CN113207687A - Tissue culture and rapid propagation method for clematis - Google Patents
Tissue culture and rapid propagation method for clematis 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
- A01H4/001—Culture apparatus for tissue culture
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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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- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for quickly breeding Clematis by tissue culture, wherein Clematis (Clematis) is taken as a gardening ornamental plant and a traditional Chinese medicine and is popular with people all the time, because the Clematis has various varieties, the varieties are obviously different in breeding modes such as sowing, grafting, cuttage, tissue culture and the like, the Clematis selected in the experiment is introduced from Japan, the variety is 'primula', and relevant researches on the tissue culture are not reported. Therefore, the research uses clematis tender stems as test materials, and researches related aspects such as axillary bud induction, multiplication culture, rooting culture, transplantation and seedling hardening are carried out on the clematis tender stems by utilizing a tissue culture technology, so that a clematis tissue culture system with high efficiency, stability and good repeatability is established. The research result can provide practical guidance for the factory production and popularization of clematis.
Description
Technical Field
The invention belongs to the technical field of clematis propagation, and particularly relates to a clematis tissue culture rapid propagation method.
Background
Plant tissue culture (plant tissue culture), also called plant in vitro culture for short, refers to a technology of culturing in vitro plant organs (organ), tissues (tissue), cells (cell) and protoplasm (proplast) by using proper culture (medium) and giving proper culture conditions under the environment of asepsis (asepsis) and artificial control (manual control) to grow or differentiate regenerative cells into complete plants.
Plant tissue culture technology is a subject developed based on plant physiology in the beginning of the 20 th century, is a biological science theory formed by combining the gradual development of plant physiology, genetics, developmental science, botany, microorganisms and other subjects, and can be researched only if the structure, mechanism, development process and essence of cytogenetic materials of biological cells are clearly understood.
The theory was first proposed by the german famous plant chemist hublond (g.haberlandt) in 1902 that plant cells have "totipotency", which means that organs and tissues of higher plants can be continuously divided into single cells, and the single cells have potential totipotency, i.e., the plant cells have totipotency. Meanwhile, it is proposed that each somatic cell can be cultured in vitro to form a complete plant like an embryonic cell, and each cell of a plant body contains all the genetic information of the individual and has a complete set of genes for forming the complete plant, so that the growth and differentiation can be induced to form the complete plant under appropriate conditions. After that, scientists have done a lot of work. White in the united states succeeded in the in vitro culture of tomato root tissue until the 30's of the 20 th century, and the first clone, also known as "clone", was established, followed by the tissue culture of the cambium of tobacco stalks.
From the 30 s to the 50 s of the 20 th century, scientists have conducted intensive research on tissue culture and found two important links of hormone regulation and culture medium modes for tissue culture technology on the basis of the past. Kogl discovered the first phytohormone, indole-3-acetic-acid (IAA) in 1930, while White discovered the effects of B vitamins and IAA on the growth of plant roots ex vivo in 1937, establishing the first medium consisting of known compounds, White medium; british scholars f.c. steward and j.reinert test carrots for the first time to confirm the concept of Haberlandt on cell totipotency. Becomes an important node in the research history of plant tissue culture. Miller et al found that Kinetin (Kinetin) promoted the bud to produce effects nearly ten thousand times higher than adenine, so Kinetin replaced adenine, thereby establishing Kinetin and auxin ratio control organ differentiation mode.
After 60 years in the 20 world, through establishment and research of the prior theoretical basis, the tissue culture technology has established the relevant theoretical basis and technical basis, and gradually combines with practical work, and plays an important role in plant genetic improvement by combining with conventional breeding. And considerable benefit is obtained, so that the research of plant tissue culture enters a brand new stage. In 1962, Murashige and Shoog studied the components of the culture medium for promoting the rapid growth of tobacco tissue culture and published the MS culture medium which is most widely used at present, and the plant tissue culture technology has been developed into a wide field at present.
The development of the tissue culture technology is widely applied to the aspects of agriculture, forestry, medicine and the like, and remarkable social benefits are obtained. The potato, the strawberry and the like are detoxified by using a tissue culture technology, so that the growth can be promoted, the yield is increased, the occurrence of diseases can be effectively reduced by using a detoxification method for seedling propagation and planting, the growth vigor of plants is obviously enhanced, the growth vigor is regular, and the yield is increased; secondly, by utilizing the characteristics of short propagation period, high propagation rate and no influence of external environment of a tissue culture technology, a large number of seedlings can be produced in a short time, based on one stem, leaf or bud of a plant, tens of thousands or even hundreds of thousands of plants can be produced in one year by utilizing the tissue culture technology, and particularly, some seeds can not be propagated or certain ornamental flowers with specific characters are rapidly propagated, such as African daisy and the like; secondly, the tissue culture technology can also be used for inducing and breeding new varieties; secondly, some plants contain specific alkaloids, terpenoids, flavonoids, phenols and other alkaloids, and natural compounds can be synthesized and extracted by utilizing a cell culture technology; and the method can be used for storing the germplasm resources, the traditional resource storage method is high in cost and serious in planting resource loss, and by using the tissue culture technology, the cost can be reduced, the occupation of manpower and material resources can be reduced, and the germplasm resources can be stably stored for a long time.
With the improvement of living standard of people, the requirements for urban greening and living environment are gradually improved, but the clematis plants are famous ornamental plants in the world, are rich in cultivated varieties and are flowering varieties in spring, summer and autumn. Can meet the requirements of colors, seasons, ecological landscape and the like in garden gardening. At present, clematis is gradually loved by people as a new flower, but the variety and the number of the clematis provided in the market are limited. Therefore, the rapid propagation research of the clematis variety with strong ornamental value is carried out, so that a perfect rapid propagation system is established, and the important value significance is achieved for industrial seedling culture and commercial production.
Disclosure of Invention
In order to realize the purpose of tissue culture and rapid propagation of clematis, the invention is realized by the following technical scheme, and the tissue culture and rapid propagation method of clematis is characterized by comprising the following steps:
s1: seedling material: collecting 1-1.5m high plants, single petals, 3-4cm flower diameter, 5 months, and collecting the strong and disease-free young stems of the current year;
s2: pretreatment: washing the collected tender stem with distilled water to remove surface dirt, wiping the surface with a test tube brush, cutting off redundant stems and branches, cutting the clean and trimmed tender stem into stem segments with the length of 5cm, filling the stem segments into a sterile bottle, and putting the sterile bottle into a superclean workbench for convenient subsequent disinfection;
s3: explant disinfection: treating the stem segments treated in S2 with 75% ethanol solution for 0-5S, and washing with sterile water twice for 2min each time; then using HgCl2Soaking for 3-8min, and washing with sterile water for 2-3 times, each time for 2 min; cutting two ends and petioles of sterilized stemThe surplus part is kept at 1.5-2cm in length, and each stem segment is provided with two axillary buds;
s4: and (3) proliferation culture: taking a common plant culture medium as a basic culture medium, adding mitogen and auxin into the culture medium, horizontally placing stem segments on the culture medium, carrying out dark culture for 5 days, and then carrying out light culture for 30 days;
s5: rooting culture: selecting 1/2MS as a basic culture medium, and adding auxin and active carbon into the basic culture medium; transferring and inoculating the tender stem subjected to propagation culture in the S4 to an S5 culture medium for rooting culture for 30 d;
s6: transplanting and hardening seedlings: selecting the tissue culture seedlings which have rooted in the S5, putting the tissue culture seedlings in natural light for growing, and opening a bottle mouth by half after the seedlings are hardened in the bottle for 20 days so that the air convection inside and outside the bottle lasts for 3-5 days; then washing the culture medium attached to the roots with running water, disinfecting and soaking for 5min with carbendazim, and then transferring the disinfected tissue culture seedlings to a cool environment to dry the surface moisture;
s7: transplanting tissue culture seedlings: planting the tissue culture seedlings processed in the S6 into a soil matrix, putting the soil matrix into a greenhouse shed, and keeping the humidity at 75-85% and the temperature at 15-20 ℃;
preferably, the culture medium in S4 is selected from one of MS, WPM, White and B5; the mitogen is one of 6-BA and TDZ, wherein the concentration of 6-BA is 0.5-3mg/L, and the concentration of TDZ is 0.01-0.05 mg/L; the auxin is NAA, and the concentration is 0.00-0.20 mg/L;
preferably, the culture medium is MS; culturing with 6-BA and NAA at concentration of 2.0 mg/L6-BA and 0.05mg/L NAA; culturing with TDZ and NAA at concentration of TDZ 0.04mg/L and NAA 0.05 mg/L;
preferably, the auxin in the S5 is one or two of NAA and IBA, wherein the concentration of the NAA is 0.3-1.0mg/L, and the concentration of the IBA is 0.2-0.8 mg/L; the concentration of the Activated Carbon (AC) is 0.00-0.50 mg/L;
preferably, the auxin uses NAA + IBA, wherein the concentration of NAA is 0.3mg/L, the concentration of IBA is 0.5mg/L, and the concentration of Activated Carbon (AC) is 0.3 mg/L;
preferably, in S7, covering a shading net according to the sunshine condition, gradually opening the shed after 20 days for ventilation, and spraying nutrient solution once every 5 days;
preferably, the soil matrix in the S7 is one or more of laterite, humus and vermiculite which are matched in proportion;
preferably, the laterite, humus soil and vermiculite are matched in proportion as follows: 1:1 of laterite and humus soil, 1:1 of laterite and vermiculite, and 1:2:2 of laterite and humus soil and vermiculite.
The invention has the beneficial effects that:
the research takes clematis 'blume button' introduced from Japan as an experimental material, collects the current-year tender stem as an explant, combines different types of exogenous induction regulators and concentration ratios, and establishes a stable tissue culture rapid propagation system of the clematis 'blume button' by exploring the processes of axillary bud starting, proliferation culture, rooting culture, transplanting and seedling hardening and the like, thereby providing technical support for mass production of high-quality seedlings;
the technology of the invention is used for cultivating clematis tissue, the rooting rate is high, the root seedling is strong, and the survival rate after transplanting is high.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the tissue culture and propagation steps of clematis chinensis according to the present invention;
FIG. 2 is a schematic diagram of the tissue culture and propagation technique of clematis;
FIG. 3 is a graph of explant induction in MS medium;
FIG. 4 is a diagram of 6-BA and NAA proliferation cultures;
FIG. 5 is a drawing of a culture of TDZ and NAA proliferations;
FIG. 6 is a pictorial view of a tissue culture seedling at a high concentration of mitogen;
FIG. 7 is a schematic diagram of a callus of a basal part;
FIG. 8 is a rooted shoot cultured on 1/2MS medium.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
Example 1
The effect and effect experiments of different disinfection treatments on survival rate, pollution rate and death rate are as follows:
the experiment is divided into 9 treatment groups, the survival rate, the pollution rate and the death rate of clematis have obvious influence among different disinfection treatments, the pretreated stem segments are treated by 75 percent ethanol solution for 0 to 5 seconds and then washed twice by sterile water, and each time lasts for 2 min; then using HgCl2Soaking for 3-8min, and washing with sterile water for 2-3 times, each time for 2 min; cutting off the redundant parts at two ends and petiole of the sterilized stem segment, maintaining the length at 1.5-2cm, each stem segment has two axillary buds, performing normal process for culture, and counting the results in Table 1-1
TABLE 1-1 Effect analysis between different Sterilization treatments
As shown in table 1-1, the survival rate of treatment 5 was 73.84% at the highest in the survival rate, which was the highest group among 9 treatments, and the multiple comparison results showed that: treatment 5 differed significantly from the remaining groups; the survival rate of treatment 1 was 30.23%, which was the lowest group of 9 treatments, and multiple comparison results showed: treatment 1 differed significantly from the remaining groups; the contamination rate of treatment 1 was 62.65% at the highest, which was the highest among 9 treatments, in terms of contamination rate, and multiple comparisons showed that: treatment 9 differed significantly from the remaining groups; the contamination rate of treatment 6 was 5.51%, the contamination rate of treatment 7 was the lowest 6.67%, and the survival rate of treatment 9 was 7.85%, and multiple comparison results showed that: the differences between treatments 6, 7, and 9 were not significant; the pollution rate in 9 treatments is in a descending trend on the whole; from the mortality perspective, treatment 9 had a mortality of up to 40.75%, and multiple comparisons showed: the difference between the remaining groups was significant at 9 months of treatment, with treatment 2 having a mortality rate of 5.16%, the lowest of 9 treatments, treatment 1 having a mortality rate of 5.87%, and treatment 4 having a mortality rate of 4.84%, and multiple comparisons showing: the differences between these three treatments were not significant.
The survival rate is increased gradually and then decreased along with the increase of the ethanol disinfection time and the HgCl2 disinfection time; the pollution rate is gradually reduced; mortality rates are on a gradual rising trend, as can be seen: the control of the contamination rate is evident with increasing time, but for the survival rate, too long a time of sterilization damages the cell activity of the ex vivo tissue, so that the survival rate decreases gradually from after treatment 5 and the mortality rate increases gradually from after treatment 5.
In conclusion, the optimal scheme that the treatment 5 with the higher survival rate and the sterilization of the clematis explants are selected in combination with the survival rate, the pollution rate and the death rate, namely the treatment 5: soaking in 75% ethanol for 3s, and soaking in HgCl2 for 5 min.
From the range analysis of the different factor levels in tables 1-2, we can see that the range analysis in terms of survival rate indicates: the average value of the A factors (K1-K3) is changed from 134.49 to 162.92, and the effect on the survival rate is gradually increased with the increase of the level value of the A factors, while the average value of the B factors (K1-K3) is changed from 126.75 to 161.20, and the effect on the survival rate is gradually increased with the increase of the level value of the B factors.
Whereas very poor analysis in terms of contamination rate showed: the average value of the A factors (K1-K3) varied from 94.35 to 25.05, and it can be seen that the effect on the contamination rate was gradually reduced as the level of the A factors increased, and the average value of the B factors (K1-K3) varied from 121.06 to 29.82, and it can be seen that the effect on the contamination rate was gradually reduced as the level of the B factors increased.
The average value of the A factors (K1-K3) varied from 24.34 to 95.67 in terms of mortality, and it can be seen that the effect on mortality was gradually increased as the level of the A factors was increased, and the average value of the level of the B factors (K1-K3) varied from 38.39 to 64.63, and it can be seen that the effect on contamination rate was gradually increased as the level of the B factors was increased.
TABLE 1-2 range analysis of different factor levels
The experiment shows that different factors (A, B) have different degrees of influence on the survival contamination rate, survival rate and death rate of the clematis explants through range analysis of different factor levels from table 1 to table 3, and that the range value (R value) of the A factor is 15.28 and the range value (R value) of the B factor is 20.51 in the aspect of survival rate, which shows that the influence degree of the B factor on the survival rate is higher than that of the A factor; the difference value (R value) of the factor a was 23.1 and the difference value (R value) of the factor B was 30.41 in terms of the contamination rate, indicating that the factor B had a higher degree of influence than the factor a in terms of the contamination rate; in terms of mortality, the difference value (R value) of factor a was 23.78 and the difference value (R value) of factor B was 8.78, indicating that factor B had a higher degree of influence on the contamination rate than factor a.
Meanwhile, excellent treatment is obtained by analyzing the difference value of all factors, wherein the excellent treatment of the survival rate is A3B3 (5 s + HgCl28min), which shows that the disinfection can be effectively carried out when the disinfection time of the ethanol and HgCl2 is horizontal; the optimal treatment of the contamination rate is A3B3 (ethanol 5s + HgCl28min), which shows that the prolongation of the disinfection time of ethanol and HgCl2 is helpful for reducing the contamination rate, the contamination rate can be controlled within a reasonable value, and the optimal treatment of the mortality rate is A1B1 (ethanol 0s + HgCl23min), which shows that the damage to the tissue of the seed material can be reduced when the disinfection time of the combination of ethanol and HgCl2 is shorter; however, the disinfection of explants cannot be considered from a certain index alone, and all factors must be considered. In summary, the optimal disinfection treatment is considered from the three aspects of survival rate, pollution rate and death rate, and the final optimal treatment for explant disinfection is as follows: 75% ethanol 3s + 0.1% HgCl25 min.
1-3 range analysis of different factor levels
Example 2
The induction effect experiment of different culture media on axillary buds:
the test uses four culture media of MS, WPM, White and B5 which are commonly used as basic culture media, the tender stem after disinfection treatment is inoculated on four culture media added with 1.0 mg/L6-BA +0.1mg/L NAA, the length of the stem segment is kept about 1.5cm, each stem segment is provided with 2 axillary buds, the stem segment is horizontally placed on the culture media, dark culture is firstly carried out for 5d, then the stem segment is transferred to a light culture stage, 4 treatment groups are totally used in the experiment, 15 bottles are used in each treatment group, 2 bottles are inoculated in each bottle, and 3 times are repeated in each treatment group. After 30 days, the axillary bud induction rate of the explant is counted, and the average seedling height and the germination rate are counted and shown in the table 2-1
TABLE 2-1 explant Induction results in different matrices
From the experimental results shown in table 2-1, the germination rates, seedling heights and germination growth vigor of different culture media are obviously different, the germination rates of the four culture media are respectively 84.32%, 46.61%, 31.67% and 35.67%, and multiple comparison results show that: the differences between treatment 1 and treatment 2 were significant from each group, and the differences between treatment 3 and treatment 4 were not significant. From the perspective of seedling height, multiple comparison results show that: the height of the treated 1 seedling is 2.53cm, the height of the treated 2 seedling is 1.86cm, the difference between the treated 1 seedling and the treated 2 seedling is not significant, the height of the treated 3 seedling is 0.90cm, the height of the treated 4 seedling is 1.08cm, and the difference between the treated 3 seedling and the treated 4 seedling is significant. From the aspect of germination growth, the explant on the MS culture medium germinates faster, leaves relax better and leaves are bright green; the sprouts on the WPM and B5 culture media are short and fine, and the leaf tips or stem sections are light yellow; the sprouting stem nodes on the White medium are short, the color loss rate of the leaves and the growth potential difference. In summary, the following steps: and selecting an MS culture medium as an optimal culture medium for tissue culture of clematis.
The test is carried out by variance analysis of different culture mediums on the induction effect of the explant, and the results in the table 2-2 show that: the different culture mediums have obvious influence on survival rate (P is 0.003< 0.05); the different culture substrates differed significantly in seedling height (P ═ 0.043< 0.05).
TABLE 2-2 analysis of variance of explant induction on different culture substrates
Example 3
Experiment for influence of 6-BA + NAA on clematis proliferation culture
Respectively adding 6-BA mitogen and NAA auxin with different concentrations into a culture medium, flatly placing stem segments on the culture medium, carrying out dark culture for 5 days, and then carrying out light culture for 30 days; the experiment has 9 treatment groups, each treatment group has 2 bottles, each bottle is inoculated with 4 plants, each group is repeated for 3 times, and after 30 days, the statistics of proliferation coefficient and seedling growth vigor are shown in a table 3-1
TABLE 3-16-Effect of treatment with NAA on the multiplication culture of clematis
As seen from Table 3-1, different concentrations of 6-BA and NAA in combination have a significant effect on the proliferation culture of clematis, and multiple comparison results show that: the proliferation factors for treatments 1-5 ranged from 0.64 to 0.74, and multiple comparisons showed that: the differences between the five treatments were not significant, the proliferation coefficients of treatments 6-10 ranged from 1.67 to 1.65, and multiple comparisons showed: the same differences between the five treatments were not significant; the proliferation factors of treatments 11-15 ranged from 3.06-3.74, and multiple comparisons showed that: the same differences between the five treatments were not significant; the proliferation factors of treatments 16-20 ranged from 4.13 to 4.14, and multiple comparisons showed: the same differences between the five treatments were not significant; the proliferation coefficients of treatments 21-25 ranged from 3.95 to 3.36, and multiple comparisons showed: the same differences between the five treatments were not significant; this indicates that as the 6-BA concentration increased between treatments, it appeared that the differences between treatments at the same 6-BA concentration level were not significant; meanwhile, in the 25 treatments, the proliferation coefficient shows a trend of increasing firstly and then decreasing, wherein the proliferation coefficient of the treatment 17 is 4.28 at most, and meanwhile, when the NAA concentration is 0.0mg/L, the seedlings of the treatments 1, 6, 11, 16 and 21 are not callus, but grow slowly, and the seedling height is short; the seedling height also grows gradually with the increase of the NAA concentration, but the excessive callus can affect the normal growth of seedlings with the formation of green callus accompanied by the excessive NAA concentration, and the NAA concentration is maintained at a reasonable level when being 0.05mg/L through experiments.
And (3) selecting treatment 17 as an optimal multiplication culture scheme by integrating the multiplication coefficient and the growth vigor of seedlings, namely: MS +2.0 mg/L6-BA +0.05mg/L NAA.
Example 4
Experiment for influence of TDZ + NAA on clematis proliferation culture
Adding TDZ mitogen and NAA auxin with different concentrations into a culture medium respectively, flatly placing stem segments on the culture medium, performing dark culture for 5 days, and then transferring to light culture for 30 days; the experiment has 9 treatment groups, each treatment group has 2 bottles, each bottle is inoculated with 4 plants, each group is repeated for 3 times, and after 30 days, the statistics of proliferation coefficient and seedling growth vigor are shown in table 4-1
TABLE 4-1 Effect of TDZ and NAA treatment on Clematis Caulifera proliferation culture
In combination with Table 4-1, the proliferation factor for treatment 17 was up to 4.84, and multiple comparisons showed: the differences among 10 treatments 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 are not significant; the proliferation factor for treatment 1 was a minimum of 1.65, and multiple comparisons showed: the differences among the 5 treatments 1, 2, 3, 4 and 5 are not obvious, and the five treatments 1, 6, 11, 16 and 21 have no callus formation in the growth, which may be the reason that the NAA concentration is 0.0mg/L, and the calluses of the other treatments are obviously increased along with the increase of the NAA concentration, thus showing that the NAA has obvious influence on the callus formation; from the growth, the yellow flowers appear on the leaves of the seedlings, and the leaves and the leaf tips are dry. It can be seen from the table that the effect of TDZ on clematis proliferation culture is obvious, with the increase of TDZ concentration, the proliferation coefficient is gradually increased from 1.65 to 4.84 of treatment 17, and then is gradually reduced to 3.31, it can be seen that the too high TDZ will shift to the effect of proliferation culture, when the TDZ concentration is above 0.05mg/L, the seedling will have vitrification, which shows that the stem is transparent and bright, and yellows, which affects the proliferation effect in later period.
And (3) selecting treatment 17 as an optimal multiplication culture scheme by integrating the multiplication coefficient and the growth vigor of seedlings, namely: MS + TDZ 0.04mg/L +0.05mg/L NAA.
Example 5
Experiment for comparing influence of different growth factors on root culture
Selecting 1/2MS as a minimal medium, and adding auxin and Activated Carbon (AC) into the minimal medium; transferring and inoculating the tender stem subjected to propagation culture in the S4 to an S5 culture medium for rooting culture for 30 d; the auxin is one or two of NAA and IBA, wherein the concentration of NAA is 0.3-1.0mg/L, and the concentration of IBA is 0.2-0.8 mg/L; the concentration of the Activated Carbon (AC) is 0.00-0.50 mg/L; the experiment had 9 treatment groups, each treatment group had 2 bottles, each bottle was inoculated with 4 plants, each group was repeated 3 times, and the rooting rate and rooting condition were counted after 30 days as shown in Table 5-1
TABLE 5-1 Effect of different auxins on rooting
As can be seen from Table 5-1, the rooting rate of treatment 4 was at most 39.24%, and multiple comparisons showed: treatment 4 differed significantly from the rest of the treatments; when the IBA concentration is 0.8mg/L, the rooting rates of the treatment 3, the treatment 7, the treatment 8 and the treatment 9 are all 0.00 percent, and it can be seen that the IBA and NAA proportion can not promote rooting at the level, the rooting effect of clematis is low as a whole from the aspect of rooting, wherein the callus does not appear on the root of the treatment 5 and the treatment 6, which is unfavorable for the transplanting and seedling hardening in the later period;
in conclusion, treatment group 4 was selected as the best protocol for rooting, 0.5mg/L IBA and 0.3mg/L NAA were added to 1/2MS medium, and the rooting rate was 39.24%.
Example 6
Contrast experiment of different culture mediums on transplanting and seedling hardening effects
Selecting rooted tissue culture seedlings, putting the rooted tissue culture seedlings in natural light for growing, and after hardening seedlings in a bottle for 20 days, opening a bottle mouth by half to enable air convection inside and outside the bottle to be continued for 3-5 days; then washing the culture medium attached to the roots with running water, disinfecting and soaking for 5min with carbendazim, and then transferring the disinfected tissue culture seedlings to a cool environment to dry the surface moisture; planting the treated tissue culture seedling into a soil matrix, placing the matrix into a greenhouse shed, and keeping the humidity at 75-85% and the temperature at 15-20 ℃; covering a shading net according to the sunshine condition, gradually opening the shed for ventilation after 20 days, and spraying nutrient solution every 5 days; 6 groups of different culture mediums were designed in total in the experiment, and the survival rate and growth after 30 days were counted as shown in Table 6-1
TABLE 6-1 comparison of the effects of transplantation in different substrates
As seen from Table 6-1, the multiple comparison results show: the survival rates of different transplanting matrixes are remarkably different, the highest survival rate of the treatment 6 is 58.12%, the survival rate of the treatment 2 is 12.35%, the survival rate of the treatment 3 is 6.54%, and multiple comparison results show that: the difference between the two is not significant; the survival rate of the treatments 1, 4 and 5 is 0.00%, and multiple comparison results show that: the difference between the three is not obvious. In summary, the most 6 transplanting seedling hardening substrates should be selected and processed, namely laterite: humus soil: the vermiculite is 1:2:2, and the survival rate is 58.12%.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (9)
1. A clematis tissue culture rapid propagation method is characterized by comprising the following steps:
s1: seedling material: collecting 1-1.5m high plants, single petals, 3-4cm flower diameter, 5 months, and collecting the strong and disease-free young stems of the current year;
s2: pretreatment: washing the collected tender stem with distilled water to remove surface dirt, wiping the surface with a test tube brush, cutting off redundant stems and branches, cutting the clean and trimmed tender stem into stem segments with the length of 5cm, filling the stem segments into a sterile bottle, and putting the sterile bottle into a superclean workbench for convenient subsequent disinfection;
s3: explant disinfection: treating the stem segments treated in S2 with 75% ethanol solution for 0-5S, and washing with sterile water twice for 2min each time; then using HgCl2Soaking for 3-8min, and washing with sterile water for 2-3 times, each time for 2 min; cutting off the redundant parts at two ends and petioles of the sterilized stem segment, keeping the length at 1.5-2cm, and providing two axillary buds for each stem segment;
s4: and (3) proliferation culture: taking a common plant culture medium as a basic culture medium, adding mitogen and auxin into the culture medium, horizontally placing stem segments on the culture medium, carrying out dark culture for 5 days, and then carrying out light culture for 30 days;
s5: rooting culture: selecting 1/2MS as a basic culture medium, and adding auxin and active carbon into the basic culture medium; transferring and inoculating the tender stem subjected to propagation culture in the S4 to an S5 culture medium for rooting culture for 30 d;
s6: transplanting and hardening seedlings: selecting the tissue culture seedlings which have rooted in the S5, putting the tissue culture seedlings in natural light for growing, and opening a bottle mouth by half after the seedlings are hardened in the bottle for 20 days so that the air convection inside and outside the bottle lasts for 3-5 days; then washing the culture medium attached to the roots with running water, disinfecting and soaking for 5min with carbendazim, and then transferring the disinfected tissue culture seedlings to a cool environment to dry the surface moisture;
s7: transplanting tissue culture seedlings: planting the tissue culture seedling treated in the S6 in a soil matrix, placing the soil matrix in a greenhouse shed, and keeping the humidity at 75-85% and the temperature at 15-20 ℃.
2. The method for tissue culture and rapid propagation of clematis chinensis according to claim 1, wherein the culture medium in S4 is selected from one of MS, WPM, White and B5; the mitogen is one of 6-BA and TDZ, wherein the concentration of 6-BA is 0.5-3mg/L, and the concentration of TDZ is 0.01-0.05 mg/L; the auxin is NAA, and the concentration is 0.00-0.20 mg/L.
3. The method for tissue culture and rapid propagation of clematis chinensis according to claim 2, wherein the culture medium is MS; culturing with 6-BA and NAA at concentration of 2.0 mg/L6-BA and 0.05mg/L NAA; the culture was carried out using TDZ and NAA at concentrations of TDZ 0.04mg/L and NAA 0.05 mg/L.
4. The method for tissue culture and rapid propagation of clematis chinensis according to claim 1, wherein the auxin in S5 is one or two of NAA and IBA, wherein the concentration of NAA is 0.3-1.0mg/L, and the concentration of IBA is 0.2-0.8 mg/L; the concentration of the Activated Carbon (AC) is 0.00-0.50 mg/L.
5. The method for tissue culture and rapid propagation of clematis chinensis according to claim 4, wherein the auxin is NAA + IBA, wherein the concentration of NAA is 0.3mg/L, the concentration of IBA is 0.5mg/L, and the concentration of Activated Carbon (AC) is 0.3 mg/L.
6. The method for tissue culture and rapid propagation of clematis chinensis according to claim 1, wherein in S7, a shading net is covered according to sunshine conditions, a shed is opened gradually after 20 days for ventilation, and nutrient solution is sprayed every 5 days.
7. The method for tissue culture and rapid propagation of clematis chinensis according to claim 1, wherein the soil matrix in the S7 is one or more of laterite, humus and vermiculite in proportion.
8. The method for tissue culture and rapid propagation of clematis chinensis according to claim 7, wherein the laterite, humus soil and vermiculite are matched in proportion as follows: 1:1 of laterite and humus soil, 1:1 of laterite and vermiculite, and 1:2:2 of laterite and humus soil and vermiculite.
9. The clematis tissue culture rapid propagation method according to any one of claims 1 to 8, which discloses application in the technical field of clematis propagation.
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