Tissue culture method for promoting differentiation and regeneration of soybean cotyledon node explant by using nano material

A technology of nanomaterials and tissue culture, applied in botany equipment and methods, plant regeneration, horticultural methods, etc., can solve the unstudied problems of the influence of carbon nanotubes on the differentiation and growth of soybean cotyledonary node explants, and achieve organizational Improve the culture efficiency, the effect is obvious, and the effect of the high-efficiency tissue culture system

Inactive Publication Date: 2011-11-02
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +1
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AI-Extracted Technical Summary

Problems solved by technology

[0004] CN101822140 discloses the method of using carbon nanotubes to promote soybean seed germination under drought stress conditions, but does not study the influence of carbon nano...
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Method used

Soybean Zhonghuang 35 cotyledon node explants, on different inducing differentiation medium SINMO, SINM4, SINM40, on SINM400, there is significant difference in differentiation frequency (as shown in Figure 2), statistics can produce 3-30 clusters of buds The number of differentiated explants, the results show that SINM4, SINM40, and SINM400 have significantly better induction and differentiation effects than SINM0; and SINM40 has the best effect, and the difference is extremely significant compared with SINM0. The induction and differentiation frequency of SINM4 and SINM400 is slightly higher than that of SINM0 (See Table 1). The differentiation frequency of SINM40 and SINM0 was repeatedly compared, and the result showed that the differentiation frequency of explants in the medium of SINM40 was 12.5% ​​higher than that of SINM0 (see Table 2). The experimental results show that the addition of nanomaterials to the medium formula can si...
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Abstract

The invention relates to the field of crop planting, and in particular relates to a tissue culture method for promoting differentiation and regeneration of a soybean cotyledon node explant by using a nano material. The method comprises the following steps: 1) disinfecting seeds; 2) germinating the seeds; 3) preparing a cotyledon explant; 4) putting the obtained explant on an induction medium containing a carbon nano tube at a concentration of 4-400mg/L to induce the explant to be differentiated; 5) transferring the differentiated soybean cotyledon node explant to an elongating and rooting medium containing a carbon nano tube at a concentration of 4-400mg/L; and 6) separating the tissue culture seedlings. The tissue culture method has the following beneficial effects: the method is easy tooperate and has an obvious effect; an efficient tissue culture system can be established; the tissue culture workload is not increased; and the method is of great importance to improvement of the soybean tissue culture efficiency.

Application Domain

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  • Tissue culture method for promoting differentiation and regeneration of soybean cotyledon node explant by using nano material
  • Tissue culture method for promoting differentiation and regeneration of soybean cotyledon node explant by using nano material
  • Tissue culture method for promoting differentiation and regeneration of soybean cotyledon node explant by using nano material

Examples

  • Experimental program(1)

Example Embodiment

[0022] Example 1
[0023] Test seed: Soybean variety Zhonghuang 35,1500 grains.
[0024] Nanomaterial: Carbon Nanotube (NM) (Single Wall)
[0025] Medium formula:
[0026] Seed germination medium: B5+sucrose 20g/L+agar 7.5g/L
[0027] Differentiation medium
[0028] SINM0: B5+6-BA1.50mg/L+sucrose 30g/L+agar 7.5g/L
[0029] SINM4: B5+6-BA1.50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​4mg/L
[0030] SINM40: B5+6-BA1.50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​40mg/L
[0031] SINM400: B5+6-BA1.50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​400mg/L
[0032] Regeneration medium
[0033] SENM0:
[0034] B5+ZT1mg/L+IAA0.1mg/L+GA 3 0.5mg/L+L-pyrogltamicacid100mg/L+asparagines
[0035] 50mg/L+sucrose 30g/L+agar 7.5g/L
[0036] SENM4:
[0037] B5+ZT1mg/L+IAA0.1mg/L+GA 3 0.5mg/L+L-pyrogltamicacid100mg/L+asparagines
[0038] 50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​4mg/L
[0039] SENM40:
[0040] B5+ZT1mg/L+IAA0.1mg/L+GA 3 0.5mg/L+L-pyrogltamicacid100mg/L+asparagines
[0041] 50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​40mg/L
[0042] SENM400:
[0043] B5+ZT1mg/L+IAA0.1mg/L+GA 3 0.5mg/L+L-pyrogltamicacid100mhg/L+asparagines
[0044] 50mg/L+sucrose 30g/L+agar 7.5g/L+NM ​​400mg/L
[0045] 1. Experimental method
[0046] 1.1. Seed disinfection
[0047] Pick 1,000 full soybean seeds, put them in sterilized Erlenmeyer flasks, wash with 75% alcohol for 1 minute, 10% sodium hypochlorite for 20 minutes, and wash 3 times with distilled water, then put the soybean seeds in ultra-clean typhoon to dry the excess water ,spare.
[0048] 1.2. Seed germination
[0049] Plant the sterilized soybean seed hilum down in the seed germination medium, 10 seeds per dish, placed in a 26℃ incubator, cultured in 16/8 light and dark, five days later, the germinated soybean seeds are cut into cotyledon nodes. Prepare explants.
[0050] 1.3. Preparation of explants
[0051] Five days after the seed germination, cut the hypocotyl 3mm close to the cotyledon node, remove the hypocotyl and the radicle, and then cut longitudinally along the middle of the two cotyledons, and cut 3-5 longitudinally and transversely at the cotyledon node for use.
[0052] 1.4, induce differentiation
[0053] Insert the cut explants obliquely into differentiation medium SINM0, SINM4, SINM40, and SINM400, and place them in an incubator at 26°C in 16/8 light and dark culture. After about two weeks, the explants differentiated into clusters of buds. Statistics The number of explants that differentiated into clusters of buds, and then the cotyledons were cut off and transferred to regeneration medium.
[0054] 1.5. Obtain regenerated plants
[0055] Transfer the differentiated explants with clumping buds to regeneration medium SENM0, SENM4, SENM40, SENM400, 25℃ incubator, 16/8 light and dark culture, subculture once every two weeks, pay attention to The yellow and dead tissues on the explants are cut off, and the cluster buds to be differentiated are elongated into small plants and rooted, and the number of explants of regenerated plants is counted. Then gradually loosen the bottle cap or sealing film for the strong seedlings. After the strong seedlings are about one week later, wash off the agar, transfer them to pots, and transplant the greenhouse.
[0056] 2. Experimental results
[0057] 2.1. Seed disinfection, germination, preparation of explants
[0058] Plant the sterilized soybean Zhonghuang 35 seeds on the seed germination medium, germinate after five days, and cut to prepare explants (such as figure 1 ).
[0059] 2.2, induce differentiation
[0060] Soybean Zhonghuang 35 cotyledon node explants, on different inducing differentiation media SINM0, SINM4, SINM40, SINM400, there are obvious differences in differentiation frequency (such as figure 2 ), counting the number of differentiated explants that can produce 3-30 clumping buds, the results show that the differentiation effect of SINM4, SINM40, and SINM400 medium is significantly better than SINM0; and SINM40 has the best effect, and the difference is very significant compared with SINM0 The frequency of induced differentiation of SINM4 and SINM400 is slightly higher than that of SINM0 (see Table 1). The differentiation frequency of SINM40 and SINM0 was repeatedly compared, and the results showed that the differentiation frequency of explants in SINM40 medium was 12.5% ​​higher than that of SINM0 (see Table 2). The experimental results show that the addition of nanomaterials in this medium formula can significantly promote the differentiation of soybean Zhonghuang 35 cotyledon node explants, and the optimal concentration of nanomaterials is 40mg/L.
[0061] Table 1: Comparison of the differentiation frequency of soybean Zhonghuang 35 cotyledon node explants in the differentiation medium containing different concentrations of nanomaterials (three repetitions)
[0062]
[0063] Table 2: Comparison of differentiation frequency of soybean Zhonghuang 35 cotyledon node explants in SINM0 and SINM40 induction differentiation medium for one week (12 repetitions, about 43 explants per treatment, summary results)
[0064]
[0065] 2.3. Obtain regenerated plants
[0066] The differentiated soybean Zhonghuang 35 cotyledon node explants were transferred to regeneration medium SENM0, SENM4, SENM40, SENM400. After two weeks, it can be seen that the explants in the SENM40 medium are the first to grow large buds and simultaneously grow fibrous roots (such as image 3 ), the explants that can differentiate and grow to form regenerated plants reach 48.1% (see Table 3), and each explant can produce 1-7 regenerated plants. This significantly increased the regeneration frequency of soybean Zhonghuang 35 cotyledon node explants and shortened the time from differentiation to seedling formation. The regenerated soybean plants obtained from the separation will be strong seedlings for one week, then transplanted into the soil, cultivated in the greenhouse, and the plants grow normally (such as Figure 4 ).
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