A tobacco adventitious root induction medium and a tobacco adventitious root cultivation method

Abstract

The present application relates to the technical field of plant tissue culture, and specifically to an induction medium for tobacco adventitious roots and a method for cultivating tobacco adventitious roots. An induction medium for tobacco adventitious roots, each liter of induction medium includes the following components by weight: 4.74 g of MS medium dry powder, 0.5‑1 mg of IBA, 10‑20 mg of hop extract, 25‑30 g of sucrose, and 7.5‑8 g of agar. The induction medium of the present application is added with IBA and hop extract. Under the synergistic effect of the two, compared with the prior art, tobacco tissue can grow more adventitious roots and reduce the contamination rate of tobacco tissue after culture.

Description

Technical Field

[0001] The present application relates to the technical field of plant tissue culture, and in particular to an induction culture medium for tobacco adventitious roots and a culture method for tobacco adventitious roots. Background Art

[0002] Tobacco is one of the important economic crops in my country. The growth and development of tobacco roots are closely related to the yield and quality of tobacco leaves. The development of adventitious roots is an important part of the entire root development. Usually, tobacco plants with more adventitious roots and higher root activity grow more vigorously, and the content of compounds such as nicotine is also relatively high.

[0003] Tissue culture can not only maintain the excellent traits of tobacco, but also provide a large number of seedlings for the market, playing a positive role in tobacco breeding. In tissue culture technology, the induction of tobacco adventitious roots has always been the focus of researchers, and the number of roots and root activity of tobacco adventitious roots can be increased by improving the formula of the culture medium.

[0004] In the prior art, the formula of tobacco culture medium is mostly based on MS culture medium, and auxin and auxin analogs are added to promote rooting, such as IAA, IBA, NAA, etc., and the optimal culture medium formula is determined by adjusting their content. At present, the induction effect of tobacco tissue culture medium on tobacco adventitious roots is difficult to meet the higher induction requirements of technicians, and a new tobacco adventitious root induction medium is needed to further improve the rooting effect of tobacco adventitious roots. Summary of the invention

[0005] In order to solve the problem that the culture medium in tobacco tissue culture is difficult to satisfy the technicians' requirements for a higher induction effect of tobacco adventitious roots, the present application provides a tobacco adventitious root induction culture medium and a tobacco adventitious root culture method. The tobacco adventitious root induction culture medium has a better induction effect than the prior art, and can also reduce the contamination rate during tobacco tissue culture.

[0006] In a first aspect, the present application provides a tobacco adventitious root induction medium, which adopts the following technical solution:

[0007] The invention discloses an induction medium for adventitious roots of tobacco. The induction medium comprises the following components by weight per liter: 4.74g of MS medium dry powder, 0.5-1mg of IBA, 10-20mg of hop extract, 25-30g of sucrose and 7.5-8g of agar.

[0008] By adopting the above technical scheme, IBA is a common hormone for promoting rooting of tobacco. The reason why IBA promotes rooting is that on the one hand, it activates the expression of rooting-related genes, regulates the process of cell division and differentiation, thereby promoting the formation of plant roots, and on the other hand, it increases the activity of cell wall relaxants to relax the cell walls and promote the elongation and division of root cells; in the process of promoting tobacco rooting, hydrogen peroxide is an important signal molecule. Some IBA promotes tobacco to produce hydrogen peroxide, and some IBA promotes the expression of other rooting-related genes; hop extract contains a certain amount of xanthohumol, which can increase NA to a certain extent The activity of DPH oxidase and NADPH oxidase can also promote the production of hydrogen peroxide; thus, in the process of IBA promoting rooting, hop extract increases the production of hydrogen peroxide. On the one hand, a large amount of hydrogen peroxide can accelerate the rate of tobacco rooting process and enhance the rooting effect of tobacco; on the other hand, with the increase of hydrogen peroxide production, the amount of IBA used to promote hydrogen peroxide production is reduced through the negative feedback process in tobacco, allowing more IBA to promote the expression of other rooting-related genes, further improving the rooting effect of tobacco; in addition, hop extract has a certain antiseptic effect and can improve the antibacterial ability of the induction culture medium;

[0009] Compared with the prior art, the induction culture medium of the present application can not only grow more adventitious roots in tobacco tissue under the synergistic effect of IBA and hop extract, but also reduce the contamination rate of tobacco tissue after culture.

[0010] Preferably, the hop extract contains 2.5-5 wt % of xanthohumol.

[0011] By adopting the above technical scheme, when the content of xanthohumol in the hop extract is too low, it is difficult to increase the production of hydrogen peroxide through NADPH oxidase, and thus it is difficult to promote the rooting effect of tobacco tissue; when the content of xanthohumol in the hop extract is too high, the production of hydrogen peroxide is too much, which can easily lead to programmed cell death and reduce the rooting effect of tobacco tissue; for this reason, the applicant finally determined after a lot of research and experimental verification that the content of xanthohumol in the hop extract of the present application is preferably as above.

[0012] Preferably, the mass ratio between the IBA and the hop extract is 1:15-25.

[0013] By adopting the above technical scheme, when the content of hop extract is too low relative to the content of IBA, it is difficult for the hop extract to synergistically promote the rooting effect of IBA, thereby reducing the amount of tobacco adventitious roots and root length; when the content of hop extract is too high, the amount of hydrogen peroxide generated is too much, which can easily lead to programmed cell death and reduce the rooting effect of tobacco tissue; for this reason, the applicant finally determined after a lot of research and experimental verification that the mass ratio between IBA and hop extract in this application is preferably the above.

[0014] Preferably, the pH value of the induction medium is 5.5-6.0.

[0015] By adopting the above technical scheme, if the pH of the induction medium is too low, the induction medium is difficult to solidify, which is not conducive to the formation and growth of tobacco adventitious roots; if the pH of the induction medium is too high, the solidification degree of the induction medium is too high, which is also not conducive to the formation and growth of tobacco adventitious roots; for this reason, the applicant finally determined after a lot of research and experimental verification that the pH value of the induction medium of this application is preferably the above.

[0016] In a second aspect, the present application provides a method for cultivating tobacco adventitious roots, which adopts the following technical solution:

[0017] A method for cultivating tobacco adventitious roots comprises the following steps:

[0018] Insertion: cutting the tobacco sterile seedlings into stem segments, and inserting the stem segments into any one of the above-mentioned induction mediums;

[0019] Cultivation: the stem segments are cultured under the following conditions: temperature of 24-26° C. and light intensity of 1900-2100 lx.

[0020] By adopting the above technical scheme, during cultivation at 24-26°C, the tobacco stem segments have stronger vitality, the amount of IBA and hop extract absorbed by the stem segments in the induction medium is increased, and as the amount of IBA and hop extract absorbed increases, the ability of the stem segments to generate adventitious roots is stronger; under a light intensity of 1900-2100lx, the photosynthesis efficiency of the stem segments is higher, and the nutrient elements in the induction medium can be better utilized to continue to grow, while providing more nutrients and energy for the generation of tobacco adventitious roots.

[0021] Preferably, in the step of inserting, the length of the stem segment is 3-5 cm.

[0022] By adopting the above technical solution, when the length of the stem segment is too short, the low cell content in the stem segment can easily cause the activity of the stem segment to decrease, thereby reducing the amount of tobacco adventitious roots generated and the root length; when the length of the stem segment is too long, the activity of the stem segment is already high enough, and the growth of tobacco adventitious roots and root length has tended to be stable. In order to achieve more splicing, there is no need to continue to increase the length of the stem segment; for this reason, the applicant has finally determined after a lot of research and experimental verification that the length of the stem segment of this application is preferably as above.

[0023] Preferably, in the culturing step, after the adventitious roots induced by the stem segments reach a length of 0.5-1 cm and the number of adventitious roots is ≥ 80% of the total number of roots, the stem segments are inserted into a blank induction medium for further induction.

[0024] By adopting the above technical scheme, if the root length of the adventitious roots induced by the stem segments is too low, the activity of the adventitious roots induced by the stem segments is low, thereby reducing the number and length of the adventitious roots of the stem segments after continued induction; if the root length of the adventitious roots induced by the stem segments is too high, the IBA and hop extract in the induction medium are consumed, and it is difficult to continue to induce the stem segments to take root; for this reason, the applicant has finally determined after a lot of research and experimental verification that if the adventitious roots induced by the stem segments of the present application meet the above conditions, the stem segments can be inserted into a blank induction medium for continued induction.

[0025] Preferably, in the culturing step, hardening of the seedlings begins when the adventitious roots induced by the stem segments reach a length of 2-3 cm and a root number of 40-50.

[0026] Through the above technical scheme, the tobacco seedlings are hardened under this standard and the survival rate is high.

[0027] Preferably, in the culturing step, the illumination time is 12-16 h / d.

[0028] By adopting the above technical scheme, when the daily light exposure time is too short, the photosynthesis of the stem segment is low, reducing the generation and elongation of adventitious roots; when the daily light exposure time is too long, the photosynthesis of the stem segment is too long, and a large amount of free radicals are accumulated, which can easily lead to stem segment cell death and reduce the generation and elongation of adventitious roots; for this reason, the applicant finally determined after a lot of research and experimental verification that the daily light exposure time of this application is preferably as above.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. Since IBA and hop extract are added to the induction medium of the present application, under the synergistic effect of the two, the tobacco tissue can grow more adventitious roots and reduce the contamination rate of the tobacco tissue after culture;

[0031] 2. The present application defines the standard for transferring stem segments to a new blank induction medium during the cultivation process in the tobacco adventitious root cultivation method, thereby improving the induction effect of tobacco adventitious roots; at the same time, it defines the standard for hardening tobacco adventitious roots, thereby ensuring the survival rate of tobacco. DETAILED DESCRIPTION

[0032] The raw materials in this application include the following parts:

[0033] The MS culture medium adopts a commercial dry powder product without sucrose and agar; the IBA adopts a commercial product with a CAS number of 133-32-4 and an effective content of ≥99%; the hop extract adopts a commercial product with a CAS number of 6745-58-1 and a xanthohumol content of ≥2.5%; the sucrose adopts a commercial product with a CAS number of 57-50-1 and an effective content of ≥99%; and the agar adopts a commercial product with an effective content of ≥99%.

[0034] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0035] Example 1

[0036] Prepare induction medium: dissolve 4.74 g MS medium, 0.8 mg IBA, 16 mg hop extract (xanthohumol 4 wt%), 30 g sucrose and 8 g agar in 980 mL deionized water by heating, adjust the pH of the solution to 5.8 with 1 mol / L HCl solution and 1 mol / L KOH solution, then make the volume to 1 L, and sterilize at 121° C. for 20 min to obtain the induction medium;

[0037] Aliquot: Aliquot the induction medium into tissue culture bottles, with 50 mL of induction medium in each tissue culture bottle;

[0038] Grafting: Cut the sterile tobacco seedlings into 5 cm stem segments, and graft 2 stem segments into each tissue culture bottle to obtain stem segments;

[0039] Cultivation: The stem segments were cultured under the following conditions: temperature 25°C, light intensity 2000 lx, and photoperiod 14 h / day;

[0040] Transfer: Randomly sample the stem segments from the same batch, with a sampling rate of 5%. Take out the sampled stem segments and measure the average number of adventitious roots, which is 15. When the number of roots with a root length of 0.8 cm is at least 12, insert the stem segments into a blank induction medium (referring to "induction medium without stem segments inserted") for continued cultivation;

[0041] Hardening of seedlings: Randomly sample stem segments from the same batch with a sampling rate of 5%. Take out the sampled stem segments and measure their adventitious root length, which is 2.4 cm (the root length is allowed to fluctuate within 2-3 cm) and the average number of roots is 43 (the number of roots is allowed to fluctuate within 40-50). This indicates that the stem segments of this batch have reached the seedling hardening standard and can begin hardening of the seedlings.

[0042] Example 2-3

[0043] In Example 2-3, based on the preparation method of Example 1, the component content, culture temperature and light intensity of the induction medium were adjusted. The specific adjustments are shown in Table 1.

[0044] Comparative Example 1-2

[0045] Comparative Example 1-2 Based on the preparation method of Example 1, the component content, culture temperature and light intensity of the induction medium were adjusted. The specific adjustments are shown in Table 1.

[0046] Comparative Example 3

[0047] Comparative Example 3 is based on the preparation method of Example 1, but no hop extract is added, and the other conditions remain unchanged.

[0048] Table 1 Data table of component contents, culture temperature and light intensity of the induction medium of Examples 1-3 and Comparative Examples 1-3

[0049]

[0050]

[0051] Performance testing

[0052] 1. Number and length of adventitious roots

[0053] The stem segments inserted in Examples 1-3 and Comparative Examples 1-3 of the present application were cultured for 20 days and 40 days, and randomly sampled from the stem segments of the same Example or Comparative Example, with a sampling rate of 5%. The sampled stem segments were taken out to measure the number of adventitious roots and the length of adventitious roots, and the total number of roots, the number of stem segments, and the total root length were counted;

[0054] Wherein, the total number of roots is the number of adventitious roots of all stem segments drawn from the same embodiment or comparative example; the number of stem segments is the number of stem segments drawn from the same embodiment or comparative example; the total root length is the length of adventitious roots of all stem segments drawn from the same embodiment or comparative example;

[0055] Calculate the number of roots and root length, where the number of roots = total number of roots / number of stem segments, and root length = total root length / total number of roots. The calculation results are shown in Table 2.

[0056] 2. Pollution rate

[0057] The tobacco stem segments inserted in Examples 1-3 of the present application and Comparative Examples 1-3 were cultured for 20 days and 40 days, and the number of contaminated bottles and the total number of bottles in the tissue culture bottles after insertion were counted to calculate the contamination rate, where the contamination rate = (number of contaminated bottles / total number of bottles) * 100%. The calculation results are shown in Table 2.

[0058] Table 2 Performance test data of Examples 1-3 and Comparative Examples 1-3

[0059]

[0060] Referring to Table 2, by comparing Examples 1-3 and Comparative Examples 1-3, it can be seen that compared with Example 1, the number of roots and the root length of the adventitious roots of Examples 2-3 are smaller than those of Example 1, and the number of roots and the root length of the adventitious roots of Comparative Examples 1 and 2 are significantly lower than those of Example 1; this indicates that the growth of tobacco adventitious roots is better under the induction medium and culture conditions of Example 1.

[0061] Embodiment 4-5

[0062] Example 4-5 Based on the preparation method of Example 1, the mass percentage of xanthohumol in the hop extract was adjusted, and the specific adjustment is shown in Table 3.

[0063] Comparative Examples 4-5

[0064] Comparative Example 4-5 Based on the preparation method of Example 1, the mass percentage of xanthohumol in the hop extract was adjusted, and the specific adjustment is shown in Table 3.

[0065] The tobacco stem segments prepared in Examples 4-5 and Comparative Examples 4-5 were subjected to the above performance tests, and the test results are shown in Table 3.

[0066] Table 3 Xanthohumol mass percentage and performance test data of Example 1, Examples 4-5 and Comparative Examples 4-5

[0067] Referring to Table 3, by comparing Example 1, Examples 4-5 and Comparative Examples 4-5, it can be seen that as the content of xanthohumol in the hop extract continues to increase, the number of roots and the length of the adventitious roots of the tobacco stem segments show a trend of first increasing and then decreasing. This may be because within a certain range, the content of xanthohumol in the hop extract continues to increase, and the amount of hydrogen peroxide generated continues to increase, which promotes the formation and growth of the adventitious roots of the tobacco stem segments; when it exceeds a certain range, the amount of hydrogen peroxide generated is too much, which can easily lead to programmed cell death and reduce the rooting effect of tobacco.

[0068] Embodiment 6-11

[0069] Examples 6-11 are based on the preparation method of Example 1, and the contents of IBA and hop extract are adjusted. The specific adjustments are shown in Table 4.

[0070] Comparative Examples 6-7

[0071] Comparative Example 6-7 Based on the preparation method of Example 1, the contents of IBA and hop extract were adjusted, and the specific adjustments are shown in Table 4.

[0072] The tobacco stem segments prepared in Examples 6-11 and Comparative Examples 6-7 were subjected to the above performance tests, and the test results are shown in Table 5.

[0073] Table 4 IBA and hop extract contents of Example 1, Examples 6-11 and Comparative Examples 6-7

[0074]

[0075] Table 5 Performance test data of Example 1, Examples 6-11 and Comparative Examples 6-7

[0076]

[0077] Referring to Table 5, it can be seen from the comparison between Example 1, Examples 6-11 and Comparative Examples 6-7 that, as the concentrations of IBA and hop extract continue to increase, the number of roots and the length of adventitious roots of tobacco stem segments first increase and then decrease. This may be because within a certain range, as the concentrations of IBA and hop extract continue to increase, the amount of hydrogen peroxide generated continues to increase, which is beneficial to the generation of adventitious roots of tobacco stem segments; when exceeding a certain range, excessive hydrogen peroxide is likely to cause programmed cell death of tobacco cells, which is not conducive to the generation of adventitious roots of tobacco stem segments;

[0078] As the content of hop extract relative to that of IBA continued to increase, the number and length of adventitious roots of tobacco stem segments showed a trend of first increasing and then decreasing. This may be because within a certain range, as the content of hop extract relative to that of IBA continued to increase, the synergistic effect between hop extract and IBA continued to increase, thereby improving the generation effect of adventitious roots of tobacco stem segments; when exceeding a certain range, excessive hydrogen peroxide easily caused programmed cell death of tobacco cells, which was not conducive to the generation of adventitious roots of tobacco stem segments.

[0079] Examples 12-13

[0080] In Examples 12-13, based on the preparation method of Example 1, the pH of the induction medium was adjusted, and the specific adjustment is shown in Table 6.

[0081] Comparative Examples 8-9

[0082] Comparative Example 8-9 Based on the preparation method of Example 1, the pH of the induction medium was adjusted, and the specific adjustment is shown in Table 6.

[0083] The tobacco stem segments prepared in Examples 12-13 and Comparative Examples 8-9 were subjected to the above performance tests, and the test results are shown in Table 6.

[0084] Table 6 pH and performance test data of the induction medium of Example 1, Examples 12-13 and Comparative Examples 8-9

[0085]

[0086] Referring to Table 6, it can be seen from the comparison of Example 1, Examples 12-13 and Comparative Examples 8-9 that as the pH of the induction medium continues to increase, the number of roots and the root length of the adventitious roots of the tobacco stem segments show a trend of first increasing and then decreasing. This may be because within a certain range, as the pH of the induction medium continues to increase, the coagulation of the induction medium continues to increase, thereby increasing the rooting effect of the adventitious roots of the tobacco stem segments; when it exceeds a certain range, the coagulation of the induction medium is too high, and the tobacco stem segments are difficult to develop and grow in the induction medium, thereby reducing the rooting effect of the tobacco stem segments.

[0087] Examples 14-15

[0088] In Examples 14-15, based on the preparation method of Example 1, the length of the tobacco stem segment was adjusted, and the specific adjustment is shown in Table 7.

[0089] Comparative Examples 10-11

[0090] Comparative Examples 10-11 Based on the preparation method of Example 1, the length of the tobacco stem segment was adjusted, and the specific adjustment is shown in Table 7.

[0091] The tobacco stem segments prepared in Examples 14-15 and Comparative Examples 10-11 were subjected to the above performance tests, and the test results are shown in Table 7.

[0092] Table 7 Stem length and performance test data of Example 1, Examples 14-15 and Comparative Examples 10-11

[0093]

[0094] Referring to Table 7, it can be seen from the comparison of Example 1, Examples 14-15 and Comparative Examples 10-11 that as the length of the stem segment continues to increase, the number of roots and the root length of the adventitious roots of the tobacco stem segment show a trend of first increasing and then tending to be stable. This may be because as the length of the stem segment continues to increase, the cell content of the stem segment continues to increase, which is conducive to the generation of adventitious roots of the tobacco stem segment; when the length of the stem segment reaches a certain range, the vitality of the stem segment is already high enough, and the generation amount and root length of the tobacco adventitious roots tend to be stable.

[0095] Examples 16-17

[0096] In Examples 16-17, based on the preparation method of Example 1, the daily illumination time is adjusted, and the specific adjustments are shown in Table 8.

[0097] Comparative Examples 12-13

[0098] In Comparative Examples 12-13, based on the preparation method of Example 1, the daily illumination time was adjusted, and the specific adjustments are shown in Table 8.

[0099] The tobacco stem segments prepared in Examples 16-17 and Comparative Examples 12-13 were subjected to the above performance tests, and the test results are shown in Table 8.

[0100] Table 8 Lighting time and performance test data of Example 1, Examples 16-17 and Comparative Examples 12-13

[0101]

[0102] Referring to Table 8, it can be seen from the comparison of Example 1, Examples 16-17 and Comparative Examples 12-13 that, as the daily illumination time continues to increase, the number of roots and the root length of the adventitious roots of the tobacco stem segments show a trend of first increasing and then decreasing. This may be because within a certain range, as the daily illumination time continues to increase, the photosynthesis of the stem segments continues to increase, thereby increasing the number of roots and the root length of the adventitious roots of the tobacco stem segments; when it exceeds a certain range, the free radicals accumulated by the photosynthesis of the stem segments cause the stem segment cells to continue to die, thereby reducing the number of roots and the root length of the adventitious roots of the tobacco stem segments.

[0103] Examples 18-19

[0104] In Examples 18-19, based on the preparation method of Example 1, the root length of the stem segment before being inserted into the blank induction medium (referred to as "root length before insertion" in the table) was adjusted, and the specific adjustments are shown in Table 9.

[0105] Comparative Examples 14-15

[0106] Comparative Examples 14-15 are based on the preparation method of Example 1, and the root length of the stem segment is adjusted before being inserted into the blank induction medium. The specific adjustment is shown in Table 9.

[0107] The tobacco stem segments prepared in Examples 18-19 and Comparative Examples 14-15 were subjected to the above performance tests, and the test results are shown in Table 9.

[0108] Table 9 Root length and performance test data before plugging of Example 1, Examples 18-19 and Comparative Examples 14-15

[0109]

[0110] Referring to Table 9, it can be seen from the comparison of Example 1, Examples 18-19 and Comparative Examples 14-15 that, as the root length of the stem segment before being inserted into the blank induction medium continues to increase, the number of roots and the root length of the adventitious roots of the tobacco stem segment show a trend of first increasing and then decreasing. This may be because within a certain range, as the root length of the stem segment before being inserted into the blank induction medium continues to increase, the adventitious root activity of the stem segment continues to increase, thereby increasing the number of roots and the root length of the adventitious roots of the tobacco stem segment; when exceeding a certain range, the IBA and hop extract in the original induction medium are exhausted, making it difficult to continue to induce the stem segment to root, thereby reducing the number of roots and the root length of the adventitious roots of the tobacco stem segment after 40 days.

[0111] All the plants in Examples 1-19 survived after hardening.

[0112] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Use of a tobacco adventitious root induction medium in tobacco adventitious root induction, characterized in that: Each liter of induction medium is composed of the following components by weight: MS medium dry powder 4.74 g, IBA 0.5-1 mg, hop extract 10-20 mg, sucrose 25-30 g, agar 7.5-8 g; The hop extract contains 2.5-5 wt% of xanthohumol; The mass ratio between the IBA and the hop extract is 1:15-25; The pH value of the induction medium is 5.5-6.

0.

2. A method for cultivating tobacco adventitious roots, characterized in that: The following steps are involved: Insertion: cutting the tobacco sterile seedlings into stem segments, and inserting the stem segments into the induction medium as claimed in claim 1; Cultivation: The stem segments are cultured under the following conditions: a temperature of 24-26° C. and a light intensity of 1900-2100 lx.

3. The method for cultivating tobacco adventitious roots according to claim 2, characterized in that: In the step of grafting, the length of the stem segment is 3-5 cm.

4. The method for cultivating tobacco adventitious roots according to claim 2, characterized in that: In the culturing step, after the adventitious roots induced by the stem segment reach a length of 0.5-1 cm and the number of adventitious roots is ≥ 80% of the total number of roots, the stem segment is inserted into a blank induction medium for further induction.

5. The method for cultivating tobacco adventitious roots according to claim 2, characterized in that: In the cultivation step, when the adventitious roots induced by the stem segments reach a length of 2-3 cm and a root number of 40-50, hardening of the seedlings begins.

6. The method for cultivating tobacco adventitious roots according to claim 2, characterized in that: During the culturing step, the light duration is 12-16 h / d.

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