A plant regeneration method for the superior clone DH32-28 of Eucalyptus urophylla

Abstract

The present invention discloses a plant regeneration method of an excellent clone of Eucalyptus grandis DH32-28: S1: branches of Eucalyptus grandis DH32-28 are collected and disinfected to obtain sterile seedlings; S2: the sterile seedlings are cultured through proliferation to obtain proliferated seedlings, and the proliferated seedlings are inoculated into a GA3 bud elongation medium to obtain elongated seedlings; S3: internode stem segments of the elongated seedlings are taken as explants to induce callus tissue; S4: the callus tissue is inoculated into a regeneration bud induction medium to induce regeneration buds; S5: the induced regeneration buds of a certain length are inoculated into a rooting medium to induce rooting, and the rooted regeneration buds are regenerated plants. The present invention can quickly and effectively regenerate Eucalyptus grandis DH32-28 plants, is easy to operate, and is convenient for mass production of seedlings through asexual reproduction.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a plant regeneration method of an excellent Eucalyptus urophylla clone DH32-28. Background Art

[0002] Eucalyptus urophylla (Eucalyptus urophylla X E. grandis), a member of the Myrtaceae family and genus Eucalyptus, is characterized by fast growth, high yield, short rotation period, high timber yield, and strong carbon sequestration capacity. It is a major timber supply species in my country and one of the main timber species cultivated in southern China. The superior clone DH32-28 of Eucalyptus urophylla, developed through years of hybridization breeding at the Guangxi State-owned Dongmen Forest Farm, is now widely commercialized in Guangxi. However, with the widespread planting of eucalyptus plantations, outbreaks of pests and diseases, as well as abiotic stresses such as frost, have emerged in recent years. This is because traditional hybridization breeding often takes a long time to develop new high-yielding and stress-resistant varieties. With the advent of the molecular breeding era, transgenic technology and gene editing have been widely applied to various agricultural and forestry crops as new methods for forest genetic improvement, offering a new solution to this dilemma. Plant regeneration is the foundation of transgenic and gene editing methods, and a stable and efficient regeneration system provides the necessary prerequisite for the establishment of a genetic transformation system for Eucalyptus urophylla. Since the 1990s, research on eucalyptus regeneration systems has made significant progress both domestically and internationally, encompassing a variety of influencing factors, including genotype, hormones, and explant type. Due to these limitations, established eucalyptus regeneration systems cannot be directly applied to the Eucalyptus urophylla DH32-28 clone. Therefore, further research is needed to explore efficient regeneration systems. This will lay a solid foundation for molecular breeding of eucalyptus, further accelerate genetic improvement and gene function research in eucalyptus, and has broad application prospects. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides an efficient plant regeneration method for the excellent asexual variety Eucalyptus grandis DH32-28. This method uses the stem segment of Eucalyptus grandis DH32-28 without axillary buds as an explant. After inducing callus tissue, it induces regenerated buds and induces rooting to finally form a complete plant, which can provide an important basis for the establishment of a genetic transformation system.

[0004] To achieve the above objectives, the present invention provides the following technical solutions:

[0005] A plant regeneration method for the superior clone DH32-28 of Eucalyptus urophylla, comprising the following steps:

[0006] S1: Collect branches of Eucalyptus grandis DH32-28 and disinfect them to obtain sterile seedlings;

[0007] S2: Obtaining proliferated seedlings and elongated seedlings: inoculating the sterile seedlings into a bud proliferating medium to obtain proliferated seedlings; then inoculating the proliferated seedlings into a bud elongation medium containing GA3 to induce elongation, thereby significantly elongating the internode stem segments and obtaining elongated seedlings;

[0008] S3: Take the internode stem segment of the elongated seedling as an explant and inoculate it into callus induction medium to obtain callus tissue;

[0009] S4: inoculating the callus tissue into the regeneration bud induction medium to induce regeneration buds;

[0010] S5: The induced regenerated buds of a certain length are inoculated into a rooting medium to induce rooting. The rooted regenerated buds are the regenerated plants.

[0011] Furthermore, in step S1, the Eucalyptus urophylla branches are collected by collecting semi-lignified 8-10 cm branches of Eucalyptus urophylla, containing 2-3 axillary buds, and immediately placing them in sterile water for moisturizing; before disinfection, the branches are shortened to 4-5 cm, each branch contains 1-2 axillary buds, and if there are side branches or leaves next to the axillary buds in the branches, the side branches or leaves should be shortened to 0.5-1 cm and rinsed under running water for 2 hours; the disinfection treatment is to cut off the tissue at the incision of the branch in an ultra-clean workbench, disinfect it with 75% volume concentration of ethanol for 30 seconds, rinse it with sterile water 4 times, and then disinfect it with 0.12% volume concentration of mercuric chloride for 8 minutes, shake it several times during the period, rinse it with sterile water 5 times, and then place the branch on sterile filter paper to dry.

[0012] Furthermore, in step S1, the tissue at the branch incision is cut off with scissors, the length of the morphological upper branch of the axillary bud point is 0.3 to 0.5 cm, and the length of the morphological lower branch is 2 to 3 cm. The lower end of the branch is inserted upright into the axillary bud induction medium, and the branch is not directly in contact with the bottom of the medium. When the axillary bud grows to 1 cm, the axillary bud is cut off to obtain a sterile seedling; the axillary bud induction medium is a mixture of 4.74 g / L MS medium components, 1 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose are added, the pH is adjusted to 5.8, and 7 g / L agar is added.

[0013] Furthermore, in step S2, the bud proliferation medium is a mixture of 4.74 g / L MS medium components, 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose are added, the pH is adjusted to 5.8, and 7 g / L agar is added.

[0014] Furthermore, in step S2, the proliferated seedlings were inoculated into a bud elongation medium containing GA3 for elongation induction, and the induction culture conditions were a light intensity of 22 μmol / (m 2 ·s), light intensity of 12h / d, and temperature of 23±1°C; the GA3-containing shoot elongation medium is a mixture of 4.74g / L MS medium components, to which 0.4mg / L 6-BA, 0.2mg / L NAA, 0.3mg / L GA3, 0.5g / L PVP40000, and 30g / L sucrose are added, the pH is adjusted to 5.8, and 7g / L agar is added.

[0015] Furthermore, in step S3, the internode stem segment of the elongated seedling of Eucalyptus grandis DH32-28 is taken, the nodes with axillary buds at both ends of the stem segment are cut off with a sterile scalpel, and the stem segment is then cross-cut into 0.3-0.5 cm explants, which are placed flat in contact with the culture medium and inoculated into callus induction medium for 7-30 days (optimally 20 days).

[0016] Furthermore, in step S3, the callus induction medium is a mixture of 2.47 g / L 1 / 2 MS medium components, 0.220 mg / L TDZ, 0.019 mg / L NAA, 30 g / L sucrose, and 7 g / L agar added thereto, with a pH of 5.8; the callus induction conditions are dark culture at a temperature of 23±1°C, and subculture every 15 days.

[0017] Furthermore, in step S4, the regeneration bud induction medium is a mixture of 2.47 g / L 1 / 2 MS medium components, 0.25 mg / L 6-BA, 0.125 mg / L NAA, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.8; the regeneration bud induction condition is a light intensity of 10 μmol / (m 2 ·s), photoperiod 12h / d, temperature 23±1℃.

[0018] Furthermore, in step S5, the regenerated buds that grew to 1 cm after induction were inoculated into a rooting medium to induce rooting; the rooting medium was a mixture of 2.47 g / L 1 / 2 MS medium components, 0.5 mg / L IBA, 30 g / L sucrose, and 6 g / L agar, with a pH of 5.8; the rooting culture conditions were a light intensity of 22 μmol / (m 2 ·s), light intensity 12h / d, temperature 23±1℃.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention uses elongated seedling stem segments without axillary buds of an excellent asexual variety of Eucalyptus grandis DH32-28 as explants, adopts a bud elongation culture medium containing GA3 (gibberellic acid) to significantly increase the number of internode stem segments of the proliferated seedlings and regenerated buds from a single internode stem segment, and adopts a unique hormone formula in the callus induction and bud regeneration culture medium, thereby obtaining a highly efficient plant regeneration method for Eucalyptus grandis DH32-28, with a rooting rate of regenerated plants of 100% and a transplant survival rate of regenerated plants of up to 93%. The method of the invention can quickly and effectively regenerate Eucalyptus grandis DH32-28 plants, is simple to operate, and is convenient for mass production of seedlings through asexual reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of hormone treatment on the elongation of Eucalyptus grandis DH32-28 plants; Figure 1 A, B, C, D, E, and F correspond to treatments A, B, C, D, E, and F in Table 1, respectively.

[0022] Figure 2 A is the callus tissue of the internode stem segment of the elongated seedlings of Eucalyptus grandis DH32-28; Figure 2 B is the regenerated bud of the internode stem segment of the elongated seedling of Eucalyptus grandis DH32-28.

[0023] Figure 3 This is the regeneration of buds from the explants of Eucalyptus grandis DH32-26; Figure 3 A, B, C, D, E, F, G, and H correspond to treatments A, B, C, D, E, F, G, and H in Table 4, respectively.

[0024] Figure 4 This is the regeneration of buds from the explants of Eucalyptus grandis DH32-28; Figure 4 A, B, C, D, E, F, G, and H correspond to treatments A, B, C, D, E, F, G, and H in Table 2, respectively.

[0025] Figure 5 This is the regeneration of buds from the explants of Eucalyptus grandis DH32-29; Figure 5 A, B, C, D, E, F, G, and H correspond to treatments A, B, C, D, E, F, G, and H in Table 5, respectively.

[0026] Figure 6 This is a regenerated plant of Eucalyptus grandis DH32-28.

[0027] Figure 7 Transplantation of regenerated plants of Eucalyptus grandis DH32-28. DETAILED DESCRIPTION

[0028] The following is a detailed description of the specific embodiments in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the examples are all commercially available. The Eucalyptus grandis DH32-28, Eucalyptus grandis DH32-26, and Eucalyptus grandis DH32-29 used in the present invention were purchased from Guangxi Bagui Seedlings High-Tech Group Co., Ltd., and the 1 / 2MS and MS culture medium component mixture was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0029] The culture media used in the following examples were prepared and dissolved in 1 L of pure water. The culture media and their formulas are as follows:

[0030] Axillary bud induction medium: add 1 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose to a 4.74 g / L mixture of MS medium components, adjust the pH to 5.8, and add 7 g / L agar.

[0031] Shoot proliferation medium: Add 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose to a 4.74 g / L mixture of MS medium components, adjust the pH to 5.8, and add 7 g / L agar.

[0032] GA3-containing shoot elongation medium: Add 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.3 mg / L GA3, 0.5 g / L PVP40000, and 30 g / L sucrose to a 4.74 g / L mixture of MS medium components, adjust the pH to 5.8, and add 7 g / L agar.

[0033] Callus induction medium: 0.220 mg / L TDZ, 0.019 mg / L NAA, 30 g / L sucrose were added to a 2.47 g / L mixture of 1 / 2 MS medium components, the pH was adjusted to 5.8, and 7 g / L agar was added.

[0034] Regeneration shoot induction medium: add 0.25 mg / L 6-BA, 0.125 mg / L NAA, 30 g / L sucrose to 2.47 g / L 1 / 2 MS medium component mixture, adjust the pH to 5.8, and add 7 g / L agar.

[0035] Rooting medium: Add 0.5 mg / L IBA and 30 g / L sucrose to a 2.47 g / L mixture of 1 / 2 MS medium components, adjust the pH to 5.8, and add 6 g / L agar.

[0036] Example 1

[0037] A plant regeneration method for the superior clone DH32-28 of Eucalyptus urophylla, comprising the following steps:

[0038] S1: Collect branches of Eucalyptus grandis DH32-28 and disinfect them to obtain sterile seedlings: Use scissors with a volume concentration of 75% alcohol to disinfect, cut off 8-10 cm semi-lignified branches of Eucalyptus grandis DH32-28 (including 2-3 axillary buds), and immediately place them in sterile water to moisturize; before disinfection, cut the branches to 4-5 cm, each branch contains 1-2 axillary buds, if there are side branches or leaves next to the axillary buds in the branches, the side branches and leaves should be cut to 0.5-1 cm, rinse under running water for 2 hours; then cut off the tissue at the incision of the branch in an ultra-clean workbench, disinfect with 75% volume concentration of ethanol for 30 seconds, and sterilize. Rinse with water 4 times, then disinfect with 0.12% mercuric chloride by volume for 8 minutes, shaking several times during the process. When the disinfection time is up, immediately pour sterile water into the disinfectant to dilute the disinfectant concentration, then pour out the water, rinse with sterile water 5 times, and place the branches on sterile filter paper to dry; use scissors to cut off the tissue at the incision of the branches placed on the sterile filter paper to dry, leaving a length of 0.3 to 0.5 cm at the morphological upper end of the axillary bud point, and a length of 2 to 3 cm at the morphological lower end of the branch, insert the lower end of the branch upright into the axillary bud induction medium, and do not let the branch directly touch the bottom of the medium. When the axillary bud grows to 1 to 2 cm, cut the axillary bud to obtain sterile seedlings;

[0039] S2: Obtaining propagation seedlings and elongated seedlings: The obtained sterile seedlings were inoculated into bud propagation medium to obtain propagation seedlings. The propagation seedlings were subcultured once every month. The stem segments of the propagation sterile seedlings of Eucalyptus grandis were generally shorter ( Figure 1 A); therefore, the obtained proliferated seedlings were inoculated into GA3-containing bud elongation medium for elongation induction, and the culture conditions were a light intensity of 22 μmol / (m 2 s), 12 h / d light intensity, 23 ± 1°C temperature, and culturing for 20 days (treatment C in Table 1) resulted in significant elongation of the internode stem segments and the production of elongated seedlings. The elongation effect and plant status were statistically analyzed, as shown in Table 1.

[0040] S3: Prepare a sterilized 90 mm glass culture dish with two filter papers, pour sterile water into the filter paper to moisten it, take out the elongated seedlings obtained in S2 and place them on the filter paper, use a sterile scalpel to cut off the nodes with axillary buds in the stem segment, which is the internode stem segment of the elongated seedling, and then cut the internode stem segment into 0.3-0.5 cm explants, inoculate the explants flatly into callus induction medium, and perform callus induction; callus induction conditions are dark culture, temperature 23±1℃, subculture every 15 days, and obtain callus after 20 days ( Figure 2 A); at this time, the callus induction rate was calculated (treatment G in Table 2);

[0041] S4: The callus obtained in S3 was inoculated into the regeneration bud induction medium to induce regeneration buds. The bud induction condition was a light intensity of 10 μmol / (m 2 s), photoperiod 12h / d, temperature 23±1℃, subculture every 15 days during the culture process; after 30 days, the regeneration rate and number of regenerated buds were counted (Table 2, Table 3, Figure 2 B. Figure 4 ).

[0042] S5: When the induced regenerated buds grow to 1 cm, cut them off and inoculate them into rooting medium to induce rooting. Make sure that only the smooth main stem is inserted into the medium. After one month, the rooted regenerated buds (the rooting rate can reach 100%) are obtained, which are the regenerated plants.

[0043] Gently take out the regenerated plants, crush the agar on the roots gently, and clean the agar on the roots under running water, while trying to avoid damaging the roots ( Figure 6 Soak the whole regenerated plant in a 1 / 1000 concentration of carbendazim solution for 30 minutes, and then place it in a planting pot with moist sterile substrate soil ( Figure 7 ). Water the substrate soil with the prepared carbendazim solution, cover the planting pot with a layer of plastic wrap, poke a few small holes in the plastic wrap to maintain soil moisture and air permeability, and then place it in the plant culture room for cultivation. During this period, constantly observe the plants, slowly expand the pore size of the plastic wrap to increase air permeability. When the plants grow new leaves, remove all the plastic wrap, which indicates that the regenerated plants have been successfully transplanted. When the regenerated plants were transplanted into the substrate soil for one month, the transplant survival rate was calculated and the transplant survival rate reached 93%. The conditions in the plant culture room are: 25±2℃, light intensity 58μmol / (m 2 ·s), photoperiod of 12h / d.

[0044] Comparative Example 1

[0045] S2: Obtaining Proliferated Seedlings: The sterile seedlings obtained were inoculated into a bud proliferation medium and cultured to obtain proliferated seedlings. The proliferated seedlings were subcultured approximately once a month (Treatment A in Table 1). The remaining steps were the same as in Example 1. The elongation effect and plant status were statistically analyzed, as shown in Table 1. In this Example, S2 only obtained proliferated seedlings, and steps S3-S5 were also performed using the proliferated seedlings obtained in S2.

[0046] Comparative Example 2

[0047] In S2, the hormones in the GA3-containing shoot elongation medium, namely "0.4 mg / L 6-BA, 0.2 mg / L NAA, and 0.3 mg / L GA3," were replaced with "0.3 mg / L GA3." The remaining components were the same as those in the GA3-containing shoot elongation medium (Treatment B in Table 1). The remaining operations were the same as in Example 1. The elongation effect and plant status in S2 were statistically analyzed, as shown in Table 1.

[0048] Comparative Example 3

[0049] In S2, the shoots were treated with a GA3-containing shoot elongation medium (Treatment C in Table 1) for 3 days and then transferred to a medium containing the hormones described in Treatment A in Table 1 (Treatment A medium: 4.74 g / L of a mixture of MS medium components, supplemented with 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.5 g / L PVP40000, 30 g / L sucrose, adjusted to a pH of 5.8, and 7 g / L agar). Culture was carried out for 17 days (Treatment D in Table 1). All other procedures were the same as in Example 1. The elongation effect and plant status in S2 were statistically analyzed, as shown in Table 1.

[0050] Comparative Example 4

[0051] In S2, the hormones in the GA3-containing shoot elongation medium, namely "0.4 mg / L 6-BA, 0.2 mg / L NAA, and 0.3 mg / L GA3," were replaced with "0.5 mg / L IBA." The remaining components were the same as those in the GA3-containing shoot elongation medium (Treatment E in Table 1). The culture was carried out for 60 days (plants in Treatment E rooted, but grew more slowly. At 60 days, the internodes and stem segments of the proliferated seedlings showed significant elongation, and at this point, they were no longer referred to as elongated seedlings, but rather as rooted seedlings). The remaining procedures were the same as in Example 1. The elongation effect and plant status of the rooted seedlings in S2 were statistically analyzed, as shown in Table 1.

[0052] Comparative Example 5

[0053] In S2, the hormones in the GA3-containing shoot elongation medium, namely "0.4 mg / L 6-BA, 0.2 mg / L NAA, and 0.3 mg / L GA3," were replaced with "0.5 mg / L IBA + 0.3 mg / L GA3." The remaining components were the same as those in the GA3-containing shoot elongation medium (Treatment F in Table 1). The culture was continued for 60 days, and the remaining procedures were the same as in Example 1. The elongation effect and plant status in S2 were statistically analyzed, as shown in Table 1.

[0054] Table 1 Effects of different hormone treatments on the elongation of Eucalyptus grandis DH32-28 plants

[0055]

[0056] The "elongation effect and plant status" described in Table 1 refers to the elongation effect and plant status of the seedlings obtained after the S2 operation is completed in each treatment: if elongated seedlings are obtained (treatment C), the elongation effect and plant status of the elongated seedlings are counted; if rooted seedlings are obtained (treatment E), the elongation effect and plant status of the rooted seedlings are counted; if the seedlings obtained do not elongate and cannot be called elongated seedlings or rooted seedlings, the elongation effect and plant status of the obtained seedlings are counted.

[0057] In Table 1, except for Treatment A, in which the hormones were used in the shoot proliferation medium, the medium used in the other BF treatments was the same as that in the GA3-containing shoot elongation medium, except that the hormones in the GA3-containing shoot elongation medium were replaced with the "hormones" listed in the second column of Table 1. The remaining components were the same as those in the GA3-containing shoot elongation medium. Specifically, the hormones "0.4 mg / L 6-BA, 0.2 mg / L NAA, and 0.3 mg / L GA3" in the GA3-containing shoot elongation medium were replaced with the "hormones" listed in the second column of Table 1. Treatment C, in particular, refers to the GA3-containing shoot elongation medium of the present invention.

[0058] As can be seen from Table 1, at 20 days, the internodes of the plants in treatment C were significantly elongated, but the plants did not take root, and were called elongated seedlings. The plants in treatment E took root, but grew slowly, and at 60 days, the internodes of the plants were significantly elongated, and were called rooted seedlings. The other three treatments with GA3 hormones inhibited the normal development of the plants ( Figure 1 , Figure 1 A, B, C, D, E, and F in Table 1 correspond to treatments A, B, C, D, E, and F in Table 1, respectively. Therefore, the explants of the elongated seedlings obtained in treatment C (i.e., the elongated seedlings obtained in S2 in Example 1) and the rooted seedlings obtained in treatment E (i.e., the rooted seedlings obtained in S2 in Comparative Example 4) were subsequently used to further optimize the regeneration system.

[0059] Comparative Example 6

[0060] In S3, leaves of the proliferated seedlings obtained in S2 (treatment A in Table 1) were taken out as explants (treatment A in Table 2, after the proliferated seedlings were obtained in S2, no elongation induction was performed, and the leaves of the proliferated seedlings were directly used as explants). The leaves were placed on filter paper, and the leaves were cut into 0.3 cm × 0.5 cm pieces perpendicular to the veins. The leaves were placed flat on the culture medium and then inoculated into the callus induction medium. The rest of the operation was the same as in Example 1. The callus induction rate and regeneration rate were calculated (Table 2, Figure 4 ).

[0061] Comparative Example 7

[0062] In S3, leaves of the elongated seedlings obtained in S2 (treatment C in Table 1) were taken as explants (treatment B in Table 2), placed on filter paper, and cut into 0.3 cm × 0.5 cm pieces perpendicular to the veins. The leaves were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as in Example 1. The callus induction rate and regeneration rate were calculated (Table 2, Figure 4 ).

[0063] Comparative Example 8

[0064] In S3, leaves of the rooted seedlings obtained in S2 (treatment E in Table 1) were taken as explants (treatment C in Table 2), placed on filter paper, and cut into 0.3 cm × 0.5 cm pieces perpendicular to the veins. The leaves were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as in Example 1. The callus induction rate and regeneration rate were calculated (Table 2, Figure 4 ).

[0065] Comparative Example 9

[0066] In S3, the petioles of the proliferated seedlings obtained in S2 were taken as explants (treatment D in Table 2), placed on filter paper, and the petioles were cut into 0.3-0.5 cm pieces. The petioles were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as in Example 1, and the callus induction rates were calculated (Table 2, Figure 4 ).

[0067] Comparative Example 10

[0068] In S3, the petioles of the elongated seedlings obtained in S2 (treatment A in Table 1) were taken out as explants (treatment E in Table 2), placed on filter paper, and the petioles were cut into 0.3-0.5 cm pieces. The petioles were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as in Example 1. The callus induction rate and regeneration rate were calculated (Table 2, Figure 4 ).

[0069] Comparative Example 11

[0070] In S3, the petioles of the rooted seedlings obtained in S2 (treatment E in Table 1) were taken as explants (treatment F in Table 2), placed on filter paper, and the petioles were cut into 0.3-0.5 cm pieces. The petioles were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as in Example 1. The callus induction rate and regeneration rate were calculated (Table 2, Figure 4 ).

[0071] Comparative Example 12

[0072] In S3, the stem segments of the rooted seedlings obtained in S2 (treatment E in Table 1) were taken out as explants (treatment H in Table 2), placed on filter paper, and the stem segments were cut into 0.3-0.5 cm pieces. The stem segments were placed flat on the culture medium and then inoculated into the callus induction medium. The remaining operations were the same as those in Example 1. In step S3, the callus induction rate (Table 2, Figure 4 ), when counting the regeneration rate in step S4, the number of regenerated buds was also counted (Table 2, Table 3).

[0073] Table 2 Effects of plant type and explant type on regeneration rate of Eucalyptus grandis DH32-28

[0074] deal with Plant type Explant type Callus induction rate (%) Regeneration rate (%) A Proliferation seedlings blade <![CDATA[100.00±0.00 a ]]> <![CDATA[20.31±1.56 d ]]> B Elongated seedlings blade <![CDATA[100.00±0.00 a ]]> <![CDATA[23.96±3.25 d ]]> C rooted seedlings blade <![CDATA[100.00±0.00 a ]]> <![CDATA[6.25±1.20 e ]]> D Proliferation seedlings petiole <![CDATA[100.00±0.00 a ]]> <![CDATA[39.58±8.00 c <!-- 6 -->]]> E Elongated seedlings petiole <![CDATA[99.41±0.59 a ]]> <![CDATA[77.72±3.08 b ]]> F rooted seedlings petiole <![CDATA[100.00±0.00 a ]]> <![CDATA[22.93±3.38 d ]]> G Elongated seedlings stem segment <![CDATA[99.29±0.71 a ]]> <![CDATA[94.29±1.70 a ]]> H rooted seedlings stem segment <![CDATA[100.00±0.00 a ]]> <![CDATA[47.75±2.89 c ]]>

[0075] Note: In Table 2, different letters in a, b, c, and d indicate significant differences.

[0076] Table 3 Comparison of the number of regenerated buds in the stem segments of the elongated and rooted seedlings of Eucalyptus grandis DH32-28

[0077] Plant type Regeneration rate (%) Number of regenerated buds per stem segment explant The elongated seedlings obtained from S2 in Example 1 94.29±1.70 8.7±0.7 Comparative Example 4: Rooted seedlings obtained from S2 47.75±2.89 4.6±0.6

[0078] As can be seen from Table 2, the regeneration rate of the elongated seedling explants was high, with the petiole regeneration rate nearly double that of the multiplication seedlings, and the stem segment regeneration rate reaching 94%. As can be seen from Table 3, there were significant differences in the stem segment regeneration rate and the number of regenerated buds per stem segment explant between the elongated seedlings and the rooted seedlings. The elongated seedlings had a stem segment regeneration rate and the number of regenerated buds per stem segment explant nearly double that of the rooted seedlings.

[0079] According to the treatment in Table 4, different genotypes of Eucalyptus grandis and different plant types and explant types were used as explants, and the rest of the operations were the same as in Example 1. For example, in treatment A, sterile seedlings of Eucalyptus grandis DH32-26 were obtained, and after the proliferation seedlings were obtained in S2, no elongation induction was performed, and the leaves of the obtained proliferation seedlings were directly used as explants. The rest of the operations were the same as in Example 1, and the callus induction rate of the leaves of the proliferation seedlings of Eucalyptus grandis DH32-26 was counted in step S3, and the regeneration rate was counted in step S4. For example, in treatment C, the proliferation seedlings obtained from Eucalyptus grandis DH32-26 were inoculated into the bud elongation medium (treatment E in Table 1) for elongation induction, and the culture conditions were a light intensity of 22 μmol / (m 2 ·s), light 12h / d, temperature 23±1℃, culture for 60 days, the plants took root, the growth rate was slow, and the internode stem segments of the proliferated seedlings were significantly elongated at 60 days. At this time, they were no longer called elongated seedlings, but rooted seedlings. Then, the leaves of the rooted seedlings were selected as explants. The rest of the operations were the same as in Example 1. In step S3, the callus induction rate of the leaves of the proliferated seedlings of Eucalyptus grandis DH32-26 was counted, and in step S4, the regeneration rate was counted. The other treatments in Table 4 and all the treatments in Table 5 were the same. The results showed that the improvement of the regeneration rate of DH32-26 and DH32-29 when the plant type was elongated seedlings was not obvious (Table 4, Table 5, Figure 3 、 Figure 5 ).

[0080] Table 4 Effects of plant type and explant type on regeneration rate of Eucalyptus grandis DH32-26

[0081]

[0082] Note: Different letters in a and b indicate significant differences

[0083] Table 5 Effects of plant type and explant type on regeneration rate of Eucalyptus grandis DH32-29

[0084]

[0085] Note: Different letters in a and b indicate significant differences

[0086] In S3, the callus induction time was changed according to Table 6. The remaining operations were the same as in Example 1. At the end of step S4, the regeneration rate was calculated to obtain Table 6. As can be seen from Table 6, the regeneration rate was not affected during the callus induction period of 15 to 30 days, and was maintained at above 90%. However, the callus induction time of 7 days significantly reduced the bud induction regeneration rate, with the regeneration rate being only 60% (Table 6). Therefore, in the regeneration of internode stem segments of Eucalyptus grandis DH32-28, 20 days is the optimal callus induction time for regeneration of internode stem segments of Eucalyptus grandis DH32-28.

[0087] Table 6 Effect of callus induction time on regeneration rate of Eucalyptus grandis DH32-28

[0088] Callus induction time (d) Regeneration rate (%) 7 63.00±4.42b 15 93.33±3.33a 20 95.00±2.89a 30 93.33±4.41a

[0089] The present invention provides a plant regeneration method for the superior clone DH32-28 of Eucalyptus urophylla. GA3-treated seedlings make it easier to obtain more stem segments, and the de novo plant regeneration process is shortened, requiring only about 50 days from callus induction to differentiate into regenerated buds, with an induction regeneration rate exceeding 90%. This method effectively addresses the regeneration issues of the superior clone DH32-28 of Eucalyptus urophylla, providing key technical support for genetic transformation, gene editing, and gene function research.

[0090] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A plant regeneration method for the superior clone DH32-28 of Eucalyptus urophylla, characterized in that: The following steps are included: S1: Collect branches of Eucalyptus grandis DH32-28 and disinfect them to obtain sterile seedlings; S2: Obtaining proliferating seedlings and elongated seedlings: inoculating sterile seedlings into bud proliferation medium for cultivation to obtain proliferating seedlings; then inoculating the proliferating seedlings into bud elongation medium containing GA3 for elongation induction to obtain elongated seedlings; the induction culture conditions are light intensity of 22 μmol / (m 2 •s), 12 h / d of light, and a temperature of 23 ± 1°C; the GA3-containing shoot elongation medium is a mixture of 4.74 g / L MS medium components, to which 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.3 mg / L GA3, 0.5 g / LPVP40000, and 30 g / L sucrose are added, the pH is adjusted to 5.8, and 7 g / L agar is added; S3: Take the stem segment of the elongated seedling and cut off the nodes with axillary buds at both ends of the stem segment. This stem segment is the internode stem segment of the elongated seedling. Then cut the internode stem segment into 0.3-0.5 cm explants, inoculate them into callus induction medium, and culture for 7-30 days to obtain callus tissue; S4: inoculating the callus tissue into the regeneration bud induction medium to induce regeneration buds; S5: The induced regenerated buds of a certain length are inoculated into a rooting medium to induce rooting. The rooted regenerated buds are the regenerated plants.

2. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S1, the collection of the Eucalyptus urophylla branches is to collect semi-lignified 8-10 cm branches of Eucalyptus urophylla, containing 2-3 axillary buds, and place them in sterile water for moisturizing; before disinfection, the branches are shortened to 4-5 cm, each branch contains 1-2 axillary buds, and if there are side branches or leaves next to the axillary buds in the branches, the side branches or leaves should be shortened to 0.5-1 cm and rinsed under running water for 2 hours; the disinfection treatment is to cut off the tissue at the branch incision in an ultra-clean workbench, disinfect with 75% volume concentration of ethanol for 30 seconds, rinse with sterile water 4 times, and then disinfect with 0.12% volume concentration of mercuric chloride for 8 minutes, shake several times during the period, rinse with sterile water 5 times, and then place the branches on sterile filter paper to dry.

3. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S1, the tissue at the branch incision is cut off, the length of the morphological upper branch of the axillary bud point is left to be 0.3-0.5 cm, and the length of the morphological lower branch is left to be 2-3 cm, the lower end of the branch is inserted into an axillary bud induction medium, and when the axillary bud grows to 1 cm, the axillary bud is cut off to obtain a sterile seedling; the axillary bud induction medium is a mixture of 4.74 g / L MS medium components, 1 mg / L 6-BA, 0.1 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose are added, the pH is adjusted to 5.8, and 7 g / L agar is added.

4. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S2, the bud proliferation medium is a mixture of 4.74 g / L MS medium components, 0.4 mg / L 6-BA, 0.2 mg / L NAA, 0.5 g / L PVP40000, and 30 g / L sucrose are added, the pH is adjusted to 5.8, and 7 g / L agar is added.

5. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S3, the callus induction medium is a mixture of 2.47 g / L 1 / 2 MS medium components with 0.220 mg / L TDZ, 0.019 mg / L NAA, 30 g / L sucrose, and 7 g / L agar added, with a pH of 5.8; the callus induction conditions are dark culture at a temperature of 23 ± 1°C, and subculture every 15 days.

6. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S4, the regeneration bud induction medium is a mixture of 2.47 g / L 1 / 2 MS medium components, 0.25 mg / L 6-BA, 0.125 mg / L NAA, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.8; the regeneration bud induction condition is a light intensity of 10 μmol / (m 2 •s), photoperiod of 12 h / d, and temperature of 23 ± 1℃.

7. The plant regeneration method of the superior clone DH32-28 of Eucalyptus urophylla according to claim 1, characterized in that: In step S5, the regenerated buds that grew to 1 cm after induction were inoculated into a rooting medium to induce rooting; the rooting medium was a 2.47 g / L mixture of 1 / 2 MS medium components with 0.5 mg / L IBA, 30 g / L sucrose, and 6 g / L agar added, with a pH of 5.8.

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