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     

Hormonal Control of Morphogenesis in Leaf Explants of Perilla frutescens Britton var. crispa Decaisne f. viridi-crispa Makino

Leaf discs of Perilla frutescens var. crispa f. viridi-crispawere cultured on a defined medium to investigate factors influencingbud and root formation, callus induction, somatic embryogenesis,and floral bud formation. Addition of naphthalene-acetic acid(NAA) to the culture medium caused compact callus whereas 2,4-dichlorophenoxyacetic acid (2,4-D) promoted soft and friable callus formationon the surface of the explants. Benzyladenine, when appliedwith auxin, suppressed callus and root formation. Somatic embryogenesisoccurred, when the explants were first grown on nutrient mediumcontaining 2,4-D and organic elements, and then transferredto the 2,4-D free medium. Treatments with cytokinins, N-phenyl-N'-(4-pyridyl)urea and its derivatives induced bud formation. A low concentrationof NAA and naphthoxy-acetic acid promoted bud development. Occasionalfloral bud formation was observed depending on the originalleaf positions on mother plants from which the leaf discs wereexcised. A gradient of floral bud forming capacity along thestem was noted. Perilla frutescens, tissue culture, embryogenesis, morphogenesis, benzyl adenine, kinetin, naphthalene-acetic acid, naphthoxy-acetic acid, 2,4-dichlorophenoxy acetic acid, indol-3yl-acetic acid, cytokinins, auxins     

Growth Behavior of a Liverwort, Jungermannia subulata Evans, in a Cell Suspension Culture. The Role of Organic Acids Required for Cell Growth

A green callus of a liverwort, Jungermannia subulata Evans,was induced from gametophytes, and a cell suspension culturewas obtained from the callus. Both callus and suspension-culturedcells grew in a modified Murashige and Skoog medium if organicacids of the TCA cycle were supplied. In the cell suspensionculture of J. subulata, ammonium was taken up preferentiallyby the cells particularly at the earliest stage of growth, whileonly a negligible amount of nitrate was utilized as long asammonium was present in the medium, and this unbalanced utilizationof the two nitrogen sources caused an abrupt drop in the pHof the medium. The organic acids of the TCA cycle supportedthe growth of this cell line by preventing the abrupt drop inthe pH of the medium. (Received June 13, 1981; Accepted October 8, 1981)     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

The Production of Plantlets from Tissue Cultures of Brussels Sprout (Brassica oleracea L. var gemmifera D.C.)

Shoots were induced on callus derived from sprout sections andpetiole slices of an inbred parent line of Brussels sprout (Brassicaoleracea L. var gemmifera D.C.). The shoots, when excised andtransferred to fresh medium, enlarged and formed roots. Theseplantlets could be transferred to soil or their number increasedby a multiplication process involving the production of newshoots from the dormant lateral buds. Some of the plantletsderived from sprout callus were grown to maturity in the fieldand their morphology and chromosome number compared to seedgrown plants. There were no significant differences in sproutsize and stem diameter but there were significant differencesin plant shape. None of the plants in the field experiment showedpolyploidy. Plants derived from callus possessed an enhanced ability toform callus and redifferentiate when sections from these plantswere placed back on to nutrient medium.     

Callus Induction and Plant Regeneration from Barley Mature Embryos

Callus cultures were induced starting from excised mature embryosin spring barley, Hordeum vulgare cv Maxima On a medium containinga high level of auxin, a first primary callus was induced whichwas friable, unorganized and capable of direct plant regenerationin the tested conditions This callus type was characterizedby fast growth and high variability in chromosome number Subsequently,a secondary callus type arose from the primary calli subculturedon the same medium in the light This callus type was white andcompact and consisted predominantly of diploid cells When transferredto hormone-free medium it gave rise to green shoots Completerooting of the shoots was achieved on half-strength basal mediumfollowed by exposure to higher light intensity Regenerated plantletscould then be transferred directly into soil without sufferingany loss in vitality Although showing different degrees in morphologicalvariability, they all maintained the diploid chromosome number Hordeum vulgare L, spring barley, morphogenic calli, organogenesis     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Embryogenesis and Plantlet Formation in Tissue Cultures of Celery

Callus cultures were initiated from sections of petioles ofcelery (Apium graveolens) var. Lathom Blanching on a modificationof the Murashige and Skoog medium. The callus, when isolatedfrom the parent explant, could be sub-cultured at regular intervalsforming both new callus and embryoids during each subculture.The embryoids appeared to be initiated on the callus surfacewhere early embryonic forms were found, but their continueddevelopment into plants only occurred when embryoids were transferredto a hormonefree medium. Embryoids were also formed in suspensionculture following inoculation of the liquid medium by differentiatingcallus, but again development of these structures was limitedto the early embryonic forms. When transferred to a solid hormone-freemedium, the embryoids, from either callus or suspension culture,developed into plantlets which could be transferred to soiland grown to maturity with only a few losses. The relevanceof this system as an aid in the breeding programme of self-blanchingcelery is discussed.     

Growth and Organogenesis in Moth Bean Callus as Affected by Paclobutrazol

Addition of relatively low concentrations (1.7 to 6.8 µM)of paclobutrazol to the culture medium decreased in vitro growthof Vigna aconitifolia (Jacqu.) Marechal cv. Jaadia (moth bean)callus. Presence of paclobutrazol increased the content of sugarsand total soluble protein in the callus. Addition of paclobutrazolto a regeneration medium reduced the differentiation of rootsand shoots in vitro. (Received October 11, 1988; Accepted May 30, 1989)     

In Vitro Multiplication of Sesame (Sesamum indicum) through Tissue Culture

Tissue cultures were established from different parts of sesame(Sesamum indicum L. cv. PT) seedlings. A callus tissue derivedfrom hypocotyl segments produced embryo like structures. Shoottips with cotyledons excised from 8 to 10-d-old seedlings producedmultiple shoot buds on a cytokinin-enriched medium. Presoakingand germination of seeds in BA or 2iP (8 mg l–1) enhancedthe development of shoot buds. Upon isolation and culture theshoots buds formed rooted plantlets in a charcoal-enriched medium. Sesamum, multiple buds, plantlets     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmoticstress, created by high agarose or carbohydrate concentrations,on the regeneration of fertile plants from protoplast-denvedcolonies of several indica (IR43, Jaya, Pusa Basmati 1) andjaponica (Taipei 309) rice varieties. Observations of the culturesdeveloped on media containing one of these carbohydrates (cellobiose,fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose),each at 88 mM, indicated that maltose was the preferential carbonsource for the proliferation of embryogenic callus and shootregeneration. Maltose-containing medium induced shoot formationin 24–66% of the protoplast-derived tissues, dependingupon the rice variety, compared to shoot regeneration from 4–32%of the tissues in sucrose-supplemented medium. Media containing288 mM maltose or an equimolar combination of 88 mM maltoseand 200 mM mannitol, caused water loss from calli and promotedthe growth of embryogenic calli. These calli formed shoots withgreater frequencies when subsequently transferred to shoot regenerationmedium with 88 mM maltose. A medium containing 88 mM maltoseand semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarosehad a similar beneficial effect on the growth of embryogeniccalli and simultaneously supported high-frequency (48–55%)shoot formation. The optimum shoot regeneration frequencies(60–78%) were obtained when protoplast-derived colonieswere serially cultured on to shoot regeneration medium containing1.0% (w/v) agarose for 4 weeks, followed by a 2-week cultureperiod on the same medium with 0.5% (w/v) agarose. Plants regeneratedon medium containing maltose and/or 1.0% (w/v) agarose werephenotypically normal and fertile. Key words: Carbohydrates, Oryza sativa L, indica and japonica rice, osmotic stress, plant regeneration, protoplast-derived colonies     

Isolation and Biochemical Characteristics of Protoplasts of Jojoba (Simmondsia chinensis (Link) Schneider)

Two types of protoplasts were isolated from leaves of shootsor callus of subcultures of jojoba (Simmondsia chinensis (Link)Schneider). Protoplasts from leaves were rich in chloro-plastsand were about half the volume of protoplasts from callus. Theviability of preparations as determined by the Evans blue techniquewas 80%. From cell cycle analysis by flow cytometry of nuclei,leaf protoplasts were uniformly in a non-proliferating phase(G0-G1), while callus protoplasts presented many phases of thecell cycle. Protoplasts from calli had only half the Chi ofthose from leaves. Yet Chi a/b ratio, as well as protein andtotal lipid content per cell, were similar in both types ofprotoplasts. A major drop in polar lipids, chiefly in mono-and digalactosyldiacylglycerol, and a parallel increase in neutrallipids occurred during protoplast isolation. The 18:2/18:3 ratiodecreased in neutral lipids concomitant with an increase intriglycerides rich in linolenic acid. Our results suggest atriggering of lipolytic acylhydrolases during the protoplastisolation, as reported for other species. Plasmolysis of thecells with high osmolarity medium and long incubation timeswere required to get a good yield of jojoba protoplasts. Inthe course of this procedure water-deficit stress takes place.A parallel with lipid changes occurring under this type of stressis discussed. (Received April 22, 1991; Accepted July 3, 1991)     

Factors Influencing Morphogenesis in Excised Roots and Suspension Cultures of Atropa belladonna

Excised root cultures of Atropa belladonna L. and A. belladonnavar. lutea Döll, established from liquid-grown seedlingsand from callus, when allowed to continue growth without subculturefor several weeks, spontaneously initiate shoot buds from smallnodules of callus which arise at the cut ends of the roots.The frequency and rapidity of formation of such buds is dependentupon the number of passages through which the roots have beensubcultured. The morphogenetic expression of callus cultures and of suspensioncultures derived from them is influenced not only by the timethat the root cultures have been maintained in culture but bythe composition of the medium used for callus initiation andsubsequent culture. The presence of elevated levels of ammoniumions in these media favours the development of incipient plantsrather than roots. Cultures have thus been obtained in whichthe predominant form of morphogenesis is embryogenesis (as establishedin a subsequent paper by Konar, Thomas, and Street, 1972).     

The Effects of Salinity upon Cellular Volume of the Marine Red Alga Porphyra purpurea (Roth) C.Ag.

Changes in cell volume of the marine red alga Porphyra purpureahave been investigated using photomicroscopic and radioisotopictechniques. There is an inverse relationship between cell volumeand external salt content. The alga responds to changes in thewater potential of its bathing medium by rapid swelling in hyposalinemedia and shrinkage in hypersaline conditions. Cells P. purpureabehave as osmometers in concentrated sea-waters, obeying theBoyle-Van't Hoff law. A non-osmotic volume, 20–25% ofthe total cell volume in sea-water, can be predicted from thelinear plot of volume versus reciprocal pressure in concentratedsea-water media. In dilute sea-waters the presence of non-rigidcell walls serves to limit any increases in cell volume. Theprimary response to dilution stress is thus an increase in turgor.Cell volume is not returned to its original value followingprolonged immersion in either hyposaline or hypersaline media,showing that the alga does not ‘osmoregulate’ sensustricto.     

Plantlet Regeneration from Callus and Parent Tissue 2Ornithogalum thyrsoides

Pieces of stem, leaf, ovary, sepals, and bulb scale of Ornithogalumall gave rise to adventitious plantlets when placed on basicMurashige and Skoog medium containing no added growth hormones.When one of the auxins, indole-3-acetic acid (IAA), -naphthaleneaceticacid (NAA), or 2, 4-diehlorophenoxyacetic acid (2, 4-D) wereadded to the medium, stem and leaf gave rise to callus fromwhich plantlets could be recovered by transfer to low auxinmedium or basic medium. Plants derived from parent tissue were diploid (2n = 12). Plantsderived from NAA callus were at first diploid but during seven5-week passages on NAA medium an increasing proportion of tetraploidplants, up to 50 per cent or more, were produced while the numberof plantlets recovered from the same amount of callus declined.     

Hormonal Control of Organogenesis and Somatic Embryogenesis inBeta vulgarisCallus

Tétu, T., Sangwan, R. S. and Sangwan-Norreel, B. S. 1987.Hormonal control of organogenesis and somatic embryogenesisin Beta vulgaris callus.—J. exp. Bot. 38: 506–517. Three main pathways of morphogenesis viz: root formation, shootformation and somatic embryogenesis, have been observed in thecallus derived from various explants of Beta vulgaris L. Growthhormones but not the basal media, determined the morphogeneticpotentiality of the callus. Auxin alone induced root formation.A combination of an auxin (naphthalene acetic acid) and a cytokinin(6-benzylaminopurine) gave only infrequent bud formation withvery low percentages (a maximum of 12%). Regular bud formationwith high percentages (52%) occurred when an anti-auxin (2,3,5-triiodobenzoicacid) with a cytokinin (BAP) was used. Shoots (2–3 cm)were transferred to a rooting medium. Roots were formed readilyin about 95% of the shoots. Histological studies showed thatcallus first formed meristematic zones and then shoot primordiadeveloped in these zones. Somatic embryos were formed only inthe calli derived from petiole explants. Multiple hormonal sequenceswere necessary for the induction and development of these somaticembryos. The embryos developed into normal plants when transferred,at the cotyledonary stage, to a hormone free basal medium. Key words: Beta vulgaris, organogenesis, somatic embryogenesis