Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)

The induction, origin, morphology, and ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C. Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%, 0.1%, 1%, or 2% sucrose. A low sucrose concentration (0.01%) and wounding of the adaxial side of the leaf significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes). Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both forms had the same ploidy level. On the basis of these findings, we propose a set of conditions which regularly and reproducibly induces apospory on most of the leaf explants of the fern P. bifurcatum.     

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500–1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

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     

Activated charcoal affects morphogenesis and enhances sporophyte regeneration during leaf cell suspension culture of Platycerium bifurcatum

In a leaf cell suspension culture of Platycerium bifurcatum, the incorporation of activated charcoal (AC) greatly increased the number of regenerated sporophytes even in growth regulator-free medium. The degree of improvement was dependent on cell aggregate sizes and medium composition. The maximal increase was observed in medium with 5.37 μM NAA and 4.44 μM BA, from 9 to 1520 sporophytes. The qualitative improvement by AC included: (1) regeneration of single sporophytes, which was relatively less frequent in non-AC media, (2) prevention of the formation of gametophyte clusters prior to sporophyte regeneration from 30- to 60-μm cells cultured in MS basal medium, (3) prevention of the formation of bud clusters, sporophytes with multiple bud primordia, `nodule'-like bud clusters in growth regulator containing media, and (4) prevention of the occurrence of hyperhydricity of regenerated sporophytes. Received: 9 December 1996 / Revision received: 18 April 1997 / Accepted: 5 July 1997     

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     

The impact of a pulse treatment of penicillin-G and streptomycin sulfate on sporophyte regeneration of Platycerium bifurcatum

 This study was aimed at investigating the impact of antibiotic pulse treatment (APT) of penicillin-G and streptomycin sulfate at a concentration of 1000 mg/l each on the regeneration ability of leaf and shoot-base explants and homogenized sporophytes of Platycerium bifurcatum. The reaction of the plant to APT, as measured by changes in sporophyte regeneration and morphology, was considered to be a stress response. Cell's susceptibility to APT was graded as: tissue homogenized prior to treatment>tissue homogenized after treatment>leaf and shoot-base explants. Biphasic inhibition in sporophyte regeneration was observed in homogenized sporophytes; a sharper decrease occurred with 20–60 min of the APT, followed by a slower decline for up to 180 min. No significant impact on the regeneration ability of leaf and shoot-base explants was found. Morphologically, APT caused leaf cleavage, mesophyll damage and hyperhydricity in some sporophytes. Except for the hyperhydric sporophytes, regenerants were able to continue their growth in soil, and no carry-over effect was found in the next generation. Received: 9 October 1998 / Revision received: 10 May 1999 / Accepted: 31 May 1999     

Regeneration of photosystem II and phycobilin pigments in photoorganotrophically grown Anabaena variabilis

Regeneration of photosynthetic activity and phycobilin pigmentswas studied with cells of Anabaena variabilis lacking photosystemII activity and phycobilin pigments. Regeneration was achievedonly when the cells were incubated in the presence of nitrateor nitrite. The addition of ammonium salts or urea was far lesseffective. Nitrate-directed regeneration was independent oflight and inhibited by chlorate. Dark-regenerated cells, however,differed from light-regenerated ones in that the former wereincapable of excitation transfer from phycocyanin to pigmentsystemII chlorophyll a, although they emitted fluorescence of pigmentsystem II chlorophyll a origin, if illuminated by the lightabsorbed by chlorophyll. The regeneration process inAnabaenacells is assumed to consist of two steps: [1] light-independent,nitratesupported synthesis of phycobilin pigments and photosystemII integrity, followed by [2] light-directed formation of excitationtransfer from phycocyanin to pigment system II chlorophyll a.An antibiotic study revealed that the former is associated withprotein synthesis, while the latter isnot. ¹ Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164, Japan. (Received November 19, 1975; )     

Rigenerazione e totipotenza

Abstract

Regeneration and totipotency.—Regeneration and totipotency of cells and nuclei in plants and animals are discussed. The paper consists of the following sections: regeneration in coelenterates and planarians; limb regeneration in amphibians; regeneration of mammalian liver; Wolffian lens regeneration in urodeles; nuclear transplantation in animal eggs; regeneration of plants from single cells (in vivo; in vitro; from somatic cell hybrids); general remarks.     

Development of mantle leaves inPlatycerium bifurcatum (Polypodiaceae)

InPlatycerium bifurcatum the leaf primordia emerge alternately right and left below the shoot apical cell on the dorsal surface of the rhizome. They arise from groups of small cells, a single large cell becoming the initial of the leaf apical cell. The longitudinal axis of the leaf apical cell is at a right angle to the rhizome axis and the leaf primordia are arranged longitudinally in two rows. The leaf apical cell gives rise to marginal initials which are responsible for leaf growth in one plain. Early marginal cells are crescent-shaped while the later ones are wedge-shaped. Hairy marginal cells appear in the very early stages of development. The interpretation of these cells as a promeristem and as initials are discussed.     

Experimental Studies of Apospory in Ferns

Apospory in Pteridium aquilinum was found to have several novelfeatures, in particular the rapidity of its occurrence. Outgrowthsfrom detached laminae were detected in as little as 3 days.There was no evidence that failure of co-ordination betweencells, senescence of the sporophytic tissue or unfavourableculture conditions effected the initiation of outgrowths asearlier workers have suggested. The response of Thelypterispalustris was notably slower and more in line with earlier reportsof apospory. Marsilea vestita appeared to be incapable of anyform of aposporous behaviour. The only feature common to all occurrences of apospory in leavesof wild-type ferns is the severance of the lamina from the mainbody of the plant. It is proposed that this may deprive thecells of regulatory substances which maintain the activationof sporophytic genes. The results are also considered in relationto the phase changes in homosporous and heterosporous plantsand the behaviour of fern protoplasts in culture. apospory, ferns, Pteridium aquilinum, Marsilea vestita, Thelypteris palustris     

Experimentally Synthesized Plant Chimeras I. In vitro Recovery of Nicotiana tabacum L. Chimeras from Mixed Callus Cultures

Experiments were conducted to develop techniques for synthesizingchimeras between plants of known genotype by utilizing in vitrotechniques Chimeral calli composed of green and albino tobaccocells were obtained by initiating callus tissue from mixturesof albino and green cotyledons, hypocotyls, callus culturesand cell suspensions The most effective mixing of genotypesoccurred when callus was derived from mixed filtered cell suspensionsUpon shoot regeneration, chimeral calli yielded 1317 non-chimeraland four chimeral plants Chimeras may have arisen as a resultof experimental procedures or possibly from spontaneous chromosomalabnormalities since leaves of some albino control plants occasionallyproduced small green islands of cells Explanations for the recoveryof a high percentage of non-chimeral shoots are presented Tobacco, callus cultures, cell suspensions, tissue culture, shoot apical meristems, somatic-crossing over     

In Vitro Selection for Salt Tolerance in Brassica juncea L. Using Cotyledon Explants, Callus and Cell Suspension Cultures

Salt-tolerant Brassica juncea L. cell lines or plants have beenselected by screening callus pieces, cell suspension culturesand cotyledon explants in vitro on high concentrations of NaCl.Callus-based selection was unsatisfactory, as only two out ofseven isolated clones retained tolerance after 3 months of subcultureon NaCl-free medium. Selections made via plated cell suspensionswere found to be more stable for salt-tolerance. AH selectedtolerant cell lines, however, failed to regenerate plantlets.A third selection method, employing cotyledon explants was basedon their high potential for regenerating multiple shoots. Outof a total of 2620 explants cultured on high salt media, threesurvived, showed sustained callus proliferation and each regeneratedone shoot. The salt-selected shoots withstood the stabilitytest after 3 months of growth and axillary bud multiplicationon NaCl-free medium. While one of these somaclones was morphologicallyabnormal and sterile, the other two could be reared to maturitywith normal seed set. Brassica juncea, tissue culture, in vitro selection, salt-tolerance, plant regeneration     

Plant regeneration from hypocotyls,cotyledons and stem segments of the C3–C4 intermediate species Moricandia arvensis

Techniques have been developed for the regeneration of Moricandia arvensis from complex explants. Hypocotyl segments and cotyledonary explants regenerated shoots, but the most efficient plant regeneration was from stem sections taken from in vitro grown shoots. Regeneration from these three explant types was tested on a range of concentrations of benzylaminopurine and either naphthylene acetic acid or indole acetic acid. Regeneration from all three explants was much higher on indole acetic acid than on naphthylene acetic acid and the ratio of auxin to cytokinin was also significant in determining the response of explants. Optimum regeneration was on 1mg/l IAA with 1mg/l BAP. Plants could be transferred to soil and grown to flowering in the glasshouse.Abbreviations GDC glycine decarboxylase - BAP benzyl aminopurine - NAA naphthalene acetic acid - IAA indole acetic acid     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phloem transdifferentiation from immature xylem cells during bark regeneration after girdling in Eucommia ulmoides Oliv

Eucommia ulmoides Oliv. (Eucommiaceae), a traditional Chinesemedicinal plant, was used to study phloem cell differentiationduring bark regeneration after girdling on a large scale. Hereit is shown that new sieve elements (SEs) appeared in the regeneratedtissues before the formation of wound cambium during bark regenerationafter girdling, and they could originate from the transdifferentiationof immature/differentiating axial xylem cells left on the trunk.Assays of water-cultured twigs revealed that girdling blockedsucrose transport until the formation of new SEs, and the regenerationof the functional SEs was not dependent on the substance providedby the axis system outside the girdled areas, while exogenousindole acetic acid (IAA) applied on the wound surface acceleratedSE differentiation. The experiments suggest that the immaturexylem cells can transdifferentiate into phloem cells under certainconditions, which means xylem and phloem cells might share someidentical features at the beginning of their differentiationpathway. This study also showed that the bark regeneration systemcould provide a novel method for studying xylem and phloem celldifferentiation. Key words: Bark regeneration, Eucommia ulmoides Oliv., immature xylem cells, sieve elements, transdifferentiation Received 19 November 2007; Revised 23 January 2008 Accepted 24 January 2008     

Experimentally Synthesized Plant Chimeras 2. A Comparison of In vitro and In vivo Techniques for the Production of Interspecific Nicotiana Chimeras

In vitro and in vivo techniques were compared for synthesizingchimeras between Nicotiana glauca Grahm and N tabacum L Interspecificchimeral callus, produced from mixed callus cultures in vitro,was placed on media which favoured only N tabacum shoot formationNone of the 474 regenerated N tabacum shoots incorporated Nglauca cells into their meristems When chimeral callus was regeneratedunder hormonal conditions favouring simultaneous organogenesis,of 397 shoots, only non-chimeral shoots of both species aroseIn vivo, reciprocal splice grafts between species were decapitatedjust above the graft union and treated with or without auxin—lanolinpastes Auxin increased callus formation but inhibited adventitiousshoot formation Three of 209 adventitious shoots arising fromthe graft union were interspecific mericlinal chimeras whichwere later stabilized as periclinal chimeras All three chimerasformed when N glauca was the understock Two of the chimerasarose on untreated shoots which produced no visible callus,indicating that excessive callus formation may be unnecessaryfor multiple cell origin of adventitious shoots to occur Chimeras, tobacco, Nicotiana glauca, Nicotiana tabacum, tissue culture, graft chimeras, callus cultures     

Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmen

Gametophyte-derived callus cultures of Platycerium coronariumcould be maintained under photoautotrophic conditions on Murashigeand Skoog medium supplemented with 2µM 2,4-dichlorophenoxyaceticacid (2,4-D) and with CO₂ enrichment. Progressive reductionof sucrose from the medium resulted in a reduction in growth,but an increase in total chlorophyll content. When subculturingwas delayed beyond 2 weeks, callus cells differentiated intogametophytes on the medium with 0.2 sucrose and no CO₂ enrichment.Enriching the photoautotrophic cultures on 2µM 2, 4-Dwith 1% CO₂ resulted in about 1.7-fold increase in fresh weightwithin 42 d. Total chlorophyll content was generally higherwith 1% CO₂ enrichment than with 10%. F_v/F_m ratio was higherfor callus on low levels of sucrose (>0.5%) than that onsucrose 1.0%. An increase in autofluorescence of chloroplasts,but not the size, was observed with decreasing sucrose levelsin the medium. Autofluorescence decreased with increase in CO₂from 0.03%. Our data are in agreement with the view that long-termexposure to high levels of decrease in photosynthetic capacity. Key words: Platycerium coronarium, stag's horn fern, autofluorescence of chloroplasts, confocal laser scanning microscope, F_v/F_m ratio, photoautotrophic callus     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luffa cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration     

Regeneration of Fertile Plants from Callus in Phaseolus polyanthus Greenman (Year Bean)

For the first time, plant regeneration of several domesticatedgenotypes of Phaseolus polyanthus Greenman (year bean) has beenachieved. Thidiazuron in combination with indole-3-acetic acidwas used to induce morphogenic, green nodular callus from explantsthat had been obtained either from greenhouse-grown plants orfrom in vitro -germinated seeds. Of the six genotypes of P.polyanthus tested, five produced shoots in vitro. Regeneratedshoots that formed roots in vitro were established in the greenhouse,whereas non-rooted shoots could be established in vitro by grafting.Morphologically normal progeny plants were obtained from thegreenhouse-established regenerants. However, by using the sameprocedure, no regeneration response was observed in two domesticatedand two wild genotypes of P. coccineus L. (runner bean). Thisprotocol should help achieve Agrobacterium - or particle bombardment-mediatedgenetic transformation to improve this important food legume.Copyright 2001 Annals of Botany Company Callus, genetic transformation, grafting, legumes, organogenesis, Phaseolus coccineus L., Phaseolus polyanthus Greenman, Phaseolus vulgaris L     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luff a cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration.