Evaluation of reproductive success strategies
The progeny of the treated inflorescence was collected, and the subsequent fruit weight determination and germination experiment were conducted. Four indicators of fruit setting rate, fruit weight, germination rate and germination energy were used to compare the quality of the progeny of different pollination treatments to evaluate the success of their reproduction.
(1) Fruit setting rate and mean fruit weight
During the peak flowering period from June 26 to July 18, 2019, all target branches were marked and the number of flowers on each branch was calculated. Then we collected the fruits of six groups (OP, WP, SSP, SP, G, X) of V. negundo var. heterophylla and divide the number of fruits by the number of flowers to get the fruit setting rate.
(2) Average fruit weight
We randomly selected 100 ripe fruits from the four treatments of OP, SP, G, and X, then weighed them. We calculated the average fruit weight of a single kernel. The above operation needed to be repeated five times for each group.
(3) Seed germination rate and germination energy
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 2019. After collection, the fruit stalks and persistent calyxes were rubbed off, and these seeds were stored in a refrigerator at 4 ℃. Greenhouse germination experiment was conducted on the seeds of OP, SP, G and X treatments in October 2019. The seeds were soaked with gibberellin solution of 1000 mg·L-1 for 24 hours, and were put on a germination bed of a 9 cm petri dish with 2 layers of moist filter paper. We placed 25 seeds in each dish evenly and there were five replicates per treatment to reduce the error. These experiments were carried out in an incubator with a relative humidity of 60%, a temperature of 25 ℃, 14 hours of light and 10 hours of darkness. 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. The germination rate (GR) and germination energy (GE) were calculated according to formulas (1-1) and (1-2).
GR (%) = (n / N) × 100% (1-1)
Where GR was the germination rate; n was the number of germinated seeds; N was the total number of tested seeds.
GE (%) = (m / N) × 100% (1-2)
GE was the germination energy; m was the number of germinated seeds when the number of germinated seeds reached the highest peak (the first 8 days in present study); N was the total number of tested seeds.