Method for rapidly inducing regeneration of adventitious buds by using multi-tillering non-heading Chinese cabbage variety and method for rapidly cultivating virus-free seedlings

Abstract

The invention discloses a method for rapidly inducing regeneration of adventitious buds by utilizing a multi-tillering non-heading Chinese cabbage variety and a method for rapidly cultivating virus-free seedlings, and the method for inducing regeneration of the adventitious buds of the non-heading Chinese cabbage comprises the following steps: culturing seeds of the multi-tillering non-heading Chinese cabbage variety into seedlings; then cutting cotyledons with stems from the seedlings to serve as explants, and carrying out bud induction culture on the explants until adventitious buds grow out from petiole wounds of the explants, so as to obtain the non-heading Chinese cabbage adventitious bud regeneration body. According to the method, a non-heading Chinese cabbage variety with a multi-tillering characteristic is innovatively found, when the cotyledons with stalks are used as explant tissues for regeneration culture, adventitious buds do not need to be differentiated through calluses, and the adventitious buds can directly grow from the wounds of the petioles, so that the explants can be directly transferred into a differential culture medium for bud induction; the regeneration time is short, and the non-heading Chinese cabbage adventitious bud regeneration body and the virus-free seedling can be rapidly obtained through the method provided by the invention.

Description

Technical Field

[0001] The invention relates to the technical field of plant seedling cultivation, and in particular to a method for rapidly inducing adventitious bud regeneration bodies by utilizing multi-tillering non-heading cabbage varieties and a method for rapidly cultivating virus-free seedlings. Background Art

[0002] Non-heading cabbage (Brassica campestris ssp.chinensis Makino), also known as small cabbage and green cabbage, is a subspecies of cabbage in the genus Brassica of the cruciferous family. It is divided into five varieties: common cabbage, collapsed cabbage, cauliflower, cauliflower and tillering cabbage, mainly including rapeseed, black cabbage, purple cabbage cauliflower, Chinese cabbage heart, and multi-stalk cabbage.

[0003] Non-heading cabbage can be made into various dishes and is deeply loved by people. It has strong adaptability, relatively low planting cost, high yield, and a wide range of uses. It plays an indispensable role in promoting the development of the agricultural economy.

[0004] At present, the establishment of tissue culture regeneration system of non-heading cabbage mainly uses petiole cotyledons or hypocotyls as explants to differentiate adventitious buds through callus. The operation process is cumbersome and the regeneration cycle is long. It usually takes at least one month to complete the process of differentiating adventitious buds from callus tissue. Subsequent rooting and other cultivation are required, and the entire seedling cycle is even longer. Therefore, how to establish a rapid regeneration system of non-heading cabbage is of great significance to obtaining virus-free seedlings of Chinese cabbage. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for rapidly inducing adventitious bud regeneration using multi-tillering non-heading cabbage varieties and a method for rapidly cultivating virus-free seedlings, so as to reduce the regeneration and cultivation cycle.

[0006] To achieve the above-mentioned purpose, the present invention provides a method for rapidly inducing adventitious bud regeneration of non-heading cabbage by using a multi-tillering non-heading cabbage variety, comprising the following steps: culturing seeds of the multi-tillering non-heading cabbage variety into seedlings, then cutting cotyledons with petioles from the seedlings as explants, performing bud induction culture on the explants until adventitious buds grow out of the petiole wounds of the explants, and obtaining adventitious bud regenerated bodies of the non-heading cabbage.

[0007] Further, the following steps are included:

[0008] Step 1: sterilizing the seeds of the multi-tillering non-heading cabbage variety to obtain sterile seeds;

[0009] Step 2: inoculating the sterile seeds onto a germination medium for germination culture to obtain seedlings, and then cutting the petioled cotyledons from the seedlings as explants;

[0010] Step 3: The explants are transferred into a differentiation medium for bud induction culture until adventitious buds grow out of the petiole wounds of the explants, thereby obtaining adventitious bud regeneration bodies of non-heading Chinese cabbage.

[0011] Furthermore, in step 1, the specific operation method of the aseptic treatment is to disinfect the seeds with 75% alcohol for 60 seconds, then soak them in 3% sodium hypochlorite for 12 minutes by shaking, and finally wash them with sterile water for 5 to 6 times, and place them on sterile filter paper to dry.

[0012] Furthermore, in step 2, the composition of the germination medium is 1 / 2MS+30g / L sucrose+8g / L agar powder, pH=5.8, the germination culture conditions are temperature 20-30°C, light intensity 2000-3000Lx, and photoperiod is light / darkness=16h / 8h.

[0013] Furthermore, in step 3, the composition of the differentiation medium is 1 / 2MS+30g / L sucrose+4mg / L6-BA+0.5mg / L NAA+5mg / L AgNO3+0.2mM Vc+8g / L agar powder, pH=5.8, and the bud induction culture conditions are temperature 20-30°C, light intensity 2000-3000Lx, and photoperiod of light / darkness = 16h / 8h.

[0014] Furthermore, the multi-tillering non-heading cabbage variety is Sutai No. 1, Zhongtai No. 1, Qiutai No. 2, or Xiangzaotai No. 4.

[0015] The present invention also provides a method for rapidly cultivating virus-free non-heading cabbage seedlings using a multi-tillering non-heading cabbage variety, comprising the following steps: culturing the non-heading cabbage adventitious bud regeneration bodies induced by the above method until leaves grow to obtain regenerated plants, and then performing rooting culture on the regenerated plants to obtain virus-free non-heading cabbage seedlings.

[0016] Further, the following steps are included:

[0017] Step 1: Cultivate the adventitious bud regeneration body of non-heading Chinese cabbage until it grows 4 to 6 leaves to obtain a regenerated plant;

[0018] Step 2: Transfer the regenerated plants into a rooting medium for rooting culture until roots are formed, thereby obtaining virus-free non-heading cabbage seedlings.

[0019] Furthermore, in step 2, the composition of the rooting medium is 1 / 2MS+30g / L sucrose+1.0mg / L NAA+5mg / L AgNO3+8g / L agar powder, pH=5.8, and the rooting culture conditions are temperature 20-30°C, light intensity 2000-3000Lx, and photoperiod of light / darkness=16h / 8h.

[0020] The beneficial effects of the present invention are embodied in:

[0021] The present invention innovatively discovers a non-heading cabbage variety with multi-tillering characteristics. When the petiole cotyledon is used as an explant tissue for regeneration culture, it is not necessary to differentiate adventitious buds through callus tissue, and adventitious buds can be grown directly from the petiole wound position, so that the explant can be directly transferred to a differentiation medium for bud induction, with a shorter adventitious bud regeneration time. Based on this principle, the present invention further optimizes the operating process and conditions of the induction method and the seedling method, significantly shortening the tissue culture regeneration time of the non-heading cabbage. Through the method provided by the present invention, the adventitious bud regeneration body and virus-free seedlings of the non-heading cabbage can be quickly obtained. The method provided by the present invention is simple to operate, can save a lot of manpower and material resources, and provides a new tissue regeneration scheme for special varieties of Chinese cabbage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are photos of the induction process of adventitious bud regeneration of Sutai No. 1 (Figure a is a photo of the seedlings, Figure b is a photo of the cotyledons transferred into the differentiation medium, Figure c is a photo of the growth of the cotyledons after three days of bud induction culture; Figure d is a photo of the budding of the cotyledons after two weeks of bud induction culture).

[0023] Figure 2 These are photos of the rooting culture of the regenerated plants of Sutai No. 1 (Figure a is a photo of the regenerated plants transferred into the rooting culture medium, and Figures b and c are photos of the rooting conditions after two weeks of rooting culture).

[0024] Figure 3 These are photos of the budding conditions of 'Huiwu 15' and 'Huiwu 121' after two weeks of bud induction culture (Figure a is a photo of the budding conditions of 'Huiwu 15', and Figure b is a photo of the budding conditions of 'Huiwu 121'). DETAILED DESCRIPTION

[0025] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0026] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods; unless otherwise specified, the methods used in the embodiments of the present invention are methods known to those skilled in the art. Among them, the seed 'Su Tai No. 1' was purchased from the official store of Ainong Youxuan (Taobao); the seed 'Zhong Tai No. 1' was purchased from Nong Lvyuan Seeds (Taobao); the seed 'Qiu Tai No. 2' was purchased from Ruihefeng Seeds (Taobao); the seed 'Xiang Zao Tai No. 4' was purchased from Hongwei Agricultural Materials (Taobao); the seed 'Huiwu 15' was purchased from Hefei Hefeng Seed Co., Ltd.; the seed 'Huiwu 121' was purchased from Hefei Hefeng Seed Co., Ltd.

[0027] Example 1

[0028] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Sutai No. 1)

[0029] The operation steps are as follows:

[0030] Step 1: Select the seeds of 'Sutai No. 1' with full seeds and uniform size, sterilize them with 75% alcohol for 60 seconds, then soak them in 3% sodium hypochlorite with shaking for 12 minutes, finally wash them with sterile water for 5 times, place them on sterile filter paper and blow dry them to obtain sterile seeds;

[0031] Step 2: using sterile tweezers to inoculate the sterile seeds onto a germination medium for germination culture, the germination medium composition is 1 / 2MS+30g / L sucrose+8g / L agar powder, pH=5.8, the germination culture conditions are temperature 25°C, light intensity 2500Lx, photoperiod light / darkness=16h / 8h, culture for 6 days to obtain seedlings, and then use sterile scissors to cut the petiole cotyledons on the seedlings as explants;

[0032] Step 3: Transfer the explants to differentiation medium for bud induction culture. The composition of the differentiation medium is 1 / 2MS+30g / L sucrose+4mg / L 6-BA+0.5mg / L NAA+5mg / L AgNO3+0.2mM Vc+8g / L agar powder, pH=5.8. The bud induction culture conditions are 25°C, 2500Lx light intensity, and 16h / 8h light / dark cycle. Cultivate for two weeks. During the process, observe whether adventitious buds grow from the petiole wound of the explants. Those with adventitious buds are the adventitious bud regeneration bodies that have been successfully induced to regenerate. See the induction process for photos. Figure 1 .

[0033] This example was repeated 5 times, with no less than 70 explants inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate obtained in each treatment are shown in Table 1.

[0034] Table 1

[0035]

[0036] As shown in Table 1, under the above culture conditions, Sutai No. 1 can rapidly regenerate adventitious buds within 14 days. In addition, we observed during tissue culture that at least 2 to 7 adventitious buds can be differentiated from the petiole wound of each explant of Sutai No. 1.

[0037] Example 2

[0038] The operation steps of rapidly cultivating virus-free seedlings of non-heading cabbage using a multi-tillering non-heading cabbage variety (Sutai No. 1) are as follows:

[0039] Step 1: Based on Example 1, the adventitious bud regeneration body continues to be cultured in the differentiation medium (culture conditions remain unchanged) until 4 to 6 leaves grow to obtain a regenerated plant;

[0040] Step 2: Transfer the regenerated plants to the rooting medium for rooting culture. The composition of the rooting medium is 1 / 2MS+30g / L sucrose+1.0mg / L NAA+5mg / L AgNO3+8g / L agar powder, pH=5.8, and the rooting culture conditions are temperature 25°C, light intensity 2500Lx, and light / darkness=16h / 8h. Cultivate for two weeks, observe the rooting situation, and obtain non-heading cabbage virus-free seedlings. See the rooting culture photos for details. Figure 2 , we can see the successful rooting of adventitious bud regeneration.

[0041] Example 3

[0042] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Zhongtai No. 1)

[0043] The operation method of this embodiment is the same as that of embodiment 1, except that the seeds used are replaced with 'Zhongtai No. 1'.

[0044] This example was repeated 3 times, and no less than 70 explants were inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate in each treatment are shown in Table 2. Under the above culture conditions, Zhongtai No. 1 can also quickly regenerate adventitious buds within 14 days. In addition, we observed during tissue culture that at least 2 to 7 adventitious buds can be differentiated from the petiole wound of each explant of Zhongtai No. 1.

[0045] Table 2

[0046]

[0047] Example 4

[0048] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Qiutai No. 1)

[0049] The operation method of this embodiment is the same as that of embodiment 1, except that the seeds used are replaced with 'Qiu Tai No. 1'

[0050] This example was repeated 3 times, and no less than 70 explants were inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate in each treatment are shown in Table 3. Under the above culture conditions, Qiutai No. 1 can quickly regenerate adventitious buds within 14 days. In addition, we observed during tissue culture that at least 2 to 7 adventitious buds can be differentiated from the petiole wound of each explant of Qiutai No. 1.

[0051] Table 3

[0052]

[0053] Example 5

[0054] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Xiang Zaotai No. 4)

[0055] The operation method of this embodiment is the same as that of embodiment 1, except that the seeds used are replaced with 'Xiang Zao Tai No. 4'.

[0056] This embodiment was repeated 3 times, and no less than 70 explants were inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate in each treatment are shown in Table 4. Under the above culture conditions, Sutai No. 1 can quickly regenerate adventitious buds within 14 days. In addition, we observed during tissue culture that at least 2 to 7 adventitious buds can be differentiated from the petiole wound of each explant of Sutai No. 1.

[0057] Table 4

[0058]

[0059] Comparative Example 1

[0060] Rapid induction of adventitious bud regeneration in non-heading cabbage using non-tillering and non-heading cabbage variety (Huiwu 15)

[0061] The operation method of this comparative example is the same as that of Example 1, except that the seeds used are replaced with 'Huiwu 15'. Figure 3 .

[0062] This comparative example was repeated 3 times, and no less than 70 explants were inoculated in each treatment. The number of adventitious bud regeneration bodies in each treatment is shown in Table 5. 'Huiwu 15' failed to grow adventitious buds at the wound of the cotyledon petiole.

[0063] Table 5

[0064]

[0065] Comparative Example 2

[0066] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using non-tillering and non-heading Chinese cabbage variety (Huiwu 121)

[0067] The operation method of this comparative example is the same as that of Example 1, except that the seeds used are replaced with 'Huiwu 121'. Figure 3 .

[0068] This comparative example was repeated 3 times, and no less than 50 explants were inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate in each treatment are shown in Table 6. 'Huiwu 121' failed to grow adventitious buds at the wound of the cotyledon petiole.

[0069] Table 6

[0070]

[0071] Comparative Example 3

[0072] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Sutai No. 1)

[0073] The operation method of this comparative example is the same as that of Example 1, except that the germination culture time in step 2 is adjusted to 4 days.

[0074] This comparative example was treated repeatedly for 3 times, with no less than 70 explants inoculated in each treatment. The number of adventitious bud regeneration bodies and the regeneration rate in each treatment are shown in Table 7. Compared with Example 1, the regeneration rate of petioled cotyledons of 4-day-old 'Sutai No. 1' seedlings was similar, but due to the young age of the seedlings and the short petioles, it was difficult to cut the explants.

[0075] Table 7

[0076]

[0077] Comparative Example 4

[0078] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Sutai No. 1)

[0079] The operation method of this comparative example is the same as that of Example 1, except that the germination culture time of step 2 is adjusted to 7 days.

[0080] This comparative example was repeated 3 times, with no less than 70 explants inoculated each time. The number of adventitious bud regeneration bodies and the regeneration rate of each treatment are shown in Table 8. Compared with Example 1, the regeneration rate of adventitious buds with petioles in 7-day-old 'Sutai No. 1' seedlings was reduced.

[0081] Table 8

[0082]

[0083] Comparative Example 5

[0084] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Sutai No. 1)

[0085] The operation method of this comparative example is the same as that of Example 1, except that the 6-BA in the differentiation medium in step 3 is replaced by TDZ.

[0086] This comparative example was repeated 3 times, with no less than 70 explants inoculated each time. The number of adventitious bud regeneration bodies and the regeneration rate of each treatment are shown in Table 9. Compared with Example 1, the regeneration rate of adventitious buds of petiolate cotyledons of 'Sutai No. 1' seedlings treated with TDZ was reduced.

[0087] Table 9

[0088]

[0089] Comparative Example 6

[0090] Rapid induction of adventitious bud regeneration in non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety (Sutai No. 1)

[0091] The operation method of this comparative example is the same as that of Example 1, except that the 6-BA in the differentiation medium in step 3 is treated with concentrations of 0, 2, 5, and 6 mg / L, respectively.

[0092] Each concentration treatment was repeated 3 times, and no less than 70 explants were inoculated for each treatment. The average bud regeneration rate (%) of the three experiments for each concentration treatment is shown in Table 10. Compared with the average regeneration rate of 15.42% obtained by the treatment with 4 mg / L 6-BA in Example 1, the regeneration rate of adventitious buds on the petioled cotyledons of the 'Sutai No. 1' seedlings treated with 0, 2, 5, and 6 mg / L 6-BA was reduced.

[0093] Table 10

[0094]

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using multi-tillering non-heading Chinese cabbage varieties, characterized in that: The following steps are involved: The seeds of a multi-tillering non-heading cabbage variety are cultured into seedlings, and then the cotyledons with petioles are cut from the seedlings as explants, and the explants are subjected to bud induction culture until adventitious buds grow out of the petiole wounds of the explants, thereby obtaining adventitious bud regeneration bodies of the non-heading cabbage.

2. The method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety as claimed in claim 1, characterized in that: The following steps are involved: Step 1: sterilizing the seeds of the multi-tillering non-heading cabbage variety to obtain sterile seeds; Step 2: inoculating the sterile seeds onto a germination medium for germination culture to obtain seedlings, and then cutting the petioled cotyledons from the seedlings as explants; Step 3: The explants are transferred into a differentiation medium for bud induction culture until adventitious buds grow out of the petiole wounds of the explants, thereby obtaining adventitious bud regeneration bodies of non-heading Chinese cabbage.

3. The method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety as claimed in claim 2, characterized in that: In step 1, the specific operation method of the aseptic treatment is to disinfect the seeds with 75% alcohol for 60 seconds, then soak them in 3% sodium hypochlorite for 12 minutes by shaking, and finally wash them with sterile water for 5 to 6 times and place them on sterile filter paper to dry.

4. The method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety as claimed in claim 2, characterized in that: In step 2, the composition of the germination medium is 1 / 2MS+30g / L sucrose+8g / L agar powder, pH=5.8, the germination culture conditions are temperature 25°C, light intensity 2000-3000Lx, and photoperiod light / darkness=16h / 8h.

5. The method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety as claimed in claim 2, characterized in that: In step 3, the composition of the differentiation medium is 1 / 2MS+30g / L sucrose+4mg / L 6-BA+0.5mg / L NAA+5mg / L AgNO3+0.2mM Vc+8g / L agar powder, pH=5.8, and the bud induction culture conditions are temperature 25°C, light intensity 2000~3000Lx, and photoperiod light / darkness=16h / 8h.

6. The method for rapidly inducing adventitious bud regeneration of non-heading Chinese cabbage using a multi-tillering non-heading Chinese cabbage variety according to any one of claims 1 to 5, characterized in that: The multi-tillering non-heading cabbage variety is Sutai No. 1, Zhongtai No. 1, Qiutai No. 2, or Xiangzaotai No.

4.

7. A method for rapidly cultivating virus-free non-heading cabbage seedlings using multi-tillering non-heading cabbage varieties, characterized in that: The method comprises the following steps: culturing the non-heading Chinese cabbage adventitious bud regeneration bodies induced by the method according to any one of claims 1 to 5 until leaves grow to obtain regenerated plants, and then performing rooting culture on the regenerated plants to obtain non-heading Chinese cabbage virus-free seedlings.

8. The method for rapidly cultivating virus-free non-heading Chinese cabbage seedlings using multi-tillering non-heading Chinese cabbage varieties as claimed in claim 7, characterized in that: The following steps are involved: Step 1: Cultivate the adventitious bud regeneration body of non-heading Chinese cabbage until it grows 4 to 6 leaves to obtain a regenerated plant; Step 2: Transfer the regenerated plants into a rooting medium for rooting culture until roots are formed, thereby obtaining virus-free non-heading cabbage seedlings.

9. The method for rapidly cultivating virus-free non-heading Chinese cabbage seedlings using a multi-tillering non-heading Chinese cabbage variety as claimed in claim 8, characterized in that: In step 2, the composition of the rooting medium is 1 / 2MS+30g / L sucrose+1.0mg / L NAA+5mg / LAgNO3+8g / L agar powder, pH=5.8, and the rooting culture conditions are temperature 20-30°C, light intensity 2000-3000Lx, and photoperiod of light / darkness=16h / 8h.

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