CN117502246B - Construction method of lotus rapid propagation system - Google Patents
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- 235000006510 Nelumbo pentapetala Nutrition 0.000 title claims abstract description 60
- 238000010276 construction Methods 0.000 title claims abstract description 7
- 230000001954 sterilising effect Effects 0.000 claims abstract description 14
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims description 23
- 238000005286 illumination Methods 0.000 claims description 19
- 238000004659 sterilization and disinfection Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 15
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- 229940088597 hormone Drugs 0.000 claims description 15
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- 238000000034 method Methods 0.000 claims description 11
- 230000035755 proliferation Effects 0.000 claims description 11
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- 239000008272 agar Substances 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 5
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- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 7
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- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
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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
-
- 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
-
- 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
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Abstract
The invention discloses a construction method of a lotus rapid propagation system, which comprises the following specific steps: sterilizing the surface of mature embryo, culturing primary seedling, propagating, inducing adventitious bud, propagating, culturing, domesticating and transplanting. The invention takes the mature lotus seeds as explants, has the advantages of convenient material taking, low pollution rate, rapid stem propagation and good propagation effect, can be used for rapid propagation production and germplasm resource preservation, and establishes a foundation for the tissue culture technology of the lotus.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a construction method of a lotus rapid propagation system.
Background
Lotus flowerNelumbo nucifera Gaertn.)For years, nelumbo of Nelumceae the raw water is used for growing flowers, is one of ten traditional famous flowers in China, has high ornamental value, economic value and unique propertyHas special cultural connotation and is deeply favored by people.
The lotus takes the asexual propagation material, namely the enlarged underground stem (lotus root), as the seedling, the traditional seedling production mode is not easy to manage, diseases and insect pests are easy to transfer, phenomena such as variety mixing and degradation (Li Maonian and the like, 2011) occur, the tissue culture rapid propagation can solve the problems of most traditional seedlings, but in the current report of lotus tissue culture rapid propagation, when the stem tip is taken as the material, the consumable is large and the sterilization is difficult, when the embryo is taken as the material, the method is limited by the green lotus seed, and the method for inducing the callus to differentiate the tissue culture seedling is less successful, so that the current situations can not meet the demands of lotus scientific research breeding, popularization and application and the like. The plant tissue culture is an important application technology, the rapid propagation, the transgenic research and the breeding all need the help of tissue culture (Liu Yujia, 2020), the plant tissue culture has the characteristics of rapid mass propagation, short period, high propagation coefficient, low cost and the like, and the establishment of a more simple and convenient tissue culture system is an effective means for expanding the sterile seedling of the lotus. The growth and development of the explant can be accurately regulated by adding plant growth regulators, particularly a combination of auxin and cytokinin.
Disclosure of Invention
The invention aims to provide a high-efficiency construction method of a lotus rapid propagation system, which provides an important technical foundation for the tissue culture technology of lotus.
The aim of the invention can be achieved by the following technical scheme:
a method for constructing a lotus rapid propagation system, which comprises the following steps:
(1) And (3) sterilization: selecting round plump lotus seeds with shells for disinfection and sterilization;
(2) Culturing primary aseptic seedlings: taking out the lotus plumule, soaking the lotus plumule in sterile water, then inoculating into an MS blank culture medium without hormone for culture until the lotus plumule emerges to obtain a first-generation aseptic seedling, and selecting the first-generation seedling with transparent sterile growth of the culture medium when the first-generation aseptic seedling exists;
the taken lotus plumule is immersed in sterile water, or is easy to brown, and can not be used immediately after being sterilized and cooled.
(3) Propagation of stem walking: inoculating the first-generation sterile seedling to a stem-walking proliferation culture medium for culture until a sterile seedling stem with buds is obtained;
(4) The stem-walking stem segment induces adventitious buds: cutting the stem of the aseptic seedling obtained in the step (3) into a plurality of stem-moving sections containing 2-3 growing points, cleaning the stems and leaves of the stem-moving sections as much as possible in order to avoid producing endophytes from dead branches and abortive leaves, inoculating the stem-moving sections to a subculture medium for culture until adventitious buds and leaves are induced in the growing points of the stem-moving sections, and obtaining the seedling;
(5) Subculture: selecting 3 or more strong adventitious bud seedlings without deformity from the small Miao Zhongshai obtained in the step (4), transferring and inoculating to a new subculture medium for proliferation culture until the seedlings grow out of roots, and taking out the seedlings for acclimatizing the tissue culture seedlings to obtain the tissue culture seedlings;
(6) Hardening and transplanting of tissue culture seedlings: and (5) selecting healthy and strong seedlings from the tissue culture Miao Zhongshai obtained in the step (5) for hardening and transplanting.
Part of the leaves die in the seedling hardening process, but new leaves grow out along with the hardening; in a specific embodiment, the transplanting is to use a 10cm×10cm nutrition pot to hold pond soil, clean the culture medium attached to the tissue culture seedling, plant the tissue culture seedling into the pot, and place the tissue culture seedling into a greenhouse for growth, and perform conventional water and fertilizer management.
Further, the recipe of the stem multiplication medium in the step (3) is MS+6-BA 0.5mg/L+ IBA 1.5 mg/L+ NAA 0.5 mg/L+GA 1 mg/L+sucrose 50g/L, agar 8 g/L, pH=5.6.
Further, the formula of the secondary culture medium in the step (4) and the step (5) is MS+6-BA 1.5 mg/L+IBA 1.2 mg/L+NAA 0.6 mg/L, the concentration of sucrose is 40-60 g/L, agar is 8 g/L, and pH=5.6.
Further, the sterilization in the step (1) is sterilization by using 75% alcohol and 3% sodium hypochlorite; preferably, the sterilization in the step (1) is carried out by sterilizing with 75% alcohol for 30s, washing with sterile water for 1 time, soaking with 3% sodium hypochlorite for 8 min, and washing with sterile water for 3 times for later use.
Further, the lotus plumule in the step (2) is soaked in sterile water for 6-8 hours; the culture time is 15-20 days in MS blank medium without hormone.
Further, the culture condition in the step (2) is that the illumination time is 14-16 hours per day, the illumination intensity is controlled to be 1500-2500Lx, and the temperature is controlled to be 25-28 ℃.
Further, the culture condition in the step (3) is that the illumination time is 14-16 hours per day, the illumination intensity is controlled to be 1500-2500Lx, and the temperature is controlled to be 25-28 ℃; preferably, fresh medium is changed every 30 days; preferably, the cultivation time in the step (3) is 2 months, and a large amount of underground stem with buds is obtained.
Further, the culture condition in the step (4) is that the illumination time is 14-16 hours per day, and the illumination intensity is 1500-2500 Lx; preferably, the culturing time in the step (4) is 1 month, and a large number of adventitious buds and leaves are induced from the growing points of the stem-removing segments after 1 month.
Further, the proliferation culture time in the step (5) is 1 month, and after the seedlings grow out of roots after 1 month, the seedlings are taken out for hardening and domestication of the tissue culture seedlings.
Further, the seedling hardening condition in the step (6) is that the culture bottle cap is firstly loosened and opened under the illumination condition of the illumination intensity of 1600-2400 lx at the temperature of 23 ℃ for 14-16 hours, then sterile water is added for culture for 24-36 hours, and then the culture bottle cap is opened for culture for 48 hours at the room temperature under the natural illumination condition.
The invention has the beneficial effects that:
the experiment is further improved, the simplest disinfection step is obtained, the operation is simple, the death rate is extremely low, the pollution rate (except for endophytes, fresh and polluted) is greatly reduced, the browning rate is extremely low, and the survival rate of the primary tissue culture seedlings can reach 100%.
The invention adopts the mature lotus embryo to cultivate aseptic seedlings, and the utilization rate of the lotus mature seeds is greatly improved and the survival rate is greatly increased by improving the disinfection method and properly treating the lotus plumule, so that the lotus tissue culture material is not limited by fresh lotus seeds.
The specific hormone proportion test is used for analyzing the induction relation between the proportions of different auxins and cytokinins and bud types, and the hormone proportion for inducing different bud types is screened out, so that a new idea is provided for the lotus tissue culture test.
Drawings
FIG. 1 different types of adventitious buds induced by sterile seedlings of lotus.
FIG. 2 effects of sucrose at different concentrations on growth of tissue culture seedlings.
FIG. 3 explant growth due to different subcultures.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
Examples
The construction method of the lotus rapid propagation system comprises the following steps:
sterilizing and breaking the shell:
test one: improvement of lotus mature seed disinfection method:
round plump lotus seeds with shells are selected, sterilized by 75% alcohol and 3% sodium hypochlorite for standby, then the shells of the lotus seeds are stripped, and lotus plumule is taken out and inoculated on a culture medium to grow into complete plants.
The results of the different disinfection treatment methods adopted in the test of this section are shown in table 1, and the test finds that except that the links of taking out the lotus plumule from the embryo and inoculating must be operated in the ultra-clean bench, the steps of early stage breaking, soaking and peeling off the seed shell can be carried out outside the ultra-clean bench, and the explant will not be polluted, and the operation is also suitable for other mature seeds of different varieties.
TABLE 1 results of different disinfection treatments
(II) culturing primary sterile seedlings:
through preliminary experiments, the situation that the plumula Nelumbinis is browned and then dies gradually is found to be the most easily caused by that seeds do not draw enough water, but endophyte outbreaks are easy to occur after the seeds are soaked for too long; the abnormal growth of lotus plumule occurs when the lotus plumule is inoculated into hormone-containing culture medium.
Therefore, firstly, mature seeds of Guangchang white lotus are treated according to the optimal disinfection step of the previous section, then lotus plumule is taken out, the lotus plumule is inoculated into an MS culture medium after being soaked differently to improve the survival rate of the lotus plumule, and the browning rate, the endophyte growth condition and the survival rate of the lotus plumule after 7d are counted to obtain the optimal time for soaking the lotus plumule; and (3) taking out lotus plumule after optimal treatment of seeds of Guangchang white lotus, inoculating the lotus plumule to different culture mediums, and counting the deformity rate and the survival rate after 7 days.
Table 2 lotus plumule soaking time treatment
Experimental results show that as the time for immersing the plumula Nelumbinis in sterile water is prolonged, the browning rate of the plumula Nelumbinis is gradually reduced, the survival rate is gradually increased, and in the longest immersing treatment period of 14 hours, the browning rate is 0, and the survival rate is 100%; however, with the extension of the soaking treatment time, endophytes of the explants are gradually appeared, endophytes begin to appear after soaking for 12 hours, the situation of the endophytes is greatly increased from 8.33% to 41.67% after soaking for 14 hours, and although the endophytes can not influence the growth situation of tissue culture seedlings in a short period, the endophytes can grow seriously more and more along with the growth of the date, so that the situation of growth inhibition, plant distortion and even death of the tissue culture seedlings can be caused, and the explosion of the endophytes should be avoided in the early inoculation operation. Therefore, the optimal soaking time of the lotus plumule should be 8 hours.
The lotus plumule is inoculated into a culture medium containing hormone to cause growth deformity, the lotus plumule does not die after the growth deformity but is seriously inhibited from growing, seedlings cannot grow, the lotus plumule is easy to grow deformity after the hormone is initially contained in the culture medium, and the deformity rate gradually increases along with the increase of the hormone concentration. Therefore, the culture medium inoculated with the plumula Nelumbinis is preferably MS culture medium containing no phytohormone.
(III) hormone combination screening for induction of adventitious buds:
and (2) testing II: in this experiment, four-factor three-level orthogonal tests were performed with 6-BA (6-benzylaminopterin) concentrations of 0.5, 1 and 1.5mg/L, IBA (indolebutyric acid) concentrations of 0.5, 1 and 1.5mg/L, NAA (α -naphthylacetic acid) concentrations of 0, 0.1 and 0.5mg/L, and GA (gibberellin) concentrations of 0, 0.5 and 1mg/L, as shown in Table 3. The lotus plumule of Guangchang white lotus is inoculated with sterile tissue culture seedlings growing for 15 days, and the seedlings are inoculated into a culture medium containing different hormone formulas and a blank control MS culture medium, and each group of seedlings is inoculated with 3 seedlings, and the test is repeated for 3 times. And (3) after 45 days, specifically counting the growth indexes such as the stem walking type, the stem walking node number, the length, the leaf number and the like.
TABLE 3 design table of orthogonal experiments on propagation medium
The buds of lotus are in the general categories of terminal buds, axillary buds, leaf buds and flower buds, wherein the terminal buds (stem) and the axillary buds (lateral buds) are growing points of asexual propagation. The results of shoots induced by different hormone formulations designed in the orthogonal test are different, as shown in FIG. 1, and the types of shoots induced by different proportion hormones and the types of shoots naturally grown in the blank MS culture medium are shown in Table 4.
TABLE 4 types of shoots induced by different hormone ratios
The results of the different hormone combinations are shown in Table 4, and after 45d of culture, the number of stem nodes and the length of the stem are the highest, the number of the stem nodes is 6.4, the length of the stem is 11.8cm, and the significant difference exists between other treatments; secondly, for treatment group 9, the number of stem nodes is 4.7, the length of the stem is 3.0cm, and the significant difference is found between the treatment group and other treatments; the number and length of the stem-walking nodes in the treatment group 6 are obviously different from those in the treatment group 9, and the growth vigor of the treatment group 6 is better than that of the treatment group 9, so that the optimal culture medium for inducing the stem-walking growth is the treatment group 6, and the formula of the culture medium is MS+0. mg/L6-BA+1.5 mg/LIBA+0.5 mg/LNAA+1 mg/LGA.
(IV) stem propagation of primary seedlings:
and (3) test III: to screen the sucrose concentration in the stock multiplication medium, 15d healthy and strong 'Guangchang white lotus' seedlings are selected and inoculated to the stock multiplication medium containing different concentrations of sucrose: MS+0.5 mg/L6-BA+1.5 mg/L IBA+0.5mg/L NAA+1mg/L GA, agar 8 g/L, pH=5.6, different sucrose concentrations are shown in Table 5, 3 seedlings were inoculated per group and the test was repeated 3 times. And after 30 days, the growth indexes such as the number, the length, the number of leaves and the like of the stem nodes are counted, and the influence of the concentration of sucrose on the proliferation of the stem nodes is explored.
As can be seen from table 5, in the statistics of proliferation factor and stalk length, when sucrose concentration was 50g/L, proliferation factor and stalk length were both highest, proliferation factor was 11.67, stalk average length was 6.90cm, and there was a significant difference from other treatment groups; in the flourishing extent of the leaves, the sucrose concentration of 50g/L is also the highest level, the average number of leaves is 24.00, and the concentration is significantly different from other concentrations. Thus, the optimal stock removal propagation medium formulation was MS+0.5 mg/L6-BA+1.5 mg/L IBA+0.5mg/L NAA+1mg/L GA, agar 8 g/L, pH=5.6, and the optimal sucrose concentration was 50g/L. The effect of sucrose at different concentrations on the growth of tissue culture seedlings is shown in figure 2.
TABLE 5 Effect of different sucrose concentrations on proliferation and growth of primary seedlings
And (fifth) subculture:
sterile material with 3 growth points of explants after 30d of culture in the previous section was inoculated into different subculture media, 2 strains were inoculated per 1 bottle, 3 bottles were inoculated per treatment, and the test was repeated 3 times. After 30d, counting growth indexes such as stem walking increasing length, leaf increasing amount, adventitious root increasing number, adventitious root increasing length, plant height increasing amount and the like, and selecting an optimal subculture medium, wherein the optimal subculture medium theoretically is as follows: MS+6-BA 1.5 mg/L+IBA 1.2 mg/L+NAA 0.6 mg/L, agar 8 g/L, pH=5.6, sucrose concentration 40-60 g/L. The effect of different subculture media on the growth of explants is shown in figure 3.
Acclimatization and transplanting of the tissue culture seedlings:
selecting healthy and strong seedlings from tissue culture Miao Zhongshai subjected to subculture for 30 days for hardening, opening a culture bottle cap and adding sterile water for culturing for 24 hours under the illumination condition of illumination intensity of 1600-2400 lx under the illumination condition of 16 hours at 23 ℃, and then opening the cover for culturing for 48 hours under the conditions of room temperature and natural illumination. The nutrient bowl with the length of 10cm multiplied by 10cm is used for containing pond soil, a culture medium attached to tissue culture seedlings is cleaned, the tissue culture seedlings are planted in the bowl, and the tissue culture seedlings are placed in a greenhouse for growth, so that conventional water and fertilizer management is performed. After 30d of domestication and transplanting, the final survival rate of the tissue culture seedlings can reach 61.67 percent.
TABLE 6
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. The construction method of the lotus rapid propagation system is characterized by comprising the following steps of:
(1) And (3) sterilization: selecting plump lotus seeds with shells for disinfection and sterilization for later use;
(2) Culturing primary aseptic seedlings: taking out the lotus plumule, soaking the lotus plumule in sterile water, and then inoculating into an MS blank culture medium without hormone for culture until the lotus plumule emerges, so as to obtain primary sterile seedlings;
(3) Propagation of stem walking: inoculating the primary sterile seedling obtained in the step (2) to a stem-moving proliferation culture medium for culture until a sterile seedling stem with buds is obtained;
(4) The stem-walking stem segment induces adventitious buds: cutting the stem of the aseptic seedling obtained in the step (3) into a plurality of stem segments containing 2-3 growing points, inoculating the cut stem segments to a secondary culture medium for culture until adventitious buds and leaves are induced in the growing points of the stem segments, and obtaining a seedling;
(5) Subculture: selecting 3 or more strong adventitious bud seedlings without deformity from the small Miao Zhongshai obtained in the step (4), transferring and inoculating the seedlings to a new subculture medium for proliferation culture until the seedlings grow out of roots, and taking out the seedlings for acclimatizing the tissue culture seedlings to obtain the tissue culture seedlings;
(6) Hardening and transplanting of tissue culture seedlings: selecting healthy and strong seedlings from the tissue culture Miao Zhongshai obtained in the step (5) for hardening off and transplanting;
the sterilization in the step (1) is sterilization by 75% alcohol and 3% sodium hypochlorite;
the lotus plumule is soaked in sterile water for 6-8h; culturing in hormone-free MS blank culture medium for 15-20 days;
the culture condition in the step (2) is that the illumination time is 14-16 hours per day, the illumination intensity is controlled to be 1500-2500Lx, and the temperature is controlled to be 25-28 ℃;
the culture condition in the step (3) is that the illumination time is 14-16 hours per day, the illumination intensity is controlled to be 1500-2500Lx, and the temperature is controlled to be 25-28 ℃;
the formula of the stem multiplication medium in the step (3) is MS+6-BA 0.5 mg/L+IBA 1.5 mg/L+NAA 0.5 mg/L+GA 1 mg/L+sucrose 50g/L, agar 8 g/L, and pH=5.6;
the secondary culture medium in the step (4) and the step (5) is MS+6-BA 1.5 mg/L+IBA 1.2 mg/L+NAA 0.6 mg/L, the concentration of sucrose is 40-60 g/L, agar is 8 g/L, and pH=5.6;
the culture condition in the step (4) is that the illumination time is 14-16 hours per day, and the illumination intensity is 1500-2500 Lx;
and (6) under the lighting condition that the seedling hardening condition is that the temperature is 23 ℃ and the lighting is carried out for 14-16 hours, under the lighting condition that the lighting intensity is 1600-2400 lx, the culture bottle cap is firstly loosened and opened, and then sterile water is added for culturing for 24-36 hours, and then the culture is carried out under the conditions of room temperature and natural lighting for 48 hours.
2. The method for constructing a rapid propagation system of lotus according to claim 1, wherein the sterilization in the step (1) is to sterilize for 30s with 75% alcohol, rinse for 1 time with sterile water, soak for 8 min with 3% sodium hypochlorite, rinse for 3 times with sterile water, and reserve.
3. The method for constructing a rapid propagation system of lotus as claimed in claim 1, wherein the culturing time in the step (3) is 2 months.
4. The method for constructing a rapid propagation system of lotus as claimed in claim 1, wherein the culturing time in the step (4) is 1 month.
5. The method for constructing a rapid propagation system of lotus seeds according to claim 1, wherein the proliferation culture time in the step (5) is 1 month.
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