CN114097616A

CN114097616A - Large cherry breeding method

Info

Publication number: CN114097616A
Application number: CN202111408366.9A
Authority: CN
Inventors: 张晓龙; 姚甜甜; 张哲民; 李勤峰
Original assignee: Lingbao Dinghong Agricultural Technology Development Co ltd
Current assignee: Lingbao Dinghong Agricultural Technology Development Co ltd
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-03-01

Abstract

The invention discloses a large cherry breeding method, which comprises the following steps: preparing a sterilized explant, primary culture, secondary culture, rooting culture, test-tube seedling domestication, bud seedling transplantation domestication and cherry seedling transplantation. The invention has the beneficial effects that: the large cherry seedlings produced by tissue culture by using biotechnology have strong disease resistance and high fruiting quality, the seedlings do not carry viruses, grafting is not needed, labor and time are saved, new varieties can be rapidly bred and popularized, and the seedling culture period is greatly shortened.

Description

Large cherry breeding method

Technical Field

The invention relates to the technical field of biological cultivation, in particular to a large cherry breeding method.

Background

The large cherry is also called Yingqing fruit, cherry pearl and cherry, is a rosaceous and Prunus plant, and is a fruit tree species with the earliest fruit maturity after Chinese cherry is relayed by a deciduous fruit tree in northern China; at present, the grafting breeding is mainly used for large cherry seedlings, but the grafting breeding is carried out by using stock seeds, so that the period is long, the labor is more, the cost is high, the grafting is required, the weather influence is great, and the survival rate is low; if cutting seedling raising is adopted, rooting is difficult, the rooting rate is low, viruses are easy to carry, the growth of cherry trees is influenced, the fruit quality is poor, and the yield is low.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a large cherry breeding method.

The purpose of the invention is realized by the following technical scheme: a large cherry breeding method comprises the following steps: preparing a sterilized explant, performing primary culture, performing secondary culture, performing rooting culture, acclimating test-tube seedlings, transplanting and acclimating bud seedlings and transplanting cherry seedlings;

the specific steps for preparing the sterilized explant are as follows: washing fresh large cherry shoots of 1.5-2cm in length with tap water for 25-35min, sterilizing with 70% alcohol on a clean bench for 30s, soaking in 0.1% L of mercury for 8-12 min while stirring, pouring out the L of mercury, injecting distilled water, brushing for 8-10 times, draining water with sterile gauze, peeling off stem tips under a dissecting mirror, cutting tip tissue of less than 0.2 mm, inoculating to a primary culture medium, and placing the sterilized explants on the primary culture medium according to the growth direction;

the formula of the culture medium for primary culture is as follows: MS + BA 1.0-2.0 mg/L + IAA 0.1-0.5 mg/L + 35% sucrose + 0.5% agar;

the formula of the culture medium for subculture is as follows: MS +6_ BA0.5-1.5 mg/L + NAA0.01-0.06 mg/L + KT0.5-1.5 mg/L + GA30.2-0.8 mg/L;

when the proliferation rate is high and the number of subcultures is small, adding a proper amount of auxin hormone, and adding 10-15 mg/L CCC or 0.5-1.0 mg/L PP 333; when the proliferation rate is low, the dosage of cytokinin is increased;

the subculture comprises the following specific steps: taking out cherry buds obtained by primary culture from a culture bottle, removing aged tissues, cutting, shearing buds and callus blocks together when cutting, wherein the size of the buds and the callus blocks is 0.5-1.0cm, and inoculating the buds and the callus blocks onto a culture medium for secondary culture; the culture temperature is 22-28 ℃; the culture illumination is 3000-;

the bud for rooting culture is stout, otherwise, strong seedling culture is carried out; adding less hormone into strong seedling culture medium, cutting off clustered buds with scissors, planting respectively with length not more than 2cm, transferring to culture medium for rooting culture after about 2 weeks when the buds grow more robustly, and inducing adventitious roots;

the formula of the culture medium for rooting culture is as follows: 1/2MS + IBA 0.5-1.5 mg/L + active carbon 0.5 mg/L;

the test-tube plantlet domestication method comprises the following specific steps: after rooting culture for 2-3 weeks, when the root length is 0.2-0.5 cm, removing the bottle cap in the culture chamber, and exposing the test-tube plantlet in the air;

the acclimatization substrate for bud seedling transplantation and acclimatization comprises, by weight, 0.5-1.5 parts of perlite, 1-3 parts of vermiculite, 1-3 parts of grass peat and 0.1-0.3 part of biological bacterial manure;

the specific steps of transplanting and domesticating the bud seedlings are as follows: after the test-tube plantlets are acclimated for 3-5 days, the bottle walls are lightly tapped to loosen the culture medium and roots, the test-tube plantlets are clamped by fingers or tweezers, the culture medium attached to the root systems is cleaned by clear water, soaked for 5-7 seconds by 0.1% carbendazim, and planted in an acclimation substrate after being dried in the air; after domestication and survival, the cherry seedlings can be transplanted to a field after being 15-20 centimeters high, and the transplanting time is 5 months;

the specific steps of cherry seedling transplantation are as follows: before transplanting, nursery land preparation is carried out, compost and stable manure are deeply turned and applied, 1500g of phoxim is used per mu, and a proper amount of fine soil is mixed and evenly dispersed in the nursery land; during transplanting, cherry seedlings are taken out from the seedling culture plate together with the matrix, planted according to the plant-row spacing of 15 multiplied by 30cm, and watered immediately after planting; and performing routine management according to a nursery land.

The invention has the following advantages: the large cherry seedlings produced by tissue culture by using biotechnology have strong disease resistance and high fruiting quality, the seedlings do not carry viruses, grafting is not needed, labor and time are saved, new varieties can be rapidly bred and popularized, and the seedling culture period is greatly shortened.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail and completely with reference to the accompanying drawings.

Thus, the following detailed description of embodiments of the invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

In addition, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Example 1: a large cherry breeding method comprises the following steps: preparing a sterilized explant, primary culture, secondary culture, rooting culture, test-tube seedling domestication, bud seedling transplantation domestication and cherry seedling transplantation.

The specific steps for preparing the sterilized explant are as follows: washing fresh cherry shoots of 1.5-2cm in length with tap water for 25-35min, sterilizing with 70% alcohol on a clean bench for 30s, soaking in 0.1% L of mercury for 8-12 min while stirring, pouring out the L of mercury, injecting distilled water, brushing for 8-10 times, draining water with sterile gauze, dissecting under a dissecting mirror, peeling off stem tip, cutting tip tissue of 0.2 mm below, inoculating to primary culture medium, and placing the sterilized explant on the primary culture medium according to growth direction.

In the embodiment, the preparation of the sterilized explant needs to be operated in a sterile room, and the air in the sterile room is sterilized; the operating tools must be autoclaved (temperature 120 ℃), for 25 minutes; the hands of the operator must be disinfected, washed clean with soap water, the nails are cut off, and the hands are scrubbed with 70% alcohol before operation; operators need to wear working clothes, wear working caps and masks, and are prohibited from talking strictly in the operation process; in operation, the test tube is held with the left hand, unfastened and the tampon removed, the test tube is held almost horizontally, close to the alcohol flame, the outside of the nozzle is burnt on the flame for several seconds, then forceps are held with the right hand to grip a piece of explant, fed into the tube, gently inserted into the culture medium, the forceps are burnt and then returned to the rack, and the tampon is gently inserted.

In this example, the inoculated culture flask was placed on a culture shelf in a culture room. The cherry sunshine-saving device is suitable for cherries, the temperature is 22-28 ℃, the sunshine length is 8-14 hours, the light intensity is 1000-3000 lx, and the humidity is 60-70%. The pollution is found and removed in time to prevent spreading and mutual infection.

The formula of the culture medium for primary culture is as follows: MS + BA 1.0-2.0 mg/L + IAA 0.1-0.5 mg/L + 35% sucrose + 0.5% agar.

In this embodiment: when preparing the culture medium, firstly, about 750mL of distilled water is put into a clean stainless steel pot, the agar is weighed and put into the pot to be boiled on an electric furnace until the agar is completely dissolved. Simultaneously, accurately measuring various mother liquids and hormones by using a measuring cylinder or a graduated pipette respectively, and putting the mother liquids and the hormones into a large beaker. Weighing sucrose, putting into melted agar solution, adding mother liquor and hormone in a beaker into the agar solution, stirring uniformly, adjusting pH to 5.8 with 1NKOH (or 1 NNaOH), putting the culture medium into a lower cup when the culture medium is hot, and quickly injecting the culture medium into bottles with 60ml per bottle under the control of a spring water stop clamp on a rubber tube of the lower cup. The culture medium does not have to be dropped onto the bottle mouth. Adding cotton plug and tipping paper. And (3) placing the packaged culture medium bottle into a pressure cooker, sterilizing at the high temperature of 121 ℃ for 20 minutes, and taking out for later use.

In this example, the MS medium stock solution is prepared by weighing in sequence according to the proportion, dissolving in a small amount of distilled water, and adding distilled water to a predetermined amount. The prepared stock solution is labeled and stored in a refrigerator for later use. The proportions of the stock solutions of MS medium are shown in Table 1.

TABLE 1 Primary Medium MS stock solution

The formula of the culture medium for subculture is as follows: MS +6_ BA0.5-1.5 mg/L + NAA0.01-0.06 mg/L + KT0.5-1.5 mg/L + GA30.2-0.8 mg/L.

The subculture comprises the following specific steps: taking out cherry buds obtained by primary culture from a culture bottle, removing aged tissues, cutting, shearing buds and callus blocks together when cutting, wherein the size of the buds and the callus blocks is 0.5-1.0cm, and inoculating the buds and the callus blocks onto a culture medium for secondary culture; the culture temperature is 22-28 ℃; the culture illumination was 3000-.

When the proliferation rate is high and the number of subcultures is small, adding a proper amount of auxin hormone, and adding 10-15 mg/L CCC or 0.5-1.0 mg/L PP 333; when the proliferation rate is low, the amount of cytokinin is appropriately adjusted.

The bud for rooting culture is stout, otherwise, strong seedling culture is carried out; adding less hormone into strong seedling culture medium, cutting off clustered buds with scissors, planting in length not more than 2cm, and transferring to rooting culture medium to induce adventitious root after about 2 weeks when the buds grow robustly.

The formula of the culture medium for rooting culture is as follows: 1/2MS + IBA 0.5-1.5 mg/L + active carbon 0.5 mg/L.

The test-tube plantlet domestication method comprises the following specific steps: after 2-3 weeks of rooting culture, when the root length is 0.2-0.5 cm, removing the bottle cap in the culture chamber, and exposing the test-tube plantlet in the air. The test-tube plantlet cherry grows and develops in a sterile high-humidity environment, and the surface of the test-tube plantlet cherry has no wax layer, so that the test-tube plantlet cherry cannot keep moisture and cannot prevent infection of mixed bacteria. Only through effectual training cultivation, can adapt to natural environment, normal growth removes the bottle lid in the culture chamber, explodes the test tube seedling in the air, lets it adapt to external environment gradually.

The acclimatization substrate for bud seedling transplantation and acclimatization comprises, by weight, 0.5-1.5 parts of perlite, 1-3 parts of vermiculite, 1-3 parts of grass peat and 0.1-0.3 part of biological bacterial manure. The acclimation substrate for the bud seedlings is suitable for being loose, good in drainage performance and good in air permeability, the acclimation substrate adopted in the embodiment is good in effect, the survival rate is high by applying the substrate in proportion, the growth is fast, and the seedlings are strong.

The specific steps of transplanting and domesticating the bud seedlings are as follows: after the test-tube plantlets are acclimated for 3-5 days, the bottle walls are lightly tapped to loosen the culture medium and roots, the test-tube plantlets are clamped by fingers or tweezers, the culture medium attached to the root systems is cleaned by clear water, soaked for 5-7 seconds by 0.1% carbendazim, and planted in an acclimation substrate after being dried in the air; after domestication and survival, the cherry seedling is 15-20 cm high and can be transplanted to a field, and the transplanting time is 5 months.

Example 2: and (4) screening mercuric chloride medicaments.

Experimental group 1: the explants were inoculated using the same procedure as in example 1; wherein, 0.1 percent mercuric chloride solution is adopted to soak the young shoots for 5 minutes;

experimental group 2: the explants were inoculated using the same procedure as in example 1; wherein, 0.1 percent mercuric chloride solution is adopted to soak young shoots for 8 minutes;

experimental group 3: the explants were inoculated using the same procedure as in example 1; wherein, 0.1 percent mercuric chloride solution is adopted to soak the young shoots for 10 minutes;

experimental group 4: the explants were inoculated using the same procedure as in example 1; wherein, 0.1 percent washing powder solution is adopted to soak young shoots for 8 minutes;

experimental group 5: the explants were inoculated using the same procedure as in example 1; wherein, 0.1 percent of detergent semen is adopted to soak young shoots for 8 minutes;

experimental group 6: the explants were inoculated using the same procedure as in example 1; wherein, the young shoots are soaked in distilled water for 8 minutes.

The results of the treatment of the mercuric chloride reagent in example 2 are shown in table 2 below.

TABLE 2 treatment results of mercuric chloride reagent

Item	Number of inoculation tubes	Pollution of the raw materials	Germinate%
				Experimental group 1	10	80	20
Experimental group 2	10	20	80
				Experimental group 3	10	0	0
Experimental group 4	10	85％	15％
				Experimental group 5	10	90％	10％
Experimental group 6	10	95％	5％

According to the results in table 2, the mercuric chloride is adopted for treating for a proper time, which is beneficial to avoiding pollution and improving germination rate, and has better sterilization effect compared with the washing powder and the detergent soaking.

Example 3: selection of a subculture medium formula and hormone dosage.

MS culture medium is used as basic culture medium, and the influence of different concentrations of 6_ BA, NAA, KT and GA3 on rapid propagation culture of cherries is tested.

Hormone concentration design

1.1 6_BA 0.5㎎/L 1.2 6_BA 1㎎/L 1.3 6_BA1.5㎎/L

2.1 NAA 0.01㎎/L 2.2 NAA0.03㎎/L 2.3 NAA0.06㎎/L

3.1 KT 0.5㎎/L 3.2 KT 1㎎/L 3.3 KT 1.5㎎/L

4.1 GA3 0.2㎎/L 4.2 GA3 0.5㎎/L 4.3 GA30.8㎎/L

Culture medium formula

1.MS+6_BA0.5㎎/L+NAA0.01㎎/L+KT0.5㎎/L+GA30.2㎎/L

2.MS+6_BA0.5㎎/L+NAA0.03㎎/L+KT1㎎/L+GA30.5㎎/L

3.MS+6_BA0.5㎎/L+NAA0.06㎎/L+KT1.5㎎/L+GA30.8㎎/L

4.MS+6_BA1㎎/L+NAA0.01㎎/L+KT1㎎/L+GA30.8㎎/L

5.MS+6_BA1㎎/L+NAA0.03㎎/L+KT1.5㎎/L+GA30.2㎎/L

6.MS+6_BA1㎎/L+NAA0.06㎎/L+KT0.5㎎/L+GA30.5㎎/L

7.MS+6_BA1.5㎎/L+NAA0.01㎎/L+KT1.5㎎/L+GA30.5㎎/L

8.MS+6_BA1.5㎎/L+NAA0.03㎎/L+KT0.5㎎/L+GA30.8㎎/L

9.MS+6_BA1.5㎎/L+NAA0.06㎎/L+KT1㎎/L+GA30.2㎎/L

The results of the multi-factor orthogonal assay are shown in Table 3.

Table 3 results of the multi-factor orthogonal assay.

	1	2	3	4	Results
						1	1	1	1	1	Is preferably used
2	1	2	2	2	Is preferably used
						3	1	3	3	3	Is preferably used
4	2	1	2	3	Bad
						5	2	2	3	1	Good taste
6	2	3	1	2	Bad
						7	3	1	3	2	Bad
8	3	2	1	3	Bad
						9	3	3	2	1	Is preferably used

According to the results of the multi-factor orthogonal test shown in the table 3, an MS +6_ BA1 mg/L + NAA0.03 mg/L + KT1.5 mg/L + GA30.2 mg/L mg is selected, the test-tube plantlet grows best, and the bud clump grows fast and sprouts more.

Example 4: the effect of different treatments on the proliferation and growth status of the subcultures.

The effect of different treatments on the proliferation and growth of the subcultures is shown in table 4.

Table 4 results of the effect of different treatments on the proliferation and growth conditions of the subcultures.

As is clear from the results in Table 4, the treated 1, 2, 3, 4 had a low proliferation fold and a poor growth state; the treatment has higher multiplication times of 6, 7, 8 and 9 but has weak buds; the treated 5 test-tube plantlets grow well, buds grow fast and sprout more, buds are strong and grow vigorously, and the culture medium MS +6_ BA1.0 mg/L + NAA0.03 mg/L + KT1.5 mg/L + GA30.2 mg/L is the optimal medium for rapid propagation culture.

When the proliferation rate is high, the number of subcultures is small, the concentration of cytokinin can be not adjusted, and a proper amount of auxin hormone can be added, and 10-15 mg/L of CCC or 0.5-1.0 mg/L of PP333 can be added. Preventing the occurrence of a large amount of vitrified seedlings. When the proliferation rate is low, the amount of cytokinin is adjusted and increased appropriately.

Example 5: effect of different temperatures on subculture.

The effect of different temperatures on subculture is shown in table 5.

Table 5 results of the effect of different temperatures on subculture.

Temperature of	Number of culture flasks	Days of culture	Growth condition cm
				16	40	30	0.8
18	40	30	0.9
				20	40	30	1
22	40	30	1.3
				24	40	30	1.2
26	40	30	1.2
				28	40	30	1.3
30	40	30	1

As can be seen from the experimental data in Table 5, the temperature of the culture room is in the range of 22-28 ℃ and the bud clumps grow fast.

Example 6: the effect of different illumination intensities on the subculture.

The effect of different light intensities on subculture is shown in table 6.

TABLE 6 results of the effects of different illumination intensities on subculture

Illuminance lx	Number of culture flasks	Days of culture	Growth height cm	Growth characteristics
					1000	40	30	1.3	Slender
3000	40	30	1.2	Rough and strong
					5000	40	30	1	Rough and strong
8000	40	30	0.7	Coarse, short and yellow hair
					10000	40	30	0.6	Coarse, short, white and yellow

As can be seen from the experimental data in Table 6, the growth of the florae is fast and good when the illumination is between 3000 and 5000 lx.

Example 7: influence of different illumination frequencies on the characteristics of subculture and large cherry.

Experimental group 1: culturing for 30 days by adopting 3000lx uninterrupted light;

experimental group 2: performing light culture and dark culture alternately for 2h by adopting 3000lx, namely performing dark culture for 2h after performing light culture for 2h, and culturing for 30 days;

experimental group 3: performing light culture and dark culture alternately for 4h by adopting 3000lx, namely performing dark culture for 4h after performing light culture for 4h, and culturing for 30 days;

experimental group 4: performing light culture and dark culture alternately for 6h by adopting 3000lx, namely performing dark culture for 6h after 6h of light culture for 30 days;

experimental group 5: 3000lx is adopted, light culture and dark culture are alternately carried out for 8h, namely 8h of dark culture is carried out after 8h of light culture, and the culture lasts for 30 days;

experimental group 6: performing light culture and dark culture alternately for 12h by adopting 3000lx, namely performing dark culture for 12h after performing light culture for 12h, and culturing for 30 days;

the effect of different light frequencies on the subculture and large cherry traits is shown in table 7.

TABLE 7 influence of different illumination frequencies on the characteristics of cherry seedlings after subculture and transplantation

From the results in table 7, it can be seen that the use of different illumination frequencies in the subculture has a certain effect on the shoots propagated by the subculture, wherein the experimental group 4 and the experimental group 5 have the highest growth height and are the most robust, but are the slowest in the subculture process; however, after the trees are transplanted, the experimental group 4 and the experimental group 5 show excellent disease resistance, which indicates that the photo culture and the case culture for 6-8h are more beneficial to the formation of disease resistance of the large cherry trees.

Example 8: in the formula of the rooting culture medium, the IBA concentration has influence on the rooting of the test-tube plantlet.

The effect of IBA concentration on tube plantlet rooting in the rooting medium formulation is shown in Table 8.

TABLE 8 results of the effect of IBA concentration in the media formulation for rooting culture on tube plantlet rooting.

From the results of Table 8 above, it can be seen that when the IBA concentration is 1.5mg/L, the rooting is fast, the roots are thick and strong, and the best use concentration is obtained.

Example 9: the temperature and humidity and the quantity of leaves in the bud seedling transplanting and domesticating process.

The temperature and humidity influence the transplanted seedlings mainly in the aspects of survival rate and growth of plants and roots; under the premise of proper temperature and humidity control, the illumination intensity and illumination time enough to ensure the photosynthesis of the seedlings to maintain the self life and growth need to be provided.

The effect of the number of active leaves and humidity on the survival rate of transplantation is shown in Table 9.

The effect of temperature on plant and root growth is shown in table 10.

TABLE 9 influence of effective leaf number and humidity on survival rate of transplantation

TABLE 10 Effect of temperature on plant and root growth results

Air temperature in the greenhouse (. degree. C.)	Ground temperature in the greenhouse (. degree. C.)	Growth status of the plant	Root growth status
				10	5	Stopping growth	Stopping growth
16	9	Slow growth	Slow growth
				22	14	Grow faster	Grow faster
30	18	Vigorous growth	Much root hair
				40	23	Scorching of leaves	Developed root system

As can be seen from the analysis of the test data shown in tables 9 and 10, the temperature of the greenhouse atmosphere is 22 to 30 ℃, the ground temperature is 14 to 18 ℃, and the relative humidity is 90% or more, which is the optimum temperature and humidity for the seedlings to be transplanted. The transplanting survival rate is high when more than 5 effective leaves are transplanted.

Example 10: a large cherry breeding method comprises the following steps: preparing a sterilized explant, primary culture, secondary culture, rooting culture, test-tube seedling domestication, bud seedling transplantation domestication and cherry seedling transplantation.

The specific steps for preparing the sterilized explant are as follows: washing fresh cherry shoots of 1.5-2cm in length with tap water for 30min, sterilizing with 70% alcohol on a clean bench for 30s, soaking in 0.1% L of mercury for 8min while stirring, pouring out the L of mercury, injecting distilled water, scrubbing for 8-10 times, sucking water with sterile gauze, peeling off stem tip under a dissecting mirror, cutting tip tissue of 0.2 mm below, inoculating onto primary culture medium, and placing the sterilized explants onto the primary culture medium according to growth direction.

In this example, the MS medium stock solution is prepared by weighing in sequence according to the proportion, dissolving in a small amount of distilled water, and adding distilled water to a predetermined amount. The prepared stock solution is labeled and stored in a refrigerator for later use. The proportions of the stock solutions of MS medium are as in Table 1 of example 1.

The formula of the culture medium for subculture is as follows: MS +6_ BA1 mg-mg/L + NAA0.03 mg-mg/L + KT1.5 mg-mg/L + GA30.2 mg-mg/L;

when the proliferation rate is high and the number of subcultures is small, adding a proper amount of auxin hormone, and adding 10-15 mg/L CCC or 0.5-1.0 mg/L PP 333; when the proliferation rate is low, the amount of cytokinin is increased.

The formula of the culture medium for rooting culture is as follows: 1/2MS + IBA1.5mg/L + activated carbon 0.5 mg/L.

The test-tube plantlet domestication method comprises the following specific steps: after 2-3 weeks of rooting culture, when the root length is 0.2-0.5 cm, removing the bottle cap in the culture chamber, and exposing the test-tube plantlet in the air.

The domesticated matrix for bud seedling transplantation and domestication comprises, by weight, 1 part of perlite, 2 parts of vermiculite, 2 parts of grass carbon and 0.2 part of biological bacterial manure.

Example 11: the same method as that in example 10 is adopted to breed fruits of Brukas variety and other varieties through tissue culture, and the economic trait ratio is shown in Table 11.

TABLE 11 comparison table of economic traits of fruits of Brukas variety and other varieties bred by tissue culture

According to the results in table 11, it can be seen that the large cherry variety brukes is bred by the tissue culture technique, and has strong disease resistance, high fruiting quality, high yield and strong seedling growth.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.

Claims

1. A large cherry breeding method is characterized in that: the method comprises the following steps: preparing a sterilized explant, primary culture, secondary culture, rooting culture, test-tube seedling domestication, bud seedling transplantation domestication and cherry seedling transplantation.

2. The large cherry breeding method according to claim 1, wherein the specific steps of preparing the sterilized explant are as follows: washing 1.5-2cm long fresh cherry tip with tap water for 25-35min, sterilizing with 70% alcohol on a clean bench for 30s, soaking in 0.1% L Hg solution for 8-12 min under stirring, pouring out the L Hg solution, injecting distilled water, brushing for 8-10 times, draining water with sterile gauze, dissecting under a dissecting mirror, peeling stem tip, cutting tip tissue of 0.2 mm below, inoculating to primary culture medium, and placing the sterilized explant on the primary culture medium according to growth direction.

3. The large cherry breeding method according to claim 1, wherein the formula of the primary culture medium is as follows: MS + BA 1.0-2.0 mg/L + IAA 0.1-0.5 mg/L + 35% sucrose + 0.5% agar.

4. The large cherry breeding method according to claim 1, wherein the formula of the culture medium for subculture is as follows: MS +6_ BA0.5-1.5 mg/L + NAA0.01-0.06 mg/L + KT0.5-1.5 mg/L + GA30.2-0.8 mg/L.

5. The large cherry breeding method according to claim 4, wherein the subculture comprises the following specific steps: taking out cherry buds obtained by primary culture from a culture bottle, removing aged tissues, cutting, shearing buds and callus blocks together when cutting, wherein the size of the buds and the callus blocks is 0.5-1.0cm, and inoculating the buds and the callus blocks onto a culture medium for secondary culture; the culture temperature is 22-28 ℃; the culture illumination was 3000-.

6. The large cherry breeding method according to claim 1, wherein the formula of the rooting culture medium is as follows: 1/2MS + IBA 0.5-1.5 mg/L + active carbon 0.5 mg/L.

7. The large cherry breeding method according to claim 1, wherein the test-tube plantlet domestication comprises the following steps: after 2-3 weeks of rooting culture, when the root length is 0.2-0.5 cm, removing the bottle cap in the culture chamber, and exposing the test-tube plantlet in the air.

8. The large cherry breeding method according to claim 1, wherein the method comprises the following steps: the acclimatization substrate for bud seedling transplantation and acclimatization comprises, by weight, 0.5-1.5 parts of perlite, 1-3 parts of vermiculite, 1-3 parts of grass peat and 0.1-0.3 part of biological bacterial manure.

9. The large cherry breeding method according to claim 8, wherein the method comprises the following steps: the specific steps of transplanting and domesticating the bud seedlings are as follows: after the test-tube plantlets are acclimated for 3-5 days, the bottle walls are lightly tapped to loosen the culture medium and roots, the test-tube plantlets are clamped by fingers or tweezers, the culture medium attached to the root systems is cleaned by clear water, soaked for 5-7 seconds by 0.1% carbendazim, and planted in an acclimation substrate after being dried in the air; after domestication and survival, the cherry seedling is 15-20 cm high and can be transplanted to a field, and the transplanting time is 5 months.

10. The large cherry breeding method according to claim 1, wherein the method comprises the following steps: the specific steps of cherry seedling transplantation are as follows: before transplanting, nursery land preparation is carried out, compost and stable manure are deeply turned and applied, 1500g of phoxim is used per mu, and a proper amount of fine soil is mixed and evenly dispersed in the nursery land; during transplanting, cherry seedlings are taken out from the seedling culture plate together with the matrix, planted according to the plant-row spacing of 15 multiplied by 30cm, and watered immediately after planting; and performing routine management according to a nursery land.