CN109496849B - Method for promoting macadamia nut stem to induce callus - Google Patents
Method for promoting macadamia nut stem to induce callus Download PDFInfo
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- CN109496849B CN109496849B CN201811413799.1A CN201811413799A CN109496849B CN 109496849 B CN109496849 B CN 109496849B CN 201811413799 A CN201811413799 A CN 201811413799A CN 109496849 B CN109496849 B CN 109496849B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to the technical field of macadimia nut cultivation, and particularly relates to a method for promoting macadimia nut stem segment to induce callus.
Description
Technical Field
The invention belongs to the technical field of macadimia nut cultivation, and particularly relates to a method for promoting macadimia nut stem segments to induce callus.
Background
Macadamia ternifolia f.muell.) belongs to the dragon eye (Proteaceae Juss.) and belongs to the perennial evergreen arbor fruit trees, namely Macadamia nut, queensland chestnut, Macadamia walnut and the like, is originally produced in australia, is rich in nutrient substances, has higher economic value and medicinal value, and enjoys the reputation of 'the king of dried fruit' in a plurality of nuts; the method is introduced into a Taipei vegetable garden to be planted in 1910 in the earliest years in China, and researches such as introduction and test of seeds and cultivation technology are really carried out only in 1979; in recent years, the variety is introduced from Australia in China to be popularized and planted in Yunnan, Guangxi and other places; with the improvement of the life quality of people, the characteristic fruit tree of macadamia nut is developed in a hot area of China, particularly in a karst high-temperature arid area, so that the fruit tree has great economic, social and ecological benefits.
The macadimia nuts are generally planted by asexual bud grafting seedlings, seedling seedlings are used as rootstocks, the cultivation time is longer, excellent rootstocks are lack of economic and efficient rapid propagation in the current planting method, the macadimia nuts are mainly propagated in grafting, cuttage and other modes on nursery gardens and orchards, the roots of seedlings propagated in cuttage, grafting and other modes are undeveloped, the plants grow slowly, the rooting time is longer, auxiliary management such as pruning and shaping is required for cutting propagation, the development of macadimia nuts in China is greatly limited, furthermore, when stem sections of the macadimia nuts are used as explants to induce callus or aseptic buds, endophytes of the commonly collected macadimia are complex and diverse, the disinfection is difficult, the pollution rate is high, the tissues are easy to necrotize, the large-scale production is difficult, in recent years, some macadimia variety of macadimia variety introduced into a fast propagation institute of Sundae, a rapid propagation system, a short propagation period, a tropical plant, a mature seedling of macadimia mature seedling, a mature, a tissue, a mature, a tissue, a mature, a root, a mature, a tissue, a root, a tissue, a mature, a tissue, a mature, a root, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a root, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue, a mature, a tissue.
Disclosure of Invention
In order to solve the technical problems in the prior art, the method adopts the Tween 20, the alcohol and the mercuric chloride to disinfect the macadimia nut explant for the first time, so that the purposes of thorough disinfection and low pollution rate are achieved, and an appropriate culture medium is added, so that the growth and development of the explant are improved, and the germination rate is improved.
A method for promoting macadimia nut stem segments to induce callus specifically comprises the following steps:
(1) collecting explants: in the growth and germination period of adult macadimia nut trees, cutting healthy stem sections without diseases, insect pests and buds on branches by using sterilized scissors to be explants, wherein the stem sections are 5-10 cm long and 0.1-0.2 cm in diameter;
(2) pretreatment of explants: quickly bringing the selected long stem sections back to the room, removing leaves, shearing the long stem sections into 3-4 cm long sections by using sterilized scissors, placing the long stem sections into a clean container, and flushing the long stem sections for 30-60 min by running water;
(3) and (3) disinfection: placing the pretreated stem segments on a sterilized ultra-clean workbench, treating for 20-40 s by using a disinfectant A, then treating for 8-12 min by using a disinfectant B, and finally washing for 4-6 min by using sterile water for 3-5 times;
(4) inoculation: cutting off 0.4-0.6 cm of each of two ends of the sterilized long stem section by using a sterilizing surgical scissors, then cutting the rest long stem section into stem sections with axillary buds with the length of about 0.8-1.2 cm, and inoculating the stem sections to a culture medium;
(5) culturing: and (3) placing the inoculated culture medium in an aseptic culture chamber for constant-temperature dark culture, wherein the culture temperature is 24-28 ℃, and the humidity is 65-75%.
The disinfectant A is prepared by adding tween 20 into alcohol according to the amount of 2 drops/500 m L and mixing.
Preferably, the concentration of the alcohol is 70-80%.
The disinfectant B is prepared by adding 2 drops of mercuric chloride per 500m L into 0.05-0.15% of mercuric chloride and mixing.
The culture medium takes MS as a basic culture medium, 0.5-2.0 mg of 6-BA, 0.1-1.0 mg/L NAA, 25-35 g/L sucrose and 5-10 g/L agar are added per liter, the pH of the mixed culture medium is adjusted to 5.8-6.0, preferably, 1.0mg of 6-BA, 0.5 mg/L NAA, 30 g/L sucrose and 8 g/L agar are added per liter, and the pH of the mixed culture medium is adjusted to 5.8-6.0.
Preferably, the cultivation temperature is 26.5 ℃ and the humidity is 70%.
Advantageous effects
① the stem of the adult macadimia nut at the growth and germination stage is used as the explant for tissue culture, and the stem of the macadimia nut at the growth and germination stage has a certain juvenile state characteristic, and has the advantages of vigorous growth, less bacteria, higher regeneration bud induction rate and less tissue necrosis.
② the young stem is used as explant to ensure the time and concentration of disinfectant treatment to be strictly screened and controlled without affecting the induction effect and destroying the tissue structure of explant, so as to control the pollution rate below 5%.
③ after culturing under constant temperature and dark culture condition, callus is induced in 10 days, and the activity of the callus is higher and the growth of regeneration bud is faster.
④ the method has the advantages of (1) disinfecting the explant by mixing the surfactant Tween 20 with alcohol and mercury bichloride for the first time, thoroughly disinfecting, and reducing the pollution rate, (2) easily obtaining the sterile explant, and (3) obtaining the callus and the regeneration bud in a short time, wherein the callus can be used as the induction material of the sterile bud, and the regeneration bud grows faster without disinfection, and the necrosis of the cell tissue is not easy to occur.
Detailed Description
The technical solution of the present invention is further limited by the following specific embodiments, but the scope of the claims is not limited to the description.
Example 1
A method for promoting macadimia nut stem segments to induce callus comprises the following steps:
(1) collecting explants: in the growth and germination period of the adult macadimia nut trees, a healthy stem section without diseases, insects and buds on branches is cut by using sterilized scissors to serve as an explant, the stem section is 5cm long, and the diameter of the stem section is 0.1 cm;
(2) pretreatment of explants: quickly taking the selected long stem back to the room, removing leaves, cutting into 3cm long sections with sterilized scissors, placing in a clean container, and flushing with running water for 30 min;
(3) and (3) disinfection: placing the pretreated stem segments on a sterilized ultra-clean workbench, treating with disinfectant A for 20s, treating with disinfectant B for 8min, and washing with sterile water for 3 times (4 min each time);
(4) inoculation: cutting off 0.4cm from each end of the sterilized long stem section by using a sterilizing surgical scissors, cutting the rest long stem section into stem sections with axillary buds, and inoculating the stem sections to a culture medium, wherein the length of the stem sections is about 0.8 cm;
(5) culturing: and (3) placing the inoculated culture medium in an aseptic culture chamber for constant-temperature dark culture, wherein the culture temperature is 25 ℃, and the humidity is 65%.
The disinfectant A is prepared by adding tween 20 into alcohol according to the amount of 2 drops/500 m L and mixing.
The alcohol concentration is 70%.
The disinfectant B is prepared by adding 2 drops of mercuric chloride per 500m L into 0.05% mercuric chloride and mixing.
The culture medium takes MS as a basic culture medium, 0.5mg of 6-BA, 0.1 mg/L NAA, 25 g/L of sucrose and 5 g/L of agar are added to each liter, and the pH value of the mixed culture medium is adjusted to 5.8-6.0.
Example 2
A method for promoting macadimia nut stem segments to induce callus comprises the following steps:
(1) collecting explants: in the growth and germination period of the adult macadimia nut trees, a healthy stem section without diseases, insects and buds on branches is cut by using sterilized scissors to serve as an explant, the stem section is 10cm long and 0.2cm in diameter;
(2) pretreatment of explants: quickly taking the selected long stem back to the room, removing leaves, cutting into 4cm long sections with sterilized scissors, placing in a clean container, and flushing with running water for 60 min;
(3) and (3) disinfection: placing the pretreated stem segment on a sterilized ultra-clean workbench, treating with disinfectant A for 40s, treating with disinfectant B for 12min, and washing with sterile water for 5 times (6 min each time);
(4) inoculation: cutting off 0.6cm from each end of the sterilized long stem section by using a sterilizing surgical scissors, cutting the rest long stem section into stem sections with axillary buds with the length of about 1.2cm, and inoculating the stem sections to a culture medium;
(5) culturing: and (3) placing the inoculated culture medium in an aseptic culture chamber for constant-temperature dark culture, wherein the culture temperature is 28 ℃, and the humidity is 75%.
The disinfectant A is prepared by adding tween 20 into alcohol according to the amount of 2 drops/500 m L and mixing.
The alcohol concentration is 80%.
The disinfectant B is prepared by adding 2 drops of mercuric chloride per 500m L into 0.15% mercuric chloride and mixing.
The culture medium takes MS as a basic culture medium, 2.0mg of 6-BA, 1.0 mg/L NAA, 35 g/L of cane sugar and 10 g/L of agar are added to each liter, and the pH value of the mixed culture medium is adjusted to 5.8-6.0.
Example 3
A method for promoting macadimia nut stem segments to induce callus comprises the following steps:
(1) collecting explants: in the growth and germination period of the adult macadimia nut trees, a healthy stem section without diseases, insects and buds on branches is cut by using sterilized scissors to serve as an explant, the stem section is 8cm in length and 0.1cm in diameter;
(2) pretreatment of explants: quickly taking the selected long stem back to the room, removing leaves, cutting into 3cm long sections with sterilized scissors, placing in a clean container, and flushing with running water for 45 min;
(3) and (3) disinfection: placing the pretreated stem segments on a sterilized ultra-clean workbench, treating with disinfectant A for 30s, treating with disinfectant B for 10min, and washing with sterile water for 5min each time for 3 times;
(4) inoculation: cutting off 0.5cm of each of the two ends of the sterilized long stem section by using a sterilizing surgical scissors, cutting the rest long stem section into stem sections with axillary buds, and inoculating the stem sections to a culture medium, wherein the length of the stem sections is about 1 cm;
(5) culturing: placing the inoculated culture medium in an aseptic culture chamber for constant-temperature dark culture, wherein the culture temperature is 26.5 ℃, and the humidity is 70%.
The disinfectant A is prepared by adding tween 20 into alcohol according to the amount of 2 drops/500 m L and mixing.
The alcohol concentration is 75%.
The disinfectant B is prepared by adding 2 drops of mercuric chloride per 500m L into 0.1% mercuric chloride and mixing.
The culture medium takes MS as a basic culture medium, 1.0mg of 6-BA, 0.5 mg/L NAA, 30 g/L of cane sugar and 8 g/L of agar are added to each liter, and the pH value of the mixed culture medium is adjusted to 5.8-6.0.
Test example 1
The disinfection mode of the scheme of the invention and other disinfection modes have influence on the pollution rate, the death rate and the callus induction rate of the explant, the explant is taken from the seedling nursery of Australian nut germplasm resources of subtropical crop research institute in Guizhou province, and the following disinfection modes are respectively adopted:
a: tween 20+ 75% alcohol 30 s-0.1% mercuric chloride 8 min-sterile water washing 5 times;
b: tween 20+ 75% alcohol 30 s-0.1% mercuric chloride 10 min-sterile water washing 5 times;
c: tween 20 and 75 percent alcohol for 30s to 0.1 percent mercury bichloride for 12min, and washing with sterile water for 5 times;
d: tween 20+ 75% alcohol 30 s-Tween 20+ 0.1% mercuric chloride 8 min-sterile water washing 5 times;
e: tween 20+ 75% alcohol 30 s-Tween 20+ 0.1% mercuric chloride 10 min-sterile water washing 5 times;
f: tween 20+ 75% alcohol 30 s-Tween 20+ 0.1% mercuric chloride 12 min-sterile water washing 5 times;
g: washing with 75% alcohol 30 s-sterile water for 5 times;
h: 0.1% mercuric chloride for 10 min-5 times of sterile water washing;
i: 75% alcohol 30 s-0.1% mercuric chloride 10 min-sterile water washing 5 times;
the different disinfection methods described above were inoculated 60 times each and repeated 3 times each, with the results shown in table 1 below:
TABLE 1
| Group of | The pollution rate% | The mortality rate is% | The inductivity is% |
| A | 13.8 | 4.2 | 76.2 |
| B | 12.7 | 6.5 | 80.5 |
| C | 12.4 | 7.6 | 82.6 |
| D | 4.3 | 0 | 96.1 |
| E | 2.4 | 0 | 99.4 |
| F | 4.9 | 0 | 95.2 |
| G | 35.7 | 3.6 | 74.6 |
| H | 28.5 | 18.4 | 75.4 |
| I | 20.1 | 12.4 | 81.3 |
As can be seen from the test results in Table 1, the disinfection method of the present invention has the characteristics of low explant contamination rate, low mortality rate and high callus induction rate.
Test example 2
The experiment adopts the technical scheme of example 3, 9 groups of experiments are set, each group only has different addition amounts of 6-BA and NAA in an MS culture medium, wherein the 6-BA concentration is respectively 0.5 mg/L, 1.0 mg/L and 2.0 mg/L, the NAA concentration is respectively 0.1 mg/L1, 0.5 mg/L2 and 1.0 mg/L, specifically 9 types of collocation, namely L-BA0.5mg/L, NAA 0.1mg/L, L-BA0.5mg/L, NAA 0.5mg/L, L-L, NAA 1.0mg/L, L-BA 1.0 mg/L, 3686-BA 1.0 mg/L, NAA 0.0mg/L and NAA 72-L, the germination rate of each group is calculated as shown in the following table, the statistics of the number of the buds generated by the number of the seedlings in each group is L-L, the number of the seedlings generated by the number of the explant, the growth of the seedlings in each group is as L-L, the following table, the statistics is as the number of the growth of the seedlings are as 0.72-L, the number of the seedlings, the seedlings are as shown in the number of the seedlings in each group is as L-L, the number of the experiment is as:
TABLE 2
| Numbering | Germination rate/% | Number of shoots generated per explant |
| 1 | 73.9 | 6.2 |
| 2 | 81.4 | 8.1 |
| 3 | 68.5 | 5.9 |
| 4 | 67.3 | 6.9 |
| 5 | 98.5 | 9.3 |
| 6 | 74.6 | 7.6 |
| 7 | 71.4 | 7.4 |
| 8 | 84.2 | 8.3 |
| 9 | 62.7 | 6.2 |
As shown in the test results in Table 2, when the addition amount of 6-BA in the MS culture medium is 1.0 mg/L and the addition amount is 0.5 mg/L, the germination and proliferation of the macadimia nuts are obviously promoted, the germination rate reaches 98.5%, and the number of sprouts generated by each explant reaches 9.3.
It should be noted that the above examples and test examples are only for further illustration and understanding of the technical solutions of the present invention, and are not to be construed as further limitations of the technical solutions of the present invention, and the invention which does not highlight essential features and significant advances made by those skilled in the art still belongs to the protection scope of the present invention.
Claims (4)
1. A method for promoting induction of callus of stem segments of macadamia nuts is characterized by comprising the following steps of (1) collecting explants, cutting healthy stem segments without plant diseases and insect pests on branches into explants with the stem lengths of 5-10 cm and the diameters of 0.1-0.2 cm in the sprouting period of adult macadamia nuts by using disinfected scissors, (2) pretreating the explants, namely quickly bringing the selected stem segments back to a room, removing leaves, shearing the stem segments into 3-4 cm long segments by using disinfected scissors, placing the segments in a clean container, flowing tap water for 30-60 min, disinfecting, namely placing the pretreated stem segments on a sterilized super-clean workbench, treating the stem segments with a disinfectant A for 20-40 s, treating the stem segments with a disinfectant B for 8-12 min, washing the stem segments for 3-5 times by using sterile water for 4-6 min each time, wherein the disinfectant A is prepared by adding the disinfectant A into a disinfected working table with 2 drops/500 m L, inoculating the stem segments with the disinfectant B for 3-5 min, and inoculating the disinfectant B into a mixed culture medium with the pH of 2.5-0.35 mg and the mixed culture medium, and inoculating the stem segments with the humidity of 2.0.35-0.35 mg of inoculated agar, and the inoculated agar 0.35-0.2 + 2.0.2.2.2 + 2.2 cm, and the humidity after the disinfection culture, and the disinfection, and the inoculated in a culture medium for inoculation of the inoculated in a culture medium, and the culture medium for 2-0.5-0-0.35 cm of the inoculated in a sterile culture medium for 2cm of the sterile culture medium for 2 cm.
2. The method of claim 1, wherein the alcohol is present in a concentration of 70-80%.
3. The method for promoting macadamia nut stem segment-induced callus according to claim 1, wherein the culture medium is MS +1.0 mg/L6-BA, +0.5 mg/L NAA, +30 g/L sucrose, +8 g/L agar, and the pH of the mixed culture medium is adjusted to 5.8-6.0.
4. The method of claim 1, wherein the culturing temperature is 26.5 ℃ and the humidity is 70%.
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