A high-efficiency rapid propagation method of polygonatum cyrtanthum with adventitious roots as explant material

Abstract

The application discloses a high-efficiency rapid propagation method of Qimen multi-flower polygonatum sibiricum with adventitious roots as explant material, and comprises the following steps: taking leaf blades of the Qimen multi-flower polygonatum sibiricum as initial materials, cutting the leaf blades into pieces after surface disinfection, inoculating the pieces into 1 / 2MS culture medium for culture after treatment with an adventitious root induction solution; cutting the induced adventitious roots into segments, inoculating the segments into MS culture medium for culture after treatment with a bud induction agent; culturing the base of the bud cluster induced by the adventitious roots in MS culture medium added with sucrose and agar after treatment with a reagent; and inoculating the adventitious buds separated from the bud cluster into 1 / 2MS culture medium for culture after treatment with an adventitious root induction agent to obtain complete regenerated plants. The application provides a rapid, efficient and high-quality Qimen multi-flower polygonatum sibiricum tissue culture propagation method which can keep the excellent characteristics of the mother plant, and provides certain technical support for germplasm preservation, large-scale propagation and variety improvement of the Qimen multi-flower polygonatum sibiricum.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, and in particular to a high-efficiency rapid propagation method of Qimen Polygonatum cyrtonema using adventitious roots as explant material. BACKGROUND

[0002] Polygonatum cyrtonema Hua is a perennial herb of the Liliaceae family and is a medicinal plant with values in medicine, food, ornamental, and health care. As a traditional medicine with both medicinal and edible properties, Polygonatum cyrtonema has the effects of invigorating the spleen, moistening the lungs, nourishing the kidney, tonifying qi and yin, delaying aging, beautifying the skin, lowering blood pressure, lowering blood sugar, lowering blood lipids, improving memory, and inhibiting the growth of tumor cells. In addition, Polygonatum cyrtonema can be processed into various health foods such as functional plant beverages, Polygonatum cyrtonema wine, and Polygonatum cyrtonema tea.

[0003] Currently, traditional methods such as sowing and tuber propagation are used for the propagation of Polygonatum cyrtonema, which has the problems of low propagation efficiency, long propagation cycle, and propagation being limited by season and time, and cannot meet the market demand of Polygonatum cyrtonema. Plant tissue culture technology has the advantages of high propagation efficiency, short propagation cycle, and no limitation on season and time, and has important significance in the conservation of medicinal plant resources and industrialized propagation. Currently, there have been reports on the tissue culture and rapid propagation of Polygonatum cyrtonema, such as the high-efficiency in vitro rapid propagation method of Qiyuan Polygonatum cyrtonema disclosed in Chinese patent application document CN115349445A and the tissue culture and rapid propagation method of Polygonatum cyrtonema disclosed in Chinese patent application document CN107864861A, which mainly focus on the rapid propagation using seeds and tubers as explants, and have the problems of long propagation cycle and high cost. Currently, there is no research on the use of adventitious roots of Polygonatum cyrtonema for plant regeneration.

[0004] Therefore, based on the selection of excellent strains of Qimen Polygonatum cyrtonema, in order to further expand the propagation methods and paths of Qimen Polygonatum cyrtonema and realize the industrialized production of Qimen Polygonatum cyrtonema seedlings, it is urgent to develop a high-efficiency rapid propagation method of Qimen Polygonatum cyrtonema using adventitious roots as explant material to meet the needs of rapid propagation and variety improvement of Qimen Polygonatum cyrtonema. SUMMARY

[0005] The technical problem to be solved by the present application is how to use plant tissue culture technology to efficiently and rapidly propagate Qimen Polygonatum cyrtonema using adventitious roots as explant material.

[0006] The present application solves the above technical problems through the following technical means:

[0007] A high-efficiency rapid propagation method of Qimen Polygonatum cyrtonema using adventitious roots as explant material, comprising the following steps:

[0008] (1) taking leaf blades of Qimen multi-flower polygonatum as initial material, cutting into pieces after surface sterilization, inoculating into 1 / 2MS medium after treatment by adventitious root induction solution to induce adventitious roots;

[0009] (2) cutting the induced adventitious roots into segments, inoculating into MS medium after treatment by bud induction agent to induce buds;

[0010] (3) transferring the base of the bud cluster induced by adventitious roots to MS medium added with sucrose and agar after treatment by agent to proliferate buds;

[0011] (4) separating the adventitious buds from the bud cluster, inoculating into 1 / 2MS medium after treatment by adventitious root induction agent to root and grow into seedlings to obtain complete regenerated plants.

[0012] Preferably, in (1), the pieces are 0.25-0.5cm 2 in length.

[0013] Preferably, in (1), the adventitious root induction solution is a solution containing 500-1000mg / L NAA and 100-300mg / L nitroprusside; the treatment time is 10-60s; and the 1 / 2MS medium is 1 / 2MS medium added with 30g / L sucrose and 7.0g / L agar.

[0014] Preferably, in (2), the induced adventitious roots are cut into segments of 0.8-1.5cm in length.

[0015] Preferably, in (2), the bud induction agent is a solution containing 500-1000mg / L TDZ; the treatment time is 5-30s; and the MS medium is MS medium added with 30g / L sucrose and 7g / L agar.

[0016] Preferably, in (3), the treatment of the base of the bud cluster induced by adventitious roots by agent includes immersing the base of the bud cluster induced by adventitious roots into a solution of TDZ with a final concentration of 50-100mg / L for 5-30s.

[0017] Preferably, in (3), the sucrose added in the MS medium is 30g / L, and the agar is 7g / L.

[0018] Preferably, in (4), the adventitious buds are adventitious buds of 2-3cm in height with 2-3 mature leaves.

[0019] Preferably, in (4), the adventitious root inducer is a mixed solution added with NAA at a final concentration of 200-500 mg / L and nitroprusside at a final concentration of 100-500 mg / L; the treatment time is 5-20 s; and the 1 / 2MS medium is a 1 / 2MS medium added with 20 g / L sucrose and 7.0 g / L agar.

[0020] Preferably, in (1), (2), (3) and (4), the culture conditions are as follows: the temperature is 25±2℃, the light intensity is 40-50 μmol m -2 s -1 , and the photoperiod is 14 / 10 h (light / dark).

[0021] The present application has the following advantages:

[0022] The present application provides a high-efficiency rapid propagation method of Qimen Polyphylla Prain var. Flava with adventitious roots as explant material, which has the following advantages: first, the adventitious roots are derived from the current-year tender leaves of a fine strain of Qimen Polyphylla Prain var. Flava, the direct regeneration approach is used in the propagation process, the germplasm characteristics of the mother plant can be maintained, and the rapid propagation of the fine single plant of Qimen Polyphylla Prain var. Flava can be realized; second, the whole rapid propagation process uses the instant treatment method of plant growth regulators, which greatly shortens the rapid propagation period and reduces the seedling cost; third, the regeneration efficiency is high, the induction rate of the adventitious roots of the leaf pieces is as high as 93.7%, the induction rate of the buds of the adventitious root pieces is as high as 100%, and the average number of buds produced after the proliferation of each adventitious root piece is as high as 34.3, and the rooting rate of the buds is as high as 100%, which provides important technical support for the germplasm preservation, large-scale propagation and later genetic improvement of the fine Qimen Polyphylla Prain var. Flava strain. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The adventitious roots are induced from the leaf pieces of Qimen Polyphylla Prain var. Flava after the light culture for 4 weeks in Example 1 of the present application;

[0024] Figure 2 The adventitious bud points are obtained from the adventitious root pieces of Qimen Polyphylla Prain var. Flava after the light culture for 1 week in Example 1 of the present application;

[0025] Figure 3 The adventitious bud clumps are induced from the adventitious root pieces of Qimen Polyphylla Prain var. Flava after the light culture for 4 weeks in Example 1 of the present application;

[0026] Figure 4 The proliferation of the adventitious root bud clumps of Qimen Polyphylla Prain var. Flava after the light culture for 4 weeks in Example 1 of the present application;

[0027] Figure 5 The complete regenerated plants with healthy root systems are obtained from the elongated buds of Qimen Polyphylla Prain var. Flava after the light culture for 3 weeks in Example 1 of the present application. DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0029] The test materials and reagents used in the following examples, and the like, can be obtained from commercial channels if not otherwise specified.

[0030] The specific techniques or conditions not specified in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0031] Example 1

[0032] A high-efficiency rapid propagation method of Qimen multi-flower polygonatum with adventitious roots as explant material is specifically as follows:

[0033] (1) The current-year tender leaves of the selected Qimen multi-flower polygonatum excellent strain are used as initial materials, which are cut into 0.25-0.5 cm 2 pieces after surface sterilization, immersed in a water solution containing 750 mg / L NAA and 200 mg / L sodium nitroprusside for 20 s, and then inoculated into 1 / 2MS medium added with 30 g / L sucrose and 7.0 g / L agar with the proximal end upwards in a constant-temperature culture room at a temperature of 25±2℃, an illumination intensity of 40-50 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark) for adventitious root induction culture. After 1 week of illumination culture, adventitious root primordia are induced around the leaf piece; after 4 weeks of illumination culture, the adventitious root induction rate is as high as 93.7%, an average of 4.2 adventitious roots are generated per leaf piece, and the average length of the adventitious roots is 6.2 cm (as shown in Figure 1 ).

[0034] (2) The induced adventitious roots are cut into 0.8-1.5 cm segments, immersed in a bud induction solution for 15 s, wherein the bud induction solution is a 750 mg / L TDZ water solution, and then inoculated into MS medium added with 30 g / L sucrose and 7 g / L agar in a constant-temperature culture room at a temperature of 25±2℃, an illumination intensity of 40-50 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark) for bud induction culture. After 1 week of illumination culture, bud points are induced at both ends of the adventitious root segment (as shown in Figure 2(see Fig. 2). After 3 weeks of subculture under illumination, the induction rate of adventitious roots reached 100%, and the average number of adventitious roots produced from each explant was 6.3 (see Fig. 3). Figure 3

[0035] (3) The base of the bud cluster induced from the adventitious roots was immersed in a TDZ aqueous solution with a final concentration of 75 mg / L for 20 s, and then transferred to a MS medium added with 30 g / L sucrose and 7 g / L agar and subcultured in a constant temperature incubator at a temperature of 25 ± 2°C, an illumination intensity of 40-50 μmol m -2 s -1 , and a light cycle of 14 / 10 h (light / dark) for bud proliferation culture. After 4 weeks of subculture under illumination, the average number of adventitious buds produced from each explant was 34.3 (see Fig. 4). Figure 4

[0036] (4) After the adventitious buds with a height of 2-3 cm and with 2-3 mature leaves were separated from the bud cluster, the base was immersed in a mixed aqueous solution added with a final concentration of 300 mg / L NAA and a final concentration of 500 mg / L sodium nitroprusside for 10 s, and then directly inoculated into a 1 / 2MS medium added with 20 g / L sucrose and 7.0 g / L agar and subcultured in a constant temperature incubator at a temperature of 25 ± 2°C, an illumination intensity of 40-50 μmol m -2 s -1 , and a light cycle of 14 / 10 h (light / dark) for rooting and seedling culture. After 2 weeks of culture, the adventitious root primordia were produced at the base of the stem segment, and after 3 weeks of culture, the induction rate of adventitious roots reached 100%, and the average number of adventitious roots produced from each explant was 6.3 (see Fig. 3). Figure 5

[0037] Example 2

[0038] This example tested the effects of the concentrations of exogenous NAA and sodium nitroprusside on the induction of adventitious roots from the leaves of the excellent strain of P. qimenense. The specific steps were as follows:

[0039] (1) The leaf pieces (0.25-0.5 cm 2 ) of P. qimenense after surface sterilization were immersed in aqueous solutions of NAA and sodium nitroprusside with different concentrations for 10 s, and then inoculated on a 1 / 2MS medium added with 30 g / L sucrose and 7.0 g / L agar, and subcultured in a constant temperature incubator at a temperature of 25 ± 2°C, an illumination intensity of 40-50 μmol m -2 s -1 ​​​The explants were inoculated on 1 / 2MS medium containing 30 g / L sucrose and 7.0 g / L agar, and then cultured in a constant temperature incubator with a temperature of 25±2°C, a light intensity of 40-50 μmol m -2 s -1 -2, a light cycle of 14 / 10 h (light / dark) for induction of adventitious roots. The induction of adventitious roots was observed at any time during the culture, and the induction rate, the number of adventitious roots produced per explant, and the length of adventitious roots were counted after 4 weeks of illumination culture. The results are shown in Table 1, which show that the exogenous NAA and sodium nitroprusside have a synergistic promoting effect on the induction of adventitious roots of Polygonatum cyrtonema leaves within a certain concentration range, and the promoting effect gradually weakens with the increase of the concentration of the two. When the leaves are treated with 750 mg / L NAA and 200 mg / L sodium nitroprusside for 10 s, the induction effect of adventitious roots is the best, the induction efficiency of adventitious roots is 88.9%, 3.8 adventitious roots are produced per explant on average, and the average length of adventitious roots is 5.0 cm.

[0040] Table 1. Effects of different concentrations of exogenous NAA and sodium nitroprusside on the induction of adventitious roots of Polygonatum cyrtonema leaves

[0041]

[0042] Note: The data are average values, each treatment contains 240 explants, and each treatment is repeated three times.

[0043] Example 3

[0044] This example tests the effects of treatment time of exogenous NAA and sodium nitroprusside on the induction of adventitious roots of leaves of Polygonatum cyrtonema excellent strains. The specific steps are as follows: (1) the cut leaves (0.25-0.5 cm 2 ) of Polygonatum cyrtonema after surface sterilization are immersed in an aqueous solution containing 750 mg / L NAA and 200 mg / L sodium nitroprusside for different treatment times (5 s, 10 s, 15 s, 20 s, 30 s and 60 s), and then inoculated on 1 / 2MS medium containing 30 g / L sucrose and 7.0 g / L agar, and then cultured in a constant temperature incubator with a temperature of 25±2°C, a light intensity of 40-50 μmol m -2 s -1 -2, a light cycle of 14 / 10 h (light / dark) for induction of adventitious roots. The induction of adventitious roots was observed at any time during the culture, and the induction rate, the number of adventitious roots produced per explant, and the length of adventitious roots were counted after 4 weeks of illumination culture. The results are shown in Table 2.

[0045] Table 2. Effects of different treatment times of exogenous NAA and sodium nitroprusside on the induction of adventitious roots of Polygonatum cyrtonema leaves

[0046] Processing time (s) Rooting rate (%) Average root number Average root length (cm) 0 0.0 0.0 0.0 5 59.6 3.1 3.4 10 88.9 3.8 5.0 15 90.6 4.0 5.7 20 93.7 4.2 6.2 30 89.3 4.8 3.7 60 75.4 4.1 2.3

[0047] Note: The data are average values, each treatment contains 240 explants, and each treatment is repeated three times.

[0048] The results showed that the induction effect of adventitious roots was gradually enhanced with the extension of the treatment time of exogenous NAA and sodium nitroprusside during the induction of adventitious roots of Polygonatum acuminatum leaves. The induction effect of adventitious roots was the best when the leaves were treated in the induction solution with the final concentration of 750 mg / L NAA and 200 mg / L sodium nitroprusside for 20 s. The induction efficiency of adventitious roots was 93.7%, the average number of adventitious roots produced by each explant was 4.2, and the average length of adventitious roots was 6.2 cm.

[0049] Example 4

[0050] This example tested the effect of exogenous TDZ concentration on the induction of adventitious buds of Polygonatum cyrtonema adventitious root explants. The specific steps were as follows: (1) the surface-sterilized Polygonatum cyrtonema leaves were cut into pieces (0.25-0.5 cm 2 ) and immersed in a mixed aqueous solution with a final concentration of 750 mg / L NAA and 200 mg / L sodium nitroprusside for 20 s, then inoculated on 1 / 2MS medium with 30 g / L sucrose and 7.0 g / L agar, and cultured for induction of adventitious roots in a constant-temperature incubator at a temperature of 25±2°C, a light intensity of 40-50 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark). The induction of adventitious roots was observed during the culture. After 4 weeks of light culture, the obtained adventitious roots were cut into segments of 0.8-1.5 cm, immersed in an aqueous solution with different concentrations (100 mg / L, 250 mg / L, 500 mg / L, 750 mg / L, and 1000 mg / L) of TDZ for different times (5 s, 10 s, 15 s, 20 s, 30 s, and 60 s), then inoculated on MS medium with 30 g / L sucrose and 7 g / L agar at a temperature of 25±2°C, a light intensity of 40-50 μmol m -2 s -1, photoperiod of 14 / 10h (light / dark) in a constant temperature incubator. After 4 weeks of illumination culture, the induction rate of the buds of the adventitious root segments was counted. The results are shown in Table 3. The results show that the exogenous TDZ concentration and treatment time have important effects on the induction of the bud clusters on the adventitious root segments of P. polyphyllus. When the treatment time is 10s, within a certain range (100-750mg / L), the induction rate of the adventitious buds and the average number of adventitious buds produced by each explant increase gradually with the increase of the TDZ concentration. When the concentration reaches 1000mg / L, the induction rate of the buds and the number of adventitious buds decrease with the prolongation of the treatment time. Among the tested concentrations, 750mg / L TDZ treatment for 15s is the most conducive to the induction of the buds on the adventitious roots, with the induction rate of the adventitious roots reaching 100% and the average number of buds produced by each adventitious root segment being 4.7.

[0051] Table 3 Effects of exogenous TDZ concentration and treatment time on the induction of buds on the adventitious root segments of P. polyphyllus

[0052]

[0053] Note: The data are the average values, each treatment contains 240 explants, and each treatment is repeated three times.

[0054] Example 5

[0055] This example tests the effects of exogenous TDZ concentration and treatment time on the bud proliferation of P. polyphyllus adventitious roots. In this example, the induction of the adventitious roots and the bud induction process of the adventitious root segments are the same as in Example 1. The base of the bud clusters obtained in Example 1 is immersed in a TDZ aqueous solution with different final concentrations (20-100mg / L) for different times (5-60s), and then transferred to MS medium with 30g / L sucrose and 7g / L agar for bud proliferation culture under the culture conditions in Example 1. After 4 weeks of illumination culture, the proliferation of the buds of the adventitious root segments is counted. The results are shown in Table 4. The results show that a low concentration of TDZ is more conducive to the proliferation culture of the bud clusters of the adventitious root segments, and the proliferation effect of the bud clusters gradually weakens with the prolongation of the treatment time. When the TDZ concentration is 75mg / L, the treatment of the base of the bud clusters for 20s has the best proliferation effect of the buds, with the average number of adventitious buds produced by each explant being 34.3.

[0056] Table 4 Effects of exogenous TDZ concentration and treatment time on the bud proliferation on the adventitious roots of P. polyphyllus

[0057]

[0058] Note: The data are the average values, each treatment contains 240 explants, and each treatment is repeated three times.

[0059] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A rapid propagation method for *Polygonatum cyrtonema* using adventitious roots as explant material, characterized in that: The method comprises the following steps: (1) taking leaf blades of Qimen multi-flower polygonatum as initial material, cutting into pieces after surface sterilization, inoculating into 1 / 2MS medium for induction of adventitious roots after treatment with adventitious root induction solution; the adventitious root induction solution is a solution of 500-1000 mg / L NAA and 100-300 mg / L nitroprusside; the 1 / 2MS medium is 1 / 2MS medium with 30 g / L sucrose and 7.0 g / L agar; (2) cutting the induced adventitious roots into segments, inoculating into MS medium for induction of buds after treatment with bud induction agent for 5-30 s; the bud induction agent is a solution of 500-1000 mg / L TDZ; the MS medium is MS medium with 30 g / L sucrose and 7 g / L agar; (3) transferring the bud clusters induced from adventitious roots into MS medium with sucrose and agar for proliferation culture of buds after treatment with 50-100 mg / L TDZ solution for 5-30 s; the MS medium is MS medium with 30 g / L sucrose and 7 g / L agar; (4) separating the adventitious buds from the bud clusters, inoculating into 1 / 2MS medium for rooting and seedling culture after treatment with adventitious root induction agent, and obtaining complete regenerated plants; the adventitious root induction agent is a mixed solution with a final concentration of 200-500 mg / L NAA and a final concentration of 100-500 mg / L nitroprusside; the 1 / 2MS medium is 1 / 2MS medium with 20 g / L sucrose and 7.0 g / L agar.

2. The rapid propagation method of P. polyphylla var. wightianum using rhizomes as explant material according to claim 1, characterized in that: In (1), cut into 0.25-0.5 cm 2 pieces.

3. The rapid propagation method of P. polyphylla var. wightianum using rhizomes as explant material according to claim 1, characterized in that: In (1), the treatment time is 10-60 s.

4. The rapid propagation method of P. polyphylla var. wightianum using rhizomes as explant material according to claim 1, characterized in that: In (2), the induced adventitious roots are cut into segments with a length of 0.8-1.5 cm.

5. The rapid propagation method of P. polyphylla var. wightianum using rhizomes as explant material according to claim 1, characterized in that: In (4), the adventitious buds are 2-3 cm high and accompanied by 2-3 mature leaves.

6. The rapid propagation method of P. polyphylla var. wightianum using rhizomes as explant material according to claim 1, characterized in that: In (4), the treatment time is 5-20 s.

7. The rapid propagation method of P. polyphylla var. amurensis using rhizomes as explant material according to any one of claims 1-6, characterized in that: In (1), (2), (3), (4), the culture conditions are as follows: temperature 25±2℃, light intensity 40-50 μmol m -2 s -1 , photoperiod 14h light / 10h dark.

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