CN114807006A - Extraction method of sugarcane pollen spores - Google Patents

Abstract

The invention belongs to the technical field of plant tissue culture, and provides a method for extracting sugarcane pollen spores, which comprises the steps of firstly cutting the florets of sterilized sugarcane young ears into halves, and grinding the cut flowers in a mortar until young ear tissues are broken; transferring the ground young ear tissues into a centrifuge tube for oscillation, and filtering by using a 100-mesh and 400-mesh cell sieve after oscillation; centrifuging the filtered serous fluid for three times, and collecting pollen spores at the bottom; adding a maltose solution with the mass fraction of 20% into another empty centrifuge tube, re-suspending the pollen spores on the maltose solution, adding a CPW buffer solution, and performing centrifugation for the fourth time to ensure that the pollen spores form two obvious annular zones in the centrifuge tube, namely a live cell zone suspended above and a dead cell zone settled at the bottom; pollen spores that formed viable cell bands on the interface were collected. The method can efficiently extract the pollen spores with high purity and strong vitality, and lays a foundation for improving the success rate of sugarcane anther culture.

Description

A kind of extraction method of sugarcane pollen spores

technical field

The invention relates to the technical field of plant tissue culture, in particular to a method for extracting sugarcane pollen spores.

Background technique

Haploid is induced by tissue culture or inducible line, and then doubled haploid (DH) with 100% homozygosity is produced by chromosome doubling, which is a way to quickly create pure line (ie DH line). The haploid breeding technology developed on this basis can create inbred lines in only 2 to 3 generations, which can greatly shorten the breeding cycle and improve the breeding efficiency. More importantly, the mature development of haploid breeding technology has provided important support for the selective breeding of crop genomes. One of the widely used ways to obtain haploid plants in my country is anther culture. Anther culture technology is to use plant tissue culture technology to inoculate anthers that have developed to a certain stage on artificial medium to form pollen embryos or pollen callus, and finally develop directly from the embryoid to plants or differentiate the callus into plants . It is a typical representative of haploid breeding technology, with unique advantages such as short selection cycle, accurate trait selection, creation of variation and fast DH population. In recent years, double haploid materials of rape, rice, wheat, corn and other crops have promoted the improvement of cultivars. However, compared with crops such as wheat, corn, and rice, the in vitro culture of sugarcane anthers is more specific, and the success rate of sugarcane anther culture is generally low due to constraints such as the long growth cycle of sugarcane. There are relatively few theoretical studies on in vitro culture of anthers. Up to now, only Chen Zhenghua et al. (1980) have reported the successful cultivation of pollen plants, but there is no detailed description of the technical system of sugarcane flower culture. In the sugarcane flower cultivation technology system, how to efficiently extract pollen spores with high purity and strong vitality is the first step in sugarcane flower cultivation, and it is also a key step that affects the success rate of sugarcane flower cultivation.

Sugarcane inflorescences are large panicles consisting of many spikelets. Spikelet is a typical feature of Poaceae, consisting of glumes, florets and rachis. The florets are lemma, palea, sap, and 3 anthers from outside to inside. base. Dense long-stalk glandular hairs, tightly packed glumes and palea were the main obstacles to sugarcane pollen spore extraction. In order to obtain sugarcane pollen spores with less impurities, high purity and sufficient quantity, simple grinding and filtration are not enough. If only simple grinding is performed, the pollen spores in the anther are difficult to be released from the tightly packed glumes, and a large number of long-stalk glandular hairs will form a entanglement or adhesion effect on the pollen spores, which makes the filtrate in the filtered filtrate. The number of pollen spores is greatly reduced. Therefore, how to efficiently extract pollen spores with high purity and strong vitality is a technical problem to be solved urgently in this field.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for extracting sugarcane pollen spores in view of the above-mentioned problems, which can efficiently extract pollen spores with high purity and strong vitality, and lay a foundation for improving the success rate of sugarcane flower cultivation.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A method for extracting sugarcane pollen spores, comprising the following steps:

(1) the floret of the sterilized sugarcane young ear is cut in half, and in the mortar after the sterilization, it is ground until the young ear tissue is broken;

(2) transfer the ground young ear tissue to a centrifuge tube;

(3) adding CPW buffer containing 5V/V% mannitol to the centrifuge tube of step (2);

(4) use a vortex mixer or a vibrator to vibrate the centrifuge tube of step (3) to obtain a homogenate of young ears;

(5) use a 100-mesh cell sieve to filter the young panicle homogenate after the oscillation, discard the residue, and retain the filtrate;

(6) filter the filtrate obtained in the previous step with a 400-mesh cell sieve, discard the tissue fragments, and obtain the filtered slurry;

(7) packing the filtered slurry into the centrifuge tube;

(8) the centrifuge tube of step (7) is put into centrifuge and centrifuged for the first time;

(9) After the first centrifugation, discard the supernatant, add CPW buffer containing 5V/V% mannitol, and mix the pollen spores by gently pipetting with a fine pipette;

(10) the centrifuge tube of step (9) is centrifuged for the second time;

(11) After the second centrifugation, discard the supernatant, add CPW buffer containing 5V/V% mannitol, and perform the third centrifugation;

(12) after the third centrifugation, the supernatant was poured out again, and the pollen spores that settled at the bottom of the centrifuge tube were collected;

(13) Add 20% maltose solution to another empty centrifuge tube, resuspend the pollen spores in step (13) on the maltose solution, and carefully add CPW containing 5V/V% mannitol buffer;

(14) Put the centrifuge tube in step (13) into the centrifuge, and centrifuge for the fourth time or stand for 30min-90min, so that the pollen spores form two obvious annular bands in the centrifuge tube, that is, the one suspended above Live cell band and dead cell band sinking to the bottom;

(15) Collect pollen spores that form live cell bands on the interface.

Preferably, the young sugarcane ear described in step (1) is surface sterilized with 75% alcohol.

Preferably, in step (1), use sterile forceps and scissors to cut the florets of the young sugarcane ear in half.

Preferably, in step (4), the time for shaking the centrifuge tube is 1-2 min.

Preferably, the rotational speed of the first centrifugation described in step (8) is 2000r/min, and the centrifugation time is 5min, and the rotational speed of the second centrifugation described in step (10) is 2000r/min, and the centrifugation time is 5min .

Preferably, the rotational speed of the third centrifugation described in step (11) is 2000r/min, and the centrifugation time is 5min.

Preferably, the other empty centrifuge tube in step (14) is a 10mL centrifuge tube, the volume of the added maltose solution is 7ml, and the volume of the added CPW buffer containing 5V/V% mannitol is 2mL.

Preferably, the rotation speed of the fourth centrifugation in step (15) is 500r/min, and the centrifugation time is 5min.

Due to the simple grinding, the pollen spores in the sugarcane anthers are difficult to be released from the tightly packed glumes, and a large number of long-stalk glandular hairs will form a entanglement or adhesion effect on the pollen spores, making the filtered filtrate in the filtrate. The number of pollen spores was greatly reduced. In order to obtain a large amount of pollen spores, the present invention uses scissors to cut the florets and anthers in half into a mortar before grinding, the purpose is to open an outlet for the release of pollen spores, add an appropriate amount of buffer and then grind, and use vibration after grinding. Shake with a vortexer or vortexer for one minute to maximize the release of pollen spores. As a result, more pollen spores can be obtained.

In the present invention, the homogenate of the young panicle is then filtered through a 100-mesh cell sieve for the first time, which can remove larger impurities and tissue fragments, and then the second filtration is performed through a 400-mesh cell sieve, and the 400-mesh cell sieve has a pore size of 38 μm. The diameter of sugarcane pollen spores is about 35 μm, which can further improve the purity of sugarcane pollen spores. In order to improve the purity of the sugarcane pollen spores, the present invention also carries out three purification centrifugations at 2000 rpm for 5 minutes; after each centrifugation, the supernatant is removed, and the buffer is added, and the pollen spores are mixed by pipetting with a pipette tip before the next centrifugation. Thereby, the purity of pollen spores can be improved.

Through the above filtration and centrifugation steps, pollen spores with higher purity can be obtained to a certain extent, but there are still a small number of impurities that are similar in volume and weight to those of live sugarcane pollen spores that cannot be removed, and the filtration and centrifugation steps cannot distinguish dead cells. and living cells. In order to further remove impurities and distinguish dead cells from living cells, we used maltose solution to make a ring. The specific steps are: add 7mL of 20% maltose solution to an empty 10mL centrifuge tube, and resuspend the pollen spores on the maltose solution. , carefully add 2 mL of CPW buffer containing 5V/V% mannitol to the maltose solution, and a very clear pollen spore boundary line will appear between the maltose solution and the mannitol solution. At this time, due to the difference in density, living cells will form a live cell band in the upper CPW solution, and dead cells and impurity debris will settle to the bottom of the 20% maltose solution to form a dead cell band. The living cell band formed in the CPW solution in the upper layer not only ensures the purity of pollen spores, but also ensures its vitality, and achieves the effect of improving the extraction quality.

To sum up, due to the adoption of the above-mentioned technical solutions, the beneficial effects of the present invention are:

1. The present invention uses scissors to cut the florets and anthers in half into the mortar before grinding, and opens an outlet for the release of pollen spores to relieve the sugarcane pollen spores due to dense long-stalk glandular hairs, tightly wrapped glumes and inside and outside. The extraction barrier caused by the palea, so that more sugarcane pollen spores can be obtained.

2. The present invention removes impurities and tissue fragments through two filtrations, and then 3-4 times of centrifugation for purification to obtain pollen spores with higher purity, and then a certain concentration of maltose solution is used to make an annular belt to remove dead cells and cells. Live cells can be distinguished to obtain pollen spores with high purity and strong vitality, which lays the foundation for improving the success rate of sugarcane flower cultivation.

3. The sugarcane pollen spores obtained by the method of the present invention have few impurities, high purity and sufficient quantity, which provides a better foundation for the success rate of sugarcane flower cultivation, and also promotes the process of sugarcane anther and pollen haploid breeding. It also provides a basis for the genome sequencing, genetic map construction and molecular breeding of sugarcane haploid breeding.

Description of drawings

Fig. 1 is the real picture of sugarcane young ear;

Fig. 2 is the operation of cutting the floret of sugarcane in half;

Fig. 3 is grinding young ear tissue;

4 is a schematic diagram of adding CPW buffer to a centrifuge tube;

Fig. 5 is the schematic diagram of oscillating;

Figure 6 is the collection of filtrate after cell sieve filtration;

Figure 7 is the collection of pollen spores after centrifugation;

Fig. 8 is that pollen spores are resuspended on maltose solution, and CPW buffer is added;

Figure 9 shows that pollen spores form two distinct annular bands in the centrifuge tube, the upper frame is a live cell zone, and the lower frame is a dead cell zone that sinks at the bottom;

Figure 10 is a state diagram of sugarcane pollen spores after extraction and purification (centrifugation, formation of annular bands);

Figure 11 is the count of pollen spores with a hemocytometer;

Figure 12 is a state diagram of sugarcane pollen spores in Comparative Example 1;

Fig. 13 is that pollen spores are counted with a hemocytometer in Comparative Example 2;

FIG. 14 shows that pollen spores were resuspended in maltose solution in Comparative Example 3, and CPW buffer was added.

Detailed ways

In order to enable those skilled in the art to better understand the solutions of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all of them. example. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the scope of protection of the present application.

Example

A method for extracting sugarcane pollen spores, comprising the following steps:

1. Take a young sugarcane ear of the variety GT42 that has been surface-sterilized with 75% alcohol (see Figure 1), use sterile tweezers and scissors, and cut the sugarcane florets in half into a sterilized mortar. Grind in a bowl until the young ear tissue is broken (see Figure 2, 3);

2. Use sterile tweezers or a spoon to transfer the ground young ear tissue to a 50mL centrifuge tube;

3. Add CPW buffer containing 5V/V% mannitol to the 35mL mark of the 50mL centrifuge tube in step 2 (Figure 4);

4. Use a vortex mixer or a shaker to shake the 50mL centrifuge tube for 1min (Figure 5) to obtain a homogenate of young ears;

5. Use a 100-mesh cell sieve to filter the oscillated young ear homogenate, discard the residue, and keep the filtrate;

6. Use a 400-mesh cell sieve to filter the filtrate obtained in the previous step, discard the tissue fragments, and obtain the filtered slurry (Figure 6);

7. Divide the filtered slurry into two 10mL centrifuge tubes (Figure 7);

8. Put two 10mL centrifuge tubes into the suitable rotor of the centrifuge, and perform the first centrifugation at a speed of 2000r/min and a centrifugation time of 5min;

9. After the first centrifugation, discard the supernatant, add 3 mL of 5V/V% mannitol CPW buffer, and mix the pollen spores by gently pipetting with a fine pipette;

10. Combine the pollen spores from two 10ml centrifuge tubes and transfer them to the same 10ml centrifuge tube;

11. Add CPW buffer containing 5V/V% mannitol to the combined centrifuge tube to make the liquid rise to 10mL, and perform a second centrifugation at a rotational speed of 2000r/min and a centrifugation time of 5min;

12. After the second centrifugation, discard the supernatant, add CPW buffer containing 5V/V% mannitol, and perform the third centrifugation;

13. After the third centrifugation, the supernatant was poured out again, and the pollen spores that settled at the bottom of the centrifuge tube were collected;

14. Add 7 mL of 20% maltose solution to another empty 10 mL centrifuge tube, resuspend the pollen spores on the maltose solution, and carefully add 2 mL of CPW (containing 5% mannitol) buffer on the maltose solution to keep a clear interface (Figure 8);

15. Put the 10mL centrifuge tube in the previous step into a suitable rotor of the centrifuge, and centrifuge for the fourth time or stand for 30min-90min, so that the pollen spores form two obvious annular bands in the 10mL centrifuge tube (Figure 9). ), that is, the live cell band suspended above and the dead cell band sinking at the bottom;

16. Collect the pollen spores that form a band on the interface (Fig. 10). The upper box is the live cell band, and the bottom box is the dead cell band. Transfer the live microspores to the new one. 10mL sterile centrifuge tube;

17. Dilute the cells to 5mL with N6 medium, and gently pipette the cells with a pipette to disperse them evenly;

18. Take 10 μL of the cell suspension, and calculate the pollen spore density with a hemocytometer to be 2.35×10 ⁶ /mL (Fig. 11).

Comparative Example 1

A method for extracting sugarcane pollen spores, comprising the following steps:

1. Take a young sugarcane ear of the variety GT42 that has been surface-sterilized with 75% alcohol, use sterile tweezers and scissors, cut the sugarcane florets in half into a sterilized mortar, and grind them in the mortar to The young ear tissue is broken;

2. Use sterile tweezers or a spoon to transfer the ground young ear tissue to a 50mL centrifuge tube;

3. Add CPW buffer containing 5V/V% mannitol to the 35mL mark of the 50mL centrifuge tube in step 2;

4. Use a vortex mixer or a shaker to shake the 50mL centrifuge tube for 1min to obtain a homogenate of young ears;

5. Use a 100-mesh cell sieve to filter the oscillated young ear homogenate, discard the residue, and keep the filtrate;

6. Use a 400-mesh cell sieve to filter the filtrate obtained in the previous step, discard the tissue fragments, and obtain a filtered slurry.

The filtered slurry smear was observed under a microscope, and the state diagram of the sugarcane pollen spores seen was shown in Figure 12. It can be seen that only extraction and filtration were performed, but no centrifugation and ring belts were made for purification. There are many sugarcane pollen spores. Impurities, tissue fragments and dead cells, the purity of sugarcane pollen spores is very low.

Comparative Example 2

The difference between this comparative example and Example 1 is that in step 1, there is no step of "using sterile tweezers and scissors to cut the florets of sugarcane in half into a sterilized mortar". The sugarcane young ears of the variety GT42 sterilized with 75% alcohol were ground in a mortar until the young ear tissue was broken, and the remaining steps 2-17 were the same as those in the embodiment. The pollen spore density was calculated to be 1.0×10 ⁵ /mL by a hemocytometer, see FIG. 13 . It can be shown that simple grinding is difficult to release the pollen spores in the sugarcane anthers from the tightly packed glumes, and a large number of long-stalk glandular hairs will form a entanglement or adhesion effect on the pollen spores, making the filtered filtrate in the filtrate. The number of pollen spores is greatly reduced, and contains a large amount of impurities and tissue fragments, and the density and purity of the finally obtained pollen spores are greatly reduced.

Comparative Example 3

The difference between this comparative example and Example 1 is that the concentration of the maltose solution in step 14 is changed to 15% and 25%. The remaining steps 2-17 are the same as in the embodiment. When the pollen spores were resuspended on the maltose solution and 2 mL of CPW (containing 5% mannitol) buffer was carefully added to the maltose solution, the pollen spores were found to run up and down too much to form clear bands (Figure 14) , which means that the concentration of maltose solution is inappropriate and cannot form an effective gradient density. The present invention adopts a maltose solution with a concentration of 20%. The appropriate concentration of maltose can more effectively gather pollen spores and form a clear pollen spore belt (Fig. 8). It is a suitable density gradient difference. Whether it is static separation or centrifugal separation, the live cell band and the dead cell band can be separated quickly.

The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications completed under the technical spirit suggested by the present invention shall belong to This invention covers the scope of the patent.

Claims (8)

1. The extraction method of the sugarcane pollen spores is characterized by comprising the following steps:

(1) cutting the small flowers of the disinfected young sugarcane into halves, and grinding the cut flowers in a sterilized mortar until young ear tissues are broken;

(2) transferring the ground young ear tissues into a centrifuge tube;

(3) adding a CPW buffer solution containing 5V/V% of mannitol into the centrifugal tube in the step (2);

(4) oscillating the centrifuge tube in the step (3) by using a vortex mixer or an oscillator to obtain young ear homogenate;

(5) filtering the vibrated young ear homogenate by using a 100-mesh cell sieve, discarding residues, and keeping a filtrate;

(6) filtering the filtrate obtained in the last step by using a 400-mesh cell sieve, and removing tissue fragments to obtain filtered serous fluid;

(7) filling the filtered slurry into a centrifuge tube;

(8) putting the centrifugal tube obtained in the step (7) into a centrifugal machine for primary centrifugation;

(9) removing supernatant after the first centrifugation, adding a CPW buffer solution containing 5V/V% mannitol, and gently blowing and beating the pollen spores uniformly by using a fine dropping tube;

(10) performing secondary centrifugation on the centrifuge tube in the step (9);

(11) removing supernatant after the second centrifugation, adding a CPW buffer solution containing 5V/V% mannitol, and carrying out third centrifugation;

(12) after the third centrifugation, pouring out the supernatant again, and collecting pollen spores deposited at the bottom of the centrifuge tube;

(13) adding a maltose solution with the mass fraction of 20% into another empty centrifuge tube, re-suspending the pollen spores obtained in the step (13) on the maltose solution, and carefully adding a CPW buffer solution containing 5V/V% mannitol;

(14) putting the centrifugal tube in the step (13) into a centrifugal machine, and carrying out fourth centrifugation or standing for 30-90 min to enable pollen spores to form two obvious annular bands in the centrifugal tube, namely a live cell band suspended above and a dead cell band settled at the bottom;

(15) pollen spores that formed viable cell bands on the interface were collected.

2. The method for extracting sugar cane pollen spores as claimed in claim 1, which is characterized in that: and (2) carrying out surface disinfection on the sugarcane young ears in the step (1) by using 75% alcohol.

3. The method for extracting sugar cane pollen spores as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the small flowers of the young spikes of the sugarcane are cut into halves by using sterile forceps and scissors.

4. The method for extracting sugar cane pollen spores as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the time for oscillating the centrifugal tube is 1-2 min.

5. The method for extracting sugar cane pollen spores as claimed in claim 1, wherein the method comprises the following steps: the rotating speed of the first centrifugation in the step (8) is 2000r/min, the centrifugation time is 5min, the rotating speed of the second centrifugation in the step (10) is 2000r/min, and the centrifugation time is 5 min.

6. The method for extracting sugar cane pollen spores as claimed in claim 1, wherein the method comprises the following steps: the rotating speed of the third centrifugation in the step (11) is 2000r/min, and the centrifugation time is 5 min.

7. The method for extracting sugar cane pollen spores as claimed in claim 1, which is characterized in that: the other empty centrifuge tube in step (14) was a 10mL centrifuge tube, the volume of maltose solution added was 7mL, and the volume of CPW buffer containing 5V/V% mannitol added was 2 mL.

8. The method for extracting sugar cane pollen spores as claimed in claim 1, which is characterized in that: the rotating speed of the fourth centrifugation in the step (15) is 500r/min, and the centrifugation time is 5 min.

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