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Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-Seed Explants

^a Plant Science Research Center, Univ. of Toledo, 2801 W. Bancroft, Toledo, OH 43606
^b current address: Edenspace Systems Corp., 1500 Hayes Dr., Manhattan, KS 66502
^c current address: School of Science, Engineering and Technology, TL 174, Penn State Harrisburg, 777 W. Harrisburg Pike, Middletown, PA 17057-4898

View larger version (15K): [in this window] [in a new window]   Figure 1. Maps of the constructs used for particle bombardment transformation: (A) pC1301 contains the GUS gene under 35S promoter; (B) pPDSG89 contains the CBF3 gene under the control of rd29A.

View larger version (46K): [in this window] [in a new window]   Figure 2. Effect of drying split-seed explants before bombardment on transient GUS expression. Error bars represent the standard deviation. The means of GUS spots from each pressure point were analyzed by t-test (P = 0.05).

View larger version (99K): [in this window] [in a new window]   Figure 3. Production of transgenic maize plants using split-seed explant: (A) split-seed explants arranged for particle bombardment; (B) bombarded explants on selection medium; (C) shoot regeneration through first round of selection; (D) shoots after a second round of selection; (E) rooting of a CBF3 putative transformant; and (F) CBF3 transgenic plants grown in the greenhouse.

View larger version (67K): [in this window] [in a new window]   Figure 4. Southern blot analysis of T0, T1, and T2 CBF3 plants: (A) Southern blot analysis of T0 plants, showing Lane 1—kb ladder; Lane 2—untransformed plant digested with KpnI; Lanes 3, 5, and 7—Lines 7, 8, and 28 digested with Hind III; Lanes 4, 6, and 8—Lines 7, 8, and 28 digested with KpnI; and Lane 10—plasmid positive control digested with KpnI; (B) T1 plants that originated from Line 28, showing Lane 1—kb ladder, Lane 2—untransformed plant; Lanes 3 to 9—plants no. 1, 3, 4, 7, 11, 13, and 14 digested with KpnI; and Lane 11—plasmid positive control digested with KpnI; (C) T2 plants digested with KpnI, showing Lane 1—kb ladder; Lane 2—untransformed plant; Lanes 3 to 20—T2 plants no. 1 to 18; and Lane 21—plasmid positive control.

View larger version (38K): [in this window] [in a new window]   Figure 5. Reverse transcription polymerase chain reaction analysis of CBF3 gene expression in T0 Lines 7, 8, and 28 grown at 26°C; C is a wild-type plant, maize actin was used as a loading control.

View larger version (52K): [in this window] [in a new window]   Figure 6. Northern blot analysis of CBF3 transcripts after T2 plants derived from Line 28 were treated with different low temperatures. Plants were grown at 26°C and then were gradually exposed to low temperatures (20, 16, 12, 10, 4, 0, and –2°C), 3 h for each temperature. Full length of the coding region of CBF3 was used as a probe and actin was used as a loading control.

View larger version (54K): [in this window] [in a new window]   Figure 7. Comparison of growth characteristics, survival rate, and fertility rate between transgenic T2 seedlings derived from Line 28 (left tray) vs. wild-type seedlings (right tray) when kept at 10°C: (A) 5-d-old seedlings grown at 26°C (normal condition); (B) 1-wk-old seedlings; and (C) 4-wk-old seedlings. (D) Mean heights of transgenic and wild-type seedlings at different time periods, with error bars representing the standard deviation; and (E) survival and fertility rates of transgenic and wild-type plants after a 2-wk recovery period from 10°C in the greenhouse, during which surviving plants were grown to maturity.

View larger version (76K): [in this window] [in a new window]   Figure 8. Northern blot analysis of T2 plants derived from Line 28 that were exposed to 4 and 0°C for different periods of time: (A) accumulation of CBF3 transcripts in response to exposure to 4°C for 1, 3, 5, 7, 12, and 24 h (C is a wild-type plant and TN is a transgenic (T2) plant derived from Line 28 grown at 26°C); (B) T2 plants derived from Line 28 that were exposed to 0°C for 15 min, 30 min, and 1, 3, 6, 12, and 24 h (C is a wild-type plant); actin was used as a loading control.

View larger version (29K): [in this window] [in a new window]   Figure 9. Comparison of cold and freezing tolerance between transgenic T2 plants derived from Line 28 (left tray) and wild-type (right tray) plants: (A, B, and C) plants after exposure to 4, 0, and –2°C, respectively, for 48 h; (D, E, and F) after a 2-wk recovery period under greenhouse conditions (26°C). (G) Electrolyte leakage of transgenic and control plants that were grown at 26°C and were then exposed to 0, 4, and –2°C for 48 h; and (H) comparison of survival rates after cold and freezing treatments of transgenic and wild-type plants after 2 wk of recovery from 0, 4, and –2°C exposure for 48 h.

View larger version (94K): [in this window] [in a new window]   Figure 10. Northern blot analysis of maize CAT3 transcripts in transgenic T2 plants and wild-type plants grown at 26°C or exposed to 4°C for 2 d; C is wild-type plants, T is transgenic plants; actin was used as a loading control.

View larger version (59K): [in this window] [in a new window]   Figure 11. Accumulation of CBF3 transcripts in T2 plants derived from Line 28 in response to water deprivation for 3 and 7 d. The numbers 1 to 3 represent three different transgenic plants from the T2 generation; C is a wild-type plant; and actin was used as a loading control.

View larger version (66K): [in this window] [in a new window]   Figure 12. Transgenic maize T2 plants derived from Line 28 (left pots) show more tolerance to drought than wild-type plants (right pots) when not watered for (A) 7 d, (B) 14 d, and (C) 21 d. (D) Comparison of survival rates after drought treatments at the indicated periods and after a 2-wk recovery period in the greenhouse; and (E) electrolyte leakage of transgenic and wild-type plants that were grown at 26°C and were then exposed to dehydration stress for the time indicated.

View larger version (48K): [in this window] [in a new window]   Figure 13. Northern blot analysis of the CBF3 gene in T2 plants derived from Line 28 that were exposed to different concentrations of NaCl solution: 100, 200, and 400 mmol L–1 for the periods indicated; C is a wild-type plant and actin was used as a loading control.

View larger version (65K): [in this window] [in a new window]   Figure 14. Transgenic T2 plants derived from Line 28 (left trays) are more tolerant to high salt (NaCl) concentrations than wild-type plants (right trays): (A, B, and C) 100, 200, and 400 mmol L–1 NaCl treatments for 3 d; (D, E, and F) treated plants after 7 d; and (G) treated transgenic and wild-type plants after 21 d in the greenhouse. (H) Survival rate comparison between transgenic and wild-type plants after a 2-wk recovery period.