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

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.

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

2011 ◽  
Vol 42 (No. 3) ◽  
pp. 103-110 ◽  
Author(s):  
N.D. Kaur ◽  
M. Vyvadilová ◽  
M. Klíma ◽  
M. Bechyně
Keyword(s):  
Brassica Napus ◽  
Plant Regeneration ◽  
Brassica Oleracea ◽  
Growth Regulators ◽  
Protoplast Culture ◽  
Treatment Time ◽  
Simple Procedure ◽  
Enzyme Treatment ◽  
Induction Medium ◽  
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&ndash;11.2 &times; 10<sup>4 </sup>protoplasts/ml in darkness at 25&deg;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 7<sup>th</sup> and 11<sup>th</sup> 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&ndash;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 GA<sub>3</sub>, 1 mg/l 2iP) with 2% sucrose and 0.8% agar. Regeneration frequency of calli was found to be 69&ndash;75% in B. oleracea and 2&ndash;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. &nbsp;

scholarly journals  Effect of 2,4-D as a novel inducer of embryogenesis in microspores of Brassica napus L.

2011 ◽  
Vol 47 (No. 3) ◽  
pp. 114-122 ◽  
Author(s):  
S.H. Ardebili ◽  
M.E. Shariatpanahi ◽  
R. Amiri ◽  
M. Emamifar ◽  
M. Oroojloo ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Heat Shock ◽  
Microspore Embryogenesis ◽  
Control Treatment ◽  
Dichlorophenoxyacetic Acid ◽  
Brassica Napus L ◽  
High Concentrations ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
Short Time ◽  
First Time

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) applied at high concentrations for a short time was investigated as a novel stress for induction of microspore embryogenesis for the first time. Brassica napus L. cvs. Topas and Hyola 420 were used as model plants for testing this hypothesis. Microspores were subjected to 2,4-D at 4 concentrations (15, 25, 35 and 45 mg/l) for 15&ndash;45 min while the classical heat shock was used as the control treatment. Among 2,4-D treatments in Topas, the highest yield of torpedo-stage embryos was achieved at 15 mg/l 2,4-D for 30 min while more normal plantlets were produced when 2,4-D (25 mg/l for 30&nbsp;min) was applied to the microspores. In Hyola 420 the results showed a lower number of embryos and normal plantlets at all concentrations of 2,4-D. Although Hyola 420 was almost equally embryogenic as Topas after heat shock treatment, large differences between genotypes (concerning embryogenic response) occurred after 2,4-D treatment. However, the mean number of embryos and regenerants was higher in heat shock as compared to 2,4-D induced stress (one magnitude of order). According to the results obtained, 2,4-D can be introduced as a new stress for induction of embryogenesis in microspores similarly like in zygotic and somatic cells. This novel stress is very important for plant species whose microspores are extremely sensitive to classical stresses.

scholarly journals Silicon Promotes Adventitious Shoot Regeneration and Enhances Salinity Tolerance ofAjuga multifloraBunge by Altering Activity of Antioxidant Enzyme

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Iyyakkannu Sivanesan ◽  
Byoung Ryong Jeong
Keyword(s):  
Salinity Tolerance ◽  
Shoot Regeneration ◽  
Microscopic Analysis ◽  
Adventitious Shoot ◽  
Induction Medium ◽  
Shoot Induction ◽  
Electron Microscopic ◽  
Sod Activity ◽  
Scanning Electron Microscopic Analysis ◽  
Shoot Induction Medium

We investigated the effect of Si concentration on shoot regeneration and salinity tolerance ofAjuga multiflora. Addition of Si to the shoot induction medium significantly increased the frequency of shoot induction. The average number of shoots regenerated per explant decreased on the medium containing NaCl alone, while there was less decrease when the shoot induction medium was supplemented with both NaCl and Si. The shoot induction percentage increased linearly with increasing concentration of Si in the NaCl containing medium. Addition of Si to the shoot induction medium significantly increased SOD, POD, APX, and CAT activity in regenerated shoot buds as compared with the control. The inclusion of Si to the NaCl containing medium significantly increased the SOD activity in leaves and roots, while it decreased POD, APX, and CAT activity in both organs. Scanning electron microscopic analysis showed that there are no distinct differences in the structure of stomata between the control and Si-treated plants. However, NaCl treatment significantly affected the structure and number of stomata as compared to the control. Wavelength dispersive X-ray analysis confirmed the high Si deposition in trichomes of plants grown in the Si containing medium but not in plants grown in the medium without Si.

scholarly journals Chelate-assisted phytoextraction: Effect of EDTA and EDDS on copper uptake by Brassica napus L.

2010 ◽  
Vol 75 (9) ◽  
pp. 1279-1289 ◽  
Author(s):  
Tijana Zeremski-Skoric ◽  
Petar Sekulic ◽  
Ivana Maksimovic ◽  
Srdjan Seremesic ◽  
Jordana Ninkov ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Contaminated Soil ◽  
Maximum Amount ◽  
Copper Uptake ◽  
High Biomass ◽  
Brassica Napus L ◽  
Growth Depression ◽  
High Concentrations ◽  
Cu Uptake ◽  
Assisted Phytoextraction

Chelate-assisted phytoextraction has been proposed as an effective approach to removing heavy metals from contaminated soil through use of high biomass plants. The aim of the present study was to compare the efficiency of the two chelators: EDTA and biodegradable EDDS in enhancing Cu uptake and translocation by Brassica napus L. grown on moderately contaminated soil and treated with increasing concentrations of EDTA or EDDS. Increasing amounts of EDDS caused a serious growth depression of Brassica napus and an increase in shoot metal concentrations. Growth depression limited the actual amount of phytoextracted Cu at high concentrations of EDDS. The maximum amount of extracted Cu was achieved by the application of 8.0 and 4.0+4.0 mmol/kg EDDS. The shoot Cu concentrations after EDTA application were much lower than with EDDS at the same doses. According to this experiment, EDTA does not appear to be an efficient amendment if Cu phytoextraction with Brassica napus is considered but EDDS is.

Callus formation and plant regeneration from mesophyll protoplasts of rape plants (Brassica napus L. cv. Zephyr)

1974 ◽  
Vol 3 (4) ◽  
pp. 265-271 ◽  
Author(s):  
K.K. Kartha ◽  
M.R. Michayluk ◽  
K.N. Kao ◽  
O.L. Gamborg ◽  
F. Constabel
Keyword(s):  
Brassica Napus ◽  
Plant Regeneration ◽  
Callus Formation ◽  
Brassica Napus L ◽  
Mesophyll Protoplasts

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

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.

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

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.

Sign in / Sign up

Export Citation Format

Share Document