Greenhouse germination experiment
Since our knowledge of the most appropriate pre-treatment for seed germination of V. negundo var. heterophylla is unknown, we carried out pre-experiments. The mature seeds of V. negundo var.heterophylla were collected from 10 naturally distributed plants (ground diameter >10 cm, growing evenly) of the study site in September 2017. After collection, the fruit stalks and persistent calyxes were rubbed off, then these seeds were stored in a refrigerator at 4 ℃. Greenhouse germination experiment was carried out from November to December 2017. The seeds were soaked for 24 hours with gibberellin acid (GA3) of different concentrations containing zero (CK), 0.2 ‰, 0.6 ‰, 0.8 ‰, 1.0 ‰ and 0.2% peroxide hydrogen (H2O2). The 9-cm Petri dishes with moist double-decked filter papers were used as germination beds, 20 seeds were placed uniformly in each dish, and 30 dishes (six pre-treatments × five replicates) were kept in an incubator at 25℃, 60% relative humidity, 14 h light and 10 h darkness for 20 days. The filter papers on the dishes were kept moist throughout the experiment. Seeds were considered germinated at the emergence of the radicle (Bewley and Black 1994), and without new seed germination for 7 consecutive days was marked as the end of the test. Seed germination was recorded daily and expressed as a % percentage of the total number of tested seeds (germination percentage). The final germination rate (germination percentage at 20 days after the test started) and germination energy (germination percentage at 8 days after the test started) were calculated. Trough studying the effect of different concentration and type of solution on the seed growth, the optimum pre-treatment solution was obtained in the greenhouse germination process. The results of this part of the experiment are shown in Fig 1.