Intraclonal Genetic Variation for Protoplast Regenerative Ability Within Solanum tuberosum cv. Record

The potato cv. Record is recognized as a recalcitrant cultivarin tissue culture and attempts in the past to obtain regenerationfrom protoplasts continually failed, despite media and protocolalterations. By sampling a large number of Record tubers, significantdifferences between lines were obtained for regeneration fromleaf discs. Eight such lines exhibiting a range of responseto regeneration from leaf discs were, used in the present studyto examine protoplast culture response. Significant variationwas detected in protoplast plating efficiency and in the numberof regenerants produced. These results are discussed in relationto the exploitation of protoplasts in potato improvement andin terms of the role of tissue culture techniques for the maintenanceof potato cultivars. Solanum tuberosum, cv. Record, potato, protoplasts, intraclonal variation     

Genetic Variation in Potato Cv. Record: Evidence from In Vitro 'Regeneration Ability'

Regeneration ability in vitro was studied in 170 individualtubers putatively derived from several or many parent plantsof the potato cv. Record. Of these, 120 were sprouted and thesprouts used to establish in vitro shoot cultures for leaf discproduction. The other 50 were grown in a glasshouse for theproduction of leaf discs. The reliable regeneration of somaclonesfrom leaf disc calluses was successful from only 11 parentaltubers. In ten of these, somaclones were derived from in vitroshoot cultures, and from a glasshouse-grown plant in the other.Four parental tubers gave the majority of somaclones, and one,R149, produced 85% of all somaclones at 15 months from initiationof leaf disc cultures. This differential regeneration abilitymay be due to genetic differences between tubers in this potatocultivar as it was found to be maintained in subsequent tubergenerations. The results are discussed in terms of seed potatoproduction and in vitro genetic conservation of vegetativelypropagated species. Potato, Solanum tuberosum cv. Record, regeneration ability, leaf disc culture, somaclonal variation     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

Plant regeneration from protoplasts of diploid potato derived from crosses of Solanum tuberosum with wild solanum species

A new method has been developed for the obtention of protoplasts of potato (Solanum tuberosum L.) diploid clones and for the regeneration of whole plants. This procedure determined specific conditions of in vitro culture of shoots: the propagation medium, light intensity and length of illumination period. Four successive media from the protoplast level to the shoot formation have been optimized. This method is suitable for the obtention of calli from 13 diploid potato lines, among 15 tested. Regeneration of plants has been obtained for 10 clones with efficiencies from 0.5 to 4 plants regenerated from 100 plated protoplasts.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Influence of the Antibiotic Ciprofloxacin on Culture of Allium longicuspis Callus-derived Protoplasts

A major problem of in vitro plant culture techniques is chroniccontamination by microorganisms. Calli derived from basal partsof leaves of Allium longicuspis Regel (Alliaceae) and culturedin a medium without antibiotic contain most probably latentcontaminating microorganisms. These calli were used as the sourcematerial for isolation and culture of protoplasts. Isolatedprotoplasts were cultured in the presence of the antibioticciprofloxacin, and the protoplast viability, cell wall regenerationand cell division were studied as a function of the antibioticconcentration. Whatever the antibiotic concentration, protoplast-derivedcells kept significantly higher viability for at least 3 weekscompared with those cultured without antibiotic. As to cellwall regeneration after 2 d, it was not affected by the antibioticexcept at the highest concentration tested (100 mg l^-1). Sporadicfirst cell division was observed after 2-6 d of culture in thepresence of ciprofloxacin while, in its absence, cell divisionwas never apparent before 10 d of culture.Copyright 1995, 1999Academic Press Allium, bacteria, cell division, cell wall regeneration, ciprofloxacin, contamination, garlic, mycoplasma, protoplast culture, viability     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Factors influencing plant regeneration from protoplasts isolated from long-term cell suspension culture of recalcitrant Indica rice cultivar IR36

Summary Factors influencing successful establishment of embryogenic cell-suspension cultures and plant regeneration from longterm cell suspension-derived protoplasts of the recalcitrant Indica rice cultivar IR36 were studied. The factors included cell and protoplast culture medium, protoplast culture procedure, the source of nurse cells, and the regeneration procedure. Embryogenic cell suspension cultures could only be established from mature seed-derived callus of IR36 in AA-based medium (Müller and Grafe, 1978). Protoplast-derived colonies could be obtained only using the filter-membrane nurse-culture procedure when Lolium multiflorum suspension cells served as nurse, rather than wild rice (Oryza ridleyi) and Japonica rice (Oryza sativa cv. Taipei 309) cells. The utilization of a two-step regeneration procedure led to regeneration of fertile plants from protoplasts isolated from 2-yr-old cell suspensions of IR36, one of the most important but recalcitrant rice cultivars.     

Plant regeneration of mesophyll protoplasts from a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry

Mesophyll protoplasts from in vitro grown plants of a cytoplasmic albino mutant ofLycopersicon esculentum cv. Large Red Cherry were isolated with yields between 0.4 to 4.4 × 10⁶ protoplasts per gram leaf tissue. Success in the culture of these protoplasts was dependent on embedding of the protoplasts in 100 µ1 agarose droplets 0.6% (w/v). A plating efficiency of 4.0% was obtained when the protoplasts were cultured in TM-2 medium with sucrose concentrations of 8.7 to 9.6% (w/v) resulting in an osmotic pressure of 432 to 469 mOsmol kg^-1. After 14 days of protoplast culture, microcalli with a diameter of 3 mm were observed. After 3 weeks, macrocalli were obtained which were transferred to regeneration medium. Regeneration of shoot primordia, with a frequency of 19%, was obtained on TM-4 medium supplemented with 1% (w/v) sucrose. The first shoot primordia were visible 10 weeks after protoplast plating. For development of the shoot primordia into shoots it was necessary to increase the sucrose concentration to 6% (w/v). Eight out of eleven regenerants were diploid (2n = 2x = 24); the other three were tetraploid. Efficient regeneration of mesophyll albino protoplasts from tomato opens the way to select at the cellular level for the chloroplast transfers.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

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.     

In vitro morphogenesis of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>) hypocotyl protoplasts: the effects of protoplast density,haemoglobin and spermidine

Sunflower, as one of the most important oil-producing crops, represents an important target for genetic improvement through gene transfer or somatic hybridization. Unfortunately, sunflower is recognized as recalcitrant to in vitro culture. The aim of our paper was to improve sunflower protoplast regeneration. Three cultivars (Romanian hybrids) and one inbred line were used for protoplast isolation from etiolated hypocotyls. Isolated protoplasts were embedded in alginate disks and cultured in two plating densities, using two culture regimes as indicated by previous authors. Plating efficiency, callus development and plant regeneration were evaluated as well as old callus histology. In cv. ‘Select’, the effects of 1:50 haemoglobin and 1 mM spermidine were assayed on asymmetric division and/or plating efficiency. Plant regeneration from hypocotyl protoplasts was achieved for two cvs., ‘Florom 328’ and ‘Turbo’, with the former proving once more its totipotency. The best culture regime proved to be as recommended by Krasnyanski and Menczel (1993), but the best density in the culture medium was the highest ever tested, 8 × 10⁵ pp ml⁻¹. Moreover, the histology of old green compact protoplast-derived callus revealed a very well organized structure suggesting senescence. In the non-responsive cv. ‘Select’, haemoglobin was found to stimulate protoplast asymmetric division and the development of heart-shaped embryo-like structures, while spermidine stimulated overall protoplast plating efficiency.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Plant regeneration from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plasmolytic behavior of the donor cell may affect protoplast response

Protoplast donor tissues (leaves of shoots in culture) from a herbaceous plant ( Solanum etuberosum ) and two woody species ( Populus alba × P. grandidentata cv. Crandon and Betula platyphylla szechuanica ) were compared during plasmolysis in a range of osmotic agents and potentials. Cells from both Solanum and Populus , species proven to be amenable to protoplast division and regeneration, plasmolyzed readily at higher osmotic potentials than cells from Betula , a species recalcitrant to prolonged culture after protoplast isolation. Betula leaf mesophyll cells exhibited persistent membrane-to-wall attachments and many failed to plasmolyze even under extreme osmolarity. Although their leaves exhibited similar photosynthetic rates, photosynthetic capacity was lost from Betula protoplasts upon isolation, and retained by Solanum protoplasts. Differential stress after isolation was not detectable through vital staining, but only Solanum and Populus gave both high protoplast yields and high plating efficiencies in continued culture.     

Plant regeneration from mesophyll protoplasts in commercial potato cultivars (Primura,Kennebec, Spunta,Desirée)

In view of an evaluation of the relative efficiency of different in vitro systems for mutant induction and isolation in potato, a procedure of plant regeneration from protoplasts of some potato (Solanum tuberosum L.) cultivars was developed. Four cultivars (Primura, Kennebec, Spunta, Desirée) were used for isolation, culture and regeneration of leaf mesophyll protoplasts.These lines were chosen because of their economic importance in Italy and in the case of cv. Desirée for the presence of markers useful in the morphological characterization of regenerants.Abbreviations MS Murashige and Skoog's medium - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - GA ₃ gibberellic acid - ZT zeatin