Chin Bull Bot ›› 2020, Vol. 55 ›› Issue (1): 9-20.doi: 10.11983/CBB19133

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Transformation of Insect Derived Antifreeze Gene into Sweet Potato (Ipomoea batatas) and Enhanced Its Freeze-tolerance

Lai Xianjun1,Zhang Yizheng2,Gu Yinghong2,Yan Lang1,*()   

  1. 1Sichuan Key Laboratory of Potatoes, College of Agriculture Science, Xichang College, Liangshan 615013, China
    2Sichuan Key Laboratory of Molecular Biology & Biotechnology, College of Life Science, Sichuan University, Chengdu 610064, China
  • Received:2019-07-05 Accepted:2019-09-24 Online:2019-12-20 Published:2020-01-01
  • Contact: Yan Lang E-mail:langyan0807@hotmail.com

Abstract:

To explore whether the gene encoding antifreeze protein from insect can enhance the freezing tolerance of sweet potato through gene transformation, and to prepare freeze-tolerance materials for breeding purposes, we constructed a plant gene expression vector harboring an antifreeze gene TmAFP from yellow mealworms (Tenebrio molitor) and obtained transgenic freeze-tolerance sweet potato lines using Agrobacterium-mediated transformation method. A high-frequency regeneration system of sweet potato was established using the variety Huachano as the recipient material, and the embryogenic suspension cells were cultured in the somatic embryo maturation medium. The sensitivity test of embryogenic cells to herbicides indicated that the combination of MS+0.2 mg·L -12,4-D+0.8 mg·L -1GAP+100 mg·L -1Carb is the most effective medium for screening the transgenic positive plants. Seven embryogenic calli were obtained and 42 resistant seedlings were regenerated, among which 23 harbored pSUIBEV3-AFP and 19 had pCAMBIA-AFP. All resistant seedlings were examined by PCR, Southern hybridization and RT-PCR, and the results showed that the TmAFP gene was integrated into the plant genome and expressed. The transgenic and non-transgenic plants were treated at -1°C for 15 hours, and then transferred to room temperature. The results demonstrated that the freeze-tolerance of the transgenic plants was greatly improved.

Key words: sweet potato, antifreeze protein, transgenic, plant regeneration of sweet potato, molecular breeding

Figure 1

Transformation system of sweet potato (A), (B) Sweet potato embryogenic suspension cells at 18 and 22 weeks cultivated in MS+0.2 mg·L-1 2,4-D liquid medium, respectively; (C) Sweet potato embryogenic suspension cells at 8 weeks cultivated in MS+0.2 mg·L-1 2,4-D solid medium; (D) Sweet potato seedlings regenerated from somatic embryo. (A), (B), (D) Bars=1 cm; (C) Bar=4 cm"

Table 1

Effect of different somatic embryo maturation medium on the regenerated seedlings "

Treatment Components
(mg·L-1)
Number of seedlings in average * **
1 MS 44.8±5.22 a A
2 MS+ABA1.0 33.6±5.18 b AB
3 MS+GA31.0 30.2±4.73 bc B
4 MS+ABA1.0+GA31.0 23.2±6.29 c B
5 MS+ABA4.0+GA31.0 21.8±6.58 c B

Figure 2

Effects of herbicide and antibiotic in different concentrations on embryogenic callus and regenerated seedlings of sweet potato (A) Sweet potato embryogenic suspension cells at 8 weeks cultivated in selective medium. The medium from left to right was MS+0.2 mg·L-1 2,4-D with 0, 0.2, 0.4, 0.6, 0.8 mg·L-1 GAP, respectively; (B) Embryogenic suspension cells at 6 weeks (the first and second from left to right) and 8 weeks (the third and fourth). The medium were MS+0.2 mg·L-1 2,4-D (the first and third) MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb (the second and fourth); (C) Embryogenic suspension cells at 8 weeks, the medium are MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb with 0.8, 1.0, 1.2, 1.4 mg·L-1 GAP, respectively; (D) Huachano stem tips cultivated 0 day and 3 weeks on medium of MS+0.2 mg·L-1 2,4-D+0.8 mg·L-1 GAP+100 mg·L-1 Carb, respectively. (A), (B), (C) Bars=1 cm; (D) Bar=2 cm"

Figure 3

Sweet potato resistant somatic embryo and the regeneration of transgenic plants (A) Sweet potato embryogenic suspension cells cultivated in MS+0.2 mg·L-1 2,4-D liquid medium at 28°C for 32 weeks; (B) Embryogenic callus cultivated in selective and non-selective medium for 4 weeks, a-c: Embryogenic callus cultivated in selective medium (MS+0.2 mg·L-1 2,4-D+100 mg·L-1 Carb+0.8 mg·L-1 GAP), resistant callus was marked by arrows; d: Embryogenic callus cultivated in control medium without herbicide; (C) The processes of transgenic plant regeneration, a: Reproductive tissue; b: Bud; c: Leaf; d: Seedling; e: Reproductive plant. Bars=1 cm"

Figure 4

Detection of TmAFP in the transgenic sweet potato plants (A) Screening with 1.0 mg·L-1 GAP (Line 4-12, 4-11, 4-10, 4-9: Resistant seedlings; Line 47-1, 47-2: Non-resistant seedlings; CK: Non-transgenic control); (B) Amplified 353-bp fragment of AFP gene (M: D2000 molecular weight marker; +CK: pSUIBEV3-AFP vector as positive control; -CK: Non-transgenic seedlings as negative control; Line 4-9, 4-10, 4-12: GAP resistant seedlings; Line 47-2: Non-resistant seedlings); (C) PCR detection of transgenic seedlings (+CK: Amplifying AFP gene using pSUIBEV3-AFP as template; -CK: Amplifying AFP gene using non-transgenic seedling; 4-9-AFP: Amplifying AFP gene in transgenic seedling; 4-9-RPB2: Amplifying RPB2 gene in transgenic seedling; -CK-RPB2: Amplifying RPB2 gene in non-transgenic seedling); (D) Southern blotting analysis (+CK: pCAMBIA-AFP vector as positive control; -CK: Non-transgenic seedlings as negative control; The others represent different transgenic seedling lines); (E) RT-PCR detection of transgenic seedlings (RP1, RP2: Transgenic seedlings with empty pCAMBIA vector; Line 4-12, 4-11, 44-1, 54-5: Transgenic seedlings with pCAMBIA-AFP vector)."

Figure 5

Detection of freezing-tolerance ability of TmAFP transgenic sweet potato plants (A) Conductivity assay under different freeze-treatments (CK1: Non-transgenic control; CK2-4: Transgenic plants with empty vector; 4-9, 4-10, 4-11, 4-12, 44-1, 54-5: TmAFP transgenic lines; ** indicate extremely significant differences (P<0.01)); (B) Phenotypic changes of transgenic sweet potato plants and controls after 15 h treatment at -1°C (Overall: CK1, 4-9, 4-10, 4-11, 4-12 planted in the same pot; Non-transgenic: Zoomed in CK1; Transgenic: Zoomed in transgenic line 4-9). Bars=5 cm"

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