CN114009338A - Formula of sterile sowing culture medium for eupatorium donyanense and tissue culture method - Google Patents
Formula of sterile sowing culture medium for eupatorium donyanense and tissue culture method Download PDFInfo
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- CN114009338A CN114009338A CN202111283028.7A CN202111283028A CN114009338A CN 114009338 A CN114009338 A CN 114009338A CN 202111283028 A CN202111283028 A CN 202111283028A CN 114009338 A CN114009338 A CN 114009338A
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- culture medium
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- 239000001963 growth medium Substances 0.000 title claims abstract description 41
- 238000009331 sowing Methods 0.000 title claims abstract description 15
- 238000012136 culture method Methods 0.000 title claims abstract description 14
- 241000735527 Eupatorium Species 0.000 title claims description 8
- 238000010899 nucleation Methods 0.000 claims abstract description 30
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000005972 6-Benzyladenine Substances 0.000 claims abstract description 20
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000035755 proliferation Effects 0.000 claims abstract description 17
- 230000004069 differentiation Effects 0.000 claims abstract description 16
- 230000035784 germination Effects 0.000 claims abstract description 15
- 229920001817 Agar Polymers 0.000 claims abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229930006000 Sucrose Natural products 0.000 claims abstract description 12
- 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 12
- 239000008272 agar Substances 0.000 claims abstract description 12
- 235000020415 coconut juice Nutrition 0.000 claims abstract description 12
- 239000005720 sucrose Substances 0.000 claims abstract description 12
- 241000233855 Orchidaceae Species 0.000 claims abstract description 8
- 239000007640 basal medium Substances 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims abstract description 5
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 4
- 238000009472 formulation Methods 0.000 claims abstract 6
- 239000000203 mixture Substances 0.000 claims abstract 6
- 239000002775 capsule Substances 0.000 claims description 15
- 244000269722 Thea sinensis Species 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 241000201841 Celosia Species 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 239000004744 fabric Substances 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 230000010152 pollination Effects 0.000 claims description 3
- 235000008954 quail grass Nutrition 0.000 claims description 3
- 238000007790 scraping Methods 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 7
- 241000258195 Holothuria Species 0.000 description 5
- 241000732800 Cymbidium Species 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 244000301654 Cymbidium dayanum Species 0.000 description 1
- 241000233805 Phoenix Species 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to a formulation of a culture medium for sterile seeding of orchids and a tissue culture method, which are characterized by comprising a seeding culture medium and a proliferation and differentiation culture medium, wherein the formulation of the seeding culture medium comprises: KC +6-BA 1.5+ NAA 0.6+ sucrose 20+ coconut juice 100+ active carbon 1+ agar 4.7, and the PH value is adjusted to 5.6 +/-1; the components comprise: KC basal medium, 6-BA (6-benzyladenine) 1.5mg/L, NAA (naphthylacetic acid) 0.6mg/L, sucrose 20g/L, coconut juice 100mL/L, activated carbon 1g/L, agar 4.7 g/L; the proliferation and differentiation medium formula comprises the following components: 1/2MS basal medium, NAA (naphthalene acetic acid) 0.1mg/L, 6-BA (6-benzyl adenine) 2mg/L, sucrose 20g/L, coconut juice 100ml/L, agar 4.5 g/L. The method can shorten the germination period of the cochinchina yunnanensis seeds to 80 days from the germination of 3-5 months of conventional sowing, and has the advantages of high germination rate, strong proliferation capacity of protocorm, high speed and high quality.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a formula of a culture medium for sterile sowing of orchids and a tissue culture method.
Background
The Cymbidium (Cymbidium dayanum) is epiphytic orchid of orchidaceae, has a general inflorescence, a scape bent outwards or droopea, has beautiful plant shape and long flowering phase, and is a wild orchid with high development and utilization values. Due to the continuous destruction of the habitat and artificial random excavation and random mining, the wild Yufenglan resources are seriously lost, and the natural distribution is sharply reduced. Therefore, the method for expanding propagation and preserving the Dougenhei by adopting the plant tissue culture technology has important significance for the protection, development and utilization of germplasm resources of the Dougenhei.
At present, the traditional plant division propagation method is mostly adopted for the artificial cultivation of the Holothuria, the propagation coefficient is extremely low, the cultivation quantity is limited, the problems of complicated operation, low germination rate, long germination period (3-5 months) and the like exist in the conventional aseptic seeding method, and the development and utilization process of the Holothuria is seriously hindered. Aiming at the defects, the invention carries out a great deal of technical innovation, provides an aseptic seeding culture medium and a set of simplified tissue culture operation flow, can shorten the germination period of the eupatorium donianum seeds to 80 days, and has high germination rate, strong differentiation capacity, high speed and high quality.
Disclosure of Invention
Aiming at the problems in the background, the invention provides a culture medium formula for sterile seeding of the Holothuria, and a tissue culture method, so as to solve the problems of long propagation period and low propagation speed of the Holothuria and improve the quality of original bulbs of the Holothuria.
The formula of the sterile seeding culture medium for the eupatorium dongpanense is characterized by comprising a seeding culture medium and a proliferation and differentiation culture medium, wherein the seeding culture medium formula comprises:
KC +6-BA 1.5+ NAA 0.6+ sucrose 20+ coconut juice 100+ active carbon 1+ agar 4.7, and the PH value is adjusted to 5.6 +/-1;
the components comprise: KC basal medium, 6-BA (6-benzyladenine) 1.5mg/L, NAA (naphthylacetic acid) 0.6mg/L, sucrose 20g/L, coconut juice 100mL/L, activated carbon 1g/L, agar 4.7 g/L;
the proliferation and differentiation medium formula comprises the following components:
1/2MS basal medium, NAA (naphthalene acetic acid) 0.1mg/L, 6-BA (6-benzyl adenine) 2mg/L, sucrose 20g/L, coconut juice 100ml/L, agar 4.5 g/L.
Preferably, the tissue culture method comprises the steps of:
s1, picking up and wiping clean the mature and uncracked celosia angulatus capsules with 75% alcohol after pollination for 8 months;
s2, placing the wiped capsule into a seed inoculating tray of a clean bench, soaking the capsule in 75% alcohol for 30S, then disinfecting the surface with 0.1% mercury bichloride for 15min, washing with sterile water for 4 times, and sucking water with sterile filter paper for standby;
s3, cutting off two ends of the capsule by using a disinfection blade, longitudinally splitting the capsule, taking out the seeds, placing the seeds in an environment-friendly tea bag, and soaking the whole tea bag in 0.2mol/L NaClO for 15min for pretreatment;
s4, washing the pretreated seeds twice with sterile water, tearing the tea bag containing the seeds with a pair of sterilized tweezers, scraping the seeds from the tea bag with a blade, placing the seeds on the surface of a seeding culture medium, dripping 1-2 drops of sterile water, shaking up, and uniformly seeding the seeds on the surface of the seeding culture medium;
after sowing, placing the seeds in dark culture until the seeds begin to germinate;
and S5, when the seeds sprout and grow into protocorms, inoculating the protocorms into a proliferation and differentiation culture medium for proliferation, and when the protocorms proliferate to a certain amount, performing root bud differentiation culture.
Preferably, the temperature of the culture chamber is 26 +/-2 ℃, and the illumination is 1500-.
Preferably, the size of the tea bag in step S3 is 4 × 6 cm.
Preferably, the dark culture method in step S4 is: and placing the seeding culture medium on a culture frame, covering the seeding culture medium by using black canvas with the shading rate of 99%, and uncovering the black cloth after germination begins.
The invention has the beneficial effects that:
the method can shorten the germination period of the seeds of the winter phoenix orchids to 80 days from the germination of 3-5 months of conventional sowing, and the germinated rootstocks are strong and plump, and have strong differentiation capability, high speed and high quality.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows that the Yufenglan is sowed aseptically for 80 days and starts to germinate;
FIG. 2 shows the proliferation of protocorm of Douglas while differentiating yellow-green leaf buds.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention, and that those not specifically noted in the examples are performed according to the techniques or conditions described in the literature in the art or according to the product specification.
The formula of the sterile seeding culture medium for the eupatorium dongpanense is characterized by comprising a seeding culture medium and a proliferation and differentiation culture medium, wherein the seeding culture medium formula comprises:
KC +6-BA 1.5+ NAA 0.6+ sucrose 20+ coconut juice 100+ active carbon 1+ agar 4.7, and the PH value is adjusted to 5.6 +/-1;
the components comprise: KC basal medium, 6-BA (6-benzyladenine) 1.5mg/L, NAA (naphthylacetic acid) 0.6mg/L, sucrose 20g/L, coconut juice 100mL/L, activated carbon 1g/L, agar 4.7 g/L;
the proliferation and differentiation medium formula comprises the following components:
1/2MS basal medium, NAA (naphthalene acetic acid) 0.1mg/L, 6-BA (6-benzyl adenine) 2mg/L, sucrose 20g/L, coconut juice 100ml/L, agar 4.5 g/L.
A tissue culture method of a culture medium formula for sterile sowing of Dougeny orchids comprises the following steps:
s1, picking up and wiping clean the mature and uncracked celosia angulatus capsules with 75% alcohol after pollination for 8 months;
s2, placing the wiped capsule into a seed inoculating tray of a clean bench, soaking the capsule in 75% alcohol for 30S, then disinfecting the surface with 0.1% mercury bichloride for 15min, washing with sterile water for 4 times, and sucking water with sterile filter paper for standby;
s3, cutting off two ends of the capsule by using a disinfection blade, longitudinally splitting the capsule, taking out the seeds, placing the seeds in an environment-friendly tea bag with the diameter of 4 multiplied by 6 cm, and soaking the whole tea bag in 0.2mol/L NaClO for 15min for pretreatment;
s4, washing the pretreated seeds twice with sterile water, tearing the tea bag containing the seeds with a pair of sterilized tweezers, scraping the seeds from the tea bag with a blade, placing the seeds on the surface of a seeding culture medium, dripping 1-2 drops of sterile water, shaking up, and uniformly seeding the seeds on the surface of the seeding culture medium;
after sowing, placing the seeds in dark culture until the seeds begin to germinate; the dark culture method comprises the following steps: and placing the seeding culture medium on a culture frame, covering the seeding culture medium by using black canvas with the shading rate of 99%, and uncovering the black cloth after germination begins.
And S5, inoculating the protocorm to a proliferation and differentiation culture medium for proliferation when the seeds sprout and grow into stems, and performing root bud differentiation culture when the protocorm proliferates to a certain amount.
In this example, the temperature of the culture chamber is 26 + -2 deg.C, the illumination is 1500-.
The sterile sowing culture medium and the tissue culture method for the cymbidium dongii can solve the problems of extremely low propagation coefficient, limited cultivation quantity and the like of the cymbidium dongii, can shorten the germination period of the cymbidium dongii seeds to 80 days from 3-5 months of conventional sowing, and have the advantages of high germination rate, strong proliferation capacity of protocorm, high speed and high quality.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. The formula of the sterile seeding culture medium for the eupatorium dongpanense is characterized by comprising a seeding culture medium and a proliferation and differentiation culture medium, wherein the seeding culture medium formula comprises:
KC +6-BA 1.5+ NAA 0.6+ sucrose 20+ coconut juice 100+ active carbon 1+ agar 4.7, and the PH value is adjusted to 5.6 +/-1;
the components comprise: KC basal medium, 6-BA (6-benzyladenine) 1.5mg/L, NAA (naphthylacetic acid) 0.6mg/L, sucrose 20g/L, coconut juice 100mL/L, activated carbon 1g/L, agar 4.7 g/L;
the proliferation and differentiation medium formula comprises the following components:
1/2MS basal medium, NAA (naphthalene acetic acid) 0.1mg/L, 6-BA (6-benzyl adenine) 2mg/L, sucrose 20g/L, coconut juice 100ml/L, agar 4.5 g/L.
2. The formulation of sterile sowing medium of eupatorium dongplanthi according to claim 1, wherein the tissue culture method comprises the following steps:
s1, picking up and wiping clean the mature and uncracked celosia angulatus capsules with 75% alcohol after pollination for 8 months;
s2, placing the wiped capsule into a seed inoculating tray of a clean bench, soaking the capsule in 75% alcohol for 30S, then disinfecting the surface with 0.1% mercury bichloride for 15min, washing with sterile water for 4 times, and sucking water with sterile filter paper for standby;
s3, cutting off two ends of the capsule by using a disinfection blade, longitudinally splitting the capsule, taking out the seeds, placing the seeds in an environment-friendly tea bag, and soaking the whole tea bag in 0.2mol/L NaClO for 15min for pretreatment;
s4, washing the pretreated seeds twice with sterile water, tearing the tea bag containing the seeds with a pair of sterilized tweezers, scraping the seeds from the tea bag with a blade, placing the seeds on the surface of a seeding culture medium, dripping 1-2 drops of sterile water, shaking up, and uniformly seeding the seeds on the surface of the seeding culture medium;
after sowing, placing the seeds in dark culture until the seeds begin to germinate;
and S5, when the seeds sprout out of the protocorm, inoculating the protocorm into a proliferation and differentiation culture medium for proliferation, and when the protocorm is proliferated to a certain amount, performing root bud differentiation culture.
3. The formulation of sterile culture medium for sowing of Yufenglan as claimed in claim 2, wherein the temperature of the culture chamber is 26 ± 2 ℃, the illumination after germination is 1500-.
4. The formulation of sterile culture medium for sowing Yuefeng orchid and the tissue culture method according to claim 2, wherein the size of the environment-friendly tea bag in step S3 is 4 x 6 cm.
5. The formulation of sterile culture medium for sowing eupatorium dongpenii as claimed in claim 2, and the tissue culture method thereof, wherein the dark culture method in step S4 comprises: and placing the seeding culture medium on a culture frame, covering the seeding culture medium by using black canvas with the shading rate of 99%, and uncovering the black cloth after germination begins.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111283028.7A CN114009338A (en) | 2021-11-01 | 2021-11-01 | Formula of sterile sowing culture medium for eupatorium donyanense and tissue culture method |
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| CN202111283028.7A CN114009338A (en) | 2021-11-01 | 2021-11-01 | Formula of sterile sowing culture medium for eupatorium donyanense and tissue culture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115589948A (en) * | 2022-10-25 | 2023-01-13 | 云南省林业和草原科学院（Cn） | Bletilla striata non-symbiotic germination culture medium and propagation method |
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| US20160143236A1 (en) * | 2013-07-08 | 2016-05-26 | South China Botanical Garden, Chinese Academy Of Sciences | Dendrobium in vitro crossbreeding method |
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| CN113179948A (en) * | 2021-04-22 | 2021-07-30 | 广西壮族自治区农业科学院 | Formula of sterile seeding culture medium for cymbidium bicolor and tissue culture method |
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2021
- 2021-11-01 CN CN202111283028.7A patent/CN114009338A/en active Pending
Patent Citations (3)
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| US20160143236A1 (en) * | 2013-07-08 | 2016-05-26 | South China Botanical Garden, Chinese Academy Of Sciences | Dendrobium in vitro crossbreeding method |
| WO2016138688A1 (en) * | 2015-03-03 | 2016-09-09 | 佛山市顺德区今日景艺生物科技有限公司 | Rhynchostylis lateral bud inducing medium and culture method therefor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115589948A (en) * | 2022-10-25 | 2023-01-13 | 云南省林业和草原科学院（Cn） | Bletilla striata non-symbiotic germination culture medium and propagation method |
| CN115589948B (en) * | 2022-10-25 | 2023-09-01 | 云南省林业和草原科学院 | Rhizoma bletillae non-symbiotic germination medium and propagation method |
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