Bean Callus Research Articles

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.

Secretion of basic and acidic chitinases from salt‐adapted and ‐unadapted winged bean cells

Six specific proteins are secreted from salt‐adapted winged bean (Psophocarpus tetragonolobus) cells. A protein, designated S AP3, with molecular mass of 37 000 was secreted abundantly by suspension cultures of winged bean callus adapted to 0.5% (86 mM) or 1.0% (171 mM) NaCl. When the amino acid sequence of the amino‐terminal part (16 amino acid residues) of SAP3 was determined, a significant homology was found between the amino‐terminal amino acid sequence of SAP3 and that of basic chitinase from kidney bean, Arabidopsis, tobacco and pumpkin. SAP3 had chitinase activity, suggesting that basic chitinase is secreted specifically from salt‐adapted winged bean cells. A protein with molecular mass of 29000 was exclusively secreted into the culture medium at the later stage (20–30 days) of both salt‐adapted and ‐unadapted cultures. The protein was purified from the medium at day 30. One‐step ammonium sulfate fractionation was sufficient for purification to homogeneity. The protein had chitinase activity. The amino‐terminal amino acid sequence (17 amino acid residues) of the protein suggested that it was acidic chitinase. Thus, acidic chitinase with a molecular mass of 29000 is exclusively secreted at the later stage of culture of salt‐adapted and ‐unadapted winged bean cells, in contrast to pumpkin (Cucurbita) cell suspension cultures where basic chitinase with a molecular mass of 32 000 is secreted abundantly at the early culture stage.

Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.

Specific and Abundant Secretion of a Novel Hydroxyproline-Rich Glycoprotein from Salt-Adapted Winged Bean Cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl(3), but little was secreted in the presence of KCl, LiCl, CaCl(2), MgCl(2), mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp(5)) instead of tetrahydroxyproline blocks (Hyp(4)) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.

Growth and organogenesis in moth bean callus cultures as influenced by triazole growth regulators and gibberellic acid

The triazole plant growth regulators, paclobutrazol and uniconazole, reduced in vitro growth of moth bean callus by 20–25% when added to the culture medium at 1 mg/L (3.4 μM). The addition of 10 mg/L (29 μM) gibberellic acid (GA3) to the culture medium in combination with the triazoles restored callus growth to a level equivalent to that of the untreated control. GA3 alone had little effect on callus growth. When added to a regeneration medium at 1 mg/L both paclobutrazol and uniconazole reduced the percentage of cultures that formed roots, as well as the mean number of roots per culture. In contrast, GA3 increased root formation and counteracted the inhibitory effects of the triazoles on rooting. The addition of triazoles or GA3 to the regeneration medium reduced the formation of green meristematic nodules, which are precursors of shoots in moth bean callus. When callus was grown in the presence of either paclobutrazol or uniconazole, subsequent root and green meristematic nodule formation were reduced upon transfer to a growth regulator-free regeneration medium. The results of this study indicate that exposure of moth bean callus tissue to micromolar concentrations of triazoles or GA3 can significantly alter in vitro growth and differentiation.

MEDIA AND ENVIRONMENTAL INFLUENCES ON SOMATIC EMBRYOGENESIS IN COMMON BEAN (PHASEOLUS VULGARIS L.)

Opaque globules formed on bean callus induced on primary leaf explants cultured on induction media (IM) containing 10 to 30 mg/l 2,4-D. Calli with globules produce structures reminiscent of somatic embryos (embryoids) after subculture in a liquid challenge medium (LCM). Calli maintained on IM for 2, 3, 4, and 5 weeks produced significantly more (26 to 34/callus) embryoids in LCM than calli maintained on IM for one week (12/callus). Well developed embryoids only occurred after calli were subculture in liquid B5 with 0.1 to 1.0 mg/l IBA. Calli subculture in LCM with > 10 mg/l IBA turned necrotic and died. Embryoids produced in B5 with 2,4-D and NAA (0.1 to 1.0 mg/l) proliferated roots and formed “frosty” appearing structures, respectively. No differences were detected in number or quality of embryoids produced in LCM from callus maintained on IM in continuous light or darkness regardless of the induction time. Ethylene accumulation in IM cultures inhibited globule formation.

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 growth of Vigna aconitifolia (Jacqu.) Marechal cv. Jaadia (moth bean) callus. Presence of paclobutrazol increased the content of sugars and total soluble protein in the callus. Addition of paclobutrazol to a regeneration medium reduced the differentiation of roots and shoots in vitro.

Use of antisera to localize callose, xylan and arabinogalactan in the cell-plate, primary and secondary walls of plant cells

Antibodies to cellobiose, L-arabinopyranose, L-arabinofuranose, D-galactose, oligosaccharides containing β1→4 xylose, oligosaccharides containing β1→4 glucose, and oligosaccharides containing β1→3 glucose have been raised in rabbits. The antisera have been characterized to show the specificity of binding to particular polysaccharides. They have been used for immunocytology using the electron microscope to locate the polymers in dividing and differentiating cells of bean (Phaseolus vulgaris L.) root, bean callus tissue and cells of Zinnia elegans L. in vitro. Arabinogalactans have been shown to be present in the cell-plate and primary walls but not in secondary thickening. Xylan as distinct from xyloglucan was found in the primary walls but not in the cell-plate. It was present in large amounts in the secondary thickening. Callose was found in the cell plate and also in the young growing wall. In the wall it was specifically located at the plasmodesmata. The use of the antibody against L-arabinofuranose enabled a specific organelle to be detected which was membranous and which occurred within the cytoplasm and also within the vacuole of the cells. Membranes carrying polymers containing L-arabinofuranose were also found in layers just under the plasmamembrane.

In vitro Growth, Metabolism and Regeneration of Moth Bean Callus as Influenced by Flurprimidol

Summary Flurprimidol (FLP) is an experimental gibberellin biosynthesis inhibitor whose biological properties have not been fully characterized. Addition of FLP (0.5-2.0 mg 1-1) to a callus medium reduced both fresh and dry weights of moth bean (Vigna aconitifolia) callus after 21 days. The concentrations (fresh weight basis) of total sugar, proline, soluble protein, and phenols in callus tissue were all increased by FLP relative to the control. In addition, the activities of peroxidase, protease, RNase, alanine aminotransferase, and aspartate aminotransferase were all greater in FLP-treated cultures than in controls. The presence of 1 mg 1-1 FLP in a regeneration medium reduced the percentage of cultures exhibiting roots as well as the mean number of roots formed per culture. Likewise, FLP reduced the formation of green meristematic nodules which are forerunners of shoots in moth bean callus.

In Vitro Somatic Embryogenesis from Bean Callus

<i>In Vitro</i> Somatic Embryogenesis from Bean Callus

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 μM NAA and 2 μM KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 μM 2,4-D and 2.3 μM zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 μM BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 μM zeatin, 0.69 μM GA3 and 1.5 μM NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 μM NAA and 1.0 μM KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.

Metabolism of N-(purin-6-yl)glycine by Soya Bean Callus

Metabolism of N-(purin-6-yl)glycine by Soya Bean Callus

Properties and Activity of N-(purin-6-yl)glycine in the Soya Bean Callus Bioassay

Properties and Activity of N-(purin-6-yl)glycine in the Soya Bean Callus Bioassay

Antiviral Activity of 2-(α-Hydroxybenzyl)-Benzimidazole and other 2-Substituted Benzimidazoles against TMV in Tomato Leaf Discs

Abstract 2-(α-hydroxybenzyl)-, 2-benzyl-, 2-phenyl-, 2-methyl-and 2-hydroxymethyl-benzimidazoles were screened for antiviral activity against TMV in tomato leaf discs, for cytokinin activity in the amaranthus betacyanin bioassay and the soya bean callus bioassay and for antagonism in the latter bioassay. None of the test compounds showed either cytokinin activity or antagonism. 2-(α-hydroxybenzyl)-benzimidazole at 23.3 μᴍ inhibited infectious virus production by 100% and no virus antigen was detectable. None of the other compounds showed similar antiviral activity. The realationship between the substituent at the 2-position and antiviral activity was as previously reported for 2-substituted benzimidazoles in animal virus chemotherapy.

Control of hemicellulose and pectin synthesis during differentiation of vascular tissue in bean (Phaseolus vulgaris) callus and in bean hypocotyl

Membrane fractions from bean hypocotyl or callus incorporate arabinose from UDP-β-L-arabinose into arabinan and xylose from UDP-α-D-xylose into xylan. The control of these syntheses has been studied during xylogenesis in stele and in xylogenesis induced in callus tissue. Induction of arabinan synthetase activity occurs during division and extension growth while that of xylan synthetase occurs subsequently during the period of secondary thickening of the cell wall. The xylan synthetase induction is correlated with the induction of phenylalanine ammonia-lyase and with lignin synthesis.

Cytokinins in Root-Nodules of the Nitrogen-Fixing Non-Legume Myrica gale L.

Cytokinin activity was detected using the soy bean callus bioassay, in extracts of rootnodules, free of «nodule-roots», in the Actinomycete-nodulated non-legume Myrica gale (bog myrtle).

The Effect of Adenine and Mevalonic acid on the Endogenous Cytokinins of a Cytokinin-dependent Strain of Soya Bean Callus

The Effect of Adenine and Mevalonic acid on the Endogenous Cytokinins of a Cytokinin-dependent Strain of Soya Bean Callus

The Metabolism of Zeatin and Zeatin Riboside by Soya Bean Callus

The Metabolism of Zeatin and Zeatin Riboside by Soya Bean Callus

Correlation of the induction of various enzymes concerned with phenylpropanoid and lignin synthesis during differentiation of bean callus (Phaseolus vulgaris L.)

The activities of phenylalanine ammonia lyase and caffeate-0-methyl transferase in bean callus tissue, during differentiation, were induced in a coordinated manner over a period of 21 days. Neither enzyme activity could be induced to increase when differentiation was repressed by the addition of abscisic acid to the induction medium. The formation of soluble phenols was also increased during the induction of differentiation but it was also increased by the transfer to induction medium of an old callus which could no longer differentiate. PAL activity was more directly correlated with xylogenesis and nodule induction than with the formation of soluble phenols. Peroxidase activity in the callus tissue was always high but the level decreased during the induction of differentiation. A new isoenzyme of peroxidase was induced during callus formation and this was probably a response to the presence of auxins in the medium used to grow the callus.

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.