A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

Millam, S.; Burns, Alan T. H.; Hocking, T. J.

doi:10.1007/bf00044264

Cited by 5 publications

(2 citation statements)

References 18 publications

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“…Different mannitol concentrations affected the yield and viability of protoplasts, while different organs of the same plant have different adaptabilities to mannitol [26,27,28]. In this study, the appropriate mannitol concentration for hypocotyls was lower than that for mesophyll [13].…”

Section: Discussionmentioning

confidence: 93%

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

Sun

Qiao

Zheng

et al. 2019

Plants

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In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

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Section: Discussionmentioning

confidence: 93%

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

Sun

Qiao

Zheng

et al. 2019

Plants

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“…In rice, protoplasts isolated from cell cultures were densely cytoplasmic immediately after isolation (Tang et al 2001). In mesophyll protoplasts of rapeseed, sub-populations were characterised as vacuolated (containing few or no chloroplasts and a large vacuole) or chloroplastic (containing chloroplasts and no large vacuole) (Millam et al 1991). As far as we know, this is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.…”

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confidence: 98%

Efficient plant regeneration from protoplasts of Kalanchoe blossfeldiana via organogenesis

Castelblanque

García‐Sogo

Pineda

et al. 2009

Plant Cell Tiss Organ Cult

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A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 9 10 5 protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial density of 1 9 10 5 protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 lM 2,4-dichlorophenoxy acetic acid (2,4-D), 5.4 lM 1-naphthaleneacetic acid (NAA) and 2.2 lM 6-benzyladenine (BA). Cell wall regeneration was observed within 4 days of culture and cell division began after 5-7 days. When cultured in a liquid medium with 5.4 lM NAA and 8.9 lM BA, protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 lM indole-3-acetic acid (IAA) and successfully established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions found gave a plant regeneration efficiency of 6.4 plants per 1 9 10 5 protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species.

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Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

1993

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We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10(7) protoplasts.ml(-1) packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10(6) protoplasts.ml(-1)) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20-40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.

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A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

Cited by 5 publications

References 18 publications

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

Efficient plant regeneration from protoplasts of Kalanchoe blossfeldiana via organogenesis

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

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