A method for promoting poplar tissue culture regeneration using carbon nanotubes

Abstract

The invention discloses a method for promoting poplar tissue culture regeneration by using carbon nanotubes, comprising the following steps: 1) preparing an explant; 2) inducing the explant in a callus induction medium; 3) taking well-growing callus and transferring it to an adventitious bud induction medium for adventitious bud induction; 4) cutting off 0.5-1 cm adventitious buds and transferring them to a rooting medium for rooting induction until a complete plant is obtained; wherein the callus induction medium, the adventitious bud induction medium and the rooting medium are all added with carbon nanotubes to a WPM medium; the method can shorten the culture period of poplar regenerated plants, and the callus obtained from the plant added with carbon nanotubes has good quality, sprouts and roots first, and can better stabilize the callus induction rate, the adventitious bud induction rate and the rooting rate at above 85%.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and biotechnology, and particularly relates to a method for promoting poplar tissue culture regeneration by utilizing carbon nanotubes. Background Art

[0002] Carbon nanotubes (CNTs) are one-dimensional nanoscale carbon structures with an aspect ratio greater than 1000. They were first discovered in 1993 by Iijima et al. using a high-resolution transmission electron microscope. CNTs possess unique mechanical, thermal, and electrical properties. As a chemical material, they can alter chemical processes and biological structures in experimental reactions. They have applications in the chemical industry, aerospace, biomedicine, wastewater treatment, electronics, metallurgy, sensors, and other fields, demonstrating enormous economic and social benefits. CNTs are classified into single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) based on the number of graphene layers. Previous studies have shown that carbon nanomaterials can penetrate cell walls, transport plant growth regulators, accelerate dedifferentiation and callus formation, promote water absorption, and enhance seedling growth and rooting.

[0003] Poplars are a key afforestation species in my country, characterized by rapid growth, strong adaptability, and excellent wood quality. They are the country's primary fast-growing, high-yielding industrial timber species. Tissue culture technology, a key component of modern biotechnology, is widely used in Populus species, providing an effective avenue for genetic transformation and industrial propagation. Improving the efficiency and quality of poplar tissue culture is crucial. Current techniques for regeneration through tissue culture of poplar plants involve adding varying concentrations and types of growth regulators to induce dedifferentiation and redifferentiation of poplar explants, thereby producing sterile seedlings.

[0004] As a new type of material, how to use carbon nanotubes to effectively improve the efficiency and quality of poplar tissue culture regeneration is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for promoting poplar tissue culture regeneration by utilizing carbon nanotubes.

[0006] The technical solution of the present invention is summarized as follows:

[0007] A method for promoting poplar tissue culture regeneration using carbon nanotubes comprises the following steps:

[0008] 1) Explant preparation:

[0009] Sterile poplar seedlings were cultured in WPM medium for 30-40 days, and leaves from the 4th to 5th phyllotaxy position were taken from top to bottom. The leaves were divided into two sections along the direction perpendicular to the main vein, and the leaves with the edges cut off near the petiole were used as explants.

[0010] 2) Callus induction:

[0011] The explants are transferred to a callus induction medium to induce callus tissue; the first subculture is performed every 4 days, and the subsequent subculture is performed every 14 days until callus tissue is produced;

[0012] The callus induction medium is WPM medium supplemented with 0.75 mg / L 2,4-D, 0.5 mg / L KT, and 40-60 mg / L CNTs;

[0013] 3) Induction of adventitious buds:

[0014] Take the callus tissue with good growth and transfer it to the adventitious bud induction medium for adventitious bud induction, subculture once every 14 days to grow adventitious buds;

[0015] The adventitious bud induction medium is WPM medium with 0.02 mg / L TDZ and 40-60 mg / L CNTs added;

[0016] 4) Root induction:

[0017] Cut off 0.5-1cm adventitious buds and transfer them to rooting medium. Subculture every 14 days for rooting induction until complete plants are obtained.

[0018] The rooting medium is WPM medium with 40-60 mg / L of CNTs added;

[0019] The 2,4-D is the abbreviation of 2,4-dichlorophenoxyacetic acid;

[0020] The KT is the abbreviation of 6-glycosylaminopurine;

[0021] The CNTs is the abbreviation of carbon nanotubes;

[0022] The TDZ is the abbreviation of thidiazuron;

[0023] The culture conditions of the aseptic poplar seedlings in the WPM culture medium are preferably: temperature 22-23° C., relative humidity 70%-80%, light 16 h / dark 8 h, and light intensity 2000-3000 lux.

[0024] The explants are transferred to the callus induction medium and the induction conditions are preferably a temperature of 27-28° C., a relative humidity of 70%-80%, and continuous darkness.

[0025] The callus tissue is transferred to the adventitious bud induction medium for adventitious bud induction. The conditions for adventitious bud induction are preferably a temperature of 24-25° C., a relative humidity of 70%-80%, and continuous illumination with a light intensity of 2000-3000 lux.

[0026] The conditions for rooting induction of adventitious buds in rooting medium are preferably a temperature of 24-25° C., a relative humidity of 70%-80%, a light period of 16 h / dark period of 8 h, and a light intensity of 2000-3000 lux.

[0027] CNTs are SWCNTs or MWCNTs; SWCNTs is the abbreviation of single-walled carbon nanotubes; and MWCNTs is the abbreviation of multi-walled carbon nanotubes.

[0028] Advantages of the present invention:

[0029] The method of the present invention can shorten the culture period of poplar regeneration plants. The callus tissue obtained from the plants added with carbon nanotubes is of good quality and is the first to sprout and take root. Furthermore, the callus tissue induction rate, the regeneration bud induction rate and the rooting rate are better stabilized at above 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 2 shows the callus induction after adding carbon nanotubes (CNTs) (at 35 days of culture). a: no CNTs added; b: 50 mg / L SWCNTs added; c: 50 mg / L MWCNTs added.

[0031] Figure 2 The rooting status of adventitious buds 14 days after being transferred to the rooting medium. Among them: a is without carbon nanotubes; b is with 50 mg / L SWCNTs; c is with 50 mg / L WCNTs. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific examples.

[0033] The abbreviations of the various raw materials in the embodiments of the present invention are summarized as follows:

[0034] The 2,4-D is the abbreviation of 2,4-dichlorophenoxyacetic acid;

[0035] The KT is the abbreviation of 6-glycosylaminopurine;

[0036] The CNTs is the abbreviation of carbon nanotubes;

[0037] The TDZ is the abbreviation of thidiazuron;

[0038] SWCNTs is the abbreviation of single-walled carbon nanotubes;

[0039] MWCNTs is the abbreviation of multi-walled carbon nanotubes.

[0040] The poplars used in the embodiments of the present invention are Nanlin 895 Poplar or 84K Poplar as examples. However, this does not limit the present invention in any way. Other varieties of poplars can also be used in the present invention.

[0041] Example 1

[0042] A method for promoting poplar tissue culture regeneration using carbon nanotubes comprises the following steps:

[0043] 1) Explant preparation:

[0044] Sterile poplar (Nanlin 895 Poplar) seedlings were cultured in WPM medium for 35 days (any number of days between 30 and 40 days could be selected), and leaves from the 4th and 5th phyllotaxy positions from top to bottom were taken, the leaves were divided into two sections along the direction perpendicular to the main vein, and the leaves with the edges cut off near the petiole were used as explants;

[0045] 2) Callus induction:

[0046] The explants were transferred to callus induction medium to induce callus tissue; the first subculture was performed every 4 days, and the subsequent subculture was performed every 14 days until callus tissue was produced;

[0047] The callus induction medium is WPM medium supplemented with 0.75 mg / L 2,4-D, 0.5 mg / L KT and CNTs;

[0048] There are 7 groups of callus induction medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added; Figure 1 ;

[0049] 3) Induction of adventitious buds:

[0050] Take the callus tissue with good growth and transfer it to the adventitious bud induction medium for adventitious bud induction, subculture once every 14 days to grow adventitious buds;

[0051] The adventitious bud induction medium is WPM medium supplemented with 0.02 mg / L TDZ and CNTs;

[0052] There are 7 groups of adventitious bud induction medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added;

[0053] 4) Root induction:

[0054] Cut off 0.5-1cm adventitious buds and transfer them to rooting medium. Subculture every 14 days for rooting induction until complete plants are obtained.

[0055] The rooting medium is WPM medium with CNTs added;

[0056] There are 7 groups of rooting medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added; see Figure 2 ;

[0057] The conditions for culturing the sterile poplar seedlings in WPM medium were: temperature 22° C., relative humidity 70%, light 16 h / dark 8 h, and light intensity 2000 lux.

[0058] The explants were transferred to callus induction medium and induced under the conditions of temperature 27°C, relative humidity 70%, and continuous darkness.

[0059] The callus tissue was transferred to the adventitious bud induction medium for adventitious bud induction under the conditions of temperature 24°C, relative humidity 70%, continuous light conditions, and light intensity 2000 lux.

[0060] The conditions for rooting induction of adventitious buds in rooting medium were temperature 24°C, relative humidity 70%, light 16h / dark 8h, and light intensity 2000 lux.

[0061] SWCNTs is the abbreviation for single-walled carbon nanotubes; MWCNTs is the abbreviation for multi-walled carbon nanotubes.

[0062] Implementation Effect

[0063] The callus growth was observed regularly, and the number of explants producing callus after 35 days of culture was recorded to calculate the callus induction rate, which was calculated as (the number of explants producing callus / the number of explants inoculated) × 100%.

[0064] After 35 days of culture, the callus was removed, the adhering medium was washed off with sterile water, and the water was absorbed with filter paper. The fresh weight of the callus produced in each leaf was then weighed using an analytical balance.

[0065] The callus tissue with good growth was transferred to the adventitious bud induction medium, and the adventitious bud induction rate was recorded after 25 days of culture. The adventitious bud induction rate = (the number of explants producing adventitious buds / the number of explants inoculated) × 100%;

[0066] The adventitious buds were transferred to the rooting induction medium and the rooting induction rate was recorded after 14 days of culture. The rooting induction rate = (number of rooted explants / number of inoculated explants) × 100%;

[0067] SPSS analysis showed that callus growth in Nanlin 895 poplars treated with 40-60 mg / L SWCNTs or MWCNTs significantly improved callus fresh weight compared to the control group. Buds and roots formed earlier. Buds and roots began about a week earlier than in the control group without carbon nanotubes. Furthermore, callus induction rates, adventitious bud induction rates, and rooting rates remained stable at over 85%. (See Table 1.)

[0068] Table 1

[0069]

[0070] Example 2

[0071] A method for promoting poplar tissue culture regeneration using carbon nanotubes comprises the following steps:

[0072] 1) Explant preparation:

[0073] Poplar (84K Poplar) was used to replace the poplar (Nanlin 895 Poplar) in step 1) of Example 1, and the rest was the same as step 1) of Example 1;

[0074] 2) Callus induction:

[0075] The explants were transferred to callus induction medium to induce callus tissue; the first subculture was performed every 4 days, and the subsequent subculture was performed every 14 days until callus tissue was produced;

[0076] The callus induction medium is WPM medium supplemented with 0.75 mg / L 2,4-D, 0.5 mg / L KT and CNTs.

[0077] There are 7 groups of callus induction medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added;

[0078] 3) Induction of adventitious buds:

[0079] Take the callus tissue with good growth and transfer it to the adventitious bud induction medium for adventitious bud induction, subculture once every 14 days to grow adventitious buds;

[0080] The adventitious bud induction medium is WPM medium supplemented with 0.02 mg / L TDZ and CNTs;

[0081] There are 7 groups of adventitious bud induction medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added;

[0082] 4) Root induction:

[0083] Cut off 0.5-1cm adventitious buds and transfer them to rooting medium. Subculture every 14 days for rooting induction until complete plants are obtained.

[0084] The rooting medium is WPM medium with CNTs added;

[0085] There are 7 groups of rooting medium: no CNTs added; 40 mg / LSWCNTs added; 40 mg / LMWCNTs added; 50 mg / LSWCNTs added; 50 mg / LMWCNTs added; 60 mg / LSWCNTs added; 60 mg / LMWCNTs added;

[0086] The conditions for culturing the sterile poplar seedlings in WPM medium were: temperature 23° C., relative humidity 80%, light 16 h / dark 8 h, and light intensity 3000 lux.

[0087] The explants were transferred to callus induction medium and induced at a temperature of 28° C., a relative humidity of 80%, and continuous darkness.

[0088] The callus tissue was transferred to the adventitious bud induction medium for adventitious bud induction under the conditions of temperature 25°C, relative humidity 80%, continuous light conditions, and light intensity 3000 lux.

[0089] The conditions for rooting induction of adventitious buds in rooting medium were temperature 25°C, relative humidity 80%, light 16h / dark 8h, and light intensity 3000 lux.

[0090] The implementation results are shown in Table 2.

[0091] Table 2

[0092]

Claims

1. A method for promoting poplar tissue culture regeneration using carbon nanotubes, characterized in that The steps include: 1) Explant preparation: Sterile poplar seedlings were cultured in WPM medium for 30-40 days, and leaves from the 4th to 5th phyllotaxy position were taken from top to bottom. The leaves were divided into two sections along the direction perpendicular to the main vein, and the leaves with the edges cut off near the petiole were used as explants. 2) Callus induction: The explant is moved to a callus induction medium for induction to generate callus tissue; The callus induction medium is composed of WPM medium + 2,4-D 0.75 mg / L + KT 0.5 mg / L + CNTs 40-60 mg / L; 3) Induction of adventitious buds: Take the callus tissue with good growth and transfer it to the adventitious bud induction medium for adventitious bud induction to grow adventitious buds; The adventitious bud induction medium is composed of WPM medium + TDZ 0.02 mg / L + CNTs 40-60 mg / L; 4) Root induction: Cut off 0.5-1cm adventitious buds and transfer them to rooting medium for rooting induction until complete plants are obtained; The rooting culture medium consists of WPM culture medium and CNTs 40-60 mg / L.

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