Rapid Propagation and in vitro Conservation of Rheum webbianum Hook. f.

Abstract

The application belongs to the technical field of plant tissue culture, and particularly discloses a tissue culture and rapid propagation and in-vitro preservation method and application of alpine plant Rheum webbianum Hook.et Arn.; the rapid propagation method comprises the following steps: explant selection and disinfection, primary induction culture, subculture proliferation culture, rooting culture and rooting seedling domestication. The in-vitro preservation method comprises the following steps: pre-treatment culture, germplasm preservation culture and rejuvenation culture. The disinfection contamination rate is less than 19%, and the disinfection mortality rate is less than 9.0%; the primary induction rate is more than 65.7%, the highest proliferation coefficient is more than 3.5, the rooting rate after the rooting culture is more than 86.5%, and the domestication survival rate can be more than 95%, so that the in-vitro seedling successfully transits to the natural environment. The subculture period is prolonged to more than 18 months, and the longest period can be up to 22 months by using suitable concentrations of ABA and CCC, simultaneously adding suitable concentrations of mannitol as an osmotic regulator, and combining with low-temperature and weak-light culture conditions.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and specifically discloses a method for rapid tissue culture propagation and in vitro preservation of the alpine plant Rheum tigrinum, as well as its application. Background Technology

[0002] Rheum australe D. Don, a perennial alpine herb belonging to the Polygonaceae family and the Rheum genus, is mainly distributed in alpine meadows, scree slopes, and barren grasslands at altitudes of 3400-4300 meters on the Qinghai-Tibet Plateau, and is found in Tibet, Sichuan, and Yunnan provinces. Its thick rhizomes are rich in anthraquinones, tannins, and erucic acid compounds, and it is the original plant of the Tibetan medicine "Junmu Zha," which has the effects of purging heat, promoting digestion, and reducing swelling, and is used to treat febrile diseases, diarrhea, constipation, and epidemic diseases. Due to long-term over-harvesting and the shrinking of alpine habitats caused by global warming, the wild population of Rheum australe D. Don has decreased sharply, and natural regeneration is difficult. This species has a low seed germination rate and requires many years of vegetative growth before flowering and fruiting, making it difficult to maintain population stability through seed reproduction.

[0003] Plant in vitro preservation technology can significantly extend the subculture interval by adjusting the composition of the culture medium and the culture conditions, avoiding contamination and genetic variation caused by frequent transfers. It is particularly suitable for the conservation of germplasm resources of rare and endangered alpine plants.

[0004] With the development of technology, in vitro tissue culture has become an effective solution to the problems existing in traditional seed propagation methods. The publication number is CN117044627A, entitled "A method for rapid propagation and in vitro preservation of the alpine plant Rheum nobile through tissue culture". This method uses Rheum nobile seeds as culture material. After sterilization of the explants, sterile seedlings are obtained by germination culture. The seedlings are then subjected to shoot proliferation, rooting induction and in vitro preservation. A rapid propagation and in vitro preservation system for Rheum nobile has been established. After three subcultures, the proliferation coefficient is as high as 1:20.17, and the in vitro preservation subculture cycle can reach more than 18 months. Guo Chenggang (Guo Chenggang, Bi Hailin, Tang Wangwai, et al. Tissue culture and rapid propagation of Rheum palmatum in Lhasa[J]. Special Products Research, 2017, 039(003):6-11.DOI:10.16720 / j.cnki.tcyj.2017.03.002.) et al. used Rheum palmatum seeds as explant material. After seed germination, the seeds underwent primary induction, differentiation induction, and rooting induction to achieve rapid propagation of Rheum palmatum in Lhasa. In the above method, both Rheum palmatum and Rheum palmatum belong to the Rheum genus of Polygonaceae. However, this method uses seeds as explant material. The problem is that Rheum genus of Polygonaceae usually grows in alpine rocky beaches and wet grasslands at an altitude of 4000-4800 meters. The harsh environment of high-altitude regions meant that the specialized bract structure of these plants evolved under such extreme conditions. Furthermore, *Rheum nobile* requires 5-6 years to flower and produce seeds, while *Rheum sarmentosum* requires three years. This presents a significant challenge in tissue culture propagation due to the difficulty in obtaining suitable materials, severely limiting the timeframe for rapid in vitro propagation. Moreover, although *Rheum nobile*, *Rheum sarmentosum*, and *Rheum sibiricum* belong to the same family and genus, the rapid propagation and preservation methods used for *Rheum sibiricum* cannot be directly applied to the tissue culture and germplasm preservation of *Rheum sibiricum*.

[0005] Currently, there are no complete and systematic reports on tissue culture and in vitro preservation methods for Rheum palmatum from Tibetan border regions, especially no publicly available in vitro preservation systems with long subculture cycles capable of long-term preservation. Therefore, establishing efficient in vitro rapid propagation and medium- to long-term in vitro preservation technologies is of great significance for the protective development and introduction and domestication of this wild medicinal plant resource. Summary of the Invention

[0006] The purpose of this invention is to address the lack of a complete and systematic method for tissue culture and in vitro preservation of Rheum palmatum, an alpine plant, by providing a method and application for rapid tissue culture propagation and in vitro preservation of Rheum palmatum.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a rapid propagation method for Tibetan rhubarb tissue culture, comprising the following steps:

[0008] (1) Selection and disinfection of explants: Take the tender leaves and stems of Tibetan rhubarb, wash them, soak them in 1% potassium permanganate solution for 20 minutes, rinse them with sterile water 5-6 times, then disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 3-4 times, disinfect them with 0.1% benzalkonium chloride solution for 3-5 minutes, rinse them with sterile water 3-4 times, disinfect them with 0.02% mercuric chloride solution for 3-5 minutes, rinse them with sterile water 7-8 times. When disinfecting with 0.1% benzalkonium chloride solution and 0.02% mercuric chloride solution, use 25-30 kHz ultrasound.

[0009] (2) Primary induction culture: The sterilized explants were wiped dry with sterile paper, and the wounds at both ends were cut off. They were then inoculated into the primary induction culture medium and cultured for 30 to 35 days under alternating light and dark conditions with a temperature of 25±2℃, a light intensity of 2000~3000Lx, and 12h of sunlight.

[0010] The formulation of the primary culture medium is as follows: MS + NAA 0.3-0.5 mg / L + 6-BA 1.0-2.0 mg / L + sucrose 30.0-50.0 g / L + agar 7.0-9.0 g / L, pH 6.0-6.2;

[0011] (3) Subculture: The sterile seedlings were cut into single buds and the leaves were cut off at the same time. The single buds and leaves were inoculated into the subculture culture medium and cultured for 35 to 45 days under alternating light and dark conditions with a temperature of 25±2℃, a light intensity of 2000~3000Lx and 12h of sunlight.

[0012] The formulation of the subculture proliferation medium is as follows: MS + 6-BA 0.5-1.0 mg / L + KT 1.0-1.5 mg / L + ZT 0.5-1.0 mg / L + 2,4-D 0.5-1.0 mg / L + sucrose 30.0-50.0 g / L + agar 7.0-9.0 g / L, pH 6.0-6.2;

[0013] (4) Rooting culture: Cut the proliferating buds into single buds, inoculate them into rooting culture medium, and culture them for 40 to 50 days in an alternating light and dark environment with a temperature of 25±2℃, a light intensity of 2000~3000Lx, and 12h of sunlight.

[0014] The rooting medium formula is as follows: 1 / 2 MS + NAA 1.0-2.0 mg / L + IBA 0.8-1.2 mg / L + CH 50.0-80.0 mg / L + methyl jasmonate 0.3-0.4 mg / L + coconut milk 60.0-80.0 mg / L + activated charcoal 0.1-1.0 g / L, pH 6.0-6.2;

[0015] (5) Acclimation of rooted seedlings: Transplant the tissue culture rooted seedlings into the rooting substrate. The acclimation environment is a greenhouse with a shading rate of 65-75%. After transplanting, spray with 30-40 mg / L salicylic acid (SA) once, and then spray with 30-40 mg / L salicylic acid (SA) once every 5-7 days.

[0016] Furthermore, in step (2), the formulation of the primary culture medium is: MS + NAA 0.3 mg / L + 6-BA 1.0 mg / L + sucrose 40.0 g / L + agar 8.0 g / L, pH 6.0.

[0017] Furthermore, in step (3), the formulation of the subculture proliferation medium is: MS + 6-BA 1.0 mg / L + KT 1.5 mg / L + ZT 1.0 mg / L + 2,4-D 1.0 mg / L + sucrose 40.0 g / L + agar 8 g / L, pH 6.0.

[0018] Furthermore, in step (4), the rooting medium formula is: 1 / 2MS + NAA 1.5mg / L + IBA 1.0mg / L + CH 65.0mg / L + methyl jasmonate 0.4mg / L + coconut milk 70.0mg / L + activated carbon 0.1g / L, pH 6.0.

[0019] The second objective of this invention is to provide a method for the in vitro preservation of Tibetan rhubarb, comprising the following steps:

[0020] (1) Pretreatment culture: The cleaned and disinfected explant material was inoculated into the pretreatment culture medium and cultured under light and dark alternation for 15 to 20 days at 10±2℃, light intensity of 1000~2000Lx, and light duration of 10h per day.

[0021] The pretreatment culture medium formulation is: 1 / 2 MS + sucrose 50.0-60.0 g / L + mannitol 1.0-5.0 g / L;

[0022] (2) Germplasm preservation culture: The pretreated explant material was inoculated into the germplasm preservation culture medium and cultured under alternating light and dark conditions at 10±2℃, light intensity of 1000~2000Lx, and light duration of 6~8h per day.

[0023] The formula for the germplasm preservation medium is: 1 / 2 MS + mannitol 30.0-50.0 g / L + ABA 2.0-5.0 mg / L + CCC 1.2-1.6 mg / L + agar 7.0-8.0 g / L;

[0024] (3) Rejuvenation culture: After germplasm preservation culture, aseptic buds are inoculated into rejuvenation culture medium and cultured in alternating light and dark conditions for 30 to 40 days at 25±2℃, light intensity of 2000~3000Lx, and daily light duration of 12h.

[0025] The formulation for the rejuvenation culture is: MS + 6-BA 2.0~3.0 mg / L + KT 2.0~3.0 mg / L + ZT 1.0~2.0 mg / L + 2,4-D 1.0~2.0 mg / L + sucrose 30.0~50.0 g / L.

[0026] Furthermore, the pretreatment culture medium is formulated as follows: 1 / 2 MS + sucrose 50.0 g / L + mannitol 3.0 g / L.

[0027] Furthermore, the formula of the germplasm preservation medium is: 1 / 2 MS + mannitol 40.0 g / L + ABA 2.5 mg / L + CCC 1.5 mg / L + agar 7.0 g / L.

[0028] Furthermore, the formulation for the rejuvenation culture is: MS + 6-BA 2.5 mg / L + KT 2.5 mg / L + ZT 1.5 mg / L + 2,4-D 1.5 mg / L + sucrose 40.0 g / L.

[0029] The beneficial technical effects of this invention are:

[0030] 1. This invention uses the tender leaves and stems of Rheum palmatum from Tibetan border regions as explant material, providing a more abundant source of explant material. For explant disinfection, the explants are first soaked in potassium permanganate solution, followed by a combined disinfection method using four disinfectants: alcohol, benzalkonium chloride solution, and mercuric chloride solution. Both the disinfection concentration and time are relatively lower than conventional disinfection methods used in tissue culture. Furthermore, ultrasound is used in conjunction with mercuric chloride solution disinfection to minimize disinfection contamination while reducing damage to the explants. The disinfection contamination rate is 16%–19%, and the disinfection mortality rate is 7.8%–9.0%. During primary induction, using NAA 0.3–0.5 mg / L and 6-BA 1.0–2.0 mg / L, the primary induction rate reaches 65.7%–75.6%, with abundant shoot differentiation, vigorous growth, and no vitrification. For subculture propagation, using 6-BA 0.5–1.0 mg / L and KT... At concentrations of 1.0–1.5 mg / L, ZT 0.5–1.0 mg / L, and 2,4-D 0.5–1.0 mg / L, the proliferation coefficient reached a maximum of 3.5–4.7, with no vitrification phenomenon. The clustered buds grew vigorously, and the rooting rate after rooting culture reached over 86.5%. The provided hardening substrate had a reasonable ratio, and the transplant survival rate could reach over 95%, achieving a smooth transition of in vitro seedlings to the natural environment.

[0031] 2. In this invention, an appropriate concentration of ABA and CCC is used in the in vitro preservation medium, and an appropriate concentration of mannitol is added as an osmotic regulator. Combined with low temperature (10±2℃) and low light (1000-1500 lx) culture conditions, the subculture cycle is extended to more than 18 months, and can reach up to 22 months, which far exceeds the preservation time of conventional tissue culture. At the same time, the subculture proliferation, rooting culture and acclimatization are carried out using the method of this invention, with a proliferation rate of more than 4.0 and a rooting rate of 80.0%, which greatly reduces the cost of artificial maintenance. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a diagram showing the results of subculture proliferation in Example 1 of the present invention.

[0034] Figure 2 This is a diagram showing the rooting culture results of Example 1 of the present invention.

[0035] Figure 3 This is a diagram showing the results of subculture proliferation in Example 2 of the present invention.

[0036] Figure 4 This is a diagram showing the rooting culture results of Example 2 of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] (a) Disinfection test

[0039] Soak in 1% potassium permanganate solution for 5, 10, 15, 20, 25, and 30 minutes, then disinfect with 75% alcohol for 5, 10, 15, 20, 25, and 30 seconds, followed by disinfection with 0.1% benzalkonium chloride solution for 1, 2, 3, 4, and 5 minutes, and then disinfect with 0.02% mercuric chloride solution for 1, 2, 3, 4, and 5 minutes.

[0040] Table 1 Disinfection Experiment Design

[0041] serial number Soaking time (min) in 1% potassium permanganate solution Disinfection time (s) with 75% alcohol Disinfection time (min) with 0.1% benzalkonium chloride Disinfection time (min) with 0.02% mercuric chloride Should it be combined with ultrasound? 1-1 5 5 1 1 no 1-2 10 10 2 2 no 1-3 15 15 3 3 no 1-4 20 20 4 4 no 1-5 20 30 5 5 Yes (the best solution) 1-6 25 25 5 5 no 1-7 30 30 5 5 no 1-8 20 30 5 3 yes 1-9 20 30 3 5 yes

[0042] Table 2 Disinfection test results

[0043] serial number Disinfection contamination rate (%) Disinfection mortality rate (%) 1-1 45.0 2.5 1-2 38.0 4.2 1-3 32.0 6.8 1-4 28.0 8.5 1-5 16.0 9.0 1-6 22.0 12.5 1-7 20.0 15.0 1-8 19.0 7.8 1-9 18.0 8.2

[0044] As shown in Tables 1 and 2 above, the disinfection method using 75% alcohol for 30 seconds, combined with soaking in 1% potassium permanganate solution for 20 minutes, disinfection with 0.1% benzalkonium chloride solution for 3-5 minutes, and disinfection with 0.02% mercuric chloride solution for 3-5 minutes, and with ultrasonic-assisted disinfection, resulted in a disinfection contamination rate of 16.0%-19.0% and a disinfection mortality rate of 7.8%-9.0%, which is the optimal disinfection method for the primary culture in this experiment.

[0045] (ii) Primary induction test

[0046] Using MS as the basic culture medium, 0.1–0.5 mg / L NAA was added to conduct a combination experiment of 6-BA to optimize the optimal hormone ratio for inducing primary shoots of rhubarb tender leaf stem explants. The specific experimental design is shown in Tables 3 and 4.

[0047] Table 3 Initial Induction Experiment Design

[0048] serial number NAA (mg / L) 6-BA (mg / L) serial number NAA (mg / L) 6-BA (mg / L) 2-1 0 0 2-6 0.3 1.5 2-2 0.1 0.5 2-7 0.3 2.0 2-3 0.1 1.0 2-8 0.5 1.0 2-4 0.1 1.5 2-9 0.5 1.5 2-5 0.3 1.0

[0049] Table 4 Results of the initial induction test

[0050] serial number Induction rate (%) other serial number Induction rate (%) other 2-1 0 A small amount of pale green callus tissue was produced, but no bud differentiation was observed. 2-6 68.2 Bud differentiation is normal, no vitrification. 2-2 42.5 Few buds differentiated, resulting in weak growth. 2-7 65.7 Moderate bud differentiation, with a small amount of vitrification. 2-3 56.8 The bud differentiation rate is moderate, and the growth is good. 2-8 70.1 Numerous buds differentiated, and the plant is growing well. 2-4 61.3 Bud differentiation is moderate, without vitrification. 2-9 67.5 Bud differentiation is moderate, with a small amount of vitrification. 2-5 75.6 Numerous bud differentiations, vigorous growth, and no vitrification. - - -

[0051] As shown in Table 3-4, when NAA 0.3-0.5 mg / L and 6-BA 1.0-2.0 mg / L are used, the primary induction rate reaches 65.7-75.6%; when NAA 0.3 mg / L and 6-BA 1.0 mg / L are used, the primary induction rate reaches 75.6%.

[0052] (III) Subgeneration Proliferation Experiment

[0053] Using MS as the basic culture medium, a combination experiment of 6-BA, KT, ZT and 2,4-D was conducted to optimize the optimal hormone ratio for the subculture proliferation of rhubarb shoots from Tibetan border areas and reduce the vitrification rate. The specific experimental design is shown in Tables 5 and 6.

[0054] Table 5 Orthogonal Design Table for Subgeneration Proliferation Experiments

[0055] serial number 6-BA (mg / L) KT (mg / L) ZT (mg / L) 2,4-D (mg / L) 3-1 0 0 0 0 3-2 0.5 1.0 0.5 0.5 3-3 0.5 1.5 1.0 0.5 3-4 1.0 1.0 1.0 0.5 3-5 1.0 1.5 1.0 1.0 3-6 1.0 2.0 0.5 0 3-7 2.0 1.0 0.5 0 3-8 2.0 1.5 1.0 0 3-9 2.0 2.0 0.5 0.5

[0056] Table 6 Results of Propagation Experiment

[0057] serial number Proliferation coefficient other serial number Proliferation coefficient other 3-1 1.2 No vitrification, slow growth 3-6 2.1 No vitrification, low proliferation rate 3-2 3.5 Small amount of vitrification 3-7 3.2 Vitrification is more severe 3-3 3.8 Small amount of vitrification 3-8 2.5 Small amount of vitrification 3-4 4.2 Small amount of vitrification 3-9 3.0 Severe vitrification 3-5 4.7 No vitrification, vigorous growth of clustered buds - - -

[0058] As shown in Tables 5-6, the proliferation coefficient reached a maximum of 3.5-4.7 when using 6-BA 0.5-1.0 mg / L, KT 1.0-1.5 mg / L, ZT 0.5-1.0 mg / L, and 2,4-D 0.5-1.0 mg / L, with no vitrification and vigorous growth of the clustered shoots. When 6-BA 1.0 mg / L, KT 1.5 mg / L, ZT 1.0 mg / L, and 2,4-D 1.0 mg / L, the proliferation coefficient reached a maximum of 4.7, which was the optimal ratio of proliferation hormones for this experiment.

[0059] (iv) Rooting test

[0060] Using 1 / 2 MS as the basal medium, orthogonal experiments were conducted with different concentrations of NAA and IBA, combined with hydrolyzed casein (CH), methyl jasmonate, and coconut milk, to screen the optimal hormone and additive ratios suitable for the rooting culture of rhubarb buds from Tibetan border. The rooting rate, average number of roots, root growth, and plant status of each group were statistically analyzed. The experimental design and results are as follows.

[0061] Table 7 Rooting Experiment Design Table

[0062] serial number NAA (mg / L) IBA (mg / L) CH (mg / L) Methyl jasmonic acid (mg / L) Coconut milk (mg / L) 4-1 0 0 0 0 0 4-2 1.0 0.8 50.0 0.3 60.0 4-3 1.0 1.2 80.0 0.4 80.0 4-4 1.5 1.0 65.0 0.4 70.0 4-5 1.5 1.2 50.0 0.3 80.0 4-6 2.0 0.8 80.0 0.3 70.0 4-7 2.0 1.2 65.0 0.4 60.0

[0063] Table 8 Rooting Test Results

[0064] serial number Rooting rate (%) Average number of roots per plant growth status 4-1 18.2 1.1 The root system is thin and weak, and the number of roots is very small, resulting in slow seedling growth. 4-2 86.5 3.2 The root system is rather thin, and some plants show slight yellowing. 4-3 92.1 4 The root system is robust, the growth is good, and there are no deformed roots. 4-4 95.8 4.6 The root system is well-developed, thick, and uniform; the seedlings are dark green and robust, with no deformed roots or browning. 4-5 91.3 3.9 The root system is growing well, with a small number of roots being relatively short. 4-6 88.7 3.5 The root system is relatively long but lacks toughness; some plants show browning at the base. 4-7 93.2 4.2 The root system is robust, with some roots twisting and turning.

[0065] As shown in Tables 7 and 8, the experimental results indicate that in 1 / 2 MS basal medium, a combination of NAA 1.0–2.0 mg / L, IBA 0.8–1.2 mg / L, supplemented with hydrolyzed casein, methyl jasmonic acid, and coconut milk can effectively induce rooting of rhubarb shoots from Tibetan border, with an overall rooting rate exceeding 86.5%. The optimal ratio is 1 / 2 MS + NAA 1.5 mg / L + IBA 1.0 mg / L + CH 6 5.0 mg / L + methyl jasmonic acid 0.4 mg / L + coconut milk 70.0 mg / L. Under this combination, the rooting rate reaches as high as 95.8%, with an average of 4.6 roots per plant. The roots are robust and uniform, the tissue culture seedlings grow vigorously, and there are no adverse phenomena such as browning or deformed roots, resulting in the best overall rooting effect.

[0066] The experiment also revealed that excessively low concentrations of plant growth regulators could not effectively initiate the rooting process, resulting in low rooting rates and poor root quality. Conversely, excessively high concentrations of regulators could cause root entanglement and browning at the base of the seedlings, which was detrimental to subsequent hardening-off and transplanting. The rooting culture medium formulation determined in this experiment balances rooting efficiency and seedling quality, and can meet the needs of large-scale tissue culture production.

[0067] Example 1

[0068] A rapid propagation method for Tibetan rhubarb through tissue culture, comprising the following steps:

[0069] (1) Selection and disinfection of explants: Take the tender leaves and stems of Tibetan rhubarb, wash them, soak them in 1% potassium permanganate solution for 20 minutes, rinse them with sterile water 6 times, then disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 4 times, disinfect them with 0.1% benzalkonium chloride solution for 5 minutes, rinse them with sterile water 4 times, disinfect them with 0.02% mercuric chloride solution for 5 minutes, rinse them with sterile water 8 times. When disinfecting with 0.1% benzalkonium chloride solution and 0.02% mercuric chloride solution, use 30kHz ultrasound.

[0070] (2) Primary induction culture: The sterilized explants were blotted dry with sterile paper, and the wounds at both ends were cut off. They were then inoculated into the primary induction culture medium (formulation: MS + NAA 0.3 mg / L + 6-BA 1.0 mg / L + sucrose 40.0 g / L + agar 8.0 g / L, pH 6.1) and cultured for 35 days under alternating light and dark conditions at a temperature of 25±2℃, a light intensity of 2500 Lx, and 12 h of sunlight.

[0071] (3) Subculture: Aseptic seedlings were cut into single buds, and leaves were also cut off. The single buds and leaves were inoculated into subculture culture medium (formulation: MS + 6-BA 1.0 mg / L + KT 1.5 mg / L + ZT 0.5 mg / L + 2,4-D 1.0 mg / L + sucrose 40.0 g / L + agar 8 g / L, pH 6.1). The medium was cultured for 40 days under alternating light and dark conditions at a temperature of 25 ± 2℃, a light intensity of 2500 Lx, and 12 h of daylight. The culture results are as follows. Figure 1 As shown.

[0072] (4) Rooting culture: The proliferating shoots were cut into single shoots and inoculated into rooting medium (formulation: 1 / 2 MS + NAA 1.5 mg / L + IBA 1.0 mg / L + CH 65.0 mg / L + methyl jasmonate 0.4 mg / L + coconut milk 70.0 mg / L + activated carbon 0.1 g / L, pH 6.1). The medium was cultured for 45 days under alternating light and dark conditions at a temperature of 25 ± 2℃, a light intensity of 2500 Lx, and 12 h of daylight. The culture results are as follows: Figure 2 As shown.

[0073] (5) Acclimation of rooted seedlings: Transplant the tissue culture rooted seedlings into the rooting substrate (soil: sand: earthworm castings: coconut coir = 2:1:1:1) and acclimate them in a greenhouse with 70% shade. Spray with 35 mg / L salicylic acid (SA) once after transplanting, and then spray with 35 mg / L salicylic acid (SA) once every 6 days thereafter.

[0074] Example 2

[0075] A rapid propagation method for Tibetan rhubarb through tissue culture, comprising the following steps:

[0076] (1) Selection and disinfection of explants: Take the tender leaves and stems of Tibetan rhubarb, wash them, soak them in 1% potassium permanganate solution for 20 minutes, rinse them with sterile water 5 times, then disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 3 times, disinfect them with 0.1% benzalkonium chloride solution for 5 minutes, rinse them with sterile water 4 times, disinfect them with 0.02% mercuric chloride solution for 3 minutes, and rinse them with sterile water 6 times. When disinfecting with 0.1% benzalkonium chloride solution and 0.02% mercuric chloride solution, use 25kHz ultrasound.

[0077] (2) Primary induction culture: The sterilized explants were blotted dry with sterile paper, and the wounds at both ends were cut off. They were then inoculated into the primary induction culture medium (MS + NAA 0.5mg / L + 6-BA 1.0mg / L + sucrose 30.0g / L + agar 7.0g / L, pH 6.0) and cultured for 35 days under alternating light and dark conditions at a temperature of 25±2℃, a light intensity of 3000Lx, and 12h of sunlight.

[0078] (3) Subculture: Aseptic seedlings were cut into single buds, and leaves were also cut off. The single buds and leaves were inoculated into subculture culture medium (formulation: MS + 6-BA 1.0 mg / L + KT 1.0 mg / L + ZT 1.0 mg / L + 2,4-D 0.5 mg / L + sucrose 30.0 g / L + agar 7.0 g / L, pH 6.0) and cultured for 45 days under alternating light and dark conditions at a temperature of 25±2℃, a light intensity of 3000 Lx, and 12 h of daylight. The culture results are as follows. Figure 3 As shown.

[0079] (4) Rooting culture: The proliferating shoots were cut into single shoots and inoculated into rooting medium (formulation: 1 / 2 MS + NAA 1.0 mg / L + IBA 0.8 mg / L + CH 50.0 mg / L + methyl jasmonate 0.3 mg / L + coconut milk 60.0 mg / L + activated carbon 1.0 g / L, pH 6.0). The medium was cultured for 50 days under alternating light and dark conditions at a temperature of 25 ± 2℃, a light intensity of 2500 Lx, and 12 h of daylight. The culture results are as follows: Figure 4 As shown.

[0080] (5) Acclimation of rooted seedlings: Transplant the tissue culture rooted seedlings into the rooting substrate (soil: sand: earthworm castings: coconut coir = 2:1:1:1) and acclimate them in a greenhouse with a 65% shading rate. After transplanting, spray with 30 mg / L salicylic acid (SA) once, and then spray with 30 mg / L salicylic acid (SA) once every 5 days.

[0081] Example 3

[0082] A rapid propagation method for Tibetan rhubarb through tissue culture, comprising the following steps:

[0083] (1) Selection and disinfection of explants: Take the tender leaves and stems of Tibetan rhubarb, wash them, soak them in 1% potassium permanganate solution for 20 minutes, rinse them with sterile water 5 times, then disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 3 times, disinfect them with 0.1% benzalkonium chloride solution for 3 minutes, rinse them with sterile water 3 times, disinfect them with 0.02% mercuric chloride solution for 5 minutes, and rinse them with sterile water 6 times. When disinfecting with 0.1% benzalkonium chloride solution and 0.02% mercuric chloride solution, use 25kHz ultrasound.

[0084] (2) Primary induction culture: The sterilized explants were blotted dry with sterile paper, and the wounds at both ends were cut off. They were then inoculated into the primary induction culture medium (formulation: MS + NAA 0.3 mg / L + 6-BA 1.5 mg / L + sucrose 50.0 g / L + agar 9.0 g / L, pH 6.2) and cultured for 30 days under alternating light and dark conditions at a temperature of 25±2℃, a light intensity of 2000 Lx, and 12 h of sunlight.

[0085] (3) Subculture: The sterile seedlings were cut into single buds and the leaves were cut off at the same time. The single buds and leaves were inoculated into the subculture culture medium (formulation: MS + 6-BA 0.5mg / L + KT 1.5mg / L + ZT 1.0mg / L + 2,4-D 0.5mg / L + sucrose 50.0g / L + agar 9.0g / L, pH 6.2) and cultured for 35 days under alternating light and dark conditions at a temperature of 25±2℃, a light intensity of 2000Lx, and 12h of sunlight.

[0086] (4) Rooting culture: The proliferating buds were cut into single buds and inoculated into rooting medium (formulation: 1 / 2MS + NAA 2.0mg / L + IBA 1.2mg / L + CH 80.0mg / L + methyl jasmonate 0.4mg / L + coconut milk 80.0mg / L + activated carbon 0.5g / L, pH 6.2). The medium was cultured for 40 days under alternating light and dark conditions with a temperature of 25±2℃, a light intensity of 2000Lx, and 12h of sunlight.

[0087] (5) Acclimation of rooted seedlings: Transplant the tissue culture rooted seedlings into the rooting substrate (soil: sand: earthworm castings: coconut coir = 2:1:1:1) and acclimate them in a greenhouse with 75% shade. Spray with 40 mg / L salicylic acid (SA) once after transplanting, and then spray with 40 mg / L salicylic acid (SA) once every 7 days.

[0088] The disinfection contamination rate, disinfection mortality rate, primary induction rate, proliferation coefficient, rooting rate, and acclimatization survival rate of Examples 1-3 were statistically analyzed, and the results are shown in Table 9 below.

[0089] Table 9 Statistical Results

[0090] Disinfection contamination rate (%) Disinfection mortality rate (%) Primary induction rate (%) Proliferation coefficient Rooting rate (%) Domestication survival rate (%) Example 1 16.0 9.0 75.6 4.7 95.8 95.3 Example 2 19.0 7.8 70.1 4.2 86.5 94.3 Example 3 18.0 8.2 68.2 3.8 94.5 95.1

[0091] Example 4

[0092] A method for in vitro preservation of rhubarb from Tibetan border regions, comprising the following steps:

[0093] (1) Pretreatment culture: The explant material cleaned and disinfected according to step (1) of Example 1 was inoculated into the pretreatment culture medium (formulation: 1 / 2MS + sucrose 50.0g / L + mannitol 5.0g / L, pH 6.0) and cultured for 15 days under light-dark alternation at 10±2℃, light intensity 1000Lx, and daily light duration 10h.

[0094] (2) Germplasm preservation culture: The pretreated explant material was inoculated into germplasm preservation medium (formulation: 1 / 2MS + mannitol 30.0g / L + ABA 5.0mg / L + CCC 1.2mg / L + agar 7.0g / L, pH 6.0) and cultured for 15 months under light-dark alternation at 10±2℃, light intensity 1000Lx, and daily light duration of 6h.

[0095] (3) Rejuvenation culture: After germplasm preservation culture, aseptic buds were inoculated into rejuvenation medium (formulation: MS + 6-BA 2.0 mg / L + KT 3.0 mg / L + ZT 1.0 mg / L + 2,4-D 2.0 mg / L + sucrose 30.0 g / L, pH 6.0) and cultured for 30 days under light-dark alternation at 25±2℃, light intensity 3000 Lx, and daily light duration 12 h.

[0096] After rejuvenation, the subculture, rooting culture and acclimatization were carried out in steps (3) to (5) of Example 1. After one generation of subculture, the proliferation coefficient was only 4.3 and the rooting rate was 93.4%.

[0097] Example 5

[0098] A method for in vitro preservation of rhubarb from Tibetan border regions, comprising the following steps:

[0099] (1) Pretreatment culture: The explant material cleaned and disinfected according to step (1) of Example 1 was inoculated into the pretreatment culture medium (formulation: 1 / 2MS + sucrose 60.0g / L + mannitol 1.0g / L, pH 6.0) and cultured for 17 days under light-dark alternation at 10±2℃, light intensity 1500Lx, and daily light duration 10h.

[0100] (2) Germplasm preservation culture: The pretreated explant material was inoculated into germplasm preservation medium (formulation: 1 / 2MS + mannitol 50.0g / L + ABA 2.0mg / L + CCC 1.6 mg / L + agar 8.0g / L, pH 6.0) and cultured for 18 months under light-dark alternation at 10±2℃, light intensity 1500Lx, and daily light duration of 8h.

[0101] (3) Rejuvenation culture: After germplasm preservation culture, aseptic buds were inoculated into rejuvenation medium (formulation: MS + 6-BA 3.0 mg / L + KT 2.0 mg / L + ZT 2.0 mg / L + 2,4-D 1.0 mg / L + sucrose 50.0 g / L, pH 6.0) and cultured for 40 days under light-dark alternation at 25±2℃, light intensity 3000 Lx, and daily light duration 12 h.

[0102] After rejuvenation, the subculture, rooting culture and acclimatization were carried out in steps (3) to (5) of Example 1. After one generation of subculture, the proliferation coefficient could be stabilized at more than 4.1, and the rooting rate was 92.6% when rooting.

[0103] Example 6

[0104] A method for in vitro preservation of rhubarb from Tibetan border regions, comprising the following steps:

[0105] (1) Pretreatment culture: The explant material cleaned and disinfected according to step (1) of Example 1 was inoculated into the pretreatment culture medium (formulation: 1 / 2MS + sucrose 50.0g / L + mannitol 3.0g / L, pH 6.0) and cultured for 20 days under light-dark alternation at 10±2℃, light intensity 2000Lx, and daily light duration 10h.

[0106] (2) Germplasm preservation culture: The pretreated explant material was inoculated into germplasm preservation medium (formulation: 1 / 2MS + mannitol 40.0g / L + ABA 3.0mg / L + CCC 1.5mg / L + agar 7.0g / L, pH 6.0) and cultured for 22 months under light-dark alternation at 10±2℃, light intensity 2000Lx, and daily light duration of 7h.

[0107] (3) Rejuvenation culture: After germplasm preservation culture, aseptic buds were inoculated into rejuvenation medium (formulation: MS + 6-BA 2.5mg / L + KT 2.5mg / L + ZT 1.5mg / L + 2,4-D 1.5mg / L + sucrose 40.0g / L, pH 6.0) and cultured in alternating light and dark conditions for 35 days at 25±2℃, light intensity 2500Lx, and daily light duration 12h.

[0108] After rejuvenation, the subculture, rooting culture and acclimatization were carried out in steps (3) to (5) of Example 1. After one generation of subculture, the proliferation coefficient could be stabilized at more than 4.0, and the rooting rate was 92.8% when rooting.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A rapid propagation method for Tibetan rhubarb through tissue culture, characterized by the following steps: include: (1) Selection and disinfection of explants: Take the tender leaves and stems of Tibetan rhubarb, wash them, soak them in 1% potassium permanganate solution for 20 minutes, rinse them with sterile water 5-6 times, then disinfect them with 75% alcohol for 30 seconds, rinse them with sterile water 3-4 times, disinfect them with 0.1% benzalkonium chloride solution for 3-5 minutes, rinse them with sterile water 3-4 times, disinfect them with 0.02% mercuric chloride solution for 3-5 minutes, rinse them with sterile water 7-8 times. When disinfecting with 0.1% benzalkonium chloride solution and 0.02% mercuric chloride solution, use 25-30 kHz ultrasound. (2) Primary induction culture: The surface moisture of the sterilized explants was absorbed with sterile paper, and the wounds at both ends were cut off. The explants were then inoculated into the primary induction culture medium and cultured for 30 to 35 days under alternating light and dark conditions with a temperature of 25±2℃, a light intensity of 2000~3000Lx, and 12h of sunlight. The formulation of the primary culture medium is as follows: MS + NAA 0.3-0.5 mg / L + 6-BA 1.0-2.0 mg / L + sucrose 30.0-50.0 g / L + agar 7.0-9.0 g / L, pH 6.0-6.2; (3) Subculture: The sterile seedlings were cut into single buds and the leaves were cut off at the same time. The single buds and leaves were inoculated into the subculture culture medium and cultured for 35 to 45 days under alternating light and dark conditions with a temperature of 25±2℃, a light intensity of 2000~3000Lx and 12h of sunlight. The formulation of the subculture proliferation medium is as follows: MS + 6-BA 0.5-1.0 mg / L + KT 1.0-1.5 mg / L + ZT 0.5-1.0 mg / L + 2,4-D 0.5-1.0 mg / L + sucrose 30.0-50.0 g / L + agar 7.0-9.0 g / L, pH 6.0-6.2; (4) Rooting culture: Cut the proliferating buds into single buds, inoculate them into rooting culture medium, and culture them for 40 to 50 days in an alternating light and dark environment with a temperature of 25±2℃, a light intensity of 2000~3000Lx, and 12h of sunlight. The rooting medium formula is as follows: 1 / 2 MS + NAA 1.0-2.0 mg / L + IBA 0.8-1.2 mg / L + CH 50.0-80.0 mg / L + methyl jasmonate 0.3-0.4 mg / L + coconut milk 60.0-80.0 mg / L + activated charcoal 0.1-1.0 g / L, pH 6.0-6.2; (5) Acclimation of rooted seedlings: Transplant the tissue culture rooted seedlings into the rooting substrate. The acclimation environment is a greenhouse with a shading rate of 65-75%. After transplanting, spray with 30-40 mg / L salicylic acid (SA) once, and then spray with 30-40 mg / L salicylic acid (SA) once every 5-7 days.

2. The method for rapid propagation of Tibetan rhubarb through tissue culture according to claim 1, characterized in that, In step (2), the formulation of the primary culture medium is: MS + NAA 0.3 mg / L + 6-BA 1.0 mg / L + sucrose 40.0 g / L + agar 8.0 g / L, pH 6.

0.

3. The method for rapid propagation of Tibetan rhubarb through tissue culture according to claim 1, characterized in that, In step (3), the formulation of the subculture proliferation medium is: MS + 6-BA 1.0 mg / L + KT 1.5 mg / L + ZT 1.0 mg / L + 2,4-D 1.0 mg / L + sucrose 40.0 g / L + agar 8 g / L, pH 6.

0.

4. The method for rapid propagation of Tibetan rhubarb through tissue culture according to claim 1, characterized in that, In step (4), the rooting medium formula is: 1 / 2MS + NAA 1.5mg / L + IBA 1.0mg / L + CH 65.0mg / L + methyl jasmonate 0.4mg / L + coconut milk 70.0mg / L + activated carbon 0.1g / L, pH 6.

0.

5. The application of the tissue culture rapid propagation method according to any one of claims 1-4 in the rapid propagation of rhubarb in Tibetan border areas.

6. A method for in vitro preservation of Tibetan rhubarb, characterized by the following steps: include: (1) Pretreatment culture: The cleaned and disinfected explant material was inoculated into the pretreatment culture medium and cultured under light and dark alternation for 15 to 20 days at 10±2℃, light intensity of 1000~2000Lx, and light duration of 10h per day. The pretreatment culture medium formulation is: 1 / 2 MS + sucrose 50.0-60.0 g / L + mannitol 1.0-5.0 g / L; (2) Germplasm preservation culture: The pretreated explant material was inoculated into the germplasm preservation culture medium and cultured under alternating light and dark conditions at 10±2℃, light intensity of 1000~2000Lx, and light duration of 6~8h per day. The formula for the germplasm preservation medium is: 1 / 2 MS + mannitol 30.0-50.0 g / L + ABA 2.0-5.0 mg / L + CCC 1.2-1.6 mg / L + agar 7.0-8.0 g / L; (3) Rejuvenation culture: After germplasm preservation culture, aseptic buds are inoculated into rejuvenation culture medium and cultured in alternating light and dark conditions for 30 to 40 days at 25±2℃, light intensity of 2000~3000Lx, and daily light duration of 12h. The formulation for the rejuvenation culture is: MS + 6-BA 2.0~3.0 mg / L + KT 2.0~3.0 mg / L + ZT 1.0~2.0 mg / L + 2,4-D 1.0~2.0 mg / L + sucrose 30.0~50.0 g / L.

7. The method for in vitro preservation of Tibetan rhubarb according to claim 6, characterized in that, The pretreatment culture medium formula is: 1 / 2 MS + sucrose 50.0 g / L + mannitol 3.0 g / L.

8. The method for in vitro preservation of Tibetan rhubarb according to claim 6, characterized in that, The formula of the germplasm preservation medium is: 1 / 2 MS + mannitol 40.0 g / L + ABA 2.5 mg / L + CCC 1.5 mg / L + agar 7.0 g / L.

9. The method for in vitro preservation of Tibetan rhubarb according to claim 6, characterized in that, The formulation for the rejuvenation culture was: MS + 6-BA 2.5 mg / L + KT 2.5 mg / L + ZT 1.5 mg / L + 2,4-D 1.5 mg / L + sucrose 40.0 g / L.

10. The application of the in vitro preservation method according to any one of claims 6-9 in the preservation of rhubarb germplasm resources in Tibetan border areas.

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