CN104145812A - Low-temperature over-summering preservation and cultivation method of brassica napus L microspore culture embryoid - Google Patents
Low-temperature over-summering preservation and cultivation method of brassica napus L microspore culture embryoid Download PDFInfo
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- CN104145812A CN104145812A CN201410287443.3A CN201410287443A CN104145812A CN 104145812 A CN104145812 A CN 104145812A CN 201410287443 A CN201410287443 A CN 201410287443A CN 104145812 A CN104145812 A CN 104145812A
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Abstract
The invention discloses a low-temperature over-summering preservation and cultivation method of brassica napus L microspore culture embryoid. The method comprises the following steps: culturing free microspores in an NLN-13 culture medium for 25-28 days to obtain an embryoid at the age of 25-28 days, and arranging the obtained embryoid in a novel NLN-13 culture medium; and preserving the embryoid in a refrigerator at the temperature of 3 DEG C for 60 days; performing transition culture on the embryoid in an incubator, replacing the culture medium with an NLN-10 shallow liquid culture medium, culturing in the incubator again, and preserving the embryoid in the refrigerator at the temperature of 3 DEG C for 120-150 days, wherein the culture medium is replaced with a NLN-10 culture medium once per 60 days in the period of preservation. According to the method, the free microspores of brassica napus L are cultivated to obtain the embryoid at a proper age, which is then preserved at a low temperature, the transfer time is greatly saved, lots of transfer expenditures and manpower resources are saved, and the method has significance for innovating germplasm resources, particularly excellent and specific germplasm resources.
Description
Technical field
The present invention relates to agricultural planting field, especially a kind of Microspore of Brassica napus is cultivated the low temperature of embryoid and is got over summer preservation cultural method.
Background technology
Cabbage type rape
brassica napusl is the highest kind of grain yield in rape (turnip type rape, mustard type rape, cabbage type rape) for three kinds of oil, in trip basin, China the Changjiang river, plants in a large number at present, and be the most important oil crop of China.
Under normal circumstances, after the microspore of cabbage type rape produces embryo, embryoid is inoculated on MS solid culture medium, need to be in blake bottle every 30-45 days subculture once.From March to October, minimumly need subculture 4 times.The pollution in summer can cause a large amount of seedling losses, is also a large amount of wastes of time, labour and financial resources simultaneously.As inoculate 10000 embryos, and according to 10 embryo/bottles, to inoculate, each inoculation needs 1000 blake bottles, 50000ml medium (every bottle of 50mL), just can complete 1 working day.
Summary of the invention
The object of the invention is: the low temperature that provides a kind of Microspore of Brassica napus to cultivate embryoid is got over the summer and preserved cultural method, to solve Microspore of Brassica napus in prior art, cultivate the problem that embryoid or seedling are got over summer difficulty.
The present invention is achieved in that Microspore of Brassica napus cultivates the low temperature of embryoid and get over the summer and preserve cultural method, Isolated microspore is cultivated after 25～28 days in NLN-13 medium, obtain embryo and be age the embryoid of 25～28 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, then by being placed in the refrigerator of 3 ℃, preserve 15～150 days; Within every 60 days during this time, change a NLN medium, first uses NLN-13 medium for 60 days, and all using afterwards NLN-10 medium, PH is 5.5.
It is preserved 15～120 days in the refrigerator of 3 ℃.
The culture density of described embryoid is, the glass culture dish that is 6cm at diameter, and every ware is deposited 150～200 embryoids.
When embryo is preserved after 60 days in 3 ℃ of refrigerators, when medium need to be replaced by NLN-10 medium from NLN-13 medium, from NLN-13 medium is replaced by NLN-10 medium, embryoid is shifted the incubator of 8 ℃ excessive 24 hours from the low temperature refrigerator of 3 ℃, and then taking-up is replaced by NLN-10 medium, and in the incubator of 8 ℃ excessive 24 hours, be then placed in the refrigerator of 3 ℃ and preserve; Within every 60 days afterwards, use the same method and change liquid, the medium at unreal night is NLN-10 medium.
In order further to verify, carried out following experiment by experiment effect of the present invention:
To with a collection of Isolated microspore, cultivate the embryoid grouping obtaining afterwards at 25～28 days, and after processing according to technical scheme of the present invention, respectively they are placed into the Refrigerator store of 3 ℃ after 15 days, embryoid starts flavescence, as depicted in figs. 1 and 2; When taking out and to change new NLN-13 medium into from refrigerator, see that light cultivates embryo after 3-5 days and be just transformed into immediately green.The time of preserving in refrigerator is longer, and the color of embryo is more yellow, but this can't affect growth of the embryo, when low temperature, preserves about 90 days, sees that light cultivates embryo after 5 days and be just transformed into green completely, as shown in Figure 3; When embryo, at low temperature refrigerator, preserve after 120d, see after light is cultivated and can obviously see that radicle is longer, as shown in Figure 4; When low temperature is preserved after 150d, the complete flavescence of color of embryo, as shown in Figure 5, once see that light cultivation is more than 5 days, the color of embryo transfers again green to, but radicle is very long.The performance that radicle extends, is that low temperature has suppressed the growth of trophosome because embryo is at continuous development growth, but the polar growth at two ends is also continuing.
Therefore, experiment of the present invention shows, rataria at least can be saved in about 150 days in 3 ℃ of low temperature refrigerators, reduced the repeatedly successive propagation of test-tube plantlet in summer completely, avoided summer test-tube plantlet to be difficult to be transplanted to the situation in land for growing field crops, as long as take out and cultivate from refrigerator when needs are transplanted or transferred embryo, then induce seedling, can realize the breeding of embryoid seedling.But, in refrigerator the holding time long, radicle is refinement and growing more, this is unfavorable for that embryo is transferred on solid culture medium, also can reduce the planting percent of embryo.Therefore the holding time should not be over 120 days in low temperature refrigerator, to it is considered herein that embryo.
Under normal circumstances, after microspore produces embryo, embryoid is inoculated on MS solid culture medium, need to be in blake bottle every 30-45 days subculture once.From March to October, minimumly need subculture 4 times.The pollution in summer can cause a large amount of seedling losses, is also a large amount of wastes of time, labour and financial resources simultaneously.As inoculate 10000 embryos, and according to 10 embryo/bottles, to inoculate, each inoculation needs 1000 blake bottles, 50000ml medium (every bottle of 50mL), just can complete 1 working day.If adopt low temperature to preserve, only need to then be put into 3 ℃ of refrigerators and just can save with 50 culture dishes dress embryos (200 embryos/ware), need to every culture fluid of replacing in 60 days just can, whole process can be saved nearly 5000 yuan.
Owing to adopting above-mentioned technical scheme, compared with prior art, the present invention cultivates the Isolated microspore of cabbage type rape, obtain the suitable embryo embryoid in age, it is preserved at low temperatures, from having saved to a great extent transit time, having saved a large amount of switching funds and human resources, simultaneously, also can reduce the pollution rate in switching process, improve largely Microspore of Brassica napus and cultivated the efficiency of Innovation Germplasm resource, for Innovation Germplasm resource, especially special excellent and Special germplasm resources, there is important function.Method of the present invention is simple, easily implements, and with low cost, result of use is good.
Accompanying drawing explanation
It is left that accompanying drawing 1 is that 3 ℃ of the ratarias in 25 ~ 28d embryo age in experiment of the present invention are preserved 15d() and the 26d(right side) after state;
It is left that accompanying drawing 2 is that 3 ℃ of the ratarias in 27 ~ 28d embryo age in experiment of the present invention are preserved 36d() and the 60d(right side) after state;
Accompanying drawing 3 is that 3 ℃ of the ratarias in 25 ~ 28d embryo age in experiment of the present invention are preserved after 91d, sees that light cultivates the state after 5 days;
Accompanying drawing 4 is that 3 ℃ of the ratarias in 27d embryo age in experiment of the present invention are preserved after 120d, sees that light cultivates the state after 5 days;
Accompanying drawing 5 is that 3 ℃ of the ratarias in 27d embryo age in experiment of the present invention are preserved (left side) after 150d, sees that light cultivates the state on (right side) after 5 days.
Embodiment
Embodiments of the invention 1: Microspore of Brassica napus is cultivated the low temperature of embryoid and got over summer preservation cultural method, Isolated microspore is cultivated after 26 days in NLN-13 medium, obtain embryo and be age the embryoid of 26 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, the glass culture dish that embryoid is 6cm with diameter is placed, and every ware is deposited 150～200 embryoids; Then by being placed in the refrigerator of 3 ℃, preserve, preserve after 60 days, need to from 3 ℃ of refrigerators, take out excessively in the incubator of 8 ℃ excessive 24 hours, the NLN-13(PH then more renewing is 5.5) liquid nutrient medium; After replacing, put back to again in the incubator of 8 ℃ excessive 24 hours, be just placed into afterwards in the refrigerator of 3 ℃ and be saved to 120 days; Within every 60 days during this time, change a NLN-10 medium, change liquid method the same, the PH of NLN-10 medium is 5.5.
Embodiments of the invention 2: Microspore of Brassica napus is cultivated the low temperature of embryoid and got over summer preservation cultural method, Isolated microspore is cultivated after 25 days in NLN-13 medium, obtain embryo and be age the embryoid of 25 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, the glass culture dish that embryoid is 6cm with diameter is placed, and every ware is deposited 150～200 embryoids; Then by being placed in the refrigerator of 3 ℃, preserve 60 days.
Embodiments of the invention 3: Microspore of Brassica napus is cultivated the low temperature of embryoid and got over summer preservation cultural method, Isolated microspore is cultivated after 28 days in NLN-13 medium, obtain embryo and be age the embryoid of 28 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, the glass culture dish that embryoid is 6cm with diameter is placed, and every ware is deposited 150～200 embryoids; Then by being placed in the refrigerator of 3 ℃, preserve, preserve after 60 days, need to from 3 ℃ of refrigerators, take out excessively in the incubator of 8 ℃ excessive 24 hours, the NLN-13(PH then more renewing is 5.5) liquid nutrient medium; After replacing, put back to again in the incubator of 8 ℃ excessive 24 hours, be just placed into afterwards in the refrigerator of 3 ℃ and be saved to 90 days; Within every 60 days during this time, change a NLN-10 medium, change liquid method the same, the PH of NLN-10 medium is 5.5.
Embodiments of the invention 4: Microspore of Brassica napus is cultivated the low temperature of embryoid and got over summer preservation cultural method, Isolated microspore is cultivated after 27 days in NLN-13 medium, obtain embryo and be age the embryoid of 27 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, the glass culture dish that embryoid is 6cm with diameter is placed, and every ware is deposited 150～200 embryoids; Then by being placed in the refrigerator of 3 ℃, preserve, preserve after 60 days, need to from 3 ℃ of refrigerators, take out excessively in the incubator of 8 ℃ excessive 24 hours, the NLN-13(PH then more renewing is 5.5) liquid nutrient medium; After replacing, put back to again in the incubator of 8 ℃ excessive 24 hours, be just placed into afterwards in the refrigerator of 3 ℃ and be saved to 150 days; Within every 60 days during this time, change a NLN-10 medium, change liquid method the same, the PH of NLN-10 medium is 5.5.
Claims (4)
1. the low temperature of a Microspore of Brassica napus cultivation embryoid is got over the summer and is preserved cultural method, it is characterized in that: Isolated microspore is cultivated after 25～28 days in NLN-13 medium, obtain embryo and be age the embryoid of 25～28 days, the embryoid obtaining is placed in to new NLN-13 medium, and the PH of new NLN-13 medium is reduced to 5.5, then by being placed in the refrigerator of 3 ℃, preserve 15～150 days; Within every 60 days during this time, change a NLN medium, first uses NLN-13 medium for 60 days, and all using afterwards NLN-10 medium, PH is 5.5.
2. the low temperature of Microspore of Brassica napus cultivation embryoid according to claim 1 is got over the summer and is preserved cultural method, it is characterized in that: it is preserved 15～120 days in the refrigerator of 3 ℃.
3. the low temperature that Microspore of Brassica napus according to claim 1 is cultivated embryoid is got over the summer and is preserved cultural method, it is characterized in that: the culture density of described embryoid is, the glass culture dish that is 6cm at diameter, and every ware is deposited 150～200 embryoids.
4. the low temperature of Microspore of Brassica napus cultivation embryoid according to claim 1 is got over the summer and is preserved cultural method, it is characterized in that: when embryo is preserved after 60 days in 3 ℃ of refrigerators, when medium need to be replaced by NLN-10 medium from NLN-13 medium, from NLN-13 medium is replaced by NLN-10 medium, embryoid is shifted the incubator of 8 ℃ excessive 24 hours from the low temperature refrigerator of 3 ℃, and then taking-up is replaced by NLN-10 medium, and in the incubator of 8 ℃ excessive 24 hours, be then placed in the refrigerator of 3 ℃ and preserve; Within every 60 days afterwards, use the same method and change liquid, the medium at unreal night is NLN-10 medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105557514A (en) * | 2015-12-14 | 2016-05-11 | 李操 | Low temperature preservation method for roxburgh anoectochilus terminal bud germplasm resources |
| CN105601386A (en) * | 2016-01-15 | 2016-05-25 | 华中农业大学 | Hydroponic culture solution and oversummer culture method for cabbage type rape microspore seedlings |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640704A (en) * | 2012-05-23 | 2012-08-22 | 贵州省油料研究所 | Method used for improving brassica napus L pollen microspore culturing embryo yield |
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2014
- 2014-06-25 CN CN201410287443.3A patent/CN104145812A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102640704A (en) * | 2012-05-23 | 2012-08-22 | 贵州省油料研究所 | Method used for improving brassica napus L pollen microspore culturing embryo yield |
Non-Patent Citations (2)
| Title |
|---|
| 朱文祥等: "植物组织培养中外植体褐变研究进展", 《安徽农业科学》 * |
| 李书宇等: "甘蓝型油菜小孢子培养胚状体长期保存技术", 《中国油料作物学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105557514A (en) * | 2015-12-14 | 2016-05-11 | 李操 | Low temperature preservation method for roxburgh anoectochilus terminal bud germplasm resources |
| CN105557514B (en) * | 2015-12-14 | 2018-11-13 | 李操 | A kind of Cryopreservation of bud germ plasm resource |
| CN105601386A (en) * | 2016-01-15 | 2016-05-25 | 华中农业大学 | Hydroponic culture solution and oversummer culture method for cabbage type rape microspore seedlings |
| CN105601386B (en) * | 2016-01-15 | 2019-05-17 | 华中农业大学 | A kind of Microspore of Brassica napus seedling water planting culture solution and the method for more summer culture |
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Application publication date: 20141119 |