CN115486369A - Method for improving induction rate of embryonic callus of myrtle anther - Google Patents
Method for improving induction rate of embryonic callus of myrtle anther Download PDFInfo
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- CN115486369A CN115486369A CN202211185472.XA CN202211185472A CN115486369A CN 115486369 A CN115486369 A CN 115486369A CN 202211185472 A CN202211185472 A CN 202211185472A CN 115486369 A CN115486369 A CN 115486369A
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- 240000005125 Myrtus communis Species 0.000 title claims abstract description 28
- 235000013418 Myrtus communis Nutrition 0.000 title claims abstract description 28
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 25
- 230000006698 induction Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000000408 embryogenic effect Effects 0.000 claims abstract description 11
- 230000001954 sterilising effect Effects 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000008223 sterile water Substances 0.000 claims abstract description 7
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims abstract description 5
- 238000011282 treatment Methods 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 4
- 238000001035 drying Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 240000007994 Rhodomyrtus tomentosa Species 0.000 description 3
- 235000007234 Rhodomyrtus tomentosa Nutrition 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000003630 growth substance Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000023409 microsporogenesis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
Images
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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for improving the embryogenic callus induction rate of myrtle anther, comprising the steps of selecting myrtle anther in a certain development stage, sterilizing, drying buds, pretreating at 4-6 ℃ for 1-4 days at low temperature by controlling the transverse diameter of the buds to be 5-9mm and the longitudinal diameter to be 5-8mm, sterilizing the buds after low-temperature pretreatment, and then 0.1-0.2 percent of HgCl 2 Sterilizing the solution; taking out the sterilized flower buds, and rinsing with sterile water; taking out the rinsed flower buds, stripping off anthers, inoculating on an induction culture medium, culturing in dark for 35-80 days, and transferring once every 20 days in the middle to obtain embryonic callus. The preferable culture medium formula of the invention is as follows: MS +0.5-3.0mg/L2,4-D +0.5-3mg/L6-BA +0.1-0.8mg/LNAA, and simultaneously combines with other conditions to culture the flower buds, thereby remarkably improving the induction rate of the callus to 30.82 percent.
Description
Technical Field
The invention relates to the technical field of plant tissue culture and rapid propagation, in particular to a method for improving the induction rate of embryonic callus of myrtle anther.
Background
Rhodomyrtus tomentosa (Rhodomyrtus tomentosa) is a perennial deciduous shrub, also known as Rhodomyrtus tomentosa, lotus, and the like, grows in red soil hilly lands widely in China, and is an acid soil indicator plant. The myrtle is the only species of myrtle naturally distributed in China, has bright and colorful colors, blooms in summer, can be applied to garden landscape and hillside ecological restoration, has the same period of flowers and fruits, has high fruit nutritive value and has certain medicinal value. The seed propagation is generally the main point of the myrtle seedling propagation. The seed germination rate is low, and meanwhile, the excellent properties of the variety cannot be maintained. Anther culture provides a new idea for screening excellent strains of the myrtle. The culture conditions of inducing anther embryogenic callus by the myrtle are optimized, and a myrtle anther embryogenic callus culture system is established, so that a theoretical basis is provided for establishing a tissue culture and rapid propagation system of the myrtle, and the method has important significance in promoting the healthy development of the myrtle industry.
For diploid plants, the anthers only have one set of chromosome group, so that the induction rate of anther embryogenic callus is improved, the percentage of haploid plants can be greatly improved, and the breeding process is shortened. The formation of anther embryogenic callus is influenced by explant morphology, culture medium, various combinations of growth regulator concentrations, the period of bud growth and development, and the pretreatment of plant material. The myrtle has more ecological researches, the related breeding research is relatively lagged, and particularly the related research of tissue culture and rapid propagation is not reported. The anther culture is important in plant breeding, the culture condition of inducing anther embryogenic callus by myrtle is researched, a myrtle anther embryogenic callus culture system is established, and a theoretical basis is provided for establishment of a myrtle tissue culture and rapid propagation system.
In view of the defects, the invention uses the myrtle as a material, researches the influence of different microspore development periods and bud sizes, different growth regulators and different pretreatments on anther callus induction, and screens out the bud size, culture medium and optimal time for low-temperature pretreatment suitable for the anther callus induction of the myrtle.
Disclosure of Invention
Based on the analysis, the invention provides a method for improving the induction rate of the calli of the myrtle anther. The invention is realized by the following means:
a method for improving the induction rate of embryonic callus of myrtle anther comprises the following steps:
(1) Firstly, controlling the size of buds;
(2) After the buds are recovered, low-temperature pretreatment is carried out;
(3) The buds pretreated at low temperature are sterilized and then 0.1-0.2% of HgCl 2 Sterilizing the solution;
(4) Taking out the sterilized flower buds, and rinsing with sterile water;
(5) Taking out the rinsed flower bud, stripping off anther, inoculating on the induction culture medium, dark culturing for 35-80 days, and transferring every 20 days to obtain embryogenic callus.
Further, before the bud is controlled in size in step (1), the method further comprises: sterilizing picked flower bud with 75% alcohol for 30-60 s, and further processing at 0.1-0.2% by HgCl 2 Sterilizing for 10-12min, rinsing with sterile water for 4-5 times, and sucking water from flower bud with sterile filter paper.
Further, the size of the flower buds obtained in the step (1) is as follows: the transverse diameter is 5-9mm, and the longitudinal diameter is 5-8mm.
Further, the low-temperature treatment in the step (2) is: pretreating for 1-4 days at 4-6 deg.C.
Further, the sterilization treatment time in the step (3) is as follows: 10-12min.
Further, the sterile water in the step (4) is rinsed for 4-5 times, and each time lasts for 2-3min.
Further, the embryogenic callus induction medium in the step (5) is as follows: MS +0.5-3.0mg/L2,4-D +0.5-3mg/L6-BA +0.1-0.8mg/LNAA.
Further, the dark culture conditions in step (5): culturing at 25 + -2 deg.C under light for 20 days, and illuminating for 12h each day.
The invention has the beneficial effects that:
in the invention, the myrtle anther at a certain development stage is selected, and is subjected to low-temperature treatment for 1-4 days, and is cultured for 20 days in MS +0.5-3.0mg/L2,4-D +0.5-3mg/L6-BA +0.1-0.8mg/L NAA, and the formation percentage of anther callus is remarkably higher than that of other treatments. The comprehensive treatment can be seen, and the callus induction rate is remarkably improved to 30.82%.
Drawings
FIG. 1 shows the flower buds of Myrtus communis at different developmental stages.
FIG. 2 shows the induction of calli from myrtle anthers under different treatments.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for improving the induction rate of embryonic callus of myrtle anther comprises the following steps:
(1) Firstly, controlling the bud size: the transverse diameter of the bud is 6mm, and the longitudinal diameter of the bud is 6mm.
(2) And further low-temperature treatment: and 2 days.
(3) Providing a certain condition culture condition: culturing at 25 deg.C under light for 20 days, and illuminating for 12 hr per day.
(4) Optimizing the formula of the culture medium: MS +1.5 mg/L2,4-D +1.5 mg/L6-BA +0.4mg/L NAA. The inductivity was 30.82%.
Example 2
A method for improving induction rate of embryonic callus of myrtle anther comprises the following steps:
(1) Firstly, controlling the size of a flower bud: the transverse diameter of the bud is 5mm, and the longitudinal diameter is 5mm.
(2) Further low-temperature treatment: for 1 day.
(3) Providing a certain condition culture condition: culturing at 23 deg.C under light for 20 days for 12 hr per day.
(4) Optimizing the formula of the culture medium: MS +0.5 mg/L2,4-D +0.5 mg/L6-BA +0.1mg/L NAA. The inductivity was 30.82%.
Example 3
A method for improving the induction rate of embryonic callus of myrtle anther comprises the following steps:
(1) Firstly, controlling the size of a flower bud: the transverse diameter of the bud is 9mm, and the longitudinal diameter of the bud is 8mm.
(2) And further low-temperature treatment: 4 days.
(3) Providing a certain condition culture condition: culturing at 27 deg.C under light for 20 days for 12 hr per day.
(4) Optimizing the formula of the culture medium: MS +3.0 mg/L2,4-D + 3mg/L6-BA +0.8mg/L NAA. The inductivity was 30.82%.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A method for improving the induction rate of embryonic callus of myrtle anther comprises the following steps:
controlling the size of flower buds;
after the buds are recovered, low-temperature pretreatment is carried out;
the buds pretreated at low temperature are sterilized and then 0.1-0.2% of HgCl 2 Sterilizing the solution;
taking out the sterilized flower buds, and rinsing with sterile water;
taking out the rinsed flower bud, stripping off anther, inoculating on the induction culture medium, dark culturing for 35-80 days, and transferring every 20 days to obtain embryogenic callus.
2. The method of claim 1, wherein:
before controlling the size of the flower buds in the step (1), the method further comprises the following steps: sterilizing picked flower bud with 75% alcohol for 30-60 s, and further processing at 0.1-0.2% by HgCl 2 Sterilizing for 10-12min, rinsing with sterile water for 4-5 times, and sucking water from flower bud with sterile filter paper.
3. The method of claim 1, wherein:
the bud size in the step (1) is as follows: the transverse diameter is 5-9mm, and the longitudinal diameter is 5-8mm.
4. The method of claim 1, wherein:
the low-temperature treatment in the step (2) comprises the following steps: pretreating for 1-4 days at 4-6 deg.C.
5. The method of claim 4, wherein:
the sterilization treatment time in the step (3) is as follows: 10-12min.
6. The method of claim 1, wherein:
rinsing with the sterile water for 4-5 times, each time for 2-3min.
7. The method of claim 1, wherein:
the embryogenic callus induction culture medium in the step (5) comprises: MS +0.5-3.0mg/L2,4-D +0.5-3mg/L6-BA +0.1-0.8mg/LNAA.
8. The method of claim 1, wherein:
the dark culture condition of the step (5): culturing at 25 + -2 deg.C under light for 20 days, and illuminating for 12h each day.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211185472.XA CN115486369A (en) | 2022-09-27 | 2022-09-27 | Method for improving induction rate of embryonic callus of myrtle anther |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211185472.XA CN115486369A (en) | 2022-09-27 | 2022-09-27 | Method for improving induction rate of embryonic callus of myrtle anther |
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|---|---|
| CN115486369A true CN115486369A (en) | 2022-12-20 |
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| CN202211185472.XA Pending CN115486369A (en) | 2022-09-27 | 2022-09-27 | Method for improving induction rate of embryonic callus of myrtle anther |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104247662A (en) * | 2014-09-29 | 2014-12-31 | 梁彩英 | Rapid propagation technology for tissue culture of rhodomyrtus tomentosa (ait.) hassk |
| CN111543317A (en) * | 2019-02-12 | 2020-08-18 | 山东农业大学 | Method for culturing and inducing callus by using platycodon grandiflorum anther |
| CN113455400A (en) * | 2021-08-20 | 2021-10-01 | 闽南师范大学 | Inducing method for Longboat anther callus |
| CN115039696A (en) * | 2022-05-09 | 2022-09-13 | 中国科学院华南植物园 | Method for inducing calluses and regenerating seedlings of myrtle |
-
2022
- 2022-09-27 CN CN202211185472.XA patent/CN115486369A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104247662A (en) * | 2014-09-29 | 2014-12-31 | 梁彩英 | Rapid propagation technology for tissue culture of rhodomyrtus tomentosa (ait.) hassk |
| CN111543317A (en) * | 2019-02-12 | 2020-08-18 | 山东农业大学 | Method for culturing and inducing callus by using platycodon grandiflorum anther |
| CN113455400A (en) * | 2021-08-20 | 2021-10-01 | 闽南师范大学 | Inducing method for Longboat anther callus |
| CN115039696A (en) * | 2022-05-09 | 2022-09-13 | 中国科学院华南植物园 | Method for inducing calluses and regenerating seedlings of myrtle |
Non-Patent Citations (3)
| Title |
|---|
| LING YANG等: "A plant regeneration protocol from callus cultures of medicinal and ornamental shrub Rhodomyrtus tomentosa", 《RESEARCH SQUARE》 * |
| 沈火林: "《园艺植物育种学》", 30 April 2011, 中央广播电视大学出版社 * |
| 王尚显等: "桃金娘组织培养初步研究", 《湖南农业科学》 * |
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