Tissue culture method of castanopsis hystrix

Abstract

The invention provides a tissue culture method of castanopsis hystrix. The method comprises the following steps: selecting a semi-lignified castanopsis hystrix scion as an explant; sequentially performing disinfection treatment, primary induction culture, first multiplication culture, degerming treatment and rooting culture on the explant; the degerming treatment comprises strong seedling culture and second multiplication culture which are sequentially carried out; a culture medium for the first multiplication culture and a culture medium for the second multiplication culture comprise a first basic culture medium, and further comprise 0.4-0.6 mg / L of 6-BA, 14-16 g / L of sucrose and 5-7 g / L of carrageenan on the basis of the first basic culture medium; the first basic culture medium is WPM. By exploring the influence of different basic culture media, the concentration of 6-BA, the concentration of sucrose and the like on proliferation and rooting, the formula of the culture media is improved, so that the proliferation rate of castanopsis hystrix tissue culture reaches 2.74 or above, and the rooting rate reaches 95% or above.

Description

Technical Field

[0001] The present application relates to the field of plant propagation technology, and in particular to a tissue culture method of Castanopsis hystrix. Background Art

[0002] Castanopsis hystrix is ​​an evergreen tree belonging to the genus Castanopsis in the family Fagaceae. It is the fastest-growing precious tree species with the largest planting area in southern my country. Its wood is hard and has a large proportion of heartwood. It can be used to cultivate large-diameter precious timber. In 2017, it was included in the "Reference List of Major Cultivated Precious Tree Species in China" by the National Forestry and Grassland Administration.

[0003] As the area of ​​​​promotion of red castanets increases year by year, the problem of imbalance between supply and demand of high-quality seedlings has become increasingly prominent. Common propagation methods of red castanets include seeds, grafting and cuttings, but due to the limited number of seeds and cuttings, the large-scale breeding and promotion of high-quality seedlings have been seriously affected.

[0004] Therefore, it is urgent to improve the traditional breeding method of Hystrix erythroxylum. Summary of the invention

[0005] Based on this, one or more embodiments of the present application provide a tissue culture method for Castanopsis hystrix having both high proliferation rate and rooting rate.

[0006] According to a first aspect of the embodiments of the present application, a method for culturing Castanopsis erythroxylum is provided, comprising the following steps:

[0007] Selecting semi-lignified Castanopsis hystrix spikelets as explants; and

[0008] The explants are sequentially subjected to disinfection treatment, primary induction culture, first proliferation culture, sterilization treatment and rooting culture;

[0009] The sterilization treatment includes the seedling culture and the second proliferation culture in sequence;

[0010] The culture medium for the first proliferation culture and the second proliferation culture includes a first basal culture medium, and based on the first basal culture medium, further includes 0.4 mg / L~0.6 mg / L 6-BA, 14 g / L~16 g / L sucrose and 5 g / L~7 g / L carrageenan; the first basal culture medium is selected from WPM.

[0011] In one embodiment, the culture medium for primary induction culture includes a second basal culture medium, and based on the second basal culture medium, further includes 1.8 mg / L~2.2 mg / L 6-BA, 0.04 g / L~0.06 g / L NAA, 14 g / L~16 g / L sucrose and 6.5 g / L~7 g / L carrageenan; the second basal culture medium is selected from WPM.

[0012] In one embodiment, the culture medium for seedling cultivation includes a third basal culture medium, and based on the third basal culture medium, it also contains 0.4 mg / L~0.6 mg / L 6-BA, 14 g / L~16 g / L sucrose and 5 g / L~7 g / L carrageenan; the third basal culture medium is selected from 1 / 2WPM.

[0013] In one embodiment, the culture medium for rooting culture includes a fourth basal culture medium, and based on the fourth basal culture medium, further includes 1.8 mg / L~2.2 mg / L IBA, 18 g / L~22 g / L sucrose and 5 g / L~7 g / L carrageenan; the fourth basal culture medium is selected from 1 / 2WPM.

[0014] In one embodiment, the degerming process is performed twice or more.

[0015] In one embodiment, after the rooting culture, the following step is also included: transplanting the rooted seedlings obtained by the rooting culture to a seedbed for cultivation.

[0016] In one embodiment, the substrate in the seedbed includes peat soil and vermiculite.

[0017] In one embodiment, the volume ratio of the peat soil to vermiculite is 1:(1.8-2.2).

[0018] In one embodiment, the disinfection process comprises the following steps:

[0019] Rinse the explants with sterile water;

[0020] Treating the explant with alcohol for 4s to 6s; and

[0021] The explants were immersed in mercuric chloride for 4 to 6 minutes.

[0022] In one embodiment, the tissue culture method satisfies at least one of the following conditions:

[0023] (1) The light intensity for primary induction culture is 2400 lux ~ 3000 lux;

[0024] (2) The light intensity for proliferation culture is 2400 lux~3000 lux; and

[0025] (3) The light intensity for rooting culture is 2400lux~3000lux.

[0026] Compared with the traditional technology, this application has the following beneficial effects:

[0027] This application explores the effects of different basic culture media, 6-BA concentrations, sucrose concentrations, etc. on proliferation and rooting, and improves the culture medium formula, so that the proliferation rate of red cone tissue culture reaches more than 2.75 and the rooting rate reaches more than 95%. Compared with the traditional method, the method of this application has the advantages of high proliferation rate, high rooting rate, short reproduction cycle, and can maintain the excellent traits of the mother plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a schematic diagram of the process of the tissue culture method of Castanopsis erythrorhizon in one embodiment of the present application;

[0030] Figure 2 This is a schematic diagram of the degerming process in one embodiment of the present application. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present application. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present application, etc. can be purchased from the market or can be prepared by existing methods.

[0033] Some embodiments of the present application provide a method for culturing Castanopsis erythroxylum, comprising the following steps:

[0034] Step S10: selecting semi-lignified Castanopsis hystrix spikelets as explants;

[0035] Step S20: Disinfecting the explant;

[0036] Step S30: primary induction culture;

[0037] Step S40: first proliferation culture;

[0038] Step S50: sterilization treatment: including seedling cultivation and second proliferation cultivation in sequence;

[0039] Step S60: rooting culture.

[0040] In some embodiments, the culture medium for the first proliferation culture and the second proliferation culture includes a first basal culture medium, and based on the first basal culture medium, further includes 0.4 mg / L to 0.6 mg / L 6-BA, 14 g / L to 16 g / L sucrose, and 5 g / L to 7 g / L carrageenan; the first basal culture medium is selected from WPM. It is understood that the concentrations of 6-BA, sucrose, and carrageenan in the culture medium for the first proliferation culture and the second proliferation culture can be independent of each other.

[0041] In some specific embodiments, the culture medium for proliferation culture includes a first basal culture medium, and based on the first basal culture medium, further includes 0.5 mg / L 6-BA, 15 g / L sucrose and 6 g / L carrageenan, and the first basal culture medium is selected from WPM.

[0042] It should be noted that, in the present application, “WPM” refers to Woody Plant Medium; “6-BA” refers to 6-Benzylaminopurine; “IBA” refers to Indole-3-Butyric Acid; and “NAA” refers to Naphthalene Acetic Acid.

[0043] In some of the embodiments, in step S10, semi-lignified Castanopsis hystrix cuttings are selected as explants.

[0044] In some embodiments, in step S20, the explant is sterilized in a sterile environment.

[0045] In some embodiments, in step S20, the disinfection process includes the following steps:

[0046] Rinse the explants with sterile water;

[0047] Treat the explants with alcohol for 4s-6s; and

[0048] Soak the explants in mercuric chloride for 4 to 6 minutes.

[0049] In some specific examples, in step S20, the disinfection process includes steps S21 to S25:

[0050] Step S21: Rinse the explant 3 to 4 times with sterile water;

[0051] Step S22: treating the explant with 75% sperm for 4s to 6s;

[0052] Step S23: Rinse the explant once with sterile water;

[0053] Step S24: Soak the explant in 0.1% mercuric chloride for 4 to 6 minutes;

[0054] Step S25: Rinse the explant 3 to 4 times with sterile water.

[0055] In some embodiments, in step S30, the culture medium for primary induction culture includes a second basal culture medium, and based on the second basal culture medium, further includes 1.8 mg / L~2.2 mg / L 6-BA, 0.04 g / L~0.06 g / L NAA, 14 g / L~16 g / L sucrose and 6.5 g / L~7 g / L carrageenan; the second basal culture medium is selected from WPM.

[0056] In some specific embodiments, in step S30, the primary induction culture includes a second basal culture medium, and based on the second basal culture medium, further includes 2 mg / L 6-BA, 0.05 g / L NAA, 15 g / L sucrose and 6.8 g / L carrageenan; the second basal culture medium is selected from WPM.

[0057] In some embodiments, in step S30, the illumination intensity of the primary induction culture is 2400 lux to 3000 lux. Understandably, the stem segments of Castanopsis rubrum can germinate under the illumination intensity of 1000 to 3000 lux and show a high survival rate, but Castanopsis rubrum is a strong positive plant, and the induction effect under high light intensity is significantly better than that under lower light intensity, so the illumination intensity of the primary induction culture is controlled to be 2400 lux to 3000 lux in this application.

[0058] In some specific embodiments, in step S30, the illumination intensity of the primary induction culture is 2500 lux.

[0059] In some embodiments, in step S40, the illumination intensity of the proliferation culture is 2400 lux to 3000 lux.

[0060] In some specific embodiments, in step S40, the illumination intensity of the proliferation culture is 2500 lux.

[0061] In some embodiments, in step S50, the number of times of degerming treatment is 2 times or more.

[0062] In some examples, in step S50, the explant is degermed in a sterile environment.

[0063] Specifically, in a sterile environment, the young shoots obtained by proliferation culture are transferred multiple times, thereby reducing the contamination of endophytes on the young shoots.

[0064] like Figure 2 As shown, a sterilization treatment includes the following steps: cutting the young shoots ( Figure 2 a), strong seedling cultivation ( Figure 2 Middle b), strong single bud ( Figure 2 Middle c), cutting the young shoots ( Figure 2 Middle d), proliferation culture ( Figure 2 Middle e), cluster budding ( Figure 2 (middle f).

[0065] In some embodiments, in step S50, the culture medium for culturing seedlings includes a third basal culture medium, and based on the third basal culture medium, further includes 0.4 mg / L~0.6 mg / L 6-BA, 14 g / L~16 g / L sucrose and 5 g / L~7 g / L carrageenan; the third basal culture medium is selected from 1 / 2WPM.

[0066] In some specific embodiments, in step S50, the culture medium for culturing seedlings includes a third basal culture medium, and based on the third basal culture medium, further includes 0.5 mg / L 6-BA, 15 g / L sucrose and 6 g / L carrageenan; the third basal culture medium is selected from 1 / 2WPM.

[0067] In some embodiments, in step S60, the culture medium for rooting culture includes a fourth basal culture medium, and based on the fourth basal culture medium, further includes 1.8 mg / L~2.2 mg / L IBA, 18 g / L~22 g / L sucrose and 5 g / L~7 g / L carrageenan; the fourth basal culture medium is selected from 1 / 2WPM.

[0068] In some specific embodiments, in step S60, the culture medium for rooting culture includes a fourth basal culture medium, and based on the fourth basal culture medium, further includes 2 mg / L IBA, 20 g / L sucrose and 6 g / L carrageenan; the fourth basal culture medium is selected from 1 / 2WPM.

[0069] In some embodiments, in step S60, the illumination intensity of the rooting culture is 2400 lux to 3000 lux.

[0070] In some specific embodiments, in step S60, the illumination intensity for rooting culture is 2500 lux.

[0071] In some embodiments, after the rooting culture, the step S70 is further included: transplanting the rooted seedlings obtained by the rooting culture to a seedbed for cultivation.

[0072] In some embodiments, the substrate in the seedbed includes peat soil and vermiculite.

[0073] In some embodiments, the volume ratio of peat soil to vermiculite is 1:(1.8-2.2).

[0074] In some specific embodiments, the volume ratio of peat soil to vermiculite is 1:2.

[0075] The method of tissue culture of red cones using the above method of the present application has at least the following advantages: (1) the use of the proliferation culture medium with a specific formula of the present application can make the proliferation rate of red cone tissue reach above 2.75, and the grown plants are strong, have many buds, and have stretched leaves; (2) the use of the rooting culture medium of the present application can make the rooting rate of red cones reach above 95%; (3) the culture method of the present application has the advantages of high reproduction efficiency, short cycle, and the ability to maintain the genetic stability of the excellent traits of the mother plant.

[0076] The present application will be further described below in conjunction with specific examples and comparative examples, but they should not be construed as limiting the scope of protection of the present application. The raw materials involved in the following specific examples, unless otherwise specified, can all be sourced from commercial sources, the instruments used, unless otherwise specified, can all be sourced from commercial sources, and the processes involved, unless otherwise specified, are all conventionally selected by those skilled in the art.

[0077] Example 1

[0078] (1) In the dry and sunny weather in autumn, go to the bud collection nursery of Castanopsis hystrix (such as Figure 1 Select the semi-lignified young spikelets of Castanopsis hystrix (as shown in a) Figure 1 As explants, cut the spikelets obliquely into stem segments with buds, retaining 1 / 2 leaves for later use.

[0079] (2) Disinfection

[0080] The explants were rinsed three times with sterile water in an ultra-clean workbench to clean the surface dust and pathogens, then treated with 75% alcohol for 5 seconds, rinsed once with sterile water, then soaked in 0.1% mercuric chloride for 5 minutes, and finally rinsed three times with sterile water to reduce the residual disinfectant on the surface of the stem segment.

[0081] (3) Primary induction culture

[0082] The sterilized explants were inoculated into WPM medium (WPM medium was used as the basic medium and contained 2.0 mg / L 6-BA, 0.05 mg / L NAA, 15 g / L sucrose and 6.8 g / L carrageenan), and transferred to a 25°C culture room for constant temperature culture with a light intensity of 2500 lux. Newly induced axillary buds (such as Figure 1 (as shown in c).

[0083] (4) First proliferation culture

[0084] (4.1) Investigate the effects of different basal culture media on proliferation culture

[0085] 1 / 2MS, WPM and MS were selected as basal culture media respectively, and 0.3 mg / L 6-BA was added to each basal culture medium. 1cm~2cm long shoots were inoculated into the culture medium and cultured in a constant temperature of 25℃ in a culture room for 30 days with a light intensity of 2500lux. Three repeated experiments were set up for each treatment group.

[0086] The results are shown in Table 1. When 1 / 2MS was used as the basic culture medium, the trunk of the plant was weak, the leaves were yellowish, and the leaves gradually fell off as the culture time prolonged. When MS was used as the basic culture medium, although the trunk of the plant was strong, it became vitrified after multiple subcultures, and the top of the tender shoots turned yellow and died. When WPM was used as the basic culture medium, the plants were strong, grew well, and had the highest proliferation rate, which could reach 2.47. Therefore, this application selected WPM as the basic culture medium for proliferation culture.

[0087] Table 1 Effects of different basal culture media on proliferation culture

[0088]

[0089] It should be noted that the data such as proliferation rate and rooting rate in this application form are all mean ± standard error; letters such as "a", "b", and "c" are used to indicate the significance of differences between different treatment groups. If the marks of two groups are different, it means that there is a statistically significant difference between the two groups (P>0.05); if the marks of two groups are the same, it means that there is no statistically significant difference between the two groups (P>0.05).

[0090] (4.2) Investigate the effects of different concentrations of 6-BA on proliferation culture

[0091] Using WPM as the basal culture medium, 6-BA was added at concentrations of 0.25 mg / L, 0.5 mg / L, 1.0 mg / L and 2.0 mg / L respectively, and 1 cm~2 cm long shoots were inoculated into the culture medium. The culture was cultured at a constant temperature of 25℃ in a culture room for 30 days with a light intensity of 2500 lux. Three repeated experiments were set up for each treatment group.

[0092] The results are shown in Table 2. Adding 6-BA can promote the occurrence of buds. With the increase of concentration, the number of buds increases and the number of leaves decreases. When the concentration of 6-BA is higher than 1.0 mg / L, the callus tissue at the base increases. Although there are many buds, they do not open leaves. After multiple subcultures, the buds become shorter and the proliferation rate decreases. When the concentration of 6-BA is 0.5 mg / L, the plants are strong, with many buds and stretched leaves. The proliferation rate reaches 2.77, and after multiple subcultures, they can still maintain good growth ( Figure 1 d). Therefore, the concentration of 6-BA added to the basal culture medium in this application is 0.4 mg / L~0.6 mg / L.

[0093] Table 2 Effects of different concentrations of 6-BA on proliferation culture

[0094]

[0095] (4.3) Investigate the effects of different culture media on proliferation culture

[0096] Proliferation medium A: Modified WPM was used as the basal medium, and 3.0 mg / L 6-BA, 1.0 mg / L NAA, 15 mg / L VC, 20 mg / L aspartic acid, 30 g / L sucrose and 6.0 g / L carrageenan were added.

[0097] The formula of the modified WPM is as follows: NH 4 NO 3 390mg / L, CaCl 2 ·2H 2 O 82mg / L, MgSO 4 7H 2 O 265mg / L, KH 2 PO 4 170mg / L, Ca(NO 3 ) 2 ·4H 2 O 165mg / L, KI 0.83mg / L, MnSO 4 ·H 2 O 22.4mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 4 ·2H 2O 0.25mg / L, CuSO 4 ·5H 2 O 0.025mg / L, Na 2 EDTA 33.4mg / L, FeSO 4 7H 2 O 27.8 mg / L, inositol 200 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 1.0 mg / L and glycine 2.0 mg / L.

[0098] Proliferation medium B: Modified WPM was used as the basal medium, and 7.0 mg / L 6-BA, 2.5 mg / L NAA, 1.0 mg / L ZT (Zeatin), 15 mg / L VC, 20 mg / L aspartic acid, 30 g / L sucrose and 6.0 g / L carrageenan were added.

[0099] Proliferation medium C: WPM was used as the basal medium, and 0.5 mg / L 6-BA, 30 g / L sucrose and 6.0 g / L carrageenan were added.

[0100] The formula of WPM is as follows: NH 4 NO 3 400mg / L, H 3 BO 3 6.2mg / L, CaCl 2 ·2H 2 O 72.5mg / L, Ca(NO 3 ) 2 ·4H 2 O 386mg / L, MgSO 4 7H 2 O 180mg / L, MnSO 4 ·H 2 O 22.3mg / L, K 2 SO 4 990mg, KH 2 PO 4 170mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 4 ·2H 2 O 0.25mg / L, CuSO 4 ·5H 2 O 0.25mg / L, Na 2 EDTA 37.3mg / L, FeSO 4 7H 2O 27.8 mg / L, inositol 100 mg / L, niacin 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 1.0 mg / L and glycine 2.0 mg / L.

[0101] Take 1cm~2cm long shoots and inoculate them into the above-prepared proliferation medium A, proliferation medium B and proliferation medium C respectively, and culture them in a constant temperature culture room at 25℃ for 30 days with a light intensity of 2500lux; set three repeated experiments for each treatment group.

[0102] The results are shown in Table 3. The proliferation rates of the three proliferation media are similar, with no significant difference; but the growth performance is different. Specifically, the plants in the proliferation medium C are strong, with many buds and leaves stretching, while the proliferation media A and B have more base callus, many buds, and unstretched leaves. Multiple subcultures are prone to short plants, shortened internodes, and leaves that cannot be pulled out normally. This may be because too much plant hormone is not conducive to the normal growth of the plant. Therefore, the present application adopts the formula of proliferation medium C for proliferation culture.

[0103] Table 3 Effects of different culture media on proliferation culture

[0104]

[0105] (5) Debacterialization

[0106] During the proliferation and cultivation process, some explant materials were contaminated by endophytes to varying degrees, and as the number of subcultures increased, endophytes seriously affected plant growth and the proliferation rate was greatly reduced. The method of multiple transfers of young shoots can gradually remove bacteria and regain sterile seedlings. The specific steps are as follows:

[0107] According to Figure 2 In the process shown, in the clean bench, use sterile scissors to cut the 1 cm long tender shoots at the end, transfer them to the seedling medium for seedling culture for 30 days, and after the tender shoots grow taller, use sterile scissors to cut the 1 cm long tender shoots at the end, transfer them to the proliferation medium for the second proliferation culture for 30 days to obtain clustered buds. Repeat the seedling culture and the second proliferation culture for multiple times to obtain sterile proliferation seedlings.

[0108] The formula of the seedling medium includes: 1 / 2WPM as the basic medium, and 0.5mg / L 6-BA, 15g / L sucrose and 6.0g / L carrageenan are added.

[0109] The formula of the proliferation medium includes: WPM+0.5mg / L 6-BA+30g / L sucrose+6.0g / L carrageenan.

[0110] (6) Rooting culture

[0111] (6.1) Effects of different basal culture media on rooting culture

[0112] 1 / 8MS, 1 / 6MS, 1 / 5MS, 1 / 4MS, 1 / 2MS and 1 / 2WPM were selected as basal culture media respectively, and 1.0 mg / L IBA was added to each basal culture medium. Tender shoots of about 2 cm in length were inoculated into the culture medium and cultured in a constant temperature of 25°C in a culture room for 30 days with a light intensity of 2500 lux. Three repeated experiments were set up for each treatment group.

[0113] The results are shown in Table 4. As the concentration of inorganic salts in the basal culture medium decreases, the root system becomes stronger, the number of fibrous roots increases, and the rooting rate increases, indicating that a low-salt environment is more conducive to the occurrence of adventitious roots of Castanopsis erythrorhizon. Among them, the rooting rate of Castanopsis erythrorhizon was the highest when 1 / 2WPM was used as the basal culture medium, reaching 74.44%, which was significantly higher than that of other treatment groups and was suitable as the basal culture medium for rooting culture.

[0114] Table 4 Effects of different basal culture media on the rooting of Castanopsis hystrix

[0115]

[0116] (6.2) Effects of different concentrations of IBA on rooting culture

[0117] 1 / 2WPM was used as the basic culture medium, and 0.25mg / L IBA, 0.5mg / L IBA, 1.0mg / L IBA, 2.0mg / L IBA, 4.0mg / L IBA, 0.25mg / L NAA, 0.5mg / L NAA, 1.0mg / L NAA, 2.0mg / L NAA, 4.0mg / L NAA were added respectively, for a total of ten culture media, to explore the effects of different types and concentrations of plant hormones on rooting effect. Tender shoots of about 2cm in length were inoculated in the culture medium and cultured in a constant temperature culture room at 25℃ for 30 days with a light intensity of 2500lux; three replicates were set for each treatment group.

[0118] The results are shown in Table 5. The rooting rate of Castanopsis erythrorhizon in the medium with IBA added was higher than that with NAA added, indicating that IBA was better than NAA in inducing rooting of Castanopsis erythrorhizon. Under low hormone concentration, adventitious roots germinated late and the root system was thin and weak. With the increase of exogenous hormone concentration, adventitious roots germinated faster, and the root system was strong and developed, and the number of fibrous roots increased. When IBA was 2.0 mg / L, the rooting rate was the highest, reaching 91.67%, which was significantly higher than other treatment groups (such as Figure 1 Therefore, the present application adds IBA at a concentration of 1.8 mg / L to 2.2 mg / L to the rooting medium.

[0119] Table 5 Effects of different types and concentrations of hormones on rooting effect

[0120]

[0121] (6.3) Effects of different concentrations of sucrose on rooting culture

[0122] 1 / 2WPM was used as the basic culture medium, and 2.0mg / L IBA was added. At the same time, sucrose at concentrations of 0g / L, 5g / L, 10g / L, 15g / L, 20g / L and 25g / L was added to the culture medium. Tender shoots of about 2cm in length were inoculated into the culture medium and cultured in a constant temperature culture room at 25℃ for 30 days with a light intensity of 2500lux. Three replicates were set for each treatment group.

[0123] The results are shown in Table 6. As the sucrose concentration increases, the rooting rate increases, the adventitious root germination period shortens, the number of fibrous roots increases, and the root system is strong; when the sucrose concentration reaches 25g / L, the rooting rate is the highest, reaching 95.83%; when the sucrose concentration reaches more than 25g / L, the callus at the base of the stem segment grows and the cortex is too swollen. Therefore, the present application adds sucrose at a concentration of 18g / L to 22g / L in the rooting medium.

[0124] Table 6 Effects of different sucrose concentrations on rooting effect

[0125]

[0126] (6.4) Effects of different culture media on rooting culture

[0127] Rooting medium A: 1 / 2 modified WPM was used as the basic medium, and 2.0 mg / L 6-BA, 1.5 mg / L NAA, 20 g / L sucrose and 6.0 g / L carrageenan were added.

[0128] Rooting medium B: 1 / 2WPM was used as the basic medium, and 2.0 mg / L 6-BA, 1.5 mg / L NAA, 20 g / L sucrose and 6.0 g / L carrageenan were added.

[0129] Rooting medium C: 1 / 2WPM was used as the basic medium, and 2.0 mg / L IBA, 20 g / L sucrose and 6.0 g / L carrageenan were added.

[0130] The results are shown in Table 7. Although the addition of 6-BA and NAA to the rooting medium made the plants strong, it did not promote rooting. The plants using the rooting medium C grew well, adventitious roots sprouted in about 2 weeks, and the rooting rate was high. Therefore, the present application selected the formula of the rooting medium C for rooting culture.

[0131] Table 7 Effects of different culture media on the rooting of Castanopsis hystrix

[0132]

[0133] (7) Acclimation and transplanting

[0134] Take 30-day-old rooted seedlings, loosen the bottle cap and place them in a sunny location for about 10 days to harden the seedlings. Take out the rooted seedlings with well-developed root systems, wash the culture medium, and disinfect it with 500 times carbendazim for later use. Use a 32-hole plug tray for transplanting (such as Figure 1 As shown in f), the matrix formula is peat soil: vermiculite = 1:2, and the survival rate can reach more than 85%. In the early stage of seedling transplanting, a layer of white transparent agricultural film should be covered for heat preservation and moisture retention, and a layer of 75%~80% shade net should be covered outside the film. After 1-2 weeks, the seedlings will develop new roots. When the seedlings are 3~4 weeks old, the seedlings grow strong, water and ventilate in time, remove the insulation film, and apply 0.1% dilute fertilizer every week. When the seedlings grow to 5cm, transplant the seedlings in black plastic bags with a specification of 8cm*10cm. The matrix is ​​yellow mud, and the shade net is replaced. The shading rate is preferably 40%~60%. When the height of the seedlings grows to 15cm~20cm in the later stage of transplanting, the seedlings can grow under natural environment conditions.

[0135] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A tissue culture method of Castanopsis erythroxylum, characterized in that: The steps include: Selecting semi-lignified Castanopsis hystrix spikelets as explants; and The explants are sequentially subjected to disinfection treatment, primary induction culture, first proliferation culture, sterilization treatment and rooting culture; The sterilization treatment includes the seedling culture and the second proliferation culture in sequence; The culture medium for the first proliferation culture and the second proliferation culture includes a first basal culture medium, and based on the first basal culture medium, further includes 0.4 mg / L~0.6 mg / L 6-BA, 14 g / L~16 g / L sucrose and 5 g / L~7 g / L carrageenan; the first basal culture medium is selected from WPM.

2. The tissue culture method of Castanopsis erythrorhizon according to claim 1, characterized in that: The culture medium for primary induction culture includes a second basal culture medium, and based on the second basal culture medium, further includes 1.8 mg / L~2.2 mg / L 6-BA, 0.04 g / L~0.06 g / L NAA, 14 g / L~16 g / L sucrose and 6.5 g / L~7 g / L carrageenan; the second basal culture medium is selected from WPM.

3. The tissue culture method of Castanopsis erythrorhizon according to claim 1, characterized in that: The culture medium for seedling cultivation includes a third basal culture medium, and based on the third basal culture medium, further includes 0.4mg / L~0.6mg / L 6-BA, 14g / L~16g / L sucrose and 5g / L~7g / L carrageenan; the third basal culture medium is selected from 1 / 2WPM.

4. The tissue culture method of Castanopsis erythrorhizon according to claim 1, characterized in that: The culture medium for rooting culture includes a fourth basic culture medium, and based on the fourth basic culture medium, further includes 1.8mg / L~2.2mg / L IBA, 18g / L~22g / L sucrose and 5g / L~7g / L carrageenan; the fourth basic culture medium is selected from 1 / 2WPM.

5. The tissue culture method of Castanopsis erythrorhizon according to claim 1, characterized in that: The number of sterilization treatments is 2 or more times.

6. The tissue culture method of Castanopsis erythrifera according to any one of claims 1 to 5, characterized in that: After the rooting culture, the method further comprises the following step: transplanting the rooted seedlings obtained by the rooting culture to a seedbed for cultivation.

7. The tissue culture method of Castanopsis erythrorhizon according to claim 6, characterized in that: The substrate in the seedbed includes peat soil and vermiculite.

8. The tissue culture method of Castanopsis erythroxylum according to claim 7, characterized in that: The volume ratio of the peat soil to vermiculite is 1:(1.8-2.2).

9. The tissue culture method of Castanopsis erythrifera according to any one of claims 1 to 5 and 7 to 8, characterized in that: The disinfection process includes the following steps: Rinse the explants with sterile water; Treating the explant with alcohol for 4s to 6s; and The explants were immersed in mercuric chloride for 4 to 6 minutes.

10. The tissue culture method of Castanopsis erythrifera according to any one of claims 1 to 5 and 7 to 8, characterized in that: The tissue culture method meets at least one of the following conditions: (1) The light intensity for primary induction culture is 2400 lux ~ 3000 lux; (2) The light intensity for proliferation culture is 2400 lux~3000 lux; and (3) The light intensity for rooting culture is 2400lux~3000lux.

Images