CN114946653A - Pineapple seedling regeneration method and axillary bud growth point transformation method - Google Patents
Pineapple seedling regeneration method and axillary bud growth point transformation method Download PDFInfo
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- CN114946653A CN114946653A CN202210498092.5A CN202210498092A CN114946653A CN 114946653 A CN114946653 A CN 114946653A CN 202210498092 A CN202210498092 A CN 202210498092A CN 114946653 A CN114946653 A CN 114946653A
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- 241000234671 Ananas Species 0.000 title claims abstract description 96
- 235000007119 Ananas comosus Nutrition 0.000 title claims abstract description 94
- 238000011069 regeneration method Methods 0.000 title claims abstract description 25
- 238000011426 transformation method Methods 0.000 title claims abstract description 10
- 230000028446 budding cell bud growth Effects 0.000 title claims description 9
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 241000196324 Embryophyta Species 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 8
- 230000001580 bacterial effect Effects 0.000 claims abstract description 7
- 239000012881 co-culture medium Substances 0.000 claims abstract description 7
- 238000005286 illumination Methods 0.000 claims abstract description 7
- 238000002791 soaking Methods 0.000 claims abstract description 5
- 239000008223 sterile water Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 230000008929 regeneration Effects 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 12
- 229930027917 kanamycin Natural products 0.000 claims description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 9
- 229960000318 kanamycin Drugs 0.000 claims description 9
- 229930182823 kanamycin A Natural products 0.000 claims description 9
- 208000035143 Bacterial infection Diseases 0.000 claims description 8
- 241000589158 Agrobacterium Species 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 7
- 208000015181 infectious disease Diseases 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 230000002458 infectious effect Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000001172 regenerating effect Effects 0.000 claims description 3
- 230000009545 invasion Effects 0.000 claims description 2
- 230000012010 growth Effects 0.000 description 9
- 230000009466 transformation Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 241000208223 Anacardiaceae Species 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 241000234670 Bromeliaceae Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000008303 genetic mechanism Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 229940027257 timentin Drugs 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
- A01H4/008—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
Abstract
The invention discloses a pineapple seedling regeneration method and an axillary bud growing point transformation method, which comprise the following steps: stripping off leaves of the whole pineapple seedling plants in the strong year period one by one in an aseptic operation table until all the leaves are removed and roots are left; slightly cutting roots in parallel by using a small knife until a growing point is exposed, slightly cutting the growing point by using the small knife for one time, and soaking the root in 1ml of bacterial liquid for 20 min; cleaning the soaked pineapple seedlings with sterile water for 2-3 times, and putting the pineapple seedlings in an empty dish padded with filter paper for airing; inserting the treated pineapple seedlings into a co-culture medium, and culturing under illumination, wherein small round white protrusions, namely new cluster buds, are generated at the original growing points after 14 days; cutting off new cluster buds with a small knife, transferring the pineapple seedlings to a screening culture medium for culture, and growing new calluses from primary growing points after 19 days; and after 70 days, new callus buds grow out from the calluses, and the callus buds and the calluses are respectively identified to select positive pineapple seedling plants.
Description
Technical Field
The invention belongs to the technical field of pineapple seedling culture, and particularly relates to a pineapple seedling regeneration method and an axillary bud growth point transformation method.
Background
Pineapple (Ananas comosus (L.) Merr.) also called pineapple belongs to Anacardiaceae (Bromeliaceae) Ananas herbaceous plant, and is one of tropical fruits. China is one of the world major pineapple producing countries, and the planting area and the total yield respectively account for 7.2 percent and 8.1 percent of the total amount of the world. Pineapple is a characteristic and efficient tropical economic crop and also an important economic source for farmers in hot areas. The pineapple can be eaten fresh, and can be processed into a plurality of byproducts such as canned syrup, fruit juice and the like.
However, the natural propagation coefficient of the pineapple is low, so that the popularization of new varieties is slow due to the shortage of seedlings, and the research system of the planting technology is not perfect, so that the market demand cannot be met. Therefore, the technology improvement of the pineapple tissue culture seedlings is also extremely important. Different from the traditional pineapple seedling breeding, the regeneration system of the axillary bud primordium of the pineapple can obtain a large amount of aseptic pineapple seedlings in a short period by a tissue culture technology, and has great application value for the breeding of the pineapple seedlings.
Due to the restriction of factors such as long growth period of the pineapple, unclear genetic mechanism of a plurality of characters and the like, the gene transformation technology of the pineapple becomes an important factor for restricting the research and development of gene engineering of the pineapple, and the process of cultivating the fast-growing high-yield new variety of the pineapple by the traditional pineapple breeding means is seriously hindered. Due to this limitation, the economically valuable transgenic pineapples available to date are extremely limited. Compared with other transformation methods, the agrobacterium-mediated transformation of the axillary bud growth points of the pineapples has the characteristics of simple operation, short culture period and the like, and has important theoretical significance for researching the genetic transformation of the pineapples.
Disclosure of Invention
The invention aims to provide a pineapple seedling regeneration method and an axillary bud growing point transformation method.
The technical scheme adopted by the invention for solving the technical problems is as follows: a regeneration method of pineapple seedlings comprises the following steps:
1) selecting complete pineapple seedling plants growing in the strong year period, wherein the height of the complete pineapple seedling plants can be 5-8 cm, and peeling off the pineapple seedling leaves one by one in a sterile operating platform by using tweezers until the leaves are all peeled off to be used as new pineapple seedlings;
2) cutting the fresh pineapple seedlings in parallel in an aseptic operation table by using a knife until a growing point is exposed, and slightly cutting the growing point by using the knife;
3) placing the treated new pineapple seedlings into a regeneration culture medium for culture, and performing illumination culture, wherein after 14 days, small round white protrusions are generated at the primary growing points, namely new cluster buds;
4) culturing in regeneration culture medium for 20 days, and growing callus from the primary growing point;
5) after culturing in the regeneration culture medium for 70 days, new pineapple seedlings can grow on the callus.
Specifically, the formula of the regeneration culture medium is 1/2MS solid culture medium +0.5mg/LNAA +2 mg/L6-BA.
An axillary bud growing point transformation method comprises infection bacterial liquid preparation and pineapple seedling regeneration treatment; the preparation of the infection bacterial liquid comprises the following steps:
1) and (3) culturing bacteria: smearing agrobacterium GV3101 carrying target gene vector on solid LB culture medium containing 50mg/L kanamycin at 28 deg.c for 36-48 hr;
2) shaking the bacteria: selecting a monoclonal, putting the monoclonal into an LB liquid culture medium containing 50mg/L kanamycin, culturing at 28 ℃ and 200rpm overnight for 12-16 h;
3) expanding culture: 2 times of activation were carried out at a ratio of 1:15, and the cells were cultured until the OD600 became 0.6 to 0.8.
4) Centrifuging the bacterial liquid cultured in the step 3), and then re-suspending twice with an isovolumetric infectious liquid to obtain an infectious bacterial liquid;
5) the centrifugation procedure of the step 4) is that the temperature is 25 ℃, the rotating speed is 10000rpm, and the time is 10 min.
Specifically, the regeneration treatment of the pineapple seedlings comprises the following steps:
1) stripping off leaves of the whole pineapple seedling plants in the strong year period one by one in an aseptic operation table until all the leaves are removed and roots are left;
2) slightly cutting roots in parallel by using a knife until a growing point is exposed, slightly cutting the growing point by using the knife for one time, and soaking the root in 1ml of infection bacterial liquid for 20 min;
3) cleaning the soaked pineapple seedlings with sterile water for 2-3 times, and putting the pineapple seedlings in an empty dish padded with filter paper for airing;
4) inserting the treated pineapple seedlings into a co-culture medium, culturing by illumination, and generating small round white protrusions at the original growing points after 14 days, namely new cluster buds;
5) cutting off new cluster buds with a small knife, transferring the pineapple seedlings to a screening culture medium for culture, and growing new calluses from primary growing points after 19 days;
6) and after 70 days, new callus buds grow out from the calluses, and the callus buds and the calluses are respectively identified to select positive pineapple seedling plants.
Specifically, the invasion liquid is 1/2MS liquid culture medium +1 mg/LAS.
Specifically, the co-culture medium is 1/2MS solid culture medium +0.5mg/LNAA +2 mg/L6-BA +200mg/L TM.
Specifically, the screening culture medium is 1/2MS solid culture medium +0.5mg/LNAA +2 mg/L6-BA +200mg/L TM +50mg/L kanamycin.
The invention has the following beneficial effects: the method utilizes the axillary bud growth points of the pineapples to transform the agrobacterium, so that the experimental operation is simple and convenient, the transformation time is shortened, the transformation cost is reduced, the propagation of pineapple seedlings and the updating and updating of new and old varieties are facilitated, and the popularization of new varieties in the market is accelerated.
Drawings
FIG. 1 is a diagram showing the growth state of the present invention.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings.
And (4) regenerating pineapple seedlings by using axillary bud growing points.
A regeneration method of pineapple seedlings comprises the following steps:
1) selecting complete pineapple seedling plants with proper size and growing in a strong year period, wherein the height of the plants can be 5-8 cm (figure 1.a), and peeling the leaves of the pineapple seedlings one by one in a sterile operating platform by using tweezers until the leaves are completely peeled (figure 1. b).
2) The sterile table was cut in parallel with a knife until the growth point was exposed, and the growth point was lightly scratched with a knife (fig. 1.c, d).
3) The treated pineapple seedlings are put into a regeneration medium for culture, and are subjected to illumination culture, and after 14 days, small round white protrusions, namely new cluster buds, are generated at the primary growing points (figure 1. f).
4) After further culturing in regeneration medium for 20 days, the primary growing point will grow callus (FIG. 1.g, h, i).
5) After culturing for 70 days in the regeneration medium, new pineapple seedlings will grow on the callus (FIG. 1. j).
The regeneration medium formula comprises: 1/2MS solid culture medium +0.5mg/LNAA +2 mg/L6-BA
Agrobacterium-mediated transformation of axillary bud growth sites
An axillary bud growing point transformation method comprises the following steps:
1) preparing infection bacterial liquid:
culturing bacteria: agrobacterium GV3101 carrying the target gene vector was spread on solid LB medium containing 50mg/L kanamycin at 28 ℃ for 36-48 hours.
Shaking the bacteria: the single clone is picked up and put into LB liquid culture medium containing 50mg/L kanamycin, and cultured for 12-16 h at 28 ℃ and 200rpm overnight.
Thirdly, expanding culture: 2 times of activation were carried out at a ratio of 1:15, and the cells were cultured until the OD600 became 0.6 to 0.8.
Fourthly, centrifuging the bacterial liquid cultured in the third step, and then re-suspending twice by using the same volume of the infection liquid to obtain the infection bacterial liquid.
The centrifugation procedure of the step (iv) is 25 ℃, 10000rpm and 10 min.
The formula of the infection liquid comprises: 1/2MS liquid medium +1mg/LAS (acetosyringone).
2) Pineapple seedling treatment:
firstly, the whole pineapple seedling plants (figure 1.a) which are proper in size and grow in the strong year period are peeled off one by one in an aseptic operation platform until the leaves are completely removed, and roots are left (figure 1. b).
And secondly, slightly cutting the sterile operating platform in parallel by using a knife until a growing point is exposed, slightly cutting the growing point by using the knife (figures 1.c and d), and soaking the cut sterile operating platform in 1ml of infection bacterial liquid for 20min (figure 1. e).
Thirdly, the soaked pineapple seedlings are washed for 2 to 3 times by sterile water and then put in an empty dish padded with three layers of filter paper for drying.
Fourthly, the treated pineapple seedlings are inserted into a co-culture medium and are cultured by illumination, and small round white protrusions, namely new cluster buds, are generated at the original growing points after 14 days (figure 1. f).
Fifthly, cutting off new cluster buds by using a scalpel, transferring the pineapple seedlings to a screening culture medium for culture, and growing new calluses after 19 days (figure 1. g-i).
Sixthly, after 70 days, new callus buds (figure 1.j) grow out from the calluses, and the callus buds and the calluses are respectively identified to select positive pineapple seedling plants.
The formula of the co-culture medium in the step (IV) is as follows: 1/2MS solid culture medium +0.5mg/LNAA +2 mg/L6-BA +200mg/L TM, step (c) screening the formula of the culture medium: 1/2MS solid medium +0.5mg/LNAA +2 mg/L6-BA +200mg/LTM +50mg/L kanamycin.
Screening is carried out through a screening culture medium, resistant pineapple seedlings are preliminarily screened, the pineapple seedlings successfully screened preliminarily are further screened by utilizing a portable GFP excitation light source, and the pineapple seedlings emitting green fluorescence in the dark are transgenic positive pineapple plants (figure 1. k).
A method for regenerating pineapple seedlings and transforming the growth points of axillary buds includes such steps as cutting the growth points of axillary buds to regenerate a great number of tissue cultured pineapple seedlings, peeling off the leaves of pineapple seedlings to expose the growth points of axillary buds, and cutting the growth points to transform them to obtain transgenic pineapple plants. The method has the advantages that the experimental operation technology is easy to master, a large number of pineapple regenerated plants can be obtained in a short time, the method is used for genetic engineering research of pineapples, and the method widens the approach for quality improvement and resistance research of pineapples.
In FIG. 1, a: one complete pineapple seedling; b: removing leaves of pineapple and leaving roots; c: cutting the main view behind the growing point after b; d: cutting a top view of the growing point after b; e: soaking the pineapple in the bacterial liquid; f: cluster buds grow on the pineapples; g-i: pineapple grows out and is callused; j: pineapple grows out and heals the damaged seedlings; k: the positive plants are detected by the portable GFP excitation light source, the green fluorescence is transgenic plants (dark arrows), and the red light is plants which are not successfully transferred (light arrows).
MS is a common culture medium, NAA and 6-BA are artificially synthesized plant growth regulators, and are often used as hormone analogues in tissue culture; TM is an abbreviation for timentin, an antibiotic that inhibits the growth of Agrobacterium.
The present invention is not limited to the above embodiments, and any structural changes made under the teaching of the present invention shall fall within the scope of the present invention, which is similar or similar to the technical solutions of the present invention.
The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Claims (7)
1. The regeneration method of the pineapple seedlings is characterized by comprising the following steps:
1) selecting complete pineapple seedling plants growing in the strong year period, wherein the height of the complete pineapple seedling plants can be 5-8 cm, and peeling off the pineapple seedling leaves one by one in a sterile operating platform by using tweezers until the leaves are all peeled off to be used as new pineapple seedlings;
2) cutting the fresh pineapple seedlings in parallel in an aseptic operation table by using a knife until a growing point is exposed, and slightly cutting the growing point by using the knife;
3) placing the treated new pineapple seedlings into a regeneration culture medium for culture, and performing illumination culture, wherein after 14 days, small round white protrusions are generated at the primary growing points, namely new cluster buds;
4) culturing in regeneration culture medium for 20 days, and growing callus from the primary growing point;
5) after culturing in the regeneration culture medium for 70 days, new pineapple seedlings can grow on the callus.
2. The method for regenerating pineapple seedlings as claimed in claim 1, wherein the regeneration medium formula is 1/2MS solid medium +0.5mg/LNAA +2 mg/L6-BA.
3. The method for transforming axillary bud growing points according to claim 1, comprising infection bacteria liquid preparation and pineapple seedling regeneration treatment; the preparation of the infection bacterial liquid comprises the following steps:
1) and (3) culturing bacteria: smearing agrobacterium GV3101 carrying target gene vector on solid LB culture medium containing 50mg/L kanamycin at 28 deg.c for 36-48 hr;
2) shaking the bacteria: selecting a monoclonal, putting the monoclonal into an LB liquid culture medium containing 50mg/L kanamycin, culturing at 28 ℃ and 200rpm overnight for 12-16 h;
3) expanding culture: 2 times of activation were carried out at a ratio of 1:15, and the cells were cultured until the OD600 became 0.6 to 0.8.
4) Centrifuging the bacterial liquid cultured in the step 3), and then re-suspending twice by using an isovolumetric infectious liquid to obtain an infectious bacterial liquid;
5) the centrifugation procedure of the step 4) is that the temperature is 25 ℃, the rotating speed is 10000rpm, and the time is 10 min.
4. The method for transforming axillary bud growth points according to claim 3, wherein the regeneration treatment of pineapple seedlings comprises the following steps:
1) stripping off leaves of the whole pineapple seedling plants in the strong year period one by one in an aseptic operation table until all the leaves are removed and roots are left;
2) slightly cutting roots in parallel by using a knife until a growing point is exposed, slightly cutting the growing point by using the knife for one time, and soaking the root in 1ml of infection bacterial liquid for 20 min;
3) cleaning the soaked pineapple seedlings with sterile water for 2-3 times, and putting the pineapple seedlings in an empty dish padded with filter paper for airing;
4) inserting the treated pineapple seedlings into a co-culture medium, culturing by illumination, and generating small round white protrusions at the original growing points after 14 days, namely new cluster buds;
5) cutting off new cluster buds with a small knife, transferring the pineapple seedlings to a screening culture medium for culture, and growing new calluses from primary growing points after 19 days;
6) and after 70 days, new callus buds grow out from the calluses, and the callus buds and the calluses are respectively identified to select positive pineapple seedling plants.
5. The method for transforming axillary bud growing points according to claim 3, wherein the invasion solution is 1/2MS liquid medium +1 mg/LAS.
6. The axillary bud growth point transformation method of claim 4, wherein the co-culture medium is 1/2MS solid medium +0.5mg/L NAA +2 mg/L6-BA +200mg/L TM.
7. The axillary bud growth point transformation method of claim 4, wherein the selection medium is 1/2MS solid medium +0.5mg/L NAA +2 mg/L6-BA +200mg/L TM +50mg/L kanamycin.
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| CN202210498092.5A CN114946653A (en) | 2022-05-09 | 2022-05-09 | Pineapple seedling regeneration method and axillary bud growth point transformation method |
| ZA2022/13140A ZA202213140B (en) | 2022-05-09 | 2022-12-05 | A method of pineapple seedlings regeneration and axillary buds growth point transformation |
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