CN110521591B - Rapid culture method of anthurium - Google Patents
Rapid culture method of anthurium Download PDFInfo
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- CN110521591B CN110521591B CN201910525562.0A CN201910525562A CN110521591B CN 110521591 B CN110521591 B CN 110521591B CN 201910525562 A CN201910525562 A CN 201910525562A CN 110521591 B CN110521591 B CN 110521591B
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- 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
- A01H4/001—Culture apparatus for tissue culture
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- 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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Abstract
The invention relates to a rapid culture method of anthurium andraeanum, which comprises aseptic seedling culture, protoplast separation, protoplast culture and protoplast plant regeneration, wherein the osmotic pressure and the cell density are reduced by adding a second MS culture medium with the same quantity in the regeneration stage of the protoplast plant and selecting and proportioning the components of the culture medium, and the combined use of a culture plate and a third MS culture medium ensures that plants come from single cells and improves the germination rate and the regeneration speed.
Description
Technical Field
The application belongs to the technical field of plant culture, and particularly relates to a method for culturing anthurium.
Background
The anthurium isClass MonocotyledonaeAraceae (Araceae family)Huazhu genusEvergreen herbaceous plants are grown for many years. The stem nodes are short; the leaves emerge from the base, green, leathery, full-limbed, oblong heart-shaped or oval heart-shaped. Slender leaf stalk, flat spat, leathery texture with waxy luster, orange red or scarlet; the panicle inflorescence is yellow, and can continuously bloom throughout the year. The anthurium andraeanum is originally produced in the heat zone rainforest areas of costabia, columbia and the like. It usually attaches to trees, sometimes it attaches to rocks or directly grows on the ground, and the nature likes warm, humid and semi-yin environment, and it is forbidden to direct sunlight. Flower candle flower appearance is peculiar, beautiful and beautiful. The flowering period is long, and the method is suitable for potted plant, cut flower or garden shading place cluster planting and beautifying.
Protoplasts are naked, viable protoplasts that have been freed of plant cell walls by a special process. Has no cell wall, but has all the characteristics of a living cell. It has no cell wall barrier, can be conveniently used for genetic manipulation, and can be used for treatingFilm，OrganelleEtc. to conduct basic research; has totipotency, and can be cultured and developed completelyPlant, its production method and use(ii) a Protoplasts are suitable for induction fusion to form hybrid cells.
MSCulture mediumIs currently the most commonly used medium. It has high inorganic salt concentration, and can ensure tissue growthMineral nutritionCan also accelerateCallus tissueThe growth of (2). Due to high ion concentration in the formula, the composition can be prepared, stored and eliminatedEven if some components are slightly mixed in and out in the course of toxicity, the balance among ions is not affected. MS (Mass Spectrometry)Solid culture mediumCan be used for inducing callus, and can also be used for embryo, stem segment,Stem tipAnd cultivation of anthers, whichLiquid culture mediumSignificant success was achieved when used in cell suspension culture. The amount and proportion of the inorganic nutrients in the MS culture medium are proper enough to meet the needs of plant cells in nutrition and physiology. Therefore, in general, no further addition is necessaryAmino acids、Casein proteinHYPERLINK "https://baike.baidu.com/item/%E6%B0%B4%E8%A7%A3"HydrolysisOrganic additives such as yeast extract and coconut milk. And othersCulture mediumIn MS medium, in comparison with the basic componentsNitrate saltPotassium, andammonium saltIs high, which is a remarkable characteristic.
In the prior art, a method for separating protoplasts is adopted to culture anthurium andraeanum, a single type of culture medium is adopted, the method is low in regeneration speed, low in germination rate and unstable in bud growth, and therefore how to provide a method for quickly culturing anthurium andraeanum is an urgent problem to be solved in the field.
Disclosure of Invention
The invention aims to provide a method for quickly culturing anthurium andraeanum, which has the advantages of high regeneration speed, high germination rate and stable bud growth. Specifically, the anthurium culturing method sequentially comprises the following steps:
(1) and (3) sterile seedling culture: taking anthurium seedlings cultured outside a hormone-free MS culture medium as a material, and performing illumination for 20 hours; (2) protoplast separation: selecting young leaves, cutting into strips, placing in a flask containing enzyme solution containing 5% (w/v) cellulase R-10, 0.5% (w/v) pectinase Y-23 and 10% (w/v) mannitol, stirring, allowing the enzyme solution to permeate through a vacuum pump, pouring the enzyme solution and the young leaf strips into a culture dish, culturing at 25 deg.C for 30 min, stirring for 10s, and culturing at 25 deg.C for 30 min; the separated protoplasts were filtered through a screen and centrifuged at 100 x g for 10 minutes to suspend the protoplasts in a 20% (w/v) sucrose solution, 10% (w/v) mannitol solution was gently dropped on top of the suspension, and after centrifugation at 50 Xg for 10 minutes, the intact protoplasts at the interface were aspirated through a pipette and washed twice in 10% (w/v) mannitol; (3) protoplast culture: culturing protoplasts in a culture dish in a first MS medium comprising MS salts, thiamine hydrochloride in an amount of 0.3 mg/L, NAA in an amount of 0.5mg/L, BAP in an amount of 0.1 mg/L, glucose in an amount of 10% (w/v) and sucrose in an amount of 6% (w/v), the pH of the first MS medium being 5.5; (4) regeneration of protoplast plants: adding an equal amount of a second MS medium containing MS salt, 0.3 mg/L thiamine hydrochloride, 0.5mg/L NAA, 0.1 mg/L BAP, 5% (w/v) glucose and 3% (w/v) sucrose to the culture dish of step (3) every 5 days until the colony grows to 0.2-0.3mm, then transferring the colony to a culture plate comprising a filter paper substrate and a transfer plate, which is placed on a third MS medium for 10-20 days, then placing the individual callus on the third MS medium for 14-20 days to produce 2-6 mm callus, transferring the callus to HSM medium for regeneration, after germination, transferring to hormone-free MS medium, and then transplanting into soil after 7-10 days. Preferably, the third MS medium contains MS salts, vitamins, 0.5mg/L BAP, 1 mg/L NAA, 3% (w/v) sucrose and 0.6% (w/v) agar, and the pH of the third MS medium is 5.8. Preferably, the HSM medium contains MS salts, vitamin B5, 1 mg/L BAP, 0.1 mg/L NAA and 3% (w/v) sucrose, and the pH of the HSM medium is 5.8.
The beneficial effects of the invention are that the second MS culture medium with the same quantity is added in the regeneration stage of the protoplast plant, the osmotic pressure and the cell density are reduced by the component selection and the proportion of the culture medium, and the combination of the culture plate and the third MS culture medium ensures that the plant comes from a single cell, and the germination rate and the regeneration speed are improved.
Detailed Description
Example 1
As a preferred embodiment, the cultivation of anthurium is carried out by the following steps: (1) and (3) sterile seedling culture: taking anthurium seedlings cultured outside a hormone-free MS culture medium as a material, and performing illumination for 20 hours; (2) protoplast separation: selecting young leaves, cutting into strips, placing in a flask containing enzyme solution containing 5% (w/v) cellulase R-10, 0.5% (w/v) pectinase Y-23 and 10% (w/v) mannitol, stirring, allowing the enzyme solution to permeate through a vacuum pump, pouring the enzyme solution and the young leaf strips into a culture dish, culturing at 25 deg.C for 30 min, stirring for 10s, and culturing at 25 deg.C for 30 min; the separated protoplasts were filtered through a screen and centrifuged at 100 x g for 10 minutes to suspend the protoplasts in a 20% (w/v) sucrose solution, 10% (w/v) mannitol solution was gently dropped on top of the suspension, and after centrifugation at 50 Xg for 10 minutes, the intact protoplasts at the interface were aspirated through a pipette and washed twice in 10% (w/v) mannitol; (3) protoplast culture: culturing protoplasts in a culture dish in a first MS medium comprising MS salts, thiamine hydrochloride in an amount of 0.3 mg/L, NAA in an amount of 0.5mg/L, BAP in an amount of 0.1 mg/L, glucose in an amount of 10% (w/v) and sucrose in an amount of 6% (w/v), the pH of the first MS medium being 5.5; (4) regeneration of protoplast plants: adding an equal amount of a second MS medium containing MS salt, 0.3 mg/L thiamine hydrochloride, 0.5mg/L NAA, 0.1 mg/L BAP, 5% (w/v) glucose and 3% (w/v) sucrose to the culture dish of step (3) every 5 days until the colony grows to 0.2-0.3mm, then transferring the colony to a culture plate comprising a filter paper substrate and a transfer plate, which is placed on a third MS medium for 10-20 days, then placing the individual callus on the third MS medium for 14-20 days to produce 2-6 mm callus, transferring the callus to HSM medium for regeneration, after germination, transferring to hormone-free MS medium, and then transplanting into soil after 7-10 days. Wherein the third MS medium comprises MS salts, vitamins, 0.5mg/L BAP, 1 mg/L NAA, 3% (w/v) sucrose and 0.6% (w/v) agar, and the pH of the third MS medium is 5.8; HSM medium contains MS salt, vitamin B5, 1 mg/L BAP, 0.1 mg/L NAA and 3% (w/v) sucrose, and has pH of 5.8.
Comparative example 1
As a comparative example, the cultivation of anthurium was carried out by the following procedure: (1) and (3) sterile seedling culture: taking anthurium seedlings cultured outside a hormone-free MS culture medium as a material, and performing illumination for 20 hours; (2) protoplast separation: selecting young leaves, cutting into strips, placing in a flask containing enzyme solution containing 5% (w/v) cellulase R-10, 0.5% (w/v) pectinase Y-23 and 10% (w/v) mannitol, stirring, allowing the enzyme solution to permeate through a vacuum pump, pouring the enzyme solution and the young leaf strips into a culture dish, culturing at 25 deg.C for 30 min, stirring for 10s, and culturing at 25 deg.C for 30 min; the separated protoplasts were filtered through a screen and centrifuged at 100 x g for 10 minutes to suspend the protoplasts in a 20% (w/v) sucrose solution, 10% (w/v) mannitol solution was gently dropped on top of the suspension, and after centrifugation at 50 Xg for 10 minutes, the intact protoplasts at the interface were aspirated through a pipette and washed twice in 10% (w/v) mannitol; (3) protoplast culture: culturing protoplasts in a culture dish in a first MS medium comprising MS salts, thiamine hydrochloride in an amount of 0.3 mg/L, NAA in an amount of 0.5mg/L, BAP in an amount of 0.1 mg/L, glucose in an amount of 10% (w/v) and sucrose in an amount of 6% (w/v), the pH of the first MS medium being 5.5; (4) regeneration of protoplast plants: adding an equal amount of the first MS medium to the culture dish of step (3) every 5 days until the colony grows to 0.2-0.3mm, then transferring the colony to a culture plate comprising a filter paper substrate and a transfer plate, the culture plate being placed on the third MS medium for 10-20 days, then placing the single callus on the third MS medium for 14-20 days to produce 2-6 mm callus, then transferring the callus to HSM medium for regeneration, after germination, transferring to hormone-free MS medium, and then transplanting into soil after 7-10 days. Wherein the third MS medium comprises MS salts, vitamins, 0.5mg/L BAP, 1 mg/L NAA, 3% (w/v) sucrose and 0.6% (w/v) agar, and the pH of the third MS medium is 5.8; HSM medium contains MS salt, vitamin B5, 1 mg/L BAP, 0.1 mg/L NAA and 3% (w/v) sucrose, and has pH of 5.8.
Comparative example 2
As another comparative example, the cultivation of anthurium was performed by the following procedure: (1) and (3) sterile seedling culture: taking anthurium seedlings cultured outside a hormone-free MS culture medium as a material, and performing illumination for 20 hours; (2) protoplast separation: selecting young leaves, cutting into strips, placing in a flask containing enzyme solution containing 5% (w/v) cellulase R-10, 0.5% (w/v) pectinase Y-23 and 10% (w/v) mannitol, stirring, allowing the enzyme solution to permeate through a vacuum pump, pouring the enzyme solution and the young leaf strips into a culture dish, culturing at 25 deg.C for 30 min, stirring for 10s, and culturing at 25 deg.C for 30 min; the separated protoplasts were filtered through a screen and centrifuged at 100 x g for 10 minutes to suspend the protoplasts in a 20% (w/v) sucrose solution, 10% (w/v) mannitol solution was gently dropped on top of the suspension, and after centrifugation at 50 Xg for 10 minutes, the intact protoplasts at the interface were aspirated through a pipette and washed twice in 10% (w/v) mannitol; (3) protoplast culture: culturing protoplasts in a culture dish in a first MS medium comprising MS salts, thiamine hydrochloride in an amount of 0.3 mg/L, NAA in an amount of 0.5mg/L, BAP in an amount of 0.1 mg/L, glucose in an amount of 10% (w/v) and sucrose in an amount of 6% (w/v), the pH of the first MS medium being 5.5; (4) regeneration of protoplast plants: adding an equal amount of a second MS medium containing MS salt, 0.3 mg/L thiamine hydrochloride, 0.5mg/L NAA, 0.1 mg/L BAP, 5% (w/v) glucose and 3% (w/v) sucrose to the culture dish of step (3) every 5 days until the colony grows to 0.2-0.3mm, then transferring the colony to a third MS medium for 10-20 days, then placing the individual callus on the third MS medium for 14-20 days to produce 2-6 mm callus, then transferring the callus to HSM medium for regeneration, after germination, transferring to hormone-free MS medium, and then transplanting into soil after 7-10 days. Wherein the third MS medium comprises MS salts, vitamins, 0.5mg/L BAP, 1 mg/L NAA, 3% (w/v) sucrose and 0.6% (w/v) agar, and the pH of the third MS medium is 5.8; HSM medium contains MS salt, vitamin B5, 1 mg/L BAP, 0.1 mg/L NAA and 3% (w/v) sucrose, and has pH of 5.8.
The ratio of germination, the germination time and the growth of sprouts in example 1, comparative example 1 and comparative example 2 were compared and the results are shown in the following table:
| protoplast regeneration time | Proportion of sprouts | |
| Example 1 | 35-40 days | 78% |
| Comparative example 1 | 60-80 days | 55% |
| Comparative example 2 | 45-70 days | 65% |
From the above table, it can be seen that the invention reduces osmotic pressure and cell density by adding the same amount of the second MS culture medium in the regeneration stage of the protoplast plant and by selecting and proportioning the components of the culture medium, and moreover, the combination of the culture plate and the third MS culture medium ensures that the plant comes from a single cell, and improves germination rate and regeneration speed.
Claims (1)
1. A rapid culture method of anthurium andraeanum is characterized by comprising the following steps: (1) and (3) sterile seedling culture: taking anthurium seedlings cultured outside a hormone-free MS culture medium as a material, and performing illumination for 20 hours; (2) protoplast separation: selecting young leaves, cutting into strips, placing in a flask containing enzyme solution containing 5% (w/v) cellulase R-10, 0.5% (w/v) pectinase Y-23 and 10% (w/v) mannitol, stirring, allowing the enzyme solution to permeate through a vacuum pump, pouring the enzyme solution and the young leaf strips into a culture dish, culturing at 25 deg.C for 30 min, stirring for 10s, and culturing at 25 deg.C for 30 min; the separated protoplasts were filtered through a screen and centrifuged at 100 x g for 10 minutes to suspend the protoplasts in a 20% (w/v) sucrose solution, 10% (w/v) mannitol solution was gently dropped on top of the suspension, and after centrifugation at 50 Xg for 10 minutes, the intact protoplasts at the interface were aspirated through a pipette and washed twice in 10% (w/v) mannitol; (3) protoplast culture: culturing protoplasts in a culture dish in a first MS medium comprising MS salts, thiamine hydrochloride in an amount of 0.3 mg/L, NAA in an amount of 0.5mg/L, BAP in an amount of 0.1 mg/L, glucose in an amount of 10% (w/v) and sucrose in an amount of 6% (w/v), the pH of the first MS medium being 5.5; (4) regeneration of protoplast plants: adding an equal amount of a second MS medium containing MS salts, 0.3 mg/L thiamine hydrochloride, 0.5mg/L NAA, 0.1 mg/L BAP, 5% (w/v) glucose and 3% (w/v) sucrose to the culture dish of step (3) every 5 days until the colony grows to 0.2-0.3mm, transferring the colony to a culture plate comprising a filter paper substrate and a transfer plate, the culture plate being placed on a third MS medium for 10-20 days, then placing the individual callus on the third MS medium for 14-20 days to produce 2-6 mm callus, transferring the callus to HSM medium for regeneration, after germination, transferring to hormone-free MS medium, and then transplanting into soil after 7-10 days, Vitamins, 0.5mg/L BAP, 1 mg/L NAA, 3% (w/v) sucrose and 0.6% (w/v) agar, the pH of the third MS medium was 5.8, the HSM medium contained MS salts, vitamin B5, 1 mg/L BAP, 0.1 mg/L NAA and 3% (w/v) sucrose, the pH of the HSM medium was 5.8.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103181326A (en) * | 2013-04-10 | 2013-07-03 | 苏州大学 | In vitro tissue cultivation method of potted anthurium andraeanum varieties |
| CN105010141A (en) * | 2015-07-09 | 2015-11-04 | 虞龙 | Anthurium rapid propagation method |
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| CN107099526A (en) * | 2017-05-09 | 2017-08-29 | 福建省林业科技试验中心 | Red palm somatic hybridization breeding method based on protoplast asymmetric fusion technology |
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2019
- 2019-06-18 CN CN201910525562.0A patent/CN110521591B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103181326A (en) * | 2013-04-10 | 2013-07-03 | 苏州大学 | In vitro tissue cultivation method of potted anthurium andraeanum varieties |
| CN105010141A (en) * | 2015-07-09 | 2015-11-04 | 虞龙 | Anthurium rapid propagation method |
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| CN107099526A (en) * | 2017-05-09 | 2017-08-29 | 福建省林业科技试验中心 | Red palm somatic hybridization breeding method based on protoplast asymmetric fusion technology |
Non-Patent Citations (2)
| Title |
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| 7个红掌品种产生愈伤组织的影响因素;张军等;《中国园艺文摘》;20111231(第2期);第21-22,52页 * |
| Effect of enzyme concentrations on protoplast isolation and protoplast culture of Spathiphyllum and Anthurium;Barbara Duquenne等;《Plant Cell,Tissue and Organ Culture》;20071231;第91卷;第165-173页 * |
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