CN116548307A - Leaf-induction-based alfalfa regeneration method and culture medium thereof - Google Patents
Leaf-induction-based alfalfa regeneration method and culture medium thereof Download PDFInfo
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- CN116548307A CN116548307A CN202310538350.2A CN202310538350A CN116548307A CN 116548307 A CN116548307 A CN 116548307A CN 202310538350 A CN202310538350 A CN 202310538350A CN 116548307 A CN116548307 A CN 116548307A
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- 241000219823 Medicago Species 0.000 title claims abstract description 98
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 89
- 239000001963 growth medium Substances 0.000 title claims abstract description 46
- 238000011069 regeneration method Methods 0.000 title claims abstract description 6
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 49
- 230000006698 induction Effects 0.000 claims abstract description 33
- 229920001817 Agar Polymers 0.000 claims abstract description 8
- 229930006000 Sucrose Natural products 0.000 claims abstract description 8
- 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 abstract description 8
- 239000008272 agar Substances 0.000 claims abstract description 8
- JPMIIZHYYWMHDT-UHFFFAOYSA-N octhilinone Chemical compound CCCCCCCCN1SC=CC1=O JPMIIZHYYWMHDT-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000005720 sucrose Substances 0.000 claims abstract description 8
- 230000008929 regeneration Effects 0.000 claims abstract description 4
- 230000012010 growth Effects 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 11
- 238000002791 soaking Methods 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- 230000028446 budding cell bud growth Effects 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 2
- 229930003451 Vitamin B1 Natural products 0.000 claims description 2
- 229960000367 inositol Drugs 0.000 claims description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 235000010374 vitamin B1 Nutrition 0.000 claims description 2
- 239000011691 vitamin B1 Substances 0.000 claims description 2
- 235000019158 vitamin B6 Nutrition 0.000 claims description 2
- 239000011726 vitamin B6 Substances 0.000 claims description 2
- 229940011671 vitamin b6 Drugs 0.000 claims description 2
- 230000001902 propagating effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 6
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 230000001476 alcoholic effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940064880 inositol 100 mg Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
Classifications
-
- 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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture medium combination for alfalfa regeneration, which comprises an alfalfa callus induction culture medium and an alfalfa rooting and sprouting culture medium, wherein the alfalfa callus induction culture medium comprises 0.8-1.2mg/L IAA, 5-9g/L agar, 25-35g/L sucrose and 0.12-0.16g/L kathon, the PH is 5.8+/-0.5, the alfalfa rooting and sprouting culture medium comprises 0.08-0.15mg/L ZT, 0.03-0.10mg/L IBA, 5-9g/L agar, 25-35g/L sucrose and 0.12-0.16g/L kathon, and the PH is 5.8+/-0.5. Through the culture medium combination, the leaf explants of the alfalfa can be cultured into alfalfa plants for propagating the alfalfa plants.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and relates to a method for regenerating alfalfa based on leaf induction and a culture medium thereof.
Background
Alfalfa (Medicago) is the most important leguminous forage with wide planting area, has wide adaptability, is rich in nutrients such as protein, mineral substances, vitamins and the like, has high yield, and is known as the 'forage king'.
The development of the alfalfa industry in China is limited by germplasm resources, and in order to meet the requirements of rapid development of the animal husbandry, the expansion of planting areas and the cultivation and propagation of different alfalfa varieties become very important.
The tissue culture of alfalfa can rapidly expand and propagate alfalfa germplasm, is important for alfalfa breeding, and is necessary to explore an efficient alfalfa tissue culture method.
Disclosure of Invention
In order to solve the problems existing in the prior art, a first aspect of the present invention provides a culture medium combination for regeneration of alfalfa, wherein the culture medium combination comprises an alfalfa callus induction culture medium and an alfalfa rooting sprouting culture medium;
the alfalfa callus induction culture medium is based on a WPM culture medium, and comprises 0.8-1.2mg/L IAA;
the alfalfa rooting and sprouting culture medium takes a WPM culture medium as a basic culture medium, and comprises 0.08-0.15mg/L ZT and 0.03-0.10mg/L IBA.
In some embodiments, the alfalfa callus induction medium includes 0.8-1.2mg/L IAA, 5-9g/L agar, 25-35g/L sucrose, and 0.12-0.16g/L kathon, with a pH of 5.8+ -0.5.
In some embodiments, the alfalfa rooting and sprouting medium comprises 0.08-0.15mg/L ZT, 0.03-0.10mg/L IBA, 5-9g/L agar, 25-35g/L sucrose and 0.12-0.16g/L kathon, and has a pH of 5.8+ -0.5.
In some embodiments, the alfalfa callus induction medium includes 1.0mg/L IAA.
In some embodiments, the alfalfa rooting and sprouting medium includes 0.1mg/L ZT and 0.08mg/L IBA.
In some embodiments, the WPM medium comprises: 400mg/L NH 4 NO 3 、386mg/L Ca(NO 3 ) 2 、990mg/L K 2 SO 4 、72.5mg/L CaCl 2 、170mg/L KH 2 PO 4 、0.25mg/L Na 2 MoO 4 ·2H 2 O、180.7mg/L MgSO 4 、22.3mg/L MnSO 4 、8.6mg/L ZnSO 4 ·7H 2 O、27.85mg/L Fe 2 (SO 4 ) 3 ·7H 2 O、0.25mg/L CuSO 4 ·5H 2 O、37.3mg/L Na 2 EDTA, 100mg/L inositol, 0.1mg/L vitamin B1, 0.5mg/L niacin, 0.5mg/L vitamin B6 and 2.0mg/L glycine.
In a second aspect, the present invention provides a method for regenerating alfalfa, the method comprising the steps of:
s1: callus induction
Inoculating the alfalfa explant into an alfalfa callus induction culture medium for induction culture to form alfalfa callus;
the alfalfa callus induction medium is the alfalfa callus induction medium in the culture medium combination according to the first aspect of the invention;
s2: rooting and bud growth induction
Cutting the alfalfa callus into callus groups, inoculating the callus groups into an alfalfa rooting and bud growing culture medium for rooting and bud growing culture to form alfalfa plants with buds and roots;
the alfalfa rooting and sprouting culture medium is the alfalfa rooting and sprouting culture medium in the culture medium combination according to the first aspect of the invention.
In some embodiments, in step S1, the induction culture is a dark culture at a temperature of 18-25℃for a period of 2-3 weeks.
In some embodiments, in step S2, the rooting and sprouting culture is performed at a culture temperature of 20-25 ℃, a light-dark period of (14-18)/(6-10) h/d, an illumination intensity of 1500-3500lx, and a culture time of 35-50 days.
In some embodiments, the alfalfa explant is an alfalfa leaf.
In some embodiments, the alfalfa leaf is an alfalfa leaf obtained by hydroponic alfalfa shoots.
In some embodiments, the alfalfa leaves are sterilized prior to the callus induction.
In some embodiments, the sterilization treatment comprises 3-5 times of sterile water washing, alcohol solution soaking, 3-5 times of sterile water washing, sodium hypochlorite solution soaking, and 3-5 times of sterile water washing in sequence.
In some embodiments, the alcoholic solution has a volume concentration of 70-80% and the alcoholic solution is soaked for a period of 25-35s.
In some embodiments, the sodium hypochlorite solution has a mass concentration of 0.8-1.5% and a time period of 4-6min for soaking.
In some embodiments, the method further comprises the steps of:
s3: hardening off seedlings
And hardening and culturing the alfalfa plants with buds and roots.
In some embodiments, the conditions of the acclimatization culture are: hardening off the seedlings under 1500-3500lx light for 5-12 days, wherein the light and dark period is (14-18)/(6-10) h/d, and the culture temperature is 25+/-3 ℃.
In some embodiments, the method further comprises the steps of:
s4: transplanting
And taking the alfalfa seedlings after seedling hardening out from the bottles, and transplanting the alfalfa seedlings into soil.
In some embodiments, the alfalfa seedling growth environment within 10-20 days after transplanting is: the temperature is 20-25deg.C, and the humidity is 80+ -5%.
Drawings
FIG. 1 is a photograph showing the condition of the alfalfa callus culture of the present invention.
FIG. 2 is a photograph showing the rooting and bud growing condition of alfalfa according to the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The steps not described in detail in the present invention are all routine in the art, and the materials not described in detail are all routine in the art.
The alfalfa used in the invention is alfalfa, and the specific varieties are as follows: SY4D.
Example 1: preparation of culture Medium
A formulation for a set of alfalfa tissue culture systems comprising a culture medium of the following formulation.
(1) Basal medium
WPM medium, specifically comprising inorganic salts: NH (NH) 4 NO 3 400mg/L、Ca(NO 3 ) 2 386mg/L、K 2 SO 4 990mg/L、CaCl 2 72.5 mg/L、KH 2 PO 4 170mg/L、Na 2 MoO 4 ·2H 2 O 0.25mg/L、MgSO 4 180.7 mg/L、MnSO 4 22.3mg/L、ZnSO 4 ·7H 2 O 8.6mg/L、Fe 2 (SO 4 ) 3 ·7H 2 O 27.85mg/L、CuSO 4 ·5H 2 O 0.25mg/L、Na 2 EDTA 37.3mg/L, organics: inositol 100mg/L, vitamin B1.1 mg/L, nicotinic acid 0.5mg/L, vitamin B6.5 mg/L, and glycine 2.0mg/L.
(2) Alfalfa leaf induced callus culture medium
The basic culture medium is WPM culture medium, and the formula also comprises 7g/L agar, 30g/L sucrose and 0.14g/L kathon, and the pH is 5.8+/-0.1. The hormone in the medium was IAA, and the content of the hormone was shown in Table 1.
(3) Alfalfa rooting and bud growing culture medium
The basic culture medium is WPM culture medium, and the formula also comprises 7g/L agar, 30g/L sucrose and 0.14g/L kathon, and the pH is 5.8+/-0.1. The hormones in the medium were ZT and IBA, and the content of the hormones is shown in Table 2, respectively.
Example 2: optimization of alfalfa tissue culture conditions
Callus induction culture
Collecting alfalfa twigs collected in 5 months, removing old leaves, and washing with tap water for 24h.
Soaking the washed alfalfa twigs in tap water at room temperature for 2d at 20-25deg.C, performing water culture, and changing water every day until new twigs grow.
Sterilizing in an ultra-clean bench. Placing the newly-developed tender branches after preliminary cleaning in a beaker in an ultra-clean bench, washing for 5 times with sterile water, sterilizing for 30s with 75% alcohol by volume fraction, washing for 5 times with sterile water, soaking for 5min with 1.0% sodium hypochlorite aqueous solution by mass fraction, continuously stirring during the period, washing for 5 times with sterile water, cutting leaves (including petioles) on the branches into small pieces with about (0.4X0.4) cm, and inoculating to 6 callus induction culture mediums shown in table 1 respectively, wherein the number of bottles of each culture medium is 10. Callus was differentiated from leaf edges by culturing in a dark room at 22.+ -. 2 ℃ for 2-3 weeks, one bottle of photographs is shown in FIG. 1, and the callus induction rate is shown in Table 1.
From this, the method of the present invention can effectively induce alfalfa explants to produce callus.
TABLE 1 alfalfa leaf induced callus culture medium and culture result statistics
| Numbering device | IAA(mg/L) | Callus induction rate (%) | Callus growth status |
| 1 | 0.2 | 21.6 | ++ |
| 2 | 0.5 | 22.3 | ++ |
| 3 | 0.8 | 52.6 | ++ |
| 4 | 1.0 | 78.3 | ++++ |
| 5 | 1.2 | 32.6 | ++ |
| 6 | 1.5 | 18.8 | + |
Note that: "+" indicates the status of callus growth.
The "++ + + +" is best in growth state, short in healing time, fast in growth, large in callus, granular, loose in structure and light yellow;
the "++ + +" indicates that the callus growth state is good, the callus growth time is short, the callus growth is fast, and the particle shape is light yellow;
"++" indicates that the callus is in a general growth state, and has slow growth and a blocky yellow green color;
"+" indicates that the callus has poor growth state, long healing time, slow growth, massive structure, compact structure, hard texture and yellow-green color.
(II) rooting and bud formation induction
The callus with good growth condition in the step (II) is placed in a sterile ultra-clean workbench, a sterile scalpel is used for properly cutting the callus with the growth width of (0.5 multiplied by 0.5) cm, the callus is respectively transferred into 8 rooting and bud growth induction culture mediums shown in the table 2, 5 to 15 callus are cultured for 42 days under the conditions that the culture temperature is 22+/-2 ℃ and the light and dark period is 16/8h/d and the illumination intensity is 1800-3000lx, buds are differentiated, simultaneously rooting is carried out, the bud growth rate and the seedling growth condition after bud growth are recorded, the number of each culture medium is 10, one bottle of the photographs is shown in the table 2, and the bud growth rate is shown in the table 2.
Therefore, the method can effectively induce alfalfa calli to root and bud.
TABLE 2 alfalfa rooting and sprouting culture medium and culture result
Hardening off seedlings
The alfalfa plants with buds and roots are subjected to seedling hardening culture, a culture bottle is opened, clear water with the thickness of 0.5cm is filled into the culture bottle, the culture bottle is replaced every day, a bottle cap is gradually opened, the alfalfa plants are subjected to seedling hardening under 2000-3000lx of illumination for 7-10 days, the light-dark period is 16/8h/d (namely 16h of illumination every day and 8h of dark culture), and the culture temperature is 25+/-1 ℃.
(IV) transplanting
Taking out the alfalfa seedling after 7-10 days of seedling hardening from the bottle, washing the culture medium with clear water, transplanting the alfalfa seedling into the mixed soil of turfy soil and vermiculite, removing bottom end leaves, retaining top buds, and covering a transparent plastic cover for moisturizing. And (5) keeping the temperature in the cover at 21-23 ℃ and the humidity at 80+/-5% within 15d after transplanting, and removing the cover after transplanting for 15d to obtain the alfalfa seedlings.
It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.
Claims (10)
1. A combination of media for alfalfa regeneration, the combination of media comprising alfalfa callus induction media and alfalfa rooting and sprouting media;
the alfalfa callus induction culture medium is based on a WPM culture medium, and comprises 0.8-1.2mg/L IAA;
the alfalfa rooting and sprouting culture medium takes a WPM culture medium as a basic culture medium, and comprises 0.08-0.15mg/L ZT and 0.03-0.10mg/L IBA.
2. The combination of media of claim 1, wherein the alfalfa callus induction media comprises 0.8-1.2mg/L IAA, 5-9g/L agar, 25-35g/L sucrose, and 0.12-0.16g/L kathon, PH 5.8±0.5; and/or
The alfalfa rooting and sprouting culture medium comprises 0.08-0.15mg/L ZT, 0.03-0.10mg/L IBA, 5-9g/L agar, 25-35g/L sucrose and 0.12-0.16g/L kathon, and the PH is 5.8+/-0.5.
3. The combination of media of claim 1, wherein the alfalfa callus induction media comprises 1.0mg/L IAA; and/or
The alfalfa rooting and sprouting culture medium comprises 0.1mg/L ZT and 0.08mg/L IBA.
4. The combination of media of claim 1, wherein the WPM media comprises: 400mg/L NH 4 NO 3 、386mg/L Ca(NO 3 ) 2 、990mg/L K 2 SO 4 、72.5mg/L CaCl 2 、170mg/L KH 2 PO 4 、0.25mg/L Na 2 MoO 4 ·2H 2 O、180.7mg/L MgSO 4 、22.3mg/L MnSO 4 、8.6mg/L ZnSO 4 ·7H 2 O、27.85mg/L Fe 2 (SO 4 ) 3 ·7H 2 O、0.25mg/L CuSO 4 ·5H 2 O、37.3mg/L Na 2 EDTA, 100mg/L inositol, 0.1mg/L vitamin B1, 0.5mg/L niacin, 0.5mg/L vitamin B6 and 2.0mg/L glycine.
5. A method of alfalfa regeneration, the method comprising the steps of:
s1: callus induction
Inoculating the alfalfa explant into an alfalfa callus induction culture medium for induction culture to form alfalfa callus;
the alfalfa callus induction medium is the alfalfa callus induction medium in the medium combination of any one of claims 1-4;
s2: rooting and bud growth induction
Cutting the alfalfa callus into callus groups, inoculating the callus groups into an alfalfa rooting and bud growing culture medium for rooting and bud growing culture to form alfalfa plants with buds and roots;
the alfalfa rooting and sprouting medium is the alfalfa rooting and sprouting medium in the medium combination according to any one of claims 1-4.
6. The method according to claim 5, wherein in step S1, the induction culture is a dark culture at a temperature of 18 to 25℃for a period of 2 to 3 weeks; and/or
In the step S2, the culture temperature of rooting and sprouting culture is 20-25 ℃, the light-dark period is (14-18)/(6-10) h/d, the illumination intensity is 1500-3500lx, and the culture time is 35-50 days.
7. The method of claim 5, wherein the alfalfa explant is an alfalfa leaf.
8. The method of claim 7, wherein the alfalfa leaf is an alfalfa leaf obtained by hydroponic cultivation of alfalfa shoots;
the alfalfa leaves are preferably subjected to the callus induction after being subjected to the sterilization treatment;
the sterilization treatment preferably comprises 3-5 times of sterile water washing, alcohol solution soaking, 3-5 times of sterile water washing, sodium hypochlorite solution soaking and 3-5 times of sterile water washing in sequence;
the volume concentration of the alcohol solution is preferably 70-80%, and the soaking time of the alcohol solution is 25-35s; and/or
The mass concentration of the sodium hypochlorite solution is preferably 0.8-1.5%, and the soaking time of the sodium hypochlorite solution is 4-6min.
9. The method of claim 5, further comprising the step of:
s3: hardening off seedlings
Performing seedling hardening culture on the alfalfa plants with the buds and the roots;
the culture conditions for hardening seedlings are preferably as follows: hardening off the seedlings under 1500-3500lx light for 5-12 days, wherein the light and dark period is (14-18)/(6-10) h/d, and the culture temperature is 25+/-3 ℃.
10. The method of claim 9, further comprising the step of:
s4: transplanting
Taking out the alfalfa seedlings after seedling hardening from the bottle, and transplanting the alfalfa seedlings into soil;
the growth environment of alfalfa seedlings within 10-20 days after transplanting is preferably: the temperature is 20-25deg.C, and the humidity is 80+ -5%.
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Citations (11)
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