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First, chosen flowers were bagged to exclude insects.
70 galls were marked for monitoring and 50 of these were bagged to measure emergence of fundatrigeniae.
All the flowers were bagged to prevent cross-pollination, and when sampled in the field, all the samples were frozen in liquid nitrogen as quickly as possible and then stored at −80°C until needed.
40 galls were bagged to measure alate emergence at each site, giving 160 bagged galls across all the sites.
Before syconia on a raceme became receptive, they were bagged to prevent wasp entry.
Before anthesis, spikes were bagged to ensure self-pollination.
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The flowers were bagged one day prior to anthesis to prevent hybridization with other varieties and tagged on the day of anthesis after the removal of the bag.
In addition, in each population five individuals with unopened flowers were bagged with a fine-meshed cloth to exclude pollinators to test for spontaneous autogamy.
For interspecific crosses, plants were emasculated and hand pollinated, the flowers were bagged and the plants allowed to fully mature.
Finally, floral capitula were bagged as in breeding system experiments to retain florets and achenes.
All flowers were bagged in PET foil (Toppits® 'Bratschlauch', Melitta, Minden, Germany) to prevent direct induction of the flowers by any airborne cue that might be released from the leaves in response to these treatments.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com