A high-efficiency rapid propagation method of carya illinoensis using embryo bud as explant material

Abstract

The application discloses a high-efficiency rapid propagation method of Carya illinoinensis by using embryo as explant material, and comprises the following steps: taking the seed of Carya illinoinensis as initial material, peeling off the shell, and inoculating the seed in a germination medium after surface disinfection to carry out germination culture of the embryo; inoculating the germinated embryo in an adventitious bud induction medium to carry out induction culture of the adventitious bud after peeling off the germinated embryo; inoculating the separated adventitious bud in an elongation medium to carry out elongation culture of the adventitious bud; cutting the elongated bud into stem segments with at least one leaf and one bud, treating the stem segments in a rooting promoting liquid, inoculating the stem segments in an adventitious root induction medium to carry out induction culture of the adventitious root, and obtaining complete regenerated plants. The method has the advantages of high regeneration efficiency and convenient material taking, the induction rate of the adventitious bud reaches 83.6%, an average of 5.3 adventitious buds can be generated from each explant, and the rooting rate of the bud reaches 86.5% at the highest.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology, and in particular to a high-efficiency rapid propagation method of Carya illinoinensis using embryo buds as explant materials. BACKGROUND

[0002] Carya illinoinensis, also known as American hickory, is a deciduous tree of the Juglandaceae family and is native to the United States and northern Mexico. It is one of the world's famous oil-bearing nut trees, with a kernel oil content of over 70%. Carya illinoinensis has the advantages of large fruit, thin shell, and high flesh content. It is rich in unsaturated fatty acids and essential mineral elements for the human body, and is deeply loved by people. At the same time, Carya illinoinensis is also a good economic tree species and a greening tree species for fruit and wood use. Its wood is hard, has beautiful texture, and is resistant to corrosion, making it widely used in the military, shipping, and construction industries. In addition, Carya illinoinensis has strong adaptability and can adapt to a wide range of soil pH, making it a very good ecological and economic tree species in China, with a broad market prospect.

[0003] Carya illinoinensis has been introduced and cultivated in China for more than a hundred years. Currently, Carya illinoinensis has become one of the preferred economic tree species for afforestation in the mountainous areas of southern China. Currently, in production, Carya illinoinensis is mainly propagated using traditional methods such as sowing, grafting, and root cutting. However, these methods have low propagation efficiency, long propagation cycles, and are limited by season and material, making it difficult to meet the current domestic market demand for Carya illinoinensis seedlings of excellent varieties. In addition, the tissue culture technology for Carya illinoinensis also has technical bottlenecks, and there are few relevant reports at home and abroad. The lack of excellent varieties and effective variety breeding techniques has become a bottleneck problem limiting the healthy development of the Carya illinoinensis industry. Therefore, there is an urgent need to develop a high-efficiency rapid propagation method of Carya illinoinensis using embryo buds as explant materials to meet the demand for rapid propagation and variety improvement of Carya illinoinensis seedlings. SUMMARY

[0004] The technical problem to be solved by the present application is how to use plant tissue culture technology to efficiently and rapidly propagate Carya illinoinensis.

[0005] The present application solves the above technical problems by the following technical means:

[0006] A high-efficiency rapid propagation method of Carya illinoinensis using embryo buds as explant materials, comprising the following steps:

[0007] (1) Obtaining embryo bud materials: using Carya illinoinensis seeds as the initial material source, after removing the outer shell, surface sterilizing, and inoculating in germination culture medium for embryo bud germination culture;

[0008] (2) Induction culture of adventitious buds: the germinating embryo is peeled off from the seed and inoculated into an induction medium for induction of adventitious buds;

[0009] (3) Elongation culture of adventitious buds: the adventitious buds are separated from the adventitious bud cluster and transferred into an elongation medium for elongation culture of the adventitious buds;

[0010] (4) Induction culture of adventitious roots: the elongated buds are cut into stem segments with at least one leaf and one bud, treated with a rooting promoting solution, and inoculated into an induction medium for induction of adventitious roots to obtain complete regenerated plants.

[0011] Preferably, in (1), the Carya illinoensis is Carya illinoensis 'Pony'.

[0012] Preferably, in (1), the germination medium is 1 / 2DKW solid medium added 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; and in (4), the culture time is 3 weeks.

[0014] Preferably, in (2), the induction medium for adventitious buds is 1 / 2DKW medium added 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 added 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 IBA at a concentration of 500-1000 mg / L and NAA at a concentration of 200-500 mg / L.

[0017] Preferably, in (4), the induction medium for adventitious roots is 1 / 4DKW medium added 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-2s-1, and a 16h light / 8h dark photoperiod.-2 s -1 , the photoperiod is 14 / 10h (light / dark).

[0020] The present application has the advantages of:

[0021] The efficient rapid propagation method of Carya illinoensis with embryo bud as explant material has the advantages of high regeneration efficiency and convenient material taking, the induction rate of adventitious buds reaches 83.6%, an average of 5.3 adventitious buds can be generated from each explant, and the rooting rate of the buds reaches 86.5% at the highest, which provides important technical support for the large-scale propagation and later genetic improvement of Carya illinoensis seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Carya illinoensis seeds inoculated on germination medium and cultured under light for 2 weeks in Example 1 of the present application;

[0023] Figure 2 Carya illinoensis bud sprouts induced by embryo buds inoculated on induction medium and cultured under light for 3 weeks in Example 1 of the present application;

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

[0025] Figure 4 Carya illinoensis adventitious buds inoculated on elongation medium and cultured for 2 weeks in Example 1 of the present application;

[0026] Figure 5 Carya illinoensis stem segments inoculated on rooting medium after treatment with adventitious root induction solution in Example 1 of the present application;

[0027] Figure 6 Carya illinoensis complete plants obtained by 3 weeks of light culture in Example 1 of the present application. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in a clear and complete manner in combination with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0029] In the following examples, the test materials and reagents used, etc., can be obtained from commercial channels unless otherwise specified.

[0030] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0031] Example 1

[0032] A highly efficient and rapid propagation method for thin-shelled pecans using embryos as explants is described below:

[0033] (1) Using the seeds of thin-shelled pecan 'Boni' as the initial material, after removing the outer shell, the seeds were disinfected with 75% ethanol for 2.5 seconds, rinsed 3 times with sterile water, then disinfected with 0.1% mercuric chloride solution for 10 minutes, rinsed 6 times with sterile water, and the surface moisture was dried before inoculation with 0.5 mg / L GA3 and 0.2 mg / L GA3.

[0034] In 1 / 2 DKW solid medium containing 6-BA, 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 Seed germination culture was conducted 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 stage. Figure 1 ).

[0035] (2) After separating the germinating embryos 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, and incubated at a temperature of 25 ± 2 °C and a light intensity of 40 μmol / m². - 2 s -1 Adventitious buds were induced and cultured in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 3 weeks of light culture, adventitious bud clusters were induced from the embryos. Figure 2 After 3 weeks of continued culture, the adventitious bud induction rate reached 83.6%, with an average of 5.3 adventitious buds produced per explant. Figure 3 ).

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

[0037] (4) After 2 weeks of continuous culture, the elongated shoots were cut into stem segments (about 1.0 cm) with at least one leaf and one bud, and the stem segments were treated with a mixed aqueous solution of IBA and NAA at final concentrations of 1000 mg / L and 400 mg / L for 3 min, and then inoculated into 1 / 4 DKW medium added with 20 g / L sucrose and 7.0 g / L agar, and subjected to rooting induction culture in a greenhouse at a temperature of 25±2°C, a light intensity of 40 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark). Figure 5 After 2 weeks of illumination culture, the stem segment base induced root primordia, and after 1 week of continuous culture, the induction rate of adventitious roots reached 86.5%, and the average number of adventitious roots produced by each explant was 3.1. Figure 6

[0038] Example 2

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

[0040] (1) After the germinated embryos separated from the mature embryos according to the steps of (1) in Example 1 were inoculated into 1 / 2 DKW medium added 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, and subjected to induction culture of adventitious buds in a greenhouse at a temperature of 25±2°C, a light intensity of 40 μmol m -2 s -1 , and a photoperiod of 14 / 10 h (light / dark), the induction of adventitious buds was observed at any time during the culture. After 6 weeks of illumination culture, the induction rate of adventitious buds and the average number of adventitious buds produced by each explant were counted. The research results are shown in Table 1, and the research results show that in the process of induction of adventitious buds of Carya illinoinensis ‘Pony’, exogenous TDZ and meta-Topolin have a synergistic promoting effect within a certain concentration range, and with the increase of the concentrations of the two, the induction of adventitious buds is gradually inhibited. When the germinated embryos are cultured in the adventitious bud induction medium added with TDZ at a final concentration of 0.1 mg / L and meta-Topolin at a final concentration of 0.5 mg / L for 6 weeks, the induction effect of adventitious buds is the best, the induction efficiency of adventitious buds is 83.6%, and the average number of adventitious buds produced by each explant is 5.3.

[0041] Table 1 Effects of concentrations of exogenous TDZ and meta-Topolin on induction of adventitious buds of Carya illinoinensis embryos

[0042]

[0043] ​

[0044] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0045] Example 3

[0046] This embodiment tested the effects of exogenous ZT and GA3 on the elongation of adventitious buds in pecan. The specific steps are as follows:

[0047] (1) The adventitious buds obtained in Example 1 were separated from the adventitious bud cluster and then transferred to plants treated with different concentrations (0.1, 0.2, ...).

[0048] ZT (0.5 and 1.0 mg / L) and GA3 (0.1, 0.2, 0.5 and 1.0 mg / L), 30 g / L sucrose and 7 g / L agar in 1 / 2 DKW medium at a temperature of 25 ± 2 °C and a light intensity of 40 μmol m. -2 s -1 Bud elongation culture was conducted in a greenhouse with a photoperiod of 14 / 10h (light / dark). After 4 weeks of light culture, the elongation of adventitious buds was recorded. Table 2 shows that the addition of ZT and GA3 to the culture medium within a certain concentration range can effectively promote the extension of pecan leaves and the elongation of stem segments, and the two have a certain synergistic promoting effect. Among them, the adventitious buds showed the best elongation effect when inoculated into a culture medium supplemented with 0.5 mg / L ZT and 0.2 mg / L GA3.

[0049] Table 2 Effects of exogenous ZT and GA3 on adventitious bud elongation in pecan.

[0050]

[0051] Note: Data are averages. Each treatment contained 120 explants, and each treatment was repeated three times.

[0052] Example 4

[0053] This example tested the effects of exogenous IBA and NAA concentrations and treatment time on adventitious root induction in pecan. The specific steps are as follows: The elongated shoots obtained in Example 3 were cut into stem segments approximately 1.0 cm long, each with at least one leaf and one bud. The base of the stem segments was immersed in aqueous solutions containing different concentrations (500, 750, and 1000 mg / L) of IBA and different concentrations (200, 400, and 500 mg / L) of NAA for different times (1, 2, 3, and 5 min). After treatment, the segments were inoculated into 1 / 4 DKW medium supplemented 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 / m². -2 s -1The rooting induction culture was carried out in a greenhouse with a photoperiod of 14 / 10 h (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 illumination culture. The results in Table 3 show that the soaking treatment of exogenous IBA and NAA combined with in-vitro rooting can effectively promote the formation of adventitious roots of C. illinoensis. Among them, the stem segments inoculated after the base was treated with a mixed aqueous solution of IBA and NAA with a final concentration of 1000 mg / L IBA and 400 mg / L NAA for 3 min had the highest induction effect of adventitious roots. After 3 weeks of illumination culture, the induction rate of adventitious roots was as high as 86.5%, and the average number of adventitious roots produced by each explant was 3.1.

[0054] Table 3 Influence of different treatment times of exogenous IBA and NAA on the induction of adventitious roots of elongated buds of C. illinoensis

[0055]

[0056] Note: The data are average values, each treatment contains 120 explants, and each treatment is repeated three times.

[0057] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for rapid propagation of Carya illinoinensis using embryo axis as explant material, characterized by: The method comprises the following steps: (1) obtaining of embryo material: taking the seed of Carya glabra as the initial material source, after the outer shell is peeled off, the surface is sterilized, and the embryo is cultured in germination medium; the germination medium is 1 / 2 DKW solid medium added 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; (2) induction culture of adventitious buds: the germinated embryo is peeled off from the seed and inoculated in the adventitious bud induction medium for induction culture of adventitious buds; the adventitious bud induction medium is 1 / 2 DKW medium added 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; (3) elongation culture of adventitious buds: the adventitious buds are separated from the adventitious bud cluster and transferred to the elongation medium for elongation culture of the adventitious buds; the elongation medium is 1 / 2 DKW medium added with 0.1-1 mg / L ZT, 0.1-1.0 mg / L GA3, 30 g / L sucrose and 7 g / L agar; (4) induction culture of adventitious roots: the elongated buds are cut into stem segments with at least one leaf and one bud, treated with a rooting promoting liquid, inoculated in the adventitious root induction medium for induction culture of adventitious roots to obtain complete regenerated plants; the rooting promoting liquid is an aqueous solution with a concentration of 500-1000 mg / L IBA and 200-500 mg / L NAA; the adventitious root induction medium is 1 / 4 DKW medium added with 20 g / L sucrose and 7.0 g / L agar.

2. The method for rapid propagation of Carya illinoensis using plumule as explant material according to claim 1, characterized in that: In (1), the Carya glabra is Carya glabra 'Pony'.

3. The method for rapid propagation of Carya illinoensis using plumule as explant material 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; and in (4), the culture time is 3 weeks.

4. The method for rapid propagation of Carya illinoensis using plumule as explant material according to claim 1, characterized in that: In (4), the treatment time of the rooting promoting liquid is 1-5 min.

5. The method for rapid propagation of Carya illinoensis using embryo bud as explant material according to any one of claims 1-4, characterized in that: In (1), (2), (3) and (4), the culture conditions are: temperature is 25 ± 2 ℃, light intensity is 40 μmol m -2 s -1 , photoperiod is 14h light / 10h dark.

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