A method for precisely cultivating Populus adenopoda tissues

Abstract

This application relates to the technical field of plant tissue culture, and particularly to a method for precisely cultivating Populus adenopoda tissue; the method includes: picking young Populus adenopoda leaves as explants; performing pretreatment on the explants to obtain non-toxic explants; inducing callus from the non-toxic explants to obtain callus; using a composite medium containing Populus adenopoda enzymolysate to induce adventitious buds from the callus; during the induction of adventitious buds, continuously photographing the adventitious buds formed by the callus to obtain variation data of the adventitious buds; determining the supplementation amount of Populus adenopoda enzymolysate according to the variation data of the adventitious buds; supplementing Populus adenopoda enzymolysate into the composite medium according to the supplementation amount of Populus adenopoda enzymolysate to obtain adventitious buds; wherein, Populus adenopoda enzymolysate has the vitamin and trace element components of Populus adenopoda itself; culturing the adventitious buds for rooting to obtain Populus adenopoda tissue. This method can accelerate the speed of callus forming adventitious buds and improve the propagation rate of Populus adenopoda tissue.

Description

Technical Field

[0001] This application relates to the technical field of plant tissue culture, and particularly relates to a method for precisely cultivating Populus adenopoda tissue. Background Art

[0002] Populus adenopoda Populus adenopoda Maxim is a plant of the genus Populus in the family Salicaceae and belongs to arbor; Populus adenopoda has the characteristics of a straight main trunk, high branching, and rapid growth. Its leaves can be used as raw materials for feed, and its trunk or resin can be used as raw materials for preparing furniture and paper. Populus adenopoda is generally dioecious, and female plants have the characteristics of a tall main trunk, extremely small seeds, and low germination rate. It is difficult to maintain the excellent properties of the mother plant using traditional seed propagation, and there are defects such as difficulty in taking root when using the conventional field cutting propagation method. Therefore, at present, it can be cultivated by inducing leaf explants, but the cycle of this cultivation method is relatively long, generally close to 90 days; in addition, the prior art can also obtain regenerated plants of Populus adenopoda through stem segment tissue culture, but this method is similar to aseptic cutting and has problems such as low propagation efficiency.

[0003] At present, although the rapid propagation of Populus adenopoda can be achieved through tissue culture, generally a specific culture medium is added for the tissue culture of Populus adenopoda, and then nutrients are supplemented according to the consumption of nutrients. The consumption of nutrients requires actual measurement of these specific culture media, which undoubtedly increases the cultivation cycle and operation complexity of Populus adenopoda. In addition, the process of measuring these nutrients is interfered by a large number of factors, resulting in inaccurate supplementation amounts of subsequent nutrients and affecting the final propagation rate of Populus adenopoda tissue. Summary of the Invention

[0004] This application provides a method for precisely cultivating Populus adenopoda tissue to solve the following technical problems: how to improve the induction efficiency and propagation rate in the process of Populus adenopoda tissue cultivation.

[0005] In a first aspect, an embodiment of this application provides a method for precisely cultivating Populus adenopoda tissue, and the method includes:

[0006] Picking young leaves of Populus adenopoda as explants;

[0007] Performing pretreatment on the explants to obtain non-toxic explants;

[0008] Performing callus induction on the non-toxic explants to obtain callus;

[0009] Using a composite culture medium containing Populus adenopoda enzymolysate to induce adventitious buds of the callus;

[0010] During the induction of the adventitious buds, continuously photograph the adventitious buds formed by the callus to obtain the variation data of the adventitious buds;

[0011] Determine the supplementary amount of Populus adenopoda enzyme hydrolysate according to the variation data of the adventitious buds;

[0012] Supplement the Populus adenopoda enzyme hydrolysate into the composite culture medium according to the supplementary amount of the Populus adenopoda enzyme hydrolysate to obtain adventitious buds; wherein, the Populus adenopoda enzyme hydrolysate has the vitamin and trace element components of Populus adenopoda itself;

[0013] Cultivate the adventitious buds for rooting to obtain Populus adenopoda tissue.

[0014] Optionally, determining the supplementary amount of Populus adenopoda enzyme hydrolysate according to the variation data of the adventitious buds includes the steps of:

[0015] Determine the length variation data ΔL of the adventitious buds per unit time according to the variation data of the adventitious buds, wherein the length variation data of the adventitious buds per unit time is the length difference between the tip of the bud leaf of the adventitious buds before and after the unit time;

[0016] Judge whether it is necessary to supplement the Populus adenopoda enzyme hydrolysate according to the length variation data ΔL;

[0017] If the length variation data ΔL ≤ 2 mm, it is necessary to supplement the Populus adenopoda enzyme hydrolysate, and calculate the supplementary amount of the Populus adenopoda enzyme hydrolysate according to the length variation data ΔL; wherein, the calculation formula for the supplementary amount of the Populus adenopoda enzyme hydrolysate is: m = (0.2 - 0.4) × ΔL + 0.2. If the unit of m is g, the unit of ΔL is mm.

[0018] Optionally, during the induction of the adventitious buds, continuously photographing the adventitious buds formed by the callus to obtain the variation data of the adventitious buds includes the steps of:

[0019] During the induction of the adventitious buds, use a time-lapse camera to continuously photograph the adventitious buds formed by the callus to obtain adventitious bud photos;

[0020] Calculate the size variation data of the adventitious buds according to the differences before and after the adventitious bud photos.

[0021] Optionally, the interval time of the continuous photographing is 24 h to 48 h, and the total duration of the continuous photographing is from the 7th day to the 28th day of the induction of the adventitious buds; and / or

[0022] The temperature for the induction of the adventitious buds is 24°C to 26°C, and the induction time of the adventitious buds is 35 d to 45 d.

[0023] Optionally, the preparation method of the Populus adenopoda enzyme hydrolysate includes:

[0024] Mix the leaves of the Populus adenopoda and the stem segments of the Populus adenopoda to obtain an enzymatic hydrolysis raw material;

[0025] Use cellulase to enzymatically hydrolyze the enzymatic hydrolysis raw material to obtain an enzymatic hydrolysate;

[0026] Vacuum dry the enzymatic hydrolysate to obtain the Populus adenopoda enzyme hydrolysate.

[0027] Optionally, the weight m2 of the leaves of the Populus adenopoda and the weight m3 of the stem segments of the Populus adenopoda satisfy the relationship: m2:m3 = 1:(1.5 - 2.5); and / or

[0028] The temperature of the enzymatic hydrolysis is 25°C - 35°C, and the time of the enzymatic hydrolysis is 1h - 2h; and / or

[0029] The temperature of the vacuum drying is 50°C - 70°C, and the time of the vacuum drying is 25min - 35min.

[0030] Optionally, the weight m1 of the Populus adenopoda enzyme hydrolysate and the volume V1 of the composite culture medium satisfy the relationship: m1:V1 = (4 - 8):1. If the unit of m1 is g, then the unit of V1 is L.

[0031] Optionally, the components of the composite culture medium further include: MS medium, 3-indolebutyric acid, thiabendazole, sucrose, agar, and Plant Preservative Mixture II; the mass concentration of 3-indolebutyric acid is 0.1mg / L, the mass concentration of thiabendazole is 0.03mg / L, the mass concentration of sucrose is 30g / L, the mass concentration of agar is 6g / L, and the volume concentration of Plant Preservative Mixture II is 0.5mL / L.

[0032] Optionally, the temperature of the rooting culture is 24°C - 26°C, and the time of the rooting culture is 10d - 14d.

[0033] Optionally, the pretreatment of the explant to obtain a non-toxic explant includes the steps of:

[0034] Rinse and disinfect the explant to obtain a non-toxic explant; wherein, the disinfectant used in the disinfection treatment includes medical alcohol and / or sodium hypochlorite.

[0035] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0036] A method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application. Based on the induction process of callus induction into adventitious buds, by continuously photographing the adventitious buds formed by the callus, the morphological change data of the adventitious buds during the induction process can be clarified. Then, according to this change data, the addition amount of Populus adenopoda enzymolysate can be determined. Due to the vitamin and trace element components of Populus adenopoda itself in the Populus adenopoda enzymolysate, it can be quickly absorbed by the callus, thereby accelerating the speed of callus forming adventitious buds. In addition, the Populus adenopoda enzymolysate also contains more sugars and plant hormones, which can quickly enter the callus to further accelerate the induction efficiency of callus forming adventitious buds. Moreover, by determining the addition amount of Populus adenopoda enzymolysate through the change data of adventitious buds, the cumbersome operation of direct sampling and the interference of various factors can be avoided, with a certain degree of accuracy, thus improving the propagation rate of Populus adenopoda tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings herein are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present application and, together with the specification, are used to explain the principles of the present application.

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0039] Figure 1 It is a schematic flow chart of a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application;

[0040] Figure 2 It is a detailed schematic flow chart of a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application;

[0041] Figure 3 It is a result diagram of the callus obtained by a method for precisely cultivating Populus adenopoda tissue provided by Embodiment 1 of the present application;

[0042] Figure 4 It is a result diagram of adventitious buds obtained after 15 days of induction using the adventitious bud induction medium of Group C25 provided by Embodiment 1 of the present application;

[0043] Figure 5 It is a result diagram of adventitious buds obtained after 15 days of induction using the adventitious bud induction medium of Group C24 provided by Embodiment 1 of the present application;

[0044] Figure 6 It is a result diagram of Populus adenopoda tissue obtained by a method for precisely cultivating Populus adenopoda tissue provided by Embodiment 1 of the present application;

[0045] Figure 7 This is the result diagram of the Populus adenopoda seedlings provided in Embodiment 1 of the present application. Detailed implementation manners

[0046] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the protection scope of the present application.

[0047] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0048] In this text, terms such as "comprising" mean "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or", which describes the relationship between related objects, indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "a plurality" means two or more; "at least one kind", "at least one of the following items", or similar expressions refer to any combination of these items, including any combination of single items or plural items; for example, "at least one of a, b, or c", or, "at least one of a, b, and c", can both represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively. The "parts representation method", such as parts by weight, parts by mass, etc., represents the proportional relationship between each component. In the proportional relationships involved in this text, the parameters that need to be described by proportion should be understood as the antecedents of the proportion formula in the order of description, and the proportional numbers should be understood as the consequents of the proportion formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the proportional numbers in the proportion formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0049] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in this text can be obtained through market purchases or can be prepared by existing methods.

[0050] It should be noted that at the present stage, the process of callus transformation into adventitious buds needs to be carried out in a sterile environment. The culture medium required for this process is generally prepared in advance and cannot be added subsequently; however, the nutrient components of these culture media are generally in excess to ensure the transformation of callus into adventitious buds. However, existing research shows that excessive nutrients will affect the growth of adventitious buds. Therefore, at the present stage, there are relevant equipment for feeding nutrient components, but most of these feeding equipment cannot be provided with sampling ports to accurately analyze the components of the culture medium. Therefore, opening sampling ports will increase the risk of these culture media being infected by miscellaneous bacteria.

[0051] Currently, there is research showing that the amount of nutrient components will directly affect the morphology of callus forming adventitious buds and the growth state of the bud leaves of adventitious buds. Therefore, if the growth state of the bud leaves of adventitious buds can be used to intuitively reflect the consumption of a certain type of nutrient component in the culture medium, it can effectively accelerate the efficiency of callus transformation into adventitious buds and avoid the defects of direct sampling.

[0052] Figure 1 Exemplarily shown is a schematic flow chart of a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application;

[0053] As Figure 1 shown, an embodiment of the present application provides a method for precisely cultivating Populus adenopoda tissue. The method includes:

[0054] S1. Picking young Populus adenopoda leaves as explants;

[0055] S2. Pretreating the explants to obtain non-toxic explants;

[0056] S3. Inducing callus from the non-toxic explants to obtain callus;

[0057] S4. Using a composite medium containing Populus adenopoda enzymolysate to induce adventitious buds from the callus;

[0058] S5. During the induction of adventitious buds, continuously photographing the adventitious buds formed by the callus to obtain variation data of the adventitious buds;

[0059] S6. Determining the supplementary amount of Populus adenopoda enzymolysate according to the variation data of the adventitious buds;

[0060] S7. According to the supplementary amount of Populus adenopoda enzymolysate, supplementing the Populus adenopoda enzymolysate into the composite medium to obtain adventitious buds; wherein, the Populus adenopoda enzymolysate has vitamin and trace element components of Populus adenopoda itself;

[0061] S8. Culturing the adventitious buds for rooting to obtain Populus adenopoda tissue.

[0062] It should be noted that the components of the medium used for callus induction include: woody plant medium (WPM), 1.5 mg / L of auxin (NAA), 3 mg / L of zeatin (ZT), 30 g / L of sucrose, 0.5 mL / L of Plant Preservative Mixture II, and 6 g / L of agar.

[0063] Figure 2 Exemplarily shown is a detailed schematic flow chart of a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application;

[0064] In some alternative embodiments, as Figure 2 shown, determining the supplementary amount of Populus adenopoda enzymolysate according to the variation data of the adventitious buds includes the steps of:

[0065] S601. Determine the length change data ΔL of the adventitious buds per unit time according to the change data of the adventitious buds, where the length change data of the adventitious buds per unit time is the length difference between the tips of the bud leaves of the adventitious buds before and after the unit time;

[0066] S602. Determine whether to supplement the Populus adenopoda enzyme hydrolysate according to the length change data ΔL;

[0067] If the length change data ΔL ≤ 2 mm, then it is necessary to supplement the Populus adenopoda enzyme hydrolysate, and calculate the supplement amount of the Populus adenopoda enzyme hydrolysate according to the length change data ΔL; where the calculation formula for the supplement amount of the Populus adenopoda enzyme hydrolysate is: m = (0.2 - 0.4) × ΔL + 0.2. If the unit of m is g, then the unit of ΔL is mm;

[0068] In these embodiments, the specific object of the change data of the adventitious buds is the length change data ΔL of the adventitious buds per unit time, which can clarify the growth rate of the adventitious buds in two adjacent time periods. The change of the tip of the bud leaf of the adventitious buds is relatively obvious during the growth process. Therefore, it can be roughly deduced whether there is a lack of nutrient components through the length change data ΔL of the tip of the bud leaf of the adventitious buds, and further, the amount to be supplemented can be calculated according to this length change data ΔL.

[0069] It should be noted that generally, the callus is in a rapid growth period within one month after turning into adventitious buds. At this time, it is necessary to strictly control the nutrient components to promote the rapid propagation of adventitious buds.

[0070] It should be noted that the value of 2 mm is an empirical value based on the process of the callus of Populus adenopoda turning into adventitious buds, and this value indicates the general growth rate of adventitious buds during the rapid propagation period.

[0071] In some alternative embodiments, during the induction process of the adventitious buds, continuously photograph the adventitious buds formed by the callus to obtain the change data of the adventitious buds, including the steps of:

[0072] S501. During the induction process of the adventitious buds, use a timing camera to continuously photograph the adventitious buds formed by the callus to obtain adventitious bud photos;

[0073] S502. Calculate the size change data of the adventitious buds according to the differences before and after the adventitious bud photos;

[0074] In these embodiments, through the changes in the photos before and after, the length change data of the adventitious buds on the photos can be obtained. Then, based on the actual shooting position and angle, the true length change data of the adventitious buds can be calculated. Specifically, the lens of the time-lapse camera can be set to face the plane where the adventitious buds are located, and the true length change data can be directly calculated from the magnification of the lens and the length change data on the photos, thus avoiding the need for sampling detection.

[0075] It should be noted that this process of comparing the photos of adventitious buds before and after can be directly carried out through the controller or directly in computer software to further shorten the cycle of the overall method. Additionally, if it is carried out under the control of the controller or the computer, a sterile storage tank and a flow control valve for the Populus adenopoda enzyme hydrolysate can be set. Through the control of the PLC chip of the controller or the computer, the supplementary amount of the Populus adenopoda enzyme hydrolysate can be directly calculated based on the size change data of the adventitious buds, and then the flow control valve can be controlled to control the supplementary amount of the Populus adenopoda enzyme hydrolysate to achieve the automated control of the entire process.

[0076] In some alternative embodiments, the interval time between consecutive shootings is 24h to 48h, and the total shooting duration of consecutive shootings is from the 7th day to the 28th day of the induction of the adventitious buds; and / or

[0077] the temperature for the induction of the adventitious buds is 24°C to 26°C, and the time for the induction of the adventitious buds is 35d to 45d;

[0078] In these embodiments, the interval time between consecutive shootings can be 24h to 48h, and the total shooting duration of consecutive shootings can be from the 7th day to the 28th day of the induction of the adventitious buds, so as to make the consecutive shooting process occur during the rapid growth period of the adventitious buds, facilitating the subsequent sufficient feeding process of the Populus adenopoda enzyme hydrolysate; additionally, the temperature for the induction of the adventitious buds can be 24°C to 26°C, and the time for the induction of the adventitious buds can be 35d to 45d, enabling the callus to rapidly transform into callus under suitable temperature conditions.

[0079] The interval time between consecutive shootings can be 24h, 30h, 36h, 42h or 48h.

[0080] The temperature for the induction of the adventitious buds can be 24.0°C, 24.5°C, 25.0°C, 25.5°C or 26.0°C.

[0081] The time for the induction of the adventitious buds can be 35d, 36d, 37d, 38d, 39d, 40d, 41d, 42d, 43d, 44d or 45d.

[0082] In some alternative embodiments, the method for preparing the Populus adenopoda enzyme hydrolysate includes:

[0083] S101. Mix the leaves of the Populus adenopoda and the stem segments of the Populus adenopoda to obtain an enzymatic hydrolysis raw material;

[0084] S102. Use cellulase to enzymatically hydrolyze the enzymatic hydrolysis raw material to obtain an enzymatic hydrolysate;

[0085] S103. Vacuum dry the enzymatic hydrolysate to obtain a Populus adenopoda enzymatic hydrolysate;

[0086] In these embodiments, the preparation of the Populus adenopoda enzymatic hydrolysate can use the mixture of the leaves of the Populus adenopoda and the stem segments of the Populus adenopoda to obtain an enzymatic hydrolysis raw material, and then use cellulase to enzymatically hydrolyze the leaves of the Populus adenopoda and the stem segments of the Populus adenopoda to dissociate the plant cells of these leaves and stem segments, as well as the corresponding vitamins, trace element components, sugars, and plant hormones, etc.; in addition, through the method of vacuum drying, the nutritional components of the enzymatic hydrolysate can be retained to the greatest extent while removing a part of the water to form a fluid-state Populus adenopoda enzymatic hydrolysate.

[0087] In some alternative embodiments, the weight m2 of the leaves of the Populus adenopoda and the weight m3 of the stem segments of the Populus adenopoda satisfy the relationship: m2:m3 = 1:(1.5 - 2.5); and / or

[0088] The temperature of the enzymatic hydrolysis is 25°C - 35°C, and the time of the enzymatic hydrolysis is 1h - 2h; and / or

[0089] The temperature of the vacuum drying is 50°C - 70°C, and the time of the vacuum drying is 25min - 35min;

[0090] In these embodiments, the weight m2 of the leaves of the Populus adenopoda and the weight m3 of the stem segments of the Populus adenopoda can satisfy the relationship: m2:m3 = 1:(1.5 - 2.5), which can facilitate the subsequent full enzymatic hydrolysis to obtain sufficient nutritional components such as vitamins, trace element components, sugars, and plant hormones, etc.; in addition, the temperature of the enzymatic hydrolysis can be 25°C - 35°C, and the time of the enzymatic hydrolysis can be 1h - 2h, which can promote the full enzymatic hydrolysis to fully enzymatically hydrolyze sufficient nutritional components such as vitamins, trace element components, sugars, and plant hormones, etc.; furthermore, the temperature of the vacuum drying can be 50°C - 70°C, and the time of the vacuum drying can be 25min - 35min, which can remove part of the water in the Populus adenopoda enzymatic hydrolysate through the method of vacuum drying to obtain a fluid-state Populus adenopoda enzymatic hydrolysate.

[0091] The value of the weight m3 of the stem segments of the Populus adenopoda can be 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5.

[0092] The temperature for enzymatic hydrolysis can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

[0093] The time for enzymatic hydrolysis can be 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h or 2.5 h.

[0094] The temperature for vacuum drying can be 50°C, 55°C, 60°C, 65°C or 70°C.

[0095] The time for vacuum drying can be 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min or 35 min.

[0096] In some alternative embodiments, the weight m1 of the Populus adenopoda enzymatic hydrolysate and the volume V1 of the composite culture medium satisfy the relationship: m1:V1 = (4 - 8):1. If the unit of m1 is g, then the unit of V1 is L.

[0097] In these embodiments, the weight m1 of the Populus adenopoda enzymatic hydrolysate and the volume V1 of the composite culture medium can satisfy the relationship: m1:V1 = (4 - 8):1, which can ensure that the composite culture medium contains a sufficient amount of Populus adenopoda enzymatic hydrolysate. A sufficient amount of Populus adenopoda enzymatic hydrolysate can maintain most of the nutrients required for the transformation of callus into adventitious buds.

[0098] The value of the weight m1 of the Populus adenopoda enzymatic hydrolysate can be 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

[0099] In some alternative embodiments, the components of the composite culture medium further include: MS medium, 3-indolebutyric acid, thiabendazole, sucrose, agar and Phytocide II. The mass concentration of 3-indolebutyric acid is 0.1 mg / L, the mass concentration of thiabendazole is 0.03 mg / L, the mass concentration of sucrose is 30 g / L, the mass concentration of agar is 6 g / L, and the volume concentration of Phytocide II is 0.5 mL / L.

[0100] In these embodiments, the components of the composite culture medium may further include: MS medium, 3-indolebutyric acid, thiabendazole, sucrose, agar, and Phytocide II type; and the mass concentration of 3-indolebutyric acid may be 0.1 mg / L, and the mass concentration of thiabendazole may be 0.03 mg / L, and the mass concentration of sucrose may be 30 g / L, and the mass concentration of agar may be 6 g / L, and the volume concentration of Phytocide II type may be 0.5 mL / L, so as to make the composite culture medium have sufficient nutrients to accelerate the transformation of callus into adventitious buds.

[0101] In some alternative embodiments, the temperature for rooting culture is 24°C to 26°C, and the time for rooting culture is 10 d to 14 d.

[0102] In these embodiments, the temperature for rooting culture may be 24°C to 26°C, and the time for rooting culture may be 10 d to 14 d, so as to make the rooting culture carried out at a suitable temperature and for a sufficient long time, thereby promoting the rooting of adventitious buds to form Populus adenopoda tissue.

[0103] The temperature for this rooting culture may be 24.0°C, 24.5°C, 25.0°C, 25.5°C or 26°C.

[0104] The time for this rooting culture may be 10 d, 11 d, 12 d, 13 d or 14 d.

[0105] It should be noted that the culture medium used for this rooting culture may be: 1 / 2 woody plant medium (WPM), 30 g / L of sucrose, 6 g / L of agar, and 0.5 mL / L of Phytocide II type, where 1 / 2 woody plant medium (WPM) means that all the macroelements of the existing woody plant medium are halved.

[0106] In some alternative embodiments, the pretreatment of the explant to obtain a non-toxic explant includes the steps of:

[0107] S201. Rinse and disinfect the explant to obtain a non-toxic explant; wherein, the disinfectant used for the disinfection treatment includes medical alcohol and / or sodium hypochlorite;

[0108] In these embodiments, using medical alcohol (alcohol with a mass concentration of 75%) and sodium hypochlorite as disinfectants, the disinfection method using the combination of medical alcohol and sodium hypochlorite can reduce the contamination rate of explants and has the least harm to explants. Compared with the operation of disinfecting explant seeds by the traditional combination of ethanol and mercuric chloride, the combination of medical alcohol and sodium hypochlorite has less environmental pollution and does not affect the safety of technicians.

[0109] It should be noted that if the surface of Populus adenopoda is dirty, a small amount of mild detergent can be added for rinsing.

[0110] The following further elaborates on this application in combination with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are usually determined according to national standards / industry standards; if there are no corresponding national or industry standards, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0111] Example 1

[0112] The following process is carried out at the Modern Agricultural R & D Base of Sichuan Agricultural University:

[0113] As Figure 2 shown, a method for precisely cultivating Populus adenopoda tissue includes:

[0114] S1. Pick young Populus adenopoda leaves as explants and place them in clean water for rinsing for 2 h to 3 h;

[0115] S2. Immerse the explants in ethanol with a mass concentration of 75% for 30 s, then rinse them with sterile water 6 times, and then treat them with sodium hypochlorite with a mass concentration of 2% for 2 min, or treat them with benzalkonium chloride with a mass concentration of 0.5% for 18 min, and then rinse them with sterile water 6 times. Finally, cut the young leaves into small pieces of 1 cm 2 to obtain non-toxic explants;

[0116] The non-toxic explants are placed in the W1 - W13 callus induction medium, and the total contamination rate and mortality rate of 13 groups within 7 days are respectively counted. The results are shown in Table 1.

[0117] Table 1 Effects of different disinfection methods on tissue culture of Populus adenopoda

[0118] ,

[0119] As can be seen from Table 1, considering the two different disinfection treatment methods, the disinfection method combining ethanol with a mass concentration of 75% and sodium hypochlorite solution with a mass concentration of 2% has the best effect, the lowest contamination rate, and reaches the most ideal effect.

[0120] S3. Under sterile conditions, inoculate the non-toxic explants in the callus induction medium for callus induction, and after dark culture at 20°C - 22°C for 7 d - 14 d, the callus as shown in Figure 3 is obtained; the formula of the callus induction medium is: WPM (purchased from Shandong Top Biological Engineering Co., Ltd.), 1.5 mg / L of NAA, 3 mg / L of ZT, 30 g / L of sucrose, 0.5 mL / L of Plant Culture Net II (Mumu Biology), and 6 g / L of agar, and its pH value is 5.8; the specific process:

[0121] Using WPM as the basic medium, a Plackett-Burman design experiment of three plant growth regulators (6-BA, ZT, NAA) was conducted using Minitab 19. All groups were cultured in the dark at 20 °C - 22 °C for 7 - 14 days, and the callus rate was counted (only the callus area accounting for 3 / 4 of the entire leaf area was counted). The results are shown in Table 2.

[0122] Table 2 Effects of different callus media on the induction effect of Populus adenopoda

[0123] ,

[0124] Based on the 13 groups of treatments, as can be seen from Table 2, the callus induction rate of group W4 is the highest. That is, the optimal callus induction medium is: WPM, 1.5 mg / L of NAA, 3 mg / L of ZT, 30 g / L of sucrose, 0.5 mL / L of Plant Tissue Culture Disinfectant II, and 6 g / L of agar. The pH value of this callus induction medium is 5.8.

[0125] S101. Mix the leaves and stem segments of Populus adenopoda to obtain the enzymatic hydrolysis raw material;

[0126] S102. Use cellulase to enzymatically hydrolyze the enzymatic hydrolysis raw material to obtain an enzymatic hydrolysate;

[0127] S103. Vacuum dry the enzymatic hydrolysate to obtain the Populus adenopoda enzymatic hydrolysate;

[0128] S501. During the induction of adventitious buds, continuously photograph the adventitious buds formed from the callus using a time-lapse camera to obtain adventitious bud photos;

[0129] S502. Calculate the size change data of the adventitious buds based on the differences before and after the adventitious bud photos;

[0130] S601. Determine the length change data ΔL of the adventitious buds per unit time based on the change data of the adventitious buds. Among them, the length change data of the adventitious buds per unit time is the length difference between the tip of the bud leaf of the adventitious buds before and after the unit time;

[0131] S602. Determine whether to supplement the Populus adenopoda enzymatic hydrolysate based on the length change data ΔL;

[0132] If the length change data ΔL ≤ 2 mm, then the Populus adenopoda enzymatic hydrolysate needs to be supplemented. Calculate the supplementation amount of the Populus adenopoda enzymatic hydrolysate based on the length change data ΔL. Among them, the calculation formula for the supplementation amount of the Populus adenopoda enzymatic hydrolysate is: m = 0.3×ΔL + 0.2. If the unit of m is g, then the unit of ΔL is mm;

[0133] S7. According to the supplementary amount of Populus adenopoda enzymatic hydrolysate, add Populus adenopoda enzymatic hydrolysate to the complex medium to obtain adventitious buds; among them, the Populus adenopoda enzymatic hydrolysate has the vitamin and trace element components of Populus adenopoda itself;

[0134] The interval time of continuous shooting is 36 h, and the total shooting duration of continuous shooting is from the 7th day to the 15th day of the induction of adventitious buds;

[0135] The temperature for the induction of adventitious buds is 25 °C, and the induction time of adventitious buds is 40 d.

[0136] The weight m2 of the leaves of Populus adenopoda and the weight m3 of the stem segments of Populus adenopoda satisfy the relationship: m2:m3 = 1:2;

[0137] The temperature for enzymatic hydrolysis is 30 °C, and the enzymatic hydrolysis time is 1.5 h;

[0138] The temperature for vacuum drying is 60 °C, and the vacuum drying time is 30 min.

[0139] The weight m1 of Populus adenopoda enzymatic hydrolysate and the volume V1 of the complex medium satisfy the relationship: m1:V1 = 7:1. If the unit of m1 is g, then the unit of V1 is L.

[0140] Use the above process to investigate the effect of different induction media of adventitious buds on Populus adenopoda. The induction time of adventitious buds in this experimental process is 15 d. Among them, for the treatment numbers named with the letter W at the beginning, the basic medium used is WPM, and for the treatment numbers named with the letter C at the beginning, the basic medium used is MS. The results are shown in Table 3.

[0141] Table 3 Effects of different induction media of adventitious buds on the formation of adventitious buds of Populus adenopoda

[0142] ,

[0143] ,

[0144] ,

[0145] In Table 3, Populus adenopoda enzymatic hydrolysate was added to both the C24 and C25 groups, but only the C25 group used the Populus adenopoda enzymatic hydrolysate feeding control method provided in the examples of the present application. The obtained adventitious bud tissues are respectively Figure 5 and Figure 4 as shown.

[0146] After integrating 46 groups of treatments and culturing for 35 d to 45 d, the callus quality of group C24 was the best and the plantlets grew normally. That is, the optimal adventitious bud induction medium was: MS medium, 0.1 mg / L IBA, 0.03 mg / L TDZ, 30 g / L sucrose, 6 g / L agar, 0.5 mL / L Plant Tissue Culture Disinfectant II type, and 7 g / L Populus adenopoda enzyme hydrolysate. The pH value of this induction medium was 5.8.

[0147] S8. Under the conditions of 3000 lx light for 14 h per day and sterile conditions, the adventitious buds were cultured for rooting to obtain the Populus adenopoda tissue as shown in Figure 6 . The medium used for rooting culture was: 1 / 2 WPM, 30 g / L sucrose, 6 g / L agar, 0.5 mL / L Plant Tissue Culture Disinfectant II type. The pH value of this medium was 5.8. The specific process was as follows:

[0148] Under sterile conditions, the adventitious buds were inoculated into different rooting media and cultured for 10 d to 14 d at 24 °C to 26 °C under the condition of 3000 lx light for 14 h per day for rooting culture. After 7 d to 10 d, the root growth was observed. The results are shown in Table 4.

[0149] Table 4 Effects of different rooting media on the rooting induction of Populus adenopoda

[0150] ,

[0151] After integrating 4 groups of treatments, as can be seen from Table 4, after culturing for 7 d to 10 d, the rooting of group G2 was the best and the plantlets grew normally. That is, the optimal rooting induction medium was: 1 / 2 WPM, 30 g / L sucrose, 6 g / L agar, 0.5 mL / L Plant Tissue Culture Disinfectant II type. The pH value of this medium was 5.8.

[0152] The above-obtained Populus adenopoda tissue was continuously cultured to obtain the Populus adenopoda seedlings as shown in Figure 7 .

[0153] In summary, the method for precisely culturing Populus adenopoda tissue provided in the embodiment of the present application, based on the induction process of callus induction and transformation into adventitious buds, continuously photographs the adventitious buds formed by the callus to clarify the morphological change data of adventitious buds during the induction process. Then, according to this change data, the addition amount of Populus adenopoda enzyme hydrolysate can be determined, which can accelerate the speed of callus formation into adventitious buds and improve the propagation rate of Populus adenopoda tissue.

[0154] In addition, a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application is not affected by environmental factors such as weather and season during the process of producing Populus adenopoda seedlings, and a large number of seedlings can be obtained in a short period; this method belongs to the category of asexual reproduction, enabling the genetic traits of Populus adenopoda to be stably passed on to the greatest extent.

[0155] In addition, a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application uses the leaves of Populus adenopoda as raw materials. By inducing young tender leaves to form callus and then transforming them into adventitious buds, problems such as long cultivation cycles and low reproduction rates existing in the existing proliferation technology of Populus adenopoda can be solved, providing certain technical support for the industrialized breeding of Populus adenopoda.

[0156] In addition, for a method for precisely cultivating Populus adenopoda tissue provided by an embodiment of the present application, the product of Phytocide II is added to the culture medium used in each step. Different from traditional single antibacterial means, the addition of the product of Phytocide II neither affects the normal growth of Populus adenopoda nor can it avoid the explants being contaminated in the later stage of cultivation.

[0157] The above are only specific implementation manners of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but rather to the broadest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A method for accurately cultivating Populus sibiricum tissue, the method comprising: Pick young and tender leaves of Populus siliquae as explants; Pre-treating the explant to obtain a non-toxic explant; The non-toxic explants are callus induced to obtain callus tissue; the medium formula for callus induction is: WPM, 1.5 mg / L NAA, 3 mg / L ZT, 30 g / L sucrose, 0.5 mL / L Zhipeijing Type II and 6 g / L agar, and its pH is 5.8; Using a composite culture medium containing enzymatic hydrolysate of Populus scolopendra, inducing adventitious buds from the callus tissue to obtain adventitious buds; the induction temperature of the adventitious buds is 24° C. to 26° C., and the induction time of the adventitious buds is 35 days to 45 days; The components of the composite culture medium are: MS culture medium, 0.1 mg / L IBA, 0.03 mg / L TDZ, 30 g / L sucrose, 6 g / L agar, 0.5 mL / L Zhipeijing II and 7 g / L of Populus scolopendra enzymatic hydrolysate, and the pH of the culture medium is 5.8; The preparation method of the enzymatic hydrolysate of Populus sphenanthera comprises: The leaves of the Populus striata and the stem segments of the Populus striata are mixed to obtain an enzymatic hydrolysis raw material; Using cellulase to enzymolyze the enzymolysis raw material to obtain an enzymolysis solution; The enzymatic hydrolysate is vacuum dried to obtain an enzymatic hydrolysate of Populus scolopendra; The weight m2 of the leaf of the Populus sibiricus and the weight m3 of the stem of the Populus sibiricus satisfy the relationship: m2:m3=1:(1.5-2.5); The enzymatic hydrolysis temperature is 25°C to 35°C, and the enzymatic hydrolysis time is 1h to 2h; The vacuum drying temperature is 50°C to 70°C, and the vacuum drying time is 25min to 35min; The adventitious buds are subjected to rooting culture to obtain Populus scolopendra tissue; the temperature of the rooting culture is 24°C to 26°C, and the time of the rooting culture is 10d to 14d; the culture medium used for the rooting culture is: 1 / 2WPM, 30g / L sucrose, 6g / L agar, 0.5mL / L Zhipeijing Type II, and the pH value of the culture medium is 5.

8.

2. The method according to claim 1, wherein the explant is pre-treated to obtain a non-toxic explant, comprising the steps of: The explant is rinsed and disinfected to obtain a non-toxic explant; among which, The disinfectant used in the disinfection process includes medical alcohol and / or sodium hypochlorite.

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