Vacuole development in evacuolated oat mesophyll ptotoplasts
Vacuole development in evacuolated oat mesophyll ptotoplasts
Protoplasts were prepared from oat leaf mesophyll cells and subsequently evacuolated by high speed centrifugation through a continuous Percoll gradient. The effects of a variety of factors, including medium composition, plant growth conditions and leaf preparation methods, on the isolation, evacuolation and subsequent culture of oat mesophyll protoplasts were examined. Protocols were modified in order to maximise the yield, viability and extent of evacuolation of protoplasts. Factors found to be of importance include plant growth temperature, medium pH and the inclusion of a vacuum infiltration step in protoplast digestion. Evacuolated protoplasts were cultured successfully following trials of a limited range of media and culture conditions.
Vacuole formation was characterised using conventional light and fluorescence microscopy, transmission electron microscopy and confocal laser scanning microscopy (CLSM). Vacuoles able to take up neutral red were formed within 3d and cell wall formation was detected after 5d culture. Within the first hour, CLSM revealed a network of interconnected tubules and some small vesicles. These showed acridine orange uptake, and subsequently underwent expansion, forming a series of small interconnected vacuoles (of the order of 3μm diameter at 24h) which were also visible in electron micrographs.
Western blot analysis of microsomal membrane preparations from intact, freshly evacuolated and 5d old cultured evacuolated protoplasts was carried out using antibodies to a range of tonoplast proteins. The tonoplast H+ATPase and H+PPase were present in intact and cultured evacuolated protoplasts, but not freshly evacuolated protoplasts. Control blots showed the plasma membrane H+ATPase was present in all protoplast samples. It was concluded that protoplast evacuolation is complete and de novo vacuole formation occurs in cultured evacuolated oat mesophyll protoplasts.
University of Southampton
1994
Newell, Jane Marie
(1994)
Vacuole development in evacuolated oat mesophyll ptotoplasts.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Protoplasts were prepared from oat leaf mesophyll cells and subsequently evacuolated by high speed centrifugation through a continuous Percoll gradient. The effects of a variety of factors, including medium composition, plant growth conditions and leaf preparation methods, on the isolation, evacuolation and subsequent culture of oat mesophyll protoplasts were examined. Protocols were modified in order to maximise the yield, viability and extent of evacuolation of protoplasts. Factors found to be of importance include plant growth temperature, medium pH and the inclusion of a vacuum infiltration step in protoplast digestion. Evacuolated protoplasts were cultured successfully following trials of a limited range of media and culture conditions.
Vacuole formation was characterised using conventional light and fluorescence microscopy, transmission electron microscopy and confocal laser scanning microscopy (CLSM). Vacuoles able to take up neutral red were formed within 3d and cell wall formation was detected after 5d culture. Within the first hour, CLSM revealed a network of interconnected tubules and some small vesicles. These showed acridine orange uptake, and subsequently underwent expansion, forming a series of small interconnected vacuoles (of the order of 3μm diameter at 24h) which were also visible in electron micrographs.
Western blot analysis of microsomal membrane preparations from intact, freshly evacuolated and 5d old cultured evacuolated protoplasts was carried out using antibodies to a range of tonoplast proteins. The tonoplast H+ATPase and H+PPase were present in intact and cultured evacuolated protoplasts, but not freshly evacuolated protoplasts. Control blots showed the plasma membrane H+ATPase was present in all protoplast samples. It was concluded that protoplast evacuolation is complete and de novo vacuole formation occurs in cultured evacuolated oat mesophyll protoplasts.
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Published date: 1994
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Local EPrints ID: 458477
URI: http://eprints.soton.ac.uk/id/eprint/458477
PURE UUID: ae3af9eb-873a-495d-a0cb-845bad0ad09b
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Date deposited: 04 Jul 2022 16:49
Last modified: 04 Jul 2022 16:49
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Author:
Jane Marie Newell
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