Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

Plant regeneration from protoplasts of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via somatic embryogenesis

A protocol for plant regeneration from protoplasts of Musa acuminata cv. Mas (AA) via somatic embryogenesis was developed. Viable protoplasts were isolated from embryogenic cell suspensions at a yield of 1.2 × 10⁷ protoplasts/ml packed cell volume (PCV). Liquid and feeder layer culture systems with medium-A and medium-B were used for protoplast culture. In liquid culture system, medium-B was more efficient for inducing cell division (17.5% at 14 days) and colony formation (6.7% at 28 days) than medium-A. However, all protoplast-derived cell colonies (PDCC) obtained from liquid culture system could not develop further. In feeder layer culture system, there was no significant difference between medium-A and medium-B on cell division and colony formation of the cultured protoplasts, and the cell division frequency at 14 days and colony formation frequency at 28 days were 24.5% and 11.2%, respectively, in medium-B. Comparative study on the effects of BAP (2.2 μM, 4.4 μM, 8.8 μM), zeatin (0.4 μM, 0.8 μM, 1.2 μM) and TDZ (0.2 μM, 0.4 μM, 0.6 μM) on embryo formation of PDCC from feeder-layer culture indicated that TDZ was best. TDZ at 0.4 μM induced 7906 mature embryos per ml PCV PDCC, which was 4-fold the frequency as with BAP at 4.4 μM, 7.5-fold as with zeatin at 0.8 μM and 150-fold as control medium (no mentioned cytokinins) after 45 days on M3 medium. About 44% of the mature embryos were converted into plantlets with poor root system after subculture on M4 medium. Root further development of regenerated plantlets was promoted by addition of activated charcoal (AC) to MS basal medium.     

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Plant regeneration from leaf protoplasts of evening primrose (Oenothera hookeri)

A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture in B5 medium containing 3 mg l^–1 α-naphthaleneacetic acid (NAA) and 1 mg l^-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4) plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l^–1 NAA and 1 mg l^–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency of the procedure for protoplast to cell line formation is high (about 80%). Received: 17 February 1997 / Revision received: 6 November 1997 / Accepted: 15 November 1997     

An Efficient Protocol for Ulmus minor Mill. Protoplast Isolation and Culture in Agarose Droplets

We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma^®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 10⁷ protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 10⁵ and 2 × 10⁵ protoplasts ml^?1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 10⁵ protoplasts ml^?1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Somatic embryogenesis in Clematis integrifolia × C. viticella

Nodal sections of Clematis integrifolia × C. viticellawere cultured at 24 °C in darkness on a medium containing the salts and vitamins of Murashige and Skoog (1962) supplemented with sucrose (30 g l⁻¹), 2 μM 6-benzylaminopurine (BAP) and 0.5 μM 4-indole-3-yl-butyric acid (IBA). These explants produced a white friable callus on their surfaces. Somatic embryos were first observed on the surface of the callus after eighteen months in culture. Thereafter, pieces (125 mm³) of the embryogenic mass were subcultured every 4 weeks and continued to produce somatic embryos over the two-year period of observation. A mean (± SE) of 64±4 cotyledonary stage embryos were observed per 125 mm³ callus four weeks after transfer to fresh medium. Comparison of the effect of growth regulators on conversion showed that the highest frequency of unipolar and bipolar conversions occurred on medium containing 10 μM kinetin and 1 μM BAP. Shoots were excised from embryos and inserted in Sorbarods saturated with liquid medium containing 0.05 μM IBA and 0.05 μM 1-naphthalenacetic acid. After four weeks 88% had formed root and survived transfer to compost in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell regeneration and sustained division of protoplasts from cotton (Gossypium hirsutum L.)

Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K₃ media of Kao et al., 1974 with 0.9 M BAP, 5 M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5×10⁵ protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.Abbreviations IAA indoleacetic acid - BAP 6-benzyl-adenine Arizona Experimental Station Publication No. 4373     

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. The planting density was 2.5–3.0×10⁵ protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo'Green Delica'

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumismelo‘Green Delica’ were used as explants for protoplastisolation and culture. Protoplasts isolated from cotyledonsand etiolated half-expanded leaves cultured in Durand, Potrykusand Donn (DPD) medium supplemented with 0.9 µMbenzylaminopurine(BAP), 3.6 µM2,4-dichlorophenoxyacetic acid (2,4-D) and1% sucrose, using the agarose bead culture method, were ableto form cell walls and subsequently go through cell division.Pretreatment of half-expanded leaf explants in the dark for14 d provided the best material for protoplast isolation andcell division. Approximately one third of protoplasts from etiolatedhalf-expanded leaves formed microcolonies. For hypocotyl protoplasts,none of the treatments used were suitable to induce cell division.There was no significant difference between sucrose, glucose,and sucrose plus glucose, in culture media on the plating efficiencyof leaf protoplasts ofC. melo‘Green Delica’; however,bigger colonies were formed in media supplemented with 1% sucrose.No shoot or whole plant regeneration was achieved. However,the methods reported here provide further information onC. meloprotoplastculture.Copyright 1998 Annals of Botany Company Cucumis melo,protoplast culture, 2,4-D, BAP, yeast extract, casein hydrolysate.     

Isolation and culture of Lens culinaris Medik. cv. Eston epicotyl protoplasts to calli

Protoplasts of Lens culinaris Medik. cv. Eston were isolated from epicotyl tissues of seedlings grown on Murashige & Skoog basal medium. For isolating the protoplasts, epicotyl tissues were digested for 12–14 h at 25°C in an isolation mixture (pH 5.7) containing 1% Cellulase RS, 0.5% Driselase, 0.25% Pectolyase Y23, 0.2M calcium chloride, 10 mM mannitol and 10 mM MES. Protoplasts were purified by flotation over 20% sucrose and washed with 0.2 M calcium chloride solution supplemented with 10 mM mannitol. Purified protoplasts were cultured at a density of 10⁵ ml^-1 in agarose (Seaplaque, 0.6%) blocks which were suspended in an identical but liquid KM8P culture medium lacking amino acids, ammonium nitrate, and coconut water but containing 0.35 M glucose and a growth regulator complement of either 2.2 M 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 M naphthaleneacetic acid (NAA), 2.3 M N-(2-furanylmethyl)-1H-purine-6-amine (kinetin), 2.2 M benzylamino purine (BAP), 2.3 M 2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol (zeatin), and 1.4 M gibberellic acid (GA₃), or 5.4 M NAA and 2.2 M each of 2,4-D and BAP. The osmotic potential of the liquid culture medium was gradually reduced over a period of 3 weeks by replacing the spent medium with a fresh medium containing 0.25, 0.1 and 0 M glucose at weekly intervals. About 6% of the dividing protoplasts developed into cell colonies after 3 weeks of culture at 25°C in diffuse light (10 E m^-2s^-1). In 35–42 days the microcolonies were about 1 mm in diameter and developed into calli on transfer to agar-solidified B5 medium supplemented with growth regulators used in the protoplast culture medium and 5 mM glutamine. Attempts to regenerate plants from protoplast-derived calli have so far been unsuccessful.Department of Applied Microbiology and Food Science, University of Saskatchewan     

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 protoplasts isolated from friable embryogenic callus of cassava

Protoplasts were isolated from friable embryogenic callus (FEC) and from suspensions derived from FEC of cassava genotype TMS60444. Suspensions yielded the highest number of protoplasts (1.5×10⁶ protoplasts/g fresh weight). Protoplasts plated at a density of 10⁵–10⁶/ml in a medium supplemented with 0.5 mg/l α-naphthaleneacetic acid and 1 mg/l zeatin began dividing after 3 days, and after 30 days this resulted in an absolute plating efficiency as high as 2.5%. After 2 months of culture, 60% of the developed calli were highly friable and in appearance identical to the original FEC. The protoplast derived FEC was first purified through two rounds of selection of 3 weeks each before beeing cultured for regeneration of plants. This was done by culturing the protoplast-derived FEC for 11 weeks on maturation medium, yielding a maximum of 184 organized embryos per 10.000 initially cultured protoplasts. Most of the organized embryos were torpedo shaped and matured after they had been isolated from the calli and transferred to fresh medium. Mature embryos were multiplied by secondary somatic embryogenesis at high efficiency (>90%) on a medium supplemented with 8 mg/l 2,4-dichlorophenoxyacetic acid. About 30% of the mature secondary somatic embryos developed into shoots after transfer to a medium supplemented with 1 mg/l N⁶-benzylaminopurine (BAP). Shoots rooted readily on a medium without BAP. Received: 30 August 1996 / Revision received: 9 June 1997 / Accepted: 1 October 1997     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Winged-bean protoplasts: Isolation and culture to callus

A system was developed for protoplast isolation and culture from suspension cultured cells of winged bean,Psophocarpus tetragonolobus. Cells from a three-day-old suspension were incubated in an enzyme mixture containing 6% cullulysin, 1% Macerase, 1% desalted Rhozyme, 0.4M sorbitol, and 0.1M CaCl₂ at pH 5.5. Average yields of protoplasts were 6.5 × 10⁶ per gram fresh weight of cells. Protoplasts were cultured in modified B5 medium containing 68.4 g/l glucose, 250 mg/l xylose, 0.1 mg/l 2,4-D, 0.5 mg/l BAP, 250 mg/l N-Z amine type AS, and 20 ml/l coconut water. After 24 h of culture, the protoplasts had synthesized a new wall, and in three days had begun division. The optimum plating density was 1–2 × 10³ protoplasts/ml. The division frequency ranged between 40%–60% for most experiments with a high of 72% in one experiment. After three weeks, cell colonies could be transferred to solid MS medium containing N-Z amine and coconut water where callus developed. This protoplast system is technically comparable to soybean for experiments concerned with genetic manipulation involving legumes.     

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     

Direct and callus-mediated protocorm-like body induction from shoot-tips of <Emphasis Type="Italic">Dendrobium chrysotoxum</Emphasis> Lindl. (Orchidaceae)

An efficient method of mass propagation of Dendrobium chrysotoxum Lindl. was developed using a shoot-tip culture system. Both direct and callus-mediated formation of protocorm-like bodies (PLBs) occurred from the basal cut surface of explants. Frequency of callusing was best in the presence of 2 μM thidiazuron (TDZ) or N⁶-benzylaminopurine (BAP). The callus exhibited complete hormone autonomy for growth and differentiation of PLBs and was maintained for 18 months without any exogenous growth regulators, an aspect important for minimising somaclonal variation. However, the rate of callus growth and PLB formation varied with application of cytokinin and auxin. In addition, the callus exhibited a differential sensitivity to the exogenous cytokinins. While BAP promoted callus growth and PLB differentiation, TDZ was inhibitory to callus mediated PLB formation and caused extensive necrosis of callus. Although α-naphthaleneacetic acid (NAA) had no significant effect on the induction of callus, subsequent growth was best in its presence. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 μM NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential.     

Shoot multiplication and adventitious regeneration in Sorbus aucuparia

In vitro mother plants initiated from a mature tree of Sorbus aucuparia, produced numerous propagules on a medium containing 2 μM 6-benzylaminopurine (BAP) and 0.2 μM 1-naphthaleneacetic acid (NAA). These were rooted on a medium containing 0.25 μM NAA and 0.25 μM indole−3-butyric acid. Adventitious shoots were produced on excised leaves and internodes on media containing 10 μM thidiazuron and 0.3–1.0 μM NAA. They formed by direct regeneration in the axils of leaflets of intact leaves. They also developed indirectly, from callus that developed on the rachis of intact leaves, and the cut ends of petioles and internodes. Somatic embryos were produced on cotyledons of zygotic embryos on medium containing 1 μM BAP, 1 μM kinetin, 0.5 μM NAA, 500 mg l⁻¹ casein hydrolysate and 250 mg l⁻¹ glutamine. On basal medium, 69% developed cotyledons and 20% germinated after pre-treatment at 4 °C on medium containing 30 g l⁻¹ maltose.