In vitro propagation of banana (Musa spp.): initiation,proliferation and development of shoot-tip cultures on defined media

In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana cultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher if apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.     

Sepecial symposium: In vitro plant recalcitrance in vitro plant recalcitrance: An introduction

Summary In vitro recalcitrance is the inability of plant cells, tissues and organs to respond to tissue culture manipulations. With respect to plant regeneration, recalcitrance can be a major limiting factor for the biotechnological exploitation of economically important plant species and it can also impair the wider application of in vitro conservation techniques. This first paper introduces a compilation of Symposium papers, collectively entitled “Do we understand in vitro plant recalcitrance?”, presented at the 1999 Congress of the Society for In Vitro Biology. The Symposium reviewed recalcitrance in the context of genetic predeterminism, molecular markers and gene expression patterns, whole and explant physiology, stress physiology, habituation, neoplastic progression and plant cancer. The symposium contributors present fundamental and applied investigative approaches which have the potential to enhance our current understanding of in vitro recalcitrance and to assist in overcoming the problems associated with nonresponsive plant cultures. This introductory paper presents the general concept of recalcitrance in relation to whole-plant and explant physiology and considers basic aspects of tissue culture manipulations in the context of recalcitrance problems.     

Benzyladenine-induced inhibition of flowering in Chenopodium rubrum in vitro is not related to the levels of isoprenoid cytokinins

The effect of 6-benzylaminopurine (BAP) on floweringand on endogenous levels of isoprenoid cytokinins wasinvestigated in explanted terminal shoots of Chenopodium rubrum cultivated in vitro. Themother plants were grown under continuous light andexplants were cut off when the 6th leaf primordiumoriginated at the shoot apex. The explants wereexposed to one dark period of 13 hours inductive forflowering or to continuous light on medium with orwithout BAP (0.05;0.2;0.4 mg.l^-1). Undernon-inductive conditions no flowering was observedeither in the control or after BAP treatment. Afterreceiving one inductive dark period, the controlexplants flowered. However, BAP application either atthe beginning of the inductive dark period and/orduring the following light cultivation inhibitedflowering and stimulated initiation and growth of leafprimordia. In the case of the most efficient BAPconcentration (0.05 mg.l^-1) flowering wasinhibited by 80% and the number of leaf primordia wasincreased by 3. Explantation caused a significantincrease in the total amount of endogenous cytokininsin the explants within first 13 h, provided they werekept in light. When explants were kept in darkness,only a slight increase in cytokinin levels wasobserved. BAP treatment had no influence on the levelsof endogenous cytokinins either in light or indarkness. We may thus conclude, that BAP applicationinhibited flowering of photoperiodically inducedterminal shoot explants and stimulated leaf primordiaformation with no significant effect on changes inlevels of endogenous isoprenoid cytokinins. This maysuggest the direct ability of BAP to regulate morphogenesis.     

Somatic embryogenesis from callus cultures ofNardostachys jatamansi

Callus cultures ofNardostachys jatamansi DC, an endangered medicinal and aromatic plant, were established using petiole explants on MS medium supplemented with 16.1 µM -naphthaleneacetic acid and 1.16 µM kinetin. Embryogenesis in these callus cultures took place only upon sequential subculture of the callus on media having gradually decreasing auxin (16.1 to 1.34 µM NAA) and simultaneously increasing cytokinin (1.16 to 9.30 µM kinetin) concentrations over a period of 7 months. Somatic embryo to plantlet conversion took place on a medium containing 9.30 µM kinetin and 1.34 µM NAA.     

Ontogeny of in vitro rooting processes in Eucalyptus globulus

Summary In the present work, histological changes observed at the base of Eucalyptus globulus shoots in in vitro culture are described. Shoots were placed on solidified Murashige and Skoog medium containing half the original salt concentration, the complete vitamin composition, 9.8 μM indolebutyric acid (IBA), and 30 gI⁻¹ agar, and were incubated in the dark for the first 7 d, followed by a 16-h photoperiod. In vitro-generated roots could be originated either from old vascular tissue or from newly formed xylem. The influence of the preexistent tissues on the neoformation process appeared to be varied. The medulla did not intervene directly, although there were abundant cellular divisions in response to the induction medium. On the other hand, the interruptions observed in the vascular cylinder of the stem suggested an influence of the interfascicular parenchyma, and therefore the medulla could have participated in the differentiation process. However, the cortical parenchyma showed most of the changes that lead to the formation of adventitious roots of E. globulus growing in vitro. Histological analysis suggests that vascular rays can also be formed in direct contact with the central cylinder of the stem, although they mainly originate from the cortical parenchyma.     

Age and Physiological Condition of Donor Plants Affect in vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa

The regeneration potential of D.alata L. germplasm preserved in vitro was compared with the micropropagation of fresh material. Nodal cuttings were conserved for 9 months in different treatments based on D-571 culture medium modified, using several variable components (mannitol, benzylaminopurine and activated charcoal). Regeneration at 8 weeks, assessed by means of percentage of explant regenerating and the multiplication at 5 weeks through the shoot length and de novo bud count formation per explant were determined. The results showed high rates (100 and 98%) of explant regeneration and micropropagation from in vitro material maintained in D-571 medium with 1.5% of mannitol + 0.1 or 1 mg l^–1 of benzylaminopurine + 2 g l^–1 of activated charcoal, respectively.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis>: influence of cytokinins

The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA₃) was applied post-flower-emergence.     

Somaclonal Variation in Rice after Two Successive Cycles of Mature Embryo Derived Callus Culture in the Presence of NaCl

Two successive cycles of mature embryo-derived callus culture separated by one cycle of sexual reproduction of R₀ regenerated plants were performed using two rice (Oryza sativa L.) cultivars in order to gain information upon the nature of somaclonal variation in this species. Plants regenerated after one cycle of tissue culture exhibited higher variability and lower performances than those of initial cultivar. A second cycle performed using R₁ embryos as explants showed that the cellular component of salt resistance in terms of growth and regenerating abilities selected during the first cycle could be transmitted to the progenies. The extent and the nature of somaclonal variation depended on the identity of R₀ mother plant and culture conditions, somaclonal variation being strongly reduced in some families obtained from salt-treated calli.     

Alterations in the pathogenicity of oneParacoccidioides brasiliensis isolate do not correlative with itsin vitro growth

Thein vitro subcultivation of some microorganisms for long periods causes measurable loss of their pathogenicity, which can be reverted by reisolation from infected hosts. We compared the pathogenicity and thein vitro growth pattern of oneP. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuousin vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B 10.A mice (RS). 4) Pb 18 (AT) reisolated from resistant A/SN mice (RR). Pathogenicity was evaluated by anatomopathology and mortality of mice infected i.p. with 5×10⁶ fungi. Median survival times of mice infected with OP ranged from 74 to 117 days during the first 51 months of subculturing; with more cycles of subculturing the median survival time increased, reaching 250 days at the 64^th month. This indicated decreasing virulence of OP during this period of subculturing. Survival of mice infected with RS and RR was respectively 112 and 123 days, which is similar to the behavior of the OP variant. Thein vitro growth curve profile of RR showed significantly higher numbers of total and viable yeasts than the other studied variant. These results show that: 1) Pb 18 isolate loses its pathogenicity by continuous subcultivation. This phenomenon is reverted by reisolation from mice, independently from their susceptibility to the fungus; 2) thein vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the host's environment.     

Preliminary results on in vitro rearing of the endoparasitoid Cardiochiles nigriceps from egg to second instar

The composition of an artificial medium and technical procedures used for in vitro rearing of the endophagous larval parasitoid Cardiochiles nigriceps Viereck (Hymenoptera, Braconidae), from post-germ band egg to the 2nd instar larva, are described. Amino acids, carbohydrates, salts, and vitamins were supplied in defined amounts as an aqueous solution which, when supplemented with 20 mg/ml of bovine albumin, 5 mg/ml of lactalbumin (enzymatic hydrolysate), 20% (v/v) fetal bovine serum, 20% (v/v) milk and 10% (v/v) chicken egg yolk, allowed for parasitoid growth and molting to the 2nd instar. Molting to the final instar was never observed.     

Development of dormancy in different lily genotypes regenerated in vitro

Dormancy development in four Lilium genotypes,L. speciosum, Star Gazer, C. King and Snow Queenregenerated in vitro was compared. Major factorsinfluencing dormancy development were the same for different genotypes andespecially L. speciosum and Star Gazer, that are closelyrelated, reacted similarly. Temperature was the main factor in dormancyinduction and breaking. The range of temperatures that induced dormancy and thelevel of dormancy that developed differed per genotype. In Star Gazer, dormancydeveloped gradually but in Snow Queen, dormancy developed very fast. Thereactions to temperature, reflected the climate in the area of origin. Abscisicacid deepened the level of dormancy induced by temperature but had no effectunder non-inductive temperature conditions. When abscisic acid synthesis wasblocked, no dormancy developed. Dormancy in all genotypes was broken by coldincubation for severalweeks. The cold requirement of the genotypes differed in line with the naturalwinter conditions in their habitat. The effect of hormones on dormancy breakingwas also investigated. A gibberellin treatment of 24 h brokedormancy in L. speciosum, Star Gazer and Snow Queen.     

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

Micropropagation of Cardiospermum Halicacabum

The in vitro studies with Cardiospermum halicacabum indicated that the different explants, i.e cotyledon, hypocotyl, cotyledonary node, leaf, internode and node had the potential to produce calli on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) and napthalene acetic acid (NAA). Calli of different explant origin showed variable growth responses on different BAP concentrations. The shoots were favourably formed from the calli of leaf and cotyledon explants. The maximum number of shoots were produced from calli subcultured on MS + BAP (17.8 µM). The roots were initiated on growth regulator free MS medium.     

An ultrastructural and electrophysiological study of the development of neuro-muscular junctions between previously dissociated foetal rat cells in vitro

Summary The development of neuro-muscular junctions between previously dissociated foetal rat spinal cord and somatic muscle has been investigated. The first indications of junction formation, both ultrastructurally and electrophysiologically, were observed after circa 18 days in vitro. The junctions contained numerous vesicles, but no secondary folds were developed even after 6 weeks in culture, and synaptic densities were not well marked. Functional endplates were found, and action potentials, endplate potentials and miniature endplate potentials recorded.The authors wish to thank Mr. D. Fraser, B. Sc., for valued technical help, and Mr. S. Waterman for photographic printing.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.