High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Changes in cytokinin and auxin concentrations in seaweed concentrates when stored at an elevated temperature

Two seaweed concentrates were made from the kelps Ecklonia maxima and Macrocystis pyrifera using a cell burst method. Cytokinin- and auxin-like activities were measured using the soybean callus and mungbean bioassays, respectively. Cytokinin-like activity was detected in both seaweed concentrates, being equivalent to approximately 50 μg L⁻¹ kinetin. Auxin-like activity was also detected in both concentrates with the E. maxima derived concentrate having higher biological activity, equivalent to 10⁻⁵–10⁻⁴ M indole-butyric acid. Two replicates of each concentrate were stored at 54 °C for 14 days to accelerate the effects of storage. Both fresh and stored samples of the two seaweed concentrates were analysed for their endogenous cytokinin and auxin content. The samples were purified using a combined DEAE-Sephadex octadecylsilica column and immunoaffinity chromatography based on wide-range cytokinin and IAA specific monoclonal antibodies. These extracts were analysed by HPLC linked to a Micromass single quadrupole mass spectrophotometer. Eighteen and nineteen different cytokinins were detected, respectively, in the two concentrates, with trans-zeatin-O-glucoside being the main cytokinin present. Accelerated storage of the concentrates caused an increase in the total cytokinin concentration with a large increase in the aromatic meta-topolin. Indole-3-acetic acid was the main auxin in both seaweed concentrates. Indole conjugates, including amino acid conjugates, were also quantified. The total auxin concentration decreased with accelerated storage for both concentrates. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.     

Functional Expression of <Emphasis Type="Italic">Spirulina</Emphasis>-Δ<Superscript>6</Superscript> Desaturase Gene in Yeast, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ⁶ desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ⁶-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Δ⁶-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Δ⁶ desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ⁶ desaturation in yeast is more likely to be cytochrome b₅, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

Alteration of lipid composition,Na+, K+-ATPase and Δ5-β-hydroxy steroid dehydrogenase activities in microsomal membranes of mature toad ovary in different seasons

Microsomal membranes isolated by sucrose density gradient centrifugation from mature toad ovary has been found to vary significantly in lipid composition and various enzyme activities in different seasons. Na⁺, K⁺—ATPase activity is the highest in breeding season (rainy season). Significantly the optimum temperature for enzyme activity is 30°C. The other enzyme Δ⁵-3β-hydroxysteroid dehydrogenase activity is also lower in hibernation period than other seasons. The total phospholipid, sterol and fatty acid contents differ significantly between seasons. The poly-unsaturated fatty acid, except arachidonic acid content in hibernation period is much lower than that during other seasons. The sterol content is also the lowest in this season. The present findings indicate that during hibernation period the membrane is more rigid and the metabolic activity of the animal is slow because of a lower level of various functionally important enzyme activities. Part of this work was presented at the 13th International Union of Biochefstry Congress, held at Amsterdam in August 1985.     

NMR natural abundance estimation of13C-metabolic solutes in normal and habituated sugarbeet cell lines

Summary NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring¹³C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of¹H,¹³C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the¹H and¹³C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these¹³C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.Abbreviations DQF-COSY double-quantum filter correlation spectroscopy - HO habituated organogenous - HNO habituated nonorganogenous - HMBC heteronuclear multiple-bond correlation - HMQC heteronuclear multiple-quantum coherence - N normal - NMR nuclear magnetic resonance - TSP sodium tetradeutero-3-(trimethylsilyl)-propionate     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Summary Callus tissue ofHaworthia mirabilis Haw. was irradiated with⁶⁰Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N⁶-(Δ²-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed. This is paper 78-10-124 of the Kentucky Agricultural Experiment Staton and is published with approval of the Director.     

Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

ω³-fatty acid desaturase and Δ¹²-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω³-fatty acid desaturase were replaced with corresponding region of Δ¹²-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of ω³-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore, the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of different fatty acid desaturases. Dongsheng Wei is the partake of first-author’s profits.     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

Habituation of plant cells does not mean insensitivity to plant growth regulators

Summary Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.     

Micropropagation ofTribulus terrestris L., an important medicinal plant

Direct regeneration of shoots and roots through juvenile expiants has been achieved inTribulus terrestris. Cotyledonary leaves along with epicotyl segment from young seedlings were cultured on MS medium containing various concentrations of auxin with cytokinin and glutamine. A combination of 0.2 mgL⁻¹ NAA, 0.5 mgL⁻¹ BAP and 50 mgL⁻¹ glutamine induced high frequency of shoot and root differentiation in 10 weeks. The callus also could be induced on the above medium from the cut end of radical segments. Morphogenic response such as per cent shoot and root differentiation was recorded at regular intervals.     

Substituted nitroguanidines provide cytokinin activity during in vitro cultivation of plant tissues

Synthetic nitroguanidine derivatives can be used as alternatives to the traditional adenine-containing cytokinins used in plant tissue culture. First, nitroguanidine derivatives (NG) mimicked the typical activity of two standard cytokinins, 6-benzylaminopurine (BAP) and 2-isopentenyladenine (2iP) in the soybean callus (Glycine max) growth bioassay. NGs caused unanticipated responses as well, as demonstrated in three lines of tobacco (Nicotiana tabacum), when the auxin concentration was reduced from the standard concentration of 2 ug/ml NAA, to much lower concentrations of 0.01 ug/ml NAA or 0.02 ug/ml IAA. At the low auxin concentrations, kinetin lost the ability to promote either growth or differentiation, while the NG cytokinins were fully able to promote both. NGs promoted growth and differentiation in the presence of 0.01 ug/ml NAA in a newly initiated, totipotent line of Coker 319 tobacco. NGs plus 0.02 ug/ml IAA also promoted callus growth in a cytokinin-habituated tobacco line, Havana 425-CH. Lastly, NGs stimulated the outgrowth of healthy callus from aged callus that had been allowed to deteriorate through lack of subculture. Upon transfer of aged NTP callus to fresh media with NGs and 0.02 ug/ml IAA, healthy cell clusters were rapidly produced. In all three cases cited above, kinetin was ineffective at the low auxin concentrations. The NGs are therefore cytokinins, with the additional possibility of reducing the level of auxin required for their activity to be expressed.Abbreviations BAP 6-benzylaminopurine or N⁶- benzyladenine - IAA indoleacetic acid - 2iP N⁶-(²- isopentenyl)adenine - NAA -naphthaleneacetic acid - NG nitroguanidine derivative     

Sterol composition of nystatin-resistantCandida maltosa mutants

Composition of sterol fractions of nystatin-resistantCandida maltosa strains was determined. Using UV-spectrometry, TLC and GLC-MS it was demonstrated that resistance to nystatin is connected with the composition alterations of yeast cell sterols. Block of different stages of ergosterol biosynthesis was revealed in some mutants,viz. C-24-transmethylation, Δ⁸→Δ⁷, 14α-demethylation, C-5(6)-dehydrogenation, reduction of C-14(15) and C-24(28) double bonds.     

In vitro selection and characterization of Ni-tolerant callus lines of Setaria italica L

Nickel tolerant callus lines of Setaria italica L. were developed from callus cultures grown on MS medium supplemented with 0.5 mg·dm⁻³ kinetin+2.0 mg·dm⁻³ 2,4-D+2.0 mg·dm⁻³ Ni⁺². Standard growth parameters such as callus fresh and dry weight, growth tolerance index were used as indicators of nickel toxicity. Measurements as early as 2 weeks after the beginning of the treatments did not yield consistent results. However, growth tolerance index at 4, and 8 weeks after the beginning of treatments yielded significant differences among the non-tolerant and tolerant calli. The tolerant calli has enhanced growth at 2.0 mg·dm⁻³ Ni⁺² while non-tolerant calli showed a reverse trend in growth in the presence of 2.0–2.5 mg·dm⁻³ of nickel. The tolerant calli differentiated into mass of embryogenic calli within 4 weeks of culture which could be maintained for prolonged period without loss of regenerative capacity.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.