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     

Plant regeneration from mesophyll protoplasts of Moricandia arvensis

Moricandia arvensis is of interest as it is a dicotyledonous species which has C₃ — C₄ intermediate photosynthesis, a mechanism which results in enhanced recapture of photorespired CO₂. Leaves from cultured shoot tips were used as a source for mesophyll cell protoplasts. Approximately 1% of the protoplasts which survived the first few days of culture produced calli. On a suitable regeneration medium, 30–60% of the calli regenerated one or more shoots. From among the regenerating shoots eight were selected, transferred to soil and grown to flowering in the glasshouse; all were fertile. The development of a protoplast regeneration system provides the opportunity to use transformation and somaclonal variation as tools in the genetic analysis of the C₃–C₄ character in this species.Abbreviations GDC glycine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzyl aminopurine - NAA naphthalene acetic acid - ABA abscisic acid     

Plant regeneration from mesophyll protoplasts of Lactuca perennis

Cultured protoplasts of young, unexpanded leaves of the wild lettuce, Lactuca perennis, divided to produce cell colonies in an agarose-solidified, modified MS medium with reduced levels of inorganic salts, together with 2,4-d, NAA and zeatin at 0.2, 0.1 and 0.5 mg 1^-1 respectively. Organogenesis followed the initial transfer of protoplastderived colonies to modified MS medium with 2,4-d, NAA and zeatin (0.1, 1.0 and 0.2 mg 1^-1 respectively) and then to full-strength MS medium with 6-BA and NAA (0.4 and 0.05 mg 1^-1). Shoots were rooted on agar-solidified MS medium lacking growth regulators. Regenerated shoots were established ex vitro, 21 weeks after protoplast isolation.Abbreviations 6-BA 6-benzyladenine - BSA bovine serum albumin - d days - 2.4-d 2,4-dichlorophenoxyacetic acid - f. wt. fresh weight - IAA indoleacetic acid - MES 2 [N-morpholino]ethane sulphonic acid - MS Murashige & Skoog (1962)     

Plant regeneration from mesophyll protoplasts in Isatis indigotica

Protoplasts of an accession of Isatis indigotica Fort. were isolated from mesophyll tissue by enzymatic digestion and cultured using a feeder cell system. Shoot regeneration efficiency was 100% via organogenesis among 627 isolated calluses within 30–37 days. Among these shoot initiating calluses, 162 (22.6%) developed normal shoots with multiple (2–5) shoots per callus. The remaining calluses developed only vitreous shoots. High concentration (5 μM) of indole-3-butyric acid had a positive effect on rooting compared to low concentration (0.5 μM) of indole-3-butyric acid and α-naphthalene-acetic acid. The average rooting efficiency of regenerated shoots of two experiments was higher on LS medium with 5 μM indole-3-butyric acid than on LS medium without growth regulators. Twenty-nine plantlets, with 2–3 expanded leaves and roots were potted in soil and 22 developed normally to maturity in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of plants from mesophyll protoplasts of the wild crucifer Eruca sativa Lam.

Protoplasts isolated from mesophyll cells of Eruca sativa Lam., cultured on suitable medium, underwent sustained cell divisions to form calli. The plating efficiency was found to be 0.4%. The protoplast-derived calli subsequently produced plantlets through organogenesis (15.71%) and somatic embryogenesis (11.25%). Regenerated plants exhibited normal appearance. These results indicate potential to introgress desirable traits from this wild crucifer into important oilseed and cole Brassicas by protoplast fusion and hybrid recovery.Abbreviations B5 Gamborg et al., 1968 - K3 Kao and Michayluk, 1974 - MS Murashige and Skoog, 1962 - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - GA₃ Gibberellic acid     

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides Pall. was here developed. The protoplasts were isolated directly from the leaves of the hairy root-induced plants. The highest yield of protoplasts was obtained from fully expanded leaves of young plants. Their viability was up to 72 ± 2.3 %. The highest division frequency (32.4 ± 0.13 %) and sustained divisions were obtained in Durand, Potrykus and Donn (DPD) medium supplemented with 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid, 0.2 mg dm⁻³ 6-benzylaminopurine, 0.3 M mannitol, 2 % sucrose and 500 mg dm⁻³ casein hydrolysate at the plating density of 3.0 × 10⁵ cm⁻³. The frequency of shoot differentiation from protocalli reached to 91.75 ± 3.1 %. Opine synthesis and polymerase chain reaction analysis confirmed that T-DNA still existed in the protoplast regenerated plants.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

Different regeneration potential of mesophyll protoplasts from cultivated and a wild species of tomato

Mesophyll protoplasts were isolated from leaves of three cultivars of Lycopersicon esculentum (L.) Mill., namely Hilda 72, Rutgers and Rentita, and from the wild tomato species Lycopersicon peruvianum (L.) Mill. Protoplasts from L. peruvianum divided and grew actively in a liquid medium according to Zapata et al. (1977), whereas protoplasts from the tomato cultivars Hilda 72 and Rutgers showed comparable rates for cell division only, when the content of major elements in this medium was reduced to one half of the original concentration and when mannitol as osmoticum was replaced by glucose. In Rentita protoplasts no cell division could be observed in about 15 different modifications of the five basic culture media tested. The morphogenetic potential of these tomato cells was assessed by comparing the root and shoot formation of protoplasts and of leaf explants. L. peruvianum exhibited the highest potential. Calli derived from protoplasts regenerated roots on Murastrige-Skoog agar containing 1 M benzylaminopurine (BAP) plus 10 M indole-3-acetic acid (IAA) and 0.1 M BAP plus 1 M IAA. Shoot formation occurred in the combinations of 10 M BAP with 0.1, 1.0, and 10 M IAA. Plantlets regenerated from the L. peruvianum calli could be grown in soil. No shoots or roots were regenerated from calli of Hilda 72 and Rutgers protoplasts in all combinations of BAP and IAA tested in the range from 0.1 M to 100 M, thus indicating the rather low morphogenetic potential of these protoplasts as compared to protoplasts from L. peruvianum leaves.Abbreviations BAP benzylaminopurine - IAA indole-acetic acid - TMV tobacco mosaic virus     

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 capacity of mesophyll protoplasts from Brassica napus and related species

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

Plant regeneration from mesophyll protoplasts of the tree legume Pithecellobium dulce benth

Conditions were developed for the isolation, culture and regeneration of mesophyll protoplasts of the tree legume, Pithecellobium dulce Benth. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) was essential to induce initial cell divisions and addition of naphthaleneacetic acid (NAA) improved the response. Sustained division and cell colony formation were achieved from the protoplasts cultured in a modified KM8P medium containing 2,4-D (2.3 μM), NAA (3 μM) and benzyladenine (BA) (2.3 μM). Dilution of the osmotica included in the protoplast culture medium was necessary to induce sustained proliferation of the protoplast-derived cells. Differentiation of shoots from the protoplast-derived calli occurred on Murashige and Skoog (MS) medium supplemented with BA (5 μM) and indole-3-acetic acid (1 μM). Omission of 2,4-D from the culture medium, after the initial 2 weeks of protoplast culture, was obligatory to induce shoot morphogenesis.     

Rapid plant regeneration from Nicotiana mesophyll protoplasts

A procedure is described for rapid plant regeneration from tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Six to seven days after protoplast isolation, colonies are placed on double filter feeder plates that consist of a strong regeneration medium containing 7.5 mg/l 6-(γ,γ-dimethylallylamino)-purine (2iP) and 0.1 mg/l p-chlorophenoxyacetic acid (pCPA). Complete plants are regenerated in about 5 weeks after transfer to a rooting medium (hormone-free Murashige and Skoog (MS) medium). However, upon remaining on shoot regeneration medium, 50–75 shoots are regenerated from single colonies derived from individual protoplasts. This procedure may reduce the amount of somaclonal variation (as measured by ploidy level) which is usually expressed in plants obtained by conventional regeneration techniques.     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl^-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.     

Plant regeneration from protoplasts of a wild lettuce species (Lactuca saligna L.)

Protoplasts isolated from young, unexpanded leaves of the wild lettuce species, Lactuca saligna, divided to give colonies, when plated at low density in KP8 medium solidified with 1% (w/v) agarose. Sustained colony development was dependent upon the incorporation of the bead culture approach. Plant regeneration, via organogenesis, followed the transfer of colonies firstly to K8 medium and then to M/S medium supplemented with IAA and 6-BAP, both solidified with 1% (w/v) agarose. Excised shoots were rooted on agar-solidified M/S medium lacking phytohormones. The potential of this species as a source of race-non-specific mildew resistance for transfer into lettuce (L. sativa) via somatic hybridisation is discussed.Abbreviations 6-BAP 6-benzylamine purine - IAA indoleacetic acid - M/S Murashige and Skoog (1962) - fwt fresh weight     

Plant regeneration from protoplasts of wild barley (Hordeum murinum L.)

Summary We have produced a large number of plants regenerated from protoplasts originally isolated from embryo-derived cell suspensions of wild barley, Hordeum murinum L.. Suspensions initially allowed protoplast isolation and culture 5.5 to 9 months from the date of callus initiation. Colony formation efficiencies ranged from 1.5 to 3.0 % and from 0.1 to 1.4 % for protoplast cultures with and without nurse cells, respectively. Both nurse and non-nurse techniques allowed efficient embryogenesis and plant regeneration. More than 400 shoots/plantlets have been obtained from 6 independent experiments. Over 150 plants have been transferred to the greenhouse. Protoplasts isolated from the youngest suspensions (5.5 months old) gave rise to the largest number of plants. Protoplasts isolated from suspensions as old as 15 months were also regenerable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - L1, L2 medium according to Lazzeri et al. 1991 - L3 medium medium according to Jähne et al. 1991a     

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

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

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)     

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.