Method for creating and identifying excellent drought-enduring germplasm of macadamia nuts

Abstract

The invention relates to a method for creating and identifying excellent drought-enduring germplasm of macadamia nuts. The method comprises the following steps: (1) selecting seeds; (2) seed soaking treatment; (3) chemical mutagenesis; (4) performing ray irradiation mutagenesis; (5) raising seedlings; (6) culturing in a culture medium; (7) identifying selected products; more germplasm with various shapes can be obtained under the combination of chemical mutagenesis and low, high and medium three-level ray irradiation mutagenesis, and then a germplasm culture medium is optimized so as to identify and screen excellent drought-resistant macadamia germplasm; the first culture medium is optimized, meanwhile, the temperature and the impermeability of the culture medium are controlled, and macadimia nut germplasm with the better drought resisting effect can be preferably screened out; the second culture medium is optimized, then small molecule active substances are added, meanwhile, parameters such as impermeability of the culture medium are controlled, macadamia nut germplasm with the better drought resistance effect is further screened out, macadamia nut seedling root growth can be effectively promoted, and the drought resistance of macadamia nut seedlings can be conveniently calculated according to the rooting amount of seedling root systems.

Description

Technical Field

[0001] The present invention relates to the field of agricultural planting technology, and in particular to a method for creating and identifying excellent drought-resistant macadamia nut germplasm. Background Art

[0002] Macadamia nuts are also known as Queensland chestnuts, Australian walnuts, macadamia nuts, and Queensland nuts. They are a type of tree nut native to Australia. Macadamia nuts are evergreen trees and dicotyledonous plants. The tree has a tall crown, with 3 to 4 whorled leaves, which are lanceolate, leathery, smooth, and have thorn-like serrations on the edges. The racemes are axillary, the flowers are beige, the fruit is spherical, the fruit leather is leathery, the endocarp is hard, and the seed kernel is beige to light brown. It is suitable for growing in mild, humid, and windy areas. Among the many dried fruits in the world, macadamia nuts have the highest economic value and have always enjoyed the reputation of "the king of dried fruits". The kernel of macadamia nuts is rich in nutrients (milky white), its outer peel is green, the endocarp is hard and brown, and the single fruit weighs 15-16 grams, with an oil content of about 70%, protein of 9%, and 17 kinds of amino acids including 8 essential amino acids for the human body. It is also rich in minerals and vitamins. Macadamia nuts are crispy, tender and delicious, with a unique creamy aroma. They are the best quality edible fruit in the world and are known as the "Queen of Dried Fruits" and "King of Nuts in the World". Their flavor and taste are far better than cashews. In addition to being used to make dried fruits, macadamia nuts can also be used to make cakes, chocolate, edible oil, cosmetics, etc. In addition, macadamia nuts also have high nutritional and medicinal value.

[0003] At present, there are literature records that 36 macadamia varieties were used as research objects, and 9 physiological indicators were measured under continuous drought stress. The results of comprehensive analysis of the drought resistance of the varieties using the membership function method showed that Yunao 7', Yunao 24', Yunao 36', Yunao 32', and Yunao 17' were highly drought-resistant varieties, Yunao 29', Yunao 2', and Yunao 19 were weakly drought-resistant varieties, and the remaining varieties were moderately drought-resistant. Grey correlation analysis and principal component analysis were used to analyze the correlation between the drought resistance of 36 varieties and 9 indicators. The results showed that leaf relative water content, soluble sugar content, soluble protein content, superoxide dismutase, and free proline content can be used as important drought resistance identification indicators for 36 macadamia varieties. However, only screening for drought-resistant varieties of existing macadamia varieties can only screen out some drought-resistant varieties. To obtain macadamia varieties with higher drought resistance, it is necessary to create and identify excellent drought-resistant germplasms. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for creating and identifying excellent drought-resistant germplasm of macadamia nuts with good drought resistance.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for creating and identifying excellent drought-resistant germplasm of macadamia nuts, comprising the following steps:

[0006] (1) Seed selection: Select mature, shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds;

[0007] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them to obtain soaked seeds;

[0008] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution, taking them out and drying them, to obtain mutated soaked seeds; the chemical mutagen solution uses sterile water as a solvent and contains ethanol at a concentration of 100 to 120 g / kg, nitrosoethyl urea at a concentration of 2.5 to 3.5 g / kg, imidazoquinolinic acid at a concentration of 0.3 to 0.5 g / kg, and 2-methylquinoline at a concentration of 0.15 to 0.25 g / kg;

[0009] (4) Radiation mutagenesis:

[0010] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 15 to 20 Gy to obtain single irradiated seeds;

[0011] 4.1) Secondary irradiation: The soaked seeds are irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 200 to 250 Gy to obtain secondary irradiated seeds;

[0012] 4.1) Three-time irradiation: The soaked seeds are irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 80 to 100 Gy to obtain three-time irradiated seeds;

[0013] (5) Seedling cultivation: The seeds irradiated three times are placed in an artificial seedling bed and grown until the seedlings are 20 to 30 cm tall to obtain seedlings;

[0014] (6) Culture medium:

[0015] 6.1) placing the created seedlings in a first culture medium and culturing them at 10-15° C. for 20-25 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 80-100 parts of coconut bran; 30-35 parts of bagasse; 20-25 parts of vermiculite; 20-25 parts of peat soil; and 6-9 parts of sodium chloride;

[0016] 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 30-35 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 80-100 parts of coconut bran; 30-35 parts of bagasse; 20-25 parts of vermiculite; 20-25 parts of peat soil; 14-17 parts of sodium chloride; 3-5 parts of soybean isoflavones; and 0.3-0.5 parts of soybean saponin;

[0017] (7) Identification and selection: The rooting volume of the secondary cultured seedlings is used as the drought resistance standard.

[0018] Furthermore, in step (1), the mature shelled macadamia nuts are fresh fruits picked in the current year, and the variety of the mature shelled macadamia nuts is "Guire No. 1".

[0019] Furthermore, in step (2), the seed soaking agent solution uses sterile water as a solvent and contains carbendazim at a concentration of 2 to 3 mg / kg and gibberellin at a concentration of 250 to 350 mg / kg.

[0020] Furthermore, in the step (3), the soaking time is 12 to 18 hours.

[0021] Furthermore, in step (3), the chemical mutagen solution uses sterile water as a solvent and contains ethanol at a concentration of 110 to 120 g / kg, nitrosoethyl urea at a concentration of 3 to 3.5 g / kg, imidazoquinolinic acid at a concentration of 0.4 to 0.5 g / kg, and 2-methylquinoline at a concentration of 0.2 to 0.25 g / kg.

[0022] Furthermore, in step (3), the concentration ratio of imidazoquinolinic acid to 2-methylquinoline in the chemical mutagen solution is 2:1.

[0023] Furthermore, in step (4), the radiation irradiation mutagenesis is:

[0024] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 17 to 20 Gy to obtain single irradiated seeds;

[0025] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 to 250 Gy to obtain secondary irradiated seeds;

[0026] 4.1) Three-time irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 90 to 100 Gy to obtain three-time irradiated seeds.

[0027] Furthermore, in the step (5), the seeds irradiated three times are placed in an artificial seedbed and grown at 20-25°C until the seedlings are 20-30 cm in height to obtain created seedlings.

[0028] Furthermore, in step (6), the culture medium is cultured:

[0029] 6.1) placing the created seedlings in a first culture medium and culturing them at 12-15° C. for 22-25 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90-100 parts of coconut bran; 32-35 parts of bagasse; 23-25 ​​parts of vermiculite; 22-25 parts of peat soil; and 7-9 parts of sodium chloride;

[0030] 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 to 35 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 to 100 parts of coconut bran; 32 to 35 parts of bagasse; 23 to 25 parts of vermiculite; 22 to 25 parts of peat soil; 15 to 17 parts of sodium chloride; 4 to 5 parts of soybean isoflavones; and 0.4 to 0.5 parts of soybean saponins.

[0031] Furthermore, in the step (7), the rooting amount of the secondary cultured seedling root system is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0032] The beneficial effects of the present invention are as follows: at present, only relevant research on the screening of drought-resistant varieties of existing macadamia nut varieties has been seen. This type of method can only screen out some drought-resistant varieties. In order to obtain macadamia nut varieties with higher drought resistance, it is necessary to create and identify excellent drought-resistant germplasm; the present invention provides a method for creating and identifying excellent drought-resistant germplasm of macadamia nut, firstly adopts a combination of chemical mutagenesis and irradiation mutagenesis to carry out mutagenesis breeding, and more germplasms with various shapes can be obtained by combining chemical mutagenesis and low, high and medium three-level irradiation mutagenesis. Please optimize the germplasm culture medium in order to identify and screen out excellent drought-resistant macadamia germplasm; specifically, this application specially optimizes the first culture medium, and controls the temperature and the osmotic resistance of the culture medium, so as to preferentially screen out macadamia germplasm with better drought resistance; and then optimize the second culture medium, add small molecule active substances to the culture medium, and control the osmotic resistance and other parameters of the culture medium, so as to further screen out macadamia germplasm with better drought resistance, and effectively promote the root growth of macadamia seedlings, so as to facilitate the calculation of the drought resistance of macadamia seedlings based on the rooting amount of the seedling roots.

[0033] The invention discloses a method for creating and identifying excellent drought-resistant macadamia nut germplasm, which combines chemical mutagenesis with radiation mutagenesis. The chemical mutagen uses nitrosoethyl urea, which is an alkylating agent. The mechanism of action is alkylation. By replacing hydrogen atoms of other molecules through alkyl substitution, the action focuses on nucleic acids, which can cause DNA breakage, deletion or repair. A certain ratio of imidazoquinolinic acid and 2-methylquinoline is added to the chemical mutagen. Imidazoquinolinic acid and 2-methylquinoline are heterocyclic compounds. Imidazoquinolinic acid is an alkylating agent that is a catalytic inhibitor of acetolactate synthase or acetohydroxyacid synthase. Enzyme inhibitors inhibit the synthesis of acetolactate in plants and can be used as herbicides. 2-Methylquinoline can be used as insecticides, fungicides, etc., but there is no literature disclosing the use of imidazoquinoline acid and 2-methylquinoline in the creation of germplasm. The present application combines nitrosoethyl urea, imidazoquinoline acid and 2-methylquinoline to accelerate the mutation speed of macadamia seeds and increase the mutation rate; subsequently combined with multiple irradiation mutagenesis, the mutagenesis effect is further enhanced, the mutagenesis of macadamia seeds is promoted, and the yield of drought-resistant macadamia seedlings can be effectively increased. DETAILED DESCRIPTION

[0034] The following embodiments may help those skilled in the art to more fully understand the present invention, but shall not limit the present invention in any way.

[0035] The invention discloses a method for creating and identifying excellent drought-resistant macadamia nut germplasm. The seed soaking agent solution can be used to effectively sterilize seeds according to actual conditions; and gibberellin can be selectively added.

[0036] The present invention provides a method for creating and identifying excellent drought-resistant germplasm of macadamia nuts, and an example of a method for calculating the drought resistance standard. The number of young roots of the created seedlings cultivated in step (5) is 100, and then after culturing in the culture medium in step (6), the number of young roots of the secondary cultured seedlings is 120. It can be calculated that the rooting amount is 20%, confirming that the drought resistance of the created seedlings is average.

[0037] Example 1

[0038] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0039] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0040] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2 mg / kg of carbendazim and 250 mg / kg of gibberellin;

[0041] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 12 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 100 g / kg of ethanol, 2.5 g / kg of nitrosoethyl urea, 0.3 g / kg of imidazoquinolinic acid, and 0.15 g / kg of 2-methylquinoline;

[0042] (4) Radiation mutagenesis:

[0043] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 15 Gy to obtain single irradiated seeds;

[0044] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 200 Gy to obtain secondary irradiated seeds;

[0045] 4.1) Three-time irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 80 Gy to obtain three-time irradiated seeds;

[0046] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 20°C until the seedlings were 20 cm tall to obtain seedlings;

[0047] (6) Culture medium:

[0048] 6.1) placing the created seedlings in a first culture medium and culturing them at 10° C. for 20 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 80 parts of coconut bran; 30 parts of bagasse; 20 parts of vermiculite; 20 parts of peat soil; and 6 parts of sodium chloride;

[0049] 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 30 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 80 parts of coconut bran; 30 parts of bagasse; 20 parts of vermiculite; 20 parts of peat soil; 14 parts of sodium chloride; 3 parts of soybean isoflavones; and 0.3 parts of soybean saponin;

[0050] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0051] Example 2

[0052] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0053] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0054] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 3 mg / kg of carbendazim and 350 mg / kg of gibberellin;

[0055] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 18 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 120 g / kg of ethanol, 3.5 g / kg of nitrosoethyl urea, 0.5 g / kg of imidazoquinolinic acid, and 0.25 g / kg of 2-methylquinoline;

[0056] (4) Radiation mutagenesis:

[0057] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 20 Gy to obtain single irradiated seeds;

[0058] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 250 Gy to obtain secondary irradiated seeds;

[0059] 4.1) Three-time irradiation: The seeds that were soaked for mutagenesis were irradiated with 137Cs-γ rays, with an irradiation dose rate of 0.75 Gy / min and an irradiation dose intensity of 100 Gy to obtain three-time irradiated seeds;

[0060] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 25°C until the seedlings were 30 cm tall to obtain seedlings;

[0061] (6) Culture medium:

[0062] 6.1) placing the created seedlings in a first culture medium and culturing them at 15° C. for 25 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 100 parts of coconut bran; 35 parts of bagasse; 25 parts of vermiculite; 25 parts of peat soil; and 9 parts of sodium chloride;

[0063] 6.2) The seedlings cultured once were placed in a second culture medium and cultured for 35 days to obtain seedlings cultured twice; the first culture medium had the following proportions by weight: 100 parts of coconut bran; 35 parts of bagasse; 25 parts of vermiculite; 25 parts of peat soil; 17 parts of sodium chloride; 5 parts of soybean isoflavones; and 0.5 parts of soybean saponin;

[0064] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0065] Example 3

[0066] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0067] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0068] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0069] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol, 3 g / kg of nitrosoethyl urea, 0.4 g / kg of imidazoquinolinic acid, and 0.2 g / kg of 2-methylquinoline;

[0070] (4) Radiation mutagenesis:

[0071] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 17 Gy to obtain single irradiated seeds;

[0072] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 Gy to obtain secondary irradiated seeds;

[0073] 4.1) Three-time irradiation: The seeds soaked for mutagenesis were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 90 Gy to obtain seeds irradiated three times;

[0074] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 23°C until the seedlings were 25 cm tall to obtain seedlings;

[0075] (6) Culture medium:

[0076] 6.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 7 parts of sodium chloride;

[0077] 6.2) The seedlings cultured once were placed in a second culture medium and cultured for 32 days to obtain seedlings cultured twice; the first culture medium had the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 15 parts of sodium chloride; 4 parts of soybean isoflavones; and 0.4 parts of soybean saponin;

[0078] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0079] Example 4

[0080] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0081] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0082] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0083] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol, 3 g / kg of nitrosoethyl urea, 0.45 g / kg of imidazoquinolinic acid, and 0.25 g / kg of 2-methylquinoline;

[0084] (4) Radiation mutagenesis:

[0085] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 18 Gy to obtain single irradiated seeds;

[0086] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 240 Gy to obtain secondary irradiated seeds;

[0087] 4.1) Three-time irradiation: The seeds soaked for mutation were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 95 Gy to obtain seeds irradiated three times;

[0088] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 23°C until the seedlings were 25 cm tall to obtain seedlings;

[0089] (6) Culture medium:

[0090] 6.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 8 parts of sodium chloride;

[0091] 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 16 parts of sodium chloride; 4.5 parts of soybean isoflavones; and 0.45 parts of soybean saponin;

[0092] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0093] Comparative Example 1

[0094] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0095] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0096] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0097] (3) Radiation mutagenesis:

[0098] 3.1) Primary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 17 Gy to obtain primary irradiated seeds;

[0099] 3.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 Gy to obtain secondary irradiated seeds;

[0100] 3.1) Three-time irradiation: The seeds that were soaked for mutagenesis were irradiated with 137Cs-γ rays, with an irradiation dose rate of 0.75 Gy / min and an irradiation dose intensity of 90 Gy to obtain seeds that were irradiated three times;

[0101] (4) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 23°C until the seedlings were 25 cm tall to obtain seedlings;

[0102] (5) Culture medium:

[0103] 5.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 7 parts of sodium chloride;

[0104] 5.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 15 parts of sodium chloride; 4 parts of soybean isoflavones; and 0.4 parts of soybean saponin;

[0105] (6) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0106] Comparative Example 2

[0107] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0108] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0109] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0110] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol, 3 g / kg of nitrosoethyl urea, 0.4 g / kg of imidazoquinolinic acid, and 0.2 g / kg of 2-methylquinoline;

[0111] (4) Seedling cultivation: The induced seeds were placed in an artificial seedbed and grown at 23°C until the seedlings were 25 cm tall to obtain the created seedlings;

[0112] (5) Culture medium:

[0113] 5.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 7 parts of sodium chloride;

[0114] 5.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 15 parts of sodium chloride; 4 parts of soybean isoflavones; and 0.4 parts of soybean saponin;

[0115] (6) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0116] Comparative Example 3

[0117] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0118] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0119] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0120] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol, 3 g / kg of nitrosoethyl urea, 0.4 g / kg of imidazoquinolinic acid, and 0.2 g / kg of 2-methylquinoline;

[0121] (4) Radiation mutagenesis: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 Gy to obtain irradiated seeds;

[0122] (5) Seedling cultivation: The irradiated seeds are placed in an artificial seedbed and grown at 23°C until the seedlings are 25 cm tall to obtain seedlings;

[0123] (6) Culture medium:

[0124] 6.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 7 parts of sodium chloride;

[0125] 6.2) The seedlings cultured once were placed in a second culture medium and cultured for 32 days to obtain seedlings cultured twice; the first culture medium had the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 15 parts of sodium chloride; 4 parts of soybean isoflavones; and 0.4 parts of soybean saponin;

[0126] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0127] Comparative Example 4

[0128] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0129] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0130] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0131] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol, 3 g / kg of nitrosoethyl urea, 0.4 g / kg of imidazoquinolinic acid, and 0.2 g / kg of 2-methylquinoline;

[0132] (4) Radiation mutagenesis:

[0133] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 17 Gy to obtain single irradiated seeds;

[0134] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 Gy to obtain secondary irradiated seeds;

[0135] 4.1) Three-time irradiation: The seeds soaked for mutagenesis were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 90 Gy to obtain seeds irradiated three times;

[0136] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 23°C until the seedlings were 25 cm tall to obtain seedlings;

[0137] (6) Identification and selection: The rooting volume of the seedling root system cannot be used as a criterion for drought resistance.

[0138] Comparative Example 5

[0139] A method for creating and identifying excellent drought-resistant macadamia germplasm, comprising the following steps:

[0140] (1) Seed selection: Select mature shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; the mature shelled macadamia nuts are fresh fruits picked that year, and the variety of mature shelled macadamia nuts is "Guire No. 1";

[0141] (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them, thereby obtaining soaked seeds; the soaking agent solution uses sterile water as a solvent and contains 2.5 mg / kg of carbendazim and 280 mg / kg of gibberellin;

[0142] (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution for 16 hours, taking them out and drying them to obtain soaked seeds for mutagenesis; the chemical mutagen solution uses sterile water as a solvent and contains 110 g / kg of ethanol and 3 g / kg of nitrosoethyl urea;

[0143] (4) Radiation mutagenesis:

[0144] 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 18 Gy to obtain single irradiated seeds;

[0145] 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 240 Gy to obtain secondary irradiated seeds;

[0146] 4.1) Three-time irradiation: The seeds soaked for mutation were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 95 Gy to obtain seeds irradiated three times;

[0147] (5) Seedling cultivation: The seeds irradiated three times were placed in an artificial seedling bed and grown at 23°C until the seedlings were 25 cm tall to obtain seedlings;

[0148] (6) Culture medium:

[0149] 6.1) placing the created seedlings in a first culture medium and culturing them at 12° C. for 22 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; and 8 parts of sodium chloride;

[0150] 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 parts of coconut bran; 32 parts of bagasse; 23 parts of vermiculite; 22 parts of peat soil; 16 parts of sodium chloride; 4.5 parts of soybean isoflavones; and 0.45 parts of soybean saponin;

[0151] (7) Identification and selection: The rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the rooting amount of the secondary cultured seedlings is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

[0152] The experimental results of the creation and identification of cold-resistant germplasm of macadamia nuts in Examples 1-4 and Comparative Examples 1-5 are statistically analyzed:

[0153] Method for calculating the number of deformed seedlings: observe the morphology of the seedlings and the special traits of the seedlings, such as short plants, thin plants, deformed leaves, etc., collect data on the special traits, and calculate the number of deformed seedlings;

[0154] Drought resistance detection method: relative water content, soluble sugar content, soluble protein content, superoxide dismutase, and free proline content of leaves are used as drought resistance identification indicators; at the same time, the obtained seedlings are placed in a drought environment for cultivation, and the survival of the seedlings is used to determine the drought resistance of the seedlings;

[0155] Table 1 below gives the statistical results of the germination rate, survival rate of seedlings, number of deformed seeds, and number of drought-resistant seedlings in Examples 1-4 and Comparative Examples 1-5;

[0156] Table 1

[0157]

[0158] In Example 1-4, the rooting amount of the seedling root system of the secondary culture can be used as the drought resistance standard; the rooting amount of the seedling root system of the secondary culture is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak. The drought-resistant seedlings selected according to the drought resistance standard in Example 1-4 are consistent with the results of the drought-resistant seedlings selected by using the relative water content of the leaves, the soluble sugar content, the soluble protein content, the superoxide dismutase, and the free proline content as drought resistance identification indicators.

[0159] In Examples 1-4, the drought-resistant seedlings selected according to the drought tolerance standard are consistent with the results of the drought-resistant seedlings selected by placing the seedlings in a drought environment and cultivating them using the survival of the seedlings as the drought resistance identification index.

[0160] In comparative example 4, the rooting amount of the seedling root system cannot be used as a drought resistance standard; only the relative water content of the leaves, the soluble sugar content, the soluble protein content, the superoxide dismutase, the free proline content, and the survival of the seedlings in a drought environment can be used as drought resistance identification indicators to screen out drought-resistant seedlings.

[0161] It can be seen from the above experimental results that the creation and identification method of excellent drought-resistant macadamia germplasm of the present invention first adopts a combination of chemical mutagenesis and radiation mutagenesis to carry out mutagenesis breeding. Under the combination of chemical mutagenesis and low, high and medium three-level radiation mutagenesis, more germplasms with various shapes can be obtained. The present application further optimizes the germplasm culture medium to identify and screen out excellent drought-resistant macadamia germplasm; specifically, the present application specially optimizes the first culture medium, and at the same time controls the temperature and the osmotic resistance of the culture medium, so as to preferentially screen out macadamia germplasm with better drought resistance; and then optimizes the second culture medium, adds small molecule active substances to the culture medium, and at the same time controls parameters such as the osmotic resistance of the culture medium, so as to further screen out macadamia germplasm with better drought resistance, and at the same time can effectively promote the root growth of macadamia seedlings, so as to facilitate the calculation of the drought resistance of macadamia seedlings according to the rooting amount of the seedling root system; chemical mutagenesis and radiation mutagenesis are combined, and the chemical mutagen adopts nitroso Ethyl urea and nitrosoethyl urea are alkylating agents with an action mechanism of alkylation. They replace hydrogen atoms of other molecules through alkyl substitution. The action focuses on nucleic acids, which can cause DNA breakage, deletion or repair. A certain ratio of imidazoquinolinic acid and 2-methylquinoline is added to the chemical mutagen. Imidazoloquinolinic acid and 2-methylquinoline are heterocyclic compounds. Imidazoloquinolinic acid is an inhibitor of acetolactate synthase or acetohydroxyacid synthase, which inhibits the acetolactate synthesis of plants and can be used as a herbicide. 2-methylquinoline can be used as an insecticide, a fungicide, etc. However, there is no document publicly showing the use of imidazoquinolinic acid and 2-methylquinoline in the creation of germplasm. The present application combines nitrosoethyl urea, imidazoquinolinic acid and 2-methylquinoline to accelerate the mutation speed of macadamia seeds and increase the mutation rate. Subsequently, multiple ray irradiation mutagenesis is combined to further enhance the mutagenic effect, promote the mutagenesis of macadamia seeds, and effectively increase the yield of drought-resistant macadamia seedlings.

[0162] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for creating and identifying excellent drought-resistant macadamia germplasm, characterized in that: The following steps are involved: (1) Seed selection: Select mature, shelled macadamia nuts that are free of pests and diseases and have smooth, undamaged shells as seeds; (2) Seed soaking treatment: placing the seeds in a soaking agent solution for soaking and sterilizing the seeds, taking them out and drying them to obtain soaked seeds; (3) Chemical mutagenesis: soaking the soaked seeds in a chemical mutagen solution, taking them out and drying them, to obtain mutated soaked seeds; the chemical mutagen solution uses sterile water as a solvent and contains ethanol at a concentration of 100 to 120 g / kg, nitrosoethyl urea at a concentration of 2.5 to 3.5 g / kg, imidazoquinolinic acid at a concentration of 0.3 to 0.5 g / kg, and 2-methylquinoline at a concentration of 0.15 to 0.25 g / kg; (4) Radiation mutagenesis: 4.1) Primary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 15 to 20 Gy to obtain primary irradiated seeds; 4.1) Secondary irradiation: The soaked seeds are irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 200 to 250 Gy to obtain secondary irradiated seeds; 4.1) Three-time irradiation: The soaked seeds are irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 80 to 100 Gy to obtain three-time irradiated seeds; (5) Seedling cultivation: The seeds irradiated three times are placed in an artificial seedling bed and grown until the seedlings are 20 to 30 cm tall to obtain seedlings; (6) Culture medium: 6.1) placing the created seedlings in a first culture medium and culturing them at 10-15° C. for 20-25 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 80-100 parts of coconut bran; 30-35 parts of bagasse; 20-25 parts of vermiculite; 20-25 parts of peat soil; and 6-9 parts of sodium chloride; 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 30-35 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 80-100 parts of coconut bran; 30-35 parts of bagasse; 20-25 parts of vermiculite; 20-25 parts of peat soil; 14-17 parts of sodium chloride; 3-5 parts of soybean isoflavones; and 0.3-0.5 parts of soybean saponin; (7) Identification and selection: The rooting volume of the secondary cultured seedlings is used as the drought resistance standard.

2. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (1), the mature shelled macadamia nuts are fresh fruits picked in the current year, and the variety of the mature shelled macadamia nuts is "Guire No. 1".

3. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (2), the seed soaking agent solution uses sterile water as a solvent and contains carbendazim at a concentration of 2 to 3 mg / kg and gibberellin at a concentration of 250 to 350 mg / kg.

4. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (3), the soaking time is 12 to 18 hours.

5. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (3), the chemical mutagen solution uses sterile water as a solvent and contains ethanol at a concentration of 110 to 120 g / kg, nitrosoethyl urea at a concentration of 3 to 3.5 g / kg, imidazoquinolinic acid at a concentration of 0.4 to 0.5 g / kg, and 2-methylquinoline at a concentration of 0.2 to 0.25 g / kg.

6. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (3), the concentration ratio of imidazoquinolinic acid to 2-methylquinoline in the chemical mutagen solution is 2:

1.

7. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (4), the radiation irradiation induces mutagenesis: 4.1) Single irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 17 to 20 Gy to obtain single irradiated seeds; 4.1) Secondary irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 230 to 250 Gy to obtain secondary irradiated seeds; 4.1) Three-time irradiation: The soaked seeds were irradiated with 137Cs-γ rays at a dose rate of 0.75 Gy / min and a dose intensity of 90 to 100 Gy to obtain three-time irradiated seeds.

8. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (5), the seeds irradiated three times are placed in an artificial seedbed and grown at 20-25°C until the seedlings are 20-30 cm tall, thereby obtaining created seedlings.

9. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (6), the culture medium is cultured: 6.1) placing the created seedlings in a first culture medium and culturing them at 12-15° C. for 22-25 days to obtain primary cultured seedlings; the first culture medium has the following proportions by weight: 90-100 parts of coconut bran; 32-35 parts of bagasse; 23-25 ​​parts of vermiculite; 22-25 parts of peat soil; and 7-9 parts of sodium chloride; 6.2) placing the first cultured seedlings in a second culture medium and culturing them for 32 to 35 days to obtain second cultured seedlings; the first culture medium has the following proportions by weight: 90 to 100 parts of coconut bran; 32 to 35 parts of bagasse; 23 to 25 parts of vermiculite; 22 to 25 parts of peat soil; 15 to 17 parts of sodium chloride; 4 to 5 parts of soybean isoflavones; and 0.4 to 0.5 parts of soybean saponins.

10. The method for creating and identifying excellent drought-resistant macadamia germplasm according to claim 1, characterized in that: In the step (7), the rooting amount of the secondary cultured seedling root system is used as the drought resistance standard; the drought resistance standard is: when the rooting amount reaches 30-40%, the drought resistance is strong; when the rooting amount is above 20% and <30%, the drought resistance is general; when the rooting amount is <20%, the drought resistance is weak.

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