, and culturing in differentiation culture medium of MS +6-BA1.0mg/L + IAA0.5mg/L for 3-10 d; when the callus begins to form green bud points, taking out and cutting the callus into 1.0-3.0cm, uniformly placing the callus in a culture dish filled with 4-5 layers of sterile filter paper, and then blowing the callus on a super clean bench for 1-5 hours;
(2) mutagenesis treatment
Soaking the callus processed in the step (1) with 1.0-3.0mmol/L calcium chloride solution for 30 min; then soaking in water with concentration of 0.1%Directly transferring into MS without cleaning in colchicine solution for 30-90min0Co-culturing at 4 deg.C for 72 hr, taking out, cleaning for 5-6 times, placing on sterile filter paper, draining water, blowing on an ultra-clean bench for 10-60min, inoculating on solid culture medium MS + BA2.0mg/L + NAA0.2mg/L + paclobutrazol 2.0mg/L containing colchicine with concentration of 1.0-5.0mg/L, treating for 20-60d, culturing at 25 deg.C, illumination intensity of 2000LX, and illumination time of 10-12 hr. The conventional long-time soaking treatment (48-72 hours) is used as a control.
(3) Morphological and chromosomal characterization
When the plant in the step (2) grows to 3-4cm and the root grows to 0.5-1cm, carrying out comparative analysis with a diploid control plant, preliminarily screening the plant with obvious phenotypic variation for rooting according to the difference of the width, the thickness and the stem thickness of the leaves and the size and the density of stomatal guard cells of the diploid control plant, and then taking the part of the root tip of the variation seedling (20 sweet wormwood herbs, 10 dioscorea zingiberensis and dioscorea composita) containing the meristem region part for carrying out chromosome karyotype identification; the polyploidy is obtained by multiplying the chromosome number by taking a chromosome set as a unit;
(4) transplantation management
Opening a bottle cap of a plant identified as tetraploid, hardening off the seedling, taking out and cleaning a root culture medium, planting the plant in an incubator filled with an autoclaved culture medium, watering thoroughly after planting, covering the plant, starting ventilation for 1-2 times/7 d after 20d of transplantation, gradually increasing the ventilation time from 10min to the whole day, not watering before uncovering the plant, only slightly spraying some clear water on the leaf surface during ventilation, gradually removing the cover after 30-35d, carrying out normal management on the seedling, and achieving the transplanting survival rate of 95%.
(II) results and analysis
2.1 Induction of callus cell polyploidy by colchicine solution Long-time soaking treatment
A small amount of polyploid cells (Table 1) were obtained from peltate yam and Dioscorea composita by soaking in colchicine solution for a long time, and Artemisia annua was 0. The occurrence frequency of dioscorea zingiberensis and dioscorea composita polyploidy increases along with the increase of treatment concentration and the extension of treatment time, the effect of colchicine concentration treatment with concentration less than 0.1% on inducing polyploidy is small, and only 2 chimeras are obtained; treatment at concentrations of 0.1% and above has a good effect on inducing polyploidy, but the toxic effect of colchicine on explants increases with increasing treatment concentration, and especially the highest concentration (0.2%) and treatment time (48h or 72h) are selected to kill all explants. Experiments prove that the effect is best when the combination of the treatment concentration is 0.1% and the treatment time is 72h, but the death rate of the callus is increased along with the prolonging of the culture time, only 5 viable effective seedlings are obtained, 4 are dioscorea zingiberensis, and 1 is dioscorea composita. All southernwood explants were killed in all soaking treatments.
TABLE 1 colchicine soaking mutagenesis treatment under in vitro culture conditions
The long-time soaking treatment method is the most commonly used method for polyploid mutagenesis, but the long-time soaking treatment method of colchicine solution has direct damage to the callus, especially the long-time soaking treatment can kill all explants and influence the mutagenesis efficiency.
2.2 Induction of callus cell polyploidy by short-time soaking in colchicine solution and Co-culture treatment
Selecting the soaking time and Ca2+Concentration, concentration of mutagen in solid medium, and co-culture time were determined by 4 factors, 3 levels were set for each factor (Table 1), and L9(3 was used4) And (4) orthogonal experimental design.
TABLE 2 orthogonal experimental design parameters
TABLE 3 colchicine soak time, Ca2+Concentration, solid medium mutagen concentration and co-culture time combination scheme
TABLE 4 mutagenesis effect of short-time soaking of colchicine solution + Co-culture treatment method
After the callus is mutagenized by the No. 1-9 treatment method, subcultured in a differentiation medium, the surface of a bud block appears browning phenomenon in the initial culture period (15-20 days), and is continuously cultured for 30 days to gradually grow a small amount of green buds (figure 1, 1-3), and the obtained buds have higher secondary subculture activity. Compared with the control tube plantlet (figures 1, 4-6), part of the tube plantlet has the defects of plant stout, increased leaf thickening, slight crease on the surface, dark green leaf color and partial leaf deformity (figures 1, 7-9). The method has the advantages that the number 1-9 treatments all obtain survival mutation seedlings, and the survival rate is high.
A large number of southernwood, dioscorea zingiberensis and dioscorea composita polyploid cells can be obtained by soaking the colchicine solution for a short time and co-culturing (table 4), the toxic effect of the colchicine on the explants is gradually increased along with the increase of the treatment concentration, and the occurrence frequency of the polyploids is increased along with the increase of the treatment concentration and the extension of the treatment time. The optimal treatment method is No. 6 treatment method, which comprises selecting callus with green bud point formed by differentiation culture for 7-10 days, air drying on superclean bench for 2 hr, and further 3.0mmol/LCa2+Soaking in solution for 30min, soaking in 0.1% colchicine solution for 60min, and transferring into MS without washing0The culture medium is co-cultured for 72h, taken out and cleaned for 5-6 times, placed on sterile filter paper to drain water, blown on an ultra-clean workbench for 30min, and then inoculated on a solid differentiation medium MS + BA2.0mg/L + NAA0.2mg/L + paclobutrazol 2.0mg/L with the concentration of 1.0mg/L for culture for 40d, the browning and mortality of explants are lower than 20%, and the induction rates of sweet wormwood and dioscorea reach 60% and 55% respectively. The method of the invention has obviously better effect than the colchicine soaking treatment for a long time.
2.3 identification of polyploids
2.3.1 morphological characterisation
Compared with the control test-tube plantlet, the treated test-tube plantlet shows plant morphological variation such as twist dwarfing, slow growth, thick stalk, thick and large leaf, wide leaf, dark leaf color, rough and bubbly leaf surface and the like. The tetraploid plants which are successfully mutagenized still show organ giant, the stem segments are thick, the leaves are larger and thicker than the diploid, and even the leaf shapes are obviously changed.
After the artemisia apiacea test-tube plantlet is transplanted, compared with a diploid plant in the current year (fig. 2 and 2-3), the morphological character of the tetraploid plant is found to be greatly different from that of the diploid plant, and most of the tetraploid plants show super-paternity. According to the difference of characters, the content of artemisinin in the diploid plants is 14.5 per mill, the morphological characters of the tetraploid plants cultured show super-parent advantages in the characters of plant height, leaf color, leaf width, leaf thickness, stem thickness and the like, particularly the plant height reaches more than 3.8 meters, the content of artemisinin reaches 16.5 per mill and is 2.0 per mill higher than that of the diploid plants, and the application value is high.
After the dioscorea zingiberensis test-tube plantlet is transplanted, compared with a diploid plant in the current year (fig. 2, 5-6) in a control diploid plant (fig. 2 and 4), the morphological characters of the tetraploid plant after 2 years of growth are found to be greatly different from those of the diploid plant, and most of the tetraploid plants show super-paternity. According to the difference of characters, the peltate yam saponin content of a control diploid plant is 12.4 per mill, the morphological characters of the cultivated tetraploid plant show super-paternity advantages in the characters of leaf color, leaf width, leaf thickness, stem thickness, seed pod, seeds and the like, particularly, the yield of a single tuber exceeds more than half, the saponin content is up to 13.6 per mill and exceeds 1.2 per mill of the diploid, and the application value is high.
After the dioscorea composita test-tube plantlet is transplanted, compared with a diploid plant in the current year (fig. 2, 8-9) by comparing a diploid plant (fig. 2, 7), the morphological characters of the tetraploid plant after 2 years of growth are found to be greatly different from those of the diploid plant, and most of the tetraploid plants show super-paternity. According to the difference of characters, the peltate yam saponin content of a control diploid plant is 15.6 per mill, the morphological characters of the cultivated tetraploid plant show super-parent advantages in the characters of leaf color, leaf width, leaf thickness, stem thickness and the like, particularly, the yield of a single tuber exceeds more than half, the saponin content reaches 17.3 per mill and exceeds 1.7 per mill of the diploid, and the application value is high.
2.3.2 chromosome slide-making technical detection
The cytological chromosome counting method can directly observe and count chromosomes, and is a reliable and intuitive method for identifying ploidy. The diploid material of artemisia apiacea (fig. 3, 1) has 2 n-2 x-18 (x-9), the diploid material of dioscorea zingiberensis (fig. 3, 2) has 2 n-2 x-20 (x-10), the diploid material of dioscorea composita (fig. 3, 3) has 2 n-2 x-20 (x-10), and the screened variant plants have 50% -60% of chromosomes over 18 or 20, but because of small chromosomes, overlapping chromosomes, etc., 54 artemisia apiacea tetraploids (fig. 3, 4) have 2 n-4 x-36 (x-9), and 55 production plants clearly show dioscorea tetraploids (fig. 3, 5-6) have 2 n-4 x-40 (x-10), wherein dioscorea zingiberensis 32 and dioscorea composita 23 are present. According to the condition that the number of the countable chromosomes is close to 36 or 40, 10-60% of the initially screened variant plants are tetraploids. Chromosome diversity is also found in mutant plants, such as 1 octaploid plant from Artemisia annua, Dioscorea zingiberensis and Dioscorea composita, and chromosome increase or decrease, chromosome overlap, etc. The method has less chimera plants.
The method can quickly realize the breeding of new species of the sweet wormwood, the dioscorea zingiberensis and the dioscorea composita, shorten the breeding period, further effectively improve the breeding speed of high-quality seedlings of the sweet wormwood, and overcome the problems that the sweet wormwood, the dioscorea zingiberensis and the dioscorea composita are easy to separate in conventional breeding, excellent characters are difficult to stably inherit, and the bred new species is easy to mix and degenerate.
Claims (9)
1. A method suitable for cultivating medicinal plants of sweet wormwood herb and yam polyploid varieties is characterized by comprising the following steps:
(1) explant selection
Selecting green bud formed embryonic callus of Artemisia annua or Dioscorea opposita, taking out and cutting into small pieces;
(2) mutagenesis treatment
1.0-3.0mmol/LCa of the callus processed in the step (1)2+Solution soaking partC, processing; then soaking in 0.1% colchicine solution for 30-90 min; then directly transferring the cells into a solid culture medium without cleaning, co-culturing at 4 ℃, taking out and cleaning, and drying moisture on an ultra-clean workbench; inoculating on solid culture medium MS containing colchicine with concentration of 1.0-5.0mg/L, BA2.0mg/L, NAA0.2mg/L and paclobutrazol 2.0mg/L, and treating for 20-60 d;
(3) morphological and chromosomal characterization
When the plant in the step (2) grows to 3-4cm and the root grows to 0.5-1cm, comparing and analyzing the plant with a diploid control plant, preliminarily screening the plant with obvious phenotypic variation for rooting according to the difference of the width, the thickness and the stem thickness of the leaves in appearance morphology and the size and the density of stomatal guard cells of the plant and the diploid control plant, and then taking a part of the root tip of the variation seedling, which comprises a meristem part, for karyotype identification; the polyploidy is obtained by multiplying the chromosome number by taking a chromosome set as a unit;
(4) transplantation management
Opening a bottle cap of the plant identified as tetraploid, hardening off seedlings, planting, ventilating and carrying out normal management on the seedlings.
2. The method as claimed in claim 1, wherein the yam is dioscorea zingiberensis or dioscorea composita which is suitable for breeding the medicinal plants Artemisia annua and rhizoma dioscoreae polyploid cultivars.
3. The method for cultivating medicinal plants Artemisia annua and rhizoma Dioscoreae polyploid variety according to claim 1 or 2, wherein the embryogenic callus of Artemisia annua or rhizoma Dioscoreae is selected in step (1), transferred into differentiation medium for cultivation, and cut into 1.0-3.0cm pieces when the callus begins to form green bud.
4. The method for cultivating medicinal plants Artemisia annua and Dioscorea rhizome polyploid variety according to claim 3, wherein the embryogenic callus of high quality single plant Artemisia annua, Dioscorea zingiberensis or Dioscorea composita is selected in step (1), and transferred into MS +6-BA1.0mg/L + IAA0.5mg/L differentiation medium for 3-10 days; when the callus begins to form green bud, taking out and cutting the callus into 1.0-3.0cm, uniformly placing the callus in a culture dish filled with 4-5 layers of sterile filter paper, and then blowing the callus on a super clean bench for 1-5 hours.
5. The method for cultivating the medicinal plants Artemisia annua and rhizoma Dioscoreae polyploid variety according to claim 1 or 2, wherein the callus treated in step (1) is applied with 1.0-3.0mmol/LCa in step (2)2+Soaking in the solution for 20-40 min.
6. The method for breeding the medicinal plants Artemisia annua and Dioscorea polysploid variety according to claim 1 or 2, wherein Ca in step (2)2+Soaking the solution treated callus in 0.1% colchicine solution for 60 min.
7. The method for cultivating medicinal plants Artemisia annua and Dioscorea polysloid variety according to claim 1 or 2, wherein the callus soaked with colchicine in step (2) is transferred into MS without washing0Co-culturing at 4 deg.C for 72 hr, taking out, cleaning for 5-6 times, placing on sterile filter paper, draining water, and blowing on an ultra-clean bench for 10-60 min.
8. The method for cultivating medicinal plants Artemisia annua and Dioscorea polysome variety according to claim 1 or 2, wherein the callus of step (2) is cultured in solid culture medium containing colchicine at 25 deg.C, illumination intensity of 2000LX, and illumination time of 10-12 h.
9. The method for cultivating the medicinal plants sweet wormwood herb and Chinese yam polyploid varieties according to the claim 1 or 2, wherein the plant identified as tetraploid in the step (4) is opened and hardened, the root culture medium is taken out and cleaned, the plant is planted in an incubator filled with the culture medium after autoclaving, the plant is watered thoroughly, the cover is covered, ventilation is started for 1-2 times/7 d after 20d of transplantation, the ventilation time is gradually increased from 10min to the whole day, watering is not needed before uncovering, a small amount of clear water is sprayed on the leaf surface during ventilation, the cover is gradually removed after 30-35d, and normal management of seedlings is carried out.
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