CN115581202A - Method for regenerating and in-vitro seedling of new variety of polygonatum cyrtonema - Google Patents
Method for regenerating and in-vitro seedling of new variety of polygonatum cyrtonema Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
Abstract
The invention discloses a method for regenerating and in-vitro seedling formation of a new polygonatum cyrtonema variety, relates to the technical field of plant biology, and aims at solving a plurality of problems in the propagation of the new polygonatum cyrtonema variety Qi Yuan, namely the polygonatum cyrtonema variety, by taking tender leaves of seedlings with tubers germinating as explant sources and utilizing the tissue culture technology in combination with the in-vitro seedling formation technology, an efficient and short-period Qi Yuan polygonatum in-vitro propagation technology is developed. The invention has the beneficial effects that: simplifies the steps of adventitious bud proliferation, elongation, rooting and domestication of the sealwort, realizes the synchronization of adventitious bud proliferation and elongation, the synchronization of rooting and domestication, and can obtain a complete Qi Yuan sealwort in vitro regeneration plant after culturing for 11 weeks. The invention provides technical support for large-scale propagation, popularization and application and variety improvement of the late stage Qi Yuan sealwort seedlings.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a method for regenerating and in-vitro seedling of a new variety of polygonatum cyrtonema.
Background
Polygonatum cyrtonema (Polygonatum cyrtonema) is a perennial herb medicinal plant in Polygonatum of Liliaceae, and the rhizome of Polygonatum cyrtonema contains abundant medicinal compounds such as saccharides, steroid saponin, cardiac glycoside, alkaloid, flavone and the like, and has the effects of tonifying qi and yin, strengthening spleen, moistening lung, tonifying kidney and the like. In recent years, with the improvement of living standard of people and the increase of the market demand for polygonatum cyrtonema, the development of industrialized cultivation of polygonatum cyrtonema faces unprecedented opportunities. Because the wild polygonatum sibiricum resources are excessively harvested and gradually exhausted, the consumption of polygonatum cyrtonema in the market is gradually increased, large-area artificial cultivation is forced to supplement the resources, and the phenomenon of shortage of polygonatum cyrtonema seedling supply in the recent market is caused. Meanwhile, one fine variety can drive one industry, and the quality and the surplus of polygonatum cyrtonema seedlings can directly affect the quality and the yield of polygonatum cyrtonema and also relate to the healthy sustainable development of the traditional Chinese medicine industry and the life safety of people. Therefore, on the basis of polygonatum cyrtonema germplasm resource breeding, the research on the high-efficiency, short-period and low-cost rapid propagation technology of the excellent variety of polygonatum cyrtonema has important significance for the sustainable development of polygonatum cyrtonema industry.
Qi Yuan rhizoma Polygonati as a new variety of bred Polygonatum cyrtonema (examined and numbered: wanpin Jiandeng character 1806007) has larger nutritive organs and higher content of effective components compared with the conventionally planted Polygonatum cyrtonema, and has higher medicinal value and market demand. At present, qi Yuan rhizoma polygonati is mainly propagated by adopting seeds and roots as raw materials, and propagation by a sowing mode has the problems of long seed germination period, low germination rate, limitation of propagation season and time interval, difficulty in maintaining the genetic stability of a maternal plant line and the like; the tuber is used for breeding, although the excellent agronomic characters of the maternal plant line can be maintained, the problems of limited breeding materials and low breeding coefficient exist, and the requirements of industrial planting, popularization and application of Qi Yuan polygonatum are difficult to meet. The plant tissue culture technology as a modern biotechnology has the advantages of high propagation efficiency, short propagation period, no limitation of seasons and time on propagation and the like, and plays an important role in large-scale propagation of plant seedlings. At present, researches on breeding polygonatum cyrtonema by using a plant tissue culture technology have been reported, for example, a patent with the publication number of CN 109302983A discloses a rapid breeding method of polygonatum cyrtonema by using terminal buds as explants, but the method has the problems of long breeding period, high cost and poor repeatability, and is not suitable for rapid breeding of a new variety of polygonatum cyrtonema, qi Yuan.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method suitable for high-efficiency regeneration and in-vitro seedling establishment of a new variety of polygonatum cyrtonema and having short seedling establishment time, high propagation coefficient, high adventitious bud induction rate and high adventitious root induction rate.
The invention solves the technical problems through the following technical means:
a method for regenerating and in-vitro seedling of a new variety of polygonatum cyrtonema, which is Qi Yuan polygonatum, comprises the following steps: taking young leaves of the germinated seedlings of the Qi Yuan rhizoma polygonati tubers as explants, sterilizing the surfaces of the young leaves, shearing the young leaves into blocks with proper sizes, and finally obtaining Qi Yuan rhizoma polygonati complete regeneration plants through adventitious bud induction, adventitious bud proliferation and elongation, adventitious root induction treatment outside a bottle, field planting and seedling management;
the leaf age of the young and tender leaves is 13-17d;
the culture medium adopted for inducing adventitious buds contains 0.5-2.0mg/L TDZ and 0.5-2.5mg/LAgNO 3 DKW medium of 3.0% (w/v) sucrose and agar;
the culture medium for proliferation and elongation of adventitious bud contains 0.05-1.0mg/L TDZ, 0.05-0.25mg/L IAA, and 0.5-2.5mg/L GA 3 DKW medium of sucrose and agar 3.0% (w/v);
the culture medium adopted by the adventitious root induction treatment outside the bottle is a rooting promoting liquid containing 500-1000mg/L K-IBA, 100-500mM melatonin and 200-500mg/L NAA.
Has the advantages that: the method is suitable for Qi Yuan rhizoma polygonati, and has the advantages of less material acquisition, convenient material acquisition and abundant material; the propagation efficiency is high, the adventitious bud inductivity is up to 93.4%, and the adventitious root inductivity is up to 97.4%. The seedling time is short, the seedlings can be grown in 11 weeks, the propagation coefficient is high, and the technical support is provided for the rapid propagation of Qi Yuan sealwort plants and the large-scale production of later-stage excellent lines.
TDZ and AgNO 3 Combined and combinedThe addition of the extract to DKW culture medium can improve the induction effect of adventitious bud, and low-concentration TDZ combines low-concentration IAA and GA 3 The number of the adventitious buds is increased, and the length of the adventitious buds is increased.
According to the research of the invention, the regeneration capacity of the leaves at different ages is different, and the induction rate of the adventitious bud can reach the highest when the leaves are 13-17d old.
Preferably, the adventitious bud induction adopts a culture medium containing 0.5mg/L TDZ and 2.0mg/L AgNO 3 The DKW medium of (1), wherein the medium used for the proliferation and elongation of adventitious buds is a medium containing 0.2mg/L TDZ,0.05mg/L IAA and 1.0mg/L GA 3 The DKW medium of (1).
Has the beneficial effects that: when the above medium is used, the efficiency of induction of adventitious buds, the proliferation of adventitious buds, and the effect of induction can be further improved.
Preferably, the surface disinfection comprises the steps of: sealing the explant in a container, washing with tap water, placing in a sterile operating platform, washing with sterile water, sterilizing with anhydrous ethanol, washing with sterile water, and washing with 0.1% (v/v) HgCl 2 Sterilizing the solution for 2-3min, washing with sterile water, and drying the surface water of the seeds with sterile filter paper.
Has the advantages that: different disinfectants and disinfection time have important influence on the Qi Yuan sealwort leaf pollution rate and the induction rate of adventitious buds. The present invention adopts 0.1% of HgCl 2 The disinfection effect and the induction condition of the adventitious bud of the disinfection are superior to those of hydrogen peroxide and sodium hypochlorite, and the induction rate of the adventitious bud is up to 93.4 percent.
Preferably, the cutting into the proper size refers to cutting the blade into 0.25-1.0cm after removing the blade tip 2 Size, ready for use.
Preferably, the adventitious bud multiplication and elongation means that the elongated Qi Yuan polygonatum adventitious buds after 4 weeks of light culture are inoculated to a culture medium for adventitious bud multiplication and elongation.
Preferably, the adventitious root induction treatment outside the bottle is to separate the elongated adventitious buds of polygonatum sibiricum from the adventitious bud cluster after being cultured for 4 weeks by illumination, wash the adventitious buds by running water, absorb the surface moisture, and immerse the morphological lower ends of the elongated buds in a culture medium adopted for the adventitious root induction treatment outside the bottle to perform the adventitious root induction treatment, wherein the induction treatment time is 30-120s.
Preferably, the permanent planting is to fixedly plant the morphological lower end of the Qi Yuan polygonatum tissue culture extended bud after the adventitious root induction treatment in a nutrient medium disc filled with a nutrient medium for adventitious root induction; the nutrient medium comprises nutrient soil, perlite and vermiculite, wherein the mass ratio of the nutrient soil to the perlite to the vermiculite is 2.
Preferably, the seedling management is to spray the Qi Yuan rhizoma polygonati tissue culture extended buds after field planting with nutrient solution once, cover the buds, place the buds in a greenhouse with the temperature of 20-23 ℃ and the relative humidity of 85-90% for seedling culture, pour water for 1 time every 5-7 days, and remove the plastic cover after 1 month.
Preferably, the nutrient solution comprises 30% -50% of Hoagland nutrient solution prepared by double distilled water.
The invention has the advantages that: the method is suitable for Qi Yuan rhizoma polygonati, and has the advantages of less material acquisition, convenient material acquisition and abundant material; the propagation efficiency is high, the adventitious bud inductivity is up to 93.4%, and the adventitious root inductivity is up to 97.4%. The seedling forming time is short, the seedlings can be formed in 11 weeks, the propagation coefficient is high, and technical support is provided for the rapid propagation of Qi Yuan sealwort plants and the large-scale production of later-stage excellent lines.
TDZ and AgNO 3 The combination and addition to DKW culture medium can improve the induction effect of adventitious bud, and the combination of low-concentration TDZ and low-concentration IAA and GA 3 The number of the adventitious buds is increased, and the length of the adventitious buds is increased.
Through the research of the invention, the regeneration capability of the leaves at different ages is different, but the induction rate of the adventitious bud can reach the highest when the leaves are 13-17d old.
When a specific medium is used, the efficiency of induction of adventitious buds, the proliferation of adventitious buds, and the induction effect can be further improved.
Different disinfectants and disinfection time have important influence on the Qi Yuan sealwort leaf pollution rate and the induction rate of adventitious buds. The present invention adopts the disinfection effect by 0.1% of HgCl2 and the induction of adventitious buds is superior to that of hydrogen peroxide and sodium hypochlorite, and the induction rate of adventitious buds reaches up to 93.4%.
By adopting the method, the adventitious bud induction rate is up to 93.4%, 9.4 adventitious buds are generated on average in each explant, and 30.1 adventitious buds with the length of 2.8cm are generated on average in each explant after proliferation and elongation culture; the induction rate of the adventitious roots is as high as 97.4%, and on average, 4.3 adventitious roots are generated per explant.
Drawings
FIG. 1 is a diagram of tubers of Qi Yuan from example 1 of this invention sprouting seedlings;
FIG. 2 is a cut-out photograph of a leaf piece inoculated on an adventitious bud induction medium for 4d in example 1 of the present invention;
FIG. 3 is an adventitious bud induction map of Qi Yuan leaves of Polygonatum sibiricum of example 1 of the present invention; in the figure, a and b are both adventitious bud regeneration (non-callus);
FIG. 4 is a diagram of the proliferation and elongation of adventitious buds of Qi Yuan Polygonatum sibiricum produced in example 1 of the present invention;
FIG. 5 is a diagram of the tissue culture extended bud rooting planting of Qi Yuan rhizoma Polygonati in example 1 of the present invention;
FIG. 6 is a diagram of in vitro rooting and seedling growth of Qi Yuan rhizoma Polygonati in example 1 of the present invention;
FIG. 7 is a diagram of Qi Yuan whole regenerated plants of Polygonati officinalis rhizoma in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
A method for regenerating and in-vitro seedling formation of a new variety of polygonatum cyrtonema includes the following steps:
(1) A seedling (figure 1) of a new variety of polygonatum cyrtonema grown in a greenhouse, namely Qi Yuan polygonatum cyrtonema which sprouts in 1-year-old tubers, is taken as an explant source, and a leaf with the leaf age of 15d is selected as the explant.
(2) Placing the leaves in the step (1) in a 250ml triangular flask, sealing the opening with gauze, washing the leaves under tap water for 20min, placing the leaves in a sterile operating platform, and washing the leaves with sterile water for 4 times; then using 75% absolute ethyl alcohol to disinfect for 45s, and then using sterile water to wash for 6 times; then sterilized with 0.1% (v/v) mercuric chloride solution for 3min, and washed with sterile water for 6 times. And finally, absorbing the water on the surface of the seeds by using sterile filter paper for later use.
(3) Cutting the disinfected blade in the step (2) into 0.25-1.0cm after removing the blade tip 2 And (3) inoculating the culture medium into a glass culture dish containing adventitious bud induction culture medium for adventitious bud induction culture (figure 2), wherein the adventitious bud induction culture medium comprises: DKW +0.5mg/L TDZ +2.0mg/L AgNO 3 +3.0% (w/v) sucrose +0.7% (w/v) agar. After 2 weeks of illumination culture, the blade cuts are formed with expansion bulges; after 4 weeks of light culture, adventitious buds were formed at the leaf cut (FIG. 3), the adventitious bud induction rate was 93.4%, and on average 9.4 adventitious buds were produced per explant.
(4) Transferring the leaf cut blocks with the adventitious buds induced in the step (3) into an adventitious bud multiplication culture medium for multiplication and elongation culture of the adventitious buds, wherein the adventitious bud multiplication culture medium comprises: DKW +0.2mg/L TDZ +0.05mg/L IAA +1.0mg/L GA 3 +3.0% (w/v) sucrose +0.7% (w/v) agar. After 4 weeks of light culture, 30.1 adventitious buds are generated on average per explant, and the multiplication coefficient is 3.2; the average shoot length was 2.8cm (FIG. 4).
(5) Separating the adventitious buds of the polygonatum cyrtonema stretched in the step (4) from the adventitious bud cluster, washing the buds with running water, absorbing the surface water, and immersing the morphological lower ends of the stretched buds in rooting promoting liquid added with 750mg/L K-IBA, 500mg/L melatonin and 200mg/L IBA to perform adventitious root induction treatment for 60s;
(6) Planting the morphological lower end of the Qi Yuan sealwort tissue culture extended bud after adventitious root induction treatment in a nutrient medium disc filled with a nutrient medium (figure 5), wherein the nutrient medium comprises nutrient soil, perlite and vermiculite, and the mass ratio of the nutrient soil, the perlite and the vermiculite is 2.
(7) Spraying 40% Hoagland's nutrient solution prepared from tap water once on the tissue culture extended buds of Qi Yuan rhizoma Polygonati after field planting, covering, placing in a greenhouse with the temperature of 20-23 ℃ and the relative humidity of 85-90% for seedling culture, watering for 1 time every 5-7 days, and removing the plastic cover after 1 month. After 3 weeks of light culture, the adventitious root induction rate was as high as 97.4%, and on average 4.3 adventitious roots were produced per explant (fig. 6).
Example 2
Influence of different disinfection methods on Qi Yuan in vitro regeneration of sealwort leaves
Taking young leaves of the germinated seedlings of tubers of the new polygonatum cyrtonema variety with the seedling age of 15 days as explants, placing the leaves in a triangular flask according to the embodiment 1, sealing the leaves with gauze, washing the leaves in tap water for 20min, placing the leaves in an aseptic operation table, and washing the leaves with aseptic water for 4 times; then using 75% absolute ethyl alcohol to disinfect for 45s, and then using sterile water to wash for 6 times; then by 10% H 2 O 2 Solution, 0.1% (v/v) HgCl 2 The solution and 20% NaClO solution were sterilized for 2-10min, respectively, and washed with sterile water for 6 times. Finally, the leaf surface water was blotted with sterile filter paper and inoculated into the adventitious bud induction medium as mentioned in example 1 to conduct adventitious bud induction culture. And (4) counting the leaf pollution rate and the adventitious bud induction rate after 1 month of illumination culture.
The results are shown in Table 1. Research results show that different disinfectants and disinfection time have important influence on the Qi Yuan sealwort leaf pollution rate and the adventitious bud induction rate. In the disinfectant species tested and the disinfection time, 0.1% of HgCl 2 The disinfection effect of 3min and the induction condition of adventitious buds are optimal, the pollution rate is only 2.3%, and the induction rate of the adventitious buds is as high as 93.4%. With time of sterilisationThe pollution rate is further reduced after the elongation, but the leaf blade is also gradually necrotic, and the adventitious bud induction rate is also gradually reduced. Therefore, in the subsequent studies, 0.1% of HgCl was used 2 Can be used as disinfectant.
TABLE 1 influence of different disinfectant species and disinfection time on Qi Yuan sealwort leaf contamination and adventitious bud induction
Example 3
Influence of leaf age on Qi Yuan adventitious bud induction rate of sealwort
Leaves of different leaf ages (5, 10, 15, 30, 45 and 60 d) were selected as explants, sterilized by the method of example 1, and inoculated into the adventitious bud induction medium of example 1 for adventitious bud induction. The induction rate of adventitious buds and the average number of adventitious buds generated per explant were measured after 4 weeks of light culture. The results of the study are shown in Table 2.
Research results show that the leaf age has certain influence on the induction of the adventitious buds of the leaves, and the regeneration capacity of the adventitious buds of the leaves tends to increase first and then decrease along with the increase of the leaf age. Among the tested leaves with different leaf ages, the leaf with the leaf age of 15d has the strongest regeneration capability, the adventitious bud induction rate is up to 93.4%, 9.4 adventitious buds are generated on average in each explant, the adventitious bud induction rate and the number of the adventitious buds are gradually reduced along with the increase of the leaf age, the adventitious bud induction rate is only 11.2% in the leaf age of 60d, and only 1.3 adventitious buds are generated on average in each explant. We speculate that the difference in the regeneration capability of leaves of different leaf ages may be due in part to the difference in the regeneration capability of leaves of different leaf ages due to the difference in their physiological states in their internal secondary metabolite content, production or transport of phytohormones, or responses to phytohormones.
TABLE 2 influence of leaf age on the induction of adventitious buds of Qi Yuan leaves of Polygonatum sibiricum
| Age of leaf (d) | Adventitious bud induction ratio (%) | Adventitious bud number/explant |
| 5 | 37.8 | 3.6 |
| 10 | 62.5 | 5.1 |
| 15 | 93.4 | 9.4 |
| 30 | 32.6 | 2.7 |
| 60 | 11.2 | 1.3 |
Example 4
Influence of different kinds and concentrations of plant growth regulators and culture medium types on induction of adventitious buds of Qi Yuan rhizoma Polygonati leaf
According to the method of example 1, the leaf of 15 days old leaf is used as explant, sterilized and inoculated into different types of minimal medium supplemented with different kinds and concentrations of plant growth regulating substances for adventitious bud induction culture in a thermostatic culture chamber. The details are shown in Table 3.
Research results show that the species, concentration and culture medium type of the plant growth regulating substances play a vital role in the induction process of the adventitious bud of Qi Yuan polygonatum. Wherein, under the same concentration, the adventitious bud induction effect of TDZ is better than that of 6-BA, and further research finds that TDZ and AgNO 3 The combination of (2) can further improve the induction efficiency of the adventitious buds and the number of the adventitious buds. The induction effect was best with DKW as the minimal medium among the 3 cultures and types tested. Therefore, the optimal induction medium for Qi Yuan adventitious buds of rhizoma Polygonati leaf is added with 0.5mg/L TDZ and 2.0mg/LAgNO 3 The DKW medium of (1).
TABLE 3 influence of culture Medium type and plant growth regulating substances on the induction of adventitious buds of Qi Yuan leaves of Polygonatum sibiricum
Example 5
In order to further improve the proliferation efficiency and the height of the adventitious buds of Qi Yuan polygonatum sibiricum leaves, TDZ, IAA and GA are tested 3 Effect of concentration on adventitious bud proliferation and elongation.
The adventitious bud clumps obtained in example 1 were transferred to a medium supplemented with different concentrations of TDZ, IAA and GA 3 The adventitious bud was grown and elongated under the same conditions as in example 1.
Research results show that the concentration of 3 plant growth regulators in the culture medium has important influence on the proliferation and elongation of adventitious buds, the use of low-concentration TDZ is favorable for the proliferation of the adventitious buds, and GA in the culture medium 3 The use of (3) can promote the elongation of adventitious buds, and the addition of IAA at a low concentration in the medium can increase the number of adventitious buds. Low concentration TDZ in combination with Low concentration IAA and GA 3 While increasing the number of adventitious buds and increasing the length of the adventitious buds, 0.2mg/L TDZ,0.05mg/L IAA and 1.0mg/L GA are added into the culture medium 3 The adventitious bud proliferation and elongation effects were best, with an average of 30.1 adventitious buds per explant, and the average height of adventitious buds was 2.8cm.
TABLE 4 influence of plant growth regulator species and concentrations on adventitious bud proliferation and elongation
Example 6
Influence of variety, concentration and treatment time of adventitious root inducer on formation of adventitious root of Qi Yuan rhizoma Polygonati tissue culture elongation bud
Only the species and concentration of the rooting inducer in the rooting promoting liquid are changed, qi Yuan rhizoma polygonati tissue culture extended buds with uniform growth and plant height of 2-3cm are used as materials, the base parts of the tissue culture extended buds which are washed by running water and absorb water are treated for 60s by the rooting promoting liquid added with the rooting inducer of different species and concentrations (the steps are the same as the step 1). After 3 weeks of culture, the adventitious root induction rate of the Qi Yuan rhizoma polygonati tissue culture extended bud is counted.
The results in Table 5 show that the variety and concentration of the adventitious root inducing liquid play a key role in inducing the tissue culture elongation bud adventitious root of Qi Yuan rhizoma polygonati, and the tested 3 plant growth regulators can improve the induction rate of Qi Yuan rhizoma polygonati adventitious roots by being used alone, and a certain synergistic promotion effect exists between the two. The induction effect of the adventitious roots of the rooting promoting liquid added with 750mg/L K-IBA, 500mM melatonin and 200mg/L IBA is optimal, and the induction rate of the adventitious roots is as high as 97.4%.
TABLE 5 influence of variety and concentration of adventitious root inducing solution on the induction of adventitious root in Qi Yuan rhizoma Polygonati tissue culture
Secondly, only the treatment time of Qi Yuan elongated sealwort buds in the rooting promoting liquid is changed, qi Yuan elongated sealwort buds which are 2-3cm long and have uniform and robust growth vigor are used as explants, and after the elongated buds are separated from adventitious bud clusters and washed by running water and surface sterilized, the treatment time is different from that of the rooting promoting liquid involved in the embodiment 1 (15s, 30s,45s,60s,90s and 120 s). The rest of the procedure was the same as in example 1. After 3 weeks of culture, the adventitious root induction rate of the Qi Yuan rhizoma polygonati tissue culture extended bud is counted. The results in Table 6 show that the treatment time of the adventitious root inducing solution plays an important role in inducing the adventitious roots of Qi Yuan rhizoma Polygonati. Within a certain treatment time, the induction rate of the adventitious roots is gradually increased along with the increase of the treatment time, and the induction rate of the adventitious roots is up to 97.4% when the adventitious roots are treated for 60 s. The induction rate of adventitious roots tended to decrease with the increase in treatment time. Specifically, the results are shown in Table 6.
TABLE 6 influence of adventitious root inducing solution treatment time on the induction of adventitious roots in Qi Yuan rhizoma Polygonati tissue culture elongation buds
| Treatment of | Treatment time(s) | Adventitious root induction ratio (%) |
| 1 | 15 | 31.6 |
| 2 | 30 | 70.4 |
| 3 | 45 | 81.7 |
| 4 | 60 | 97.4 |
| 5 | 90 | 91.1 |
| 6 | 120 | 83.6 |
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for regenerating and in-vitro seedling formation of a new variety of polygonatum cyrtonema is characterized by comprising the following steps: the new variety of polygonatum cyrtonema is Qi Yuan polygonatum cyrtonema, and the method comprises the following steps: taking young leaves of the germinated seedlings of the Qi Yuan rhizoma polygonati tubers as explants, sterilizing the surfaces of the young leaves, shearing the young leaves into blocks with proper sizes, and finally obtaining Qi Yuan rhizoma polygonati complete regeneration plants through adventitious bud induction, adventitious bud proliferation and elongation, adventitious root induction treatment outside a bottle, field planting and seedling management;
the leaf age of the young and tender leaves is 13-17 days;
the culture medium adopted for inducing adventitious buds contains 0.5-2.0mg/L TDZ and 0.5-2.5mg/LAgNO 3 DKW medium of 3.0% sucrose and agar;
the culture medium for proliferation and elongation of adventitious bud contains 0.05-1.0mg/L TDZ, 0.05-0.25mg/L IAA, and 0.5-2.5mg/L GA 3 DKW medium of 3.0% sucrose and agar;
the culture medium adopted by the adventitious root induction treatment outside the bottle is a rooting promoting liquid containing 500-1000mg/L K-IBA, 100-500mM melatonin and 200-500mg/L NAA.
2. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema according to claim 1, which is characterized in that: the surface disinfection comprises the following steps: sealing the explant in a container, washing under tap water, placing in a sterile operating station, washing with sterile water, sterilizing with absolute ethanol, washing with sterile water, and adding 0.1% of HgCl 2 Sterilizing the solution for 2-3min, washing with sterile water, and drying the surface water of the seeds with sterile filter paper.
3. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema according to claim 1, which is characterized in that: the cutting into pieces with proper size refers to cutting the blade into pieces with 0.25-1.0cm after the blade tip is removed 2 Size, ready for use.
4. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema according to claim 1, which is characterized in that: the adventitious bud proliferation and elongation means that the elongated Qi Yuan polygonatum adventitious buds cultured for 4 weeks by illumination are inoculated to a culture medium adopted by the adventitious bud proliferation and elongation.
5. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema as claimed in claim 1, which is characterized in that: the adventitious root induction treatment outside the bottle is to separate the elongated adventitious buds of the polygonatum sibiricum from the adventitious bud cluster after the illumination culture for 4 weeks, soak the morphological lower ends of the elongated buds in a culture medium adopted by the adventitious root induction treatment outside the bottle to carry out adventitious root induction treatment after the adventitious buds are washed by running water and the surface moisture is absorbed, wherein the induction treatment time is 30-120s.
6. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema as claimed in claim 1, which is characterized in that: the permanent planting is to fixedly plant the morphological lower end of the Qi Yuan polygonatum tissue culture extended bud after the adventitious root induction treatment in a nutrient medium disc filled with a nutrient medium for adventitious root induction; the nutrient medium comprises nutrient soil, perlite and vermiculite, wherein the mass ratio of the nutrient soil to the perlite to the vermiculite is 2.
7. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema according to claim 1, which is characterized in that: and the seedling management is to spray the Qi Yuan rhizoma polygonati tissue culture extended buds after field planting with a nutrient solution once, cover the buds, place the buds in a greenhouse with the temperature of 20-23 ℃ and the relative humidity of 85-90% for seedling culture, pour water for 1 time every 5-7 days, and remove a plastic cover after 1 month.
8. The method for regenerating and in vitro seedling of new variety of polygonatum cyrtonema according to claim 7, wherein: the nutrient solution comprises 30-50% of Hoagland nutrient solution prepared by double distilled water.
9. The method for regenerating and in vitro seedling of a new variety of polygonatum cyrtonema according to claim 1, which is characterized in that: the culture medium adopted for inducing the adventitious bud contains 0.5mg/L TDZ and 2.0mg/LAgNO 3 The DKW medium of (1), wherein the medium used for the proliferation and elongation of adventitious buds is a medium containing 0.2mg/L TDZ,0.05mg/L IAA and 1.0mg/L GA 3 The DKW medium of (1).
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