Callus Pieces Research Articles

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL(-1) and 26 mgL(-1) ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.

Solute concentrations of the phloem and parenchyma cells present in squash callus

Abstract. Glutaraldehyde fixation was used to determine the solute concentrations in the various cell types present in tissue cultures of squash (Cucurbita pepo). Small pieces of callus were plasmolyzed in a graded series of mannitol solutions and fixed in 20 kg m−3 glutaraldehyde adjusted to be isosmotic with the particular plasmolysing solution. The callus samples were further processed using standard electron microscopy techniques. Using this procedure, mature sieve elements that form in squash callus have an osmotic potentional of ‐2.4MPa. The osmotic potential of the callus sieve elements was comparable to values reported for the sieve tube members of the phloem in intact plants. This ability of callus sieve elements to develop high internal hydrostatic pressures demonstrates that they are capable of phloem loading. However, the osmotic potentials of the surrounding parenchymatous cells and companion cells were only –1.15 and –1.5 MPa, respectively. In contrast to the companion cells of the phloem in intact plant tissues, the osmotic potential of the callus companion cells indicated that they were not directly involved in phloem loading. Several immature sieve elements containing distinct nuclei and vacuoles were observed in the callus granules. These immature sieve elements were plasmolyzed in weaker mannitol solutions (below 0.6kmol m−3) than the enucleate sieve elements (1.01 kmol m−3 mannitol). The low solute concentrations in immature sieve elements indicated that the ability to load sugars occurs concomitantly with the maturation of the sieve element protoplast.

Cytodifferentiation during the development of friable embryogenic callus of maize (Zea mays)

Immature embryos of Zea mays L. were cultured on N6 medium to obtain embryogenie callus. Cultured tissue fragments with various developmental stages of friable callus were sampled and prepared for light microscopy and transmission electron microscopy. Pieces of compact callus were also prepared for light microscopy to compare the structural organization of both callus types. Friable callus develops from a thin layer of abaxial scutellum cells, including the epidermis. During further development the callus cells dissociate, owing to the breakdown of the middle lamellae, while older vacuolated cells degenerate. This results into long cell aggregates separated by large intercellular spaces, giving the callus its friable appearance. The microscopical sections showed a striking difference between friable and compact callus. Vascular elements were not found in the friable callus. On the contrary, vascular bundles were prominent in compact callus. Friable callus is therefore correlated with a less differentiated state than compact callus. The embryogenic potential of friable callus is situated in embryogenic units. These are aggregates of small isodiametric cells containing a central nucleus, an electron-dense cytoplasm, and many organelles. Proliferation was only observed in these cells, which are therefore presumed to generate new embryogenic units, somatic embryos, and vacuolated callus cells. The results further indicate that discrete masses of embryogenic cells, possibly early embryoids, have a unicellular origin. Key words: in vitro culture, callus, somatic embryogenesis, ultrastructure, Zea mays.

Somatic embryogenesis and plant regeneration from immature inflorescences of several elite inbreds of maize

Somatic embryogenesis and plant regeneration was obtained from immature inflorescence segments of maize ( Zea mays L.). Immature tassels and ears (0.5–3.0 cm) were excised, cut into segments (1 – 2 mm), and place onto media supplemented with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), sucrose and l-proline. After 2–3 weeks, the segments produced compact, nodular, white, embryogenic callus as well as soft, granular, friable, non-embryogenic callus. Embryogenic callus further developed somatic embryos with distinct scutellar- and coleoptile-like structures, which produced normal, fertile plants with no apparent morphological abnormalities. Maximum embryogenic callus formation was observed when segments, cut from 0.6–1.0 cm inflorescences, were cultured on Murashige and Skoog (MS) medium with 2 mg/l 2,4-D, 3% sucrose, and 24 mM l-proline. Although embryogenic callus production was observed in all inbreds tested, significant genotype effects were apparent. The inbreds A619, H99, LH39, LH39, MBS501, MS71, Oh43, U94 and U98 all displayed high frequencies of embryogenic callus production (45–80% of cultured segments) and plant regeneration (up to 50% of callus pieces transferred). Thus, immature inflorescences can be used as explants for high frequency somatic embryogenesis and plant regeneration from several commercially-important maize inbreds.

Transfection of germinating barley seed electrophoretically with exogenous DNA

A method is described for transfection (genetic transformation) of barley caryopsis electrophoretically with DNA. β-Glucuronidase activity was detected after the electrophoretic transfection with plasmid pBI221 DNA carrying the cauliflower mosaic virus promotor and bacterial β-glucuronidase coding sequence. Electrophoretic transfection is evidently effective with pieces of callus and seeds of many plants.

Evaluation of soybean resistance to Phialophora gregata culture filtrate in tissue culture

Resistance to the fungal pathogen, Phialophora gregata (Allington and Chamberlain) W. Gams, the cause of brown stem rot (BSR) in soybean [Glycine max (L.) Merr.], is an important trait for cultivars grown in the northern USA. A novel tissue culture method was developed where ten soybean cultivars were differentiated on the ability of their excised cotyledons to remain green and initiate callus in a tissue culture medium containing P. gregata culture filtrate. Cultivar BSR classifications by the cotyledon method corresponded to greenhouse root-dip assay classifications in 80%, 100%, and 90% of the three P. gregata isolate treatments. Another method, employing pieces of somatic callus exposed to the culture filtrate, had a 70% average correspondence to the greenhouse results. Physiologic specialization was demonstrated in parallel in vivo/in vitro assays for the first time. These data suggest that the cotyledon method would accurately identify soybean lines resistant to certain aberrant or wild-type P. gregata isolates.

Protoplast culture and plantlet regeneration in stock (Matthiola incana R. Br.).

Protoplasts were isolated from callus derived from excised cotyledons of stock (Matthiola incana R. Br.), by using an enzyme mixture of 1.5% Meicelase and 0.05% Macerozyme R-10. Modified Murashige-Skoog major elements, Ringe and Nitsch minor elements and modified vitamins supplemented with 0.2mg/l 2, 4-D, 1mg/l NAA, 1mg/l BAP, 8.1% mannitol, 1% glucose and 1% sucrose supported protoplast division at 25°C. The frequency of colony formation 10 days after culture was the highest (17.9% of isolated protoplasts) at protoplast density of 0.5×105/ml. Colonies developed to the callus-stage 2 months after initial culture. When callus pieces were cultured in full strength MS medium supplemented with 1mg/l BAP or 1mg/l zeatin, adventitious buds were induced in 9%-16% of callus explants. The buds elongated with leaves open at 20°C and rooted in medium supplemented with 0.1mg/l or 5mg/l IBA.

Somatic embryogenesis and plant regeneration from cultured mature and immature embryos of Paspalum notatum (gramineae)

Plants were regenerated from mature and immature embryos of Paspalum notatum ( 2n = 4x = 40) cultured in vitro. Explants were induced to form callus when aseptically cultured on Murashige and Skoog medium (MS), 0.8% agar and 1 mg/l 2,4-D. Well organized somatic embryos were formed on the surface of the proliferating scutellar callus derived from both mature and immature embryos. Regeneration of multiple shoots was readily achieved by transferring pieces of callus onto fresh media containing both NAA (0.1 or 1 mg/l) and BAP (1 or 3 mg/l). Shoots were induced to form roots upon transfer to MS with 8% sucrose, but lacking hormones.

Somatic embryogenesis and plant regeneration in Cyphomandra betacea (Cav.) Sendt

Callus cultures with globular proembryogenic structures were induced from zygotic embryos and hypocotyl segments of Cyphomandra betacea on MS medium supplemented with 2,4-D. Proembryogenic structures produced somatic embryos and plantlets on regulator-free basal medium. Pieces of embryogenic callus subcultured on medium with the same original composition gave rise to new globular structures and the potential for plantlet regeneration has been maintained for over a year. The histological examination of these proembryogenic structures suggested that somatic embryos arise from single cells. Regenerated plants are phenotypically normal, having diploid chromosome numbers (2n = 24).

An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus

The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 x 10(-13) mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium.

Proline accumulation and sodium sulfate tolerance in callus cultures of Brassica napus L. cv. Westar

Callus cultures of Brassica napus L. cv. Westar were selected which contained 5 - 6 times more proline than unselected callus. Callus pieces from these cultures were able to survive much better after subculture to medium containing 105 mM Na2SO4 than unselected callus, or unselected callus cultured on exogenous proline before or during transfer to the salt. Exogenous proline was rapidly absorbed. In unselected callus there was a peak in proline accumulation ca. 2 days after transfer to Na2SO4, followed by a decline. In contrast proline accumulation in tolerant callus was linear with time, reaching maximum levels at 8 days. Proline levels induced by exposure to salt were maintained in the absence of stress.

A study of the interaction of sodium dodecyl sulphate with the proteins of human heel stratum corneum

Synopsis The aim of this research is to study the in vitro interaction of the anionic surfactant sodium dodecyl sulphate with human heel callus with special regard to the callus proteins. The sorption of SDS by callus is analysed at different pH values, demonstrating the expected maximum binding of the anionic surfactant at low pH. In both surfactant and blank solutions, swelling of the callus pieces occurs but to different extents. The sorption of SDS by callus is accompanied by a loss of free amino acids and proteins. The protein composition of the callus residues after chemical treatment and of the corresponding treatment baths is examined by amino acid analysis and shows differences in the respective molar amino acid ratios. Results obtained with more specific techniques, such as gel electrophoresis and immunoblotting, demonstrate identical as well as different protein components in the treatment baths depending on the experimental conditions (pH, blank or SDS). Although effects due to the surfactant treatment are in principle more distinct than with blank experiments, those of the latter cannot be neglected.

Cytogenetic and isozymic characterization of anther-panicle culture derived tall fescue aneuploids

Chromosome associations of 44 tall fescue (Festuca arundinaceaSchreb., 2n=6x=42) plants derived from anther-panicle culture of ‘Kentucky 31’ were evaluated to determine if new genetic stocks could be identified. Seventeen of the plants were euploids (21 II), two were monosomic (20II+1I), 22 were double monosomic (19II+2I) and three were triple monosomic (18II+3I). Zymograms were obtained for phosphoglucoisomerase (PGI), glutamate oxaloacetate transaminase (GOT), acid phosphatase (ACPH), malate dehydrogenase (MDH) and 6-phosphogluconate dehydrogenase (6-PGD). The zymograms were identical for GOT, ACPH, and 6-PGD but different patterns were found for MDH and PGI in some of the double monosomics and euploids from a different piece of callus than the majority of the plants.

Plant regeneration and thebaine content of plants derived from callus culture of Papaver bracteatum

Plants have been regenerated from embryogenic callus cultures of two varieties of Papaver bracteatum and successfully transplanted to soil. Regeneration occurred in good yield, around 40 plants to soil within 5 months per 0.4g piece of original callus. Thebaine concentrations comparable to those in seed-grown plants were obtained in callus-derived plants. It is suggested that there is potential for mass micropropagation of P. bracteatum, which may be useful in developing agriculturally-improved lines.

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.

Tissue Culture in Paspalum (Gramineae): Plant regeneration from Cultured Inflorescences

Summary Plants were regenerated from inflorescences of Paspalum spp. (P. intermedium, P. wettsteinii, P. virgatum, P. quadrifarium, P. notatum, P. almum, P. equitans, P. ovale, P. dilatatum x P. regnellii) cultured in vitro . Explants from young, unemerged inflorescences (at premeiotic stage) were induced to form callus when aseptically cultured on Murashige and Skoog medium (MS), 0.8 % agar, 5 mg · 1 −1 2,4-D and 2 mg · 1 −1 BA. Regeneration of multiple shoots was readily achieved by transferring pieces of callus onto fresh media containing both NAA (0.1 or 1 mg · 1 −1 ), and BA (1 or 3 mg · 1 −1 ). Shoots were induced to form roots upon transfer to MS with 8 % sucrose, lacking hormones. Calli from P. rufum failed to regenerate shoots. Explants from inflorescences of P. arundinellum, P. brunneum, P. conspersum, P. durifolium, P. ionanthum, P. simplex , and P. malacophyllum , did not produce callus when cultured in vitro .

Cytokinin contents and specific characteristics of tissue strains from three sexual genotypes of Mercurialis annua

Analyses of the endogenous cytokinin contents of established tissue strains of Mercurialis annua are reported. The strains were derived from three individuals (strong male, weak male, female), differing by one of the three genes determining sex. The data are compared with the endogenous cytokinins of male and female shoot apices. Tissue strains are characterized by the disappearance of natural cytokinin metabolites in the female; in both males, Δ(2)-isopentenyl-adenosine and only trans-ribosylzeatin exist but in different quantities. Benzyladenine and ribosylbenzyladenine were identified in the three strains but the quantities also differed as a function of the genotype. The marked differences in cytokinin metabolism of tissue strains indicate that sex genes continue to function in the dedifferentiated state. Each strain also exhibited persistent morphological and histological characteristics, and a different sensitivity to the withdrawal of 2-4-dichlorophenoxyacetic acid or benzyladenine from the medium. Each had a specific and characteristic effect on the organogenesis of nodes cultivated in close proximity to callus pieces. These data complement the above results and show that sex genes act at the callus-tissue level. The possibility that these genes act at the early stages of embryogenesis of male and female individuals is also discussed.

Retention of shoot regeneration capacity of tobacco callus by Na2SO4

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown in the light on shoot-forming medium in the presence of Na2SO4 for over a year. An increase in Na2SO4 concentration resulted in decreasing callus growth, decreasing percentage of calli producing shoots and number of shoots per callus, and increasing callus percent dry weight. Regeneration of shoots from callus grown in the absence of Na2SO4 began to decline after 14 months in culture, and shoot regeneration capacity was completely lost after 18 months. In contrast, 18-month old callus continuously grown in the presence of Na2SO4 retained the ability to form shoots. The highest percent of callus pieces that formed shoots and the maximum number of shoots per callus occurred at 70 mM (1%) Na2SO4. All plants arising from the 18-month old callus were polyploid. Both 9- and 18-month old callus exhibited more negative water and osmotic potentials in the presence of increasing Na2SO4 concentration.

Organogenesis and fine structure in megagametophyte callus lines of Picea abies

Differentiation and fine structure were studied in 63 callus lines originating from the haploid megagametophyte of Picea abies (L.) Karst. Developing cones were collected from 27 trees growing in 13 localities in Finland. Vernalization of cones for 12–42 days at 4°C was optimal for callus initiation from the immature megagametophyte (primary endosperm). Five combinations of media based on the macronutrient elements of Chu et al. (1975; Sci. Sin. 18: 659–668) and the micronutrient elements and vitamins of Murashige and Skoog were tested for callus induction, growth and differentiation. Only about 1.5% of the megagametophytes produced subculturable callus (which may be partly due to the high frequency of lethal genes), although in certain mother trees callus production was as high as 20%. In most of the trees sampled, polyamines could not replace casein hydrolysate and glutamine in induction of megagametophyte callus. About half of the originally haploid, diploid and mixoploid callus lines were able to differentiate. A combination of three polyamines (putrescine 0.25, spermidine 0.1 and spermine 0.025 mM) favoured development of roots. In five callus lines shoots and roots developed in the same piece of callus, but these organs usually had no connection with each other. The fine structure of the callus cells was normal, but their starch stores were rather abundant. Mesophyll cells of needles differentiated from originally haploid callus had chloroplasts with fairly well‐developed grana. Secondary metabolites were observed in the vacuoles of some callus cells and in organ initials. Plasmodesmata were very rare in callus cells but they were characteristic of those of the needles. The electron microscope observations showed that the poor capacity for differentiation of P. abies callus cultures was not due to cytological instability.

Regeneration of plants from callus tissue of the forage legume Stylosanthes guianensis

Plants were regenerated from leaflets of Stylosanthes guianensis (Aubl.) Sw. cultured in vitro. Explants from both mature and immature leaflets were induced to form callus when aseptically cultured on Murashige and Skoog salts containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and three combinations of napthaleneacetic acid (NAA) and benzyladenine (BA). Regeneration of shoots was readily achieved by transferring pieces of callus, even after subculturing for 6 months, onto fresh medium containing a low NAA/BA ratio. Shoots were induced to form roots upon transfer to medium devoid of hormones.