Senescence is delayed when ramie (Boehmeria nivea L.) is transformed with the isopentyl transferase (ipt) gene under control of the SAG12 promoter

Abstract

Ramie is an economically important industrial fiber crop widely planted in China, India, and other Southeast Asian and Pacific Rim countries. It plays an important role in China's economy, where ramie farming, industry, and trade provide livelihood support to about five million people. However, poor fiber production resulting from leaf senescence and leaf abscission is a significant problem. In this study, we report the successful production of transgenic ramie plants which delayed leaf senescence and enhanced biomass. Transgenic ramie plants were obtained via transformation with the Agrobacterium tumefaciens strain harboring the binary vector pSG529 containing the isopentyl transferase (ipt) gene under control of the SAG12 promoter (P_SAG12-ipt construct). Agrobacterium tumefaciens strain EHA105 was used for the midrib explant transformation. The transformation frequency was 28.29%. Southern blot confirmed the integration of 1-4 copies of the NPTII gene into the ramie genome in the tested lines. At the fiber maturation stage, the transgenic plants had higher photosynthesis rates, chlorophyll content (SPAD values), and stronger resistance to exogenous ethylene compared with wild-type plants.

Keywords: ethylene; ipt; leaf senescence; ramie.

Figure 1

Poor production resulting from leaf senescence (A) and leaf abscission (B) is a significant problem.

Figure 2

Generation of transgenic ramie plants. (A) 40‐day‐old plants grown on MS medium supplemented with 0.01 mg·L⁻¹ NAA were used for leaf midrib explant preparation. The triangular flask is 6 cm in diameter and 10 cm in height. (B) Leaf midrib explants were infected with EHA105 harboring pSG529 and cultured on solid cocultivation medium (MS medium + 0.2 mg·L⁻¹ TDZ + 0.04 mg·L⁻¹ 2,4‐D + 50 mg·L⁻¹ AS). The diameter of Petri dish is 9 cm. (C) After 2 days’ coculture, leaf midrib explants were incubated on selection medium (MS + 0.2 mg·L⁻¹ TDZ + 0.04 mg·L⁻¹ 2,4‐D + 40 mg·L⁻¹ kanamycin+750 mg·L⁻¹ cefotaxime). (D) Infected leaf midrib explants on selection medium produced Km‐resistant shoots after 3 weeks of selection culture. (E) Transgenic plants were cultured on elongation medium (MS medium + 250 mg·L⁻¹ cefotaxime + 0.01 mg·L⁻¹ NAA). (F) Transgenic ramie plants rooted. (G) Transgenic shoots propagated on MS medium supplemented with 0.01 mg·L⁻¹ NAA. (H) Transgenic plants grown in Hoagland's solution. The cylindrical bottle is 4 cm in diameter and 10 cm in height. (I) Transgenic ramie plants were transplanted to small plastic pots in the greenhouse. The length, width, and height of the pots are 11, 11, and 14 cm, respectively.

Figure 3

The regeneration of transgenic plants. The diameter of Petri dish is 9 cm.

Figure 4

The vector construct and molecular characterization of transgenic ramie plants. (A) Schematic drawings of the T‐DNA region in the pSG529 plasmid vector. RB, T‐DNA right border; P_{SAG 12} ‐ipt, the cytokinin biosynthesis gene ipt, derived from Agrobacterium tumefaciens, under the control of the SAG12 promoter from Arabidopsis; CaMV35S, cauliflower mosaic virus; NPTII, neomycin phosphotransferase II gene; NosT, nopaline synthase terminator; LB, T‐DNA left border. Xbal was the restriction enzyme site. (B) PCR analysis of the transgenic plants using primers specific to the ipt and NPTII genes. M, molecular size marker [the first lane is the DNA marker (TIANGEN Biotech Co. Ltd., Beijing, China)]. The fragment lengths of the DNA marker were 100 bp, 300 bp, 500 bp, 700 bp, 900 bp, and 1200 bp. P, positive control (plasmid DNA); C, control (untransformed plant). Lanes 1–22 were transgenic lines 1–22, respectively. Arrows point to the expected size bands. (C) Total RNA was isolated from WT and transgenic lines 1, 5, 7, 8, 9, 13, 15, 17, 19, 20, and 21. RT‐PCR was performed with ipt‐specific or GAPDH‐specific primers. The GAPDH gene was used as an internal control. (D) In the Southern blot analysis of the transgenic plants, the number of bands reflects the number of transgene insertions. Lane 9 was a negative control. Lanes 1–8 are transgenic lines 1, 5, 8, 9, 15, 17, 19, and 20, respectively.

Figure 5

Transgenic plants expressing ipt had higher SPAD values and photosynthesis rates than WT plants. The SPAD values (A) and photosynthesis rates (B) of these seedlings were measured accordingly with triplicates. Asterisks in (A) and (B) indicated significant differences (P < 0.05) between the transgenic lines and WT plants.

Figure 6

Effect of ethephon‐induced leaf senescence. Plants were subjected to 1200 ppm ethephon treatments for 10 days and photographed. The diameter of Petri dish is 15 cm. See ‘Materials and methods’ for further details.

Figure 7

The phenotype of transgenic and WT plants. Plants at (A) the beginning (control) and (B) the end (treatment) of the fiber maturation stage. Subsequently, their stem basts were peeled (C) and dried (D). Bars are 10 cm.

Figure 8

The biomass traits of WT and transgenic plants. Asterisks indicated significant differences (P < 0.05) between the transgenic lines and WT plants.