A method for proliferating blackberry tissue culture by two rounds of proliferation

Abstract

The application discloses a method for two-step proliferation of blackberry tissue culture and rapid propagation. The method comprises the following steps: (1) inserting a blackberry stem into a primary proliferation induction medium to induce axillary bud germination and differentiation, thereby obtaining a sterile explant containing blackberry primary regenerated buds; (2) cutting the explant stem containing the primary regenerated buds from the sterile explant containing the blackberry primary regenerated buds obtained in the step (1) and placing the explant stem into a secondary proliferation induction medium to induce secondary differentiation and proliferation, thereby obtaining blackberry regenerated cluster seedlings; and (3) cutting the blackberry regenerated cluster seedlings obtained in the step (2) and inoculating the blackberry regenerated cluster seedlings into a rooting medium to perform rooting culture, thereby obtaining blackberry regenerated plants. The differentiation and proliferation coefficient of the regenerated seedlings reaches about 30, the rooting rate is close to 100%, and the survival rate of the seedlings is very high.

Description

TECHNICAL FIELD

[0001] The present application relates to plant tissue culture technology, in particular to a two-round proliferation method for blackberry tissue culture and rapid propagation. BACKGROUND

[0002] Blackberry (Rubus spp.) is a perennial berry tree of the genus Rubus L. in the Rosaceae family. Blackberry fruit is a purple nearly black aggregate fruit, soft and juicy, with a strong aroma. Numerous studies have shown that blackberry is rich in anthocyanins, proanthocyanidins, phenolic acids, and functional active ingredients such as vitamin C and vitamin E, and has outstanding health care effects such as antioxidant, anti-aging, cholesterol reduction, and immune enhancement. It is loved by high-end consumers, especially European and American consumers, and is known as "life fruit" and "black diamond". It is estimated that the global blackberry planting area has exceeded 27,000 hectares (Strik BC, Clark JR, Finn CE, et al. 2007. Worldwide blackberry production. Acta Horticulturae, 777:205-213.), and has developed into the third generation of emerging small berries. China is the main planting area of blackberry in Asia and the third largest blackberry cultivation country in the world, and the planting and promotion heat is still increasing, and the demand for good seedlings is extremely high.

[0003] In production, the seedling breeding of blackberry is mainly realized through layering, cutting and root sprouting techniques. However, the layering and root sprouting have problems of uneven emergence (difference in stem segment strength), easy disease (soil around the roots is infected), and inconvenient operation (straight plant type is difficult to bend) (Liu Xiaowei. 2018. Establishment of tissue culture and rapid propagation technology system of blackberry 'Arapaho' and preparation of leaf protoplasts. Sichuan Agricultural University. Ya'an.). Although the cutting has high propagation coefficient and controllable operation consistency, the seedling management after cutting requires high requirements for environmental conditions and management techniques, and is time-consuming and labor-intensive. Therefore, since the 1970s, technical workers have begun to explore the establishment of a blackberry tissue culture and rapid propagation technology system, and currently, regenerated plants can be induced and differentiated from stem segments, leaves, petioles and cotyledons, which effectively supports the demand of the industry for high-quality seedlings. However, the existing blackberry tissue culture and rapid propagation technology system usually consists of multiple links, including external plantlet disinfection, primary axillary bud germination induction, proliferation (some have a dark culture link), subculture (stem elongation induction), strong seedling and rooting, each link needs to be configured with different culture media, and the culture medium usually needs to be replaced once every 20-30 days, which is complicated and has high cost consumption. At the same time, the regeneration seedlings of the existing system have low proliferation coefficient, and under the commonly used stem segment induction method, the proliferation coefficient is about 5-6 (He Jiatao. 2007. Evaluation of fruit quality of raspberry and blackberry and establishment of efficient shoot tip regeneration system. Central China Agricultural University. Wuhan.) or less than 10 (He Liheng. 2005. Detoxification and rapid propagation of American red raspberry and blackberry and factory seedling breeding. China Agricultural University. Beijing.), and the leaves of the regenerated seedlings are yellowish and weak, and the overall rapid propagation efficiency is low. SUMMARY

[0004] Therefore, the present application provides a two-round proliferation type blackberry tissue culture and rapid propagation method which is simple in operation and efficient in proliferation.

[0005] The two-round proliferation type blackberry tissue culture and rapid propagation method provided by the present application comprises the following steps:

[0006] (1) inserting a blackberry stem segment into a primary proliferation induction culture medium to perform axillary bud germination and differentiation induction culture, so as to obtain a sterile explant containing blackberry primary regenerated buds;

[0007] (2) cutting an explant stem segment containing the primary regenerated buds from the sterile explant containing the blackberry primary regenerated buds obtained in the step (1) and placing it in a secondary proliferation induction culture medium to perform secondary differentiation and proliferation induction culture, so as to obtain blackberry regenerated cluster seedlings;

[0008] (3) cutting the blackberry regenerated cluster seedlings obtained in the step (2) and inoculating them into a rooting culture medium for rooting culture, so as to obtain blackberry regenerated plants.

[0009] Optionally, the primary proliferation induction medium comprises the following concentrations of components: NH4NO31.65 g / L, KNO31.9 g / L, KH2PO40.17 g / L, CaCl2·2H2O 0.88 g / L, MgSO4·7H2O 0.37 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 3 mg / L to 4 mg / L, indole-3-butyric acid (IBA) 0.5 mg / L to 1 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L; and the pH of the primary proliferation induction medium is 5.6 to 5.8.

[0010] Optionally, the secondary proliferation induction medium comprises the following concentrations of components: NH4NO30.4 g / L, KH2PO40.17 g / L, K2SO40.99 g / L, CaCl2·2H2O 0.096 g / L, Ca(NO3)2·4H2O 0.556 g / L, MgSO4·7H2O 0.37 g / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl2·6H2O 0.025 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 2 mg / L to 3 mg / L, indole acetic acid (IAA) 0.5 mg / L to 1.0 mg / L, gibberellin (GA3) 2 mg / L to 3 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L; and the pH of the secondary proliferation induction medium is 5.6 to 5.8.

[0011] Optionally, the rooting medium comprises the following concentrations of ingredients: NH4NO30.825 g / L, KNO30.95 g / L, KH2PO40.085 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.185 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine-HCl 0.1 mg / L, pyridoxine-HCl 0.5 mg / L, glycine 2.0 mg / L; and sucrose 10 g / L to 30 g / L, indole-3-butyric acid (IBA) 0.1 mg / L to 0.3 mg / L, and carrageenan 0 g / L or 7.0 g / L; and the pH of the rooting medium is 5.6 to 5.8.

[0012] Optionally, in step (1), the blackberry stem segments are lignified current-year blackberry stem segments; the blackberry stem segments are 6-7 cm stem segments, each containing 1-2 bud points; before the blackberry stem segments are inserted into the primary proliferation induction medium, the blackberry stem segments are subjected to sterilization by the following method: the blackberry stem segments are soaked in 75% alcohol for 30-40 s, then washed with sterile water for 3-5 times, and shaken and soaked for 3-5 min each time; after the water is absorbed, the explants are soaked in 3% sodium hypochlorite for 10-15 min, then washed with sterile water for 5-6 times, and shaken and soaked for 8-10 min each time; the stem segments are inserted into the primary proliferation induction medium in a manner that the morphological upper part of the stem segments faces upward, and one of the bud points is in full contact with the primary proliferation induction medium, immersed in the medium or leveled with the medium.

[0013] Optionally, in step (1), the axillary bud germination and differentiation induction culture is carried out at a temperature of 24-28°C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours of light / 10 hours of darkness for 25-35 days.

[0014] Optionally, in step (2), when the explant stem segments containing the primary regenerated buds are cut, the regenerated buds are not cut off from the stem segments, but only the stem segments other than the part where the regenerated buds grow are cut off; the cut explant stem segments containing the primary regenerated buds are placed horizontally in the secondary proliferation induction medium, and the remaining stem segments remaining at the part where the regenerated buds grow are immersed in the secondary proliferation induction medium.

[0015] Optionally, in the step (2), the secondary proliferation induction culture is carried out at a temperature of 24-28 DEG C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours light / 10 hours dark for 40-50 days.

[0016] Optionally, in the step (3), the rooting culture is carried out by vertically inoculating the regenerated cluster seedlings with a height of 4-5 cm into the rooting medium in a single plant form; and the lower leaves of the plant are removed when inoculated into the rooting medium, and 2-3 top leaves are reserved.

[0017] Optionally, in the step (3), the rooting culture is carried out at a temperature of 24-28 DEG C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours light / 10 hours dark for 20-30 days.

[0018] The method for the two-round proliferation type blackberry tissue culture and rapid propagation provided by the application comprises three steps of explant disinfection, primary proliferation induction, secondary proliferation induction, and rooting induction, the differentiation proliferation coefficient of the regenerated seedlings is about 30, the rooting rate is close to 100%, and the survival rate is extremely high. Meanwhile, the method provided by the application starts the proliferation induction from the explant, which is different from the conventional idea of first inducing the axillary bud germination and then using the germinated axillary bud for proliferation differentiation, greatly saves the time consumption of seedling raising, and shortens the seedling raising period; in addition, the method provided by the application in the rooting link can adopt liquid rooting, avoids the washing root link before transplanting, reduces the root breaking phenomenon and labor consumption, and is the first of the blackberry seedling raising system.

[0019] The application makes the factory tissue culture seedling raising process of blackberry extremely simple, and a complete set of production can be completed by the circulation use of only three kinds of culture media; the proliferation seedling raising can be started from the first step of the explant, the seedling raising period is greatly shortened, the super-high proliferation rate further improves the seedling raising efficiency of the whole system, and the profit space of seedling raising is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0020] For the purpose of illustration and not limitation, the application will now be described with the preferred embodiments of the application, with particular reference to the accompanying drawings, in which:

[0021] Figure 1 The left drawing is a tissue culture growth schematic diagram of the blackberry aseptic explant, and the right drawing is a near view of the effect of the blackberry axillary bud in the S2 primary proliferation induction.

[0022] Figure 2 The blackberry explant (single plant) in the S10 secondary proliferation induction (subculture proliferation) medium shown in Table 2.

[0023] Figure 3 Fig. 2 shows the proliferation status of blackberry in the secondary proliferation induction (subculture proliferation) medium shown in Table 2; wherein, the left side graph is a front view of the proliferation status of blackberry in the corresponding medium; and the right side graph is a corresponding bottle bottom view thereof.

[0024] Figure 4 Fig. 3 shows the growth of blackberry in the rooting medium shown in Table 3; wherein, the left side graph is a schematic view of the growth status of blackberry in the corresponding rooting medium; and the right side graph is a close-up view of the corresponding root system growth effect thereof.

[0025] Figure 5 Fig. 4 shows the effect of induction of blackberry in the G8 shallow liquid rooting medium shown in Table 3 for 15 days; wherein, A is a schematic view of the growth status of blackberry in the liquid rooting medium; B is a close-up view of the corresponding root system growth effect thereof; and C is the blackberry regenerated seedling obtained by the shallow liquid rooting culture, which can be used for transplanting.

[0026] Figure 6 Fig. 5 shows a schematic view of the growth status of the blackberry regenerated seedling obtained by the shallow liquid rooting culture after transplanting. DETAILED DESCRIPTION

[0027] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0028] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0029] Example 1, Optimization of Blackberry Two-Round Proliferation Type Tissue Culture Rapid Propagation Method

[0030] I. Disinfection of explants and induction of primary proliferation

[0031] Fresh, lignified current-year shoots (with leaves removed) of ‘Prime-Ark Freedom’ blackberry were cut into 6-7 cm stem segments with buds (each segment containing at least one bud point) from the Golden Sun Farm, washed with dishwashing liquid water, and then rinsed with running water for 1-2 h. The explants were then sterilized on a clean bench. The stem segments were soaked in 75% alcohol for 30-40 s, then rinsed with sterile water 3-5 times, and shaken for 3-5 min each time. After the water stains on the explants were absorbed with sterile filter paper, the explants were soaked in 3% available chlorine sodium hypochlorite for 10-15 min, then rinsed with sterile water 5-6 times, and shaken for 8-10 min each time. Thereafter, the water stains on the surface of the explants were absorbed with sterile filter paper, and the 0.5-1 cm thick tissue at the cut end of the stem segment was removed with a sterilized knife blade. The remaining stem segment was then inserted vertically (with the morphological upper part facing upwards) into the primary proliferation induction medium ( Figure 1 ).

[0032] Because the stem section cut surface is directly exposed to the air, it is more bacteria; and the stem section cut surface is most seriously damaged by disinfectant, the cells are seriously damaged, removing it not only helps to improve the sterility rate, but also helps the cells to contact the culture medium, and is conducive to regeneration induction.

[0033] The primary proliferation induction culture of this embodiment sets four culture schemes S1-S4, and the primary proliferation induction medium used in the four schemes is shown in Table 1.

[0034] Table 1 Screening of blackberry primary proliferation induction medium

[0035]

[0036] The primary proliferation mother liquor in Table 1 is composed of the following components at the following concentrations: NH4NO31.65 g / L, KNO31.9 g / L, KH2PO40.17 g / L, CaCl2·2H2O 0.88 g / L, MgSO4·7H2O 0.37 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L.

[0037] Prepare the blackberry primary proliferation induction medium:

[0038] Prepare the primary proliferation induction medium according to Table 1. First, accurately weigh each compound in turn, melt one by one with a small amount of distilled water, and mix thoroughly; then weigh and add an appropriate amount of sucrose, stir to dissolve; then accurately add the corresponding amount of hormone with a pipette, and add distilled water to the required volume; then use a pH meter to adjust the pH of the solution to 5.6-5.8 by adding a small amount of HCl or NaOH; then transfer the solution to a heating appliance and add the specified amount of carrageenan, stir and heat until the carrageenan is fully dissolved. Then, divide the prepared primary proliferation induction medium equally into glass tissue culture bottles, about 30 mL per bottle, and tighten the cap. Then, place it in a tissue culture special high-temperature high-pressure sterilization pot, sterilize at 121°C for 20 min. After sterilization, take out the medium and let it cool and solidify, then transfer it to a clean bench, sterilize it under ultraviolet light for 20 min, and then use it.

[0039] When the explants are inserted into the culture medium, try to make one bud point immerse into the culture medium or be level with the culture medium.

[0040] The initial proliferation culture is carried out at a temperature of 25°C, a light intensity of 1200Lx, and a light cycle of 14 hours light / 10 hours darkness for 25-35 days.

[0041] As shown in Table 1, the initial proliferation effect of the axillary buds induced by S2 culture medium is the best. Thus, the optimal culture medium for the initial proliferation induction of blackberry is as follows: Figure 1

[0042] The optimal initial proliferation induction culture medium consists of the following components at the following concentrations: NH4NO31.65g / L, KNO31.9g / L, KH2PO40.17g / L, CaCl2·2H2O 0.88g / L, MgSO4·7H2O 0.37g / L, KI 0.83mg / L, Na2MoO4·2H2O 0.25mg / L, H3BO36.2mg / L, CuSO4·5H2O 0.025mg / L, MnSO4·4H2O 22.3mg / L, CoCl2·6H2O 0.025mg / L, ZnSO4·7H2O 8.6mg / L, Na2EDTA·2H2O 37.3mg / L, FeSO4·7H2O 27.8mg / L, nicotinic acid 0.5mg / L, myo-inositol 100mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, 6-benzylaminopurine (6-BA) 4mg / L, indole-3-butyric acid (IBA) 0.5mg / L, sucrose 25g / L, and carrageenan 7.0g / L, with a pH value of 5.6.

[0043] Secondary proliferation induction (proliferation subculture)

[0044] The sterile explants obtained in step one are cut to obtain the primary regenerated buds obtained after the initial proliferation induction culture using sterilized tools in the clean bench. The excess stem structure at the upper and lower ends of the regenerated buds is removed, and the explant stem where the regenerated buds grow is retained and transferred to the secondary proliferation culture medium, i.e., the proliferation subculture medium. When placed, the retained stem is placed horizontally and covered in the proliferation subculture medium to ensure that the cut ends of the stem are in full contact with the culture medium to better absorb nutrients and hormones. At the same time, the horizontal placement allows the regenerated buds to grow vertically and better support the cluster of buds that will sprout later.

[0045] The proliferation subculture of this embodiment is set up in 12 culture schemes S1-S12. The secondary proliferation culture media used in these 12 schemes are shown in Table 2. ​

[0046] Table 2. Screening of blackberry secondary proliferation induction medium

[0047]

[0048] The MS mother liquor in the above Table 2 is composed of the following components at the following concentrations:

[0049] NH4NO31.65 g / L, KNO31.90 g / L, KH2PO40.17 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.37 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, glycine 2.0 mg / L, and carrageenan 7.0 g / L;

[0050] The 2 / 3MS mother liquor in the above Table 2 is composed of the following components at the following concentrations: NH4NO31.1 g / L, KNO31.27 g / L, KH2PO40.113 g / L, MgSO4·7H2O 0.25 g / L. The remaining components and concentrations are the same as those of the MS mother liquor.

[0051] The modified DKW mother liquor in the above Table 2 is composed of the following components at the following concentrations:

[0052] NH4NO3 1.416 g / L, KH2PO4 0.265 g / L, K2SO4 1.559 g / L, CaCl2 2H2O 0.113 g / L, Ca(NO3)2 4H2O 1.367 g / L, MgSO4 7H2O 0.361 g / L, Na2MoO4 2H2O 0.25 mg / L, H3BO3 6.2 mg / L, CuSO4 5H2O 0.025 mg / L, MnSO4 4H2O 22.3 mg / L, ZnSO4 7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl 6H2O 0.025 mg / L, Na2EDTA 2H2O 37.3 mg / L, FeSO4 7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine-HCl 0.1 mg / L, pyridoxal-HCl 0.5 mg / L, glycine 2.0 mg / L, and carrageenan 7.0 g / L.

[0053] The secondary propagation mother liquor in Table 2 above is composed of the following concentrations of ingredients:

[0054] NH4NO3 0.4 g / L, KH2PO4 0.17 g / L, K2SO4 0.99 g / L, CaCl2 2H2O 0.096 g / L, Ca(NO3)2 4H2O 0.556 g / L, MgSO4 7H2O 0.37 g / L, Na2MoO4 2H2O 0.25 mg / L, H3BO3 6.2 mg / L, CuSO4 5H2O 0.025 mg / L, MnSO4 4H2O 22.3 mg / L, ZnSO4 7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl 6H2O 0.025 mg / L, Na2EDTA 2H2O 37.3 mg / L, FeSO4 7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine-HCl 0.1 mg / L, pyridoxal-HCl 0.5 mg / L, glycine 2.0 mg / L, and carrageenan 7.0 g / L.

[0055] A secondary propagation induction medium for blackberry was prepared:

[0056] The secondary proliferation induction medium was prepared according to Table 2. First, each compound was accurately weighed in turn, and then was melted and mixed uniformly with a small amount of distilled water. Then, an appropriate amount of sucrose was weighed and added, and was fully stirred and dissolved. Subsequently, the corresponding amount of hormone was accurately added by using a pipette, and then distilled water was added to the required volume. Then, the pH value of the solution was adjusted to 5.6-5.8 by adding a small amount of HCl or NaOH using a pH meter. Thereafter, the solution was transferred to a heating device, and the specified amount of carrageenan was added, and was stirred and heated until the carrageenan was fully dissolved. Then, the prepared secondary proliferation induction medium was evenly divided and packaged into glass tissue culture bottles, about 30 mL per bottle, and the cap was screwed tightly. Then, it was placed in a high-temperature and high-pressure sterilization pot specially used for tissue culture, and was sterilized at 121°C for 20 min. After sterilization, the medium was taken out and placed, and after it was cooled and solidified, it was transferred to an ultraclean bench, and was sterilized by ultraviolet light for 20 min, and was ready for use.

[0057] The secondary proliferation culture was carried out at a temperature of 24°C, a light intensity of 1800 Lx, and a light cycle of 14 hours of light / 10 hours of darkness for 40-50 days.

[0058] The results of the secondary proliferation induction culture of blackberry are shown in Tables 2 and Figure 2 Figure 3 According to the results in Tables 2 and Figure 3 , the treatment of S10 has the best effect on the secondary proliferation induction of axillary buds. Therefore, the optimal secondary proliferation induction medium for blackberry is as follows:

[0059] The optimal secondary proliferation induction medium is composed of the following components at the following concentrations: NH4NO30.4 g / L, KH2PO40.17 g / L, K2SO40.99 g / L, CaCl2·2H2O 0.096 g / L, Ca(NO3)2·4H2O 0.556 g / L, MgSO4·7H2O 0.37 g / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl·6H2O 0.025 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 3 mg / L, indole acetic acid (IAA) 0.5 mg / L, gibberellin (GA3) 3 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L, and the pH value is 5.6.

[0060] ​III. Rooting culture

[0061] The cluster buds obtained from the secondary propagation culture in Step II were cut open and inoculated into the rooting culture medium. Eight treatments G1-G8 were set for the rooting culture of the present example, and the rooting culture media of the eight treatments were as shown in Table 3.

[0062] Table 3. Screening of rooting culture media for blackberry

[0063]

[0064]

[0065] The rooting mother liquor in Table 3 above was composed of the following components at the following concentrations: NH4NO30.825 g / L, KNO30.95 g / L, KH2PO40.085 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.185 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, and glycine 2.0 mg / L.

[0066] The rooting culture medium was prepared according to Table 3. First, each compound was accurately weighed, and then melted and mixed uniformly with a small amount of distilled water. Appropriate amount of sucrose was then weighed and added, and dissolved by stirring. Then, the corresponding amount of hormone was accurately added using a pipette, and the solution was quantitatively diluted to the required volume with distilled water. The pH value of the solution was then adjusted to 5.6-5.8 by adding a small amount of HCl or NaOH using a pH meter. Thereafter, the solution was transferred to a heating device, and the specified amount of carrageenan was added, and stirred and heated until the carrageenan was fully dissolved. Then, the prepared secondary propagation induction medium was evenly dispensed into glass tissue culture bottles, about 30 mL per bottle, and the caps were tightly screwed. Then, the bottles were placed in a tissue culture special high-temperature high-pressure sterilization pot, and sterilized at 121°C for 20 min. After sterilization, the medium was taken out and allowed to cool and solidify, and then transferred to a clean bench, and sterilized under UV light for 20 min, and then ready for use.

[0067] The culture was carried out at a temperature of 28°C, a light intensity of 1500 Lx, and a light cycle of 14 hours light / 10 hours darkness for 20-30 days, until 6-10 roots grew out.

[0068] The rooting results of blackberry are shown in Table 3 and Figure 4 、 Figure 5 Table 3 and Figure 4 The results of Table 3 and Figure 6 Table 4 show that the rooting effect of blackberry is best in G2 and G8 medium. Among them, G8 medium avoids the addition of gel, which can reduce the cost investment; and because it is a liquid medium for rooting, it avoids the root washing step before transplanting, which can reduce labor input, reduce root damage and breakage, and is conducive to further reducing cost consumption and improving transplanting survival rate. The transplanting effect of blackberry shallow liquid rooting seedlings is shown in , the plant leaves are dark green and grow well. Therefore, the optimal rooting medium for blackberry is as follows:

[0069] The optimal rooting medium is composed of the following concentrations of components: NH4NO30.825 g / L, KNO30.95 g / L, KH2PO40.085 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.185 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L; and sucrose 20 g / L, indole-3-butyric acid (IBA) 0.3 mg / L, carrageenan 7.0 g / L or 0, pH 5.6.

[0070] Example 2, two-round proliferation type tissue culture and rapid propagation method of blackberry

[0071] I. Disinfection of explants and induction of primary proliferation

[0072] Fresh, lignified current year shoots (leaves removed) of 'Prime-Ark Freedom' blackberry were excised from the Golden Sun Farm, washed with dish soap water, and rinsed with running water for 1 h. The explants were surface sterilized by immersing the stems in 75% ethanol for 40 s, followed by 5 rinses in sterile water with shaking for 5 min each. After the water was blotted from the explants, they were immersed in 3% NaClO for 11 min, followed by 5 rinses in sterile water with shaking for 10 min each. The water was blotted from the explants, and a 0.5 cm thick section of tissue was removed from the cut end of the stem with a sterile scalpel. The remaining stem was inserted vertically (with the morphological upper end facing up) into the primary proliferation induction medium and incubated at 25°C under 1200 Lx light for 14 h light / 10 h dark for 25 days.

[0073] The primary proliferation induction medium consisted of the following components at the indicated concentrations: NH4NO31.65 g / L, KNO31.9 g / L, KH2PO40.17 g / L, CaCl2-2H2O 0.88 g / L, MgSO4-7H2O 0.37 g / L, KI 0.83 mg / L, Na2MoO4-2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4-5H2O 0.025 mg / L, MnSO4-4H2O 22.3 mg / L, CoCl2-6H2O 0.025 mg / L, ZnSO4-7H2O 8.6 mg / L, Na2EDTA-2H2O 37.3 mg / L, FeSO4-7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine-HCl 0.1 mg / L, pyridoxine-HCl 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 4 mg / L, indole-3-butyric acid (IBA) 0.5 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L, pH 5.6.

[0074] Secondary proliferation induction (proliferation subculture)

[0075] The sterile explants obtained in Step 1 were used to excise the primary regenerated shoots obtained after the primary proliferation induction culture in a sterile hood. The excess stem structure at the top and bottom of the regenerated shoots was removed, and the explant stem where the regenerated shoots were attached was retained. The retained stem was placed horizontally in the secondary proliferation medium. The retained stem was submerged in the medium to ensure that the cut ends of the stem were in contact with the medium. The explants were incubated at 25°C under 1200 Lx light for 14 h light / 10 h dark for 45 days.

[0076] The secondary proliferation induction medium is composed of the following components at the following concentrations: NH4NO30.4 g / L, KH2PO40.17 g / L, K2SO40.99 g / L, CaCl2·2H2O 0.096 g / L, Ca(NO3)2·4H2O 0.556 g / L, MgSO4·7H2O 0.37 g / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl·6H2O 0.025 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 3 mg / L, indole acetic acid (IAA) 0.5 mg / L, gibberellin (GA3) 3 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L, with a pH value of 5.6.

[0077] III. Rooting culture

[0078] The cluster buds obtained by the secondary proliferation culture are cut open one by one and inoculated into a rooting medium, and cultured at a temperature of 25°C, a light intensity of 1200 Lx, and a light cycle of 14 hours of light / 10 hours of darkness for 20 days, until 6-10 roots grow out, and then transplanted to grow seedlings.

[0079] The rooting medium is composed of the following components at the following concentrations: NH4NO30.825 g / L, KNO30.95 g / L, KH2PO40.085 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.185 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, sucrose 20 g / L, indole butyric acid (IBA) 0.3 mg / L, and carrageenan 7.0 g / L or 0, with a pH value of 5.6.

[0080] The proliferation coefficient of the two-round proliferation type tissue culture and rapid propagation method of blackberry provided by the application is about 30, which is much higher than the existing system, and the regenerated seedling stem is strong and solid, and the leaf color is dark green, which is a convenient and efficient seedling rapid propagation technical method, can be applied to factory large-scale seedling raising in production, can greatly shorten the production cycle, and greatly reduce the cost consumption.

[0081] The two-round proliferation type tissue culture and rapid propagation technical method of blackberry provided by the application includes three steps: the first step is to obtain a sterile explant, and the first round of proliferation induction is completed at the same time, at this time, the plant phenotype cannot see the differentiation dynamic, but the internal hormone adjustment is completed. The second step is proliferation subculture, that is, the proliferation differentiation of axillary buds and the subculture elongation of regenerated seedlings are combined, the secondary proliferation can directly make the axillary buds of the explant high-differentiation, the proliferation coefficient is 20-30, the seedling height is 3cm-5cm, and the seedling reaches the standard of seedling for rooting. In the large-scale production of tissue culture factory, the first and second steps or the second step operation is repeated at intervals of 30-40 days, so that a large number of blackberry regenerated seedlings can be obtained in a short time. The third step is rooting, and the rooting induction of regenerated seedlings can be completed through the shallow liquid rooting method, and the cultivation and cultivation link is entered. The method for factory seedling raising of blackberry provided by the application has simple culture medium formula, low replacement frequency, short production cycle, and super-high proliferation efficiency and high seedling rate.

[0082] The above specific embodiments do not constitute a limitation on the protection scope of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can occur depending on design requirements and other factors. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for two-step proliferation of blackberry tissue culture, comprising the following steps: (1) inserting a blackberry stem section into a primary proliferation induction medium to induce axillary bud germination and differentiation, thereby obtaining a sterile explant comprising blackberry primary regenerated shoots; the blackberry stem section is a lignified current-year blackberry stem section; the blackberry stem section is a 6-7 cm stem section comprising 1-2 bud points; the insertion into the primary proliferation induction medium is performed in such a way that the morphological upper part of the stem section faces upward, and one of the bud points is in full contact with the primary proliferation induction medium, immersed in the medium or leveled with the medium; (2) cutting a stem section comprising the primary regenerated shoots from the sterile explant comprising blackberry primary regenerated shoots obtained in step (1) and placing the stem section into a secondary proliferation induction medium to induce secondary differentiation and proliferation, thereby obtaining blackberry regenerated clump seedlings; the cutting of the stem section comprising the primary regenerated shoots is performed without cutting the regenerated shoots from the stem section, but only cutting the stem section other than the part where the regenerated shoots grow; the placement into the secondary proliferation induction medium is performed by placing the cut stem section comprising the primary regenerated shoots horizontally into the secondary proliferation induction medium, and immersing the remaining stem section where the regenerated shoots grow into the secondary proliferation induction medium; (3) cutting the blackberry regenerated clump seedlings obtained in step (2) and inoculating them into a rooting medium for rooting culture, thereby obtaining blackberry regenerated plants; The primary proliferation induction medium consists of the following concentrations of components: NH4NO31.65 g / L, KNO31.9 g / L, KH2PO40.17 g / L, CaCl2·2H2O 0.88 g / L, MgSO4·7H2O 0.37 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 3-4 mg / L, indole-3-butyric acid (IBA) 0.5-1 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L; the pH value of the primary proliferation induction medium is 5.6-5.

8. The secondary proliferation induction medium is composed of the following components at the following concentrations: NH4NO30.4 g / L, KH2PO40.17 g / L, K2SO40.99 g / L, CaCl2·2H2O 0.096 g / L, Ca(NO3)2·4H2O 0.556 g / L, MgSO4·7H2O 0.37 g / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, KI 0.83 mg / L, CoCl·6H2O 0.025 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, 6-benzylaminopurine (6-BA) 2 mg / L to 3 mg / L, indole acetic acid (IAA) 0.5 mg / L to 1.0 mg / L, gibberellin (GA3) 2 mg / L to 3 mg / L, sucrose 25 g / L, and carrageenan 7.0 g / L; The pH value of the secondary proliferation induction medium is 5.6 to 5.8; The rooting medium is composed of the following components at the following concentrations: NH4NO30.825 g / L, KNO30.95 g / L, KH2PO40.085 g / L, CaCl2·2H2O 0.44 g / L, MgSO4·7H2O 0.185 g / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, H3BO36.2 mg / L, CuSO4·5H2O 0.025 mg / L, MnSO4·4H2O 22.3 mg / L, CoCl2·6H2O 0.025 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, nicotinic acid 0.5 mg / L, myo-inositol 100 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxal hydrochloride 0.5 mg / L, glycine 2.0 mg / L, and sucrose 10 g / L to 30 g / L, indole butyric acid (IBA) 0.1 mg / L to 0.3 mg / L, and carrageenan 0 g / L or 7.0 g / L; The pH value of the rooting medium is 5.6 to 5.

8.

2. The method of tissue culture propagation of two-walled proliferative blackberry as claimed in claim 1, wherein: In the step (1), before the blackberry stem segments are inserted into the primary proliferation induction medium, the blackberry stem segments are sterilized by the following method: the blackberry stem segments are soaked in 75% alcohol for 30 to 40 seconds, then washed with sterile water for 3 to 5 times, and shaken and soaked for 3 to 5 minutes each time; after the water stains are absorbed, the blackberry stem segments are dried, The explants are soaked with 3% sodium hypochlorite for 10-15 min, then washed with sterile water for 5-6 times, and soaked for 8-10 min each time.

3. The method of claim 1 or 2, wherein the two-walled proliferative blackberry tissue culture micropropagation method is characterized by: In the step (1), the axillary bud germination and differentiation induction culture is carried out at a temperature of 24-28°C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours light / 10 hours dark for 25-35 days.

4. The method of claim 1, wherein the two-walled proliferative blackberry tissue culture micropropagation method is characterized by: In the step (2), the secondary proliferation induction culture is carried out at a temperature of 24-28°C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours light / 10 hours dark for 40-50 days.

5. The method of tissue culture propagation of blackberry according to claim 1, wherein: In the step (3), the blackberry regenerated cluster seedlings are 4-5 cm high, and are vertically inoculated into the rooting culture medium in the form of single plant. When inoculated into the rooting culture medium, the lower leaves of the plants are removed, and 2-3 top leaves are reserved.

6. The method of tissue culture rapid propagation of two-walled proliferative blackberry as claimed in claim 1 or 5, characterized in that: In the step (3), the rooting culture is carried out at a temperature of 24-28°C, a light intensity of 1200-1800 Lx, and a light cycle of 14 hours light / 10 hours dark for 20-30 days. ​

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