7. An inverted fluorescent microscope is recommended, since
after transformation the cells can be incubated overnight in
glass-bottom cell culture plates while expressing the fusion
genes, and then observed directly. Transferring protoplasts to
microscopic slides after incubation is complete should be
avoided because protoplasts can be destroyed during pipetting.
If an inverted microscope is not available, leave the samples in
their tubes for overnight incubation. For upright microscopy,
on the following day use cut tips to transfer the samples to
concave glass slides, instead of standard slides, to avoid breakage of the large and fragile protoplasts.
8. Light does not affect transformation efficiency, but bright light
is not recommended since etiolated protoplasts do not have
developed photosystems. A controlled temperature chamber is
recommended in order to avoid any unexpected temperature
changes that might occur if the samples are left on a lab bench
overnight.
Acknowledgments
Research in the Wurtzel laboratory has been funded by the
National Institutes of Health (grant GM081160), National Science
Foundation, American Cancer Society, Rockefeller Foundation
International Rice Biotechnology Program, McKnight Foundation, USDA, DOD, New York State, The City University of
New York, and Lehman College. MS acknowledges support from
Kean University, NJ.
References
1. Moise AR, Al-Babili S, Wurtzel ET (2014)
Mechanistic aspects of carotenoid biosynthesis.
Chem Rev 114(1):164–193
2. Giuliano G (2017) Provitamin A biofortification of crop plants: a gold rush with many
miners. Curr Opin Biotechnol 44:169–180.
https://doi.org/10.1016/j.copbio.2017.02.
001
3. Wurtzel ET (2018) Changing form and function through carotenoids and synthetic biology. Plant Physiol 179(3):830–843. https://
doi.org/10.1104/pp.18.01122
4. Schaub P, Al-Babili S, Drake R, Beyer P (2005)
Why is golden rice golden (yellow) instead of
red? Plant Physiol 138(1):441–450. https://
doi.org/10.1104/pp.104.057927
5. Shumskaya M, Wurtzel ET (2013) The carotenoid biosynthetic pathway: thinking in all
dimensions. Plant Sci 208(1):58–63
6. Shumskaya M, Bradbury LMT, Monaco RR,
Wurtzel ET (2012) Plastid localization of the
key carotenoid enzyme phytoene synthase is
altered by isozyme, allelic variation, and activity. Plant Cell 24:3725–3741. https://doi.
org/10.1105/tpc.112.104174
7. Li F, Vallabhaneni R, Yu J, Rocheford T, Wurtzel ET (2008a) The maize phytoene synthase
gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and
thermal stress-tolerance. Plant Physiol
147:1334–1346
8. Beltra ´n J, Kloss B, Hosler JP, Geng J, Liu A,
Modi A, Dawson JH, Sono M, Shumskaya M,
Ampomah-Dwamena C, Love JD, Wurtzel ET
(2015) Control of carotenoid biosynthesis
through a heme-based cis-trans isomerase.
Nat Chem Biol 11(8):598–605
9. Ampomah-Dwamena C, Driedonks N,
Lewis D, Shumskaya M, Chen X, Wurtzel ET,
Elucidating Carotenoid Enzyme Localization and Interactions
233
after transformation the cells can be incubated overnight in
glass-bottom cell culture plates while expressing the fusion
genes, and then observed directly. Transferring protoplasts to
microscopic slides after incubation is complete should be
avoided because protoplasts can be destroyed during pipetting.
If an inverted microscope is not available, leave the samples in
their tubes for overnight incubation. For upright microscopy,
on the following day use cut tips to transfer the samples to
concave glass slides, instead of standard slides, to avoid breakage of the large and fragile protoplasts.
8. Light does not affect transformation efficiency, but bright light
is not recommended since etiolated protoplasts do not have
developed photosystems. A controlled temperature chamber is
recommended in order to avoid any unexpected temperature
changes that might occur if the samples are left on a lab bench
overnight.
Acknowledgments
Research in the Wurtzel laboratory has been funded by the
National Institutes of Health (grant GM081160), National Science
Foundation, American Cancer Society, Rockefeller Foundation
International Rice Biotechnology Program, McKnight Foundation, USDA, DOD, New York State, The City University of
New York, and Lehman College. MS acknowledges support from
Kean University, NJ.
References
1. Moise AR, Al-Babili S, Wurtzel ET (2014)
Mechanistic aspects of carotenoid biosynthesis.
Chem Rev 114(1):164–193
2. Giuliano G (2017) Provitamin A biofortification of crop plants: a gold rush with many
miners. Curr Opin Biotechnol 44:169–180.
https://doi.org/10.1016/j.copbio.2017.02.
001
3. Wurtzel ET (2018) Changing form and function through carotenoids and synthetic biology. Plant Physiol 179(3):830–843. https://
doi.org/10.1104/pp.18.01122
4. Schaub P, Al-Babili S, Drake R, Beyer P (2005)
Why is golden rice golden (yellow) instead of
red? Plant Physiol 138(1):441–450. https://
doi.org/10.1104/pp.104.057927
5. Shumskaya M, Wurtzel ET (2013) The carotenoid biosynthetic pathway: thinking in all
dimensions. Plant Sci 208(1):58–63
6. Shumskaya M, Bradbury LMT, Monaco RR,
Wurtzel ET (2012) Plastid localization of the
key carotenoid enzyme phytoene synthase is
altered by isozyme, allelic variation, and activity. Plant Cell 24:3725–3741. https://doi.
org/10.1105/tpc.112.104174
7. Li F, Vallabhaneni R, Yu J, Rocheford T, Wurtzel ET (2008a) The maize phytoene synthase
gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and
thermal stress-tolerance. Plant Physiol
147:1334–1346
8. Beltra ´n J, Kloss B, Hosler JP, Geng J, Liu A,
Modi A, Dawson JH, Sono M, Shumskaya M,
Ampomah-Dwamena C, Love JD, Wurtzel ET
(2015) Control of carotenoid biosynthesis
through a heme-based cis-trans isomerase.
Nat Chem Biol 11(8):598–605
9. Ampomah-Dwamena C, Driedonks N,
Lewis D, Shumskaya M, Chen X, Wurtzel ET,
Elucidating Carotenoid Enzyme Localization and Interactions
233