A Simple Procedure for Mesophyll Protoplast Culture and Plant Regeneration in Brassica oleracea L. and Brassica napus L.

An improved protocol for Brassica protoplast culture and plant regeneration was developed. Isolated protoplasts from four-weeks-old in vitro shoot tip culture of Brassica oleracea var. botrytis cv. Siria F1 and Brassica napus doubled haploid of breeding line OP-1 were cultured at a density of 9.8–11.2 × 104 protoplasts/ml in darkness at 25°C in a modified medium containing 2% glucose, 0.25 mg/l 2,4-D, 1 mg/l BAP and 1 mg/l NAA. The first divisions of protoplasts were observed on the third day of culture in B. oleracea and on the fourth day in B. napus. The protoplast cultures were diluted with low osmotic medium on 7th and 11th day. The frequency of dividing cells was about 80% in B. oleracea and 50% in B. napus. After one month, the microcalli of approximately 0.5–1 mm in size were transferred into an induction medium with various combinations of growth regulators. Minimum duration of enzyme treatment time and extended dark period in the initial phase of culture increased the survival rate of protoplasts. Organogenesis started when the calli enlarged in size on an induction medium (1 mg/l NAA, 0.02 mg/l GA3, 1 mg/l 2iP) with 2% sucrose and 0.8% agar. Regeneration frequency of calli was found to be 69–75% in B. oleracea and 2–3% in B. napus. Well-developed shoots were transferred for rooting to a half-strength MS medium without growth regulators. More than 100 B. oleracea regenerants were transferred into soil, and they produced normal heads and set seeds. This very simple procedure is efficient and suitable mainly for B. oleracea var. botrytis and represents a background for fusion experiments.

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Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Frontiers in Plant Science ◽

10.3389/fpls.2021.680859 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xueyuan Li ◽

Sjur Sandgrind ◽

Oliver Moss ◽

Rui Guan ◽

Emelie Ivarson ◽

...

Keyword(s):

Brassica Napus ◽

Shoot Regeneration ◽

Gene Editing ◽

Protoplast Culture ◽

Callus Formation ◽

Protoplast Regeneration ◽

Induction Medium ◽

Shoot Induction ◽

Brassica Napus L ◽

High Concentrations

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

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Protoplast culture and plant regeneration of different agronomically important Brassica species and varieties

Agricultural and Food Science ◽

10.23986/afsci.72416 ◽

1991 ◽

Vol 63 (5) ◽

pp. 371-378

Author(s):

János Pauk ◽

Sándor Fekete ◽

Juha Vilkki ◽

Seppo Pulli

Keyword(s):

Brassica Napus ◽

Plant Regeneration ◽

Silver Nitrate ◽

Protoplast Culture ◽

Gradient Centrifugation ◽

Brassica Species ◽

Regeneration Medium ◽

Physical Damage ◽

Brassica Napus L ◽

Culture Techniques

Protoplast cultures were prepared from 6-day-old hypocotyls of six spring, seven winter cultivars of Brassica napus L. and one line of Brassica campestris L. The molarity of enzyme solution was raised to 0,714 M mannitol resulting in well manipulable, cytoplasm dense protoplasts. In the protoplast purification procedure density gradient centrifugation was used to minimize physical damage of protoplasts. Three different protoplast culture systems —(1) liquid, (2) 2nd day embedded, (3) directly embedded in low melting agarose were compared. The two different protoplast embedding techniques resulted in the same efficiency of cell division as the liquid culture method and over this fact the colony browning was avoided. Using protoplast agarose-embedding and culture techniques, healthy calli were obtained for plant regeneration experiments. Incorporation of silver nitrate into the regeneration medium improved the efficiency of plant regeneration in responsive genotypes and the regeneration was induced in three nonresponsive (without silver nitrate) genotypes, too. The supplement of silver nitrate in regeneration medium was especially advantageous in plant regeneration of B. campestris. Out of fourteen commercial cultivars of Brassica napus and B. campestris, there is only one recalcitrant genotype in obtaining plantlets from protoplast-derived calli.

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Cotyledon-derived diploid and haploid protoplast culture and diploid plant regeneration in Brassica napus cv. ' Topas '

Canadian Journal of Botany ◽

10.1139/b98-022 ◽

1998 ◽

Vol 76 (3) ◽

pp. 530-541

Author(s):

M Sun ◽

H Kieft ◽

AAM van Lammeren

Keyword(s):

Cell Division ◽

Brassica Napus ◽

Plant Regeneration ◽

Protoplast Culture ◽

Shoot Formation ◽

Protoplast Isolation ◽

Brassica Napus L ◽

Successful Isolation ◽

Young Leaves ◽

Haploid Embryos

The present paper describes a simple and reliable protocol for the successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L. cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32°C instead of 25°C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.Key words: cotyledon protoplast culture, haploid culture, plant regeneration.

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