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

[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 ).