Strategies for promoting division of cultured plant protoplasts: Beneficial effects of oxygenated perfluorocarbon

Summary Assessments have been made of physical and chemical options, alone and in combination, for gaseous manipulation of cassava (Manihot esculenta Crantz.) leaf protoplast cultures. Protoplasts were cultured for 25 d in liquid medium at an initial plating density of 4 × 10⁵ ml^–1 overlying (1) agar-solidified medium (control), (2) agar medium under an oxygen-enriched (10 mbar, 1 min) atmosphere, (3) agar medium supplemented with perfluorodecalin (Flutec ^® PP5), or (4) agar medium supplemented with oxygenated (10 mbar, 15 min) perfluorodecalin. Similar experimental treatments were also set up, with glass rods embedded in the agar medium. The mean initial plating efficiency (IPE) of protoplasts following culture with oxygenated perfluorodecalin without glass rods (6.7 ± 0.6%; n = 3) was over 2-fold greater (P < 0.05) than that of control cultures (2.6 ± 0.2%; n = 3). The mean IPE of protoplasts cultured with oxygenated perfluorodecalin in the presence of glass rods (5.8 ± 0.2%; n = 3) was also over 2-fold greater (P < 0.05) than controls. There was no significant difference between the IPE of protoplasts cultured under an increased oxygen atmosphere or with oxygenated perfluorodecalin, irrespective of the presence of glass rods.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

A reproducible procedure for plant regeneration from seedling hypocotyl protoplasts of Vigna sublobata L.

Viable protoplasts of Vigna sublobata L. were isolated enzymatically from hypocotyls of axenic seedlings. Protoplast yields were dependent upon seedling age, with maximum yields (2.25 ± 0.35 × 10⁶ g fwt^–1) from seedlings aged 6 d. Protoplasts regenerated cell walls and underwent sustained divisions when cultured in either agarose-solidified or liquid K8P medium. The plating density affected the division frequency and plating efficiency; the division frequency (68 ±0 6.0%) was maximum at 4.0 × 10⁴ ml^–1 while plating efficiency was maximum (1.3 ± 0.1%) at 5.0 × 10⁴ ml^–1. Dividing protoplasts developed into microcalli, which produced glossy green compact nodular calli on transfer to 8.0 gl^–1 w/v agar-solidified medium containing MS salts, B5 organic components, 30 g l^–1 sucrose, NAA (0.2–0.5 mg l^–1), zeatin riboside (0.5–2.0 mg l^–1) and GA₃ (0.5–1.0 mg l^–1). These calli, after sub-culture on the same medium, produced shoot buds which underwent elongation following transfer of tissues to 6.0 g l^–1 agar-solidified B5 medium containing 30g l^–1 sucrose, IBA (0.01 mg l^–1) and BAP (1.0 mg l^–1). Elongated shoots developed roots after transfer to 8.0g l^–1 agar-solidified, hormone-free MS medium with 30 g l^–1 sucrose.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyladenine or benzylaminopurine - B5 medium after Gamborg et al (1968) - 2,4-D 2,4-dichlorophenoxyacetic acid - GA³ gibberellic acid - 2,i-P 6-(--dimethylallylamino) purine - MS medium after Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

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 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°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-d (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.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

Plant regeneration from leaf mesophyll protoplasts of the tropical woody plant,passionfruit (Passiflora edulis fv flavicarpa Degener.): the importance of the antibiotic cefotaxime in the culture medium

Enzymatic digestion of newly expanded leaves of glasshouse-grown seedlings of passionfruit released protoplasts which exhibited highest division frequency (38.6%) when plated at a density of 1.5×10⁵ ppts ml^–1 in agarose-solidified droplets of KM8P medium containing the antibiotic cefotaxime (250 g ml^–1). Cefotaxime was essential for sustained cell division. Protoplast-derived calli were cultured on agarsolidified MS medium with 5.0 mg H NAA, 0.25 mg l^–1 BAP and additional vitamins. These calli regenerated shoots on transfer to MS medium with 1.0 mg l^–1 BAP. Regenerated shoots were rooted in half-strength MS medium with 3.0 mg l^–1 IBA and 0.5 mg l^–1 NAA (7 d), followed by sub-culture to MS medium lacking growth regulators. The ability to regenerate plants from protoplasts of passionfruit is discussed in relation to the application of somatic cell techniques for the genetic improvement of this economically important tropical woody plant.Abbreviations B5 medium after Gamborg et al. (1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - d day - FDA fluorescein diacetate - FPE final plating efficiency - f. wt fresh weight - h hour - 1BA 4-indole-3yl-butyric acid - IPE initial plating efficiency - MES 2-N-morpholinoethane sulphonic acid - MS medium after Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (M. Wt. 10,000) - rpm rotations per minute     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

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     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×10⁴ ml^-1 in Kao's medium supplemented with 1.0 mgl^-12,4-D, 0.1 mgl^-1 NAA and 0.5 mgl^-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 Em^-2s^-1) in a 16/8 h ligø ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl^-1 benzyladenine. On the fifteenth day, microcalli were plated on K₃ 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.     

Callus formation from protoplasts of Artemisia sphaerocephala Krasch and some factors influencing protoplast division

Round wormwood (Artemisia sphaerocephala Krasch) seeds were germinated on Murashige & Skoog (1962) medium without plant growth regulators. The hypocotyls of seedlings were sliced and cultured on M1 medium with 2,4-dichlorophenoxyacetic acid (9.05 M) to induce callus. The induced calluses were subcultured on the same medium. Ten day old calluses were used to isolate protoplasts in an enzyme solution with 0.65 M mannitol. Protoplast yield strongly depended upon the state of callus cultures. Certain amount of hemicellulase could improve protoplast isolation. Purified protoplasts were cultured in modified Kao & Michayluk (1975) medium with 0.60 M mannitol as osmoticum, suggesting that protoplasts of A. sphaerocephala need a high initial osmolarity. Protoplasts generally divided evenly and the percentage of first division could reach 10%. Kinetin exhibited a positive effect on initial cell division. Furthermore, we studied the effect of protoplast density and vitamin C on sustained growth of protoplasts. After forty days, 1 mm calluses in diameter formed.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - KM8P Kao & Michayluk (1975) protoplast medium - MS Murashige & Skoog (1962) medium - MES-2 (N-morpholino)ethanesulfonic acid     

Regeneration of protoplast-derived green plants of Kentucky blue grass (Poa pratensis L.)

Summary Green plants were repeatedly regenerated from suspension-derived protoplasts of Kentucky blue grass (Poa pratensis L.) cv. Geronimo. One suspension was capable of donating competent protoplasts during long-term culture i. e. 10–16 months after its establishment. The plating efficiency of the protoplasts from three different suspension lines varied from 0.004% in the lowest up to 1.5% in the highest responding line, using agarose-bedding in nutrition medium devoid of nurse or feeder cultures. Green plants germinated from polyembryos, which developed from 0.4–2.7% of the protoplast-derived microcolonies. A total of 127 plants were successfully transferred to soil.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - PE plating efficiency     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

Plant regeneration from cotyledon protoplasts of Brassica carinata

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.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R₂ medium modified by the addition of 560 mg l^–1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l^–1) and -naphthaleneacetic acid (0.5 mg 1^–1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GPFs growth promoting factors - NAA -naphthaleneacetic acid On leave from Department of Genetics, Haryana Agricultural University, Hisar, IndiaOn leave from Biotechnology Centre, Punjab Agricultural University, Ludhiana, India     

Somatic embryogenesis from mesophyll protoplasts of Trigonella corniculata (leguminosae)

Mesophyll protoplasts isolated enzymatically from Trigonella corniculata divided to form callus, with a plating efficiency of 49% in Kao (1977) medium. Protoplast-derived tissues formed somatic embryoids at high frequency on MS medium with 2.0 mg L^–7 NAA and 0.5 mg L^–7 BAP. Embryoids developed into plants on MS medium lacking hormones.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - MES 2-(N-morpholine) ethanesulphonic acid - NAA -naphthaleneacetic acid On leave from Genetics Institute, Academia Sinica, Beijing, China     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate