CN111937747B - Breeding method for forming strong and healthy rootstocks of polygonatum cyrtonema with multiple buds - Google Patents
Breeding method for forming strong and healthy rootstocks of polygonatum cyrtonema with multiple buds Download PDFInfo
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Abstract
A breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds mainly comprises the following steps: 1) selection and pretreatment of explants, 2) propagation and strong seedling culture, 3) differentiation culture and 4) rooting culture. The breeding method is ideal in design, researches are carried out on cultivating the seed rhizome and breaking the dormancy of the rhizome bud body, strong rhizomes with multiple buds developing simultaneously and bud body planting are cultivated, the quality is good, the growth speed is high, domesticated seedlings capable of being subjected to leaf-drawing in the same year are domesticated and cultivated in a greenhouse for one year, the seed stem standard for production can be achieved, the problem of shortage of high-quality seedlings is solved, the seedling cultivation time is shortened, the cost is reduced, and the planting yield can be increased in multiples.
Description
Technical Field
The invention relates to a plant tissue culture breeding technology, in particular to a breeding method for forming strong and developing rootstocks of polygonatum cyrtonema's multiple buds.
Background
The Polygonatum sibiricum is a perennial herb of Polygonatum of Liliaceae, and has a medicinal history of more than two thousand years. Many varieties of sealwort are provided, tubers of three types of sealwort, polygonatum kingianum and polygonatum cyrtonema are used as original medicines according to the specification of the pharmacopoeia 2005 edition of the people's republic of China, wherein the polygonatum cyrtonema Hua is the best in quality, mainly contains saccharides, steroid saponin, flavone and anthraquinone compounds, alkaloid, cardiac glycoside, lignan, vitamins, various amino acids and trace elements which are useful for human bodies and the like, has the effects of prolonging the service life, resisting aging, influencing the cardiovascular system, regulating the immunity, resisting inflammation and resisting pathogenic microorganisms, resisting fatigue, improving the learning memory, inhibiting tumor cells and the like, and is safe, nontoxic and free of mutagenicity. With the gradual and deep research of people on pharmacology and pharmacotization of polygonatum, the polygonatum is more and more widely applied in the aspects of clinical application, health products, foods, cosmetics, ornamental value and the like.
In recent years, the demand of polygonatum is increasing day by day, wild resources can not meet the production requirements far away, and artificial cultivation is a main channel for solving the shortage of polygonatum medicinal materials. The sealwort propagation is carried out by vegetative propagation (wild rhizomes) and sexual propagation (seeds), the propagation period of the seeds is long, the seeds are rarely used in production, the wild rhizomes are mostly used for cultivation, the propagation coefficient of the method is low, the germplasm character degradation is easy to cause, the genetic stability of the variety is influenced, in addition, the wild resources are limited, and the problem of high-quality seedling is the bottleneck of large-area cultivation of the sealwort. In addition, polygonatum cyrtonema belongs to a single-bud development dominant type, only one rhizome can be formed in one year, and the planting yield is extremely low after 3-4 years of planting, so that the market demand is difficult to meet. Specifically, referring to the attached figure 2 of the specification, a growth effect diagram of the rhizomes of polygonatum cyrtonema planted for more than 10 years is shown, and the rhizomes are of a single-bud development type and low in planting yield.
At present, many reports about tissue culture and rapid propagation of polygonatum cyrtonema are carried out based on bud culture and all adopt the traditional tissue culture steps: selecting and disinfecting explants → culturing in an induction culture medium → culturing in a proliferation culture medium → culturing in a strong seedling culture medium → culturing in a rooting culture medium, wherein the tissue culture period of the method is at least more than half a year. For the multiplication medium culture, Xuzhou et al 2006, in which the cluster buds obtained by induction are cut into 2-3 clusters (i.e., 2-3 buds) and then inoculated into a serial multiplication medium for multiplication, Xuhongmei et al 2003 believes that the hormone combination TDZ 1.5mg/L + 2, 4-D1.0mg/L is most advantageous for rhizome bud multiplication of Polygonatum sibiricum, and Xuzhou et al 2006 believes that the hormone combination 6-BA4.0mg/L + NAA0.2mg/L not only enables rapid multiplication of buds but also has good comprehensive quality of adventitious bud multiplication and no abnormal leaf formation. For the strong seedling rooting culture stage, Xuzhou dao 2006 means that the differentiated cluster buds are inoculated on a culture medium MS + 6-BA1.0 mg/L + NAA0.2mg/L, after 30 days, the cluster buds are separated, a strong single root stem bud is taken and then inoculated on a rooting culture medium MS + NAAO.5mg/L. In a word, the existing research is only limited to the culture of bud proliferation and single-root-stem bud rooting by using different hormone combinations, the obtained seed rhizome is small, the growth speed of the seedling at the later stage is low, the seedling culture period is at least 2 years, the cost is high, and the large-scale production of the high-quality sealwort seedling cannot be met.
The test-tube plantlets are transplanted to a greenhouse, most rootstocks are not subjected to leaf-pulling in the current year and are in a dormant state, and the leaf-pulling can be performed only after the next year after winter, so that the seedling culture period of polygonatum cyrtonema is prolonged. Reports about breaking the dormancy of polygonatum cyrtonema buds are few, and only Zhangwang reports GA3Bud dormancy can not be broken, the bud dormancy breaking is optimally processed for 12 hours by 6-BA200PPm, and the emergence rate can reach 90%. Too high concentration of 6-BA and too long treatment time can cause excessive growth of polygonatum cyrtonema buds, abnormal leaves, rapid seedling pouring and failure of rhizome growth. It is therefore important to find a suitable 6-BA concentration and treatment time.
Disclosure of Invention
The invention provides a breeding method for forming strong and developing rootstocks of polygonatum cyrtonema with multiple buds, and aims to overcome the defects that the existing breeding method for polygonatum cyrtonema is not ideal enough, or the rhizome of polygonatum cyrtonema is small, the growth speed of later-stage seedlings is slow, the cost is high, or the polygonatum cyrtonema bud grows excessively, the leaves are deformed, the rootstocks cannot grow, the large-scale production of high-quality polygonatum cyrtonema seedlings cannot be met, and the like.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds comprises the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema as explants; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and sterilizing the explant, wherein the excess materials are used for cutting redundant rhizomes of the explant, removing bracts on buds and keeping middle rhizomes with buds.
b) And (3) proliferation and strong seedling culture: cutting the bud top of the disinfected rootstock blocks with buds by using a scalpel, and inoculating the rootstock blocks with buds to a propagation and strong seedling culture medium for culture until tubers are expanded, bud bodies are thick and the color of the tubers is deepened to obtain a first rootstock block; the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, and the pH value of said culture medium is controlled at 5.5-5.7.
c) Differentiation culture: cutting the first rhizome block into uniform first unit rhizome blocks (the volume is about 1.0cm multiplied by 0.5 cm) and inoculating the first unit rhizome blocks into a differentiation culture medium for culturing until the bud body extends and leaves are extracted to obtain a second rhizome block; the differentiation medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + sugar 30g/L, and the pH value of the differentiation culture medium is controlled at 5.5-5.7; and cutting bud points of the second rhizome block by using a scalpel, cutting the second rhizome block into second unit rhizome blocks of 0.5cm multiplied by 0.5cm, and inoculating the second unit rhizome blocks into the proliferation strong seedling culture medium for cyclic culture for several generations to obtain a large amount of second rhizome blocks.
d) Rooting culture: cutting the second rhizome block into third rhizome blocks with uniform volume (the volume is about 1.5cm multiplied by 1.5 cm), ensuring that each third rhizome block has at least three rootstocks above the healthy bud, transferring the third rhizome blocks to a rooting culture medium for culturing until obtaining a plurality of buds and three or more seed rhizome blocks with strong development and root seedlings, wherein the rooting culture medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + active carbon 0.5g/L sugar 60g/L, the pH value of said rooting culture medium is controlled at 5.5-5.7.
Further, greenhouse transplanting is required after the step d): planting the root tuber blocks into a seedling growing sieve filled with a matrix (peat soil: sand =2: 1), shading by using a single-layer sunshade net with 70% in winter and spring, and applying the nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is needed when the surface of a soil layer is whitish, and water and fertilizer are applied at intervals in summer.
Further, before transplanting in the greenhouse, the treatment of seed and rhizome during seedling emergence is required; and (3) hardening off the seedlings of the rhizomes in an open culture bottle for 3 days, then taking out the rhizomes, flushing the agar with tap water, soaking the rhizomes in 800 times of liquid chlorothalonil for 15 minutes, and then soaking the rhizomes in 50 mg/L of 6-BA for 3 hours.
Further, in the step a), underground rhizomes of polygonatum cyrtonema planted for 2-3 years are selected as explants in 9-10 months of the current year; the volume of the rooted rootstock blocks with buds is about 1.0cm multiplied by 0.5 cm.
Further, the time for inoculating the stem block with the bud root in the step b) into a culture medium for proliferating strong seedlings to culture is controlled to be 42-47 days; the time for inoculating the first unit rhizome block into the differentiation culture medium in the step c) for culture is also controlled to be 42-47 days; the time for inoculating the third rhizome block into the rooting culture medium in the step d) is also controlled to be 42-47 days.
A breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds comprises the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema as explants; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and sterilizing the explant, wherein the excess materials are used for cutting redundant rhizomes of the explant, removing bracts on buds and keeping middle rhizomes with buds.
b) And (3) proliferation and strong seedling culture: cutting the bud top of the disinfected rootstock blocks with buds by using a scalpel, inoculating the rootstock blocks with buds into a proliferation and strong seedling culture medium for culturing, and expanding tubers until roots grow to obtain first rootstock blocks; the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, the pH value of said culture medium is controlled at 5.5-5.7; the first rhizome block is obtained after the culture time is more than 3 months.
c) Treating seed and rhizome during seedling emergence; and (3) hardening off the seedlings of the rhizomes in an open culture bottle for 3 days, then taking out the rhizomes, flushing the agar with tap water, soaking the rhizomes in 800 times of liquid chlorothalonil for 15 minutes, and then soaking the rhizomes in 50 mg/L of 6-BA for 3 hours.
d) Transplanting in a greenhouse: planting the root tuber blocks into a seedling growing sieve filled with a matrix (peat soil: sand =2: 1), shading by using a single-layer sunshade net with 70% in winter and spring, and applying the nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is needed when the surface of a soil layer is whitish, and water and fertilizer are applied at intervals in summer.
From the above description of the present invention, it can be seen that the advantages of the present invention over the prior art are: the breeding method is ideal in design, researches are carried out on cultivating the seed rhizome and breaking the dormancy of the rhizome bud body, strong rhizomes with multiple buds developing simultaneously and bud body planting are cultivated, the quality is good, the growth speed is high, domesticated seedlings capable of being subjected to leaf-drawing in the same year are domesticated and cultivated in a greenhouse for one year, the seed stem standard for production can be achieved, the problem of shortage of high-quality seedlings is solved, the seedling cultivation time is shortened, the cost is reduced, and the planting yield can be increased in multiples. The second unit rhizome block is inoculated into the culture medium for multiplication and strong seedling for circulation culture for a plurality of generations, the operation is simple, the trouble of separating a single plant is saved, and the rootstocks with multiple buds and strong development can be obtained within 90 days.
Drawings
FIG. 1 is a schematic diagram of the breeding process of the present invention.
FIG. 2 is a diagram showing the growth effect of the rhizome of Polygonatum cyrtonema which has been planted for more than 10 years, is of a single-bud development type, and has a low planting yield.
FIG. 3 is a graph showing the effect of sucrose concentration of 20g/L on the growth in Table 1, the rootstocks grow for 20 days, and the leaves are tender yellow and slender.
FIG. 4 is a graph showing the effect of sucrose concentration of 20g/L on the growth of the rootstocks of Table 1, which are grown for 30 days, yellow and dead leaves, white in color and weak in vitality.
FIG. 5 is a graph showing the effect of the growth of 30g/L sucrose concentration in Table 1, which is observed in the case of 30 days of growth, and the leaves are yellow-green and slender.
FIG. 6 is a graph showing the effect of sucrose concentration 30g/L on growth in Table 1, wherein the rootstocks were grown for 45 days, some of the leaves were yellow and dead, the rootstocks were white, and the vitality was weak.
FIG. 7 is a graph showing the growth effect of sucrose concentration of 30g/L in Table 1, which is a proliferated seedling.
FIG. 8 is a graph showing the growth effect of sucrose concentration of 30g/L in Table 1, as a root seedling.
FIG. 9 is a graph showing the effect of sucrose concentration of 60g/L on the growth of the rootstocks in 45-day-old seedlings in Table 1.
FIG. 10 is a graph showing the effect of sucrose concentration of 60g/L on the growth of a rooted seedling with multiple shoots simultaneously developed 45 days after the growth of the rootstock, and the leaf slimness in Table 1.
FIG. 11 is a graph showing the effect of sucrose concentration of 90g/L on the growth of the rootstocks in the state where only the rootstocks were grown after 45 days of growth without leaf pulling and the rootstocks were strong.
FIG. 12 is a graph showing the effect of sucrose concentration of 90g/L on the growth of rhizomes growing for 3 months or more in Table 1.
FIG. 13 is a graph showing the effect of the first-unit rootstalk block on the differentiation medium for 30 days, and the multiple shoots are strong.
FIG. 14 is a diagram showing the growth effect of the third rhizome after growing for 45 days in a rooting medium, forming a rooted seedling with multiple shoots and strong development, with dark green leaf color, large rhizome and strong bud body.
FIG. 15 shows a root and stem of the present species (i.e., a test-tube plantlet) planted for 5 years.
Detailed Description
Example one
Refer to the description attached fig. 1, fig. 11, fig. 12. A breeding method for forming strong and developing rootstocks of polygonatum cyrtonema with multiple buds can quickly form multiple-bud development breeding of polygonatum cyrtonema tubers, and comprises the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema planted for 2-3 years as explants in 9-10 months of the current year; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and disinfecting the explant, and specifically comprises the following steps: cleaning underground rhizomes (namely explants) of polygonatum cyrtonema with water, removing leaves and roots, cutting off redundant rhizomes around, removing bracts which can be stripped from buds, and taking middle rootstock blocks with buds, wherein the volume of the rootstock blocks with buds is about 1.0cm multiplied by 0.5 cm; then washing the rhizomes with buds with flowing tap water for 1-2 hours, and airing the rhizomes; soaking in 75% ethanol for 15s on a clean bench, washing with sterile water for 2-4 times, adding into 0.1% mercuric chloride, shaking for 8-10min, taking out, washing with sterile water for 5-8 times, and drying with sterile filter paper.
b) And (3) proliferation and strong seedling culture: cutting off bud tops of the rootstock blocks with buds by using a scalpel, inoculating the rootstock blocks with buds to a proliferation and strong seedling culture medium for culture, expanding the periphery of the rootstock after 10 days to grow a callus, then sequentially emerging bud points, and continuously expanding tubers until the tubers grow to grow roots to obtain a first rootstock block, wherein the culture time is more than 3 months to obtain seed rootstocks, and each rootstock has more than 3 vigorously developed rootstock seedlings; the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, the pH value of said culture medium is controlled at 5.5-5.7 (optimum pH value is 5.6); . The culture medium for proliferating strong seedlings has the characteristics that: the method can effectively inhibit the leaf extraction of the rootstock blocks with buds, ensure the dormancy of buds, avoid the leaf extraction, remove the top advantages, supply nutrition to the rootstocks in a centralized way, and ensure that the rootstocks expand at a high speed and the lateral buds of the tubers are strong and expand obviously.
c) Treating seed and rhizome during seedling emergence; taking out the culture bottle after hardening off seedlings for 3 days, flushing agar with tap water, soaking the culture bottle in 800 times of liquid chlorothalonil for 15 minutes, and then soaking the culture bottle in 50 mg/L6-BA (namely cytokinin) for 3 hours to obtain the seedlings for planting; by adopting 50 mg/L6-BA and soaking the seed rhizome for 3 h, the physiological effects of effectively breaking the dormancy of the bud body of the seed rhizome, inducing the growth of dormant buds, breaking the apical dominance, promoting the growth of lateral buds and the like can be achieved.
d) Transplanting in a greenhouse: planting the root tuber blocks into a seedling growing sieve filled with a matrix (peat soil: sand =2: 1), shading by using a single-layer sunshade net with 70% in winter and spring, and applying the nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is needed when the surface of a soil layer is whitish, and water and fertilizer are applied at intervals in summer; the matrix and shading mode of greenhouse transplanting can adapt to the growth and development of polygonatum cyrtonema test-tube plantlets.
Example two
Reference is made to the description accompanying fig. 1, fig. 2-fig. 11, fig. 13-fig. 15. A breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds comprises the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema planted for 2-3 years as explants in 9-10 months of the current year; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and disinfecting the explant, and specifically comprises the following steps: cleaning underground rhizomes (namely explants) of polygonatum cyrtonema with water, removing leaves and roots, cutting off redundant rhizomes around, removing bracts which can be stripped from buds, and taking middle rootstock blocks with buds, wherein the volume of the rootstock blocks with buds is about 1.0cm multiplied by 0.5 cm; then washing the rhizomes with buds with flowing tap water for 1-2 hours, and airing the rhizomes; soaking the mixture on an ultra-clean workbench for 15s with 75% alcohol, washing with sterile water for 2-4 times, adding into 0.1% mercuric chloride, shaking continuously for 8-10min, taking out, washing with sterile water for 5-8 times, and sucking out sterile water with sterile filter paper;
b) inducing the rootstock blocks with buds: treating the rootstock blocks with the buds by using a uniform electric field, specifically, placing the rootstock blocks with the buds into an induction container, and then carrying out induction treatment on the rootstock blocks with the buds in the induction container by using a 100 kv-300 kv/m uniform electric field (optimally 200 kv/m) emitted by a high-voltage electrostatic generator, wherein the induction treatment time is controlled to be 2-3 minutes; the research shows that: the rhizomes with buds are placed in a high-voltage electrostatic field, the induction treatment time is within 2-3 minutes, the germination potential, the germination rate and the dehydrogenase activity of the rhizomes with buds are remarkably improved along with the increase of the treatment time, the treatment effect is optimal when the treatment time is 3 minutes, and the treatment effect is gradually weakened along with the continuous increase of the treatment time after the treatment time exceeds 3 minutes.
c) And (3) proliferation and strong seedling culture: cutting off bud tops of the induced and treated rootstock blocks with buds by using a scalpel, inoculating the rootstock blocks with buds into a proliferation and strong seedling culture medium for culture, expanding the periphery of rootstocks after 10 days, growing callus, and then successively emitting bud points until tubers expand, the buds are thick and the color of the tubers is deepened to obtain a first rootstock block; culturing on the proliferation strong seedling culture medium for 42-47 days (the optimal days are 45 days); the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, the pH value of said culture medium is controlled at 5.5-5.7 (optimum pH value is 5.6); the culture medium for proliferating strong seedlings has the characteristics that: the method can effectively inhibit the leaf extraction of the rootstock blocks with buds, ensure the dormancy of buds, avoid the leaf extraction, remove the top advantages, supply nutrition to the rootstocks in a centralized way, and ensure that the rootstocks expand at a high speed and the lateral buds of the tubers are strong and expand obviously.
d) Differentiation culture: cutting the first rootstock block into a first unit rootstock block with uniform volume (the volume is about 1.0cm multiplied by 0.5 cm), inoculating the first unit rootstock block into a differentiation culture medium for culture, expanding the periphery of the rootstock block after 7 days, and emitting a plurality of buds on the periphery until the buds extend and leaves are extracted to obtain a second rootstock block; the differentiation medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + sugar 30g/L, the pH value of the differentiation culture medium is controlled at 5.5-5.7 (optimum pH value is 5.6); and cutting bud points of the second rhizome block by using a scalpel, cutting the second rhizome block into second unit rhizome blocks of 0.5cm multiplied by 0.5cm, inoculating the second unit rhizome blocks into the proliferation strong seedling culture medium, and performing circulating culture for several generations, so that a large number of second rhizome blocks (namely high-quality bottle seedlings) can be obtained in a short time. The differentiation culture medium has the characteristics that: the growth speed of the rootstocks is high, and the buds can be stretched and pulled out of the leaves.
e) Rooting culture: cutting the second rhizome block into third rhizome blocks with uniform volume (the volume is about 1.5cm multiplied by 1.5 cm), ensuring that each third rhizome block has at least three rootstocks above the healthy bud, transferring the third rhizome blocks to a rooting culture medium for culturing until obtaining a plurality of buds and three or more seed rhizome blocks with strong development and root seedlings, wherein the rooting culture medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + activated carbon 0.5g/L sugar 60g/L, the pH value of the rooting medium is controlled between 5.5 and 5.7 (optimum pH value is 5.6). The rooting culture medium has the characteristics that: can obtain the rooted seedlings with multiple buds and strong development at the same time, and can realize the increase of the planting yield by times.
f) Treating seed and rhizome during seedling emergence; and (3) hardening seedlings in an open culture bottle for 3 days, taking out, flushing agar by using tap water, soaking for 15 minutes by using 800 times of liquid chlorothalonil, and soaking for 3 hours by using 50 mg/L6-BA to plant. By adopting 50 mg/L6-BA and soaking the seed rhizome for 3 h, the physiological effects of effectively breaking the dormancy of the bud body of the seed rhizome, inducing the growth of dormant buds, breaking the apical dominance, promoting the growth of lateral buds and the like can be achieved.
g) Transplanting in a greenhouse: planting the root tuber blocks into a seedling growing sieve filled with a matrix (peat soil: sand =2: 1), shading by using a single-layer sunshade net with 70% in winter and spring, and applying the nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is carried out when the surface of a soil layer is whitish, water and fertilizer are applied once at intervals in summer, and the matrix and shading mode for greenhouse transplanting can adapt to the growth and development of polygonatum cyrtonema test-tube seedlings.
The sugar in the proliferation and seedling strengthening culture medium, the differentiation culture medium and the rooting culture medium is sucrose, and the other components are MS +6-BA 1.0 mg.L-1 +NAA 0.5 mg·L-1 +IBA+agar5.0 g·L-1。
The applicant researches and discovers that the rhizome size and quality of polygonatum cyrtonema proliferation test-tube plantlet are key influence factors for later-stage field cultivation, the dormancy of the test-tube plantlet bud prolongs the seedling cultivation time of polygonatum cyrtonema, and the single-bud development of polygonatum cyrtonema is a direct reason for low cultivation yield. Therefore, on the basis of previous researches, a breeding method for rapidly and massively forming polygonatum cyrtonema tuber multiple-bud development is discussed, and the influence of the sucrose concentration in a culture medium on polygonatum cyrtonema seed rhizomes and the influence of the 6-BA concentration and the treatment time on breaking bud dormancy are mainly studied. The polysaccharide is used as an important supply of carbon source in tissue culture, and plays a role in providing energy source and stabilizing osmotic pressure in the tissue culture, and the adding concentration of the polysaccharide in a culture medium is particularly important.
Remarking: table 1 shows the effect of different sucrose concentrations on rhizome of Polygonatum cyrtonema species
| Sucrose concentration (g/L) | Multiplication factor (times) | Leaf extraction Rate (%) | Growth conditions |
| 20.00 | 2.38 | 93.23 | After 7 days, the buds grow, 15 days, the leaves are formed, the leaves are bright yellow and slender, and 30 days later, the leaves grow Yellow and dead, swollen rootstalk, whitish color and weak vitality. |
| 30.00 | 3.10 | 80.13 | Bud growth is about 0d, leaves are formed after 20d, the leaves are yellow green and long, and partial leaves after 45d Yellow and dead, swollen rootstalk, white color and weak vitality. |
| 60.00 | 5.69 | 43.78 | Part of rootstocks grow about 15d, buds grow 30d, leaves are formed, the leaves are large and normally green, the rhizome is enlarged, after 45 days, the rhizome is dark in color, obviously enlarged, sufficient in nutrition and sends out Root system, robust plant. Part of the rootstocks are obviously expanded, leaves are not grown, and the rootstocks are robust. |
| 90.00 | 4.38 | 0.00 | The leaves are not taken out, the rootstocks are rapidly expanded to fill the whole culture bottle, the color of the rootstocks is dark after 30 days, the bud is strong and has sufficient nutrition, and the root system can be generated after the culture time is long. |
Remarking: table 2 shows the effect of 6-BA concentration and treatment time on the dormancy of broken sprouts
| 6－BA(mg/L) | Treatment time (h) | Leaf extraction Rate (%) | Growth conditions |
| 200 | 12 | 100 | After 3d, the buds uniformly extend, overgrow, the leaves are deformed, and after 7d, the seedlings are poured out, and the roots are killed. |
| 100 | 12 | 100 | After 3d, the buds uniformly extend, overgrow, the leaves are deformed, and after 7d, the seedlings are poured out, and the roots are killed. |
| 50 | 12 | 100 | After 3d, the buds uniformly extend, overgrow, the leaves are deformed, and after 7d, the seedlings are poured out, and the roots are killed. |
| 50 | 6 | 100 | After 7d, the buds uniformly extend, overgrow, the leaves are deformed, and after 15d, the seedlings are poured, the roots and stems do not grow, and the seedlings can also grow in the next year. |
| 50 | 3 | 100 | After 10 days, the buds uniformly extend, and the plants normally grow in dark green. |
| 20 | 3 | 63 | After 10 days, the buds extend continuously, and the plants grow normally in dark green. |
Remarking: table 3 shows the yield and number of shoots taken after 5 years of planting of the multi-shoot test-tube plantlet
| Variety of (IV) C | Observation of number of plants (plants) | Average original tuber weight (g) | Average number of shoots after 5 years | Average tuber weight (g) after 5 years |
| Multiple bud test-tube plantlet | 30 | 1.28 | 3.8 | 375.33 |
| Wild single-bud tuber | 30 | 15.03 | 1 | 150.15 |
| Seed of corn | 30 | 0.15 | 1 | 50.23 |
The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.
Claims (3)
1. A breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds is characterized by comprising the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema as explants; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and sterilizing the explant, wherein the excess materials are obtained by cutting excess rhizomes of the explant, stripping bracts on buds and reserving middle rhizomes with buds; selecting underground rhizomes of polygonatum cyrtonema planted for 2-3 years as explants in 9-10 months of the year; the volume of the root block with buds is about 1.0cm multiplied by 0.5 cm; the method specifically comprises the following steps: washing underground rhizomes of polygonatum cyrtonema with water, removing leaves and roots, cutting off redundant rhizomes around, peeling off bracts which can be peeled off from buds, and taking middle rhizomes with buds, wherein the volume of the rhizomes with buds is about 1.0cm multiplied by 0.5 cm; then washing the rhizomes with buds with flowing tap water for 1-2 hours, and airing the rhizomes; soaking the mixture on an ultra-clean workbench for 15s with 75% alcohol, washing with sterile water for 2-4 times, adding into 0.1% mercuric chloride, shaking continuously for 8-10min, taking out, washing with sterile water for 5-8 times, and sucking out sterile water with sterile filter paper;
b) and (3) proliferation and strong seedling culture: cutting the bud top of the disinfected rootstock blocks with buds by using a scalpel, and inoculating the rootstock blocks with buds to a propagation and strong seedling culture medium for culture until tubers are expanded, bud bodies are thick and the color of the tubers is deepened to obtain a first rootstock block; the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, the pH value of said culture medium is controlled at 5.5-5.7;
c) differentiation culture: cutting the first rhizome block into uniform first unit rhizome blocks, inoculating the first unit rhizome blocks into a differentiation culture medium, and culturing until the bud body is elongated and leaves are extracted to obtain a second rhizome block; the differentiation medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + sugar 30g/L, and the pH value of the differentiation culture medium is controlled at 5.5-5.7; cutting bud points of the second rhizome block by using a scalpel, cutting the second rhizome block into second unit rhizome blocks of 0.5cm multiplied by 0.5cm, and inoculating the second unit rhizome blocks into the proliferation strong seedling culture medium for cyclic culture for several generations to obtain a large amount of second rhizome blocks;
d) rooting culture: cutting the second rhizome block into third rhizome blocks with uniform volume, ensuring that each third rhizome block has at least three healthy buds, transferring the third rhizome blocks to a rooting culture medium for culturing until a plurality of buds with more than three healthy and strong root-bearing seedlings are obtained, wherein the rooting culture medium is as follows: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + 0.2mg/L IBA + active carbon 0.5g/L sugar 60g/L, the pH value of said rooting culture medium is controlled at 5.5-5.7;
e) treatment of seed rootstocks at emergence: hardening off the seedling of the rhizomes in an open culture bottle for 3 days, then taking out the rhizomes, flushing the agar with tap water, soaking the rhizomes in 800 times of liquid chlorothalonil for 15 minutes, and then soaking the rhizomes in 50 mg/L of 6-BA for 3 hours;
f) transplanting in a greenhouse: planting the root and stem blocks into a seedling growing sieve filled with a matrix, shading by using a single-layer sunshade net with 70 percent in winter and spring, and applying a nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is needed when the surface of a soil layer is whitish, and water and fertilizer are applied at intervals in summer.
2. The breeding method for forming the healthy and strong rootstocks of polygonatum cyrtonema as claimed in claim 1, wherein: the stem block with bud roots in the step b) is inoculated into a culture medium for strong seedling proliferation, and the culture time is controlled to be 42-47 days; the time for inoculating the first unit rhizome block into the differentiation culture medium in the step c) for culture is also controlled to be 42-47 days; the time for inoculating the third rhizome block into the rooting culture medium for culture in the step d) is also controlled to be 42-47 days; the volume of the first unit rhizome block is about 1.0cm multiplied by 0.5 cm; the third rhizome block is about 1.5cm × 1.5 cm.
3. A breeding method for forming strong and developed rootstocks of polygonatum cyrtonema with multiple buds is characterized by comprising the following steps:
a) selecting and pretreating explants: selecting underground rhizomes of polygonatum cyrtonema as explants; the explant pretreatment comprises the steps of sequentially cleaning, cutting excess materials and sterilizing the explant, wherein the excess materials are obtained by cutting excess rhizomes of the explant, stripping bracts on buds and reserving middle rhizomes with buds;
b) and (3) proliferation and strong seedling culture: cutting the bud top of the disinfected rootstock blocks with buds by using a scalpel, inoculating the rootstock blocks with buds to a proliferation and strong seedling culture medium for culture, expanding the periphery of the rootstock after 10 days, growing callus, continuously emerging bud points, expanding tubers until the roots grow, and culturing for more than 3 months to obtain seed rootstock, wherein more than 3 developed and strong rootstock seedlings are arranged on each rootstock; the culture medium for proliferating strong seedlings comprises: MS + 1.0 mg/L6-BA + 0.5mg/L NAA + sugar 90g/L, the pH value of said culture medium is controlled at 5.5-5.7;
c) treating seed and rhizome during seedling emergence; hardening off the seedling of the rhizomes in an open culture bottle for 3 days, then taking out the rhizomes, flushing the agar with tap water, soaking the rhizomes in 800 times of liquid chlorothalonil for 15 minutes, and then soaking the rhizomes in 50 mg/L of 6-BA for 3 hours;
d) transplanting in a greenhouse: planting the root and stem blocks into a seedling growing sieve filled with a matrix, shading by using a single-layer sunshade net with 70 percent in winter and spring, and applying a nutrient solution for 1 time every 7 days; in summer and autumn, a double-layer shading net with 70 percent is adopted for shading, watering is needed when the surface of a soil layer is whitish, and water and fertilizer are applied at intervals in summer.
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| CN113100057B (en) * | 2021-04-02 | 2022-09-13 | 安徽省林业高科技开发中心 | Seedling strengthening and rapid propagation method for polygonatum cyrtonema |
| CN114403010B (en) * | 2022-02-22 | 2022-11-15 | 福建农林大学 | Polygonatum cyrtonema micro-tuber dark energy-saving in-vitro propagation method |
| CN115191305B (en) * | 2022-08-02 | 2023-09-15 | 江西农业大学 | Method for breaking dormancy rapid leaf growth of rhizoma polygonati seeds |
| CN115250914A (en) * | 2022-08-04 | 2022-11-01 | 三明市农业科学研究院 | Seedling growing method using simple greenhouse to replace sealwort cultivation room |
| CN116530415B (en) * | 2023-05-30 | 2024-04-02 | 贵州省植物园 | Circularly operated polygonatum cyrtonema tissue culture breeding method |
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