Protoplast culture and regeneration from Brassica oleracea 'rapid cycling'and other varieties (original) (raw)
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Agricultural and Food Science
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 regenerati...
Protoplast culture and plant regeneration from Brassica carinata Braun
1987
Protoplasts isolated from hvDocotvls of three-day-old seedlinqs of Brassica carinBta (Rraun) cv R-212~ were cultured in a modified Nitsch and Nitsch l i n u i d medium containinQ 13% sucrose, 0.4% F i c o l l , 0.25 mq/l BA, 0.5 mq/l NAA and Q.5 mg/l 2,4-D. The densit~ of medium caused the DrotoDlasts and the developinn microcalli to f l o a t on the surface of the l i n u i d medium whereas a l l debris and lysed c e l l s sank to the bottom of the culture plate. After 4-6 weeks developing microcalli were approximately O. 5 mm in diameter and were transferred onto MS medium containinq 3% sucrose, ~.4% agarose, 200 m~/l casein h~drolysate~ 5 mq/l ~A and ~.5 mq/l NAA, pH 5.7. Approximately 20% of the c a l l i transferred to this medium produced plantlets.
Plant Cell, Tissue and Organ Culture, 1990
Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5 x 104 ml ~ in Kao's medium supplemented with 1.0 mg 1 ~ 2,4-D, 0.1 mg 1l NAA and 0.5mgl ~ zeatin riboside. After three days of culture in darkness at 25 + I°C, cultures were transferred to light (70 #E m-2 s J) in a 16/8 h liga ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl-~ 2,4-dichlorophenoxyacetic acid and 1.0mgl-I benzyladenine. On the fifteenth day, microcalli were plated on K 3 medium gelled with 0.5% agarose (Type 1, low EEO, Sigma). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8-10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.
Plant regeneration from cotyledon protoplasts of Brassica carinata
Plant Cell Tissue and Organ Culture, 1990
Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 104 ml-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl-1 BAP, 0.035 mgl-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.
Plant Cell, Tissue and Organ Culture, 1995
Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oteracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2-4 h following isolation in all cultivars. 30-70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60-90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4-5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24-120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1-9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.
Plant regeneration from root protoplasts of Brassica
Plant Science Letters, 1982
Protoplasts isolated enzymatically from roots of Brassica alba (White Mustard), B. campestris {Turnip), B. napus (Rape) and B. oleracea (Cabbage), divided to form callus. Plant regeneration was obtained from protoplast derived tissues of B. napus and B. oleracea, but only rhizogenesis was observed with B. campestris. Tissues of B. alba remained undifferentiated. The suitability of root protoplasts for genetic manipulations in the genus Brassica is discussed. *On leave from the Institute of Plant Physiology, Academia Sinica, Shanghai, China. Abbreviations: BAP, 6-benzylaminopurine; CPA, p-chlorophenoxyacetic acid; IAA, 3-indoleacetic acid; NAA, a-naphthaleneacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid. 0304--4211/82/0000--0000/$02.75
Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra
Plant Cell, Tissue and Organ Culture, 1993
Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-D (1 mg 1 1), NAA (0.1 mg1-1) and zeatin riboside (0.5 mg 1-1). After initial incubation for 3 days in dark at 25 _+ I°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-0 (0.1 mg 1-1) and BAP (1 mg 1 1). About 62% of the cells divided at least once and 46% of them reached 8-16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1 1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1 1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.
A simple culture method for Brassica hypototyl protoplasts
Plant Cell Reports, 1985
Hypoco~,l protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the d a r k-~ in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, l mg/l NAA and 0.5 mg/l 2-4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4-6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3-4 weeks.
Plant regeneration from mesophyll protoplasts of Rorippa indica (L.) Hiern, a wild crucifer
Curr Sci, 2003
In Rorippa indica, a wild crucifer, high-frequency plant regeneration through organogenesis (~38%) was achieved from in vitro-grown leaf explants. When the same-aged in vitro-grown leaves were utilized for protoplast isolation, ~ 4 × × 10 6 protoplasts g -1 leaf tissue were obtained after digestion with an enzyme solution containing cellulase and macerozyme. A maximum of ~ 66% first division, ~ 53% second division and ~35% plating efficiency were obtained when protoplasts were plated at 5 × × 10 4 protoplasts ml -1 plating density. All the 528 protoplast-derived calli tested for regeneration exhibited shoot organogenesis. Regenerated shoots were transferred to soil after rooting and hardening. Chromosome analysis of microspore mother cells from pot-grown protoplast regenerated plants showed that all were diploid (2n = 32).