Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

Isolation of biochemical mutants using haploid mesophyll protoplasts ofHyoscyamus muticus

Two amino-acid auxotrophic cell clones ofHyoscyamus muticus, VA5 (His^-) and VIIIB9 (Trp^-), isolated in a previous experiment have been characterised quantitatively. Studies of growth in the presence and absence of histidine and tryptophan and an examination of dose-response relationships for the two amino acids confirmed the strict auxotrophy of both cell lines. No revertants to prototrophy were detected in either cell line after more than two years in culture. N-methyl-N′-nitro-nitrosoguanidine did not induce reversion in VA5 cell populations. Wild-type aggregates mixed in various combinations with VA5 and VIIIB9 cells could be recovered after plating in selective conditions. No cross-feeding was detected, either between wild-type and auxotrophic cells or between the auxotrophic lines themselves. Both variants were recloned by protoplast culture. All protoplast-derived clones were auxotrophic. The auxotrophic phenotypes behaved as recessive traits in protoplast-fusion experiments.     

The use of agarose bead culture for the regeneration of single cell-derived colonies from protoplasts isolated from suspension cultures of Humulus lupulus

A cell-wall digestion medium has been devised to isolate protoplasts from suspension cultures of Humulus lupulus. Conditions have been developed for colony formation from protoplasts and the plating efficiency determined in three types of agar and by two culture methods. Viable calli were produced only when protoplasts embedded in Seaplaque agarose were incubated in a defined liquid medium. HPLC analysis showed that none of the isolated colonies accumulated -acids.     

Somatic hybridization between Nicotiana rustica and N. sylvestris

Protoplasts isolated from cell cultures of chlorophyll-deficient Nicotiana rustica cv. chlorotica and wild-type N. sylvestris were fused. The scheme for selection of somatic hybrids was based on the inability of the protoplast-derived colonies of the parental species to turn green; N. sylvestris protoplasts also had a very low plating efficiency in the medium employed. A total of 777 green colonies which were presumptive hybrids were isolated within four weeks of the fusion experiments. One hundred and eight green colonies formed shoots in vitro and 16 lines were rooted and grown in the greenhouse. Each of these hybrid plants displayed vegetative and floral traits intermediate to those of the parental species, except for plant height which in almost all cases was greater in the hybrids. Isozyme analyses by gel electrophoresis and isoelectric focussing of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBPCase) demonstrated that the nuclear genomes of both parents were expressed by the hybrids. Each of the eight somatic hybrid plants analyzed expressed only the N. rustica chloroplast genome as shown by isoelectric focussing of the large subunit of RUBPCase. This study demonstrated the value of N. rustica cv. chlorotica as a parental line in somatic hybridization with N. sylvestris and it might have widespread use with wild-type lines of other species.     

Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

A new procedure for increasing efficiency of protoplast plating and clone selection

Sea Prep^TM agarose (FMC corp.) was used as a gelling agent for media in the culture of potato and tomato protoplasts. The concentration and conditions appropriate for gelling and liquifaction were determined. The new method provided increased calli transfer efficiency of up to 50-fold. Shoot formation from the protoplast-derived cells occurred normally and without reduction in frequency.     

An assessment of the cultural capabilities of Trifolium repens L. (white clover) and Onobrychis viciifolia Scop. (sainfoin) mesophyll protoplasts

Mesophyll protoplasts isolated from white clover and sainfoin divided to form callus under similar cultural conditions. White clover protoplasts showed varietal differences in their plating efficiency. Sainfoin tissues regenerated readily by forming shoots, but induction of morphogenesis in white clover was only achieved after testing several media and culture sequences. Many of the white clover shoots were abnormal in being fused together to form green plate-like structures, but the latter still developed into plantlets while attached to the parent callus. The ability to isolate, culture, and regenerate mesophyll protoplasts of these two forage legumes is discussed in relation to future attempts to produce somatic hybrids between high tannin containing bloat-safe sainfoin and other major forage legumes such as alfalfa, white clover, and red clover.     

An improved polyurethane support system for monitoring growth in plant cell cultures

An improved culture system for plant cells that employs filter paper resting on polyurethane saturated with liquid medium is described. It combines a simplified version of the system outlined by Weber and Lark [1979, Theor Appl Genet 55: 81–86] with the method of growth estimation described by Horsch et al. [1980, Can J Bot 58: 2402–2406]. The growth of plated cells or callus can be conveniently monitored through repeated non-destructive fresh weight measurements of the filter paper and adhering cells, thereby allowing the construction of a complete growth curve over the course of an experiment. Experiments with 3 Nicotiana genotypes (N. plumbaginifolia Viv., N. tabacum L. ‘SC 58’ and N. tabacum ‘WI 38’) and 3 Vitis vinifera L. genotypes (‘Chenin Blanc’, ‘Dogridge’ and ‘White Riesling’) clearly demonstrate higher growth rates of plated cells on polyurethane supports compared with agar. Further experiments with N. plumbaginifolia illustrate the use of polyurethane supports for culturing cells at low pH (4.0) and the recovery of spent medium for monitoring changes in pH. These features will greatly facilitate quantitative studies of mineral nutrition and metal toxicity in cultured cells. Polyurethane supports also allow the incorporation of conditioned medium or feeder cells to support the growth of cells at low densities and facilitate the rapid recovery of variant cells.     

Investigation of novel oligoelectrolyte polymer carriers for their capacity of DNA delivery into plant cells

Development of modern agriculture and biotechnology is closely connected with the use of novel and effective genetic engineering methods. Presently, non-viral nanoparticle-mediated plant transformation methods gain more attention because of their stability, safety, and convenience of performance. In this work, new polymeric dimethylaminoethyl metacrylate (DMAEM)-based polymers were synthesized and investigated for their properties in gene delivery. Formation of stable complexes between TN 83/6, TN 84/5, DLM-9-DM and LM-8-DM polymers and plasmid DNA, as well as the DNA protection by the PDMAEM polymers against nuclease degradation were confirmed by electrophoresis in agarose gel. In addition, model organisms Allium cepa and Nicotiana tabacum L. were studied to evaluate cytotoxic effect of the PDMAEM carriers. The created PDMAEM-based carriers were effective in delivery of plasmid DNA into moss and tabacco protoplasts (obtaining stable transformants of Ceratodon purpureus moss, as well as in transient expression of the reporter yfp gene product in N. tabacum protoplasts). Thus, novel PDMAEM-based polymers were shown to be promising carriers for delivery of DNA into plant cells, and carriers possess high potential for further applications in this field.     

An improved protocol for the culture of cassava leaf protoplasts

Viable protoplasts (yield > 1.9 × 10⁷ g^–1 fresh weight; mean viability 85±2%, n=5) were isolated from leaves of axenic shoot cultures of Manihot esculenta Crantz. cv. M. Thai 8. Protoplasts were cultured for up to 50 days in liquid, ammonium-free MS medium, overlaying agarose-solidified B5 medium with short glass rods embedded perpendicularly within, and protruding from, the agarose layer. Control protoplasts were cultured identically, but without glass rods. Sustained protoplast division was observed only in the presence of glass rods, where the initial plating efficiency was almost 6-fold greater than control (p < 0.05). The mean final plating efficiency of treated cultures was 1.0±0.2% while, in contrast, significant colony formation was not observed in controls.Abbreviations BA 6-benzyladenine - CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea - MES 2[N-morpholino]ethane sulphonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - IPE initial plating efficiency - FPE final plating efficiency     

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10⁶ / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl₂ and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.Abbreviations BAP benzylaminopurine - NAA naphthaleneacetic acid - PCR Polymerase Chain Reaction - fw fresh weight     

Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus

Key message

A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets.

Abstract

Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9?% in C. intybus genotypes and efficiencies of up to 0.7?% in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5?mg?l^?1 indole-3-acetic acid-enriched medium (up to 87.5?% of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.     

Isolation of Hyphomonas Strains that Induce Normal Morphogenesis in Protoplasts of the Marine Red Alga Pyropia yezoensis

Marine macroalgae cannot develop normal morphology under axenic conditions although normal morphogenesis can be sustained when certain bacteria are present. In this study, bacteria that induced normal morphogenesis in the red alga Pyropia yezoensis (Nori) were identified. The bacteria were isolated from algal media, thalli, tissue debris, and purified protoplasts during protoplast isolation from P. yezoensis laboratory cultures. 16S rRNA gene sequence analysis showed these bacterial isolates belonged to α-Proteobacteria (12 groups), γ-Proteobacteria (3 groups), and Flavobacteria (2 groups). Axenic protoplasts of P. yezoensis generated by removing epiphytic bacteria were co-cultured along with the bacterial isolates. Most axenic protoplasts showed irregular morphogenetic and anaplastic cells; cells with normal morphology were scarce. However, inoculation with 11 strains of Hyphomonas (α-Proteobacteria) led to significantly higher normal morphogenetic rates (4.5–7.3 %, P?Hyphomonas strains were recovered from all experiments; thus, certain Hyphomonas strains can induce normal morphogenesis in P. yezoensis protoplasts. Direct inoculation of the Hyphomonas strain exhibited higher morphogenetic activity than inoculation of its extracellular and intracellular products. This is the first study demonstrating the influence of specific bacteria on protoplast morphology in marine macroalgae.     

Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum

Summary Protoplasts were derived from haploid and diploid Nicotiana sylvestris and N. tabacum. Exposure of the protoplasts to mutagenic doses of ultraviolet (U.V.) radiation prior to two selection rounds in the presence of 4 mM (or 5 mM) and 8 mM of valine, respectively, was required to obtain cell lines with persistent valine resistance. Such lines were obtained from haploid and diploid N. sylvestris protoplasts as well as from haploid protoplasts of N. tabacum but not from (1.8 × 10⁷) diploid N. tabacum protoplasts. The ratio between number of verified valine-resistant cell lines and the initial number of U.V. exposed protoplasts enabled the estimation of the following order of mutation frequency: haploid N. sylvestris > haploid N. tabacum > diploid N. sylvestris. Plants which retained the valine resistance and transmitted it to their sexual progeny were derived from the resistant cell lines.     

The use of anther-derived haploids in Nicotiana

Immature anthers from monosomic Nicotiana tabacum L. (“Purpurea”) plants were cultured on a chemically defined agar medium. Plantlets obtained from these anthers were transferred first to a rooting media and subsequently to soil in greenhouse pots. Six nullihaploid plant types have been isolated, two of which have been positively identified as nullihaploids C and E. Chromosome numbers (n?1=23) were verified somatically from root tip and corolla tissues and meiotically by observing PMC's at metaphase I. The chromosome number was doubled in one of the lines by culturing leaf midvein tissue on a modified anther culture media. The nullisomic chromosome number (2n?2=46) was determined in root tips, and the nullisomic condition of 23 bivalents was observed in metaphase I PMC's.     

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     

Induction of efficient cell division in alfalfa protoplasts

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.Contribution No. 774 Ottawa Research Station     

Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana

Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a ⁶⁰Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1–2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg^-1 doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.     

Electroporation of Tobacco Protoplasts with Homologous and Nonhomologous Transformation Vectors

A modified protoplast selection/plating technique was used to quantitate and compare the transformation efficiencies of tobacco NTl protoplasts electroporated with plasmid molecules harboring a chimeric nos-neo gene (pMON213) or both this chimeric gene and a region of homology with the host chromosome (pCPI). The latter plasmid was constructed by cloning the pMON213 EcoR I cassette carrying the nos-neo gene into pNtSS233, a pBR322 derivative harboring a 1.2-kbp piece of Nicotiana tabacum DNA comprising the 5'-end of a gene coding for 1,5-ribulose bisphosphate carboxylase-oxygenase (Rubisco). These plasmids were linearized by restriction endonuclease digestion and electroporated into tobacco protoplasts which were subsequently selected on kanamycin-containing medium. Both plasmids displayed the same transformation efficiency, indicating that the presence of a homologous region in the selectable vector had no influence on the rates of transformation.     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.