CN115644064B - Method for establishing pineapple embryogenic cell suspension system from pineapple callus - Google Patents
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Abstract
The application discloses a method for establishing pineapple embryogenic cell suspension lines from pineapple callus. And (3) screening granular callus which is vigorous in growth and crisp and fragile, performing suspension culture through a liquid culture medium, and continuously subculturing while screening out large-grain callus to establish a pineapple embryogenic suspension cell line. In the pineapple embryogenic cell suspension system, the cell shape and the cell mass are uniform, the cell dispersibility is good, the cell growth and proliferation are rapid, and the pineapple embryogenic cell suspension system can be directly used for separation, culture, hybridization, gene transfer, production of secondary metabolites and the like of protoplasts, can be used as genetic transformation materials, can effectively avoid chimeric manifestation, and greatly improves the success rate and the efficiency of pineapple biological breeding.
Description
Technical Field
The invention relates to the technical field of plant cell culture, in particular to a method for establishing a pineapple embryogenic cell suspension system from pineapple callus.
Background
The plant embryogenic suspension cell line is embryogenic cell line cultured by suspending plant cells or smaller cell mass in a liquid culture medium, has the characteristics of uniform cell shape and cell mass, good cell dispersibility, rapid cell growth and proliferation and the like, can be directly used for separation, culture, hybridization, gene transfer, production of secondary metabolites and the like of protoplast, and is a good material for genetic transformation. The establishment of embryogenic cell suspension is affected by various factors such as plant varieties, explants, callus, culture medium, environmental factors and the like, plant cells are easier to agglomerate and are more sensitive to shearing force, and various factors restrict the establishment of plant embryogenic suspension cell lines. At present, a plurality of plants at home and abroad establish a perfect suspension cell line, and directly use suspension cells as materials to carry out genetic transformation operation, protoplast culture, somatic hybridization and other researches, but in pineapple biological breeding, researches and applications related to suspension cells are rarely seen.
Pineapple is an important tropical fruit in China, and main breeding modes include crossbreeding, bud mutation seed selection, mutation breeding and genetic engineering breeding. Traditional crossbreeding and bud mutation seed selection are time-consuming, labor-consuming and large in workload, breeding efficiency is low, pineapple callus is used as a transformation material for mutation breeding and genetic engineering breeding, but a great amount of chimerism phenomenon exists in transformation offspring easily due to the fact that the callus is a mixture of cell clusters and differentiated embryoids, separation selection is difficult, target characters are gradually lost in the culturing process of transgenic offspring, and finally breeding failure is caused. If the single cell, namely embryogenic suspension cell line is used as a transformation object, the generation of chimeric manifestation can be effectively avoided, and the success rate and the efficiency of pineapple biological breeding are greatly improved.
Disclosure of Invention
The invention aims to provide a method for establishing a pineapple embryogenic cell suspension system from pineapple callus so as to make up for the technical blank.
The scheme of the invention comprises the following main contents:
a method of establishing a pineapple embryogenic cell suspension from pineapple callus, comprising the steps of:
(1) Taking an explant, culturing the explant by adopting a callus culture medium, and selecting granular callus which grows vigorously and is crisp and fragile as a material for culturing suspension cells, so that the phenomenon of browning and whitening and the phenomenon of watery callus are avoided;
(2) Placing granular callus into a container, crushing, adding a suspension cell culture medium into the container, and carrying out constant-temperature shaking culture on a shaking table;
(3) Sieving large-particle callus in the cultured suspension culture, standing, sucking the upper liquid culture medium after the fine-particle culture is precipitated, adding fresh suspension cell culture medium, and performing constant-temperature shaking culture on a shaking table;
(4) Sieving large-particle callus, repeating the operation of the step (3), and carrying out constant-temperature shaking culture on a constant-temperature shaking table;
(5) And (3) carrying out subculture on the suspension culture, and taking the culture periodically for microscopic observation until the observed suspension cells are round or oval with uniform size and are composed of single cells and small cell clusters, and establishing a pineapple embryogenic cell suspension system without other irregularly-shaped cells and large cell clusters.
Preferably, the callus selected in step (1) is callus induced with crown buds, peri-buds, suction buds or tuber buds as explants for 5 to 6 weeks. Wherein, when the bud is used as an explant, the proliferation speed of the cells is the highest.
Preferably, the callus culture medium used in step (1) is: 13. Mu.M/L6-BA, 1. Mu.M/LNAA and 7g/L agar were added to the MS medium, pH=5.8; the callus culture condition is constant temperature 28 ℃ and dark culture. Studies show that the induction rate of the callus is obviously improved and the browning rate is obviously reduced after 13 mu M/L6-BA and 1 mu M/L NAA are added into the MS culture medium. And the callus grows vigorously, is crisp and fragile, and the pineapple embryogenic cell suspension system established based on the callus has the characteristics of high growth speed, good uniformity and the like.
Preferably, the suspension cell culture medium in step (2) comprises: 500-600 mg/L of ammonium chloride, 2000-3000 mg/L of potassium nitrate, 400-500 mg/L of calcium chloride, 300-400 mg/L of magnesium sulfate heptahydrate, 150-200 mg/L of monopotassium phosphate, 0.5-1.0 mg/L of potassium iodide, 6-7 mg/L of boric acid, 22-26 mg/L of manganese sulfate, 1.5-2.0 mg/L of zinc sulfate, 0.2-0.3 mg/L of sodium molybdate, 0.02-0.03 mg/L of copper sulfate, 0.02-0.03 mg/L of cobalt chloride, 35-40 mg/L of disodium edetate, 25-30 mg/L of ferrous sulfate, 1000-1200 mg/L of glutamine, and hydrolysate (namely enzyme hydrolyzed casein, prepared by hydrolyzing trypsin, and commercially available products) 500-600 mg/L of arginine, 100-150 mg/L of arginine, 1-3 mg/L of zinc sulfate, 0.3-0.6 mg/L of thiamine hydrochloride, 0.5-0.03 mg/L of pyridoxine hydrochloride, 0.5-7.05 mg/L of nicotinic acid, 0.07-10 mg/L of nicotinic acid, 0.5-300 mg/L of boric acid, and 10.5-300 mg/L of activated carbon, and 10.07-10 mg/L of sucrose. Within the above range, similar results to those of example 1 were obtained.
More preferably, the suspension cell culture medium in step (2) is: with water as a solvent, 535mg/L ammonium chloride, 2528mg/L potassium nitrate, 440mg/L calcium chloride, 370mg/L magnesium sulfate heptahydrate, 170mg/L potassium dihydrogen phosphate, 0.83mg/L potassium iodide, 6.2mg/L boric acid, 22.3mg/L manganese sulfate, 1.72mg/L zinc sulfate, 0.25mg/L sodium molybdate, 0.025mg/L copper sulfate, 0.025mg/L cobalt chloride, 37.3mg/L disodium ethylenediamine tetraacetate, 27.8mg/L ferrous sulfate, 1000mg/L glutamine, 500mg/L casein hydrolysate, 120mg/L arginine, 2mg/L glycine, 0.4mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 100mg/L inositol, 5000mg/L sucrose, 9.9mg/L picloram, 0.053mg/L abscisic acid, 2000mg/L activated carbon, 2-300 mg/L morpholine and pH=5.8.
Preferably, the culture conditions of the suspension cells in step (2) are: the constant temperature is 28-30 ℃, the rotating speed is 80-100 rpm, and the dark culture is carried out for 7-8 days.
Preferably, the step (3) is: and (3) screening out large-particle callus in the suspension culture after 7d of culture by using a 30-mesh screen, standing, sucking the upper liquid culture medium after the fine-particle culture is precipitated, adding a fresh suspension cell culture medium, and carrying out constant-temperature shaking culture on a shaking table.
Preferably, in step (4), the large particle callus is removed by a 50 mesh screen.
Preferably, the variety of the pineapple comprises table pesticide 17, golden diamond, sweet honey or golden pineapple.
Preferably, the microscopic observation in step (5) of the present invention can be performed by directly sucking the cell suspension into a special culture dish for microscopic observation. The cell suspension may also be combined with 1% chromium trioxide (CrO) 3 ) The solutions were mixed 1:1, placed in a microscope-dedicated dish for staining observation in a water bath at 70℃for 15 minutes.
The beneficial effects obtained by the invention are as follows:
the pineapple embryogenic cell suspension system established by the method provided by the invention has the following characteristics: (1) the suspension cells have good dispersibility and uniformity, the cell shape and the cell mass size are approximately the same, the appearance of the suspension system is small particles with uniform size, and the suspension system is observed to be cells or small cell masses with approximately the same volume and shape under a microscope; the growth speed of the suspension cells is high, and the amount of the cells can be increased by 1 time in a period of 2-3 days or even shorter. The obtained embryogenic cell suspension system has stable growth, good cell activity and high cell proliferation speed.
The embryogenic suspension cell line can be directly used for protoplast separation, culture and hybridization, gene transfer, production of secondary metabolites and the like, can screen out expected mutants in a cell level in a short period, and can greatly improve the efficiency of biological breeding. The preparation method disclosed by the invention is simple in steps and does not need complicated equipment.
Drawings
FIG. 1 shows round or oval embryogenic cells dispersed in the examples
Detailed Description
In order to facilitate the understanding of the technical content of the present invention, the present invention will be described in further detail with reference to specific examples.
EXAMPLE 1 method for establishing pineapple embryogenic cell suspension System from pineapple callus
1) The crown bud stem segment of the good pineapple variety Tainong 17 is used as an explant, a callus culture medium is used for culturing for 5 weeks to obtain a large amount of calli, and granular calli which grow vigorously and are crisp in the same batch are selected as a material for culturing suspension cells, so that the phenomena of browning, whitening and watery calli are avoided.
The callus culture medium is: MS medium +13. Mu.M/L6-BA (6-benzylaminopurine) +1. Mu.M/L NAA (naphthylacetic acid) +7g/L agar, pH=5.8. The culture condition is 28 ℃ constant temperature and dark culture.
2) The callus (about 1 g) is put into a 200mL triangular flask, gently crushed by forceps, 50mL of suspension cell culture medium is sucked by a pipette and added into the triangular flask, simultaneously, larger callus blocks in the triangular flask are crushed by the pipette tip, the opening of the triangular flask is plugged by cotton wrapped by gauze, and then the opening is sequentially wrapped by a bottle sealing film and tinfoil. Dark culture was performed on a shaker at a constant temperature of 28℃and a rotation speed of 80rpm for 7d.
Wherein the suspension cell culture medium uses water as a solvent, contains 535mg/L ammonium chloride, 2528mg/L potassium nitrate, 440mg/L calcium chloride, 370mg/L magnesium sulfate heptahydrate, 170mg/L potassium dihydrogen phosphate, 0.83mg/L potassium iodide, 6.2mg/L boric acid, 22.3mg/L manganese sulfate, 1.72mg/L zinc sulfate, 0.25mg/L sodium molybdate, 0.025mg/L copper sulfate, 0.025mg/L cobalt chloride, 37.3mg/L disodium ethylenediamine tetraacetate, 27.8mg/L ferrous sulfate, 1000mg/L glutamine, 500mg/L casein hydrolysate, 120mg/L arginine, 2mg/L glycine, 0.4mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 100mg/L inositol, 5000mg/L sucrose, 9.9mg/L picloramine, 0.053mg/L abscisic acid, 2000mg/L activated carbon, 2-300 mg/L morpholine and pH=5.
3) The suspension culture after 7d was screened out large particle callus with a 30 mesh (pore size 600 μm) stainless steel screen, left to stand until fine particle culture precipitated, then the upper liquid medium was aspirated, fresh suspension cell medium was added again, and dark culture was performed on a shaker at a constant temperature of 28℃and a rotation speed of 80rpm for 7d.
4) The suspension culture was screened out of large-particle callus with a 50 mesh (pore size 280 μm) stainless steel screen, left to stand until fine-particle culture precipitated, then the upper liquid medium was aspirated, fresh suspension cell medium was added, and dark culture was performed on a shaker at a constant temperature of 28℃and a rotation speed of 80rpm for 7d.
5) Standing the suspension culture, sucking the upper liquid culture medium after the fine particle culture is precipitated, and adding a fresh culture medium for subculture. The subculture was performed once every 7d of culture.
6) 3-5 mL of culture was aspirated for microscopic observation at each subculture, and after the second subculture (culture 28 d) it was observed that there were already partially dispersed round or oval cells in the culture, but there were still many irregularly shaped cells and larger cell clusters.
7) When subcultured five to seven times (cultures 49d to 63 d), most of the cultures were round or oval single cells of uniform size or small cell clusters of 2 to 30 cells were observed.
The results are shown in FIG. 1. The results show that the pineapple embryogenic cell suspension system established by the invention has the following characteristics:
(1) the suspension cells have good dispersibility and uniformity, the cell shape and the cell mass size are approximately the same, the appearance of the suspension system is small particles with uniform size, and the suspension system is observed to be cells or small cell masses with approximately the same volume and shape under a microscope;
(2) the growth speed of the suspension cells is high, and the amount of the 2-3 d cells can be increased by 1 time.
Example 2
In this example, callus was obtained by culturing on the respective 17 varieties pineapple-bud, bud-sucking and tuber-bud explants. The other steps were the same as in example 1.
The results show that when the culture is subcultured five to seven times (49 d to 63 d), most of the culture is round or oval single cells with uniform size or small cell clusters of 2 to 30 cells, the formed embryogenic cell suspension system grows stably, the cell activity is good, and the cell proliferation speed is high. Wherein the proliferation rate of the cells is higher than that of the buds of the whole-grain system.
Example 3
In the embodiment, the used callus is callus obtained by culturing golden diamond, sweet and pineapple crown bud stem segments of golden pineapple varieties as explants. The other steps were the same as in example 1.
The results show that no obvious difference exists between varieties in the culture process, and an embryogenic cell suspension system with stable growth, good cell activity and high cell proliferation speed can be formed.
Comparative example 1
In this example, the suspension cell culture medium formulation used was MS medium +13. Mu.M/L6-BA (6-benzylaminopurine) +1. Mu.M/L NAA (naphthylacetic acid), pH=5.8. The other steps were the same as in example 1.
The results show that:
1) At the time of the secondary (culture 21 d), it was observed that there were partially dispersed cells in the culture, mostly irregularly shaped cells and larger cell clusters.
2) On secondary subculture (culture 28 d), more dispersed cells in the culture were observed, mostly irregularly shaped cells and larger cell clusters.
3) At three times of subculture (35 d), the cells appeared to be significantly brown in the culture solution, and the number of cells was not significantly increased.
Comparative example 2
In this case, the concentration of sucrose in the suspension cell culture medium used was 1g/L, 10g/L, 20g/L, 30g/L, respectively. The other steps were the same as in example 1.
The results show that:
1) After one subculture (culture 21 d), it was observed that there were partially dispersed suspension cells in each of the different sucrose concentration media, wherein the cell number in the 1g/L sucrose concentration media was the smallest, followed by 30g/L sucrose concentration media, and the cell numbers in the 10g/L and 20g/L sucrose concentrations were not significantly different from those in example 1.
2) At the secondary time (28 d culture), the cell number in the culture medium with the concentration of 1g/L sucrose is not obviously increased compared with that at the primary time of the secondary culture, and the cell numbers in the culture medium with the concentration of 10g/L, 20g/L and 30g/L sucrose are increased, but the increase amount is less than that of the example 1.
3) At five times of subculture (culture 49 d), the cell number in the 1g/L sucrose concentration medium was continuously reduced by changing the fresh medium at the time of subculture, the cell number in the 20g/L and 30g/L sucrose concentrations medium was also reduced, and many floating impurities and cell debris were present in the liquid medium.
4) Seven times (63 d) following the subculture, the medium at a sucrose concentration of only 10g/L and the number of cells in example 1 were still increasing, but the increase was still smaller than in example 1.
Comparative example 3
In this example, the concentration of abscisic acid in the suspension cell culture medium used was 0mg/L and 0.1mg/L, respectively. The other steps were the same as in example 1.
The results show that:
1) After one time of subculture (culture 21 d), the culture media with different abscisic acid concentrations all have partially dispersed suspension cells, wherein the culture media with 0mg/L abscisic acid concentration is mostly irregularly shaped cells and larger cell clusters, and the number of cells in the culture media with 0.1mg/L abscisic acid concentration is obviously smaller.
2) In the secondary culture (culture 28 d), more dispersed cells, mostly irregularly shaped cells and larger cell clusters were observed.
3) At three times of subculture (35 d), the cells in the culture solution appeared to be significantly brown, and the number of cells was not significantly increased.
The above-mentioned embodiments are only some of the preferred embodiments of the present invention, and are not intended to limit the present invention, but the scope of the present invention is not limited to the above-mentioned embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for establishing pineapple embryogenic cell suspension lines from pineapple calli, comprising the steps of:
(1) Taking an explant, culturing for 5 to 6 weeks by adopting a callus culture medium, and selecting granular callus which grows vigorously and is crisp and fragile as a material for culturing suspension cells, so as to avoid the phenomena of browning and whitening and the callus which is watered;
the explant is a Tainong 17 pineapple bud, bud sucking, tuber bud or crown bud stem segment, or the explant is a sweet pineapple crown bud stem segment, or the explant is a golden pineapple crown bud stem segment;
the callus culture medium is: 13. Mu.M/L6-BA, 1. Mu.M/L NAA and 7g/L agar were added to the MS medium, pH=5.8; the callus culture condition is constant temperature 28 ℃ and dark culture;
(2) Placing granular callus into a container, crushing, adding a suspension cell culture medium into the container, and carrying out constant-temperature shaking culture on a shaking table;
the suspension cell culture medium is as follows: taking water as a solvent, wherein the solvent comprises 535mg/L ammonium chloride, 2528mg/L potassium nitrate, 440mg/L calcium chloride, 370mg/L magnesium sulfate heptahydrate, 170mg/L potassium dihydrogen phosphate, 0.83mg/L potassium iodide, 6.2mg/L boric acid, 22.3mg/L manganese sulfate, 1.72mg/L zinc sulfate, 0.25mg/L sodium molybdate, 0.025mg/L copper sulfate, 0.025mg/L cobalt chloride, 37.3mg/L disodium ethylenediamine tetraacetate, 27.8mg/L ferrous sulfate, 1000mg/L glutamine, 500mg/L casein hydrolysate, 120mg/L arginine, 2mg/L glycine, 0.4mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 100mg/L inositol, 5000mg/L sucrose, 9.9mg/L picloram, 0.053mg/L abscisic acid, 2000mg/L activated carbon, 2-300 mg/L morpholine and pH=5.8;
(3) Sieving large-particle callus in the cultured suspension culture, standing, sucking the upper liquid culture medium after the fine-particle culture is precipitated, adding fresh suspension cell culture medium, and performing constant-temperature shaking culture on a shaking table;
(4) Sieving large-particle callus, repeating the operation of the step (3), and carrying out constant-temperature shaking culture on a constant-temperature shaking table;
and (3) carrying out subculture on the suspension culture, and taking the culture periodically for microscopic observation until the observed suspension cells are round or oval with uniform size and are composed of single cells and small cell clusters, and establishing a pineapple embryogenic cell suspension system without other irregularly-shaped cells and large cell clusters.
2. The method of claim 1, wherein the culturing conditions of the suspension cells in step (2) are: and (3) culturing in a dark way at the constant temperature of 28-30 ℃ and the rotating speed of 80-100 rpm for 7-8 d.
3. The method of claim 1, wherein step (3) is: and (3) screening out large-particle callus in the suspension culture after 7d of culture by using a 30-mesh screen, standing, sucking the upper liquid culture medium after the fine-particle culture is precipitated, adding a fresh suspension cell culture medium, and carrying out constant-temperature shaking culture on a shaking table.
4. The method of establishing a pineapple embryogenic cell suspension system from pineapple callus according to claim 1, wherein in step (4), large particle callus is removed by a 50 mesh screen.
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| US20220304267A1 (en) * | 2019-05-24 | 2022-09-29 | Galy Co. | Compositions and methods for plant cell culture |
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| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN108368517A (en) * | 2015-10-30 | 2018-08-03 | 先锋国际良种公司 | Method and composition for quick-speed plant conversion |
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