Artificial pollination experiment
We conducted the hand-pollinated experiment on 10 individuals (growing
evenly) in June to July 2019 to evaluate mating system. Since the single
flower of V. negundo var. heterophylla was small, the
inflorescence was used as a unit for treatment. On each plant, 18
inflorescences (6 treatments × 3 replicates) were randomly marked, each
single flower in each inflorescence was treated the same way. Note that
in the selection of experimental subjects, the selected inflorescence
was basically the same in terms of length, height and other dimensions,
thus reducing the possibility caused by this problem.
Pollination treatments including: (1) OP (Fig.2a):
open-pollinated by pollinators;
(2) WP (Fig.2b): emasculated and bagged with mesh bags to detect wind
pollination; (3) SSP (Fig.2c): bagged with paper bags and left
unmanipulated to detect spontaneous self-pollination; (4) SP (Fig.2d):
hand-pollinated with pollen from the same flower using a small brush and
bagged to detect manipulated self-pollination; (5) G (Fig.2e):
emasculated, hand-pollinated with pollen from another flower of the same
tree and bagged to detect geitonogamy; (6) X (Fig.2f): emasculated,
hand-pollinated with pollen from another tree and bagged to detect
xenogamy. The bagging was carried out at bud phase and all
hand-pollinated flowers were bagged with paper pollination bags to
exclude pollinators. (4) and (5) were to detect the degree of
self-compatibility, and (6) was to detect the degree of hybridization.
The number of treated flowers per labeled inflorescence was recorded
every day. 9074 flowers of V. negundo var. heterophyllawere treated in total.
Before the six treatments, stigmatic
receptivity was checked to ensure that the selected branches were
healthy and normally developed. Bagging was carried out at the bud
stage. According to Sun et al. (2020), the best pollination time was
determined to be from 10:00 am to 12:00 am in view of the
pollen viability and stigmatic
receptivity at different stages after flowering, and with the help of
tweezers, the anther directly contacted the stigma for pollination. The
phenological developmental stage of the flower at pollination was during
the anthesis. Then we followed up the post pollination development and
subsequently collected the progeny of the treated inflorescences for
subsequent fruit weight determination and germination experiments.