Method for accurately cultivating populus hopeiensis tissue

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for accurately culturing populus hopeiensis tissues. The method comprises the following steps: picking tender populus bolleana leaves as explants; the method comprises the following steps: pretreating an explant to obtain a non-toxic explant; performing callus induction on the non-toxic explant to obtain a callus; carrying out adventitious bud induction on the calluses by using a composite culture medium containing populus lanceolata zymolyte; in the induction process of the adventitious buds, continuously shooting the adventitious buds formed by the calluses to obtain change data of the adventitious buds; according to the change data of the adventitious buds, determining the supplementing amount of the populus lanceolata zymolyte; according to the supplementing amount of the populus lanceolata zymolyte, supplementing the populus lanceolata zymolyte into the composite culture medium to obtain adventitious buds; wherein the populus henryi zymolyte has vitamin and trace element components of the populus henryi; and carrying out rooting culture on the adventitious buds to obtain populus lanceolata tissues. According to the method, the speed of forming adventitious buds by the calluses can be increased, and the propagation rate of populus hopeiensis tissues can be increased.

Description

Technical Field

[0001] The present application relates to the field of plant tissue culture technology, and in particular to a method for accurately cultivating Populus scolopendra tissue. Background Art

[0002] Populus rapa Populus adenopoda Maxim ) is a plant of the genus Populus in the family Salicaceae and is a tree; Populus rapa has the characteristics of a straight trunk, high branches 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 the preparation of furniture and paper. Populus rapa is generally dioecious, and the female plant has the characteristics of a tall trunk, extremely small seeds and a low germination rate. It is difficult to maintain the quality of the mother plant if traditional seed propagation is used, and the conventional field cutting propagation method has the defect of difficulty in rooting. Therefore, at this stage, it can be cultivated by leaf explant induction, but the cycle of this cultivation method is relatively long, generally close to 90 days; in addition, the existing technology can also obtain regenerated plants of Populus rapa by stem segment tissue culture, but this method is similar to sterile cuttings and has problems such as low reproduction efficiency.

[0003] Although rapid propagation of Populus quascens can be achieved through tissue culture of Populus quascens at this stage, the tissue culture of Populus quascens generally involves adding a specific culture medium, and then supplementing nutrients based on the consumption of nutrients. The consumption of nutrients requires the actual measurement of these specific culture media, and this measurement process undoubtedly increases the cultivation cycle and operation complexity of Populus quascens. In addition, the process of measuring these nutrients will be interfered by a large number of factors, resulting in inaccurate subsequent nutrient supplementation, which will affect the final reproduction rate of the Populus quascens tissue. Summary of the invention

[0004] The present application provides a method for accurately cultivating Populus rapa tissue to solve the following technical problem: how to improve the induction efficiency and reproduction rate of the Populus rapa tissue cultivation process.

[0005] In a first aspect, the present application provides 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; Inducing callus on the non-toxic explant to obtain callus tissue; Using a composite culture medium containing enzymatic hydrolysate of Populus scolopendra, inducing adventitious buds from the callus tissue; During the induction process of the adventitious buds, continuously photographing the adventitious buds formed by the callus tissue to obtain change data of the adventitious buds; Determining the amount of the enzymatic hydrolysate to be added according to the change data of the adventitious buds; According to the added amount of the enzymatic hydrolysate of Populus sirmifolia, the enzymatic hydrolysate of Populus sirmifolia is added to the composite culture medium to obtain adventitious buds; wherein the enzymatic hydrolysate of Populus sirmifolia contains vitamins and trace elements of Populus sirmifolia itself; The adventitious buds are subjected to rooting culture to obtain Populus scolopendra tissue.

[0006] Optionally, the method of determining the amount of the Populus scolopendra enzymatic hydrolysate to be added according to the change data of the adventitious buds comprises the steps of: Determine the length change data ΔL of the adventitious bud per unit time according to the change data of the adventitious bud, wherein the length change data of the adventitious bud per unit time is the length difference of the bud leaf tip of the adventitious bud before and after the unit time; According to the length change data ΔL, judging whether it is necessary to add the enzymatic hydrolysate of Populus scolopendra; If the length change data ΔL≤2mm, it is necessary to add poplar hydrolysate, and the additional amount of the poplar hydrolysate is calculated according to the length change data ΔL; wherein, the calculation formula for the additional amount of the poplar hydrolysate is: m=(0.2~0.4)×ΔL+0.2, if the unit of m is g, the unit of ΔL is mm.

[0007] Optionally, during the induction process of the adventitious buds, continuously photographing the adventitious buds formed by the callus tissue to obtain the change data of the adventitious buds comprises the steps of: During the induction process of the adventitious buds, the adventitious buds formed by the callus tissue are continuously photographed using a time-lapse camera to obtain photographs of the adventitious buds; According to the before and after differences of the adventitious bud photos, the size change data of the adventitious bud is calculated.

[0008] Optionally, the interval time of the continuous shooting is 24h to 48h, and the total shooting time of the continuous shooting is from the 7th day to the 28th day of the induction of the adventitious buds; and / or The temperature for inducing the adventitious buds is 24° C. to 26° C., and the time for inducing the adventitious buds is 35 days to 45 days.

[0009] Optionally, the method for preparing the enzymatic hydrolysate of Populus sirmifolia 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 enzymolysis solution is vacuum dried to obtain the Populus sirmifolia enzymolysis product.

[0010] Optionally, the weight m2 of the leaf of the Populus rapa and the weight m3 of the stem of the Populus rapa satisfy the relationship: m2:m3=1:(1.5-2.5); and / or The enzymatic hydrolysis temperature is 25°C to 35°C, and the enzymatic hydrolysis time is 1h to 2h; and / or The vacuum drying temperature is 50° C. to 70° C., and the vacuum drying time is 25 min to 35 min.

[0011] Optionally, the weight m1 of the Populus sirmifolia enzymatic hydrolysate and the volume V1 of the composite culture medium satisfy the relationship: m1:V1=(4-8):1, and if the unit of m1 is g, the unit of V1 is L.

[0012] Optionally, the components of the composite culture medium also include: MS culture medium, 3-indolebutyric acid, thiabendazole, sucrose, agar and Zhipeijing type II; the mass concentration of the 3-indolebutyric acid is 0.1 mg / L, the mass concentration of the thiabendazole is 0.03 mg / L, the mass concentration of the sucrose is 30 g / L, the mass concentration of the agar is 6 g / L, and the volume concentration of the Zhipeijing type II is 0.5 mL / L.

[0013] Optionally, the temperature of the rooting culture is 24° C. to 26° C., and the time of the rooting culture is 10 days to 14 days.

[0014] Optionally, 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; wherein the disinfectant used in the disinfection includes medical alcohol and / or sodium hypochlorite.

[0015] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art: The embodiment of the present application provides a method for accurately cultivating Populus rapa tissue. The method continuously photographs the adventitious buds formed by the callus tissue on the basis of the induction process of callus tissue induction into adventitious buds to clarify the morphological change data of the adventitious buds during the induction process, and then the amount of Populus rapa hydrolysate to be added can be clarified based on the change data. The vitamins and trace elements of the Populus rapa hydrolysate itself can be quickly absorbed by the callus tissue, thereby accelerating the speed of adventitious bud formation by the callus tissue. In addition, the Populus rapa hydrolysate also has more sugars and plant hormones, which can quickly enter the callus tissue to further accelerate the induction efficiency of adventitious bud formation by the callus tissue. In addition, the method determines the amount of Populus rapa hydrolysate to be added through the change data of the adventitious buds, which can avoid the tedious operation of direct sampling and the interference of various factors, and has a certain accuracy, thereby improving the reproduction rate of Populus rapa tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 A schematic flow chart of a method for accurately cultivating Populus scolopendra tissue provided in an embodiment of the present application; Figure 2 A detailed schematic diagram of a method for accurately cultivating Populus scolopendra tissue provided in an embodiment of the present application; Figure 3 This is a result diagram of callus tissue obtained by a method for accurately cultivating Populus sibiricus tissue provided in Example 1 of the present application; Figure 4 This is a diagram of the adventitious bud results obtained after 15 days of induction using the C25 group adventitious bud induction medium provided in Example 1 of the present application; Figure 5 This is a diagram showing the adventitious buds obtained after 15 days of induction using the C24 adventitious bud induction medium provided in Example 1 of the present application; Figure 6 A result diagram of Populus sibiricus tissue obtained by a method for accurately cultivating Populus sibiricus tissue provided in Example 1 of the present application; Figure 7 This is a result diagram of the Populus scolopendra seedlings provided in Example 1 of the present application. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0020] Various embodiments of the present application may be presented 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 understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the 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, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0021] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, "plurality" means two or more; "at least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e. a and b), ac, bc, or abc, where a, b, and c can be single or plural, respectively. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional 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 to the proportional numbers in the proportional 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.

[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0023] It should be noted that at present, the process of converting callus tissue 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 later; however, the nutrients in these culture media are generally excessive to ensure the conversion of callus tissue into adventitious buds, but existing studies have shown that excessive nutrients will affect the growth of adventitious buds. Therefore, there are related equipment for flow-adding nutrients at this stage, but most of these flow-adding equipment cannot open 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 foreign bacteria.

[0024] Current studies have shown that the amount of nutrients will directly affect the morphology of adventitious buds formed by callus tissue and the growth status of buds and leaves of adventitious buds. Therefore, if the growth status of buds and leaves of adventitious buds can be used to directly reflect the consumption of certain nutrients in the culture medium, it can effectively accelerate the efficiency of callus tissue transformation into adventitious buds and avoid the defects of direct sampling.

[0025] Figure 1 A schematic diagram of a method for accurately cultivating Populus scolopendra tissue provided in an embodiment of the present application is exemplarily shown; like Figure 1 As shown, the present application embodiment provides a method for accurately cultivating Populus sibiricum tissue, the method comprising: S1. Pick young leaves of Populus sibiricus as explants; S2. pre-treating the explant to obtain a non-toxic explant; S3. inducing callus on the non-toxic explant to obtain callus tissue; S4. Inducing adventitious buds from the callus using a composite medium containing an enzymatic hydrolysate of Populus sibiricum; S5. During the induction process of the adventitious buds, continuously photographing the adventitious buds formed by the callus to obtain the change data of the adventitious buds; S6. Determine the amount of the enzymatic hydrolysate to be added according to the change data of the adventitious buds; S7. according to the amount of the enzymatic hydrolysate of Populus sirmifolia added, the enzymatic hydrolysate of Populus sirmifolia is added to the composite culture medium to obtain adventitious buds; wherein the enzymatic hydrolysate of Populus sirmifolia contains vitamins and trace elements of Populus sirmifolia itself; S8. The adventitious buds are subjected to rooting culture to obtain Populus scolopendra tissue.

[0026] It should be noted that the components of the culture medium used for callus induction include: woody plant medium (WPM), 1.5 mg / L auxin (NAA), 3 mg / L zeatin (ZT), 30 g / L sucrose, 0.5 mL / L Zhipeijing Type II and 6 g / L agar.

[0027] Figure 2A detailed schematic diagram of a method for accurately cultivating Populus scolopendra tissue provided in an embodiment of the present application is exemplarily shown; In some optional embodiments, such as Figure 2 As shown, according to the change data of the adventitious buds, determining the amount of the added Populus sibiricum enzymatic hydrolysate comprises the steps of: S601. Determine the length change data ΔL of the adventitious bud per unit time according to the change data of the adventitious bud, wherein the length change data of the adventitious bud per unit time is the length difference of the bud leaf tip of the adventitious bud before and after the unit time; S602. judging whether it is necessary to add the enzymatic hydrolysate of Populus sirmifolia according to the length change data ΔL; If the length change data ΔL≤2mm, it is necessary to add the Populus scolopendra enzymatic hydrolysate, and the additional amount of the Populus scolopendra enzymatic hydrolysate is calculated according to the length change data ΔL; wherein the calculation formula for the additional amount of the Populus scolopendra enzymatic hydrolysate is: m=(0.2-0.4)×ΔL+0.2, if the unit of m is g, the unit of ΔL is mm; 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, so that the growth rate of the adventitious buds in two adjacent time periods can be clearly determined, and the changes in the tips of the buds and leaves of the adventitious buds during the growth process are more obvious. Therefore, the length change data ΔL of the tips of the buds and leaves of the adventitious buds can be used to roughly deduce whether there is a lack of nutrients, and the amount to be supplemented can be further estimated based on this length change data ΔL.

[0028] It should be noted that generally callus tissue is in a rapid growth period within one month of transforming into adventitious buds. At this time, the nutrient content needs to be strictly controlled to promote the rapid reproduction of adventitious buds.

[0029] It should be noted that the value of 2mm is an empirical value based on the process of conversion of callus tissue of Populus rapa into adventitious buds, which indicates the general growth rate of adventitious buds during the rapid propagation period.

[0030] In some optional embodiments, during the induction process of the adventitious buds, continuously photographing the adventitious buds formed by the callus to obtain the change data of the adventitious buds comprises the steps of: S501. During the induction process of the adventitious buds, the adventitious buds formed by the callus are continuously photographed using a timer camera to obtain photographs of the adventitious buds; S502. Calculate the size change data of the adventitious buds according to the before and after differences of the adventitious buds photos; In these embodiments, the length change data of the adventitious buds in the photo can be obtained through the front and back changes of the photo, and the real length change data of the adventitious buds can be calculated based on the actual shooting position and angle. Specifically, the lens of the timing camera can be set to face the plane where the adventitious buds are located, and the real length change data can be directly calculated using the magnification of the lens and the length change data in the photo, thereby avoiding sampling detection.

[0031] It should be noted that this process can be directly performed through a controller or in a computer software based on the difference between the before and after photos of the adventitious buds, so as to further reduce the cycle of the overall method. In addition, if it is performed under the control of a controller or a computer, a sterile storage tank and a flow control valve for the hydrolyzate of Populus striata can be set, and the amount of the hydrolyzate of Populus striata can be directly calculated based on the size change data of the adventitious buds through the PLC chip control of the controller or the computer, and then the flow control valve can be controlled to control the amount of the hydrolyzate of Populus striata to be added, so as to realize the automatic control of the whole process.

[0032] In some optional embodiments, the interval time of the continuous shooting is 24h to 48h, and the total shooting time of the continuous shooting is from the 7th day to the 28th day of the induction of the adventitious buds; and / or The temperature for inducing the adventitious buds is 24°C to 26°C, and the time for inducing the adventitious buds is 35d to 45d; In these embodiments, the interval time of continuous shooting can be 24h~48h, and the total shooting time of continuous shooting can be the 7th to 28th day of the induction of adventitious buds, so as to promote the continuous shooting process during the rapid growth period of adventitious buds, so as to promote the subsequent flow addition process of the enzymatic hydrolysate of Populus quascens to be fully carried out; in addition, the temperature for inducing adventitious buds can be 24℃~26℃, and the time for inducing adventitious buds can be 35d~45d, so as to promote the callus tissue to be quickly transformed into callus tissue under suitable temperature conditions.

[0033] The interval time of the continuous shooting can be 24h, 30h, 36h, 42h or 48h.

[0034] The temperature for induction of the adventitious buds may be 24.0°C, 24.5°C, 25.0°C, 25.5°C or 26.0°C.

[0035] The time for inducing the adventitious buds may be 35d, 36d, 37d, 38d, 39d, 40d, 41d, 42d, 43d, 44d or 45d.

[0036] In some optional embodiments, the method for preparing the enzymatic hydrolysate of Populus sirmifolia comprises: S101. mixing the leaves of the Populus sirmifolia and the stem segments of the Populus sirmifolia to obtain an enzymatic hydrolysis raw material; S102. The enzymatic hydrolysis raw material is enzymatically hydrolyzed using cellulase to obtain an enzymatic hydrolyzate; S103. The enzymatic hydrolyzate is vacuum dried to obtain a Populus sirmifolia enzymatic hydrolyzate; In these embodiments, the preparation of the enzymatic hydrolysate of Populus rapa can be achieved by mixing the leaves and stem segments of Populus rapa to obtain an enzymatic hydrolysate raw material, and then enzymatically hydrolyzing the leaves and stem segments of Populus rapa by cellulase to separate the plant cells of these leaves and stem segments and the corresponding vitamins, trace elements, sugars, plant hormones, etc.; in addition, vacuum drying can be used to retain the nutrients of the enzymatic hydrolysate to the greatest extent while removing a portion of the water to form a fluid state of the Populus rapa enzymatic hydrolysate.

[0037] In some optional embodiments, the weight m2 of the leaf of the Populus rapa and the weight m3 of the stem segment of the Populus rapa satisfy the relationship: m2:m3=1:(1.5-2.5); and / or The enzymatic hydrolysis temperature is 25°C to 35°C, and the enzymatic hydrolysis time is 1h to 2h; and / or The vacuum drying temperature is 50°C to 70°C, and the vacuum drying time is 25min to 35min; In these embodiments, the weight m2 of the leaves of Populus radiata and the weight m3 of the stem segments of Populus radiata can satisfy the relationship: m2:m3=1:(1.5-2.5), which can facilitate the subsequent enzymatic hydrolysis to be fully carried out to obtain sufficient nutrients such as vitamins, trace elements, sugars and plant hormones; in addition, the enzymatic hydrolysis temperature can be 25°C-35°C, and the enzymatic hydrolysis time can be 1h-2h, which can promote the enzymatic hydrolysis to be fully carried out to fully hydrolyze sufficient nutrients such as vitamins, trace elements, sugars and plant hormones; in addition, the vacuum drying temperature can be 50°C-70°C, and the vacuum drying time can be 25min-35min, and part of the water in the Populus radiata hydrolysate can be removed by vacuum drying to obtain a fluid state of the Populus radiata hydrolysate.

[0038] The weight m3 of the stem segment of the poplar 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.

[0039] The temperature of the 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.

[0040] The enzymatic hydrolysis time 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.

[0041] The vacuum drying temperature may be 50°C, 55°C, 60°C, 65°C or 70°C.

[0042] The vacuum drying time 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.

[0043] In some optional embodiments, the weight m1 of the Populus scolopendra 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, the unit of V1 is L; In these embodiments, the weight m1 of the Populus hortensis hydrolysate and the volume V1 of the composite culture medium can satisfy the relationship: m1:V1=(4-8):1, so that the composite culture medium has a sufficient amount of the Populus hortensis hydrolysate, and the sufficient amount of the Populus hortensis hydrolysate can maintain most of the nutrients for the transformation of callus tissue into adventitious buds.

[0044] The weight m1 of the Populus sirmifolia enzymatic hydrolysate can be 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0.

[0045] In some optional embodiments, the components of the composite culture medium also include: MS culture medium, 3-indolebutyric acid, thiabendazole, sucrose, agar and Zhipeijing type II; the mass concentration of the 3-indolebutyric acid is 0.1 mg / L, the mass concentration of the thiabendazole is 0.03 mg / L, the mass concentration of the sucrose is 30 g / L, the mass concentration of the agar is 6 g / L, and the volume concentration of the Zhipeijing type II is 0.5 mL / L.

[0046] In these embodiments, the components of the composite culture medium may also include: MS culture medium, 3-indolebutyric acid, thiabendazole, sucrose, agar and Zhipeijing Type II; 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 Zhipeijing Type II may be 0.5 mL / L, so that the composite culture medium has sufficient nutrients to accelerate the rate at which callus tissue is transformed into adventitious buds.

[0047] In some optional embodiments, the temperature of the rooting culture is 24° C. to 26° C., and the time of the rooting culture is 10 days to 14 days.

[0048] In these embodiments, the rooting culture temperature can be 24° C. to 26° C., and the rooting culture time can be 10 days to 14 days, so that the rooting culture is carried out at a suitable temperature and for a sufficiently long time, thereby promoting the rooting of adventitious buds to form Populus rapa tissue.

[0049] The temperature of the rooting culture may be 24.0°C, 24.5°C, 25.0°C, 25.5°C or 26°C.

[0050] The rooting culture period can be 10 days, 11 days, 12 days, 13 days or 14 days.

[0051] It should be noted that the culture medium used for rooting culture can be: 1 / 2 woody plant medium (WPM), 30g / L sucrose, 6g / L agar and 0.5mL / L Zhipeijing Type II, wherein 1 / 2 woody plant medium (WPM) means that all the macroelements of the existing woody plant culture medium are reduced by half.

[0052] In some optional embodiments, the explant is pre-treated to obtain a non-toxic explant, comprising the steps of: S201. The explant is rinsed and disinfected to obtain a non-toxic explant; wherein the disinfectant used for the disinfection comprises medical alcohol and / or sodium hypochlorite; In these embodiments, medical alcohol (alcohol with a mass concentration of 75%) and sodium hypochlorite are used as disinfectants. The disinfection method using a combination of medical alcohol and sodium hypochlorite can reduce the explant contamination rate and the disinfection method is minimally harmful to the explants. Compared with the traditional combination of ethanol and mercuric chloride for disinfecting explant seeds, the combination of medical alcohol and sodium hypochlorite has less pollution to the environment and will not affect the safety of technicians.

[0053] It should be noted that if the surface of the poplar is dirty, you can add a small amount of mild detergent to rinse it.

[0054] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually measured in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are measured in accordance with common international standards, conventional conditions or conditions recommended by the manufacturer. Example 1

[0055] The following process was carried out at the Modern Agriculture Research and Development Base of Sichuan Agricultural University: like Figure 2 As shown, a method for accurately cultivating Populus striata tissue comprises: S1. Pick young leaves of Populus sibiricus as explants and rinse them in clean water for 2h~3h; S2. Soak the explants in 75% ethanol for 30 seconds, then rinse with sterile water for 6 times, then treat with 2% sodium hypochlorite for 2 minutes, or treat with 0.5% Sanisol for 18 minutes, then rinse with sterile water for 6 times, and finally cut the young leaves into 1 cm 2 Small pieces of non-toxic explants were obtained; The non-toxic explants were placed in callus induction medium W1-W13, and the total contamination rate and mortality rate of the 13 groups within 7 days were counted. The results are shown in Table 1.

[0056] Table 1 Effects of different disinfection methods on tissue culture of Populus sphenanthera , As shown in Table 1, based on the two different disinfection treatment methods, the disinfection method combining 75% ethanol and 2% NaClO solution has the best effect, the lowest contamination rate, and achieves the most ideal effect.

[0057] S3. Under sterile conditions, the non-toxic explants were inoculated into callus induction medium for callus induction, and cultured in the dark at 20°C to 22°C for 7 to 14 days to obtain the following Figure 3 The callus tissue shown in the figure; the formula of callus induction medium is: WPM (purchased from Shandong Top Bioengineering Co., Ltd.), 1.5mg / L NAA, 3mg / L ZT, 30g / L sucrose, 0.5mL / L Zhipeijing Type II (Mumu Biotechnology) and 6g / L agar, with a pH of 5.8; specific process: Plackett-Burman design experiments were conducted with three plant growth regulators (6-BA, ZT, and NAA) using WPM as the basic culture medium using Minitab 19. All groups were cultured at 20°C to 22°C in the dark for 7 to 14 days, and the callus rate was counted (only the callus area where the callus occupied 3 / 4 of the entire leaf area was counted). The results are shown in Table 2.

[0058] Table 2 Effects of different callus culture media on the induction of Populus scolopendra , Comprehensively considering the 13 treatments, it can be seen from Table 2 that the callus induction rate of group W4 was the highest. That is, the best callus induction medium was: WPM, 1.5 mg / L NAA, 3 mg / L ZT, 30 g / L sucrose, 0.5 mL / L Zhipeijing II and 6 g / L agar, and the pH of the callus induction medium was 5.8.

[0059] S101. The leaves and stems of Populus sibiricum are mixed to obtain an enzymatic hydrolysis raw material; S102. using cellulase to enzymatically hydrolyze the raw material to obtain an enzymatic solution; S103. The enzymatic hydrolyzate is vacuum dried to obtain a Populus sirmifolia enzymatic hydrolyzate; S501. During the induction of adventitious buds, the adventitious buds formed by the callus are continuously photographed using a time-lapse camera to obtain photographs of the adventitious buds; S502. Calculate the size change data of the adventitious buds according to the before and after differences in the adventitious bud photos; S601. Determine the length change data ΔL of the adventitious buds per unit time according to the change data of the adventitious buds, wherein the length change data of the adventitious buds per unit time is the length difference of the bud leaf tip of the adventitious buds before and after the unit time; S602. According to the length change data ΔL, it is determined whether it is necessary to add the enzymatic hydrolysate of Populus sirmifolia; If the length change data ΔL≤2mm, it is necessary to add the Populus scolopendra enzymatic hydrolysate, and the amount of the Populus scolopendra enzymatic hydrolysate added is calculated according to the length change data ΔL; wherein, the calculation formula for the amount of the Populus scolopendra enzymatic hydrolysate added is: m=0.3×ΔL+0.2, if the unit of m is g, the unit of ΔL is mm; S7. According to the amount of the added Populus sirens hydrolysate, the Populus sirens hydrolysate is added to the composite medium to obtain adventitious buds; wherein the Populus sirens hydrolysate has the vitamins and trace elements of the Populus sirens itself; The interval between continuous shooting was 36 h, and the total shooting duration of continuous shooting was from the 7th to the 15th day of adventitious bud induction; The temperature for inducing adventitious buds was 25°C, and the time for inducing adventitious buds was 40 days.

[0060] The weight m2 of the leaf of Populus striata and the weight m3 of the stem of Populus striata satisfy the relationship: m2:m3=1:2; The temperature of enzymatic hydrolysis was 30°C and the time of enzymatic hydrolysis was 1.5 h; The vacuum drying temperature was 60°C and the vacuum drying time was 30 min.

[0061] The weight m1 of the enzymatic hydrolysate of Populus scolopendra and the volume V1 of the composite culture medium satisfy the relationship: m1:V1=7:1. If the unit of m1 is g, the unit of V1 is L.

[0062] The above process was used to investigate the effects of different induction culture media for adventitious buds on Populus rapa. The induction time of adventitious buds in this test process was 15 days. Among them, the basic culture medium used for the treatment number starting with W was WPM, and the basic culture medium used for the treatment number starting with C was MS. The results are shown in Table 3.

[0063] Table 3 Effects of different adventitious bud induction medium treatments on adventitious bud formation of Populus scolopendra , , , In Table 3, both C24 and C25 groups were added with the Populus scolopendra enzymatic hydrolysate, but only C25 group used the Populus scolopendra enzymatic hydrolysate flow control method provided in the embodiment of the present application, and the adventitious bud tissues obtained were Figure 5 and Figure 4 shown.

[0064] Comprehensively considering the 44 treatments, after 35-45 days of culture, the callus quality of group C24 was the best, and the plants grew normally. That is, the best 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 Zhipeijing II and 7 g / L of Populus scolopendra enzymatic hydrolysate, and the pH of the induction medium was 5.8.

[0065] S8. Under 3000 lx light for 14 h per day and sterile conditions, the adventitious buds were cultured for rooting to obtain Figure 6 The poplar tissue shown in the figure; the culture medium used for rooting culture is: 1 / 2WPM, 30g / L sucrose, 6g / L agar, 0.5mL / L Zhipeijing II, the pH value of the culture medium is 5.8, the specific process is: Under sterile conditions, the adventitious buds were inoculated into different rooting culture media and cultured at 24°C to 26°C and 3000 lx light for 14 h per day for 10 to 14 days for rooting culture. The root growth was observed after 7 to 10 days. The results are shown in Table 4.

[0066] Table 4 Effects of different rooting media on rooting induction of Populus scolopendra , Comprehensively considering the four treatments, it can be seen from Table 4 that after 7-10 days of culture, the rooting condition of group G2 was the best and the plants grew normally. That is, the best rooting induction medium was: 1 / 2WPM, 30g / L sucrose, 6g / L agar, 0.5mL / L Zhipeijing II, and the pH of the medium was 5.8.

[0067] The above-obtained Populus sibiricum tissue was further cultured to obtain Figure 7 Seedlings of cottonwood are shown.

[0068] In summary, the embodiments of the present application provide a method for accurately cultivating Populus quascens tissue. The method continuously photographs the adventitious buds formed by the callus tissue on the basis of the induction process of callus tissue induction into adventitious buds, so as to clarify the morphological change data of the adventitious buds during the induction process. The amount of Populus quascens enzymatic hydrolysate to be added can be determined based on the change data, which can accelerate the speed of adventitious bud formation from the callus tissue and improve the reproduction rate of the Populus quascens tissue.

[0069] In addition, an embodiment of the present application provides a method for accurately cultivating Populus rapa tissue. During the production of Populus rapa seedlings, this method is not affected by environmental factors such as weather and season, and a large number of seedlings can be obtained in a short period of time. This method belongs to the category of asexual reproduction, so that the genetic traits of Populus rapa can be stably passed on to the greatest extent.

[0070] In addition, an embodiment of the present application provides a method for accurately cultivating Populus rapa tissue. This method uses Populus rapa leaves as raw materials and can solve the problems of long cultivation cycle and low reproduction rate in existing Populus rapa proliferation technologies by inducing young buds and leaves to form callus tissue and then transforming them into adventitious buds. This provides certain technical support for the factory-based breeding of Populus rapa.

[0071] In addition, the embodiment of the present application provides a method for accurately cultivating Populus rapa tissue, in which the Zhipeijing Type II product is added to the culture medium used in each step. Different from the traditional single antibacterial method, the addition of the Zhipeijing Type II product does not affect the normal growth of the Populus rapa, and can also prevent the explant from being contaminated in the later stage of cultivation.

[0072] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and 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 the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by 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; Inducing callus on the non-toxic explant to obtain callus tissue; Using a composite culture medium containing enzymatic hydrolysate of Populus scolopendra, inducing adventitious buds from the callus tissue; During the induction process of the adventitious buds, continuously photographing the adventitious buds formed by the callus tissue to obtain change data of the adventitious buds; Determining the amount of the enzymatic hydrolysate to be added according to the change data of the adventitious buds; According to the added amount of the enzymatic hydrolysate of Populus sirmifolia, the enzymatic hydrolysate of Populus sirmifolia is added to the composite culture medium to obtain adventitious buds; wherein the enzymatic hydrolysate of Populus sirmifolia contains vitamins and trace elements of Populus sirmifolia itself; The adventitious buds are subjected to rooting culture to obtain Populus scolopendra tissue.

2. The method according to claim 1, wherein the amount of the enzymatic hydrolysate of Populus striata to be added is determined according to the change data of the adventitious buds, comprising the steps of: According to the change data of the adventitious bud, the length change data ΔL of the adventitious bud per unit time is determined, wherein: The length change data of the adventitious buds per unit time is the length difference of the bud leaf tip of the adventitious buds before and after the unit time; According to the length change data ΔL, judging whether it is necessary to add the enzymatic hydrolysate of Populus scolopendra; If the length change data ΔL≤2mm, it is necessary to add poplar hydrolysate, and the additional amount of the poplar hydrolysate is calculated according to the length change data ΔL; wherein, the calculation formula for the additional amount of the poplar hydrolysate is: m=(0.2~0.4)×ΔL+0.2, if the unit of m is g, the unit of ΔL is mm.

3. The method according to claim 2, wherein during the induction process of the adventitious buds, the adventitious buds formed by the callus are continuously photographed to obtain the change data of the adventitious buds, comprising the steps of: During the induction process of the adventitious buds, the adventitious buds formed by the callus tissue are continuously photographed using a time-lapse camera to obtain photographs of the adventitious buds; According to the before and after differences of the adventitious bud photos, the size change data of the adventitious bud is calculated.

4. The method according to claim 1 or 3, wherein the interval of the continuous shooting is 24 hours to 48 hours, and the total shooting time of the continuous shooting is from the 7th day to the 28th day of the induction of the adventitious buds; and / or The temperature for inducing the adventitious buds is 24° C. to 26° C., and the time for inducing the adventitious buds is 35 days to 45 days.

5. The method according to claim 1, wherein the method for preparing the enzymatic hydrolysate of Populus sibiricum 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 enzymolysis solution is vacuum dried to obtain the Populus sirmifolia enzymolysis product.

6. The method according to claim 5, wherein 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); and / or The enzymatic hydrolysis temperature is 25°C to 35°C, and the enzymatic hydrolysis time is 1h to 2h; and / or The vacuum drying temperature is 50° C. to 70° C., and the vacuum drying time is 25 min to 35 min.

7. The method according to claim 1, wherein the weight m1 of the Populus sirmifolia enzymatic hydrolysate and the volume V1 of the composite culture medium satisfy the relationship: m1:V1=(4-8):1, and if the unit of m1 is g, the unit of V1 is L.

8. The method according to claim 7, wherein the components of the complex culture medium further include: MS culture medium, 3-indolebutyric acid, thiabendazole, sucrose, agar and Zhipeijing type II; the mass concentration of the 3-indolebutyric acid is 0.1 mg / L, the mass concentration of the thiabendazole is 0.03 mg / L, the mass concentration of the sucrose is 30 g / L, the mass concentration of the agar is 6 g / L, and the volume concentration of the Zhipeijing type II is 0.5 mL / L.

9. The method according to claim 1, wherein the temperature of the rooting culture is 24°C to 26°C, and the time of the rooting culture is 10 days to 14 days.

10. 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 sterilized to obtain a non-toxic explant; wherein, The disinfectant used in the disinfection process includes medical alcohol and / or sodium hypochlorite.

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