Anti-browning caragana korshinskii tissue culture rapid propagation method

Abstract

The invention discloses an anti-browning caragana korshinskii tissue culture and rapid propagation method. The method comprises the following steps: (1) pretreatment of explants; (2) bud induction culture; (3) bud multiplication culture; (4) rooting culture; and (5) seedling hardening and transplanting. According to the method, the primary culture browning rate is stably controlled to be 10% or below from 30% or above, the bud induction rate is stabilized to be 90% or above, the proliferation multiple reaches 4.0 times or above, bud clusters are robust and free of vitrification, the rooting rate stably reaches 85% or above, and root systems are thick, strong and developed; according to the method, the overall propagation period is shortened to 85-100 days, rapid propagation of multiple generations in one year is achieved, the system is stable, the pollution rate is low, the method is suitable for large-scale seedling raising, and reliable seedling guarantee is provided for ecological restoration engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, in particular to a tissue culture and rapid propagation method of Caragana jubata resistant to browning. BACKGROUND

[0003] The traditional propagation methods of Caragana jubata are seed propagation and cutting propagation. The seed propagation is limited by environment, and has problems such as serious insect pests, deep dormancy, low germination rate, and separation of offspring traits. The cutting propagation has problems such as difficult rooting, low survival rate, and limitation by mother plant source, and has a long cutting period, and is prone to death due to water loss in arid areas, which cannot meet the demand of large-scale seedling raising.

[0004] The tissue culture technology is considered as an effective way to solve the above problems. However, when the Caragana jubata is subjected to tissue culture and rapid propagation, the following problems exist: the browning rate of primary culture is higher than 30%, the bud induction rate is generally lower than 70%, the bud body is thin and weak, and the efficiency of proliferation and rooting stages is low and the stability is poor, which cannot realize the rapid propagation target of stability, high efficiency and being applicable to large-scale production. SUMMARY

[0005] The present application provides a tissue culture and rapid propagation method of Caragana jubata resistant to browning, which solves the problems of browning, low induction efficiency, difficult rooting, and unstable seedling quality in the prior art.

[0006] To solve the above technical problems, the present application provides a tissue culture and rapid propagation method of Caragana jubata resistant to browning, which comprises the following steps: (1) Pre-treatment of explants: select Caragana jubata stem segments with axillary buds as explants, and obtain sterile explants after disinfection for standby use; (2) Bud induction culture: inoculate the sterile explants into an induction medium, first culture in darkness, and then culture in light to induce the formation of clustered buds; the induction medium is based on MS and contains 6-BA 0.5-2.0 mg / L, NAA 0.05-0.2 mg / L, KT 0.1-0.3 mg / L, activated carbon 0.5-1.5 g / L, and ascorbic acid 50-100 mg / L; (3) Bud proliferation culture: cut the clustered buds obtained in step (2) and transfer them to a proliferation medium for culture under light to obtain a multi-bud cluster; the proliferation medium is based on MS and contains 6-BA 1.0-3.0 mg / L, KT 0.1-0.3 mg / L, and activated carbon 0.5-1.5 g / L; (4) rooting culture: taking the healthy bud segments obtained in step (3) and transferring to rooting medium to induce rooting; the rooting medium is based on 1 / 2MS and contains IBA 0.5-1.5 mg / L, NAA 0.1-0.3 mg / L and activated carbon 0.1-0.5 g / L; (5) hardening-off and transplanting: transferring the rooting seedlings obtained in step (4) into a substrate to acclimate and then transplanting.

[0007] In a preferred embodiment of the present application, in step (1), the disinfection is performed by sequentially immersing in 75% ethanol solution for 20-40 s, then treating with 0.05%-0.1% mercury chloride solution for 5-10 min, and finally rinsing with sterile water for 5-6 times.

[0008] In a preferred embodiment of the present application, the induction medium, the proliferation medium and the rooting medium in steps (2), (3) and (4) further contain 25-35 g / L sucrose and 6-8 g / L agar, and the pH value is 5.8-6.0.

[0009] In a preferred embodiment of the present application, the composition of the induction medium is: MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + KT 0.2 mg / L + activated carbon 0.8 g / L + ascorbic acid 80 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0010] In a preferred embodiment of the present application, the composition of the proliferation medium is: MS + 6-BA 2.0 mg / L + KT 0.2 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0011] In a preferred embodiment of the present application, the composition of the rooting medium is: 1 / 2MS + IBA 1.0 mg / L + NAA 0.2 mg / L + activated carbon 0.2 g / L + sucrose 28 g / L + agar 7 g / L, pH 5.8.

[0012] In a preferred embodiment of the present application, in step (2), the dark culture time is 7-10 days; the light culture condition is: temperature 25±2℃, light intensity 2000-3000 Lux, light time 12-14 h / d, and culture time 25-35 days.

[0013] In a preferred embodiment of the present application, in step (5), the acclimatization process is carried out for 7-10 days in an environment with a relative humidity of 70-80% and shading of 50-70%, and then the light is gradually increased.

[0014] In a preferred embodiment of the present application, in step (5), the substrate used for acclimatization is a mixture of peat soil and vermiculite in a volume ratio of 1:1.

[0015] In a preferred embodiment of the present application, the culture conditions of step (3) are: temperature 25±2℃, light time 12h / d, light intensity 2500-3000 Lux, and culture for 30-40 days; the culture conditions of step (4) are: temperature 25±2℃, light time 12h / d, light intensity 2000-2500 Lux, and culture for 20-30 days.

[0016] The present application has the following advantages: the method for tissue culture and rapid propagation of the caragana against browning, by using ascorbic acid and activated carbon in combination at the exogenous initiation stage and in cooperation with dark culture, forms multiple anti-browning effects, and stably controls the browning rate of primary culture to be below 10% from above 30%; by using specific ratio of 6-BA and KT and in cooperation with MS medium, the bud induction rate is as high as 94.5%, the proliferation multiple is above 4.5 times, and the bud clumps are robust and non-glassy; by using 1 / 2MS low-salt medium and cooperating with IBA and NAA mixed auxin system, the rooting rate is stably above 85%, and the root system is robust and developed; the overall propagation cycle of the present application is shortened to 85-100 days, realizing rapid propagation of'more than one generation per year', the system is stable and has low pollution rate, is suitable for large-scale seedling raising, and provides reliable seedling guarantee for ecological restoration engineering. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a state diagram of the caragana clump buds after 30 days of culture in the embodiment of the present application; Figure 2 is a growth physical diagram of the caragana test tube seedlings. DETAILED DESCRIPTION

[0018] The preferred embodiments of the present application are described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the protection scope of the present application is more clearly and definitely defined.

[0019] Example 1 A method for tissue culture and rapid propagation of caragana against browning, comprising the following steps: (1) Pretreatment of explants: Select healthy one-year-old branches without pests and diseases, remove leaves, cut into about 2 cm stem segments, brush the surface soil with a toothbrush, rinse with tap water for 60 s, then soak in a 1% concentration of carbendazim solution for 5-7 min, rinse with tap water 4 times after taking out, then transfer to a clean bench, first soak in a 75% concentration of ethanol solution for 30 s, rinse with sterile water 4 times, then treat with a 0.1% concentration of mercury chloride solution for 6 min, and finally rinse with sterile water 5-6 times. Dry the water for standby use.

[0020] (2) Bud induction culture: cut off 0.5 cm of the two ends of the treated sterile explants which contacted the mercury chloride solution, leave 1 cm for inoculation in the induction medium, first culture in the dark for 8 days, then transfer to light conditions, culture for 30 days at a temperature of 25±2℃, light intensity of 2500 Lux, and light time of 14 h / d to induce the formation of clustered buds. After the culture ends, the axillary bud induction rate of the explants reaches 94.5%, and the browning rate is only 7.2%. Most of the explants form 2-3 clustered buds which are healthy, with expanded and dark green leaves, and no vitrification.

[0021] The induction medium used has the following components: MS + 6-BA (6-benzylaminopurine) 1.0 mg / L + NAA (naphthalene acetic acid) 0.05 mg / L + KT (kinetin) 0.2 mg / L + activated carbon (AC) 0.8 g / L + ascorbic acid (VC) 80 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0022] Among them, ascorbic acid can inhibit browning, and kinetin can promote bud differentiation and increase the germination rate.

[0023] (3) Bud proliferation culture: cut the clustered buds obtained in step (2) and transfer to the proliferation medium, culture for 35 days at a temperature of 25±2℃, light time of 12 h / d, and light intensity of 2500 Lux.

[0024] The proliferation medium used has the following components: MS + 6-BA 2.0 mg / L + KT 0.2 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7 g / L, pH 5.8.

[0025] After 35 days of culture, the average multiplication rate of the bud clusters reaches 4.5 times. The bud bodies grow healthily, with dark green leaves, no vitrification and obvious etiolation, and the state diagram is shown in the accompanying Figure 1

[0026] ​(4) Rooting culture: the healthy bud segments obtained in step (3) are transferred to a rooting culture medium and cultured for 25 days at a temperature of 25±2℃, a light time of 12 h / d, and a light intensity of 2200 Lux.

[0027] The rooting culture medium used has the following components: 1 / 2MS + IBA 1.0 mg / L + NAA 0.2 mg / L + activated carbon 0.2 g / L + sucrose 28 g / L + agar 7 g / L, pH 5.8.

[0028] After 25 days of culture, the rooting rate is as high as 93.6%. On average, each plant has 4.2 roots, the root system is strong and healthy white, the average root length is about 4.8 cm, the lateral roots develop well, and the test tube seedlings are as shown in Figure 2

[0029] (5) Seedling raising and transplanting: the rooted seedlings obtained in step (4) are taken out, washed to remove the agar, and then moved into a substrate mixed by peat soil and vermiculite at a volume ratio of 1:1. First, the seedlings are acclimated for 8 days in an environment with a humidity of 80%, daily spraying for moisture retention 3 times, and a shading area of 50%, and then the shading proportion is gradually reduced. After 15 days of acclimation, the new leaves of the seedlings are unfolded, the root system is tightly combined with the new substrate, and the plants grow healthily. At this time, the seedlings are moved into a normal greenhouse for normal management, and the transplanting survival rate is more than 92%.

[0030] Example 2 Screening test for optimization of basic culture medium and anti-browning agent in the induction stage Healthy one-year-old tender branch stem segments 2 cm long are sterilized and cultured according to the method in Example 1.

[0031] In order to screen the best basic culture medium and anti-browning scheme, two factors are set: ① basic culture medium (MS, 1 / 2MS, B5); ② anti-browning treatment (add VC 80 mg / L + AC 0.8 g / L, only add AC 0.8 g / L, no anti-browning agent). All the culture media contain the induction hormone combination preferred by the application: 6-BA 1.0 mg / L + NAA 0.05 mg / L + KT 0.2 mg / L, as well as sucrose 30 g / L, agar 7 g / L, pH 5.8.

[0032] The sterilized stem segments are vertically inserted into the culture medium, 2 explants per bottle, 15 bottles per batch, and repeated 3 times. The culture is carried out according to the conditions in step (2) of Example 1, and the relevant indicators are shown in Table 1.

[0033] Table 1 ​The results showed that the type of basic medium and the anti-browning treatment had significant effects on the induction of axillary buds. Among them, the MS medium + VC 80 mg / L + AC 0.8 g / L treatment group performed the best, with a significantly higher induction rate than all other treatment groups, the lowest browning rate, and the best seedling height and bud mass. Example 3: Optimization test of hormone combination of induction medium The test materials and culture conditions were the same as in Example 1. Using MS as the basic medium, with fixed addition of VC 80 mg / L + AC 0.8 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.8, an orthogonal test design was used to explore the effects of 6-BA (0.5, 1.0, 1.5 mg / L), NAA (0.05, 0.1, 0.15 mg / L), and KT (0.1, 0.2, 0.3 mg / L) on the induction of axillary buds, with a total of 9 treatment groups. Each treatment had 15 bottles, each with 2 explants, with 3 replicates. After 30 days of culture, the induction rate, browning rate, and bud growth were statistically analyzed. The results are shown in Table 2.

[0034] Table 2 The results showed that 6-BA had the greatest effect on the induction rate of axillary buds, followed by KT, and NAA had relatively less effect. When 6-BA 1.0 mg / L + NAA 0.05 mg / L + KT 0.2 mg / L, the induction rate of axillary buds was 94.5%, the browning rate was 7.1%, and the bud mass was optimal, with all indicators better than other treatment groups. When the concentration of 6-BA exceeded 1.0 mg / L, it easily led to bud malformation; when the concentration of NAA exceeded 0.05 mg / L, it easily caused overgrowth, which was not conducive to the normal development of axillary buds.

[0035] Example 4: Optimization test of rooting medium formula Healthy bud segments from the proliferation in Example 3 were taken, with 15 bottles per batch, 2 bud segments per bottle, and 3 replicates.

[0036] Using 1 / 2MS as the base, with fixed AC 0.2 g / L and agar 7 g / L, pH 5.8, the effects of IBA and NAA ratio on rooting were explored. The culture was carried out at a temperature of 25±2℃, light intensity of 2300 Lux, and light for 12 h / d for 25 days. The results are shown in Table 3.

[0037] Table 3 The results showed that the concentration of IBA, NAA and sucrose content had significant effect on rooting. The combination of IBA 1.0 mg / L+NAA 0.2 mg / L+sucrose 28 g / L was the best, the rooting rate was 93.6%, the average root length was 4.8 cm, the lateral root was 4-5, and the root system was strong and no deformity, which was the best formula for inducing healthy root system of Caragana.

[0038] The above description is only an embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for tissue culture rapid propagation of Caragana jubata against browning, characterized in that, The method comprises the following steps: (1) Pre-treatment of explants: select Caragana korshinskii stem segments with axillary buds as explants, and obtain sterile explants after disinfection for standby; (2) Bud induction culture: inoculate the sterile explants into an induction medium, first culture in dark conditions for 5-10 days, then transfer to light conditions for 15-25 days to induce the formation of clustered buds; the induction medium is based on MS and contains 6-BA 0.5-2.0 mg / L, NAA 0.05-0.2 mg / L, KT 0.1-0.3 mg / L, activated carbon 0.5-1.5 g / L and ascorbic acid 50-100 mg / L; (3) Bud proliferation culture: cut the clustered buds obtained in step (2) and transfer to a proliferation medium for 30-40 days to obtain a multi-bud cluster under light conditions; the proliferation medium is based on MS and contains 6-BA 1.0-3.0 mg / L, KT 0.1-0.3 mg / L and activated carbon 0.5-1.5 g / L; (4) Rooting culture: cut the healthy bud segments obtained in step (3) and transfer to a rooting medium to induce rooting; the rooting medium is based on 1 / 2MS and contains IBA 0.5-1.5 mg / L, NAA 0.1-0.3 mg / L and activated carbon 0.1-0.5 g / L; (5) Seedling raising and transplanting: transfer the rooted seedlings obtained in step (4) to a substrate for acclimatization and then transplant.

2. The method of claim 1, wherein, In step (1), the disinfection is: sequentially use a 75% ethanol solution for 20-40 s, then use a 0.05%-0.1% mercury chloride solution for 5-10 min, and finally rinse with sterile water for 5-6 times.

3. The method of claim 1, wherein, The induction medium, the proliferation medium and the rooting medium in steps (2), (3) and (4) further contain 25-35 g / L sucrose and 6-8 g / L agar, and the pH value is 5.8-6.

0.

4. The method of claim 3, wherein, The composition of the induction medium is: MS + 6-BA 1.0 mg / L + NAA 0.05 mg / L + KT 0.2 mg / L + activated carbon 0.8 g / L + ascorbic acid 80 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.

8.

5. The method of claim 3, wherein, The composition of the proliferation medium is: MS + 6-BA 2.0 mg / L + KT 0.2 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + agar 7 g / L, pH 5.

8.

6. The method of claim 3, wherein, The composition of the rooting medium is: 1 / 2MS + IBA 1.0 mg / L + NAA 0.2 mg / L + activated carbon 0.2 g / L + sucrose 28 g / L + agar 7 g / L, pH 5.

8.

7. The method of claim 1, wherein, In step (2), the dark culture time is 7-10 days; the light culture conditions are: temperature 25±2℃, light intensity 2000-3000 Lux, light time 12-14 h / d, and culture time 25-35 days.

8. The method of claim 1, wherein, In step (5), the acclimatization is carried out in an environment with relative humidity 70%-80% and light shielding 50%-70% for 7-10 days, and then the light is gradually increased.

9. The method of claim 1, wherein, In step (5), the substrate used for acclimatization is mixed by peat soil and vermiculite at a volume ratio of 1:

1.

10. The method of claim 1, wherein, The culture conditions in step (3) are: temperature 25±2℃, light time 12 h / d, light intensity 2500-3000 Lux, and culture time 30-40 days; the culture conditions in step (4) are: temperature 25±2℃, light time 12 h / d, light intensity 2000-2500 Lux, and culture time 20-30 days.

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