Tissue culture regeneration method of tetrapanax papyriferus

Abstract

The invention discloses a four-ball tea tissue culture regeneration method which comprises the following steps: explant treatment: firstly, treating a branch to be collected by using Australian tea tree essential oil, sleeving the branch to be collected by using a bag, then collecting the branch to be collected, shearing, disinfecting and drying to obtain a stem section with axillary buds; the method comprises the following steps: preparing a specific culture medium and specific culture conditions according to culture requirements of each culture stage, carrying out tissue culture processes of primary culture, axillary bud multiplication culture, small clumpy bud culture, small clumpy bud rooting and the like to obtain a four-ball tea tissue culture seedling, and carrying out sealed seedling hardening and transplanting on the four-ball tea tissue culture seedling to obtain a high-quality four-ball tea seedling. Compared with the prior art, the method has the advantages that the pollution rate and the death rate of primary culture of the explant of the tetrapanax papyriferus are reduced in a manner of combining Australian tea tree essential oil treatment with physical isolation, the aseptic seedling obtaining rate is 88.33% by combining a specific disinfection method and a culture medium, and the axillary bud proliferation coefficient reaches 3.5 and the induction rate reaches 90.00% by preparing the specific culture medium and culture conditions for axillary bud proliferation; the multiplication coefficient of the small clumpy buds is 4.5, the induction rate is 90.00%, and the rooting rate is 85.00%.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for tissue culture regeneration of Camellia sinensis. Background Art

[0002] Camellia tetracocca Zhang is a species of tea in the Camellia genus, Subgen. Thea, Sect. Thea, Ser. Quiquelocularis. It is a rare ancient tea tree that lives in the mountains and forests at an altitude of 1700-1950m in Pu'an County. It was discovered and named "Four-ball Tea" by Professor Zhang Hongda in 1981. It is a unique tea species in Pu'an County, Guizhou Province. The "Golden Boat" black tea series products developed by Pu'an people using the new branches and tender leaves of Four-ball Tea are not only golden in color, mellow in taste, fragrant and soft, and sweet in flavor, but also rich in various nutrients in the tea soup. If it is mass-produced, it will be deeply loved by consumers.

[0003] Pu'an County has nearly 500 ancient tea trees and is one of the important origins of tea culture. The research and application of the unique four-ball tea tree resources in Pu'an, Guizhou, can make the related tea products of four-ball tea a characteristic business card of Pu'an area and increase local income, which is of great significance to the local economic and cultural development.

[0004] Four-ball tea belongs to a small tree plant. The current germplasm resources of four-ball tea are scarce, and there are problems such as long growth cycle, low natural fruiting rate and hybrid fruiting rate, which make it impossible to industrialize tea products related to four-ball tea. Tissue culture and rapid propagation can quickly and efficiently maintain woody genetic characteristics and obtain a large number of returning seedlings. It is one of the effective measures to solve the problems of long growth cycle, low natural fruiting rate and hybrid fruiting rate of four-ball tea. Therefore, it is urgent to develop four-ball tea tissue culture technology to realize the industrialization of four-ball tea tissue culture, seedling cultivation, research and development, and production. However, so far, no research and reports on in vitro propagation of plant organs related to four-ball tea have been found. When the inventor studied the tissue culture of this species, he found the following problems:

[0005] (1) The resources of healthy, pest-free, and robust ancient tea trees are extremely scarce, and explants are very limited. The explant collection period affects the contamination and browning of the explants. The best collection period is in spring and summer, but there is more rain in spring and summer, and the number and variety of pathogens on wild plants are relatively large. After collection and inoculation, it is very easy to be contaminated, and it is difficult to obtain sterile seedlings.

[0006] (2) During the tissue culture process, problems such as browning, vitrification, low induced germination rate, low reproduction coefficient, slow growth rate, and difficulty in rooting are very likely to occur.

[0007] To solve the above problem (1), the inventors used tea branches containing axillary buds in May and June as explants, sprayed tea tree essential oil on the explant branches to be used and bagged them one month in advance, thereby reducing the probability of explant bacteria without harming the growth of axillary buds, and added thiourea to prevent browning and inhibit bacteria in the initial culture, thereby reducing the contamination, browning and death of the explants. To solve the above problem (2), a specific culture medium and culture conditions were used to obtain tea tissue culture seedlings according to the initial culture-induction of axillary bud proliferation-small cluster bud induction and rejuvenation-small cluster bud rooting, and through tissue culture, seedling hardening and post-transplantation management, a large number of tea seedlings were obtained in a short period of time. Summary of the invention

[0008] The invention aims to provide a method for tissue culture regeneration of four-ball tea, aiming to reduce explant pollution, browning and death, improve induced germination rate and reproduction coefficient, quickly root and harden seedlings to achieve short-term and large-scale acquisition of four-ball tea seedlings.

[0009] To achieve the above object, the present invention provides a method for tissue culture regeneration of Camellia sinensis, the method comprising:

[0010] S1, explant treatment: first treat the branches to be collected with Australian tea tree essential oil and put the branches to be collected with a bag, then collect the branches to be collected, cut, disinfect and dry, and obtain stem segments with axillary buds;

[0011] S2, primary culture: taking the stem segment with axillary buds, inoculating it into a primary culture medium for primary culture to obtain axillary buds, wherein the primary culture medium comprises MS and thiourea;

[0012] S3, axillary bud proliferation culture: taking the stem segment with axillary buds in step S2 and inoculating it into an induction medium for culture;

[0013] S4, small bud cluster cultivation: cutting the axillary buds in step S3, inoculating the axillary buds into an induction medium for induction culture, obtaining bud clusters, dividing the bud clusters into small bud clusters with 3-6 adventitious buds, inoculating the small bud clusters into a rejuvenation medium for cultivation, and obtaining robust small bud clusters;

[0014] S5, rooting of small buds: inoculating the robust small buds into a rooting medium for cultivation, wherein the rooting medium comprises 1 / 2MS, ammonium ethyl hexanoate, and sodium dodecylbenzene sulfonate;

[0015] S6, sealing, hardening and transplanting.

[0016] According to the above technical scheme, the inoculation material for axillary bud proliferation culture of the present invention is the stem segment with axillary buds in the initial culture stage, which achieves axillary bud proliferation while avoiding the formation of callus tissue at the inoculation site, improves the axillary bud proliferation efficiency and shortens the proliferation time.

[0017] Preferably, in the above technical solution, the induction medium is MS+80-100 mg / L Ca(NO3)2+6-8 mg / L BA+3-4 mg / L IAA mg / L+0.01-0.05 mg / L triiodobenzoic acid+0.1-1 mg / L triacontanol.

[0018] Preferably, in the above technical solution, in step S4, the induction culture is specifically cultured at a temperature of 26-30°C and a light intensity of 800-1000 Lx for 7-10 days, and then cultured at a light intensity of 3000-6000 Lx and a light duration of 8-12 h.

[0019] Preferably, in the above technical solution, in the step of inoculating the small cluster of buds into a rejuvenation medium for culturing, the rejuvenation medium is MS+0.6-0.8 mg / L 6-BA+0.04-0.08 mg / L IBA.

[0020] Preferably, in the above technical solution, the robust small cluster of buds is inoculated into a rooting medium for culture, and the rooting medium is 1 / 2MS+40-50 mg / L Ca(NO3)2+1-1.5mg / L IAA+1-1.5mg / L IBA+0.3-0.5mg / L ampicillin+0.02-0.05mg / L sodium dodecylbenzene sulfonate.

[0021] According to the above technical scheme, by adding ammonium hexanoate and sodium dodecylbenzene sulfonate and adjusting the concentration ratio of Ca(NO3)2, IAA, IBA, ammonium hexanoate and sodium dodecylbenzene sulfonate, the rapid rooting of small buds of four-ball tea can be induced and the elongation of the root system of small buds of four-ball tea can be promoted.

[0022] Preferably, in the above technical solution, in the steps S3, S4 and S5, the culturing is specifically carried out under the conditions of a light intensity of 3000-6000Lx and a light duration of 8-12h.

[0023] Preferably, in the above technical solution, in the step of inoculating into the primary culture medium for primary culture, the primary culture medium is MS+6-8 mg / L BA+3-4 mg / L IAA+0.01-0.05 mg / L thiourea.

[0024] Preferably, in the above technical solution, in the step of inoculating into the primary culture medium for primary culture, the primary culture is specifically cultured at a temperature of 26-30°C and a light intensity of 300-500Lx for 5-10 days, and then cultured under the conditions of a light intensity of 2000-3000 Lx and a light time of 8-12 h.

[0025] Preferably, in the above technical solution, the steps of explant treatment are specifically as follows:

[0026] Australian tea tree essential oil is sprayed on the stem segments, leaves and axillary buds of the branches to be collected 1-1.5 months before explant collection, and then the branches to be collected are covered with bags. The branches to be collected are collected in May-June, and the branches to be collected are cut into stem segments with 1-2 axillary buds, which are disinfected and dried to obtain stem segments with axillary buds.

[0027] Preferably, in the above technical solution, the steps of disinfection and drying are specifically as follows:

[0028] The stem segment with axillary buds is stirred and soaked in 75% alcohol for 10-30s, and rinsed with sterile water; then soaked in 0.1% mercuric chloride solution for 6-12 min, stirring at a frequency of 2-3 min / time during the soaking period, rinsed with sterile water, and the surface moisture of the stem segment with axillary buds is absorbed with sterile filter paper, and then 0.3-0.5 cm from both ends of the stem segment with axillary buds is cut off to obtain a stem segment with 1-2 axillary buds.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) One method is to use Australian tea tree essential oil treatment combined with plastic bag physical isolation. One month before collection, Australian tea tree essential oil was sprayed and plastic bags were used to reduce the contamination rate and mortality rate of the initial culture; the other method is to use mercuric chloride disinfection, Hg 2+ It binds to negatively charged proteins, denatures bacterial proteins and inactivates enzymes. Adding thiourea to the primary culture medium has certain bactericidal and anti-browning effects, thereby reducing the contamination rate of explants and the browning mortality rate.

[0031] (2) Using specific culture medium and culture conditions, cluster-type four-ball tea seedlings are quickly obtained by inducing axillary bud proliferation, inducing and rejuvenating small cluster buds, rooting small cluster buds, sealing and hardening the seedlings, and then transplanting. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings.

[0033] Figure 1 This is a diagram of processing tea leaves before explant collection in a specific embodiment of the present invention;

[0034] Figure 2 This is a diagram of processing tea leaves after explant collection in a specific embodiment of the present invention;

[0035] Figure 3 is a diagram of a tea plant tissue culture at the initial culture stage in a specific embodiment;

[0036] Figure 4 is a diagram of a four-ball tea tissue culture at the axillary bud proliferation culture stage in a specific embodiment;

[0037] Figure 5 is a diagram of inducing clustered buds in a tissue culture of four-ball tea at the small cluster bud culture stage in a specific embodiment;

[0038] Figure 6 It is a diagram of rejuvenation of tea tissue culture at the small bud culture stage in a specific embodiment;

[0039] Figure 7 It is a diagram of rejuvenation of small bud clusters in the small bud cluster culture stage of tea tissue culture in a specific embodiment;

[0040] Figure 8 is a diagram of a tissue culture of four-ball tea in a specific embodiment;

[0041] Fig. 9 is a diagram of a tea plant tissue culture at the small bud rooting stage in a specific embodiment;

[0042] Fig.10 It is a picture of the tissue culture of Camellia sinensis at the small cluster bud rooting stage in a specific embodiment. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] A method for tissue culture regeneration of four-ball tea, the method comprising:

[0046] S1, Explant treatment:

[0047] Select healthy, pest-free, and robust wild tea tree species, spray 15% Australian tea tree essential oil on the stems, leaves, and axillary buds of the branches to be collected one month before collection, and immediately cover the branches to be collected with plastic bags to combine Australian tea tree essential oil and plastic bags for physical isolation to reduce the contamination rate and mortality rate of initial culture;

[0048] From May to June every year, when the weather is sunny for more than 3 consecutive days, plastic bags are kept to collect explants from 10:00 to 12:00. The explants are the branches to be collected that have grown robustly after being bagged. The bases of the collected branches are wrapped with clean wet towels and placed in a widened cup with ice packs for transportation to the laboratory.

[0049] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut into stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash with distilled water or purified water 2-3 times, and place on a clean bench; soak and stir the stem segments with 75% alcohol on the clean bench for 15 seconds, and rinse with sterile water 3-4 times; then soak the explants in 0.1% mercuric chloride solution for 8 minutes, stir with a glass rod every 2-3 minutes during the soaking period, and rinse with sterile water 3-4 times;

[0050] Use sterile filter paper to absorb the moisture on the surface of the sterilized stem segment, and cut off the remaining leaves and 0.5 cm from the upper and lower ends of the stem segment, and cut it into 1.5-2.5 cm long stem segments with 1-2 axillary buds for later use.

[0051] S2, initial cultivation:

[0052] Prepare the primary culture medium MS+8mg / L BA+4mg / LIAA+0.05mg / L thiourea+3% sucrose+5.5g / L agar powder, sterilize by high pressure steam, and cool to solidify;

[0053] Take the stem segments of 1-2 axillary buds, inoculate them into the primary culture medium, culture them at 26-30°C and 300Lx light intensity for 6 days, and then place them at 2500Lx light intensity and 12 h light time for primary culture until the axillary buds grow to 2 cm.

[0054] S3, axillary bud proliferation culture:

[0055] Prepare induction medium MS + 100mg / L Ca(NO3)2 + 8mg / L BA + 4mg / L IAA

[0056] mg / L+0.05mg / L triiodobenzoic acid+0.2mg / L triacontanol+3% sucrose+4.5g / L agar powder;

[0057] The axillary buds obtained in step S2 are inoculated together with the initial stem segments on the above-mentioned induction medium, and cultured under the conditions of a light intensity of 5000 Lx and a light duration of 10 h until a large number of axillary buds proliferate at the axillary buds.

[0058] S4, small cluster bud culture:

[0059] Prepare induction medium MS + 100 mg / L Ca(NO3)2 + 6 mg / L BA + 4 mg / L IAA mg / L + 0.05 mg / L triiodobenzoic acid + 0.5 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0060] Cut the axillary buds in step S3, inoculate the axillary buds into the above-mentioned induction medium, culture them at a temperature of 26-30°C and a light intensity of 800 Lx for 7 days, and then culture them at a light intensity of 5000 Lx and a light time of 10 h until a large number of adventitious buds are induced at the base of the inoculated axillary buds to obtain cluster buds.

[0061] Prepare rejuvenation medium MS + 0.8 mg / L 6-BA + 0.05 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0062] The bud clusters are divided into small bud clusters with 3-6 adventitious buds, the small bud clusters are inoculated into the rejuvenation medium, and cultured under the conditions of a light intensity of 5000 Lx and a light duration of 10 h until some of the small bud clusters are 2.0-3.5 cm tall and grow robustly;

[0063] S5, small cluster bud rooting:

[0064] Prepare rooting medium: 1 / 2MS + 50mg / L Ca(NO3)2 + 1.5mg / L IAA + 1.5mg / LIBA + 0.5mg / L ammonium hexanoate + 0.05mg / L sodium dodecylbenzene sulfonate + 1.5% sucrose + 5.5g / L agar powder;

[0065] Inoculate the small clusters of buds with a plant height of 2.0-3.5 cm and strong growth into a rooting medium for cultivation, and cultivate them under the conditions of a light intensity of 5000 Lx and a light duration of 10 h;

[0066] S6, seal and harden the seedlings: when the root system of the tissue culture seedlings is 4-5 cm long, move the rooted bottle seedlings to a seedling shed with a shading rate of 75-80% for 30 days, and then transplant them into non-woven seedling bags (specifications 9cm×3cm or 10cm×12cm). The seedling medium is a light medium prepared with coconut bran, perlite and peat soil in a ratio of 2:1:2, which is put into the non-woven seedling bag. Carefully take the four-ball tea tissue culture seedlings out of the bottle, wash off the culture medium and move them into the non-woven seedling bag. The shading rate is 75-80% and the substrate humidity is maintained at 80-90% for the first 7 days after transplanting; the shading rate is 50-75% after 7 days of transplanting, and the shading rate is 20-40% after 21 days of transplanting. Spray water once a day, and each spraying lasts 30 minutes. After the four-ball tea seedlings grow new leaves, spray the leaves with 0.1-0.3% compound fertilizer (N:P2O5:K2O=15:15:15) aqueous solution, and apply fertilizer once every 15 days.

[0067] The death and contamination conditions in the initial culture, axillary bud proliferation, cluster bud proliferation coefficient and average bud height of small cluster buds, rooting time, average root length 14 days after rooting and rooting rate of Example 1 are shown in Table 1.

[0068] Table 1 Project indicators at each stage of Example 1

[0069]

[0070]

[0071] Example 2

[0072] A method for tissue culture regeneration of four-ball tea, the method comprising:

[0073] S1, explant treatment: basically the same as in Example 1, except that the disinfection method is slightly different:

[0074] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut the stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash them with distilled water or purified water 2-3 times, and place them on a clean bench; soak and stir the stem segments with 75% alcohol for 30 seconds on the clean bench, and rinse them with sterile water for 3-4 times; then soak the explants with 0.3% mercuric chloride solution for 12 minutes, stir them with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water for 3-4 times;

[0075] S2, initial culture:

[0076] Prepare the primary culture medium MS+6mg / L BA+3mg / LIAA+0.03mg / L thiourea+3% sucrose+5.5g / L agar powder, sterilize by high pressure steam, and cool to solidify;

[0077] Take the stem segments of 1-2 axillary buds, inoculate them into the primary culture medium, culture them at 26-30°C and 500Lx light intensity for 5 days, and then place them at 3000Lx light intensity and 8h light time for primary culture until the axillary buds grow to 2cm.

[0078] S3, axillary bud proliferation culture:

[0079] Prepare induction medium MS + 100mg / L Ca(NO3)2 + 8mg / L BA + 4mg / L IAA

[0080] mg / L+0.03mg / L triiodobenzoic acid+0.3mg / L triacontanol+3% sucrose+4.5g / L agar powder;

[0081] The axillary buds obtained in step S2 are inoculated together with the initial stem segments on the above-mentioned induction medium, and cultured under the conditions of a light intensity of 4000 Lx and a light duration of 12 h until a large number of axillary buds proliferate at the axillary buds.

[0082] S4, small cluster bud culture:

[0083] Prepare induction medium MS + 100mg / L Ca(NO3)2 + 6mg / L BA + 4mg / L IAA

[0084] mg / L+0.03mg / L triiodobenzoic acid+0.8mg / L triacontanol+3% sucrose+4.5g / L agar powder;

[0085] Cut the axillary buds in step S3, inoculate the axillary buds into the above-mentioned induction medium, culture them at a temperature of 26-30°C and a light intensity of 1000Lx for 7 days, and then culture them at a light intensity of 4000Lx and a light time of 12h until a large number of adventitious buds are induced at the base of the inoculated axillary buds to obtain cluster buds.

[0086] Prepare rejuvenation medium MS + 0.8 mg / L 6-BA + 0.04 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0087] The bud clusters are divided into small bud clusters with 3-6 adventitious buds, the small bud clusters are inoculated into the rejuvenation medium, and cultured under the conditions of a light intensity of 4000 Lx and a light duration of 12 h until some of the small bud clusters are 2.0-3.5 cm tall and grow robustly;

[0088] S5, small cluster bud rooting:

[0089] Prepare rooting medium 1 / 2MS + 50mg / L Ca(NO3)2 + 1.5mg / L IAA + 1.5mg / L IBA + 0.3mg / L ammonium hexanoate + 0.02mg / L sodium dodecylbenzene sulfonate + 1.5% sucrose + 5.5g / L agar powder;

[0090] The small clusters of buds with a plant height of 2.0-3.5 cm and strong growth are inoculated into a rooting medium for cultivation under the conditions of a light intensity of 4000 Lx and a light duration of 12 h.

[0091] S6, sealing and hardening the seedlings: the same as in Example 1.

[0092] Example 3

[0093] A method for tissue culture regeneration of four-ball tea, the method comprising:

[0094] S1, explant treatment: basically the same as in Example 1, except that the disinfection method is slightly different:

[0095] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut the stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash them with distilled water or purified water 2-3 times, and place them on a clean bench; soak and stir the stem segments with 75% alcohol on the clean bench for 30 seconds, and rinse them with sterile water 3-4 times; then soak the explants with 0.1% mercuric chloride solution for 12 minutes, stir them with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water 3-4 times;

[0096] S2, initial culture:

[0097] Prepare the primary culture medium MS+8mg / L BA+4mg / LIAA+0.01mg / L thiourea+3% sucrose+5.5g / L agar powder, sterilize by high pressure steam, and cool to solidify;

[0098] Take the stem segments of the 1-2 axillary buds, inoculate them into the primary culture medium, culture them at a temperature of 26-30°C and a light intensity of 300Lx for 10 days, and then place them at a light intensity of 2000Lx and a light duration of 12h for primary culture until the axillary buds grow to 2cm.

[0099] S3, axillary bud proliferation culture:

[0100] Prepare induction medium MS + 90mg / L Ca(NO3)2 + 6mg / L BA + 3mg / L IAA

[0101] mg / L+0.05mg / L triiodobenzoic acid+0.2mg / L triacontanol+3% sucrose+4.5g / L agar powder;

[0102] The axillary buds obtained in step S2 are inoculated together with the initial stem segments on the above-mentioned induction medium, and cultured under the conditions of a light intensity of 6000 Lx and a light duration of 8 h until a large number of axillary buds proliferate at the axillary buds.

[0103] S4, small cluster bud culture:

[0104] Prepare induction medium MS + 90mg / L Ca(NO3)2 + 6mg / L BA + 4mg / L IAA

[0105] mg / L+0.05mg / L triiodobenzoic acid+0.5mg / L triacontanol+3% sucrose+4.5g / L agar powder;

[0106] Cut the axillary buds in step S3, inoculate the axillary buds into the above-mentioned induction medium, culture them at a temperature of 26-30°C and a light intensity of 800Lx for 7 days, and then culture them at a light intensity of 6000Lx and a light time of 8h until a large number of adventitious buds are induced at the base of the inoculated axillary buds to obtain cluster buds.

[0107] Prepare rejuvenation medium MS + 0.6 mg / L 6-BA + 0.04 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0108] The bud clusters are divided into small bud clusters with 3-6 adventitious buds, the small bud clusters are inoculated into the rejuvenation medium, and cultured under the conditions of a light intensity of 6000 Lx and a light duration of 8 h until some of the small bud clusters are 2.0-3.5 cm tall and grow robustly;

[0109] S5, small cluster bud rooting:

[0110] Prepare rooting medium: 1 / 2MS + 40 mg / L Ca(NO3)2 + 1 mg / L IAA + 1 mg / L IBA

[0111] +0.5mg / L ammonium hexanoate +0.05mg / L sodium dodecylbenzene sulfonate +1.5% sucrose +5.5g / L agar powder;

[0112] The small clusters of buds with a plant height of 2.0-3.5 cm and strong growth are inoculated into a rooting medium for cultivation under the conditions of a light intensity of 6000 Lx and a light duration of 8 h.

[0113] S6, sealing and hardening the seedlings: the same as in Example 1.

[0114] Comparative Example 1

[0115] A method for processing tea explants, the method being as follows:

[0116] Select healthy, pest-free, and robust wild tea tree species, spray 75% alcohol on the stems, leaves, and axillary buds of the branches to be collected one month before collection, and immediately cover the branches to be collected with plastic bags;

[0117] From May to June every year, when the weather is sunny for more than 3 consecutive days, plastic bags are kept to collect explants from 10:00 to 12:00. The explants are the branches to be collected that have grown robustly after being bagged. The bases of the collected branches are wrapped with clean wet towels and placed in a widened cup with ice packs for transportation to the laboratory.

[0118] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut the stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash them with distilled water or purified water 2-3 times, and place them on a clean bench; soak and stir the stem segments with 75% alcohol for 15 seconds on the clean bench, and rinse them with sterile water for 3-4 times; then soak the explants with 0.1% mercuric chloride solution for 8 minutes, stir them with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water for 3-4 times;

[0119] Use sterile filter paper to absorb the moisture on the surface of the sterilized stem segment, cut off the remaining leaves and 0.5 cm of the upper and lower ends of the stem segment, cut into 1.5-2.5 cm long stem segments with 1-2 axillary buds, inoculate the stem segments into the culture medium MS + 8 mg / L BA + 4mg / L IAA + 0.05mg / L thiourea, culture at a temperature of 26-30℃ and a light intensity of 300Lx for 6 days, and then culture under the conditions of a light intensity of 2500Lx and a light time of 12h.

[0120] Comparative Example 2

[0121] A method for processing tea explants, the method being as follows:

[0122] Select healthy, pest-free, and robust wild tea tree species. Collect explants in plastic bags from 10:00 to 12:00 when the weather is sunny for more than 3 consecutive days in May and June each year. The explants are the robust branches to be collected after bagging. Wrap the base of the collected branches with a clean wet towel and place them in a widened cup with an ice pack and transport them to the laboratory.

[0123] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut the stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash them with distilled water or purified water 2-3 times, and place them on a clean bench; soak and stir the stem segments with 75% alcohol for 15 seconds on the clean bench, and rinse them with sterile water for 3-4 times; then soak the explants with 0.1% sodium hypochlorite solution for 8 minutes, stir them with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water for 3-4 times;

[0124] Use sterile filter paper to absorb the moisture on the surface of the sterilized stem segment, cut off the remaining leaves and 0.5 cm of the upper and lower ends of the stem segment, and cut it into 1.5-2.5 cm long stem segments with 1-2 axillary buds; inoculate the stem segment into the culture medium MS+8mg / L BA+4mg / L IAA, culture it at a temperature of 26-30°C and a light intensity of 300Lx for 6 days, and then culture it under the conditions of a light intensity of 2500Lx and a light duration of 12h.

[0125] Comparative Example 3

[0126] A method for processing tea explants, the method being as follows:

[0127] Select healthy, pest-free, and robust wild tea tree species, spray 15% Australian tea tree essential oil on the stems and leaves of the branches to be collected one month before collection, and immediately cover the branches to be collected with plastic bags, combining Australian tea tree essential oil with physical isolation to reduce the contamination rate and mortality rate of initial culture;

[0128] From May to June every year, in the period of 10:00 to 12:00 when the weather is sunny for more than 3 consecutive days, plastic bags are used to collect explants, wherein the explants are the branches to be collected that have grown robustly after being bagged. The bases of the collected branches are wrapped with clean wet towels and placed in a widened cup with ice packs for transportation to the laboratory;

[0129] Cut off the leaves and old stem segments of the branches, wash the dust on the surface with tap water, cut the stem segments with a length of 2.5-3.0 cm and 1-2 axillary buds, wash them with distilled water or purified water 2-3 times, and place them on a clean bench; soak and stir the stem segments with 75% alcohol for 15 seconds on the clean bench, and rinse them with sterile water for 3-4 times; then soak the explants with 0.1% mercuric chloride solution for 8 minutes, stir them with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water for 3-4 times;

[0130] Use sterile filter paper to absorb the moisture on the surface of the sterilized stem segment, cut off the remaining leaves and 0.5 cm of the upper and lower ends of the stem segment, cut into 1.5-2.5 cm long stem segments with 1-2 axillary buds, inoculate the stem segments into the culture medium MS + 8mg / L BA + 4mg / L IAA, culture at a temperature of 26-30℃ and a light intensity of 300Lx for 6 days, and then culture under the conditions of a light intensity of 2500Lx and a light time of 12h.

[0131] The tea explants were treated according to the methods of Comparative Examples 1, 2 and 3, and the death and contamination of the tea explants were detected during the culture process. The mortality rate, contamination rate and sterile seedling acquisition rate were finally calculated, and compared with the mortality rate, contamination rate and sterile seedling acquisition rate in the initial culture stage of Example 1. The results are shown in Table 2 below, where the formulas for the contamination rate, browning rate and sterile seedling acquisition rate are as follows:

[0132] Contamination rate (%) = (number of contaminated strains / total number of inoculated strains) × 100%;

[0133] Mortality rate (%) = (number of browned plants and dead plants / total number of inoculated plants) × 100%;

[0134] The rate of sterile seedlings obtained (%) = (uncontaminated and unbrown / total number of inoculations) × 100%.

[0135] Table 2 Effects of different explant treatment methods on browning rate, contamination rate and sterile seedling acquisition rate

[0136] Group mortality rate(%) Contamination rate (%) Sterile seedling acquisition rate (%) Example 1 5.00(3 / 60) 6.67(4 / 60) 88.33(53 / 60) Comparative Example 1 10.00(6 / 60) 16.67(10 / 60) 73.33(44 / 60) Comparative Example 2 18.33(11 / 60) 35.00(21 / 60) 46.67(28 / 60) Comparative Example 3 15.00(9 / 60) 26.67(16 / 60) 58.33(35 / 60)

[0137] It was proved that the sterile seedlings obtained by bagging branches treated with Australian tea tree essential oil before collection were significantly better than those treated with alcohol. Further, the disinfection effect of mercuric chloride on tea tree explants was better, which may be due to Hg 2+It can bind to negatively charged proteins to denature bacterial proteins and inactivate enzymes; before collection, spray 15% Australian tea tree essential oil on the stems and leaves of the branches to be collected and bag them, which can effectively reduce the contamination rate in the initial culture stage and increase the rate of obtaining sterile seedlings; adding 0.05 mg / L thiourea to the culture medium has certain anti-browning and antibacterial effects. Its anti-browning effect may be that thiourea inhibits the activity of catalase, reduces its oxidation of phenolic substances, and achieves the reduction of browning.

[0138] Comparative Example 4

[0139] To explore the effect of explant collection time on the number of fungi and the regeneration ability of buds

[0140] The explants were collected at three stages: March-April, May-June, and July-August. The explant treatment method was the same as in Example 1. The results are shown in Table 3.

[0141] Table 3 Effects of explant collection time on mortality, contamination rate and sterile seedling acquisition rate

[0142] mortality rate Contamination rate The rate of obtaining sterile seedlings March-April 13.33(8 / 60) 6.67(4 / 60) 80.00(48 / 60) May-June 5.00(3 / 60) 6.67(4 / 60) 88.33(53 / 60) July-August 21.67(13 / 60) 28.33(17 / 60) 50.00(30 / 60)

[0143] The contamination rate in March-April and May-June is less, and the sterile seedling acquisition rate is higher, but the sterile seedling acquisition rate in May-June is better, which may be that the axillary buds have a shorter growth time in March-April, which is more fragile, and the mortality rate is higher after disinfection with alcohol and mercuric chloride, resulting in a lower sterile seedling acquisition rate. The inventor found that the four-ball tea explant maturity collected in May-June is the best, and the impact of disinfection on it is minimal, and the speed of inducing axillary bud germination is fast, efficient, and less browning. Suitable explant material is combined with the mode of using Melaleuca alternifolia essential oil+plastic bag bagging, so that the axillary buds obtain certain growth, while reducing the problem of microbial reproduction pollution in the rainy season, and finally the sterile seedling acquisition rate is the best.

[0144] Comparative Example 5

[0145] The inoculated object is the tissue culture seedling obtained by the initial culture stage culture method of Example 1, which is transferred to the induction medium of MS + 8mg / L BA + 4mg / L IAA mg / L + 2mg / L TDZ + 3% sucrose + 4.5g / L agar powder, and cultured under the conditions of light intensity 5000 Lx and light time 10h.

[0146] Comparative Example 6

[0147] The inoculated object is the tissue culture seedling obtained by the initial culture stage culture method of Example 1, which is transferred to the induction medium of MS + 8mg / L BA + 4mg / L IAA + 3% sucrose + 4.5g / L agar powder, and cultured under the conditions of light intensity 5000 Lx and light time 10h.

[0148] Comparative Example 7

[0149] The inoculated object is the tissue culture seedling obtained by the cultivation method of the initial cultivation stage of Example 1, which is transferred to the induction medium of MS + 70mg / L Ca (NO3) 2 + 8mg / L BA + 4mg / L IAA mg / L + 0.05mg / L triacontanol + 3% sucrose + 4.5g / L agar powder, and cultured under the conditions of light intensity 5000 Lx and light time 10 h.

[0150] Comparative Example 8

[0151] The inoculated object is the tissue culture seedling obtained by the cultivation method of the initial cultivation stage of Example 1, which is transferred to the induction medium of MS + 100 mg / L Ca (NO3) 2 + 8 mg / L BA + 4 mg / L IAA mg / L + 0.2 mg / L triacontanol + 3% sucrose + 4.5g / L agar powder, and cultured under the conditions of light intensity 5000Lx and light time 10h.

[0152] The axillary bud proliferation coefficients and induction rates of Comparative Examples 5, 6, 7 and 8 were statistically analyzed and compared with the axillary bud proliferation culture of Example 1. The results are shown in Table 4.

[0153] Table 4 Proliferation coefficient and induction rate of Example 1 and Comparative Examples 5-8

[0154] Proliferation coefficient Induction rate (%) Example 1 3.5 90.00％(36 / 40) Comparative Example 5 2.5 57.50％(23 / 40) Comparative Example 6 1.5 47.50％(19 / 40) Comparative Example 7 2.0 67.50％(27 / 40) Comparative Example 8 3.0 77.50％(31 / 40)

[0155] Comparative Example 9

[0156] Transfer the small clusters of buds with a height of 2.0-3.5 cm and strong growth to the rooting medium 1 / 2MS + 50 mg / LCa(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.5 mg / L riboflavin + 1.5% sucrose + 5.5 g / L agar powder, and culture them under the conditions of light intensity of 5000 Lx and light time of 10 h.

[0157] Comparative Example 10

[0158] Transfer the small clusters of buds with a height of 2.0-3.5 cm and strong growth to the rooting medium 1 / 2MS + 50 mg / LCa(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.5 mg / L aminoethyl hexanoate + 1.5% sucrose + 5.5 g / L agar powder, and culture under the conditions of light intensity of 5000 Lx and light time of 10 h.

[0159] The rooting conditions of Comparative Examples 9 and 10 were statistically analyzed and compared with the small bud rooting culture of Example 1. The results are shown in Table 5.

[0160] Table 5 Rooting of small buds in Example 1 and Comparative Examples 9-10

[0161] Rooting time 14d average root length (cm) Rooting rate (%) Example 1 25d rooting 1cm 85.00％(34 / 40) Comparative Example 9 55d rooting 0.40cm 57.50％(23 / 40) Comparative Example 10 30d rooting 0.75cm 72.50％(29 / 40)

[0162] The present invention can be implemented in various ways and is not limited to the embodiments described. A person skilled in the art can understand that the present invention can be implemented in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary and are not intended to limit the present invention.

Claims

1. A method for tissue culture regeneration of Camellia sinensis, characterized in that: The method comprises: S1, explant treatment: first treat the branches to be collected with Australian tea tree essential oil and put the branches to be collected with a bag, then collect the branches to be collected, cut, disinfect and dry, and obtain stem segments with axillary buds; S2, primary culture: taking the stem segment with axillary buds, inoculating it into a primary culture medium for primary culture to obtain axillary buds, wherein the primary culture medium comprises MS and thiourea; S3, axillary bud proliferation culture: taking the stem segment with axillary buds in step S2 and inoculating it into an induction medium for culture; S4, small bud cluster cultivation: cutting the axillary buds in step S3, inoculating the axillary buds into an induction medium for induction culture, obtaining bud clusters, dividing the bud clusters into small bud clusters with 3-6 adventitious buds, inoculating the small bud clusters into a rejuvenation medium for cultivation, and obtaining robust small bud clusters; S5, rooting of small buds: inoculating the robust small buds into a rooting medium for cultivation, wherein the rooting medium comprises 1 / 2MS, ammonium ethyl hexanoate, and sodium dodecylbenzene sulfonate; S6, sealing, hardening and transplanting.

2. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: The induction medium is MS+80-100 mg / L Ca(NO3)2+6-8 mg / L BA+3-4 mg / L IAA mg / L+0.01-0.05 mg / L triiodobenzoic acid+0.1-1 mg / L triacontanol.

3. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: In step S4, the induction culture is specifically cultured at a temperature of 26-30°C and a light intensity of 800-1000 Lx for 7-10 days, and then cultured at a light intensity of 3000-6000 Lx and a light duration of 8-12 hours.

4. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: In the step of inoculating the small bud cluster into a rejuvenation medium for culturing, the rejuvenation medium is MS+0.6-0.8 mg / L 6-BA+0.04-0.08 mg / L IBA.

5. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: The robust small cluster of buds is inoculated into a rooting medium for cultivation, wherein the rooting medium is 1 / 2MS+40-50 mg / LCa(NO3)2+1-1.5 mg / L IAA+1-1.5 mg / L IBA+0.3-0.5 mg / L ammonium hexanoate+0.02-0.05 mg / L sodium dodecylbenzene sulfonate.

6. The method for tissue culture regeneration of Camellia sinensis according to claim 1, characterized in that: In the steps S3, S4 and S5, the culturing is specifically carried out under the conditions of light intensity of 3000-6000Lx and light time of 8-12h.

7. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: In the step of inoculating into the primary culture medium for primary culture, the primary culture medium is MS+6-8 mg / L BA+3-4 mg / L IAA+0.01-0.05 mg / L thiourea.

8. The method for tissue culture regeneration of Camellia sinensis as claimed in claim 1, characterized in that: In the step of inoculating into the primary culture medium for primary culture, the primary culture is specifically cultured at a temperature of 26-30°C and a light intensity of 300-500 Lx for 5-10 days, and then cultured at a light intensity of 2000-3000 Lx and a light duration of 8-12 hours.

9. The method for tissue culture regeneration of Camellia sinensis according to claim 1, characterized in that: The steps of the explant treatment are specifically as follows: Australian tea tree essential oil is sprayed on the stem segments, leaves and axillary buds of the branches to be collected 1-1.5 months before explant collection, and then the branches to be collected are covered with bags. The branches to be collected are collected in May-June, and the branches to be collected are cut into stem segments with 1-2 axillary buds, which are disinfected and dried to obtain stem segments with axillary buds.

10. The method for tissue culture regeneration of Camellia sinensis according to claim 9, characterized in that: The steps of disinfection and drying are as follows: The stem segment with axillary buds is stirred and soaked in 75% alcohol for 10-30s, and rinsed with sterile water; then soaked in 0.1% mercuric chloride solution for 6-12min, stirring at a frequency of 2-3 min / time during soaking, rinsed with sterile water, and the surface moisture of the stem segment with axillary buds is absorbed with sterile filter paper, and then 0.3-0.5cm from both ends of the stem segment with axillary buds is cut off to obtain a stem segment with 1-2 axillary buds.

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