Litchi seedling breeding method

Abstract

The invention belongs to the technical field of litchi breeding, and particularly relates to a litchi seedling breeding method. According to the method, IBA, KT and the litchi pulp extract are combined, so that the explants are favorably induced to germinate; 6-BA, IBA and natrolite powder are added into the second culture medium, on the basis of reducing tissue culture browning, germination differentiation and development of litchi stems are promoted, and high-vitality plantlets can be obtained; 2, 4-D, zeatin, mannitol and activated carbon are added for rooting culture, so that the rooting rate of tender shoots (light green seedlings) is increased, and the survival rate of subsequent litchi seedlings is increased. Therefore, the technical scheme provided by the invention can be applied to breeding of litchi seedlings, and is simple, convenient and rapid, and the survival rate is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of litchi breeding, and particularly relates to a method for breeding litchi seedlings. Background Art

[0002] Litchi (Litchi chinensis) belongs to the genus Litchi of the family Sapindaceae. The litchi tree is an evergreen tree, 15 - 28 meters tall; its leaves are even-pinnate compound leaves, alternate, with usually 3 to 6 pairs of leaflets, elliptical or ovate in shape, and smooth edges; the flowers are small, yellowish-green, aggregated into a panicle; the litchi fruit is a drupe, which is not only delicious but also rich in nutritional value, and has a wide market prospect.

[0003] Litchi seedlings are often propagated by grafting and cutting. Although the existing methods can ensure that the new plants inherit the excellent traits of the mother plant, they are time-consuming and require a high amount of labor, often taking a lot of time and effort. At the same time, due to the low induction rate of litchi embryos and the low frequency of regenerated plants, in vitro culture of litchi is relatively difficult, which severely restricts the development of the litchi seedling industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for breeding litchi seedlings, which is simple and has a high survival rate.

[0005] The present invention provides a culture medium composition, which includes: a first culture medium, a second culture medium, and a third culture medium;

[0006] The first culture medium is composed of MS medium, IBA 0.5 - 1 mg / L, KT 0.2 - 0.4 mg / L, litchi pulp extract 1 - 1.5 mL / L, sucrose 25 - 30 g / L, and agar 5 - 7 g / L;

[0007] The second culture medium is composed of MS medium, 6 - BA 0.7 - 1 mg / L, IBA 0.2 - 0.3 mg / L, natrolite powder 0.05 - 0.1 mg / L, sucrose 25 - 30 g / L, and agar 5 - 7 g / L;

[0008] The third culture medium is composed of MS medium, 2,4 - D 0.6 - 0.8 mg / L, zeatin 0.3 - 0.4 mg / L, mannitol 80 - 100 mg / L, activated carbon 3 - 5 mg / L, sucrose 25 - 30 g / L, and agar 5 - 7 g / L.

[0009] Preferably, the preparation method of the litchi pulp extract includes:

[0010] Mix litchi pulp with water at a ratio of 1 g:10 - 12 mL, and then perform ultrasonic extraction and filtration in sequence. The filtrate is the litchi pulp extract.

[0011] Preferably, the parameters of the ultrasonic extraction include: temperature 20 - 25°C, time 45 - 55 min, and power 550 - 600 w.

[0012] The present invention provides the application of the above - described culture medium composition in the breeding of litchi seedlings.

[0013] The present invention provides a method for breeding litchi seedlings, which uses the culture medium composition described in the above - mentioned technical solution for breeding, and includes the following steps:

[0014] Disinfect the explant to obtain the disinfected explant;

[0015] Inoculate the disinfected explant into the first culture medium, the second culture medium, and the third culture medium in sequence for cultivation to obtain sterile seedlings;

[0016] Harden off and transplant the sterile seedlings to obtain litchi seedlings;

[0017] The explant includes: a litchi stem segment with axillary buds.

[0018] Preferably, the conditions for culturing in the first culture medium include: temperature 23 - 25°C, light intensity 800 - 900 LUX, light for 8 - 10 h per day, and time 5 - 7 d;

[0019] The conditions for culturing in the second culture medium include: temperature 23 - 25°C, light intensity 1000 - 1100 LUX, light for 10 - 12 h per day, and time 21 - 28 d;

[0020] The conditions for culturing in the third culture medium include: temperature 23 - 25°C, light intensity 1200 - 1300 LUX, light for 10 - 12 h per day, and time 45 - 50 d.

[0021] Preferably, the disinfection process includes:

[0022] Disinfect the litchi stem segment with an ethanol solution with a volume concentration of 70% - 75% for the first time for 30 - 45 s, and then disinfect it with a sodium hypochlorite solution with a mass concentration of 0.1% for the second time for 15 - 20 s to obtain the pretreated explant;

[0023] Wash the pretreated explant with sterile water and air - dry it to obtain the disinfected explant.

[0024] Preferably, the process of hardening off and transplanting includes:

[0025] Transplant the sterile seedlings into the substrate for hardening - off cultivation to obtain litchi seedlings;

[0026] The substrate is composed of perlite, humus soil, litchi shells and Bacillus subtilis bacterial liquid in a mass ratio of 20 - 30:20 - 30:5 - 8:0.5 - 0.8.

[0027] Preferably, the effective viable count of the Bacillus subtilis bacterial liquid is > 1×10 7 cfu / mL.

[0028] Preferably, the conditions for acclimatization culture include:

[0029] The temperature is 26 - 28°C, the humidity is 65% - 70%, the light intensity is 1000 - 1200 LUX, and the light exposure is 10 - 12 h per day.

[0030] Beneficial effects:

[0031] The present invention provides a culture medium composition, which includes: a first culture medium, a second culture medium and a third culture medium; the first culture medium is composed of MS medium, IBA 0.5 - 1 mg / L, KT 0.2 - 0.4 mg / L, litchi pulp extract 1 - 1.5 mL / L, sucrose 25 - 30 g / L and agar 5 - 7 g / L; the second culture medium is composed of MS medium, 6 - BA 0.7 - 1 mg / L, IBA 0.2 - 0.3 mg / L, natrolite powder 0.05 - 0.1 mg / L, sucrose 25 - 30 g / L and agar 5 - 7 g / L; the third culture medium is composed of MS medium, 2,4 - D 0.6 - 0.8 mg / L, zeatin 0.3 - 0.4 mg / L, mannitol 80 - 100 mg / L, activated carbon 3 - 5 mg / L, sucrose 25 - 30 g / L and agar 5 - 7 g / L. In the present invention, by combining IBA, KT and litchi pulp extract, it is beneficial to induce the germination of explants; by adding 6 - BA, IBA and natrolite powder to the second culture medium, on the basis of reducing tissue culture browning, it also promotes the sprouting, differentiation and development of litchi stem segments, which is beneficial to obtaining small seedlings with high vitality; by adding 2,4 - D, zeatin, mannitol and activated carbon for rooting culture, it not only improves the rooting rate of tender shoots (light green small seedlings), but also enhances the survival rate of subsequent litchi seedlings. Therefore, the technical solution provided by the present invention can be applied to the breeding of litchi seedlings. Specific embodiments

[0032] In the present invention, unless otherwise specified, the raw materials, methods and equipment used are conventional selections.

[0033] There are no special requirements for the litchi variety in the present invention; in the specific embodiments of the present invention, the litchi explants are derived from Feizixiao (Litchi chinensis 'Feizixiao') litchi; the explants include litchi stem segments with axillary buds, preferably young litchi stem segments with axillary buds; the length of the stem segments is preferably 1-2 cm.

[0034] The raw material source of the litchi pulp extract in the present invention is the pulp of Feizixiao litchi; the litchi shell is preferably the shell of Feizixiao litchi.

[0035] The pH values of the first medium, the second medium and the third medium in the present invention are preferably 5.8-6.0 respectively; there are no special requirements for the preparation methods of the first medium, the second medium and the third medium, and they can be prepared by the well-known methods in the art.

[0036] To further illustrate the present invention, the following describes the solutions provided by the present invention in detail with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Test Example 1

[0038] Effect of different disinfection methods on explants

[0039] Select young litchi stem segments with axillary buds and cut them into 1-2 cm as explants.

[0040] Treatment 1: In a sterile environment, soak the explants in an ethanol solution with a volume concentration of 75% for 30 s, then rinse with sterile water 2 times, and then soak the explants in a sodium hypochlorite solution with a mass concentration of 0.1% for 15 s to obtain the soaked explants; wash the soaked explants with sterile water 4 times, dry the moisture, and obtain the disinfected explants.

[0041] Treatment 2: The difference from Treatment 1 is only that the soaking time of 30 s in Treatment 1 is replaced by soaking for 45 s.

[0042] Treatment 3: The difference from Treatment 1 is only that the soaking time of 15 s in Treatment 1 is replaced by soaking for 20 s.

[0043] Treatment 4: The difference from Treatment 1 is only that the soaking time of 30 s in Treatment 1 is replaced by soaking for 1 min.

[0044] Treatment 5: The difference from Treatment 1 is only that the soaking time of 15 s in Treatment 1 is replaced by soaking for 1 min.

[0045] Treatment 6: The difference from Treatment 1 is only that the sodium hypochlorite solution with a mass concentration of 0.1% in Treatment 1 is replaced by a mercuric chloride solution with a mass concentration of 0.1%.

[0046] Respectively take the disinfected explants in Treatments 1 - 6, inoculate one explant into a culture bottle containing MS solid medium to form one replicate; conduct 20 parallel replicates for each treatment; place each treatment in the same culture environment for 14 days, and respectively count the number of contaminated explants and dead explants. The results are shown in Table 1.

[0047] Table 1 Number of contaminated explants and dead explants in different treatments

[0048] Treatment Number of contaminations Number of deaths Treatment 1 3 1 Treatment 2 3 0 Treatment 3 4 0 Treatment 4 10 5 Treatment 5 12 3 Treatment 6 10 4

[0049] Combined with the data in Table 1, it can be seen that different treatment methods have different effects on the disinfection of litchi explants; after disinfection using the methods in Treatments 1 - 3, after 14 days of culture, the number of contaminated explants is 3 - 4, and the number of dead explants is 1; while after adjusting the disinfection time and the type of disinfectant, the number of contaminated explants is as high as 10 - 12, and the number of dead explants is 3 - 5. Therefore, the methods in Treatments 1 - 3 are more conducive to the disinfection of litchi explants.

[0050] Test Example 2

[0051] Influence of various factors during the first culture process

[0052] Select tender litchi stem segments with axillary buds and cut them into 1 - 2 cm as explants.

[0053] In a sterile environment, soak the explants in an ethanol solution with a volume concentration of 75% for 30 s, then rinse with sterile water 2 times, and then soak the explants in a sodium hypochlorite solution with a mass concentration of 0.1% for 20 s to obtain the soaked explants; wash the soaked explants with sterile water 4 times, dry the moisture, and obtain the disinfected explants for standby.

[0054] Treatment 1:

[0055] (1) Prepare the first culture medium:

[0056] It consists of MS medium, 0.5 mg / L of IBA, 0.3 mg / L of KT, 1 mL / L of litchi pulp extract, 30 g / L of sucrose, and 7 g / L of agar; the pH value of the medium is 5.8;

[0057] Among them, the preparation method of the litchi pulp extract is as follows:

[0058] Mix litchi pulp and water at a ratio of 1 g:10 mL, and then use a juicer to crush the mixture to obtain a crushed product;

[0059] At 25 °C and 550 w, perform ultrasonic extraction on the crushed product for 50 min to obtain an ultrasonic extract; filter the ultrasonic extract with a filter membrane with a pore size of 0.22 μm, and the obtained filtrate is the litchi pulp extract.

[0060] (2) Culture conditions

[0061] The temperature is 25 °C, the light intensity is 800 LUX, the light exposure is 10 h per day, and the time is 5 d.

[0062] Treatment 2:

[0063] The difference from Treatment 1 is only in step (1). The process of preparing the first culture medium in step (1) is as follows:

[0064] It consists of MS medium, 0.8 mg / L of IBA, 0.4 mg / L of KT, 1.5 mL / L of litchi pulp extract, 30 g / L of sucrose, and 7 g / L of agar. The pH value of the culture medium is 5.8.

[0065] Treatment 3:

[0066] The difference from Treatment 1 is only in step (1). The preparation method of the litchi pulp extract in the process of preparing the first culture medium in step (1) is as follows:

[0067] Mix litchi pulp with water at a ratio of 1 g:12 mL, and then use a juicer to crush the mixture to obtain a crushed product;

[0068] At 20 °C and 550 w, perform ultrasonic extraction on the crushed product for 55 min to obtain an ultrasonic extract; filter the ultrasonic extract with a filter membrane with a pore size of 0.22 μm, and the obtained filtrate is the litchi pulp extract.

[0069] Treatment 4:

[0070] The difference from Treatment 1 is only in step (1). The process of preparing the first culture medium in step (1) is as follows:

[0071] It consists of MS medium, 0.3 mg / L of IBA, 0.1 mg / L of KT, 2 mL / L of litchi pulp extract, 30 g / L of sucrose, and 7 g / L of agar. The pH value of the culture medium is 5.8.

[0072] Treatment 5:

[0073] The difference from Treatment 1 is only in step (1). The process of preparing the first culture medium in step (1) is as follows:

[0074] It consists of MS medium, 0.5 mg / L of IAA, 0.3 mg / L of KT, 30 g / L of sucrose, and 7 g / L of agar. The pH value of the culture medium is 5.8.

[0075] Treatment 6:

[0076] The difference from Treatment 1 is only in step (1). The process of preparing the first culture medium in step (1) is as follows:

[0077] It is composed of MS medium, 0.5 mg / L of IBA, 0.3 mg / L of 2,4-D, 1 mL / L of litchi pulp extract, 30 g / L of sucrose and 7 g / L of agar, and the pH value of the medium is 5.8.

[0078] Treatment 7:

[0079] The difference from Treatment 1 is only in step (2), and the culture conditions in step (2) are as follows:

[0080] The temperature is 25 °C, the light intensity is 1200 LUX, the light is on for 8 h every day, and the time is 5 d.

[0081] In Treatments 1-7, each explant was inoculated into 1 culture bottle to form one replicate, and 20 parallel replicates were carried out for each treatment. The number of contaminated explants and the appearance morphology of the non-contaminated explants were respectively counted at the 5th d, and the results are shown in Table 2.

[0082] Table 2 Effects on explants in different treatments

[0083] Treatment Number of contaminations Morphology of explants Treatment 1 1 There are a few white dot-like calluses on the surface Treatment 2 1 There are a few white dot-like calluses on the surface Treatment 3 2 There are a few white dot-like calluses on the surface Treatment 4 5 No change on the surface Treatment 5 4 No change on the surface Treatment 6 2 The cut of the explant is browning, and there is no change on the surface Treatment 7 5 No change on the surface

[0084] Combined with the data in Table 2, it can be seen that when the medium in Treatments 1-3 is used for the first culture of explants, it is beneficial to induce the germination of explants, so a small amount of callus can be formed on the surface, and at the same time the contamination rate is low; although the contamination rate in Treatment 6 is not high either, browning occurred at the cut of the explants, which is not conducive to the induction of explant germination; the number of contaminated explants in Treatments 4-5 and Treatment 7 is relatively high, and there is no change on the surface of the explants, indicating that germination has not occurred yet.

[0085] Test Example 3

[0086] Influence of various factors during the second culture

[0087] Select young litchi stem segments with axillary buds and cut them into 1-2 cm as explants.

[0088] In a sterile environment, soak the explants in an ethanol solution with a volume concentration of 75% for 30 s, then rinse them 2 times with sterile water, and then soak the explants in a sodium hypochlorite solution with a mass concentration of 0.1% for 20 s to obtain the soaked explants; wash the soaked explants 4 times with sterile water, dry them in the air, and obtain the disinfected explants for standby.

[0089] Inoculate the disinfected explants into the first medium and culture them for 5 d under the culture conditions: the temperature is 25 °C, the light intensity is 800 LUX, and the light is on for 10 h every day to obtain the explants after the first culture for standby;

[0090] Among them, the culture medium for the first culture consists of MS medium, 0.5 mg / L of IBA, 0.3 mg / L of KT, 1 mL / L of litchi pulp extract, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the culture medium is 5.8;

[0091] Among them, the preparation method of the litchi pulp extract is as follows:

[0092] Mix litchi pulp with water at a ratio of 1 g:10 mL, and then use a juicer to crush the mixture to obtain a crushed product; perform ultrasonic extraction on the crushed product at 25°C and 550 w for 50 min to obtain an ultrasonic extract; filter the ultrasonic extract with a filter membrane with a pore size of 0.22 μm, and the obtained filtrate is the litchi pulp extract.

[0093] Treatment 1:

[0094] (1) Second culture:

[0095] The second culture medium consists of MS medium, 0.8 mg / L of 6-BA, 0.2 mg / L of IBA, 0.1 mg / L of natrolite powder, 30 g / L of sucrose, and 7 g / L of agar, and the pH value is 5.8;

[0096] The culture conditions include: the temperature is 25°C, the light intensity is 1000 LUX, the light is on for 12 h every day, and the time is 25 d.

[0097] Treatment 2:

[0098] The difference from Treatment 1 is only in step (1), and the second culture medium in step (1) is as follows:

[0099] The second culture medium consists of MS medium, 1 mg / L of 6-BA, 0.25 mg / L of IBA, 0.05 mg / L of natrolite powder, 30 g / L of sucrose, and 7 g / L of agar, and the pH value is 5.8.

[0100] Treatment 3:

[0101] The difference from Treatment 1 is only in step (1), and the second culture medium in step (1) is as follows:

[0102] The second culture medium consists of MS medium, 0.8 mg / L of 6-BA, 0.2 mg / L of IBA, 0.1 mg / L of activated carbon, 30 g / L of sucrose, and 7 g / L of agar, and the pH value is 5.8.

[0103] Treatment 4:

[0104] The difference from Treatment 1 is only in step (1), and the second culture medium in step (1) is as follows:

[0105] The second culture medium consists of MS medium, 6-BA 0.8 mg / L, NAA 0.2 mg / L, natrolite powder 0.1 mg / L, sucrose 30 g / L, and agar 7 g / L, with a pH value of 5.8;

[0106] Treatment 5:

[0107] The difference from Treatment 1 is only that the process of the first culture is omitted. The specific steps are as follows:

[0108] The disinfected explants are directly inoculated into the second culture medium for cultivation. The second culture medium consists of MS medium, 6-BA 0.8 mg / L, IBA 0.2 mg / L, natrolite powder 0.1 mg / L, sucrose 30 g / L, and agar 7 g / L, with a pH value of 5.8;

[0109] The culture conditions include: temperature 25°C, light intensity 1000 LUX, 12 hours of light per day, and the time is 25 days.

[0110] Treatment 6:

[0111] The difference from Treatment 1 is only in step (1). The proliferation culture conditions in step (1) are as follows:

[0112] The culture conditions include: temperature 25°C, light intensity 1400 LUX, 12 hours of light per day, and the time is 25 days.

[0113] In Treatments 1 - 6, each explant is inoculated into 1 culture bottle to form one replicate. Each treatment is carried out with 20 parallel replicates. The number of germinations occurring at 21 days is counted respectively, and the germinated shoots are described. The results are shown in Table 3.

[0114] Table 3 Effects of different treatments on the germination of explants

[0115] Treatment Number of germinations Morphology of the young shoots after germination Treatment 1 18 Change from white young shoots to light green small seedlings Treatment 2 17 Change from white young shoots to light green small seedlings Treatment 3 16 White young shoots Treatment 4 8 White young shoots Treatment 5 5 White young shoots Treatment 6 8 White young shoots

[0116] Combined with the data in Table 3, it can be seen that the culture methods in Treatments 1 - 2 are more conducive to inducing the explants to produce shoots and inducing them to transform into light green small seedlings; while the culture methods in Treatments 3 - 6 have either fewer germinations or the germinated shoots are still white and have not undergone transformation.

[0117] Test Example 4

[0118] Influence of various factors during the third culture process

[0119] Select the tender stem segments of litchi with axillary buds and cut them into 1 - 2 cm as explants.

[0120] Under aseptic conditions, the explants were immersed in an ethanol solution with a volume concentration of 75% for 30 s, then rinsed twice with sterile water, and then the explants were immersed in a sodium hypochlorite solution with a mass concentration of 0.1% for 20 s to obtain the immersed explants; the immersed explants were washed 4 times with sterile water and dried to obtain the disinfected explants for standby.

[0121] The disinfected explants were inoculated into the first medium and cultured for 5 d under the culture conditions: temperature 25 °C, light intensity 800 LUX, and 10 h of light per day to obtain the first-cultured explants for standby;

[0122] Among them, the medium for the first culture was composed of MS medium, 0.5 mg / L of IBA, 0.3 mg / L of KT, 1 mL / L of litchi pulp extract, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the medium was 5.8;

[0123] Among them, the preparation method of the litchi pulp extract was as follows:

[0124] The litchi pulp was mixed with water at a ratio of 1 g:10 mL, and then the mixture was crushed by a juicer to obtain a crushed product; the crushed product was ultrasonically extracted at 25 °C and 550 w for 50 min to obtain an ultrasonic extract; the ultrasonic extract was filtered through a filter membrane with a pore size of 0.22 μm, and the obtained filtrate was the litchi pulp extract.

[0125] The first-cultured explants were inoculated into the second medium (the medium was composed of MS medium, 0.8 mg / L of 6-BA, 0.2 mg / L of IBA, 0.1 mg / L of natrolite powder, 30 g / L of sucrose, and 7 g / L of agar, and the pH value was 5.8); cultured at a temperature of 25 °C, a light intensity of 1000 LUX, and 12 h of light per day for 25 d to obtain light green seedlings for standby;

[0126] Treatment 1:

[0127] (1) The third culture:

[0128] The third medium was composed of MS medium, 0.6 mg / L of 2,4-D, 0.4 mg / L of zeatin, 80 mg / L of mannitol, 3 mg / L of activated carbon, 30 g / L of sucrose, and 7 g / L of agar, and the pH value of the medium was 5.8.

[0129] The culture conditions included: temperature 25 °C, light intensity 1300 LUX, 10 h of light per day, and the time was 45 d.

[0130] Treatment 2:

[0131] The difference from Treatment 1 was only in step (1), and the medium in step (1) was as follows:

[0132] The third culture medium is composed of MS medium, 0.8 mg / L of 2,4-D, 0.3 mg / L of zeatin, 90 mg / L of mannitol, 3 mg / L of activated carbon, 30 g / L of sucrose and 7 g / L of agar, and the pH value of the culture medium is 5.8.

[0133] Treatment 3:

[0134] The difference from Treatment 1 is only in step (1), and the culture medium in step (1) is as follows:

[0135] The third culture medium is composed of MS medium, 0.6 mg / L of 6BA, 0.4 mg / L of zeatin, 80 mg / L of mannitol, 3 mg / L of activated carbon, 30 g / L of sucrose and 7 g / L of agar, and the pH value of the culture medium is 5.8.

[0136] Treatment 4:

[0137] The difference from Treatment 1 is only in step (1), and the culture medium in step (1) is as follows:

[0138] The third culture medium is composed of MS medium, 0.6 mg / L of 2,4-D, 0.4 mg / L of IAA, 80 mg / L of mannitol, 3 mg / L of activated carbon, 30 g / L of sucrose and 7 g / L of agar, and the pH value of the culture medium is 5.8.

[0139] Treatment 5:

[0140] The difference from Treatment 1 is only in step (1), and the culture medium in step (1) is as follows:

[0141] The third culture medium is composed of MS medium, 0.6 mg / L of 2,4-D, 0.4 mg / L of KT, 3 mg / L of activated carbon, 30 g / L of sucrose and 7 g / L of agar, and the pH value of the culture medium is 5.8.

[0142] Treatment 6:

[0143] The difference from Treatment 1 is only in step (1), and the culture conditions in step (1) are as follows:

[0144] The culture conditions include: the temperature is 25 °C, the light intensity is 1800 LUX, the light is on for 10 h every day, and the time is 45 d.

[0145] In Treatments 1 - 6, the light green seedlings obtained after the second culture are cut from the stem segments, and each seedling is inoculated into a culture bottle to form a replicate. 20 parallel replicates are carried out for each treatment, and the number of dead seedlings and the total number of rooted seedlings at 45 d are respectively counted. The results are shown in Table 4.

[0146] Table 4 Effects on rooting in different treatments

[0147] Treatment Number of deaths Total number of roots Treatment 1 1 48 Treatment 2 2 40 Treatment 3 5 28 Treatment 4 4 31 Treatment 5 6 29 Treatment 6 6 30

[0148] It can be seen from the data in Table 4 that when the method provided in Treatments 1-2 is used for the third cultivation, not only is the survival rate high, but it is also beneficial for the small seedlings to take root; while the cultivation methods provided in Treatments 3-6 reduce the survival rate of the small seedlings due to browning during the cultivation process, and at the same time are not conducive to the small seedlings taking root.

[0149] Test Example 5

[0150] Influence of various factors during the transplanting process

[0151] Select the aseptic seedlings after taking root by the method in Treatment 1 of Test Example 4 for the following tests:

[0152] Treatment 1:

[0153] (1) Prepare the substrate:

[0154] Dry and crush the litchi shells (the particle size of the crushed material < 0.5 cm) to obtain the crushed material;

[0155] Mix perlite, humus soil, the crushed material, and Bacillus subtilis bacterial liquid (the effective viable count is 1×10 7 cfu / mL) evenly according to the mass ratio of 30:30:5:0.5 to obtain the substrate;

[0156] (2) Transplant the aseptic seedlings into the above substrate, and carry out acclimatization cultivation at 28 °C, a humidity of 65%-70%, a light intensity of 1200 LUX, and 12 hours of light per day.

[0157] Treatment 2:

[0158] The difference from Treatment 1 is only in step (1). In step (1), the substrate composition is as follows:

[0159] Mix perlite, humus soil, the crushed material, and Bacillus subtilis bacterial liquid (the effective viable count is 1×10 7 cfu / mL) evenly according to the mass ratio of 20:30:8:0.5 to obtain the substrate;

[0160] Treatment 3:

[0161] The difference from Treatment 1 is only in step (1). In step (1), the substrate composition is as follows:

[0162] Mix perlite, humus soil, the crushed material, and Bacillus subtilis bacterial liquid (the effective viable count is 1×10 7 cfu / mL) evenly according to the mass ratio of 50:30:8:0.5 to obtain the substrate;

[0163] Treatment 4:

[0164] The difference from Treatment 1 is only in step (1), and the matrix composition in step (1) is as follows:

[0165] Mix perlite, humus soil, crushed material and Bacillus amyloliquefaciens bacterial liquid (effective viable count is 1×10 7 cfu / mL) evenly according to the mass ratio of 30:30:5:0.5 to obtain the matrix;

[0166] Treatment 5:

[0167] The difference from Treatment 1 is only in step (1), and the matrix composition in step (1) is as follows:

[0168] Mix perlite, humus soil and crushed material evenly according to the mass ratio of 30:30:5 to obtain the matrix;

[0169] Treatment 6:

[0170] The difference from Treatment 1 is only in step (2), and the matrix composition in step (2) is as follows:

[0171] (2) Transplant the sterile seedlings into the above matrix, and carry out acclimatization culture at 28°C, humidity of 65%-70%, light intensity of 1500 LUX, and 12 hours of light per day.

[0172] In Treatments 1-6, select sterile seedlings with basically the same growth vigor, transplant them into flower pots containing the matrix respectively, transplant 1 plant in each flower pot to form one treatment, and conduct 20 parallel repetitions for each treatment. Respectively count the survival numbers of the sterile seedlings 30 days after transplantation, and the results are shown in Table 5.

[0173] Table 5 Survival numbers of sterile seedlings in different treatments

[0174] Treatment Number of survivors Growth vigor Treatment 1 20 Good, thick Treatment 2 19 Good, thick Treatment 3 17 Good, short Treatment 4 13 Good, short Treatment 5 12 Good, short Treatment 6 10 Good, short

[0175] It can be seen from the data in Table 5 that when using the methods provided in Treatments 1-2 for acclimatization treatment, not only the survival rate is high, but also the growth vigor of the sterile seedlings in each treatment is good; while after using the methods provided in Treatments 3-6 for acclimatization, the survival rate is low, and the growth vigor of each sterile seedling is not as good as that of the sterile seedlings in Treatments 1-2.

[0176] In summary, the present invention combines IBA, KT and litchi pulp extract, which is beneficial to inducing the germination of explants; by adding 6-BA, IBA and natrolite powder to the second culture medium, on the basis of reducing tissue culture browning, it also promotes the sprouting, differentiation and development of litchi stem segments, which is beneficial to obtaining small seedlings with high vitality; by adding 2,4-D, zeatin, mannitol and activated carbon for rooting culture, it not only improves the rooting rate of tender shoots (light green small seedlings), but also enhances the survival rate of subsequent litchi seedlings. Therefore, the technical solution provided by the present invention can be applied to the breeding of litchi seedlings.

[0177] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A culture medium composition, characterized in that The culture medium composition comprises: a first culture medium, a second culture medium and a third culture medium; The first culture medium consists of MS culture medium, 0.5-1 mg / L IBA, 0.2-0.4 mg / L KT, 1-1.5 mL / L litchi pulp extract, 25-30 g / L sucrose and 5-7 g / L agar; The second culture medium consists of MS culture medium, 0.7-1 mg / L 6-BA, 0.2-0.3 mg / L IBA, 0.05-0.1 mg / L sodium zeolite powder, 25-30 g / L sucrose and 5-7 g / L agar; The third culture medium consists of MS culture medium, 0.6-0.8 mg / L 2,4-D, 0.3-0.4 mg / L zeatin, 80-100 mg / L mannitol, 3-5 mg / L activated carbon, 25-30 g / L sucrose and 5-7 g / L agar.

2. The culture medium composition according to claim 1, characterized in that The preparation method of the litchi pulp extract comprises: The litchi pulp and water are mixed at a ratio of 1 g: 10-12 mL, and then ultrasonic extraction and filtration are performed in sequence. The filtered juice is the litchi pulp extract.

3. The culture medium composition according to claim 2, characterized in that The parameters of the ultrasonic extraction include: temperature of 20-25°C, time of 45-55min, and power of 550-600w.

4. Use of the culture medium composition according to any one of claims 1 to 3 in litchi seedling breeding.

5. A method for breeding litchi seedlings, characterized in that: The method of breeding using the culture medium composition according to any one of claims 1 to 3 comprises the following steps: sterilizing the explant to obtain a sterilized explant; The sterilized explants are inoculated into the first culture medium, the second culture medium and the third culture medium in sequence for culturing to obtain sterile seedlings; Hardening and transplanting the sterile seedlings to obtain litchi seedlings; The explant comprises: a litchi stem segment with axillary buds.

6. The method according to claim 5, characterized in that The conditions for inoculating in the first culture medium for culturing include: a temperature of 23-25° C., a light intensity of 800-900 LUX, 8-10 hours of light per day, and a time of 5-7 days; The conditions for inoculating and culturing the second culture medium include: a temperature of 23-25° C., a light intensity of 1000-1100 LUX, 10-12 hours of light per day, and a time of 21-28 days; The conditions for inoculating in the third culture medium for culturing include: a temperature of 23-25° C., a light intensity of 1200-1300 LUX, 10-12 hours of light per day, and a time of 45-50 days.

7. The method according to claim 5, characterized in that The disinfection process includes: The litchi stem segments are sequentially sterilized with an ethanol solution having a volume concentration of 70% to 75% for a first time for 30 to 45 seconds, and sterilized with a sodium hypochlorite solution having a mass concentration of 0.1% for a second time for 15 to 20 seconds to obtain pretreated explants; The pretreated explants are washed with sterile water and air-dried to obtain sterilized explants.

8. The method according to claim 5, characterized in that The process of seedling hardening and transplanting comprises: Transplanting the sterile seedlings into a substrate for seedling hardening and culture to obtain litchi seedlings; The matrix is ​​composed of perlite, humus, litchi shell and Bacillus subtilis liquid in a mass ratio of 20-30:20-30:5-8:0.5-0.

8.

9. The method according to claim 8, characterized in that The effective viable count of the Bacillus subtilis liquid is >1×10 7 cfu / mL.

10. The method according to claim 8, characterized in that The conditions of the seedling hardening culture include: The temperature is 26-28℃, the humidity is 65%-70%, the light intensity is 1000-1200LUX, and the light intensity is 10-12h per day.