CN113973713A - Butterfly orchid tissue culture method - Google Patents
Butterfly orchid tissue culture method Download PDFInfo
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- CN113973713A CN113973713A CN202111262667.5A CN202111262667A CN113973713A CN 113973713 A CN113973713 A CN 113973713A CN 202111262667 A CN202111262667 A CN 202111262667A CN 113973713 A CN113973713 A CN 113973713A
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- China
- Prior art keywords
- pedicel
- tissue culture
- culture medium
- culture
- butterfly orchid
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- Pending
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- 238000012136 culture method Methods 0.000 title claims abstract description 15
- 244000079298 Epidendrum obrienianum Species 0.000 title 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 18
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 238000005520 cutting process Methods 0.000 claims abstract description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 229920000742 Cotton Polymers 0.000 claims abstract description 4
- 239000000428 dust Substances 0.000 claims abstract description 4
- 230000002062 proliferating effect Effects 0.000 claims abstract description 3
- 238000005728 strengthening Methods 0.000 claims abstract description 3
- 239000001963 growth medium Substances 0.000 claims description 30
- 244000127818 Phalaenopsis amabilis Species 0.000 claims description 15
- 238000002791 soaking Methods 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims 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 description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 235000020415 coconut juice Nutrition 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- 238000005286 illumination Methods 0.000 claims description 6
- 238000004161 plant tissue culture Methods 0.000 claims description 2
- 239000003104 tissue culture media Substances 0.000 claims description 2
- 241001505935 Phalaenopsis Species 0.000 abstract description 18
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 1
- NEAPKZHDYMQZCB-UHFFFAOYSA-N N-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]ethyl]-2-oxo-3H-1,3-benzoxazole-6-carboxamide Chemical compound C1CN(CCN1CCNC(=O)C2=CC3=C(C=C2)NC(=O)O3)C4=CN=C(N=C4)NC5CC6=CC=CC=C6C5 NEAPKZHDYMQZCB-UHFFFAOYSA-N 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000011160 research Methods 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
- A01H4/008—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
- A01H4/002—Culture media for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a phalaenopsis tissue culture method, which comprises the steps of selecting pedicel, proliferating tissue culture seedlings of phalaenopsis, strengthening seedlings, rooting and domesticating, and also comprises the step of sterilizing the pedicel, wherein the sterilizing of the pedicel specifically comprises the following steps: wiping the flower stalks with alcohol cotton to wipe dust on the surface; cutting the flower stalks into sections to obtain flower stalk sections; sterilizing the pedicel section; and (4) inoculating and culturing the pedicel. The flower stalks are adopted for in vitro culture, and the generated tissue culture seedlings can keep the excellent characters of the original variety, thereby laying the foundation for breeding and industrial production. The phalaenopsis tissue culture method is simple to operate, low in cost and suitable for rapid propagation of most phalaenopsis. This application adopts alcohol and sodium hypochlorite to carry out disinfection treatment to the pedicel, has guaranteed its sterile condition better, provides aseptic condition for following cultivation.
Description
Technical Field
The invention relates to a butterfly orchid tissue culture method, and belongs to the technical field of plant culture.
Background
The phalaenopsis is a plant of orchidaceae and phalaenopsis, has rhombus-shaped petals, red spots or fine lines, medium cleft rhombuses and yellow flesh bulges at the base. Is distributed in China, Thailand, Philippines, Malaysia, Indonesia. Borne on jungle trunks in tropical and subtropical areas at low altitudes. The butterfly orchid has beautiful and beautiful flowers, long flower watching period and many flowers, can absorb indoor harmful gas, can purify air and can be used as a pot culture for appreciation.
At present, the research on the phalaenopsis tissue culture at home and abroad is less, and patent CN104686362A discloses a phalaenopsis tissue culture method, which adopts the steps of obtaining the stem tip of phalaenopsis, sterilizing and disinfecting the stem tip of phalaenopsis, and carrying out primary culture, secondary culture and rooting culture on the sterilized stem tip of phalaenopsis, so that the rooting rate reaches 26.7%.
Patent CN107691221A discloses a method for culturing phalaenopsis tissue, which designs different culture media according to the requirements of phalaenopsis at different growth stages to shorten the growth cycle of phalaenopsis.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a butterfly orchid tissue culture method.
In order to achieve the first object, the invention is realized by the following technical scheme: a butterfly orchid tissue culture method comprises the steps of selecting pedicel, proliferating tissue culture seedlings of butterfly orchid, strengthening seedlings, rooting and domesticating, and further comprises the step of sterilizing the pedicel, wherein the sterilizing of the pedicel specifically comprises the following steps:
s1: wiping the flower stalks with alcohol cotton to wipe dust on the surface;
s2: cutting the flower stalks into sections to obtain flower stalk sections;
s3: sterilizing the pedicel section
S3.1: carrying out disinfection treatment by adopting alcohol;
s3.2: adopting sodium hypochlorite for disinfection treatment;
s3.3: and (5) carrying out pedicel treatment after disinfection.
By adopting the technical scheme, the pedicel of the phalaenopsis is selected as an explant, and a strong phalaenopsis plant of a good variety is selected, wherein the mother plant is required to be free of diseases, insect pests, malformation and virus, and is harvested and cut in the morning of fine days when 2-3 flowers bloom.
Firstly, wiping the retrieved pedicel with alcohol cotton to remove dust on the surface, and cutting the pedicel into a bud with scissors after high-temperature sterilization on a super clean workbench.
Preferably, step S3.1 specifically includes: soaking the pedicel in 70-80% ethanol for 2-3min, and washing with sterile water.
Preferably, step S3.2 specifically includes: peeling off bract of caulis Fibraureae, soaking in 2-8% sodium hypochlorite solution for 5-10min, and washing with sterile water.
Preferably, step S3.3 is specifically: cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
By adopting the technical scheme, the contact parts of the two ends of the pedicel section and the sodium hypochlorite solution are cut off by a scalpel, and the middle part of the pedicel section is inserted into a culture medium.
Preferably, the method also comprises the inoculation and culture of the pedicel, and specifically comprises the following steps: inoculating the sterilized pedicel section into a culture medium for culture.
Preferably, the culture medium is a plant tissue culture medium consisting of 6-BA, coconut juice, sucrose and agar.
Preferably, the addition amount of coconut juice in the culture medium is 80-90mL, the addition amount of sucrose is 20-30g, the addition amount of agar is 5-8g, the concentration of the 6-BA component in the culture medium is 4-6mg/L, and the pH value of the culture medium A is 5-6.
Preferably, the culture conditions for inoculating the pedicel into the culture medium are as follows: culturing at 22-25 deg.C under illumination for 8-12 hr per day for 40 days.
The invention has the beneficial effects that:
(1) the phalaenopsis tissue culture method is simple to operate, low in cost and suitable for rapid propagation of most phalaenopsis.
(2) The flower stalks are adopted for in vitro culture, and the generated tissue culture seedlings can keep the excellent characters of the original variety, thereby laying the foundation for breeding and industrial production.
(3) This application adopts alcohol and sodium hypochlorite to carry out disinfection treatment to the pedicel, has guaranteed its sterile condition better, provides aseptic condition for following cultivation.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
The method comprises the following steps: selecting flower stalks as explants
Step two: soaking the pedicel in 70% ethanol for 3min, and washing with sterile water; peeling off bract skin of the pedicel section, immersing in 8% sodium hypochlorite solution, soaking for 5min, and washing with sterile water; cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
Step three: inoculating the sterilized pedicel section into a culture medium for culture; the addition amount of coconut juice in the culture medium is 90mL, the addition amount of sucrose is 20g, the addition amount of agar is 5g, the concentration of a 6-BA component in the culture medium A is 6mg/L, and the pH value of the culture medium A is 5.5; the culture conditions were: the culture temperature is 25 ℃, the illumination is 12h every day, and the culture is carried out for 40 days.
Example 2
The method comprises the following steps: selecting flower stalks as explants
Step two: soaking the pedicel section in 75% ethanol for 3min, and washing with sterile water; peeling off bract skin of the pedicel section, immersing in 5% sodium hypochlorite solution, soaking for 5min, and washing with sterile water; cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
Step three: inoculating the sterilized pedicel section into a culture medium for culture; the addition amount of coconut juice in the culture medium is 90mL, the addition amount of sucrose is 30g, the addition amount of agar is 5g, the concentration of a 6-BA component in the culture medium A is 5mg/L, and the pH value of the culture medium A is 5.5; the culture conditions were: the culture temperature is 22 ℃, the illumination is 12h every day, and the culture is carried out for 40 days.
Example 3
The method comprises the following steps: selecting flower stalks as explants
Step two: soaking the pedicel section in 80% ethanol for 2min, and washing with sterile water; peeling off bract skin of the pedicel section, immersing in sodium hypochlorite solution with mass concentration of 2%, soaking for 10min, and washing with sterile water; cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
Step three: inoculating the sterilized pedicel section into a culture medium for culture; the addition amount of coconut juice in the culture medium is 85mL, the addition amount of sucrose is 30g, the addition amount of agar is 6g, the concentration of a 6-BA component in the culture medium A is 6mg/L, and the pH value of the culture medium A is 5.5; the culture conditions were: the culture temperature is 25 ℃, the illumination is 10 hours per day, and the culture is carried out for 40 days.
Example 4
The method comprises the following steps: selecting flower stalks as explants
Step two: soaking the pedicel section in 80% ethanol for 2min, and washing with sterile water; peeling off the bud skin of the flower stalk segment, immersing in a sodium hypochlorite solution with the mass concentration of 2%, and soaking for 10 min; cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
Step three: inoculating the sterilized pedicel section into a culture medium for culture; the addition amount of coconut juice in the culture medium is 85mL, the addition amount of sucrose is 20g, the addition amount of agar is 8g, the concentration of a 6-BA component in the culture medium A is 6mg/L, and the pH value of the culture medium A is 6; the culture conditions were: the culture temperature is 25 ℃, the illumination is 8h every day, and the culture is carried out for 40 days.
Test example 1
And (3) test groups: comparative example 1 and example 1, and comparative example 1 is different from example 1 in that the pedicel is not subjected to sodium hypochlorite sterilization treatment.
The test method comprises the following steps: observing the culture state of the flower stalks.
And (3) test results: see table 1 for details.
TABLE 1 comparison of incubation states of comparative example 1 and example 1
| Group of | Incubation state |
| Comparative example 1 | The flower stalks are rotten 7 days after inoculation |
| Example 1 | 7 days after inoculation, the lateral bud is enlarged and extended outwards, and lobules grow out in about 30 days |
Referring to table 1, it can be seen that the flower stalks are only sterilized by alcohol, so that the flower stalks cannot be sterilized completely, and the flower stalks are rotten during the cultivation process, thereby affecting the later cultivation.
Test example 2
And (3) test groups: comparative examples 2 to 6, and the media in comparative examples 2 to 6 contained 6-BA at 0mg/L, 2mg/L, 4mg/L, 6mg/L, and 8mg/L, respectively, and the other steps were the same as in example 1.
The test method comprises the following steps: observing the culture state of the flower stalks.
And (3) test results: see table 2 for details.
TABLE 2 comparison of culture conditions of flower stalks with comparative examples 2 to 6
| Group of | Incubation state |
| Comparative example 2 | After 15 days of inoculation, the lateral buds are expanded and extended outwards, and grow slowly |
| Comparative example 3 | After 10 days of inoculation, lateral buds are expanded and extended outwards, and grow slowly |
| Comparative example 4 | 7 days after inoculation, the lateral bud is enlarged and extended outwards, and lobules grow out about 40 days |
| Comparative example 5 | 7 days after inoculation, the lateral bud is enlarged and extended outwards, and lobules grow out in about 35 days |
| Comparative example 6 | 7 days after inoculation, the lateral bud is enlarged and extended outwards, and lobules grow out in about 30 days |
| Comparative example 7 | 7 days after inoculation, the lateral bud is enlarged and extended outwards, and lobules grow out in about 30 days |
Referring to Table 2, the earlier the development of the lateral bud of the pedicel was achieved with the increase of the concentration of the 6-BA ingredient in the medium, and the pedicel cultivation state was the best when the concentration of the 6-BA ingredient was 4-6 mg/L.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (8)
1. A butterfly orchid tissue culture method comprises the steps of selecting pedicel, proliferating tissue culture seedlings of butterfly orchid, strengthening seedlings, rooting and domesticating, and is characterized by further comprising the step of sterilizing the pedicel, wherein the sterilizing of the pedicel specifically comprises the following steps:
s1: wiping the flower stalks with alcohol cotton to wipe dust on the surface;
s2: cutting the flower stalks into sections to obtain flower stalk sections;
s3: sterilizing the pedicel section
S3.1: carrying out disinfection treatment by adopting alcohol;
s3.2: adopting sodium hypochlorite for disinfection treatment;
s3.3: and (5) carrying out pedicel treatment after disinfection.
2. The butterfly orchid tissue culture method according to claim 1, wherein the step S3.1 is specifically as follows: soaking the pedicel in 70-80% ethanol for 2-3min, and washing with sterile water.
3. The butterfly orchid tissue culture method according to claim 2, wherein the step S3.2 is specifically as follows: peeling off bract of the pedicel section, soaking in 2-8% sodium hypochlorite solution for 5-10 min.
4. The butterfly orchid tissue culture method according to claim 3, wherein the step S3.3 is specifically as follows: cutting off the contact part of the two ends of the pedicel section with sodium hypochlorite solution.
5. The butterfly orchid tissue culture method of claim 1, further comprising the inoculation and culture of pedicel, and specifically comprises the following steps: inoculating the sterilized pedicel section into a culture medium for culture.
6. The method for culturing the phalaenopsis amabilis tissue as claimed in claim 5, wherein the culture medium is a plant tissue culture medium consisting of 6-BA, coconut juice, sucrose and agar.
7. The butterfly orchid tissue culture method of claim 6, wherein the coconut juice in the culture medium is 80-90mL, the sucrose is 20-30g, the agar is 5-8g, the concentration of the 6-BA component in the culture medium A is 4-6mg/L, and the pH of the culture medium A is 5-6.
8. The method for culturing the phalaenopsis amabilis tissue as claimed in claim 5, wherein the conditions for inoculating the pedicel into the culture medium and culturing are as follows: culturing at 22-25 deg.C under illumination for 8-12 hr per day for 40 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111262667.5A CN113973713A (en) | 2021-10-28 | 2021-10-28 | Butterfly orchid tissue culture method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111262667.5A CN113973713A (en) | 2021-10-28 | 2021-10-28 | Butterfly orchid tissue culture method |
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| Publication Number | Publication Date |
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| CN113973713A true CN113973713A (en) | 2022-01-28 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202111262667.5A Pending CN113973713A (en) | 2021-10-28 | 2021-10-28 | Butterfly orchid tissue culture method |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1994068A (en) * | 2006-12-27 | 2007-07-11 | 李�杰 | Method for quickly breeding butterfly orchid by using pedicel nodal bud to induct pedicel |
| WO2013162485A1 (en) * | 2012-04-27 | 2013-10-31 | M.A.E. Tarim Hayvancilik Gida Turizm Pazarlama Sanayi Ve Ticaret A.S. | Orchid comprising several new tubers and the production method thereof |
| CN104082148A (en) * | 2014-07-18 | 2014-10-08 | 内蒙古农业大学 | Method for performing regeneration propagation on sterile stalks of butterfly orchids |
| CN104604687A (en) * | 2015-01-29 | 2015-05-13 | 赤峰市农牧科学研究院 | Method for inducing clustered shoots to rapidly propagate butterfly orchid by utilizing pedicle stem segments after bud cutting |
| CN106106163A (en) * | 2016-07-12 | 2016-11-16 | 成都东山兰韵农业有限公司 | A kind of iris cultivates propagation method |
| CN106258968A (en) * | 2016-08-12 | 2017-01-04 | 成都东山兰韵农业有限公司 | The iris tissue culture method that a kind of browning rate is low |
-
2021
- 2021-10-28 CN CN202111262667.5A patent/CN113973713A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1994068A (en) * | 2006-12-27 | 2007-07-11 | 李�杰 | Method for quickly breeding butterfly orchid by using pedicel nodal bud to induct pedicel |
| WO2013162485A1 (en) * | 2012-04-27 | 2013-10-31 | M.A.E. Tarim Hayvancilik Gida Turizm Pazarlama Sanayi Ve Ticaret A.S. | Orchid comprising several new tubers and the production method thereof |
| CN104082148A (en) * | 2014-07-18 | 2014-10-08 | 内蒙古农业大学 | Method for performing regeneration propagation on sterile stalks of butterfly orchids |
| CN104604687A (en) * | 2015-01-29 | 2015-05-13 | 赤峰市农牧科学研究院 | Method for inducing clustered shoots to rapidly propagate butterfly orchid by utilizing pedicle stem segments after bud cutting |
| CN106106163A (en) * | 2016-07-12 | 2016-11-16 | 成都东山兰韵农业有限公司 | A kind of iris cultivates propagation method |
| CN106258968A (en) * | 2016-08-12 | 2017-01-04 | 成都东山兰韵农业有限公司 | The iris tissue culture method that a kind of browning rate is low |
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| Title |
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| 李淑英等: "蝴蝶兰的组织培养", 《农业工程技术(温室园艺)》 * |
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