Aug 05, 2022
Experimental protocol for data collection of Datura spp.
- 1Escuela Nacional de Estudios Superiores (ENES) Unidad Morelia and Laboratorio Nacional de Análisis y Síntesis Ecológica (LANASE), Universidad Nacional Autónoma de México (UNAM), Campus Morelia, Morelia, Michoacán, Mexico;
- 22Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México (UNAM)
Protocol Citation: Eunice Kariñho-Betancourt 2022. Experimental protocol for data collection of Datura spp.. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr6b7ovmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 05, 2022
Last Modified: August 05, 2022
Protocol Integer ID: 68282
Abstract
Experimental design employed to obtain biological materials for RNA seq analysis. We collected leaf tissue to examine gene family evolution of four Datura species.
Materials
Seeds from four species of the plant genus Datura (D. stramonium, D. pruinosa, D. inoxia and D. wrightii) collected in natural populations of central and southern Mexico, and southern United States, and stored for no longer than five years, were employed to produce experimental plants.
Germination
Germination
Plants were started from seed between March-April, growing under 16:8, L:D cycle with 25:20°C (L:D) in the glasshouse of the Institute of Ecology, National Autonomous University of Mexico. Experimental plants were obtained by sowing seeds of maternal families (natural progenies). Once the true leaves appeared and seedlings reached 3-5 cm long, they were planted in single 150 mL pots (Figure 1), in sterilized soil, and watered ad libitum.
Figure 1. A) Germination and B) seedling transplant of Datura sp.
Leaves collection
Leaves collection
Nine plants (of different maternal families) were collected at two developmental stages. To capture developmental variation 10 fully expanded leaves from each plant were harvested either at the vegetative or reproductive stage as follows:
When plants reached at least 15 cm and/or started dichotomous branching (~40 days after germination), three juvenile plants were defoliated, whereas the six other plants remained undamaged until flowering.
Since leaf age of vegetative plants at the time of first harvesting differed from mature plants at the final harvesting, we controlled for leaf age differences at the two collection episodes by marking the new leaves produced after the first harvesting, in both treatment-allocated plants so that they could be compared.
When the first flower emerged, at the reproductive stage (~ 60 days after germination), leaves of the remaining six plants were harvested. All leaves were flash-frozen after collection and stored at -80°C until RNA extraction.