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In addition, RNA was isolated from open flowers, un-opened flowers, roots, seeds and fruit exocarp.
Pollen cells from different developmental stages, namely tetrads, post-meiotic microspores, and mature pollen grains from open flowers, were isolated according to existing protocols.
Nectar samples were harvested from open flowers and stored in sterile tubes, filled with 70% Ethanol until further analysis by HPLC (see [ 71] for detailed methods).
Pollen grains collected from open flowers of both wild-type and mutants bearing one copy of either HAM1 or HAM2, were examined by staining with DNA-specific dye 4',6-diamidino-2-phenylindole (DAPI).
To determine the reason why p5cr mutations were transmitted through the male germline, while p5cs1/p5cs2 double mutations were not, pollen grains collected from open flowers of p5cs and p5cr mutant plants were stained with Alexander stain.
All PCR-positive transgenic lines were assayed for GUS expression by histochemical analysis of transverse sections from leaves at approximately 30 50% of full expansion, or whole flower parts from open flowers and roots from mature plants [ 3].
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For viability assays, pollens were isolated from opened flowers of male, GyM-H and converted flowers of AgNO3 treated female plant.
Total RNA was isolated from opening flowers (stage 4 as defined by Ueyama et al. [ 9]) using a FastRNA pro GREEN kit (Qbiogene, Irvine, CA, USA).
Wider corolla tubes may be associated with increased pollinator visitation rates, because a greater range of pollinating insects can access the nectar from more open flowers with wider tubes.
The petals from the fully open flowers show distinct epidermal features at the apex, medium and base.
We selected 10 inflorescences in bud that lacked aphids and belonged to plants at least 2 m from plants with open flowers.
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