An improved micropropagation system for Chrysanthemum based on Pluronic F-68-supplemented media

Supplementation of MS-based medium containing 4.4 M 6-benzyladenine and 2.7 M -naphthaleneacetic acid with 0.001–0.1% (w/v) of the non-ionic, co-polymer surfactant, Pluronic F-68, significantly (p<0.05) increased the mean fresh weight gain of cultured leaf explants of chrysanthemum (Dendranthema grandiflora) Tone Maid and Early Charm by a maximum of 74% and 34%, respectively. The percentage of individual explants giving adventive shoots was also stimulated by Pluronic F-68; for cv. Early Charm, 0.001% (w/v) Pluronic F-68 induced the maximum response, whereas for cv. Tone Maid, the maximum response occurred with 0.01% surfactant. Shoot regeneration from explants was also enhanced at these concentrations of surfactant, compared to explants cultured in the absence of Pluronic F-68.Abbreviations BA 6-benzyladenine - MS Murashige & Skoog 1962 - NAA -naphthaleneacetic acid     

Effects of Pluronic F-68 on shoot regeneration from cultured jute cotyledons and on growth of transformed roots

The effects have been studied of the non-ionic surfactant, Pluronic F-68, on the growth in culture of jute (Corchorus capsularis L.) cotyledons with attached petioles, cotyledon explants and transformed roots. Supplementation of culture medium with 0.001–0.5% (w/v) of either commercial grade Pluronic F-68 or a purified fraction prepared by passage through silica gel, stimulated shoot production from the petioles of C. capsularis var. D154 and C134 cotyledons. This effect was most marked in C134, because of the failure of control cotyledons to produce shoots in the absence of Pluronic. Plants regenerated from Pluronic-treated cotyledons were morphologically normal. Growth of transformed roots of C. capsularis var. D154 was stimulated in medium supplemented with commercial grade or purified Pluronic F-68, with maximum increases in both fresh and dry weights with 0.1% (w/v) of the surfactant. Roots cultured in the presence of Pluronic F-68 could be maintained without sub-culture for up to 70 days, whereas roots cultured in the absence of Pluronic required subculture every 7 days, to prevent necrosis. Transformed roots also produced callus in the presence of 0.001–1.0% (w/v) of either commercial grade or purified Pluronic. The biotechnological implications of these results are discussed in relation to the potential value of non-ionic surfactants as growth-stimulating additives to plant culture media.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog (1962)     

Pluronic F‐68 Enhanced Shoot Regeneration in Micropropagated Citrus Rootstock and Passiflora Species

The promotory effects have been studied of the non‐ionic surfactant, Pluronic F‐68, on bud induction/shoot regeneration in epicotyl and cotyledon explants of Citrus depressa and on shoot regeneration from leaf segments of 4–6 week‐old axenic nodal segment‐derived in vitro plants of Passiflora mollissima, P. giberti and P. edulis var. flavicarpa. For epicotyls of C. depressa, supplementation of agar‐solidified MS‐based bud induction/shoot regeneration medium with 0.5% [w/v] Pluronic F‐68 significantly (P < 0.05) increased mean fresh weight gain of cultures, percentage of explants giving shoots and number of shoots per explant. The same Pluronic concentration also enhanced the mean percentage of cotyledons exhibiting bud induction and the number of buds regenerated per cotyledon explant. Fresh weight gain was unaffected across the range of concentrations (0.001–0.5% w/v) of Pluronic F‐68 evaluated for this latter explant source. For leaf explants from axenic shoot cultures of P. mollissima, supplementation of NN‐based medium, containing 3 mg/l 6‐benzyladenine and 2.0 mg/l kinetin with 0.001–0.5% [w/v] Pluronic F‐68, significantly (P < 0.05) increased mean (± s.e.m.) biomass gain by a maximum of 2.7 ± 0.1 g fresh weight (g f.wt.) over the control. Similarly, for leaf explants of P. giberti, 0.001–0.5% [w/v] Pluronic F‐68 in MS‐based medium, containing 1.0 mg/l 6‐BAP and 0.5 mg/l kinetin significantly (P < 0.05) increased mean percentage of explants undergoing shoot regeneration. For P. edulis leaf explants, mean f.wt. gain was also significantly (P < 0.05) higher with Pluronic F‐68 at 0.001–0.5% [w/v].     

Stimulation of multiple shoot regeneration from seedling leaves of Hypericum perforatum L. by pluronic F-68

The effects of the non-ionic surfactant, Pluronic F-68, on the growth of shoots regenerated from seedlings (14 days post-germination) of Hypericum perforatum L. were studied. The supplementation of agar-solidified medium with 0.001% (w/v) of Pluronic increased the mean fresh weight of the regenerants after 60 days by 40% and the mean number of plant regenerants recovered per seedling by 34%; a less pronounced increase in the number of regenerants occurred with 0.01% (w/v) of the surfactant. By contrast, the mean fresh weight of the regenerants cultured in the presence of 0.1% (w/v) Pluronic F-68 was 15% lower than untreated controls, although the mean number of regenerants per seedling remained unaltered. The growth of seedling leaf-derived Hypericum callus after 60 days was unaffected by all the concentrations of Pluronic tested. However, there was a tendency for callus cells grown in the presence of Pluronic to be more highly pigmented with anthocyanins. The cultivation of leaf explants with 0.001% or 0.01% (w/v) Pluronic did not affect either the mean fresh weight of the regenerants or the mean number of regenerants per explant. However, decreases in both the mean fresh weight and the mean number of regenerants (both 33.0% lower than the control) occurred following the cultivation with 0.1% (w/v) Pluronic.     

Influence of surfactants on growth and regeneration from mature internodal stem segments of sweet orange (<Emphasis Type="Italic">Citrus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis>) cv. Hamlin

The development of a reliable shoot regeneration system for mature tissue of citrus is of major importance to accelerate the evaluation of commercial traits. Three non-ionic surfactants were evaluated independently in terms of their affects on the growth and regeneration of mature internodal stem segments of sweet orange cv. Hamlin in culture. Growth and shoot development of explants were influenced by type of surfactant added to the regeneration medium DBA3, its concentration and order of flush growth used for explant preparation. Supplementation of Pluronic F-68 at 0.001% (w/v) to the medium was the superior treatment resulting in significantly higher fresh weight gain of explant, improved mean number of shoots per explant and the percentage of explants giving shoots (33.5% from first flush) and shoot yield was twofold higher compared to treatments without surfactant (17%). Triton X-100 was the least responsive in terms of its affect on the growth and regeneration of stem segments but such shoots had a normal phenotype. Explants cultured on DBA3 medium containing Tween 20 exhibited growth and shoot yield similar to treatments without surfactant, but at concentrations 0.01–0.5% (v/v), the shoots became vitrified and failed to graft successfully in vivo. Growth and shoot yield of explants showed a general decline between flushes especially from second and third harvests. Shoots derived from stem segments which were cultured on media containing Pluronic F-68 and no surfactant had a higher survival rate (70–80%, respectively) compared to treatments using Triton X-100 at 0.001–0.1% (v/v) (33% survival). All acclimatized grafts exhibited typical mature wood characteristics and flowered 14–16 months after transfer to the greenhouse.     

Effects of Pluronic F-68 on growth of transformed roots ofSolanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of transformed roots ofSolanum dulcamara L. have been studied. Growth was stimulated by addition of low concentrations (0.001–0.1% w/v) of freshly-prepared commercial grade Pluronic to liquid culture medium, with maximum increases in root fresh and dry weights at 0.01% (w/v). In contrast, higher concentrations (0.25–1.00% w/v) of freshly-prepared Pluronic inhibited growth. Freshly-prepared purified Pluronic retarded root growth, even at concentrations that were stimulatory with the commercial preparation. Similarly, commercial grade Pluronic solutions stored at 4°C or 22°C for 5 days (aged) were inhibitory to root growth.     

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.Abbreviations MS Murashige and Skoog - BAP 6-Benzylaminopurine - TDZ Thidiazuron     

Strategies for promoting division of cultured plant protoplasts: synergistic beneficial effects of haemoglobin (Erythrogen) and Pluronic F-68

Novel approaches, involving supplementation of aqueous culture medium with haemoglobin solution (Erythrogen), in the presence or absence of the copolymer surfactant, Pluronic F-68, have been evaluated to facilitate cellular oxygen availability to promote mitotic division. Cell-suspension-derived protoplasts of albino Petunia hybrida cv. Comanche were cultured for up to 45 days in KM8P medium containing 1:50–1:500 (vol:vol) Erythrogen. The mean initial protoplast plating efficiency after 9 days with 1:50 Erythrogen (18.5%) was significantly greater (P<0.05) than in untreated controls (11.3%). Supplementation of culture medium with 1:50 Erythrogen, together with 0.01% (wt/vol) Pluronic F-68, increased the mean plating efficiency after 9 days (24.4%) by 92% (P<0.05) over the control (12.7%). These treatments also produced increases in biomass of protoplast-derived cells up to 2.5-fold greater than control (P<0.01) over 80 days. Gassing the medium, containing 1:50 Erythrogen, with carbon monoxide abolished the increase in plating efficiency. There was no additional benefit of gassing Erythrogen-supplemented medium with 100% oxygen. The synergistic, beneficial effect of Erythrogen and Pluronic F-68 on protoplast division has implications for plant biotechnology utilising protoplasts. Received: 24 May 1996 / Revision received: 12 July 1996 / Accepted: 15 August 1996     

Pluronic F-68: an answer for shoot regeneration recalcitrance in microspore-derived <Emphasis Type="Italic">Brassica napus</Emphasis> embryos

Recalcitrance to tissue culture is observed in some genotypes of Brassica napus. Several studies have confirmed that Pluronic F-68 has growth-promoting effects on numerous tissue types. This work investigated the effect of the non-ionic surfactant Pluronic F-68 at four concentrations (0.1%, 0.25%, 0.5%, and 1% (w/v)) on the responsiveness of recalcitrant B. napus lines to tissue culture. Microspores from seven populations of B. napus were cultured on Nitsch and Nitsch medium with this compound. The embryos obtained were plated on solid B5 medium supplemented with zeatin for shoot induction. Pluronic F-68 had a highly significant effect on the proportion of shoot regeneration (P < 0.05) in some of the recalcitrant populations. However, no strong dose–response effect was observed. The estimated probability of a shoot occurring in the absence of Pluronic F-68 ranged from 0.04 to 0.31 depending on the genotype, while in the presence of Pluronic F-68, it ranged from 0.07 to 0.53, respectively.     

In vitro multiplication of Calophyllum apetalum (Clusiaceae), an endemic medicinal tree of the Western Ghats

Young shoots collected from mature trees of Calophyllum apetalum during the flush season (November–February) were red (1–2 weeks), pinkish-red (3–5 weeks), pale green (6–8 weeks) and dark green (9–10 weeks) coloured after different periods of growth. All the shoot tip and single node explants of the youngest 1–2-week-old shoots cultured in Murashige and Skoog's (1962) agar medium were lost due to excessive browning and necrosis; nodes of the 6–8-week-old shoots subjected to transfers twice a week in fresh medium containing 8.8 ;M BAP responded the most (68% of explants) with the formation of 3.2 shoots per explant in 7 weeks. The shoot tip was a relatively poor source of regeneration (2.3 shoots per explant; 39% of explants). Subculturing of explants from in vitro derived shoots for 5 weeks in medium containing 4.4 M BAP resulted in the formation of an increased number and percentage of shoots in the nodes (5.3 per explant; 74% of explants). The shoot cultures were transferred to 1/2 MS basal medium for 4 weeks to induce the elongation of shoots (;3.0 cm). Rooting of the microshoots (>2.0 cm) was achieved when cultured in quarter strength MS medium supplemented with 9.8 ;M IBA for 4 weeks followed by transfer to 1/4 MS basal medium for 4 weeks. The rooted plantlets transferred to clay pots filled with soil, sand and farmyard manure (1:1:1), maintained in a mist chamber at a relative humidity of 80–90%, acclimatised at a 56% rate after 6 weeks. Out of 345 plants restored to their native habitat in the forest at three locations of the institute campus, 293 plants survived and showed uniform growth free of morphological defects.     

Agrobacterium-mediated transformation of strawberry calli and recovery of transgenic plants

Transformed calli and shoots of strawberry (Fragaria × ananassa Duch.) cv. Redcoat were obtained using Agrobacterium tumefaciens carrying plasmid pB1121. Inoculated leaf explants produced transgenic calli at a frequency of 3% on selection medium containing 50 g/ml kanamycin. Twenty per cent of selected caili regenerated, giving rise to transgenic shoots. All transgenic calli and shoots expressed substantial amounts of GUS and NPT-II activity. The Southern blot analysis confirmed the insertion of both marker genes into the strawberry genome as single and multiple copy inserts. The transgenic shoots elongated on rooting medium in the presence of 25 g/ml kanamycin, but exhibited reduced rooting ability.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NPT-II neomycin phosphotransferase(EC 2.7.1.95) - GUS -glucuronidase(EC 3.2.1.31) - X-GLUC 5-bromo-4-chloro-3-indolyl-glucuronide - 4-MU 4-methylumbelliferone NRCC No. 31227     

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from half-seed explants of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.Abbreviations BA benzylamino purine - IAA indole-3-acetic acid - 2iP 6--dimethyl (allyl) amino purine - KIN kinetin - MS Murashige and Skoog medium - NAA napthaleneacetic acid - ZEA zeatin     

Stimulation of shoot regeneration from jute cotyledons cultured with non-ionic surfactants and relationship to physico-chemical properties

Summary The effects have been studied of the non-ionic surfactants, Plutonic F-68, Tween 20 or Triton X-100, on shoot regeneration from cultured jute (Corchorus capsularis L.) cotyledons with attached petioles. This group of non-ionic surfactants was selected in order to determine a possible relationship between the physico-chemical properties of individual compounds and their observable effects on plant morphogenesis in cultured jute cotyledons. Supplementation of culture medium with 0.001–0.5% (w/v) Pluronic F-68 increased the mean percentage of cotyledons producing shoots and the mean number of shoots/cotyledon, with maximal responses at 0.5% (w/v). By contrast, Tween 20 produced maximal effects at 0.001% (v/v), with inhibition of shoot formation at 0.5% (v/v). In both cases, phenotypically normal plants were recovered which could be grown to maturity. Culture of cotyledons with 0.001% (v/v) Triton X-100 similarly increased both the percentage of cotyledons producing shoots and the number of shoots/cotyledon. However, these shoots did not develop into mature plants. Additionally, shoots did not regenerate from cotyledons cultured with Triton at 0.01–0.5% (v/v). These results demonstrate mat there is an apparent relationship between the hydrophilic-hydrophobic (HLB) balance of surfactants which determine their cell permeabilising properties and consequently, their effects on morphogenesis.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

Effect of in vitro storage at 4 °C on survival and proliferation of poplar shoots

Shoot explants of in vitro proliferating cultures of Populus tremula (L.) x Populus tremuloides (L.) were stored for three months at 4°C, in dark or light, in basal culture medium with or without 2-isopentenyladenine (2iP), and in rooting medium with naphthalene acetic acid. They were transferred to cold at different times after subculturing. One hundred percent of shoots survived all tested conditions, in spite of leaf browing and necrosis. After transfer to 24°C for 2 weeks and a normal multiplication cycle, the shoots proliferated at a rate similar to controls or at a higher rate in the case of shoots introduced into the cold 7 or 14 days after subculture and stored in dark on medium containing 2iP.Abbreviations 2iP 2-isopentenyladenine - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

Enhancement of shoot regeneration from cotyledon explants of Brassica rapa ssp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors

The presence of benzyladenine or naphthaleneacetic acid in seed germination medium markedly enhanced subsequent shoot regeneration from the base of the excised cotyledon explants of Brassica rapa cv. Horizon. Cotyledon explants from younger seedlings (3 or 4-day old) produced more shoots than those from older seedlings. Addition of the ethylene inhibitor aminoethoxyvinylglycine (1.0 M) to the regeneration medium improved shoot regeneration three fold.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - MGBG methylglyoxal-bisguanylhydrazone - MSBN ms (murashige & skoog 1962) medium supplemented with 4.4 m BA & 5.4 m NAA, 2% sucrose - NAA naphthaleneacetic acid     

Propagation of Gasteria croucheri Bak. from shoot producing callus

Leaf explants of Gasteria croucheri Bak. cultured on a modified Murashige and Skoog medium supplemented with 8.8 × 10^-6M (2 mgl^-1) benzyladenine and 2.7 × 10^-5M (5 mgl^-1) -naphthalene acetic acid yielded a callus which produced adventitious shoots. This callus could be maintained on a medium with 1.9 × 10^-5M (4 mgl^-1) kinetin only and continued to produce shoots which could be rooted on a hormone free medium. Plantlets could be readily acclimatized.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Feronia limonia</Emphasis> (L.) Swingle from hypocotyl and internodal explants

The hypocotyl and internodal segments from in vitro grown seedlings of Feronia limonia (L.) Swingle (wood apple) were cultivated on Murashige and Skoogs (1962, MS) medium supplemented with N⁶-benzyladenine (BA) or adenine (ADE) or kinetin (KN) at 0.5 to 5 µM. The optimum response was recorded on the medium containing 2 µM BA. An average of 12 and 8 shoots were developed from hypocotyl and internodal explants, respectively, after eight weeks of culture. The shoots were excised, and the residual explants were transferred to fresh medium where again they developed shoots. Up to three such passages resulted in the production of shoots from repeatedly subcultured explants and an average of 24 – 36 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to half strength MS medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). The developed plantlets were successfully transferred to mixture of soil, sand and coco-peat (1:1:1) and hardened in controlled environment. Hardened plants were transplanted to soil in greenhouse.     

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. Russet Burbank. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.     

Image analysis assessments of perfluorocarbon- and surfactant-enhanced protoplast division

Image analysis has been used to assess the growth of cell suspension-derived protoplasts of Petunia hybrida cv. Comanche at an interface between aqueous culture medium (KM8P), supplemented with 0.01% (w/v) Pluronic F-68, and oxygenated (10 mbar; 10 min) perfluorodecalin. Protoplasts synthesised a new cell wall and entered normal mitotic division which was sustainable to the cell colony/callus stage. This process was accentuated by the collective and additive effects of oxygen, perfluorodecalin and surfactant media supplements. The mean area (mm³) of protoplast-derived cell colonies after 68 days of growth was increased 35 fold over control (media alone) in the presence of these combined treatments. The new cultural regime, leading to improved cell throughput from protoplasts, is discussed primarily in relation to the role of perfluorodecalin as a gas carrier and possible effects of Pluronic F-68 in stimulating cellular uptake of nutrients and/or growth regulators. Image analysis provides a novel and accurate approach to quantifying cell growth responses.Abbreviations dpi dots per inch - FPE final plating efficiency - IPE initial plating efficiency - KM Kao & Michayluk (1975) - PFC Perfluorocarbon - UM Uchimiya & Murashige (1974)