Fig 1.
Map of the T-DNA construct and identification of overexpressing TaLEA and ThbZIP transgenic birches with PCR.
(A) Schematic of the T-DNA region of the binary vector pROKII-TaLEA-ThbZIP. RB, right border; Pnos, nopaline synthase promoter; NptII, kanamycin resistance gene; Tnos, nopaline synthase terminator; P35S, CaMV 35S promoter; Sma I, Sac I, BamH I, and Kpn I, four different restriction enzyme sites; TaLEA, TaLEA gene; ThbZIP, ThbZIP gene; LB, left border. Agarose gel electrophoresis of PCR products from wild type and transgenic lines with the primer of NptII (B), TaLEA (C), ThbZIP (D). M, DNA marker; PC, positive control; 1–11, eleven Km resistant lines; L-4, 5, 7, 8, 9, five transgenic lines both containing TaLEA and ThbZIP genes; WT, wild type plantlet; H2O, double-distilled water as negative control.
Fig 2.
Regeneration of overexpressing TaLEA and ThbZIP transgenic birch.
(A) The transgenic callus (red circle) have formed on one of the cut sites in a leaf segment. (B) Close-up view of the circled area in (A). (C) Some transgenic calls gained from leaf segments. (D) transgenic cluster of shoots has formed from a callus. (E) Four transgenic shoots were transferred to rooting medium. (F) One-month-old transgenic plants were grown in sterile soil media. (G) Three-month-old transgenic plants were grown in a greenhouse.
Fig 3.
Northern blot analysis of TaLEA and ThbZIP expression in transgenic birches.
Total RNA was extracted from the aerial tissues of one-month-old wild type and transgenic lines. The full length TaLEA and ThbZIP genes labeled with DIG-dUTP were used as probes. rRNA, ribosomal RNAs from different samples; TaLEA, target band of TaLEA gene; ThbZIP, target band of ThbZIP gene; L-4, 5, 8, three transgenic lines both containing TaLEA and ThbZIP genes; WT, wild type plantlet, respectively.
Fig 4.
NaCl stress-tolerance test of transgenic birch ectopic overexpressing TaLEA and ThbZIP.
Stems of transgenic and wild type plants were cultured on WPM medium containing 2 g L-1 (A), 4 g L-1 (B) or 6 g L-1 NaCl (C). Shoots of transgenic and wild type plants were transferred to 1/2 MS root medium containing 4 g L-1 NaCl (D), or 6 g L-1 NaCl (E). Photographs were taken 20 d following stress treatment. WT, wild type; L-4, transgenic line 4; L-8, transgenic line 8.
Fig 5.
The phenotypes of WT on day 8 under different concentrations of NaCl.
From left to right were WT, the treatment of 2 g L-1, 4 g L-1, 6 g L-1 and 8 g L-1 of salt stress, respectively.
Fig 6.
Pn values of WT under different concentrations salt stress.
Fig 7.
Fv/Fm values of WT under different concentrations salt stress.
Fig 8.
Pn-PPFD and Pn-Ca curves of different transgenic lines before stress (A, and C) and 8 d after salt stress (B and D).
Table 1.
Pn- Par simulation equation and Pn, LSP, LCP values of different lines before and 8 d after stress.
Table 2.
Pn- Ca simulation equation and Pn, CSP, CCP values of different lines before and 8 d after stress.
Fig 9.
Pn (A), Gs (B), Ci (C) and Tr (D) values of different lines after different days salt stress.
Fig 10.
Fv/Fm values of different lines after different days salt stress.
Table 3.
ANOVA analysis of photosynthetic traits and Fv/Fm among different lines and time under salt stress.
Fig 11.
POD (A), SOD (B), MDA (C) and Na+ (D) values of different lines after different days salt stress.
Table 4.
ANOVA analysis of SOD, POD, MDA and Na+ Concentration among different lines and time under salt stress.
Table 5.
Average POD, SOD, MDA and Na+ content of different lines in different time under salt stress.