CN116784234A - Method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia annua - Google Patents
Method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia annua Download PDFInfo
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- CN116784234A CN116784234A CN202310767393.8A CN202310767393A CN116784234A CN 116784234 A CN116784234 A CN 116784234A CN 202310767393 A CN202310767393 A CN 202310767393A CN 116784234 A CN116784234 A CN 116784234A
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- 238000000034 method Methods 0.000 title claims abstract description 17
- 230000001939 inductive effect Effects 0.000 title claims abstract description 7
- 238000000338 in vitro Methods 0.000 title claims description 5
- 230000008929 regeneration Effects 0.000 claims abstract description 63
- 238000011069 regeneration method Methods 0.000 claims abstract description 63
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 8
- 230000001954 sterilising effect Effects 0.000 claims abstract description 8
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 235000011869 dried fruits Nutrition 0.000 claims abstract description 3
- 238000010008 shearing Methods 0.000 claims abstract description 3
- 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 31
- 229930006000 Sucrose Natural products 0.000 claims description 31
- 239000005720 sucrose Substances 0.000 claims description 31
- 229920001817 Agar Polymers 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 14
- 230000012010 growth Effects 0.000 claims description 13
- 238000005286 illumination Methods 0.000 claims description 12
- 239000002689 soil Substances 0.000 claims description 9
- 239000000499 gel Substances 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 5
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 4
- 241000360771 Coreana Species 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 239000012882 rooting medium Substances 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 abstract description 22
- 230000004083 survival effect Effects 0.000 abstract description 5
- 240000004731 Acer pseudoplatanus Species 0.000 abstract description 4
- 235000002754 Acer pseudoplatanus Nutrition 0.000 abstract description 4
- 235000006485 Platanus occidentalis Nutrition 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 15
- 229920002148 Gellan gum Polymers 0.000 description 13
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000006870 ms-medium Substances 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241001409201 Tabebuia chrysantha Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241001073507 Callicarpa Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
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- 238000011010 flushing procedure Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a method for obtaining regenerated complete plants by inducing the isolated hypocotyl of the aeolia annua. The method comprises the following steps: (1) acquisition of sterile materials: collecting mature, cracked and dried fruit pods, shearing off seed wings, sterilizing, cleaning, and inoculating to an MS culture medium for culture to obtain tissue culture seedlings; (2) hypocotyl regeneration culture: selecting hypocotyls of tissue culture seedlings, and placing the hypocotyls in a hypocotyl regeneration culture medium to induce regeneration buds; (4) subculturing regenerated buds: placing the regeneration buds into a secondary culture medium for culture, and carrying out secondary culture to obtain regeneration plants; and (5) rooting culture of regenerated seedlings: inoculating the regenerated plants into a rooting culture medium for rooting culture; (6) transplanting rooting seedlings. The invention establishes a high-efficiency regeneration system of the sycamore through a large number of experiments and clear regeneration parameters, obtains transplanted survival plants, has high transplanting survival rate, lays a foundation for high-quality resource preservation, propagation and genetic transformation research of the sycamore, and is beneficial to application and popularization of the regeneration system.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia coreana.
Background
The establishment of the isolated hypocotyl high-efficiency regeneration system of the falcate-bark falcate is an effective way for developing the research on the germplasm resource protection and genetic engineering of the falcate-bark falcate, and is also an effective way for rapid propagation. The domestic yellow aeolian bells regeneration research starts late, and the current yellow aeolian bells regeneration system is not reported yet. Along with the completion of the whole genome sequencing of the aeolian yellow, the breeding of the aeolian yellow flower must enter the molecular breeding era, the construction of the aeolian yellow flower genetic transformation system is particularly important, and the efficient regeneration system is the premise of the construction of the genetic transformation system.
The transplanting survival rate of the regenerated seedlings obtained through the hypocotyl regeneration is a key factor influencing the application and popularization of a hypocotyl regeneration system. At present, the research is focused on the rapid propagation of the suzuki, and a regeneration system of the suzuki is established to be further researched.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for inducing the in-vitro hypocotyl of the aeolian coreana to obtain a regenerated complete plant so as to obtain the resource preservation of the variety of materials.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for inducing an isolated hypocotyl of a falcate-bark tree to obtain a regenerated whole plant, comprising the following steps:
(1) Acquisition of sterile material: collecting mature, cracked and dried fruit pods, shearing off seed wings, sterilizing, cleaning, and inoculating to an MS culture medium for culture to obtain tissue culture seedlings;
(2) Hypocotyl regeneration culture: selecting hypocotyls of tissue culture seedlings, and placing the hypocotyls in a hypocotyl regeneration culture medium to induce regeneration buds;
(4) Subculturing the regenerated buds: placing the regeneration buds into a secondary culture medium for culture, and carrying out secondary culture to obtain regeneration plants;
(5) Rooting culture of regenerated seedlings: inoculating the regenerated plants into a rooting culture medium for rooting culture;
(6) Transplanting rooting seedlings.
The hypocotyl regeneration culture medium contains IBA, 6-BA, TDZ, sucrose and plant gel.
The rooting culture medium contains IBA, NAA, sucrose and agar.
The hypocotyl regeneration culture medium is MS+IBA0.5 mg/L, TDZ 0.2.2 mg/L, 6-BA5mg/L+20g/L sucrose+2.5g/L plant gel;
the secondary culture medium is MS+sucrose (30 g/L) +agar powder (6.0 g/L);
the rooting culture medium is MS+IBA 3mg/L+NAA 0.01 mg/L+sucrose 20 g/L+agar 6g/L;
preferably, the sterile material of step (1) is obtained by: dark culture is carried out for 3-7 d, and then illumination culture is carried out; the total cultivation time is 20-30 d. Although the MS medium is used for the acquisition of the sterile material, the tissue culture seedling subculture, and the regeneration bud subculture, the amount of sucrose contained in the MS medium is not required to be completely uniform.
Preferably, the condition of the hypocotyl regeneration culture is culture under the photoperiod of 12h illumination and 12h darkness.
Preferably, the condition of the secondary culture is culture under the photoperiod of 16h illumination and 8h darkness.
Preferably, the rooting culture conditions are 12h of illumination and 12h of darkness.
Preferably, the rooting seedling transplanting is to take rooting seedlings, place the rooting seedlings in a growth chamber for hardening off for 7d, clean root agar and callus, transplant the rooting seedlings into a seedling raising basin filled with nutrient soil, and the culture medium is common nutrient soil: perlite = 3:1v/v, the soil particles are not agglomerated, not dripped and are loose, and finally the asexual propagation high-quality seedling is obtained.
Preferably, the aeolian yellow croaker is Handroanthus chrysanthus (jacq.) s.o. gross.
The beneficial effects of the invention are as follows: the invention establishes a high-efficiency regeneration system of the sycamore through a large number of experiments and clear regeneration parameters, obtains transplanted survival plants, has high transplanting survival rate, lays a foundation for high-quality resource preservation, propagation and genetic transformation research of the sycamore, and is beneficial to application and popularization of the regeneration system.
Drawings
FIG. 1 is a flow chart showing the construction of a regeneration system of Callicarpa xanthophylla in example 1, wherein A is a growth state diagram of tissue culture seedlings, the hypocotyl state (preferably hypocotyl in circle) required for regeneration, B is a regeneration bud generated by the hypocotyl, C is a growth state of the regeneration bud 80D, D is a growth state of the rooting seedling 30D, and E is a growth state after transplanting the rooting seedling for 5D;
FIG. 2 is a graph showing regeneration buds induced by different regeneration media, corresponding to example 1, example 2, example 3 and comparative example 4 in sequence;
FIG. 3 shows the growth of regenerated seedlings in different rooting culture media;
FIG. 4 is a graph showing the growth of plants before transplanting different rooting media.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
MS culture medium salt, sucrose, agar powder and plant gel used in the examples of the invention are purchased from Ding national biology company (Guangzhou), 6-BA is purchased from Ding national biology company (Guangzhou), IBA and NAA are purchased from Genview company, TDZ is purchased from Sigma company, naOH and HCl are purchased from metallocene chemical reagent factory, and activated carbon A.C. is purchased from Yu Fuchen chemical company.
Example 1
In this example, the regeneration process of the isolated hypocotyl of aeolia armigera Handroanthus chrysanthus (jacq.) s.o.grose is shown in fig. 1, and the specific parameters are as follows:
(1) Acquisition of sterile Material for explants
And 2022, 4 months, collecting from a park attached to a kindergarten of agricultural university of south China, cutting off seed wings in a laboratory, soaking for 15min with 1 liter of tap water added with 3 pumps of detergent, and flushing for 30min with running water. Placing the seeds in an ultra-clean workbench, soaking the seeds in alcohol with the volume fraction of 75% for 50s, washing the seeds with sterile water for 3 times, soaking and sterilizing the seeds in mercury chloride with the mass fraction of 0.1% for 10min, and washing the seeds with sterile water for 3 times. Inoculating on a standby MS culture medium, culturing for 7d in dark, culturing for 20d in light, cleaning contaminated plants in time, observing the growth condition of the contaminated plants, and finally obtaining sterile isolated plants, namely tissue culture seedlings.
(2) Hypocotyl regeneration
Selecting, namely selecting hypocotyls (shown in figure 1A) of tissue culture seedlings which grow robustly for 20-30 days, inoculating 6 bottles of explants with about 5mm for each treatment, and inoculating 5 explants per bottle, wherein the bottles contain hypocotyl regeneration culture medium. The culture conditions are illumination culture, the illumination intensity is 2500lx, the period is illumination 12h and darkness 12h, the regeneration bud is induced (figure 1B), and the culture is carried out for 80d, so as to obtain the regeneration bud (figure 1C).
The hypocotyl regeneration culture medium is as follows: MS+IBA0.5 mg/L, TDZ 0.2mg/L, 6-BA5mg/L+20g/L sucrose+2.5g/L plant gel Phytagel is prepared by adding IBA0.5 mg, TDZ 0.2mg, 6-BA5mg, sucrose 20g and plant gel 2.5g into 1L liquid MS medium, and sterilizing for use.
(3) Subculture of regenerated shoots
Uniformly transferring the regenerated buds obtained in the step (2) together with the bottom explants and the callus into a secondary culture medium for 1 time, wherein the secondary culture medium comprises the following components: MS+sucrose (30 g/L) +agar powder (6.0 g/L), its preparation method is that 30g of sucrose and 6g of agar powder are added into 1L of liquid MS culture medium, the pH is regulated to 5.8-6.0 by 1M NaOH solution, the temperature is 121 ℃, sterilization is carried out for 20min, and the culture is carried out under the photoperiod of 16h of illumination and 8h of darkness.
(4) Rooting culture of regenerated seedlings
Removing the bottom old leaves of the regenerated plants obtained in the step (3), trimming the top leaves with scissors to be flat, and inoculating the plants with the height of about 1.5-2 cm into a rooting medium for culture (figure 1D). The rooting culture medium takes MS as a basic culture medium, the IBA content is 3mg/L, the NAA content is 0.01mg/L, the sucrose content is 20g/L, and the agar content is 6g/L, and the preparation method is that 3mg of IBA, 0.01mg of NAA, 20g of sucrose and 6g of agar are added into 1L of liquid MS culture medium, and sterilized for standby.
The culture conditions are as follows: and (3) culturing for 28d by illumination, wherein the illumination culture period is 12h by illumination and 12h by darkness.
(5) Transplanting regenerated root seedlings
Taking the rooting seedling cultured for 35d in the step (4), placing the rooting seedling in a growth chamber for hardening off for 7d, cleaning root agar and callus (preventing root rot), transplanting the rooting seedling into a seedling raising basin filled with nutrient soil, wherein the culture medium is common nutrient soil: perlite = 3:1v/v, the soil particles are not agglomerated, not dripped and are loose, and finally the asexual propagation high-quality seedling is obtained (figure 1E). In fig. 1, a is a growth state diagram of a tissue culture seedling, the hypocotyl state (preferably, the hypocotyl in a circle) required for regeneration, B is a regenerated bud generated by the hypocotyl, C is the growth state of the regenerated bud 80D, D is the growth state of a root seedling 30D, and E is the growth state after the root seedling is transplanted for 5D.
Example 2
Unlike example 1, the hypocotyl regeneration medium in step (2) had the following composition: MS+IBA0.5 mg/L, TDZ 0.2mg/L, 6-BA 0.5mg/L+20g/L Sucrose+2.5g/L Phytagel.
Example 3
Unlike example 1, the hypocotyl regeneration medium in step (2) had the following composition: MS+IBA0.5 mg/L+TDZ 0.2mg/L+6-BA 3mg/L+20g/L Sucrose+2.5g/L Phytagel.
Example 4
Unlike example 1, the hypocotyl regeneration medium in step (2) had the following composition: MS+IBA0.5 mg/L+TDZ 0.2mg/L+20g/L Sucrose+2.5g/L Phytagel.
Example 5
Unlike example 1, the composition of the rooting medium in step (4) is: MS+IBA 5mg/L+NAA0.05mg/L+20g/L sucrose+6g/L agar.
Comparative example 1
Unlike example 1, the seed sterilization mercury chloride sterilization time in step (1) was 8 minutes.
Comparative example 2
Unlike example 1, the seed disinfection mercuric chloride disinfection time in step (1) was 12 minutes.
Comparative example 3
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+1 mg/L6-BA+1 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 4
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+3 mg/L6-BA+1 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 5
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+5 mg/L6-BA+1 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 6
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+1 mg/L6-BA+3 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 7
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+3 mg/L6-BA+3 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 8
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+5 mg/L6-BA+3 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 9
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+1 mg/L6-BA+5 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 10
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+3 mg/L6-BA+5 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
Comparative example 11
Unlike example 1, the hypocotyl regeneration medium in step (2) was: MS+5 mg/L6-BA+5 mg/LNAA+20g/L sucrose+2.5g/L Phytagel.
The regeneration of hypocotyls in comparative examples 1, 2, 3 and 4 was counted, and the rate of hypocotyl regeneration was counted.
The hypocotyl regeneration rate was counted in comparative example 1 and comparative examples 3 to 11.
Rooting conditions of examples 1 and 5 are counted, specifically rooting rates after 35 days are counted, average root numbers and average root lengths are counted, and the results are shown in the following table.
Claims (9)
1. The method for inducing the in-vitro hypocotyl of the aeolia coreana to obtain the regenerated complete plant is characterized by comprising the following steps of:
(1) Acquisition of sterile material: collecting mature, cracked and dried fruit pods, shearing off seed wings, sterilizing, cleaning, and inoculating to an MS culture medium for culture to obtain tissue culture seedlings;
(2) Hypocotyl regeneration culture: selecting hypocotyls of tissue culture seedlings, and placing the hypocotyls in a hypocotyl regeneration culture medium to induce regeneration buds;
(4) Subculturing the regenerated buds: placing the regeneration buds into a secondary culture medium for culture, and carrying out secondary culture to obtain regeneration plants;
(5) Rooting culture of regenerated seedlings: inoculating the regenerated plants into a rooting culture medium for rooting culture;
(6) Transplanting rooting seedlings.
2. The method of claim 1, wherein the hypocotyl regeneration medium comprises IBA, 6-BA, TDZ, sucrose, and plant gel.
3. The method of claim 1, wherein the rooting medium comprises IBA, NAA, sucrose, agar.
4. The method of claim 1, wherein the hypocotyl regeneration medium is MS+IBA0.5 mg/L, TDZ 0.2mg/L, 6-BA5mg/L+20g/L sucrose+2.5g/L plant gel;
the secondary culture medium is MS+30 g/L of sucrose+6.0 g/L of agar powder;
the rooting culture medium is MS+IBA 3mg/L+NAA 0.01 mg/L+sucrose 20 g/L+agar 6g/L.
5. The method of claim 1, wherein the sterile material of step (1) is obtained by: dark culture is carried out for 3-7 d, and then illumination culture is carried out; the total cultivation time is 20-30 d.
6. The method according to claim 1, wherein the condition for the regeneration culture of hypocotyl is photoperiod culture of 12 hours under light and 12 hours under dark.
7. The method according to claim 1, wherein the conditions of the subculture are a culture under a photoperiod of 16h light and 8h dark.
8. The method according to claim 1, wherein the rooting culture conditions are 12 hours of light and 12 hours of darkness.
9. The method of claim 1, wherein the rooting seedling transplanting is to take rooting seedlings, placing the rooting seedlings in a growth chamber for hardening off for 7d, cleaning root agar and callus, transplanting the rooting seedlings into a seedling raising basin filled with nutrient soil, and the culture medium is common nutrient soil: perlite = 3:1v/v, the soil particles are not agglomerated, not dripped and are loose, and finally the asexual propagation high-quality seedling is obtained.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106665367A (en) * | 2017-03-21 | 2017-05-17 | 钦州市林业科学研究所 | Tabebuia chrysantha tissue culture and rapid propagation method |
| CN108391591A (en) * | 2018-01-26 | 2018-08-14 | 长江大学 | A kind of Golden Bell Tree tissue cultivation rapid breeding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106665367A (en) * | 2017-03-21 | 2017-05-17 | 钦州市林业科学研究所 | Tabebuia chrysantha tissue culture and rapid propagation method |
| CN108391591A (en) * | 2018-01-26 | 2018-08-14 | 长江大学 | A kind of Golden Bell Tree tissue cultivation rapid breeding method |
Non-Patent Citations (1)
| Title |
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| 陈丽文等: "黄花风铃木的组培快繁技术", 《现代园艺》, no. 3, pages 116 - 117 * |
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