Microtubules in cultured plant protoplasts

Simmonds, Daina H.

doi:10.1111/j.1438-8677.1991.tb01545.x

Cited by 10 publications

(7 citation statements)

References 44 publications

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“…During the incubation in enzyme mixture, various cellular structures, such as the cell wall, the protein systems and the stability of the plasmamembrane will be disturbed or lost. Because of digestion of the cell wall by enzymes, changes also occur in the orientation and organization of various elements (microtubules, microfilaments, intermediate filaments) of the cytoskeleton as well as the cell polarity, influencing cell division (Traas 1990;Simmonds 1991). Especially, APM and CB present in the enzyme mixture influence microtubule and chromosome decondensation, and microfilaments, respectively.…”

Section: Discussionmentioning

confidence: 98%

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Ramulu

Dijkhuis

Famelaer

et al. 1993

Planta

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Abstract.Results on the enhancement of the frequency of protoplasts with micronuclei, and on the isolation and enrichment of smaller sub-diploid microprotoplasts in transformed Nicotiana plumbaginifolia Viv. are reported. Suspension cells were treated with the spindle toxin amiprophos-methyl (APM) for 48 h, and subsequently incubated in a mixture of cell-wall-digesting enzymes in the presence of APM and cytochalasin-B. During enzyme incubation, the frequency of micronucleated protoplasts increased by a factor of 2-6. A shorter period (3 h) of incubation with a higher concentration of enzymes as well as a longer period (16 h) of incubation with a lower concentration of enzymes gave similar frequencies of micronucleated protoplasts and yields of micronuclei. Further, synchronization by sequential treatment with the DNA-synthesis inhibitor hydroxy urea, or aphidicolin, followed by APM and enzyme incubation, significantly increased the frequency of micronucleated protoplasts and the number of micronuclei. The suspension of protoplasts (mono-and micronucleated) obtained after enzyme incubation was fractionated throuh a continuous iso-osmotic gradient of Percoll, using high-speed centrifugation. This resulted in one large and a few small bands, which contained a heterogeneous population of microprotoplasts, protoplasts and cytoplasts. In contrast to the large band, the small bands contained a relatively higher frequency of small sub-diploid microprotoplasts. To separate the small sub-diploid microprotoplasts from the large microprotoplasts and protoplasts of the bands, discontinuous Percoll gradients and sequential filtration through nylon sieves of decreasing pore size (48--20-15-10-5 [am) were investigated. Compared with the former method, the latter gave a highly enriched fraction containing predominantly (~80%) small subdiploid microprotoplasts with DNA contents equivalent to that of one to four chromosomes, as revealed by miAbbreviations: APH =aphidicolin; APM = amiprophos-methyl; Au = arbitrary units; CB = cytochalasin B; DAPI = 4,6-diamidino-2-phenylindole; FDA = fluorescein diacetate; HU = hydroxy urea Correspondence to: K.S. Ramulu; FAX: 31 (8370) 18094 crodensitometric and flow-cytometric analyses. The application of this technique for partial genome and limited gene transfer is discussed.

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Section: Discussionmentioning

confidence: 98%

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Ramulu

Dijkhuis

Famelaer

et al. 1993

Planta

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“…The isolation of protoplasts from mature plant tissue leads, to some extent, to a disruption of the CMTs organised in parallel in the cells to randomly‐oriented CMTs (Simmonds 1991). During regeneration of the protoplast, the CMTs reorientate into parallel arrays prior to or during cell wall synthesis (Lloyd et al 1980; Simmonds and Setterfield 1986; Hasezawa et al 1988; Skagen 1998).…”

Section: Introductionmentioning

confidence: 99%

Simulated weightlessness and hyper‐g results in opposite effects on the regeneration of the cortical microtubule array in protoplasts from Brassica napus hypocotyls

Skagen

Iversen

1999

Physiologia Plantarum

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Enzymatic digestion of the cell wall of Brassica napus hypocotyls gave a heterogeneous suspension of protoplasts with the cortical microtubules (CMTs) randomly organised or CMTs organised in parallel. The effect of variable g-influences has been tested on CMT organisation. In contrast to the 1 g-protoplasts, which reorganised the CMTs into parallel arrays during the 96 h test period, the frequency of randomly-oriented CMTs in the protoplasts exposed to simulated weightlessness (0 g) on a 2-D clinostat increased significantly during the same period. The opposite effect was obtained when the protoplasts were exposed to hyper-g (7 or 10 g), where the reorganisation of the CMTs into parallel arrays was accelerated compared to the 1 and 0 g-protoplasts. These results indicate that a unidirectional gravity force is a necessity for the reorganisation of CMTs in protoplasts to parallel arrays and that CMTs act as responding elements that are able to sense different levels of gravity. Besides the inability of the protoplasts to reorganise the CMTs into parallel arrays, the quantity of CMTs in the individual protoplast decreased during 4 days of simulated weightlessness, both compared to the CMTs quantity in the protoplasts immediately after isolation and compared to the 1 g- and hyper-g-protoplasts after 24 and 48 h of g-exposure. The size of the protoplasts was also affected by the g-exposure. Protoplasts exposed to simulated 0 g increased significantly after 24 and 48 h, whereas the 1 g- and 10 g-protoplasts maintained the same size during the 48 h test period.

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“…The result presented here is also suggestive that for a good understanding of the relation ship between cytoskeletal function and cytomorphogenesis, the aging related MT configurations should be distinguished and not be intermingled with that in embryogenic/regenerative cell. On the other hand, compared with protoplast, cell suspension averts cell wall removal and regeneration, both of which were found affecting the configurations of cortical MTs (Simmonds 1991). Therefore, modifications might be made before the conclusion from this study being applied to protoplast.…”

Section: Discussionmentioning

confidence: 99%

Cell-Aging Related Configurations of Cortical Microtubules in Carrot (Daucus carota L.) Suspension Cells.

et al. 1994

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Microtubules in cultured plant protoplasts

Cited by 10 publications

References 44 publications

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Isolation of sub-diploid microprotoplasts for partial genome transfer in plants: Enhancement of micronucleation and enrichment of microprotoplasts with one or a few chromosomes

Simulated weightlessness and hyper‐g results in opposite effects on the regeneration of the cortical microtubule array in protoplasts from Brassica napus hypocotyls

Cell-Aging Related Configurations of Cortical Microtubules in Carrot (Daucus carota L.) Suspension Cells.

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