Lily tissue rapid propagation method

Abstract

The invention discloses a lily tissue rapid propagation method which is characterized by comprising the following steps: (1) disinfecting middle scales of lilies, and performing adventitious bud induction culture in a first culture medium; (2) performing multiplication culture in a second culture medium; (3) performing rooting culture in a third culture medium; (4) then carrying out seedling hardening, and then transplanting into a fourth culture medium; wherein the variety of the lilium brownii is hairy lilium brownii. By adopting the rapid propagation method for the lily tissue, the adventitious bud induction rate, the adventitious bud differentiation coefficient, the multiplication coefficient, the rooting rate and the transplanting survival rate of the Lilium lanceolata can be greatly improved, and the browning rate in the tissue culture process can be obviously reduced.

Description

Technical Field

[0003] This invention relates to the field of rapid propagation technology of plant tissues, specifically to a method for rapid propagation of lily tissues. Background Technology

[0005] Lilium dauricum, a perennial bulbous lily belonging to the genus Lilium in the family Liliaceae, is mainly distributed in Northeast China. It exhibits strong drought and cold resistance, making it a valuable candidate for cold-hardy cultivation within the Lilium genus. However, existing rapid tissue propagation methods for Lilium dauricum have resulted in relatively low adventitious bud induction rates, proliferation coefficients, rooting rates, and transplant survival rates, along with a relatively high browning rate. Therefore, a rapid tissue propagation method for Lilium dauricum is urgently needed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for rapid propagation of lily tissues to solve the problems in the prior art.

[0008] In a first aspect, the present invention provides a method for rapid propagation of lily tissues, comprising the following steps:

[0009] (1) Disinfect the middle scales of the lily and induce adventitious buds in the first culture medium;

[0010] (2) Then, proliferation culture was carried out in the second culture medium;

[0011] (3) Then carry out rooting culture in the third culture medium;

[0012] (4) Then harden off the seedlings and then transplant them into the fourth culture medium;

[0013] The variety of lily mentioned is Lilium pubescens;

[0014] The first culture medium comprises MS medium, 0.5-1 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 80-100 mg / L carboxymethyl chitosan and 200-400 mg / L sodium alginate; the pH of the first culture medium is 5.6-6.2.

[0015] The second culture medium contains MS medium, 1-1.5 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 80-100 mg / L carboxymethyl chitosan, 0.2-0.5 mg / L zeatin, and 100-200 mg / L hydrolyzed casein; the pH of the second culture medium is 5.6-6.2.

[0016] The third culture medium comprises MS medium, 1-1.5 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 100-200 mg / L carboxymethyl chitosan, 50-80 mg / L sodium alginate and 30-50 mg / L humic acid; the pH of the third culture medium is 5.6-6.2.

[0017] The fourth culture medium contains vermiculite, humus, peat moss, licorice extract and wolfberry extract in a mass ratio of (1-2):(1-2):(0.5-1):(0.1-0.3):(0.05-0.1).

[0018] The disinfection conditions include: first soaking in 70 wt%-75 wt% ethanol for 20-30 seconds, then soaking in 1-3 wt% sodium hypochlorite solution for 10-12 minutes, and finally soaking in an aqueous solution containing 80-100 mg / L carboxymethyl chitosan and 200-400 mg / L sodium alginate for 5-8 minutes, and then rinsing with sterile water at least 3 times;

[0019] The conditions for adventitious bud induction culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 12-16 hours / day, relative humidity 20%-25%, and culture time 35-45 days.

[0020] The conditions for proliferation culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 12-16 hours / day, relative humidity 20%-25%, and culture time 35-45 days;

[0021] The conditions for rooting culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 8-12 hours / day, relative humidity 20%-25%, and culture time 35-45 days;

[0022] The conditions for hardening off the seedlings include: maintaining a temperature of 21-25℃ under natural light, with a relative humidity of 30%-40% on the first 1-2 days, and then a relative humidity of 20%-25% on the 3rd-5th days;

[0023] The post-transplantation culture conditions include: a temperature of 21-25℃, a relative humidity of 20%-25%, and a relative humidity of 30%-40% for the fourth culture medium.

[0024] Preferably, the carboxymethyl chitosan has a degree of deacetylation ≥90% and a molecular weight <50 kDa.

[0025] Preferably, the steps for preparing the licorice extract include:

[0026] i. Slice the dried licorice root, add water and decoct 2-3 times, 1-2 hours each time, with a material-to-liquid ratio of 1:6-1:10;

[0027] ii. Then combine the filtrates and concentrate them to 20%-25% of their original volume;

[0028] iii. Add 90 wt%-95 wt% ethanol to adjust the ethanol content to 60 wt%-70 wt%, and let stand for 24 hours;

[0029] iv. Subsequently, the supernatant was collected by centrifugation, the ethanol was recovered, and the product was spray-dried to obtain licorice extract powder.

[0030] Preferably, the steps for preparing the wolfberry extract include:

[0031] (a) Take wolfberry stems and leaves, wash them, and add 15 wt%-20 wt% ethanol aqueous solution. The material-to-liquid ratio is 1:8-1:10.

[0032] (b) Then, ultrasonic extraction was performed at 25-35℃ for 1.5-2 hours, with an ultrasonic frequency of 50-60 kHz and an ultrasonic power of 40-50 W. The filtrate and residue were then obtained by filtration.

[0033] (c) The filtrate is concentrated under reduced pressure at 0.002-0.05 MPa and 22-25°C until the ethanol content reaches 50wt%-60wt%, to obtain the first concentrate;

[0034] (d) The filter residue is extracted twice. The extraction conditions for each extraction include: adding 9-11 times the mass of 15 wt%-20 wt% ethanol aqueous solution, ultrasonic extraction at 25-35℃ for 1.5-2 hours, ultrasonic frequency of 50-60 kHz, ultrasonic power of 40-50 W, to obtain a second concentrate.

[0035] (e) Combine the first concentrate and the second concentrate, and spray dry to obtain wolfberry extract powder.

[0036] Using appropriate temperature and power in the above ultrasonic conditions not only helps to break down plant cell walls and promote the release of cell contents, but also prevents the destruction of components such as wolfberry polysaccharides in wolfberry extract.

[0037] The culture medium used after transplanting in this invention contains licorice extract and wolfberry extract, which can enhance the adaptability of hairy lily to the transplanting environment and improve the survival rate after transplanting.

[0038] In this invention, employing appropriate disinfection conditions (especially soaking in an aqueous solution containing appropriate amounts of carboxymethyl chitosan and sodium alginate for a certain period of time) can significantly reduce the browning rate during adventitious bud induction. Preferably, the disinfection conditions include: first soaking in 75 wt% ethanol for 30 seconds, then soaking in 2 wt% sodium hypochlorite solution for 10 minutes, and finally soaking in an aqueous solution containing 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate for 6 minutes, followed by rinsing 5 times with sterile water. Using the above disinfection method, adventitious bud induction culture is performed within 15 minutes after rinsing, thus reducing the likelihood of browning.

[0039] In this invention, the use of suitable adventitious bud induction medium (especially with appropriate amounts of carboxymethyl chitosan and sodium alginate) and culture conditions can significantly improve the adventitious bud induction rate and differentiation coefficient, and reduce the browning rate. Preferably, the conditions for adventitious bud induction culture include: temperature 23°C, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days; and the first culture medium contains MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 80 mg / L carboxymethyl chitosan and 200 mg / L sodium alginate; the pH of the first culture medium is 5.8.

[0040] In this invention, the use of suitable proliferation medium and culture conditions can significantly improve the proliferation coefficient and reduce the browning rate, especially with the addition of appropriate amounts of carboxymethyl chitosan, zeatin, and hydrolyzed casein to the second culture medium. Preferably, the proliferation culture conditions include: temperature 23°C, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days; and the second culture medium contains MS medium, 1 mg / L 6-BA, 0.3 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 80 mg / L carboxymethyl chitosan, 0.2 mg / L zeatin, and 100 mg / L hydrolyzed casein; the pH of the second culture medium is 5.8.

[0041] In this invention, the use of suitable rooting culture medium and culture conditions (especially the addition of appropriate amounts of carboxymethyl chitosan, sodium alginate, and humic acid to the third culture medium) can significantly improve the rooting rate and reduce the browning rate. Preferably, the rooting culture conditions include: a temperature of 23°C, a light intensity of 1500-2000 LUX, a light duration of 8 hours / day, a relative humidity of 20%, and a culture time of 35 days; and the third culture medium contains MS medium, 1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 200 mg / L carboxymethyl chitosan, 50 mg / L sodium alginate, and 50 mg / L humic acid; the pH of the third culture medium is 5.8.

[0042] In this invention, the pH is adjusted using 10 wt%-15 wt% KOH solution and 5 wt%~10 wt% dilute acetic acid.

[0043] In this invention, the use of appropriate hardening and post-transplanting culture conditions (especially the addition of appropriate amounts of licorice extract and wolfberry extract to the fourth culture medium) can significantly improve the survival rate after transplanting.

[0044] Preferably, the conditions for hardening off the seedlings include: maintaining a temperature of 23°C under natural light, a relative humidity of 30% for the first 1-2 days, and a relative humidity of 20% for the third-5 days.

[0045] Preferably, the post-transplantation culture conditions include: a temperature of 23°C, a relative humidity of 20%, and a relative humidity of 40% for the fourth culture medium; and the fourth culture medium contains vermiculite, humus, peat moss, licorice extract, and wolfberry extract in a mass ratio of 2:2:1:0.2:0.1.

[0046] Secondly, the present invention provides the application of the above-described method in the rapid propagation of lily tissues.

[0047] Compared with the prior art, the beneficial effects of the present invention include:

[0048] The rapid propagation method of lily tissues of the present invention can not only greatly improve the adventitious bud induction rate, adventitious bud differentiation coefficient, proliferation coefficient, rooting rate and transplant survival rate of Lilium pubescens, but also significantly reduce the browning rate during tissue culture. Detailed Implementation

[0050] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following specific embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described below are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0051] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the ranges, the endpoint values ​​of the ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. As used herein, when referring to a specific numerical value, it means that the value can vary within ±5%.

[0052] In the description of this application, unless otherwise stated, the terms "multiple / a variety" and similar terms mean two / more than one. As used herein, the terms "containing," "including," or "comprising" can be open-ended, semi-closed, or closed-ended.

[0053] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, they are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commonly available materials and reagents that can be purchased commercially.

[0054] In the following examples, the MS culture medium contains 1650 mg / L ammonium nitrate, 6.2 mg / L boric acid, 350 mg / L anhydrous calcium chloride, 0.025 mg / L cobalt dichloride hexahydrate, 0.025 mg / L copper sulfate pentahydrate, 37.3 mg / L Na2EDTA, 27.8 mg / L ferrous sulfate heptahydrate, 350 mg / L anhydrous magnesium sulfate, 16.9 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 0.83 mg / L potassium iodide, 1900 mg / L potassium nitrate, 170 mg / L potassium dihydrogen phosphate, 8.6 mg / L zinc sulfate heptahydrate, 2 mg / L glycine, 100 mg / L inositol, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine hydrochloride, and 0.1 mg / L thiamine hydrochloride.

[0055] In the following embodiments, the calculation method for the relevant parameters includes:

[0056] Adventitious bud induction rate (%) = (Number of scales forming adventitious buds / Total number of explants) × 100%;

[0057] Adventitious bud differentiation coefficient = Total number of adventitious buds / Number of scales forming adventitious buds;

[0058] Proliferation coefficient = Total number of buds after proliferation / Number of inoculated buds;

[0059] Rooting rate = (Number of rooted plants / Total number of inoculated plants) × 100%;

[0060] Transplant survival rate = (Number of surviving plants after transplanting / Total number of transplanted plants) × 100%;

[0061] Browning rate = (Number of browned explants / Total number of inoculated explants) × 100%. The results are shown in the table below.

[0062] Example 1

[0063] A method for rapid propagation of lily tissues includes the following steps:

[0064] (1) Disinfect the middle scales of the lily and induce adventitious buds in the first culture medium;

[0065] (2) Then, proliferation culture was carried out in the second culture medium;

[0066] (3) Then carry out rooting culture in the third culture medium;

[0067] (4) Then harden off the seedlings and then transplant them into the fourth culture medium;

[0068] The variety of lily mentioned is Lilium pubescens;

[0069] The first culture medium contains MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 80 mg / L carboxymethyl chitosan and 200 mg / L sodium alginate; the pH of the first culture medium is 5.8.

[0070] The second culture medium contains MS medium, 1 mg / L 6-BA, 0.3 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 80 mg / L carboxymethyl chitosan, 0.2 mg / L zeatin and 100 mg / L hydrolyzed casein; the pH of the second culture medium is 5.8.

[0071] The third culture medium contains MS medium, 1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 200 mg / L carboxymethyl chitosan, 50 mg / L sodium alginate and 50 mg / L humic acid; the pH of the third culture medium is 5.8.

[0072] The fourth culture medium contains vermiculite, humus, peat moss, licorice extract, and wolfberry extract in a mass ratio of 2:2:1:0.2:0.1.

[0073] The disinfection conditions include: first soaking in 75 wt% ethanol for 30 seconds, then soaking in 2 wt% sodium hypochlorite solution for 10 minutes, and finally soaking in an aqueous solution containing 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate for 6 minutes, and then rinsing 5 times with sterile water.

[0074] The conditions for the adventitious bud induction culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0075] The conditions for the proliferation culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0076] The conditions for rooting culture include: temperature 23℃, light intensity 1500-2000 LUX, light duration 8 hours / day, relative humidity 20%, and culture time 35 days.

[0077] The conditions for hardening off the seedlings include: maintaining a temperature of 23°C under natural light, a relative humidity of 30% for the first 1-2 days, and a relative humidity of 20% for the third-to-fifth days;

[0078] The post-transplantation culture conditions include: a temperature of 23°C, a relative humidity of 20%, and a relative humidity of 40% for the fourth culture medium.

[0079] The steps for preparing the licorice extract in this embodiment include:

[0080] i. Slice the dried licorice root, add water and decoct twice, one hour each time, with a material-to-liquid ratio of 1:10;

[0081] ii. Then combine the filtrates and concentrate to 20% of the original volume;

[0082] iii. Add 90 wt% ethanol to adjust the ethanol content to 60 wt%, and let stand for 24 hours;

[0083] iv. Subsequently, the supernatant was collected by centrifugation, the ethanol was recovered, and the product was spray-dried to obtain licorice extract powder.

[0084] The steps for preparing the wolfberry extract in this embodiment include:

[0085] (a) Take wolfberry stems and leaves, wash them, and add 20 wt% ethanol aqueous solution. The material-to-liquid ratio is 1:8.

[0086] (b) Then, the mixture was ultrasonically extracted at 25°C for 1.5 hours at an ultrasonic frequency of 50 kHz and an ultrasonic power of 40 W. The filtrate and residue were then obtained by filtration.

[0087] (c) The filtrate was concentrated under reduced pressure at 0.003 MPa and 22°C until the ethanol content reached 50 wt%, to obtain the first concentrate;

[0088] (d) The filter residue was extracted twice. The extraction conditions for each extraction included: adding 9 times the mass of 20 wt% ethanol aqueous solution, ultrasonic extraction at 30°C for 2 hours, ultrasonic frequency of 60 kHz, ultrasonic power of 50 W, to obtain a second concentrate.

[0089] (e) Combine the first concentrate and the second concentrate, and spray dry to obtain wolfberry extract powder.

[0090] Example 2

[0091] A method for rapid propagation of lily tissues includes the following steps:

[0092] (1) Disinfect the middle scales of the lily and induce adventitious buds in the first culture medium;

[0093] (2) Then, proliferation culture was carried out in the second culture medium;

[0094] (3) Then carry out rooting culture in the third culture medium;

[0095] (4) Then harden off the seedlings and then transplant them into the fourth culture medium;

[0096] The variety of lily mentioned is Lilium pubescens;

[0097] The first culture medium contains MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate; the pH of the first culture medium is 5.8.

[0098] The second culture medium contains MS medium, 1 mg / L 6-BA, 0.3 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 100 mg / L carboxymethyl chitosan, 0.5 mg / L zeatin and 200 mg / L hydrolyzed casein; the pH of the second culture medium is 5.8.

[0099] The third culture medium contains MS medium, 1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 100 mg / L carboxymethyl chitosan, 80 mg / L sodium alginate and 30 mg / L humic acid; the pH of the third culture medium is 5.8.

[0100] The fourth culture medium contains vermiculite, humus, peat moss, licorice extract, and wolfberry extract in a mass ratio of 2:2:1:0.2:0.1.

[0101] The disinfection conditions include: first soaking in 75 wt% ethanol for 30 seconds, then soaking in 2 wt% sodium hypochlorite solution for 10 minutes, and finally soaking in an aqueous solution containing 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate for 6 minutes, and then rinsing 5 times with sterile water.

[0102] The conditions for the adventitious bud induction culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0103] The conditions for the proliferation culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0104] The conditions for rooting culture include: temperature 23℃, light intensity 1500-2000 LUX, light duration 8 hours / day, relative humidity 20%, and culture time 35 days.

[0105] The conditions for hardening off the seedlings include: maintaining a temperature of 23°C under natural light, a relative humidity of 30% for the first 1-2 days, and a relative humidity of 20% for the third-to-fifth days;

[0106] The post-transplantation culture conditions include: a temperature of 23°C, a relative humidity of 20%, and a relative humidity of 40% for the fourth culture medium.

[0107] The steps for preparing the licorice extract and wolfberry extract in this embodiment are the same as in Example 1.

[0108] Example 3

[0109] A method for rapid propagation of lily tissues includes the following steps:

[0110] (1) Disinfect the middle scales of the lily and induce adventitious buds in the first culture medium;

[0111] (2) Then, proliferation culture was carried out in the second culture medium;

[0112] (3) Then carry out rooting culture in the third culture medium;

[0113] (4) Then harden off the seedlings and then transplant them into the fourth culture medium;

[0114] The variety of lily mentioned is Lilium pubescens;

[0115] The first culture medium contains MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 90 mg / L carboxymethyl chitosan and 300 mg / L sodium alginate; the pH of the first culture medium is 5.8.

[0116] The second culture medium contains MS medium, 1 mg / L 6-BA, 0.3 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 90 mg / L carboxymethyl chitosan, 0.3 mg / L zeatin and 160 mg / L hydrolyzed casein; the pH of the second culture medium is 5.8.

[0117] The third culture medium contains MS medium, 1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 120 mg / L carboxymethyl chitosan, 60 mg / L sodium alginate and 40 mg / L humic acid; the pH of the third culture medium is 5.8.

[0118] The fourth culture medium contains vermiculite, humus, peat moss, licorice extract, and wolfberry extract in a mass ratio of 2:2:1:0.2:0.1.

[0119] The disinfection conditions include: first soaking in 75 wt% ethanol for 30 seconds, then soaking in 2 wt% sodium hypochlorite solution for 10 minutes, and finally soaking in an aqueous solution containing 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate for 6 minutes, followed by rinsing 5 times with sterile water.

[0120] The conditions for the adventitious bud induction culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0121] The conditions for the proliferation culture include: temperature 23℃, light intensity 1500 LUX, light duration 12 hours / day, relative humidity 20%, and culture time 35 days.

[0122] The conditions for rooting culture include: temperature 23℃, light intensity 1500-2000 LUX, light duration 8 hours / day, relative humidity 20%, and culture time 35 days.

[0123] The conditions for hardening off the seedlings include: maintaining a temperature of 23°C under natural light, a relative humidity of 30% for the first 1-2 days, and a relative humidity of 20% for the third-to-fifth days;

[0124] The post-transplantation culture conditions include: a temperature of 23°C, a relative humidity of 20%, and a relative humidity of 40% for the fourth culture medium.

[0125] The steps for preparing the licorice extract and wolfberry extract in this embodiment are the same as in Example 1.

[0126] Comparative Example 1

[0127] The first culture medium does not contain carboxymethyl chitosan and sodium alginate, and other conditions are the same as in Example 1.

[0128] Comparative Example 2

[0129] The first culture medium contains 80 mg / L carboxymethyl chitosan but does not contain sodium alginate, and other conditions are the same as in Example 1.

[0130] Comparative Example 3

[0131] The first culture medium does not contain carboxymethyl chitosan, but contains 200 mg / L sodium alginate, and other conditions are the same as in Example 1.

[0132] Comparative Example 4

[0133] The second culture medium does not contain carboxymethyl chitosan, zein, or hydrolyzed casein, and other conditions are the same as in Example 1.

[0134] Comparative Example 5

[0135] The second culture medium does not contain carboxymethyl chitosan, but contains 0.2 mg / L zeatin and 100 mg / L hydrolyzed casein, with other conditions the same as in Example 1.

[0136] Comparative Example 6

[0137] The second culture medium does not contain zeatin, but contains 80 mg / L carboxymethyl chitosan and 100 mg / L hydrolyzed casein, with other conditions the same as in Example 1.

[0138] Comparative Example 7

[0139] The second culture medium does not contain hydrolyzed casein, but contains 80 mg / L carboxymethyl chitosan and 0.2 mg / L zeatin, with other conditions the same as in Example 1.

[0140] Comparative Example 8

[0141] The third culture medium does not contain carboxymethyl chitosan, sodium alginate, or humic acid, and other conditions are the same as in Example 1.

[0142] Comparative Example 9

[0143] The third culture medium does not contain carboxymethyl chitosan, but contains 50 mg / L sodium alginate and 50 mg / L humic acid, with other conditions the same as in Example 1.

[0144] Comparative Example 10

[0145] The third culture medium does not contain sodium alginate, but contains 200 mg / L carboxymethyl chitosan and 50 mg / L humic acid, with other conditions the same as in Example 1.

[0146] Comparative Example 11

[0147] The third culture medium does not contain humic acid, but contains 200 mg / L carboxymethyl chitosan and 50 mg / L sodium alginate, with other conditions the same as in Example 1.

[0148] Comparative Example 12

[0149] The fourth culture medium does not contain licorice extract or wolfberry extract, and other conditions are the same as in Example 1.

[0150] Comparative Example 13

[0151] The fourth culture medium does not contain licorice extract, but contains wolfberry extract, and other conditions are the same as in Example 1.

[0152] Comparative Example 14

[0153] The fourth culture medium does not contain wolfberry extract, but contains licorice extract, and other conditions are the same as in Example 1.

[0154]

[0155]

[0156]

[0157]

[0158] As can be seen from Tables 1 to 4 above, the rapid propagation method of lily tissue using the embodiments of the present invention can not only greatly improve the adventitious bud induction rate, adventitious bud differentiation coefficient, proliferation coefficient, rooting rate and transplant survival rate of Lilium hairyense, but also significantly reduce the browning rate during tissue culture.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of lily tissues, characterized in that, Includes the following steps: (1) Disinfect the middle scales of the lily and induce adventitious buds in the first culture medium; (2) Then, proliferation culture was carried out in the second culture medium; (3) Then carry out rooting culture in the third culture medium; (4) Then harden off the seedlings and then transplant them into the fourth culture medium; The variety of lily mentioned is Lilium pubescens; The first culture medium comprises MS medium, 0.5-1 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 80-100 mg / L carboxymethyl chitosan and 200-400 mg / L sodium alginate; the pH of the first culture medium is 5.6-6.

2. The second culture medium contains MS medium, 1-1.5 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 80-100 mg / L carboxymethyl chitosan, 0.2-0.5 mg / L zeatin, and 100-200 mg / L hydrolyzed casein; the pH of the second culture medium is 5.6-6.

2. The third culture medium comprises MS medium, 1-1.5 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30-40 g / L sucrose, 4-5 g / L agar, and also contains 100-200 mg / L carboxymethyl chitosan, 50-80 mg / L sodium alginate and 30-50 mg / L humic acid; the pH of the third culture medium is 5.6-6.

2. The fourth culture medium contains vermiculite, humus, peat moss, licorice extract and wolfberry extract in a mass ratio of (1-2):(1-2):(0.5-1):(0.1-0.3):(0.05-0.1). The disinfection conditions include: first soaking in 70 wt%-75 wt% ethanol for 20-30 seconds, then soaking in 1-3 wt% sodium hypochlorite solution for 10-12 minutes, and finally soaking in an aqueous solution containing 80-100 mg / L carboxymethyl chitosan and 200-400 mg / L sodium alginate for 5-8 minutes, and then rinsing with sterile water at least 3 times; The conditions for adventitious bud induction culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 12-16 hours / day, relative humidity 20%-25%, and culture time 35-45 days. The conditions for proliferation culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 12-16 hours / day, relative humidity 20%-25%, and culture time 35-45 days; The conditions for rooting culture include: temperature 21-25℃, light intensity 1500-2000 LUX, light duration 8-12 hours / day, relative humidity 20%-25%, and culture time 35-45 days; The conditions for hardening off the seedlings include: maintaining a temperature of 21-25℃ under natural light, with a relative humidity of 30%-40% on the first 1-2 days, and then a relative humidity of 20%-25% on the 3rd-5th days; The post-transplantation culture conditions include: a temperature of 21-25℃, a relative humidity of 20%-25%, and a relative humidity of 30%-40% for the fourth culture medium.

2. The method for rapid propagation of lily tissues according to claim 1, characterized in that, The carboxymethyl chitosan has a degree of deacetylation ≥90% and a molecular weight <50 kDa.

3. The method for rapid propagation of lily tissues according to claim 1, characterized in that, The steps for preparing the licorice extract include: i. Slice the dried licorice root, add water and decoct 2-3 times, 1-2 hours each time, with a material-to-liquid ratio of 1:6-1:10; ii. Then combine the filtrates and concentrate them to 20%-25% of their original volume; iii. Add 90 wt%-95 wt% ethanol to adjust the ethanol content to 60 wt%-70 wt%, and let stand for 24 hours; iv. Subsequently, the supernatant was collected by centrifugation, the ethanol was recovered, and the product was spray-dried to obtain licorice extract powder.

4. The method for rapid propagation of lily tissues according to claim 1, characterized in that, The steps for preparing the wolfberry extract include: (a) Take wolfberry stems and leaves, wash them, and add 15 wt%-20 wt% ethanol aqueous solution. The material-to-liquid ratio is 1:8-1:

10. (b) Then, ultrasonic extraction was performed at 25-35℃ for 1.5-2 hours, with an ultrasonic frequency of 50-60 kHz and an ultrasonic power of 40-50 W. The filtrate and residue were then obtained by filtration. (c) The filtrate is concentrated under reduced pressure at 0.002-0.05 MPa and 22-25°C until the ethanol content reaches 50 wt%-60 wt%, to obtain the first concentrate; (d) The filter residue is extracted twice. The extraction conditions for each extraction include: adding 9-11 times the mass of 15 wt%-20 wt% ethanol aqueous solution, ultrasonic extraction at 25-35℃ for 1.5-2 hours, ultrasonic frequency of 50-60 kHz, ultrasonic power of 40-50 W, to obtain a second concentrate. (e) Combine the first concentrate and the second concentrate, and spray dry to obtain wolfberry extract powder.

5. The method for rapid propagation of lily tissues according to any one of claims 1-4, characterized in that, The disinfection conditions include: first soaking in 75 wt% ethanol for 30 seconds, then soaking in 2 wt% sodium hypochlorite solution for 10 minutes, and finally soaking in an aqueous solution containing 100 mg / L carboxymethyl chitosan and 400 mg / L sodium alginate for 6 minutes, followed by rinsing 5 times with sterile water. The conditions for inducing adventitious buds include: a temperature of 23°C, a light intensity of 1500 LUX, a light duration of 12 hours / day, a relative humidity of 20%, and a culture time of 35 days; the first culture medium contains MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 80 mg / L carboxymethyl chitosan and 200 mg / L sodium alginate; the pH of the first culture medium is 5.

8.

6. The method for rapid propagation of lily tissues according to any one of claims 1-4, characterized in that, The conditions for the proliferation culture included: a temperature of 23°C, a light intensity of 1500 LUX, a light duration of 12 hours / day, a relative humidity of 20%, and a culture time of 35 days; the second culture medium contained MS medium, 1 mg / L 6-BA, 0.3 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contained 80 mg / L carboxymethyl chitosan, 0.2 mg / L zeatin, and 100 mg / L hydrolyzed casein; the pH of the second culture medium was 5.

8.

7. The method for rapid propagation of lily tissues according to any one of claims 1-4, characterized in that, The rooting culture conditions include: a temperature of 23°C, a light intensity of 1500-2000 LUX, a light duration of 8 hours / day, a relative humidity of 20%, and a culture time of 35 days. The third culture medium contains MS medium, 1 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, 5 g / L agar, and also contains 200 mg / L carboxymethyl chitosan, 50 mg / L sodium alginate, and 50 mg / L humic acid. The pH of the third culture medium is 5.

8.

8. The method for rapid propagation of lily tissues according to any one of claims 1-4, characterized in that, The conditions for hardening off the seedlings include: maintaining a temperature of 23°C under natural light, a relative humidity of 30% for the first 1-2 days, and a relative humidity of 20% for the third-to-fifth days.

9. The method for rapid propagation of lily tissues according to any one of claims 1-5, characterized in that, The post-transplantation culture conditions include: a temperature of 23°C, a relative humidity of 20%, and a relative humidity of 40% for the fourth culture medium; and the fourth culture medium contains vermiculite, humus, peat moss, licorice extract, and wolfberry extract in a mass ratio of 2:2:1:0.2:0.

1.

10. The application of the method according to any one of claims 1-9 in the rapid propagation of lily tissues.

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