A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones

Abstract

The invention discloses a method for rejuvenating and rooting aging proliferation seedlings of asexually propagated Eucalyptus globulus, which belongs to the field of plant seedling propagation technology. The method comprises the following steps: cutting Eucalyptus globulus proliferation tissue culture seedlings into small bud clusters, and then vertically inserting the small bud clusters into a rejuvenation medium under sterile conditions, covering the culture bottle and transferring it to a light culture room for cultivation for two generations to successfully rejuvenate; inserting a single bud seedling with two or more pairs of leaves and 2-3 cm long after cultivation into a rooting medium, and then transferring the culture bottle to a light culture room for cultivation until adventitious roots grow at the base of the bud seedling. The method can rejuvenate aging proliferation seedlings, improve their proliferation ability, promote mass production of proliferation seedlings, and solve the problems existing in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of plant seedling propagation, and particularly relates to a method for rejuvenating and rooting aging and multiplying clonal seedlings of Eucalyptus grandiflora. Background Art

[0002] Eucalyptus cloeziana, a tree of the genus Eucalyptus in the Myrtaceae family, is native to Queensland, Australia. Its scientific name is Queensland Eucalyptus. It is drought-tolerant, highly resistant to pests and diseases, and grows rapidly, especially in its later stages. It is well-suited for cultivation into large-diameter timber. Its yellowish-brown wood, with straight grain and uniform structure, is heavy, strong, and highly hard, making it an excellent lumber. It has been included in the lists of precious timber species in provinces such as Sichuan and Fujian, as well as in the list of national reserve forest species.

[0003] Eucalyptus grandiflora is propagated through seedling propagation and tissue culture. Tissue culture-grown clones are highly stable and less susceptible to mutation, making them an ideal choice for intensive forestry. However, compared to mature tissue-culture clones of Eucalyptus grandis, E. grandiflora is a difficult species to propagate, especially due to its relatively difficult and unstable rooting. Only a handful of domestic facilities are capable of large-scale, factory-based cultivation of E. grandiflora clones. Insufficient seedling production capacity is a key factor limiting the development of the E. grandiflora industry.

[0004] Long-term practice has revealed that after repeated subculture, Eucalyptus grandiflora tissue culture seedlings experience a decline in bud cluster growth and poor axillary bud initiation. This is manifested by: an average proliferation coefficient dropping below 2.5; browning at the base of the bud cluster; callus particles on the leaves of new shoots; and vitrification of stem segments. To address the yield decline caused by aging seedlings, tissue culture plants establish clonal scion nurseries. When scion cuttings decline, these scion cuttings can be harvested from these nurseries to develop new clones and replace aging seedlings. However, maintaining these scion nurseries and redeveloping the tissue culture system is time-consuming and labor-intensive, making it unsuitable for large-scale production.

[0005] Therefore, using the aged propagation seedlings of Eucalyptus globulus as materials and re-cultivating them into propagation seedlings that can reproduce normally will help ensure the production of Eucalyptus globulus tissue culture seedlings, ensure the orderly development of the Eucalyptus globulus industry from the source, and provide a reference for the development of other Eucalyptus globulus asexual lines. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for rejuvenating and rooting aged proliferating seedlings of asexual lines of Eucalyptus grandiflora. This method can rejuvenate aged proliferating seedlings, improve their proliferating ability, promote mass production of proliferating seedlings, and solve the problems existing in the prior art.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, comprising the following steps:

[0009] (1) The proliferated tissue culture seedlings of Eucalyptus grandiflora were cut into small bud clusters, and then the small bud clusters were vertically inserted into the rejuvenation medium under sterile conditions. The culture bottles were covered and transferred to the light culture room for cultivation for two generations. The rejuvenation medium was made of the first basal medium, 6-benzylaminopurine, naphthaleneacetic acid, thiadiazole phenyl urea, safflower yellow, carrageenan and sucrose, wherein the concentration of 6-benzylaminopurine was 0.2-0.4 mg / L, the concentration of naphthaleneacetic acid was 0.05-0.2 mg / L, the concentration of thiadiazole phenyl urea was 0.2-0.3 mg / L, the concentration of safflower yellow was 0.5-1.0 g / L, the concentration of carrageenan was 7 g / L, and the concentration of sucrose was 30 g / L. The pH value of the rejuvenation medium was 5.8.

[0010] (2) inserting a single sprout with two or more pairs of leaves and 2-3 cm in length that has been cultivated or proliferated after cultivation into a rooting medium, and then transferring the culture bottle into a light culture room and culturing until adventitious roots grow at the base of the sprout to obtain the product;

[0011] The rooting medium consists of a second basal medium, sodium naphthylacetate, diethylaminoethyl hexanoate, carrageenan and sucrose, wherein the concentration of sodium naphthylacetate is 0.5-1.5 mg / L, the concentration of diethylaminoethyl hexanoate is 5-10 mg / L, the concentration of carrageenan is 7 g / L, the concentration of sucrose is 30 g / L, and the pH value of the rooting medium is 5.8; the sterilization temperature of the rooting medium is 115-121° C., and the sterilization time is 20-30 min.

[0012] Furthermore, in step (1), the length of the small bud cluster is 1-1.5 cm, with 2-3 branches of bud seedlings.

[0013] Furthermore, the concentration of 6-benzylaminopurine is 0.4 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of thiadiazole phenyl urea is 0.2 mg / L, and the concentration of safflower yellow is 1.0 g / L; the sterilization temperature of the rejuvenating culture medium is 115-121°C, and the sterilization time is 20-30 min.

[0014] Furthermore, in step (1), the insertion depth of the small bud cluster is 1 / 3-1 / 2 of the length of the small bud cluster.

[0015] Furthermore, the regeneration culture temperature is 23-27°C, the light duration is 8-10h / d, the light intensity is 2800-3200Lx, and the cultivation time of each generation is 20-25d.

[0016] Furthermore, the concentration of sodium naphthylacetate is 1.2 mg / L and the concentration of diethylaminoethyl hexanoate is 8 mg / L.

[0017] Furthermore, in step (2), the rooting culture temperature is 23-27° C., the light duration is 11-13 h / d, and the light intensity is 5000-8000 Lx.

[0018] Furthermore, the variety of Eucalyptus globulus is Sichuan Linzhen 7523.

[0019] The beneficial effects produced by the present invention are:

[0020] 1. The rejuvenation culture medium of the present invention can rejuvenate aged proliferated seedlings. The rejuvenated proliferated seedlings can continue to proliferate for more than 10 generations. When the proliferated seedlings that have proliferated for more than 10 generations show signs of aging again, they can be rejuvenated again.

[0021] 2. The improved rooting medium for Eucalyptus globulus seedlings provided by the present invention has a high rooting efficiency. After the seedlings are accessed to the rooting medium, adventitious roots appear at the base of the seedlings 15 days later, and the root system is fully developed at 25 days later. Each seedling has 3-5 taproots, and the root hairs of the taproots are vigorous and pure white in color. The average root length is ≥3cm, the average number of roots is ≥3, and the root system is rich in root hairs, with a root hair length of 1-2mm. The rooted seedlings have a high transplant survival rate. The average rooting rate reaches more than 95%, the root system is well-developed and strong, with good elasticity and is not easy to break; the root hairs are abundant and have strong absorption capacity. The rooted seedlings are easy to clean after hardening, the transplant survival rate is high, the afforestation survival rate is high, and the afforestation window period is long, and afforestation can be carried out from March to the end of October. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A is a photograph of aging and proliferating seedlings. Figure 1 B is a photograph of aging sprouts;

[0023] Figure 2 A is a photo taken 20 days after the bud cluster was inserted into the rejuvenation medium. Figure 2 B is a photograph of the seedlings after the second subculture in the rejuvenating medium. Figure 2 C is a photograph of the sprouts after they have grown; Figure 2 D is a photograph of the seedling after rejuvenation;

[0024] Figure 3 A is a photograph of the rooted seedlings. Figure 3 B is a photographic image of the rooted seedlings ready for transplanting after rooting;

[0025] Figure 4 This is a photo of the seedlings 1 month after transplanting;

[0026] Figure 5 This is a photo of the seedlings 2 months after transplanting;

[0027] Figure 6 This is a photo of the seedlings 4 months after transplanting;

[0028] Figure 7A is the seedling 6 months after transplanting. Figure 7 B is a close-up of the seedling substrate and root system 6 months after transplanting. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments.

[0030] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but is merely intended to represent selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0031] It should be noted that 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 actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0032] The features and performance of the present invention are further described in detail below with reference to the embodiments and drawings.

[0033] Example 1

[0034] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, comprising the following steps:

[0035] (1) The bottom and browning part of the "Chuanlinzhen 7523" large-flowered eucalyptus bud proliferation tissue culture seedlings that have been cultivated for more than 10 generations and whose bud proliferation ability has decreased to less than 2.5, the upper part of the bud has vitrification, the leaf tip has granular callus, the base has swollen and lost the stem segment shape, and the bottom has browning phenomenon were cut off, and then they were cut into small bud clusters with 2 buds. The top of the small bud cluster was flattened and the length of the small bud cluster after trimming was 1.3 cm. Then, under sterile conditions, the small bud cluster was vertically Insert the sterilized rejuvenating medium to a depth of 1 / 2 the length of the small bud cluster, cover the lid, and transfer the culture bottle to a light culture room for 2 generations, with each generation culturing time of 23 days, a cultivation temperature of 25°C, a light duration of 10 hours / day, and a light intensity of 3000Lx; during the cultivation process, the axial growth of new buds on the aging bud cluster can be promoted. During each subculture, the monarch drug will cut the proliferated seedlings into small bud clusters and flatten the top of the proliferated seedlings to remove deformed leaves with callus.

[0036] The rejuvenating medium is prepared from the first basal medium and 6-benzylaminopurine, naphthaleneacetic acid, thiadiazole phenyl urea, safflower yellow, carrageenan, and sucrose. The concentration of 6-benzylaminopurine is 0.4 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of thiadiazole phenyl urea is 0.2 mg / L, the concentration of safflower yellow is 1.0 g / L, the concentration of carrageenan is 7 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rejuvenating medium is 5.8. The sterilization temperature of the rejuvenating medium is 115° C., and the sterilization time is 30 minutes.

[0037] Each liter of the first basal culture medium contains 1900 mg of potassium nitrate, 1650 mg of ammonium nitrate, 278 mg of potassium dihydrogen phosphate, 370 mg of magnesium sulfate heptahydrate, 332 mg of anhydrous calcium chloride, 0.83 mg of potassium iodide, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 100 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 1.25 mg of niacin, 0.48 mg of glycine, 1.25 mg of ascorbic acid, 5 mg of L-cysteine, 8 mg of riboflavin, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0038] (2) transferring the cultivated seedlings to a proliferation culture medium for proliferation culture;

[0039] (3) After proliferation culture, a single bud with 2 or more pairs of leaves and 3 cm in length was used as the rooting material. Its morphological lower end was inserted into the rooting medium. The culture bottle was then transferred to a light culture room for 18 days. The rooting culture temperature was 25°C, the light duration was 12 h / d, and the light intensity was 7000 Lx. Adventitious roots grew at the base of the bud, completing the plant morphology.

[0040] The rooting medium consists of a second basal medium, sodium naphthylacetate, diethylaminoethyl hexanoate, carrageenan, and sucrose, wherein the concentration of sodium naphthylacetate is 1.2 mg / L, the concentration of diethylaminoethyl hexanoate is 8 mg / L, the concentration of carrageenan is 7 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rooting medium is 5.8, and the sterilization temperature of the rooting medium is 115° C. for 30 minutes.

[0041] Each liter of the second basal culture medium contains 247 mg of potassium nitrate, 216 mg of ammonium nitrate, 432 mg of potassium dihydrogen phosphate, 258 mg of magnesium sulfate heptahydrate, 250 mg of calcium nitrate tetrahydrate, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 5 mg of cobalt chloride, 60 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 0.75 mg of niacin, 0.288 mg of glycine, 0.8 mg of ascorbic acid, 1 mg of L-cysteine, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0042] Example 2

[0043] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, comprising the following steps:

[0044] (1) The bottom and brown part of the proliferating tissue culture seedlings of the large-flowered eucalyptus "Chuanlinzhen 7523" that have been cultivated for more than 10 generations and whose bud cluster proliferation ability has decreased to less than 2.5, vitrification phenomenon has appeared on the upper part of the bud, granular callus has appeared on the tip of the leaf, the base has swollen and lost the stem segment shape, and the bottom has browning phenomenon have been cut off, and then they are cut into small bud clusters with 2 buds. The top of the small bud cluster is flattened and the length of the small bud cluster after trimming is 1 cm. Then, under sterile conditions, the small bud cluster is vertically inserted into the sterilized rejuvenation culture medium with an insertion depth of 1 / 3 of the length of the small bud cluster. The lid is covered and the culture bottle is transferred to the light culture room for cultivation for 2 generations. The cultivation time of each generation is 20, the cultivation temperature is 27℃, the light time is 10h / d, and the light intensity is 3200Lx.

[0045] The rejuvenating medium is prepared from the first basal medium and 6-benzylaminopurine, naphthaleneacetic acid, thiadiazole phenyl urea, safflower yellow, carrageenan, and sucrose. The concentration of 6-benzylaminopurine is 0.2 mg / L, the concentration of naphthaleneacetic acid is 0.05 mg / L, the concentration of thiadiazole phenyl urea is 0.2 mg / L, the concentration of safflower yellow is 0.5 g / L, the concentration of carrageenan is 7 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rejuvenating medium is 5.8. The sterilization temperature of the rejuvenating medium is 121° C., and the sterilization time is 20 minutes.

[0046] Each liter of the first basal culture medium contains 1900 mg of potassium nitrate, 1650 mg of ammonium nitrate, 278 mg of potassium dihydrogen phosphate, 370 mg of magnesium sulfate heptahydrate, 332 mg of anhydrous calcium chloride, 0.83 mg of potassium iodide, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 100 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 1.25 mg of niacin, 0.48 mg of glycine, 1.25 mg of ascorbic acid, 5 mg of L-cysteine, 8 mg of riboflavin, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0047] (2) Use a single bud with two or more pairs of leaves and 2 cm in length as the rooting material, insert its morphological lower end into the rooting medium, and then transfer the culture bottle to the light culture room for 15 days. The rooting culture temperature is 27 ° C, the light time is 13h / d, and the light intensity is 8000Lx. Adventitious roots grow at the base of the bud, completing the plant morphology.

[0048] The rooting medium consists of a second basal medium, sodium naphthylacetate, diethylaminoethyl hexanoate, carrageenan, and sucrose, wherein the concentration of sodium naphthylacetate is 0.5 mg / L, the concentration of diethylaminoethyl hexanoate is 5 mg / L, the concentration of carrageenan is 7 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rooting medium is 5.8, and the sterilization temperature of the rooting medium is 121° C. for 20 minutes.

[0049] Each liter of the second basal culture medium contains 247 mg of potassium nitrate, 216 mg of ammonium nitrate, 432 mg of potassium dihydrogen phosphate, 258 mg of magnesium sulfate heptahydrate, 250 mg of calcium nitrate tetrahydrate, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 5 mg of cobalt chloride, 60 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 0.75 mg of niacin, 0.288 mg of glycine, 0.8 mg of ascorbic acid, 1 mg of L-cysteine, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0050] Example 3

[0051] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, comprising the following steps:

[0052] (1) The bottom and brown part of the proliferating tissue culture seedlings of the large-flowered eucalyptus "Chuanlinzhen 7523" that have been cultivated for more than 10 generations and whose bud cluster proliferation ability has decreased to less than 2.5, vitrification phenomenon has appeared on the upper part of the bud, granular callus has appeared on the tip of the leaf, the base has swollen and lost the stem segment shape, and the bottom has browning phenomenon have been cut off, and then they are cut into small bud clusters with 3 buds. The top of the small bud cluster is flattened and the length of the small bud cluster after trimming is 1.5 cm. Then, under sterile conditions, the small bud cluster is vertically inserted into the sterilized rejuvenation culture medium with an insertion depth of 1 / 2 of the length of the small bud cluster. The lid is covered and the culture bottle is transferred to the light culture room for cultivation for 2 generations. The cultivation time for each generation is 20-25 days. The cultivation temperature is 25℃, the light time is 8h / d, and the light intensity is 2800Lx.

[0053] The rejuvenating medium is prepared from the first basal medium and 6-benzylaminopurine, naphthaleneacetic acid, thiadiazole phenyl urea, safflower yellow, carrageenan, and sucrose. The concentration of 6-benzylaminopurine is 0.4 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of thiadiazole phenyl urea is 0.3 mg / L, the concentration of safflower yellow is 1.0 g / L, the concentration of carrageenan is 8 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rejuvenating medium is 6. The sterilization temperature of the rejuvenating medium is 118° C., and the sterilization time is 25 minutes.

[0054] Each liter of the first basal culture medium contains 1900 mg of potassium nitrate, 1650 mg of ammonium nitrate, 278 mg of potassium dihydrogen phosphate, 370 mg of magnesium sulfate heptahydrate, 332 mg of anhydrous calcium chloride, 0.83 mg of potassium iodide, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 100 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 1.25 mg of niacin, 0.48 mg of glycine, 1.25 mg of ascorbic acid, 5 mg of L-cysteine, 8 mg of riboflavin, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0055] (2) Use a single bud with two or more pairs of leaves and 3 cm in length as the rooting material, insert its morphological lower end into the rooting medium, and then transfer the culture bottle to the light culture room for 20 days. The rooting culture temperature is 23 ° C, the light time is 11 h / d, and the light intensity is 5000 Lx. Adventitious roots grow at the base of the bud, completing the plant morphology.

[0056] The rooting medium consists of a second basal medium, sodium naphthylacetate, diethylaminoethyl hexanoate, carrageenan, and sucrose, wherein the concentration of sodium naphthylacetate is 1.5 mg / L, the concentration of diethylaminoethyl hexanoate is 10 mg / L, the concentration of carrageenan is 8 g / L, and the concentration of sucrose is 30 g / L. The pH value of the rooting medium is 6, and the sterilization temperature of the rooting medium is 118° C. for 25 minutes.

[0057] Each liter of the second basal culture medium contains 247 mg of potassium nitrate, 216 mg of ammonium nitrate, 432 mg of potassium dihydrogen phosphate, 258 mg of magnesium sulfate heptahydrate, 250 mg of calcium nitrate tetrahydrate, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 5 mg of cobalt chloride, 60 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 0.75 mg of niacin, 0.288 mg of glycine, 0.8 mg of ascorbic acid, 1 mg of L-cysteine, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

[0058] Comparative Example 1

[0059] A method for rejuvenating and rooting aging and proliferating seedlings of Eucalyptus grandiflora clones, which differs from Example 1 in that the rejuvenating medium is adjusted to be prepared from the first basal medium and 6-benzylaminopurine, naphthaleneacetic acid, indolebutyric acid, carrageenan, and sucrose, wherein the concentration of 6-benzylaminopurine is 0.1 mg / L, the concentration of naphthaleneacetic acid is 0.25 mg / L, the concentration of indolebutyric acid is 0.1 mg / L, the concentration of carrageenan is 7 g / L, and the concentration of sucrose is 30 g / L; and the pH value of the rejuvenating medium is 5.8.

[0060] The rooting medium was adjusted to consist of the second basal medium, sodium naphthaleneacetate, indolebutyric acid, carrageenan and sucrose, wherein the concentration of sodium naphthaleneacetate was 1.0 mg / L, the concentration of indolebutyric acid was 1.5 mg / L, the concentration of carrageenan was 7 g / L, the concentration of sucrose was 30 g / L, and the pH value of the rooting medium was 5.8.

[0061] Comparative Example 2

[0062] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus grandiflora clones is provided, which differs from Example 1 in that the sterilization temperature of the rejuvenating medium and the rooting medium is adjusted to 135° C. for 20 min.

[0063] Comparative Example 3

[0064] A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, comprising the following steps:

[0065] (1) The bottom and browning part of the proliferating tissue culture seedlings of the large-flowered eucalyptus "Chuanlinzhen 7523" that had been cultivated for more than 10 generations and whose bud proliferation ability decreased to less than 2.5, had vitrification on the upper part of the bud, granular callus at the tip of the leaf, swollen base with loss of stem segment morphology, and browning at the bottom were cut off, and then cut into small bud clusters with two buds. The small bud clusters were inserted into the starting culture medium and cultured for 35 days. The first 8 days were shaded with black cloth, and then the black cloth was opened to receive normal light.

[0066] The rejuvenation medium is made of B5 medium supplemented with 0.1 mg / L 6-benzyladenine, 0.1 mg / L naphthaleneacetic acid, and 30 g / L sucrose;

[0067] (2) Cut off the side buds that sprouted during the rejuvenation culture and transfer them to the proliferation culture medium for proliferation culture;

[0068] (3) The seedlings after proliferation culture were transferred to a rooting medium for rooting culture. The seedlings were shaded with black cloth for the first 8 days after inoculation, and then the black cloth was opened to receive normal light and rooted for 30 days to obtain the product;

[0069] The rooting medium was prepared by adding 0.2 mg / L NAA, 0.2 mg / L indoleacetic acid, 1 mg / L indolebutyric acid and 20 g / L sucrose to B5 medium.

[0070] Test example

[0071] Figure 1 A. Photograph of aging and proliferating seedlings. Figure 1 B is a photograph of aging seedlings. From the picture, it can be seen that the base of the bud cluster of the proliferating seedlings is browning, there are callus particles on the leaves of the new buds, the stem segments are vitrified, and the growth of the bud cluster of the proliferating seedlings will decline, and the axillary bud germination effect is poor.

[0072] 1. The "Chuanlinzhen 7523" large-flowered eucalyptus tissue culture seedlings that had been continuously cultivated for more than 10 generations and whose bud cluster proliferation ability decreased to less than 2.5, had vitrification on the upper part of the bud, granular callus on the leaf tip, swollen base with loss of stem segment morphology, and browning at the bottom were rejuvenated according to the methods in Example 1 and Comparative Examples 1-3. The results are shown in Table 1.

[0073] Table 1: Rejuvenation results

[0074]

[0075] According to the results in the above table, except for Example 1, only the method in Comparative Example 2 can rejuvenate. The only difference between the method in Comparative Example 2 and the method in Example 1 is that the sterilization temperature of the culture medium is increased from 115°C to 135°C. It may be that the high temperature causes some nutrients in the culture medium to degrade, so the proliferation generation of Comparative Example 2 is slightly lower than that of Example 1, but the stable proliferation generation is equivalent to that of Example 2. The rejuvenated proliferated seedlings can still stably proliferate for 13-15 generations.

[0076] The aged and proliferated seedlings in Comparative Examples 1 and 3 were all unable to be rejuvenated. Some continued to age, some bud clusters grew into callus tissue, and some turned brown and died.

[0077] Figure 2 A is a photograph taken 20 days after the small bud cluster was inserted into the rejuvenation medium in the method of Example 1. Figure 2 B is a photograph of the seedlings after the second subculture in the rejuvenation medium in the method of Example 1, Figure 2 C is a photograph of the sprouts grown in the method of Example 1; Figure 2 D is a photograph of the seedlings after rejuvenation in the method of Example 1; it can be seen from the figure that the growth trend of the seedlings after rejuvenation treatment is significantly increased, the browning phenomenon at the base of the bud cluster disappears, the callus particles disappear, the vitrification of the stem segment disappears, the growth trend of the proliferating seedling bud cluster increases, and the axillary bud initiation effect is enhanced.

[0078] In summary, the method of this patent can effectively rejuvenate the aged propagated seedlings of the "Chuanlinzhen 7523" asexual line. The rejuvenated propagated seedlings can continue to proliferate for more than 10 generations on the original propagation culture medium. After aging, they can be rejuvenated using this patented method, thus constructing a recyclable large-scale breeding technology system, which is conducive to the large-scale breeding and order-based production of Eucalyptus globulus.

[0079] 2. The seedlings after the above-mentioned rejuvenation treatment were multiplied according to the DB51 / T 3027-2023 technical regulations for seedling cultivation of Eucalyptus globulus, and then the seedlings after the proliferation treatment were rooted according to the methods in Example 1 and Comparative Examples 1-3; the seedlings after the rooting treatment were hardened and transplanted. The specific rooting results and transplanting results are shown in Table 2.

[0080] Table 2: Rooting results

[0081]

[0082] According to the rooting results in Table 2, the rooting effect of Example 1 is the best, with an average rooting rate of up to 96%, the shortest rooting period (15-20 days), an average number of roots of 4.5, an average root length of 5 cm (the maximum root length is 11 cm), and a root system densely covered with root hairs. The final transplant survival rate reached 98%, and all indicators were significantly better than those of other groups (see Figure 3 A and 3B), Figure 4This is a photo of the seedlings 1 month after transplanting; Figure 5 This is a photo of the seedlings 2 months after transplanting. It can be seen that the seedlings are in good growth condition, with uniform growth rate and high survival rate. Figure 6 This is a photo of the seedlings 4 months after transplanting. It is obvious that the seedlings are growing well and their height has increased significantly. Figure 7 A is the seedling 6 months after transplanting. Figure 7 B is a close-up of the seedling substrate and root system 6 months after transplanting. It can be seen that the seedling has lush leaves, vigorous growth trend, and well-developed root system.

[0083] In comparison:

[0084] The rooting effect of Comparative Example 1 was second best, with an average rooting rate reduced to 85%, a rooting period extended by 5-10 days (20-25 days), an average number of roots reduced to 3, an average root length shortened to 3 cm (maximum root length 7 cm), and only individual adventitious roots with root hairs. The transplant survival rate dropped to 85%, and the comprehensive root characteristics (root number, root length, etc.) were significantly weaker than those in Example 1.

[0085] The rooting effect of Comparative Example 2 further decreased, because the activity of sodium naphthaleneacetate in the culture medium was severely lost during the high-temperature disinfection process, resulting in an average rooting rate of only 65%, a rooting period extended to 30-35 days, an average number of roots of only 2, an average root length of only 2 cm (the maximum root length was 3 cm), and the root system had no root hairs at all. The transplant survival rate dropped to 65%, and the key indicators (rooting rate, number of roots, root length, etc.) decreased by about 30%-60% compared with Example 1.

[0086] The rooting effect of comparative example 3 was the worst, because the nutrient composition of the basal culture medium was not suitable for root induction and growth requirements. The average rooting rate was only 58%, the rooting period was extended to 41 days, the average number of roots was 3 but the root length was only 2 cm (the maximum root length was 3 cm), the root system had no root hairs at all, and the final transplant survival rate was as low as 35%. The overall performance was significantly inferior to that of other groups.

Claims

1. A method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones, characterized in that: The following steps are involved: (1) The proliferated tissue culture seedlings of Eucalyptus grandiflora were cut into small bud clusters, and then the small bud clusters were vertically inserted into the rejuvenation medium under sterile conditions. The culture bottles were covered and transferred to the light culture room for cultivation for two generations. The rejuvenation medium was made of the first basal medium, 6-benzylaminopurine, naphthaleneacetic acid, thiadiazole phenyl urea, safflower yellow, carrageenan and sucrose, wherein the concentration of 6-benzylaminopurine was 0.2-0.4 mg / L, the concentration of naphthaleneacetic acid was 0.05-0.2 mg / L, the concentration of thiadiazole phenyl urea was 0.2-0.3 mg / L, the concentration of safflower yellow was 0.5-1.0 g / L, the concentration of carrageenan was 7 g / L, and the concentration of sucrose was 30 g / L. The pH value of the rejuvenation medium was 5.

8. Each liter of the first basal culture medium contains 1900 mg of potassium nitrate, 1650 mg of ammonium nitrate, 278 mg of potassium dihydrogen phosphate, 370 mg of magnesium sulfate heptahydrate, 332 mg of anhydrous calcium chloride, 0.83 mg of potassium iodide, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 100 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 1.25 mg of niacin, 0.48 mg of glycine, 1.25 mg of ascorbic acid, 5 mg of L-cysteine, 8 mg of riboflavin, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate. (2) Insert a single sprout with two or more pairs of leaves and 2-3 cm in length that has been cultivated or proliferated after cultivation into the rooting medium, and then transfer the culture bottle to a light culture room and culture until adventitious roots grow at the base of the sprout; The rooting medium consists of a second basal medium, sodium naphthylacetate, diethylaminoethyl hexanoate, carrageenan, and sucrose, wherein the concentration of sodium naphthylacetate is 0.5-1.5 mg / L, the concentration of diethylaminoethyl hexanoate is 5-10 mg / L, the concentration of carrageenan is 7 g / L, the concentration of sucrose is 30 g / L, and the pH value of the rooting medium is 5.8; the rooting medium is sterilized at a temperature of 115-121° C. for 20-30 minutes; Each liter of the second basal culture medium contains 247 mg of potassium nitrate, 216 mg of ammonium nitrate, 432 mg of potassium dihydrogen phosphate, 258 mg of magnesium sulfate heptahydrate, 250 mg of calcium nitrate tetrahydrate, 6.2 mg of boric acid, 22.3 mg of manganese sulfate monohydrate, 8.6 mg of zinc sulfate heptahydrate, 0.025 mg of copper sulfate pentahydrate, 0.25 mg of sodium molybdate dihydrate, 5 mg of cobalt chloride, 60 mg of inositol, 0.24 mg of pyridoxine hydrochloride, 0.75 mg of niacin, 0.288 mg of glycine, 0.8 mg of ascorbic acid, 1 mg of L-cysteine, 27.8 mg of ferrous sulfate heptahydrate, and 37.3 mg of disodium edetate.

2. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, characterized in that: Step (1) The length of the small bud cluster is 1-1.5 cm, with 2-3 branches of bud seedlings.

3. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, wherein: The concentration of 6-benzylaminopurine in the rejuvenating medium is 0.4 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of thiadiazole phenyl urea is 0.2 mg / L, and the concentration of safflower yellow is 1.0 g / L; the sterilization temperature of the rejuvenating medium is 115-121°C, and the sterilization time is 20-30 minutes.

4. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, wherein: In step (1), the insertion depth of the small bud cluster is 1 / 3-1 / 2 of the length of the small bud cluster.

5. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, wherein: The temperature for regeneration culture is 23-27℃, the light duration is 8-10h / d, the light intensity is 2800-3200Lx, and the cultivation time for each generation is 20-25d.

6. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, wherein: The concentration of sodium naphthylacetate in the rooting medium was 1.2 mg / L and the concentration of diethylaminoethyl hexanoate was 8 mg / L.

7. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus globulus clones according to claim 1, wherein: In step (2), the rooting culture temperature is 23-27°C, the light duration is 11-13h / d, and the light intensity is 5000-8000Lx.

8. The method for rejuvenating and rooting aging and multiplying seedlings of Eucalyptus grandiflora clones according to claim 1, wherein: The large-inflorescence eucalyptus variety is Sichuan Linzhen 7523.

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