A method for obtaining transgenic plants from Castanopsis chinensis and its application

Abstract

The present invention discloses a method for obtaining transgenic plants of Castanopsis chinensis. The base of the stem of Castanopsis chinensis seedlings is infected with Agrobacterium rhizogenes carrying a fluorescent reporter gene. After infection, the seedlings are cultured in pure vermiculite pots. Transgenic roots can be obtained in one month. After the transgenic roots are lignified, transgenic plants are obtained by root cutting. The method of the present invention is simple to operate, low in cost, high in efficiency, and short in cycle. The transgenic adventitious roots can be quickly verified using a handheld fluorescent protein excitation light source, and then transgenic plants are successfully obtained by transgenic root culture. The method of the present invention is an efficient method for obtaining stable transgenic plants by bypassing tissue culture. It can also be applied to the rapid verification of gene functions of Castanopsis chinensis and the genetic engineering breeding of Castanopsis chinensis, laying a good foundation for the future development of a gene editing breeding system for Castanopsis chinensis.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for obtaining transgenic plants from Castanopsis sylvestris and an application thereof. Background Art

[0002] Idesia polycarpa Maxim. is a new, high-yield, high-quality woody oil-bearing economic tree species. Both its flesh and seeds produce oil at a high oil content, making it a fully-fruited oil-producing tree species, earning it the nickname "the oil depot" of the tree. Idesia polycarpa oil is rich in linoleic acid, DHA, tocopherol, β-sitosterol, and other natural active substances, making it a highly nutritious edible vegetable oil that meets edible oil hygiene standards. Furthermore, Idesia polycarpa is suitable for large-scale cultivation on marginal lands, such as barren hills and wastelands. Therefore, developing an Idesia polycarpa industry could increase my country's edible oil supply without competing with grain production and reduce the proportion of oil imports. This is crucial for alleviating my country's increasingly severe edible oil supply security. Furthermore, developing the Idesia polycarpa industry would also increase the incomes of farmers in mountainous areas. However, the lack of high-quality Idesia polycarpa varieties has limited its rapid development.

[0003] In recent years, molecular-assisted breeding technologies, such as gene editing, have demonstrated significant advantages in the rapid improvement and cultivation of new crop varieties. The publicly available genome data for Castanopsis chinensis has laid a solid foundation for gene-editing and molecular-assisted breeding of Castanopsis chinensis. However, research on Castanopsis chinensis began relatively late, and no genetic transformation systems have been reported. This lack of a genetic transformation system has limited the application of gene editing technology in the breeding and improvement of new Castanopsis chinensis varieties. Therefore, establishing an efficient genetic transformation system for Castanopsis chinensis and obtaining transgenic plants would not only facilitate the study of gene function in Castanopsis chinensis but also lay the foundation for the use of gene editing technology to cultivate new, superior Castanopsis chinensis varieties.

[0004] Agrobacterium-mediated transformation is commonly used in the field of plant genetic transformation, with Agrobacterium tumefaciens and Agrobacterium rhizogenes being the two most common Agrobacterium strains. Traditional plant genetic transformation is achieved through co-cultivation of Agrobacterium tumefaciens under tissue culture conditions. Through the integration of exogenous genes and the regeneration of transgenic plants, the genetic characteristics of the target gene are stably maintained and expressed. However, for some difficult-to-transform plants and woody plants, obtaining regenerated shoots through the Agrobacterium tumefaciens transformation pathway is extremely difficult and time-consuming, and antibiotic screening is also required to identify transgenic plants. Summary of the Invention

[0005] In view of this, the present invention provides a simple, rapid and efficient Agrobacterium rhizogenes-mediated transformation method and its application for obtaining Castanopsis schrenkiana transgenic plants in a short period of time without the need for tissue culture.

[0006] In order to solve the above technical problems, the present invention provides a method for obtaining transgenic plants from Castanopsis sylvestris, comprising the following steps:

[0007] (1) Infecting plant materials: The stems of Castanopsis truncatula seedlings were used as plant materials, and Agrobacterium rhizogenes colonies carrying a fluorescent reporter gene were inoculated into the base of the stems to obtain infected materials;

[0008] (2) Watering the infection material: inserting the infection material into a soil pot containing a matrix, and watering the Agrobacterium rhizogenes liquid carrying the fluorescent reporter gene into the matrix along the stem;

[0009] (3) Hairy root induction: The plant materials after watering are moved to a smart greenhouse for cultivation for 25-35 days to generate transgenic hairy roots;

[0010] (4) Obtaining transgenic plants through the above-mentioned transgenic hairy roots.

[0011] Furthermore, in step (1), the Castanopsis aviculare seedlings are healthy seedlings that are 20-40 days old; and the stems of the Castanopsis aviculare seedlings are semi-lignified stems.

[0012] Furthermore, in the step (1), a sterile syringe with a volume of 1 mL is used to infect the stems of the Castanopsis schrenkiana seedlings. The syringe needle needs to be sterilized on an alcohol lamp. The inoculation and infection are carried out on the stems about 1-2 cm away from the roots of the Castanopsis schrenkiana. Four rows are inoculated in four directions of the stems of each Castanopsis schrenkiana seedling.

[0013] Furthermore, the four directions were distributed in four opposite directions on the stem of the Castanopsis chinensis seedlings, with any adjacent inoculation points spaced 2 mm apart to obtain the infection material.

[0014] Furthermore, in step (1), the Agrobacterium rhizogenes is K599 Agrobacterium rhizogenes carrying the mCherry reporter gene, and the specific preparation process of the bacterial solution during infection is as follows:

[0015] The K599 Agrobacterium strain containing mCherry was streaked and cultured in LB solid medium containing kanamycin and rifampicin, and activated and cultured in a constant temperature incubator at 28°C for 2 days; several single colonies were picked and inoculated into LB liquid medium containing kanamycin and rifampicin and 10mM calcium chloride, with a culture volume of 3-5mL, and cultured at 28°C and 200rpm for 12 hours; then 300μL of the bacterial solution was taken and smeared on LB solid medium containing kanamycin and rifampicin for proliferation and culture, and the bacterial solution was expanded at a ratio of 1:1000 to a bacterial solution OD600 = 0.8~1.0 to obtain Agrobacterium colonies and bacterial solution, respectively.

[0016] Furthermore, in step (2), the matrix in the soil pot is sterilized pure vermiculite; after the plant material is infected, it is irrigated with clean water to cultivate, and the matrix in the soil pot is kept moist without water accumulation.

[0017] Furthermore, in the step (2), the bacterial solution used for irrigation is the Agrobacterium rhizogenes bacterial solution carrying the mCherry gene, and 3-5 mL is applied to each Castanopsis chinensis seedling.

[0018] Furthermore, in step (3), the photoperiod in the intelligent greenhouse environment is 16 hours of light and 8 hours of darkness, the temperature is maintained at 24-30°C, and the humidity is maintained at 70% to 90%.

[0019] Furthermore, in step (4), after the transgenic hairy roots are lignified, they are segmented and cut into soil pots for cultivation, and transgenic plants grow on the hairy roots in 30-40 days; wherein the segmented transgenic root segments are about 4 cm-6 cm long and about 3 mm-5 mm thick, and some lateral roots can be retained.

[0020] Furthermore, in step (4), the cutting matrix is vermiculite.

[0021] The present invention also provides an application of a method for obtaining transgenic plants from Castanopsis schrenkiana. The method for obtaining transgenic plants from Castanopsis schrenkiana can be used to establish a Castanopsis schrenkiana genetic transformation system.

[0022] The present invention also provides an application of a method for obtaining transgenic plants from Castanopsis schrenkiana. The method for obtaining transgenic plants from Castanopsis schrenkiana can be used to establish stably transgenic Castanopsis schrenkiana plants.

[0023] The present invention also provides an application of a method for obtaining transgenic plants from Castanopsis schrenkiana. The method for obtaining transgenic plants from Castanopsis schrenkiana can be used for cultivating new varieties of Castanopsis schrenkiana.

[0024] The beneficial effects of the technical solution of the present invention are as follows: the method of obtaining transgenic plants from Castanopsis truncatum L. of the present invention rapidly obtains transgenic roots, and after the transgenic roots become lignified, stable transgenic plants can be obtained in just one month. The method of the present invention does not require tissue culture techniques and has the advantages of simple operation, low cost, and a short cycle time. Furthermore, the present invention utilizes the mCherry reporter gene in conjunction with a handheld fluorescent protein excitation light source to rapidly and cost-effectively perform nondestructive testing and identification of transgenic roots, and then rapidly obtains transgenic plants from the transgenic roots. The method of the present invention can be used to rapidly obtain transgenic plants and establish a genetic transformation system for Castanopsis truncatum L., providing an effective technical approach for the cultivation of new Castanopsis truncatum L. varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of the induction transformation of hairy roots of Castanopsis truncatum in the method for obtaining transgenic plants of Castanopsis truncatum in Example 1 of the present invention;

[0026] Figure 2 The handheld fluorescent protein excitation light source of Example 2 of the present invention is used to preliminarily identify the transgenic roots of Castanopsis sylvestris;

[0027] Figure 3 This is the identification of transgenic roots of Castanopsis sylvestris by laser confocal microscopy in Example 2 of the present invention (bar: 50 μm);

[0028] Figure 4A In Example 2 of the present invention, at the DNA level, conventional PCR was used to amplify and identify the target gene mCherry in the transgenic roots;

[0029] Figure 4B In Example 2 of the present invention, the RNA level was measured using semi-quantitative PCR to amplify and identify the target gene mCherry in transgenic roots;

[0030] Figure 5 This is the transgenic root segment regenerated plant of Example 4 of the present invention (bar: 1 cm);

[0031] Figure 6A The fourth embodiment of the present invention uses a handheld fluorescence excitation source to identify transgenic plants (bar: 1 cm);

[0032] Figure 6B The fourth embodiment of the present invention uses a confocal microscope (bar: 20 μm) to identify transgenic plants;

[0033] Figure 7A In Example 4 of the present invention, the target gene mCherry in the transgenic plant was amplified and identified using conventional PCR at the DNA level;

[0034] Figure 7B In Example 4 of the present invention, the RNA level was measured, and semi-quantitative PCR was used to amplify and identify the target gene mCherry in the transgenic plants. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0036] It has been reported that Agrobacterium rhizogenes can be used to transform some difficult-to-transform plants, inducing the production of transgenic hairy roots to produce composite transgenic plants. Furthermore, because hairy roots induced by Agrobacterium rhizogenes develop from a single transformed cell, the probability of chimera formation is low, making it possible to regenerate transgenic plants from transgenic roots. Therefore, given that Agrobacterium rhizogenes-mediated plant transformation does not require cumbersome tissue culture steps, has a short transformation cycle, and is simple to operate, it is highly feasible for transforming woody plants such as Castanopsis sylvestris, which lack genetic transformation systems.

[0037] Example 1

[0038] Refer to the attached Figure 1 , an embodiment of the present invention provides a method for genetic transformation of the root system of Castanopsis sylvestris, comprising the following steps:

[0039] (1) The stems of Castanopsis chinensis seedlings were used as plant materials, and K599 Agrobacterium colonies carrying the mCherry reporter gene were picked out with a syringe needle and inoculated into the base of the stems. The colonies were inoculated in four directions of the stems of each Castanopsis chinensis seedling to obtain infected materials;

[0040] (2) Insert the infected material into the seedling tray, ensuring that the inoculation wound of the plant material is covered by the matrix in the seedling tray, and inject the bacterial solution of K599 Agrobacterium carrying the mCherry reporter gene into the matrix along the stem through a syringe, keeping the matrix moist without water accumulation;

[0041] (3) The infected plant material is moved to a smart greenhouse for cultivation for 25-35 days to generate transgenic hairy roots.

[0042] Preferably, in step (1), the Castanopsis aviculare seedlings are healthy seedlings that are 20-40 days old; and the stems of the Castanopsis aviculare seedlings are semi-lignified Castanopsis aviculare stems.

[0043] Preferably, in step (1), a sterile syringe with a volume of 1 mL is used to infect the stems of Castanopsis schrenkiana seedlings. The syringe needle needs to be sterilized on an alcohol lamp, and the infection inoculation is performed on the stems about 1-2 cm away from the roots of Castanopsis schrenkiana.

[0044] Preferably, in step (1), the four directions are distributed in four opposite directions on the stem of the Castanopsis schrenkiana seedling, and any adjacent inoculation points are spaced 2 mm apart to obtain the infective material.

[0045] Preferably, in step (1), the Agrobacterium rhizogenes is K599 Agrobacterium rhizogenes carrying the mCherry reporter gene, and the specific preparation process of the bacterial solution during infection is as follows:

[0046] The mCherry K599 Agrobacterium strain was streaked and cultured in LB solid culture medium containing kanamycin and rifampicin, and activated and cultured in a constant temperature incubator at 28°C for 2 days; several single colonies were picked and inoculated into LB liquid culture medium containing kanamycin and rifampicin and 10mM calcium chloride, with a culture volume of 3-5ml, and cultured at 28°C and 200rpm for 12 hours; then 300μL of the bacterial solution was taken and smeared on LB solid culture medium containing kanamycin and rifampicin for proliferation and culture, and the bacterial solution was expanded at a ratio of 1:1000 to a bacterial solution OD600 = 0.8~1.0 to obtain Agrobacterium colonies and bacterial solution, respectively.

[0047] Preferably, in step (2), the matrix in the seedling tray is sterilized pure vermiculite, and the plant material after infection is irrigated and cultured using clean water.

[0048] Preferably, in step (2), the bacterial solution used for irrigation is the Agrobacterium rhizogenes bacterial solution carrying the mCherry gene, and 3-5 mL is applied to each stem of Castanopsis chinensis seedling.

[0049] Preferably, in step (3), the ambient light cycle in the smart greenhouse is 16 hours of light and 8 hours of darkness, the temperature is maintained at 24-30°C, and the humidity is maintained at 70% to 90%.

[0050] Example 2

[0051] After inducing hairy roots by the method of Example 1 of the present invention, positive identification of transgenic plants was performed, including fluorescence signal detection and molecular identification.

[0052] The Agrobacterium strain used in this example carries the mCherry gene, so transgenic tissue is identified by red fluorescence. Typical hairy roots that form are washed and observed using a handheld fluorescent protein excitation light source and a laser confocal microscope. The presence and expression of the mCherry gene are verified at both the DNA and RNA levels.

[0053] In the embodiment of the present invention, four groups of infected plants were selected for observation, with natural light on the left and mCherry fluorescence on the right.

[0054] Refer to the attached Figure 2 , you can see that some hairy roots show red signals under fluorescence.

[0055] In the present embodiment, a laser confocal microscope was further used to observe the fluorescence signal, and hairy roots that were not successfully transformed were selected as a control group.

[0056] Refer to the attached Figure 3 In the mCherry laser channel, red fluorescence can be clearly seen evenly distributed in the root cells of the hairy roots with red signals, while no signal was observed in the control group, indicating that mCherry was integrated into the hairy roots of Castanopsis sylvestris induced by the present invention.

[0057] The present invention further extracts the hairy root DNA and RNA induced by the method of Example 1.

[0058] The extracted DNA was amplified by PCR (DNA polymerase chain reaction) (Tables 1 and 2). Specific primers (mCherry-F / R) were used to verify the vector pFGC-mCherry. The primer sequences are shown in Table 3.

[0059] Table 1 PCR 20 μL reaction system

[0060]

[0061] Table 2 PCR reaction conditions

[0062]

[0063] Table 3 Primers

[0064]

[0065] Refer to the attached Figure 4A The target gene band was detected by 1% agarose gel. DNA of positive hairy roots of transgenic Castanopsis chinensis was extracted and a specific fragment with the expected length (500 bp) was amplified by conventional PCR.

[0066] The extracted RNA was reverse transcribed into cDNA and then amplified by semi-quantitative PCR. The expression of the mCherry gene was verified using specific primers (mCherry-F2 / R2) and an internal reference gene (UBA80-F / R). The primer sequences are shown in Table 4.

[0067] Table 4 Primers

[0068]

[0069] Refer to the attached Figure 4B , amplification was performed using semi-quantitative PCR, and mCherry was not expressed in wild-type roots, but was expressed in transgenic roots. This indicates that the method of the present invention embodiment using Agrobacterium rhizogenes to transform Castanopsis sylvestris to obtain transgenic hairy roots is feasible, and the mCherry gene can be stably expressed in the hairy roots.

[0070] Example 3

[0071] Based on the method of Example 1 of the present invention, the genetic transformation efficiency of Agrobacterium rhizogenes K599 on the roots of Castanopsis sylvestris at different developmental stages was explored.

[0072] Hairy roots are produced by plant cortical cells. For woody plants, as the physiological age increases and the degree of lignification increases, the proportion of cortical cells will gradually decrease, thereby hindering the induction of hairy roots. Therefore, in order to analyze whether the root genetic transformation efficiency of Castanopsis chinensis at different developmental stages is different, the examples of the present invention use Castanopsis chinensis with different degrees of lignification for infection, and the stems of Castanopsis chinensis seedlings with no lignification (20 days), slight lignification (30 days), semi-lignified (40 days) and fully lignified (100 days) are infected. Note: Transgenic root induction rate = positive root induced plants / hairy root induced plants, each experiment was repeated three times.

[0073] As shown in Table 5, at 20, 30, 40, and 100 days, the transformation efficiency of Castanopsis truncatum seedlings at 40 days was as high as 71.91%. However, when the degree of lignification increased to complete lignification (100 days), the transformation efficiency dropped to 0. This indicates that the transformation efficiency of Castanopsis truncatum seedlings with Agrobacterium rhizogenes is related to the degree of lignification of the stems, with the best transformation efficiency achieved when the stems were semi-lignified.

[0074] Table 5 Regeneration rates of transgenic roots and hairy roots of Castanopsis chinensis at different developmental stages

[0075]

[0076] Example 4

[0077] The embodiments of the present invention provide a method for obtaining transgenic plants from Castanopsis sylvestris and a method for identifying the transgenic plants.

[0078] After the transgenic hairy roots have become lignified, cut the lignified roots into segments 4-6 cm long and 3-5 mm thick. Some lateral roots may be retained and cultured in vermiculite.

[0079] Refer to the attached Figure 5 After one month of cultivation in vermiculite, new plants can be seen regenerating from the root segments.

[0080] The transgenic roots used in the present invention contain a vector encoding the mCherry gene. Regenerated plants were identified by mCherry red fluorescence. Observation was performed using a handheld fluorescent protein excitation light source and a laser confocal microscope. The presence and expression of mCherry were verified at both the DNA and RNA levels.

[0081] Regenerated plants from wild-type roots and regenerated plants from transgenic roots were selected for observation using a handheld fluorescence excitation source. The left side is natural light, and the right side is mCherry fluorescence.

[0082] Refer to the attached Figure 6A It can be seen that under fluorescence irradiation, the transgenic root regenerated plants have red signals as a whole.

[0083] The embodiment of the present invention further uses a laser confocal microscope to observe the fluorescence signal.

[0084] Refer to the attached Figure 6B Under the mCherry laser channel, red signals were observed in the leaves of the transgenic root regenerated plants, distributed in the leaf cells; whereas no red signals were observed in the control group, indicating that mCherry was integrated into the transgenic root regenerated plants induced by the present invention.

[0085] DNA and RNA were extracted from the leaves of the regenerated plants of the hairy roots induced in the embodiment of the present invention.

[0086] Extracted DNA was amplified by PCR (polymerase chain reaction), and extracted RNA was reverse-transcribed to cDNA and then amplified by semi-quantitative PCR. The pFGC-mCherry vector was validated by PCR and semi-quantitative PCR using specific primers (mCherry-F2 / R2). UAB80 was used as an internal reference gene for Castanopsis sylvestris (Mountain Cross section). The primer sequences are shown in Table 4.

[0087] Refer to the attached Figure 7A The target gene band was detected by 1% agarose gel. DNA of the regenerated shoots from the transgenic roots of Castanopsis truncatula was extracted and a specific fragment with the expected length (354 bp) was amplified by conventional PCR.

[0088] Refer to the attached Figure 7B RNA was extracted from the leaves of transgenic root regenerated plants and amplified using semi-quantitative PCR. mCherry was not expressed in the regenerated plants of wild-type roots, but was expressed in the regenerated plants of transgenic roots, indicating that mCherry can be stably expressed in the regenerated plants of transgenic hairy roots.

[0089] This demonstrates that the method for obtaining transgenic plants from Castanopsis truncatum according to the embodiments of the present invention can stably produce transgenic Castanopsis truncatum plants, and can be used to obtain transgenic Castanopsis truncatum plants, effectively establish a genetic transformation system for Castanopsis truncatum, and cultivate new Castanopsis truncatum varieties. The method of the embodiments of the present invention not only lays a solid foundation for future research on the gene function of Castanopsis truncatum and future gene-editing molecular breeding, but also provides strong support for the cultivation of high-quality female Castanopsis truncatum plants.

Claims

1. A method for obtaining transgenic plants from Castanopsis sylvestris, characterized by: The following steps are involved: (1) Infected plant materials: Take the stems of Castanopsis chinensis seedlings as plant materials, and inoculate the base of the stems with Agrobacterium rhizogenes carrying a fluorescent reporter gene to obtain the infected materials; the Castanopsis chinensis seedlings are healthy seedlings that are 20-40 days old; the stems of the Castanopsis chinensis seedlings are semi-lignified stems of Castanopsis chinensis seedlings; inoculate 4 rows in 4 directions of the stem of each Castanopsis chinensis seedling; (2) Watering the plant material: inserting the infected material into a soil pot containing a matrix, and pouring the Agrobacterium rhizogenes solution carrying the fluorescent reporter gene into the matrix along the stem; (3) Hairy root induction: The watered plant material is moved to a smart greenhouse for 25-35 days to generate transgenic hairy roots; (4) Obtain transgenic plants through transgenic hairy roots; after the transgenic hairy roots become lignified, they are cut into segments and cultured in soil pots; that is, the lignified roots are cut into segments 4-6 cm long and 3-5 mm thick, and some lateral roots can be retained. The segments are cultured in vermiculite, and transgenic plants grow on the hairy roots after 30-40 days.

2. The method for obtaining transgenic plants from Castanopsis sylvestris according to claim 1, characterized in that: The four directions are distributed in four opposite directions on the stem of the Castanopsis schefflera seedlings, and any adjacent inoculation points are spaced 2 mm apart to obtain the infection material.

3. The method for obtaining transgenic plants from Castanopsis sylvestris according to claim 1, wherein: In the step (2), the inoculated wound of the plant material is covered with a matrix in the soil pot, and the matrix is sterilized pure vermiculite.

4. The method for obtaining transgenic plants from Castanopsis sylvestris according to claim 1, wherein: In the step (2), 3-5 mL of Agrobacterium rhizogenes solution carrying a fluorescent reporter gene is applied to the infecting material.

5. The method for obtaining transgenic plants from Castanopsis sylvestris according to claim 1, wherein: In step (3), the photoperiod in the smart greenhouse environment is 16 hours of light and 8 hours of darkness, the temperature is 24-30°C, and the humidity is 70%-90%.

6. Use of the method for obtaining transgenic plants from Castanopsis sylvestris according to any one of claims 1 to 5, characterized in that: Used to obtain transgenic plants of Castanopsis chinensis.

7. Use of the method for obtaining transgenic plants from Castanopsis sylvestris according to any one of claims 1 to 5, characterized in that: Used to construct a genetic transformation system for Castanopsis sylvestris.

8. Use of the method for obtaining transgenic plants from Castanopsis sylvestris according to any one of claims 1 to 5, characterized in that: Used for the cultivation of new varieties of Castanopsis chinensis.

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