Transformation of passionfruit (Passiflora edulis fv flavicarpa Degener.) using Agrobacterium tumefaciens

Leaf and stem explants of passionfruit (Passiflora eadulis fv flavicarpa) were co-cultivated with a disarmed strain of Agrobacterium tunefaciens harbouring the co-integrate vector pMON200. Four plants of passionfruit were regenerated from leaf explants on agar-solidified Murashige and Skoog (1962) based medium containing 4.43 M 6-benzyl-aminopurine and supplemented with 86 M kanamycin sulphate. The four plants were rooted by transfer to MS based medium with 14.7 M 3-indolebutyric acid and 2.68 M -naphthyleneacetic acid for 7 d, followed by MS based medium lacking growth regulators. Both media used for rooting contained 172 M kanamycin sulphate. Rooted plants were potted and grown to maturity. Three of the plants synthesised nopaline and expressed neomycin phosphotransferase activity; DNA dot blot and polymerase chain reaction analyses confirmed the presence of the neomycin phosphotransferase gene in three plants.Abbreviations BAP 6-benzylaminopurine - CTAB hexadecy-Itrimethylammonium bromide - EDTA ethylenediaminetetraacetic acid - IBA 3-indolebutyric acid - MS Murashige and Skoog (1962) - NAA -naphthyleneacetic acid - NPTII neomycin phosphotransferase II - nptII neomycin phosphotransferase II gene - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl)aminomethane     

Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium

Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3–4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50–100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l^–1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control.Abbreviations BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - MS Murashige & Skoog (1962) medium - IBA indolebutyric acid - MES 2-N-morpholinoethane sulfonic acid     

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.     

Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D 2,4-dichlorphenoxyacetic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-banzylaminopurine - IPA isopentenyladenine - IPAR isopentenyladenosine - MES (2-[N-morpholino]) ethanesulfonic acid     

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.     

Efficient plant regeneration from cell suspension protoplasts of the woody medicinal plant Solanum dulcamara L. (bittersweet,woody nightshade)

Large populations of viable protoplasts were released from suspension cultured cells of the woody medicinal plant Solanum dulcamara (bittersweet, woody nightshade) when the cells were harvested 3 to 7 months after culture initiation and 4 to 5 days after transfer to fresh medium. A Bio-Gel p6 purified enzyme mixture enhanced the protoplast plating efficiency 6 fold compared to the unpurified mixture, without affecting protoplast yield. Agarose-solidified medium markedly improved protoplast division and colony formation, and enabled protoplasts to be plated at lower densities than in liquid medium. All protoplast-derived tissues produced shoots on MS based medium with 1.0 mgl^-1 zeatin. Shoots rooted readily on medium lacking phytohormones. Cytological examination revealed high chromosome stability of suspension cultured cells, of plants derived from such cells, and of protoplast-derived plants. The implication of these results is discussed in relation to the genetic manipulation of this pharmaceutically important plant.     

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.     

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

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

Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast.

Summary Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing 2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be transferred to soil after being acclimatized.     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1^–1 4-indole-3yl-butyric acid, 2.0 mg 1^–1 BAP, 0.2 mg 1^–1 gibberellic acid, 50 mg 1^–1 casein hydrolysate and 10 mg 1^–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - GA₃ gibberellic acid - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-but yric acid - IPE initial plating efficiency - NAA 1-naphthaleneacetic acid - f.wt. fresh weight - MES 2-N-morpholinoethane sulfonic acid - MS Murashige and Skoog (1962) - %PE % plating efficiency - PVP-10 polyvinylpyrrolidone (Av. MW 10,000) - FDA fluorescein diacetate     

Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration

Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 l droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).Abbreviations BAP 6-Benzylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA indole acetic acid - MES 2-(N-morpholino)- ethane sulfonic acid - NAA naphthalene acetic acid - PE plating efficiency     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

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     

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     

A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis sims. F. Flavicarpa Deg.)

Summary Mineral deficiency symptoms were observed in leaves of yellow passionfruit plantlets grown in MS medium (Murashige and Skoog, 1962) with 1.0 mg l⁻¹ (3.0 μM) gibberellic acid. Initially, leaves showed interveinal chlorosis, followed by bleaching of the leaves and retarded growth. Leaf mineral analysis was done and compared to mineral requirements suggested for passionfruit in the literature. Several modifications were made to the inorganic composition of MS medium, according to mineral deficiencies, mainly of Fe and Ca, and possible toxicity of Cl. The concentration of the elements in the new medium (MSM) was based on the mineral composition of leaves of healthy plants. The chemical equilibrium was checked using the software Geochem (Sposito and Mattigod, 1980) and final adjustments were made to ensure good availability of nutrients. To test the efficiency of the modified medium nodal segments were cultured in both MS and MSM supplemented with 3.0 mg l⁻¹ (13.3 μM) 6-benzyladenine. After three subcultures mineral analysis of the leaves was done. Severe mineral deficiency was observed on the leaves of plantlets cultured in MS, while plantlets cultivated in MSM had green leaves. A comparison of the mineral analysis of plantlets in both media showed a fairly large increase in Ca, Cu, Fe, Mg and S and decrease in levels of B and Cl in plantlets cultivated in MSM. A slight increase or decrease in other elements was also observed. Subculture of the chlorotic plantlets into MSM showed that the visual symptoms of mineral deficiency disappeared in 2–4 wk.     

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     

Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds.)

Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions.Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10^-3 to 10^-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid.     

Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue

Summary Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.Abbreviations BAP benzylaminopurine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - NOA naphthoxyacetic acid - PCR polymerase chain reaction - PVP polyvinylpyrrolidone - TDZ thidiazuron     

Highly efficlent plant regeneration from mesophyll protoplasts of Indian field cultivars of tomato (Lycopersicon esculentum)

Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (>9.0×10⁶ g f.wt.^-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - g f.wt. gram fresh weight - IAA indoleacetic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - Zea zeatin