CN111133903B - Alfalfa water culture cutting propagation method - Google Patents
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- 238000005520 cutting process Methods 0.000 title claims abstract description 90
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims description 35
- 238000000034 method Methods 0.000 title claims description 27
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- 238000012258 culturing Methods 0.000 claims description 3
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/10—Vegetative propagation by means of cuttings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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Abstract
本发明公开了一种紫花苜蓿水培扦插扩繁技术，包括扦插营养液配制，扦插苗的制备，水培扦插，扦插后管理，定植等步骤，其特征在于，其中，扦插苗的制备方法为:以水培28‑32天的紫花苜蓿为扦插苗，选取茎基直径为1.6～1.8mm的第一节茎节为插穗；扦插后管理方法为：水培扦插的第一周光照强度为180‑220μmol/m2·s，光照12h(25℃)/黑暗12h(20℃)，相对湿度70‑80％。一周后，光照强度变280‑320μmol/m2·s，光照16h(25℃)/黑暗8h(20℃)，相对湿度70‑80％，每6‑8天更换同样配方的营养液至培养24‑26天。
The invention discloses an alfalfa hydroponic cutting propagation technology, which includes the steps of cutting nutrient solution preparation, cutting seedling preparation, hydroponic cutting, post-cutting management, field planting and the like, wherein, the cutting seedling preparation method is as follows : take the alfalfa of hydroponic 28-32 days as the cutting seedling, choose the stem base diameter and be that the first stalk node of 1.6～1.8mm is the cutting; the management method after the cutting is: the first week light intensity of the hydroponic cutting is 180 ‑220μmol/m 2 ·s, 12h light (25℃)/12h dark (20℃), relative humidity 70‑80%. One week later, the light intensity changed to 280-320 μmol/m2·s, the light was 16h (25°C)/8h (20°C) dark, and the relative humidity was 70-80%. The nutrient solution of the same formula was replaced every 6-8 days until the cultivation period was 24- 26 days.
Description
Technical Field
The invention relates to the technical field of alfalfa asexual propagation, in particular to an alfalfa water culture cutting propagation method.
Background
The alfalfa is a herbaceous plant of the genus Medicago of the family Leguminosae, and is extremely low in selfing maturing rate and high in seed propagation difficulty due to the fact that the alfalfa belongs to strict cross-pollinated plants, so that the excellent quality of a single plant is maintained, the later generation of the plant is accelerated to propagate, a plant line with specific characters is formed, and the plant line is used for the subsequent comprehensive variety and the single plant breeding process. Meanwhile, after the male sterile line is found, the asexual cuttage has great significance in the three-line propagation and later-stage breeding work of the alfalfa. In addition, because the alfalfa is the isofloral tetraploid, and the genotypes of different plants of the same variety are different, the molecular correlation research interference is large after the seeds germinate and grow seedlings, and the cuttage propagation can generate plants with the consistent genotypes, so that materials with the consistent genotypes are provided for the molecular research of genome, transcriptome, proteome and the like.
At present, the common cutting method at home and abroad is mainly based on field cutting and indoor seedling tray cutting under the condition of soil culture. Although the survival rate of partial germplasm cuttage can reach 85%, field cuttage is relatively inconvenient to manage; indoor seedling tray cuttage has convenient management and labour saving and time saving's advantage relatively field cuttage, nevertheless all has common shortcoming with field cuttage: the growth consistency of the cuttage clone is poor, and the cuttage clone is particularly used for molecular breeding research and has large interference on results. Therefore, a cuttage method which is more convenient and efficient and can generate clones with high consistency needs to be invented, the research of alfalfa molecular biology can be met, and the conventional propagation and conservation of alfalfa can also be met.
The water culture cuttage uses water or nutrient solution as a medium to replace common media, has the advantages of rapidness, convenience, cleanness, no pollution, low carbon, environmental protection, easy management, low cost and the like, can accurately control the nutrient content of plants, is favorable for knowing the growth condition of the plants, is slightly limited by natural conditions, can realize a large amount of seedling culture in multiple seasons, and simultaneously has the whole process of conveniently observing the growth of root systems. However, the alfalfa water culture cutting system has not been reported all the time due to factors such as poor rooting property and the like.
Disclosure of Invention
The invention aims to provide a method for obtaining a large amount of water culture cutting seedlings of alfalfa, which has higher original rooting rate and can be used for solving the defect of the water culture cutting method of the alfalfa. The invention provides the alfalfa water culture cutting propagation technology for the first time, and provides an important propagation technology for conventional breeding and molecular level research of alfalfa. Particularly, the bottleneck that the alfalfa is difficult to be used for molecular level researches such as genome, transcriptome, proteome and the like due to large growth difference of soil culture cutting propagation can be solved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a water planting cutting propagation method of alfalfa comprises the steps of preparing a cutting nutrient solution, preparing cutting seedlings, performing water planting cutting, managing after cutting, planting and the like.
The preparation method of the cutting seedling comprises the steps of taking alfalfa which is cultured in water for 28-32 days as the cutting seedling, and selecting a first stem node with the stem base diameter of 1.6-1.8 mm as a cutting shoot;
the management method after cuttage comprises the following steps: the illumination intensity of the first week of the water culture cutting is 180-220 mu mol/m2 & s for 12h (25 ℃) in light/12 h in darkness (20 ℃), and the relative humidity is 70-80%. Changing the nutrient solution after one week, changing the illumination intensity to be 280-320 mu mol/m2 & s, changing the relative humidity to be 70-80% at the illumination intensity of 16h (25 ℃)/darkness of 8h (20 ℃), and changing the nutrient solution with the same formula every 6-8 days until the culture lasts 24-26 days; wherein the nutrient solution is prepared by using a modified Hoagland solution and/or a HB-101 natural plant vigor solution.
Preferably, the alfalfa cultured in water for 28-32 days is used as a cutting seedling and cultured in the improved Hoagland solution for 30 days.
The cutting method comprises the following steps: pouring 750mL of prepared cutting nutrient solution into a culture container, vertically and flatly cutting an upper cut of the selected cutting shoot and a branch, obliquely cutting a lower cut at an angle of 45 degrees with the branch, keeping a blade with 1 leaf bud and keeping the length of the cutting shoot at 6-8 cm, and rolling the cutting shoot with a sea surface (the length is 5-6 cm, and the width is 1.8-2.0 cm) with the thickness of 1cm to ensure that the leaf bud is exposed outside and is not wrapped by sponge. Then, it was inserted into a small hole of a PVC plate, and then placed in a culture vessel.
The water culture cutting method comprises the following steps: and (3) taking the improved 1/2Hoagland solution as a water culture base solution, adding 1/1000 HB-101 natural plant vigor solution, transferring the mixture into the improved Hoagland solution after rooting, culturing for one week, and planting.
Wherein, the water planting cuttage uses a plastic pot as a culture container, the outer side of the pot is covered by a black adhesive tape to simulate the dark growth condition of the plant root system, and can prevent the growth of green algae in the nutrient solution, and the culture container is provided with 10-20 round hole PVC plates with the diameter of 1-1.5cm on the thickness of 0.4-0.6 cm.
Wherein, the planting method comprises the following steps: after rooting for 25 days, the rooted alfalfa water culture seedlings are strengthened in the improved Hoagland solution for one week and then can be used for subsequent tests or planted in seedling raising pots.
The invention further provides a detailed method for facilitating the operation.
(1) Preparing cutting seedlings: the alfalfa seeds are cultured in water culture 7 days after germination acceleration in an incubator, and the culture conditions are as follows: light illumination for 16h (25 deg.C)/dark illumination for 8h (20 deg.C), and light intensity of 300 μmol/m2S, relative humidity 75%, change culture broth every 7 days until 30 days of culture. The improved Hoagland solution is adopted in the water culture, and the formula and the preparation method are as follows:
note: when the nutrient solution is prepared, each medicine is dissolved separately and then mixed, and the prepared mother solution is stored in a brown bottle and stored in the dark. When preparing the nutrient solution working solution, A, B mother solution is respectively diluted by 200 times, C solution is diluted by 1000 times, D solution is diluted by 200 times of iron salt, and the improved Hoagland nutrient solution is obtained after mixing.
(2) The design of cuttage device: a white plastic pot is used as a culture container, the outer side of the pot is covered by a black adhesive tape to simulate the dark growth condition of a plant root system, and the growth of green algae in the nutrient solution can be prevented. Cutting a PVC plate with the thickness of 0.5cm according to the size of the culture pot, uniformly punching into 15 round holes, and sterilizing for later use.
(3) Preparing a cutting nutrient solution: the cutting nutrient solution takes improved 1/2Hoagland as a base solution, and 1/1000 of natural plant vigor solution is added into the base solution for storage at room temperature for later use. Wherein, the formula of the Hoagland liquid is the same as that of the Hoagland liquid, and the natural plant vigor liquid is purchased from Taobao electronic commerce, such as Japanese HB-101 brand.
1/2 Hoagland solution, the formula and preparation method are as follows:
note: when the nutrient solution is prepared, each medicine is dissolved separately and then mixed, and the prepared mother solution is stored in a brown bottle and stored in the dark. When preparing the nutrient solution working solution, A, B mother solution is diluted by 400 times, C solution is diluted by 2000 times, and D solution is diluted by 400 times of iron salt. After mixing, a modified 1/2 Hoagland solution was obtained.
(4) Selecting cutting slips: using the alfalfa cultured for 30 days in the step (1) for cuttage, and selecting a first stem node of a branch with a stem base diameter of 1.6-1.8 mm as a cutting shoot;
(5) cuttage: pouring 750mL of prepared cutting nutrient solution into a culture container, vertically and flatly cutting an upper cut of the selected cutting slip and a branch, obliquely cutting a lower cut and the branch at an angle of 45 degrees, rolling the cutting slip by using cut sea noodle, wherein the length of the cutting slip is 6-8 cm, leaves are not left, and the cutting slip is provided with 1 leaf bud, and ensuring that the leaf bud is exposed outside and is not wrapped by sponge. Then inserting the PVC plate into a small hole of a PVC plate, and then placing the PVC plate into a culture container;
(6) managing after cuttage: the first week illumination intensity of water culture is 200 mu mol/m2S, light 12h (25 ℃)/dark 12h (20 ℃), relative humidity 75%. The light intensity became 300. mu. mol/m after one week 2S, 16h (25 ℃) in light/8 h (20 ℃) in darkness, 75% relative humidity, replacing the nutrient solution of the same formulation every 7 days until 25 days of culture.
(7) Planting: after 7 days of culture, the alfalfa roots are sequentially rooted, after 25 days of rooting, the rooting rate reaches 98.3%, and the rooted alfalfa water culture seedlings can be used for a subsequent test after being strengthened in the nutrient solution in the step (1) for one week, or can be planted in a seedling raising pot.
The culture pot in the step (2) has the specification of 17.5cm long, 11.5cm wide and 5cm high, and the volume is 800 mL; the PVC plate is 17.5cm long, 11.5cm wide and 0.5cm thick, and the diameter of the round hole on the plate is 1.2 cm; the sponge in the step (5) is 1cm in thickness, 5-6 cm in length and 1.8-2.0 cm in width. The PVC plate and the surface of the culture pot are wiped by 75% alcohol, and the cut sponge strips are cleaned for 1-2 times by tap water and then cleaned for 1 time by distilled water. Placing the culture pot with the sterilized surface, the PVC plate and the washed sponge in a super clean bench for ultraviolet disinfection for 4 hours for later use.
When the nutrient solution in the step (1) is prepared, each medicine is dissolved separately and then mixed, and the prepared mother solution is stored in a brown bottle and stored in the dark. And (2) when the nutrient solution of the cutting seedlings is cultured in the step (1), A, B and D mother solution are respectively diluted by 200 times, and C solution is diluted by 1000 times. The cutting nutrient solution in the step (3) can be prepared by the mother solution prepared in the step (1), A, B and D mother solutions are respectively diluted by 400 times, C solution is diluted by 2000 times, and 1/1000 of HB-101 natural plant vigor solution is added.
Compared with the existing cuttage technology, the cuttage method has the advantages and beneficial effects that:
1. the invention relates to the water culture cutting propagation of alfalfa. Compared with field and indoor seedling tray cuttage, the method has the advantages of convenience in management, time saving and labor saving, and the growing consistency of the cuttage clone is good, so that the method can be particularly used for molecular breeding research.
2. The invention takes the culture pot as a culture container, adopts alcohol surface disinfection and ultraviolet disinfection, effectively avoids the phenomenon of mildewing in the water culture process caused by bacteria carried in the culture container, has small occupied space and low requirement on working space, and is suitable for wide popularization.
3. The invention uses the water planting seedling branch to perform cuttage, the cutting shoot can be easily obtained all the year round, and the method can perform cuttage propagation of asexual seedlings all the year round without being limited by seasons and air temperatures, thereby prolonging the effective cuttage time.
4. The nutrient solution prepared by the invention has balanced and reasonable nutrition, enables the cutting seedlings to grow robustly and consistently, has simple operation, simple and convenient management, economy, practicability and high efficiency, avoids plant diseases and insect pests, saves manpower consumed by management measures such as watering, weeding, spraying and the like required by the traditional soil cutting, and saves a large amount of cost; rooting starts after 7 days of cuttage, the rooting rate reaches more than 50% after 15 days, the rooting rate reaches 98.3% after 25 days of cuttage, the root system is strong and developed, the rooting rate is high and stable, and the seedling culture period is greatly shortened.
5. The method is closely combined with the actual production, has strong practicability and operability and low cost, can be widely applied to alfalfa breeding and research, can provide reference for cuttage propagation of other plants, and has wide application prospect and technical result transformation potential.
Drawings
FIG. 1 shows a culture pot, a PVC plate and a sponge strip used in the examples.
FIG. 2 is the overall view after cuttage in the example.
FIG. 3 is an overall difference chart of the cuttage of different stem node segments in the embodiment.
FIG. 4 is a graph showing the effect of cuttage on plant height and root length of different stem node segments in examples.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. The specific experimental conditions and methods not indicated in the following examples are generally conventional means well known to those skilled in the art.
Example 1:
choose the alfalfa variety without the kerchier to test, the seed carries out the water planting after 7 days of pregermination in the incubator and cultivates, and the culture condition is: light illumination for 16h (25 deg.C)/dark illumination for 8h (20 deg.C), and light intensity of 300 μmol/m2S, relative humidity 75%, changing modified Hoagland nutrient solution every 7 days until 25 days of culture, the nutrient solution formulation for culture is as follows:
Note: when the nutrient solution is prepared, each medicine is dissolved separately and then mixed, and the prepared mother solution is stored in a brown bottle and stored in the dark. When preparing the nutrient solution working solution, A, B mother solution is respectively diluted by 200 times, C solution is diluted by 1000 times for preparation, and D solution is diluted by 200 times of iron salt.
A white plastic pot (17.5 cm in length, 11.5cm in width and 5cm in height) is used as a culture container, and the outside of the pot is covered with a black adhesive tape to simulate the dark growth condition of the plant root system and prevent the growth of green algae in the nutrient solution. After a PVC plate with the thickness of 0.5cm is cut according to the size of a basin, 15 round holes (3 multiplied by 5) with the diameter of 1.2cm are uniformly punched. And cleaning the cut sponge strips (with the thickness of 1cm, the length of 5-6 cm and the width of 1.8-2.0 cm)) for 1-2 times by using tap water, and then cleaning for 1 time by using distilled water. The culture pot with the surface disinfected by 75% alcohol, the PVC plate and the washed sponge are placed in a super clean bench for ultraviolet disinfection for 4 hours for later use.
Preparing a cutting nutrient solution: the cutting nutrient solution takes improved 1/2 Hoagland as a base solution, is added with 1/1000 of the cutting nutrient solution of HB-101 natural plant vigor solution, and is stored at room temperature for later use.
Note: when preparing the nutrient solution, each medicine is dissolved separately and then mixed, the prepared mother solution is stored in a brown bottle and stored in the dark, and when preparing the nutrient solution working solution, A, B mother solution is diluted 400 times respectively, C solution is diluted 2000 times and D solution is diluted 400 times by using ferric salt.
Selection of cutting slips: and (3) performing water culture on the AMELLESCIS AMELLIS for 30 days for cuttage, selecting a robust branch with a stem base diameter of 1.6-1.8 mm, wherein the lower node is a first stem node, the middle node is a fifth stem node, and the upper node is the topmost end of the branch.
Cuttage: pouring 750mL of prepared cutting nutrient solution into a culture container, vertically and flatly cutting an upper cut of the selected cutting slips and a branch, obliquely cutting a lower cut of the selected cutting slips and the branch at an angle of 45 degrees, rolling the cutting slips by sea noodles, wherein the cutting slips are 6-8 cm long, leaves are not left, and the cutting slips have 1 leaf bud, and the leaf buds are exposed outside and are not wrapped by sponge. Then, it was inserted into a small hole of a PVC plate, and then placed in a culture vessel.
Managing after cuttage: the first week illumination intensity of water culture is 200 mu mol/m2S, light 12(25 ℃)/dark 12h (20 ℃), relative humidity 75%. One week later, the nutrient solution was replaced, the light intensity was changed to 300. mu. mol/m 2. s, the light intensity was changed to 16h (25 ℃)/dark for 8h (20 ℃), the relative humidity was changed to 75%, and the nutrient solution of the same formula was replaced every 7 days. After 14 days, the rooting rate of the upper, middle and lower sections can respectively reach 55%, 78.3% and 51.7%. After 25 days of culture, the rooting rate is not changed any more, the rooting rate of the upper section is 78.3%, the rooting rate of the middle section is 93.3%, and the rooting rate of the lower section can reach 98.3%.
Planting: and (3) after the rooted alfalfa water culture seedlings are strengthened in the nutrient solution in the step (1) for one week, the alfalfa water culture seedlings can be used for a subsequent test or planted in a seedling raising pot.
According to the method of the embodiment, after 25 days of water culture cutting, the rooting rate is observed and counted, wherein the rooting rate is (the number of rooting single plants/the total number of the group of cuttings) multiplied by 100 percent, and the specific results are shown in table 1.
Table 1 shows the rooting rate and growth index obtained by carrying out the water culture cutting seedling culture of alfalfa for 25 days according to the method of the examples 1-6.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it will be obvious to those skilled in the art that modifications may be made in the technical solutions described in the above embodiments, or equivalents may be substituted for some of the technical features. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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| CN117981590A (en) * | 2024-03-13 | 2024-05-07 | 青岛农业大学 | A kind of semi-wet cutting propagation method of alfalfa |
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