Method for efficiently and rapidly propagating carya illinoensis by taking germs as explant materials

Abstract

The invention discloses a high-efficiency rapid propagation method of carya illinoensis by taking germs as explant materials, which comprises the following steps: taking seeds of carya illinoensis as an initial material source, peeling off shells, disinfecting the surfaces, and inoculating into a germination culture medium for germination culture of the germs; stripping germinated germs, inoculating the germinated germs into an adventitious bud induction culture medium, and carrying out induction culture on adventitious buds; separating the adventitious buds and then transferring the adventitious buds into an elongation culture medium for elongation culture of the adventitious buds; cutting the elongated bud into a stem section with at least one leaf and one bud, treating the stem section with a rooting promoting solution, inoculating the stem section into an adventitious root induction culture medium, and performing induction culture of adventitious roots to obtain a complete regenerated plant. The method provided by the invention has the advantages of high regeneration efficiency and convenience in material taking, the induction rate of the adventitious buds reaches 83.6%, each explant can generate 5.3 adventitious buds on average, and the rooting rate of the buds reaches 86.5% at most.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a method for high-efficiency and rapid propagation of thin-shell walnuts using embryos as explant materials. Background Art

[0002] Carya illinoinensis, also known as American pecan, is a deciduous tree of the genus Carya in the Juglandaceae family. It is native to the United States and northern Mexico and is one of the world's famous oil-bearing dried fruit tree species, with a kernel oil content of more than 70%. Carya illinoinensis has the advantages of large fruit, thin shell, and rich flesh. It is rich in nutritional value, rich in unsaturated fatty acids and mineral elements necessary for the human body, and is deeply loved by people. At the same time, Carya illinoinensis is also an excellent economic tree species and greening tree species for both fruit and wood. Its wood is hard, beautiful in texture, and corrosion-resistant. It is widely used in military industry, shipbuilding and construction. In addition, Carya illinoinensis also has strong applicability and can adapt to a wide range of soil pH. It is suitable for cultivation in limestone mountains. It is a very excellent ecological and economic tree species in my country with broad market prospects.

[0003] my country has introduced and cultivated thin-shelled pecans for hundreds of years. At present, thin-shelled pecans have become one of the preferred economic tree species for afforestation in southern mountainous areas of my country. At present, in production, thin-shelled pecans are mainly propagated by traditional sowing, grafting and root cutting, which have problems such as low propagation efficiency, long propagation cycle, and propagation being restricted by seasons and materials. It is difficult to meet the current domestic market demand for thin-shelled pecan seedlings of excellent varieties. In addition, thin-shelled pecan tissue culture technology also has technical bottlenecks, and there are few reports on this aspect at home and abroad. The lack of excellent varieties and effective variety breeding technology has become a bottleneck problem restricting the healthy development of the thin-shelled pecan industry. Therefore, it is urgent to develop a thin-shelled pecan efficient and rapid propagation method using embryos as explant materials to meet the needs of rapid propagation and variety improvement of thin-shelled pecan seedlings. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to use plant tissue culture technology to efficiently and quickly propagate thin-shell walnuts.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] A highly efficient and rapid propagation method for thin-shelled pecans using embryos as explant materials comprises the following steps:

[0007] (1) Acquisition of embryo materials: The seeds of Carya sylvestris were used as the initial material source. After the shells were peeled off, the surface was sterilized and inoculated into the germination medium for embryo germination culture;

[0008] (2) Induction culture of adventitious buds: The budding embryos are peeled off from the seeds and inoculated into adventitious bud induction medium for induction culture of adventitious buds;

[0009] (3) Elongation culture of adventitious buds: After separating the adventitious buds from the adventitious bud clusters, they are transferred to the elongation medium for elongation culture of the adventitious buds;

[0010] (4) Adventitious root induction culture: The elongated buds are cut into stem segments with at least one leaf and one bud, treated with rooting promotion solution, and then inoculated into adventitious root induction medium for adventitious root induction culture to obtain complete regenerated plants.

[0011] Preferably, in (1), the thin-shelled pecan is thin-shelled pecan 'Boni'.

[0012] Preferably, in (1), the germination medium is a 1 / 2DKW solid medium supplemented with 0.2-1.0 mg / L GA3, 0.2-0.5 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar.

[0013] Preferably, in (1), the culture time is 2 weeks; in (2), the culture time is 6 weeks; in (3), the culture time is 4 weeks; in (4), the culture time is 3 weeks.

[0014] Preferably, in (2), the adventitious bud induction medium is 1 / 2DKW medium supplemented with 0.05-0.5 mg / L TDZ, 0.2-1.0 mg / L meta-Topolin, 30 g / L sucrose, and 7 g / L agar.

[0015] Preferably, in (3), the elongation medium is 1 / 2DKW medium supplemented with 0.1-1 mg / L ZT, 0.1-1.0 mg / L GA3, 30 g / L sucrose and 7 g / L agar.

[0016] Preferably, in (4), the rooting promoting solution is an aqueous solution containing 500-1000 mg / L IBA and 200-500 mg / L NAA.

[0017] Preferably, in (4), the adventitious root induction medium is 1 / 4 DKW medium supplemented with 20 g / L sucrose and 7.0 g / L agar.

[0018] Preferably, in (4), the treatment time of the rooting promoting solution is 1-5 min.

[0019] Preferably, in (1), (2), (3) and (4), the culture conditions are: temperature of 25±2°C, light intensity of 40 μmol m-2 s -1 , the photoperiod was 14 / 10h (light / dark).

[0020] The advantages of the present invention are:

[0021] The invention provides a high-efficiency and rapid propagation method for thin-shell pecans using embryos as explant materials, which has the advantages of high regeneration efficiency and convenient material acquisition, an induction rate of adventitious buds of 83.6%, an average of 5.3 adventitious buds per explant, and a maximum rooting rate of buds of 86.5%, providing important technical support for large-scale propagation of thin-shell pecan seedlings and subsequent genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The thin-shelled pecan seeds inoculated on the germination medium and cultured under light for 2 weeks in Example 1 of the present invention;

[0023] Figure 2 The bud clusters induced from the embryos of Carya sylvestris inoculated on the induction medium and cultured under light for 3 weeks in Example 1 of the present invention;

[0024] Figure 3 The adventitious buds of Carya sylvestris inoculated on the induction medium and cultured under light for 6 weeks in Example 1 of the present invention;

[0025] Figure 4 The adventitious buds of Hickory nutsedge inoculated on the elongation medium and cultured for 2 weeks in Example 1 of the present invention;

[0026] Figure 5 The thin-shelled pecan stem segment inoculated on the rooting medium after being treated with the adventitious root inducing solution in Example 1 of the present invention;

[0027] Figure 6 The complete plant of Carya nutans was obtained by 3 weeks of light culture in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0030] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0031] Example 1

[0032] An efficient and rapid propagation method for thin-shell pecans using embryos as explant materials, the specific operation is as follows:

[0033] (1) The seeds of Hickory nut ‘Boni’ were used as the initial material source. After peeling the shell, they were sterilized with 75% ethanol for 30 seconds for 2 times, rinsed with sterile water for 3 times, and then sterilized with 0.1% mercuric chloride solution for 10 minutes. After that, they were washed with sterile water for 6 times. After the moisture on the surface of the seeds was absorbed, they were inoculated in a medium supplemented with 0.5 mg / L GA3 and 0.2 mg / L

[0034] The medium was 1 / 2DKW solid medium containing 6-BA, 30 g / L sucrose and 7 g / L agar at 25 ± 2 °C and a light intensity of 40 μmol m -2 s -1 , the seed germination culture was carried out in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 2 weeks of light culture, the seed contamination rate was 8.1%, and 90.5% of the seeds were in the germination state ( Figure 1 ).

[0035] (2) After the budding embryos were separated from the mature embryos, they were inoculated into 1 / 2 DKW medium supplemented with 0.1 mg / L TDZ, 0.5 mg / L meta-Topolin, 30 g / L sucrose, and 7 g / L agar at a temperature of 25 ± 2 °C and a light intensity of 40 μmol m - 2 s -1 , the adventitious buds were induced in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 3 weeks of light culture, the embryos induced adventitious bud clusters ( Figure 2 ); After 3 weeks of continuous culture, the adventitious bud induction rate was as high as 83.6%, with an average of 5.3 adventitious buds per explant ( Figure 3 ).

[0036] (3) After the adventitious buds were separated from the adventitious bud clusters, they were transferred to 1 / 2DKW medium supplemented with 0.5 mg / L ZT, 0.2 mg / L GA3, 30 g / L sucrose, and 7 g / L agar at a temperature of 25 ± 2 °C and a light intensity of 40 μmol m -2 s -1 , the elongation of the buds was carried out in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 2 weeks of light culture, the adventitious buds were strong, the leaves gradually stretched, and the average length of the buds was 2.5cm ( Figure 4 ).

[0037] (4) After culturing for another 2 weeks, the elongated buds were cut into stem segments (about 1.0 cm) with at least one leaf and one bud. The base of the stem segments was treated with a mixed aqueous solution of 1000 mg / L IBA and 400 mg / L NAA for 3 min and then inoculated in 1 / 4 DKW medium supplemented with 20 g / L sucrose and 7.0 g / L agar at 25 ± 2 °C and a light intensity of 40 μmol m -2 s -1 , rooting induction culture was carried out in a greenhouse with a photoperiod of 14 / 10 h (light / dark) ( Figure 5 After 2 weeks of light culture, adventitious root primordia were induced at the base of the stem segment. After 1 week of culture, the adventitious root induction rate was as high as 86.5%, and an average of 3.1 adventitious roots were produced per explant ( Figure 6 ).

[0038] Example 2

[0039] This example tests the effects of exogenous TDZ and meta-Topolin on the induction of adventitious buds of Carya sylvestris. The specific steps are as follows:

[0040] (1) The embryos germinated according to step (1) of Example 1 were separated from the mature embryos and inoculated into 1 / 2 DKW medium supplemented with different concentrations (0.05-0.5 mg / L) of TDZ and different concentrations (0.2-1.0 mg / L) of meta-Topolin, 30 g / L sucrose, and 7 g / L agar at a temperature of 25±2°C and a light intensity of 40 μmol m -2 s -1 , the induction culture of adventitious buds was carried out in a greenhouse with a photoperiod of 14 / 10h (light / dark). The induction of adventitious buds was observed from time to time during the culture period. After 6 weeks of light culture, the induction rate of adventitious buds was counted, and the average number of adventitious buds produced by each explant was calculated. The research results are shown in Table 1. The research results show that in the adventitious bud induction process of Hickory 'Boni', exogenous TDZ and meta-Topolin play a synergistic role within a certain concentration range. With the increase of the concentration of the two, the induction of adventitious buds is gradually inhibited. Among them, when the embryo was placed in the adventitious bud induction medium supplemented with a final concentration of 0.1mg / L TDZ and 0.5mg / Lmeta-Topolin for 6 weeks, the induction effect of adventitious buds was the best, the adventitious bud induction efficiency was 83.6%, and an average of 5.3 adventitious buds were produced per explant.

[0041] Table 1 Effects of exogenous TDZ and meta-Topolin concentrations on adventitious bud induction in embryos of Carya elata

[0042]

[0043]

[0044] Note: Data are mean values, each treatment contained 120 explants, and each treatment was repeated three times.

[0045] Example 3

[0046] This example tests the effects of exogenous ZT and GA3 on the elongation of adventitious buds of Carya sylvestris. The specific steps are as follows:

[0047] (1) The adventitious buds obtained in Example 1 were separated from the adventitious bud clusters and transferred to the culture medium supplemented with different concentrations (0.1, 0.2,

[0048] The cells were cultured in 1 / 2DKW medium containing 0.5 and 1.0 mg / L) ZT and different concentrations (0.1, 0.2, 0.5 and 1.0 mg / L) GA3, 30 g / L sucrose and 7 g / L agar at 25 ± 2 °C and a light intensity of 40 μmol m -2 s -1 , the elongation of buds was carried out in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 4 weeks of light culture, the elongation of adventitious buds was counted. The results in Table 2 show that the addition of ZT and GA3 in the culture medium can effectively promote the elongation of the leaves and stem segments of Hickory nut within a certain concentration range, and the two have a certain synergistic effect. Among them, the elongation effect of adventitious buds was the best when the adventitious buds were inoculated in the culture medium supplemented with 0.5mg / L ZT and 0.2mg / L GA3.

[0049] Table 2 Effects of exogenous ZT and GA3 on the elongation of adventitious buds of Carya sylvestris

[0050]

[0051] Note: Data are average values, each treatment contains 120 explants, and each treatment was repeated three times

[0052] Example 4

[0053] This example tests the effects of exogenous IBA and NAA concentrations and treatment time on adventitious root induction in Carya sylvestris. The specific steps are as follows: the elongated buds obtained in Example 3 were cut into stem segments of about 1.0 cm with at least one leaf and one bud, the base of the stem segments were immersed in aqueous solutions with different concentrations (500, 750 and 1000 mg / L) of IBA and different concentrations (200, 400 and 500 mg / L) of NAA for different treatment times (1, 2, 3 and 5 min), and then inoculated in 1 / 4 DKW medium with 20 g / L sucrose and 7.0 g / L agar at a temperature of 25 ± 2 ° C and a light intensity of 40 μmol m -2 s -1, rooting induction culture was carried out in a greenhouse with a photoperiod of 14 / 10h (light / dark). The induction of adventitious roots was observed every week, and the induction efficiency and average number of adventitious roots were counted after 3 weeks of light culture. The research results in Table 3 show that the immersion treatment of exogenous IBA and NAA combined with rooting in the bottle can effectively promote the formation of adventitious roots of thin-shelled pecans. Among them, the adventitious root induction effect of the stem segment inoculated after the base of the stem segment was treated with a mixed aqueous solution of a final concentration of 1000mg / L IBA and 400mg / LNAA for 3 minutes was the highest. After 3 weeks of light culture, the adventitious root induction rate was as high as 86.5%, and an average of 3.1 adventitious roots were produced per explant.

[0054] Table 3 Effects of different treatment times of exogenous IBA and NAA on the induction of adventitious roots of elongated buds in Carya sylvestris

[0055]

[0056] Note: Data are mean values, each treatment contained 120 explants, and each treatment was repeated three times.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An efficient and rapid propagation method for thin-shelled pecans using embryos as explant materials, characterized in that: The following steps are involved: (1) Acquisition of embryo materials: The seeds of Carya sylvestris were used as the initial material source. After the shells were peeled off, the surface was sterilized and inoculated into the germination medium for embryo germination culture; (2) Induction culture of adventitious buds: The budding embryos are peeled off from the seeds and inoculated into adventitious bud induction medium for induction culture of adventitious buds; (3) Elongation culture of adventitious buds: After separating the adventitious buds from the adventitious bud clusters, they are transferred to the elongation medium for elongation culture of the adventitious buds; (4) Adventitious root induction culture: The elongated buds are cut into stem segments with at least one leaf and one bud, treated with rooting promotion solution, and then inoculated into adventitious root induction medium for adventitious root induction culture to obtain complete regenerated plants.

2. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (1), the thin-shelled pecan is thin-shelled pecan 'Pony'.

3. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (1), the germination medium is a 1 / 2DKW solid medium supplemented with 0.2-1.0 mg / L GA3, 0.2-0.5 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar.

4. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (1), the culture time is 2 weeks; in (2), the culture time is 6 weeks; in (3), the culture time is 4 weeks; in (4), the culture time is 3 weeks.

5. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (2), the adventitious bud induction medium is 1 / 2 DKW medium supplemented with 0.05-0.5 mg / L TDZ, 0.2-1.0 mg / L meta-Topolin, 30 g / L sucrose, and 7 g / L agar.

6. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (3), the elongation medium is 1 / 2 DKW medium supplemented with 0.1-1 mg / L ZT, 0.1-1.0 mg / L GA3, 30 g / L sucrose and 7 g / L agar.

7. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (4), the rooting promoting solution is an aqueous solution containing 500-1000 mg / L IBA and 200-500 mg / L NAA.

8. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (4), the adventitious root induction medium is 1 / 4 DKW medium supplemented with 20 g / L sucrose and 7.0 g / L agar.

9. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to claim 1, characterized in that: In (4), the treatment time of the rooting promoting solution is 1-5 minutes.

10. The method for high-efficiency and rapid propagation of thin-shelled pecans using embryos as explant materials according to any one of claims 1 to 9, characterized in that: In (1), (2), (3) and (4), the culture conditions are: temperature of 25±2°C, light intensity of 40 μmol m -2 s -1 , the photoperiod was 14 / 10h (light / dark).

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