Gene Transformation Of P_SAG12-IPT In Gerbera Modified By Particle Bombardment And Its Anti-senescence Mechanisms

1. The study of the in vitro regeneration and transformation of GerberaThe in vitro regeneration and transformation of Gerbera jamesonii Bolus were investigated. The effects of different BA and NAA concentrations and combinations on the adventitious buds proliferation and target distance on the transformation frequency were studied. The results indicated that 1.5 mg L^-1 BA significantly increased adventitious buds proliferation. 9 cm target distance significantly improved the transformation frequency.2. Identifications of the modified gerbera by PCR and Southern blottingAnti-Kan gerbera materials were tested by PCR and Southern blotting. 26 lines from 106 materials were selected by PCR and 17 positive lines were obtained by Southern blotting. Through drought stress, 3 lines were identified to have high resistance to stress and one outstanding line from 3 was selected as the material for the following experiments.3. The effects of osmotic stress on antioxidant enzymes activities in the leaf discs of transgenic gerberaUsing an in vitro culture technique of leaf discs, the effects of osmotic stress induced by polyethylene glycol (PEG) 6000 on antioxidant enzymes activities, lipid peroxidation, and the contents of protein, proline and chlorophyll were investigated in leaf discs Gerbera modified by P_SAG12-IPT. Leaf discs of modified gerbera (GM) and control gerbera (WT) were incubated in 0, 20, 40 % (w/v) PEG 6000 nutrient solution for 20 h. The results indicated that the contents of chlorophyll a, chlorophyll b, total chlorophylls and carotenoids were significantly decreased after osmotic stress in the leaf discs of both GM and WT gerbera plants. However, this decrease extent was significantly smaller in the GM plants than in WT plants. The soluble protein content was decreased by osmotic stress and the decrease extent was considerably smaller in GM plants compared to WT plants. The osmotic stressincreased the activities of SOD, CAT, APX, GPX and DHAR in the leaf discs of both GM and WT plants, however, this increase was more considerably in GM plants than WT plants. The MDA content was considerably increased by osmotic stress in WT plants, however, was not significantly increased in GM plants. 20% PEG stress did not significantly affect the proline content and 40% PEG stress largely increased the proline content, however, the proline content in the GM plants was two folds as in the WT plants. These results indicated that GM gerbera plants showed much higher ability of anti-senescence, however, the increases of antioxidative enzymes activities may be involved in the increased ability of anti-senescence.4. The effects of photooxidative stress induced by paraquat on antioxidant enzymes activities in the leaf discs of transgenic gerberaUsing an in vitro culture technique of leaf discs, the effects of photooxidative stress induced by different concentrations of paraquat (PQ) (25, 50 uM) on leaf senescence of ?s\on-IPT modified Gerbera (GM) compared with the control Gerbera (WT). The results indicated that PQ treatment increased the leaf senescence and significantly decreased the contents of chlorophyll a, chlorophyll b, total chlorophylls and carotenoids;however, the decreases in GM plants were considerably smaller. The soluble protein content was largely decreased by PQ treatment, and the decrease extent was smaller in GM plants. The SOD activity was considerably increased by PQ treatment and increased with the increasing of PQ concentration, especially the SOD activity in GM plants was largely increased. The changes in CAT and DHAR activities had the similar tendency with the SOD activity. The activities of APX and GPX were not detected in the leaves of PQ treatments. It may indicate that APX and GPX were labile to the photooxidative stress. MDA is the product of lipid peroxidation, and the MDA content was significantly increased with the increasing of PQ concentration. In the same PQ concentration, the MDA content in the GM plants was significantly lower than in WT plants. Therefore, it could be concluded that the chimeric gene Psag\2-IPT transformed Gerbera plants had higher antioxidative potential, and therefore the senescence was delayed.5. The effects of drought stress on antioxidant enzymes activities in the leaves of transgenic gerberaIn the greenhouse, the GM and WT plants were treated by drought stress for 6 days and then irrigated again for recovery. The morphological observation indicated that the leaves of WT plants wilt after 6 days of drought stress, however, the young leaves of GM plants did not wilt till 8 days after treatment. The MDA content in the leaves was considerably increased after drought stress and decreased after 6 days of recovery, but not reduced to the level of the treatment beginning. In the same drought treatment condition, the MDA content was lower in the GM plants than WT plants. After 6 days of drought stress, the activities of SOD, CAT, APX, GPX, DHAR and GR were significantly higher in the GM plants than in the WT plants. After 6 days of recovery, the activities of SOD, CAT, APX, GPX, DHAR and GR in the GM plants were still higher than in the WT plants. The soluble protein content was significantly decreased after drought stress. However, the decrease in the WT plants was bigger than in the GM plants. After 6 days of recovery, the soluble protein content was increased and the increase in the GM plants was higher that in the WT plants. Therefore, the delay of senescence in the GM plants may be related to the increases of antioxidative enzymes activities.6. Effects of drought stress on chlorophyll fluorescence parameters and photosynthesis parameters in transgenic gerberaUsing chlorophyll fluorescence kinetics techniques, the chlorophyll fluorescence parameters (OPSII, Fv'/Fm', qP, ETR) and photosynthesis parameters (Pn, Gs) of the GM and WT plants were investigated under drought stress. The results showed that OPSII, Fv'/Fm', qP, ETR, Pn and Gs were decreased after drought stress and increased after recovery. However, their decreases by drought stress in the GM plants were lower than in the WT plants. The GM plants showed a higher resistance to drought stress.