Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six rapid-cycling brassica species. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Studies of protoplast culture types and plant regeneration from callusderived protoplasts of Asparagus officinalis L. cv. Lucullus 234

Plating efficiency and colony formation of callus-derived protoplasts of Asparagus officinalis L. cv. Lucullus 234 differed significantly with different protoplast culture media and types of culture. Osmotic conditions and hormone concentrations of liquid media produced the greatest influence on plating efficiency and colony formation in bead culture. Protoplasts grew best in bead culture with a solid modified Kao & Michayluk protoplast culture medium (KM) supplemented with 0.5 mg l^–1 -naphthaleneacetic acid (NAA), 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg l^–1 kinetin, and 0.6% agarose (KM6) and a liquid modified KM medium differing from KM6 medium in sugar content, having 0.18 M sucrose and 0.18 M mannitol (A8). An average plating efficiency of 19.1% and colony formation of 15.5% was obtained one week after isolation in bead culture with the KM6 and A8 media. The highest average shoot regeneration of 92.3% was obtained with a Murashige & Skoog medium (MS) containing 0.125 mg l^–1 NAA, 0.125 mg l^–1 2,4-D, 0.25 mg l^–1 6-benzylaminopurine (6-BAP) and 3% sucrose. Plants have been regenerated and transferred to the greenhouse.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea 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.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Plant regeneration from callus and protoplast cultures of Lotus pedunculatus Cav.

Plant regeneration from leaf- and cotyledon-derived calli and from protoplast-derived tissue has been obtained in Lotus pedunculatus. Callus induction was achieved with 2,4-D and plant regeneration required the following two media sequences: bud formation was stimulated by IAA and BA and shoot growth by kinetin. Root formation occurred in the presence of IAA. Cotyledon protoplasts showed a low plating efficiency and plant regeneration was achieved via an intervening callus phase.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2iP N^6--2-isopentenyl-adenine - NAA -naphthaleneacetic acid     

Plant regeneration from patchouli protoplasts encapsulated in alginate beads

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×10³ protoplasts per 25 l bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10^-6 M NAA and 10^-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - f. wt. fresh weight - GC gas chromatography - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthaleneacetic acid     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

Plant regeneration from stem cortex expiant and protoplast cultures of Brassica juncea (mustard)

Summary Plant regeneration from stem cortex explants of 13 genotypes of Brassica juncea was assessed. Regeneration was strongly affected by genotype, as up to 50.6 shoots were produced per 100 calli of the most responsive line (Blaze), whereas no shoots were obtained from less responsive lines (Zeml, Vniimk351). Blaze was chosen for B. juncea stem cortex protoplast isolation. After one week of culture, 11–14% of the cells had divided, and about 0.002% produced 1–2 mm colonies within 6 weeks. Up to 7% of these colonies gave rise to shoots upon transfer to plant regeneration medium.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BAP 6-benzylaminopurine - FDA fluorescein diacetate - IAA indole acetic acid - IBA indole butyric acid - 2ip 2-isopentenyl-adenine - NAA 1-naphthaleneacetic acid - TD thidiazuron     

Callus initiation and regeneration capacities in Brassica species

In order to know the genetic differences of de- and redifferentiation capacities, seven Brassica species (B. campestris, B. nigra, B. oleracea, B. hirta, B. carinata, B. juncea and B. napus) were cultured in vitro, and their response to the medium supplemented with various combinations of auxin and cytokinin hormones was compared. Important factors for callus initiation were shown to be auxin and species. Calli were induced most frequently in Murashige and Skoog (MS) medium with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), whereas -naphthaleneacetic acid (NAA) induced preferentially roots at a concentration of 2 to 5 mg/l. Callus-, root- and shoot-forming capacities from explanted cotyledon tissues were significantly different among the seven Brassica species. Calli derived from cotyledons and hypocotyls of seven species were transferred to MS media with 20g/l sucrose, 0 to 0.1 mg/l NAA and 0 to 4 mg/l kinetin to compare their regeneration capacities. Among the seven species tested, B. napus (2n=4x=38, genome AACC) had the highest shoot forming capacity (20.0%). Other amphiploid species, B. carinata (2n=4x=34, BBCC) and B. juncea (2n=4x=36, AABB) formed shoots at low frequencies (2.8% and 1.2%, respectively). A diploid species, B. oleracea (2n=2x 18, CC) also showed high shoot formation (10.2% on average). This result suggests that the gene(s) controlling shoot formation may be localized in the C genome. Differences were also found among varieties and cultivars within a species. One of the cultivars, Siberian kale (B. oleracea var. acephala) gave about 50% shoot formation. This kale was shown cytologically to be an autotetraploid (2n=4x=36, CCCC), thus probably possessing a double set of the shoot-forming gene(s).     

Callus induction and plant regeneration from gladiolus

A method for the initiation of callus capable of plant regeneration from in vivo grown cormels of gladiolus (Gladiolus x grandiflorus Hort.) is described. Sliced cormels of the large-flowering hybrid, Peter Pears were cultured in vitro on a modified Murashige and Skoog medium, supplemented with various auxins. Yellow callus, which was either friable or compact, could be induced on all media tested. Callus induced on media with naphthaleneacetic acid failed to proliferate. Callus induced on media with 9 mM 2,4-dichlorophenoxyacetic acid showed the best growth. Addition of micro-elements and vitamins increased the induction and growth of callus capable of plant regeneration. Explants taken from the middle part of the cormels had the highest competence for callus initiation. Callus was induced on several gladiolus hybrids and the South African species G. garnierii Klatt. Callus induction was genotype dependent and among the cultivars tested, Peter Pears and White Prosperity were superior with respect to callus production on the media with either 2,4-dichlorophenoxyacetic acid or picloram. Plants were regenerated from yellow compact callus of all genotypes on media containing zeatin and benzyladenine in various concentrations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog basal salt and vitamins (1962) - CI callus induction medium - NAA -naphthaleneacetic acid - BA 6-benzyladenine - picloram 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid - zeatin 6-[4-hydroxy-3-methylbut-2-enylamino]purine     

Variation amongst Brassica juncea cultivars for regeneration from hypocotyl explants and optimization of conditions for Agrobacterium-mediated genetic transformation

Summary Twelve cultivars of Brassica juncea grown in different agroclimatic regions of the world were tested for their ability to regenerate in vitro from hypocotyl explants and, accordingly, were divided into three groups. One group of cultivars regenerated on MS medium supplemented with 2,4-D, BAP and with NAA, BAP combinations; another group regenerated only on MS with 2,4-D, BAP; and the third group showed very low regeneration on both of these combinations. Inclusion of silver nitrate in the medium was essential for high frequency of regeneration. In general, Indian cultivars were more responsive than the cultivars of CIS and Australian origin. Using the media optimal for regeneration and an Agrobacterium-based binary vector carrying hpt and gus-intron genes, conditions for genetic transformation of B. juncea hypocotyl explants were optimized. Transformation frequencies, identified by GUS staining at the initial stages of growth, were lower on MS medium with 2,4-D, BAP than on MS with NAA, BAP. Plants resistant to 20 g/ml hygromycin were regenerated at a frequency of 11–36% from hypocotyl explants and were shown to be transformed by Southern blotting, GUS staining and progeny analysis.     

Protoplast culture and somaclonal variability of species of series Juglandifolia

Mesophyll protoplasts of species of series Juglandifolia (Solanum rickii, S. lycopersicoides, S. ochranthum and S. juglandifolium) were isolated and cultured in liquid nutrient media TM-2 or KM8P. The cell colonies formed were transferred onto agar-solidified media TM-3 or GM, and 10 to 15 days later onto TM-4, PRM, MS3ZG, KK or C regeneration media. Formation of the shoots for S. rickii and S. lycopersicoides was observed after 30 to 35 days on regeneration medium. The regenerated shoots were rooted on hormone-free MS medium. Morphological and cytogenetic analyses have shown that somaclonal variants might arise in the course of plant regeneration from protoplasts of these species.Abbreviations BAP 6-benzylaminopurine - 2, 4-D 2, 4 dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - B5 Gamborg medium - TRIS tris(hydroxymethyl)aminomethane     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.)

Embryogenic callus initiated from basal segments of micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable of yielding large numbers of protoplasts which underwent sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast-derived callus and are currently being grown to maturity. This is the first report of plant regeneration from protoplasts of a wild species of Oryza.Abbreviations BAP 6-benzyalamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2[N-morpholino] ethanesulphonic acid - NAA -naphthalene acetic acid - PAR photosynthetically active radiation - SCV settled-cell volume     

Protoplast culture and regeneration from Brassica oleracea ‘rapid cycling’ and other varieties

The results are reported for the first time on successful plant regeneration from mesophyll-derived protoplasts of rapid cycling B. oleracea. Comparative data were also presented on plant regeneration from mesophyll-derived protoplasts of two other varieties namely var. botrytis and var. gemmifera. It was found that a modified Pelletier (Pelletier et al. 1983) protocol is highly beneficial for protoplast culture and plant regeneration from mesophyll-derived protoplasts. The plating efficiency of B. oleracea rapid cycling protoplasts was, in the best combination of isolation method, culture technique and culture media 4.5%±0.4% and the plant regeneration frequency approximately 15%. Plant regeneration was further improved by transferring the calli from the D medium of Pelletier to a callus growth medium (MS11) and subsequently to the K₃ regeneration medium of Glimelius (Glimelius 1984). Various factors influencing plating efficiency and plant regeneration from mesophyll protoplasts of B. oleracea are discussed.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - IBA 3-indole butyric acid - NAA 1-naphtylacetic acid - 2iP N⁶-(2-isopentyl)adenine