Biosynthesis and distribution of tomatine in cultured excised Lycopersicon esculentum roots

Growth of cultured excised tomato roots in the presence of ¹⁴C-mevalonic acid lactone results in labelling of tomatine. In the main axis of the root, incorporation was greatest in the apical meristematic region. Tomatine was present in equal concentrations in all parts of the cultured root system.     

Degradation of tomatine to 3β-hydroxy-5α-pregn-16-en-20-one by ripe tomatoes

Tomatine-4-¹⁴C was prepared by foliar administration of cholesterol-4-¹⁴C and silicone oil to tomato plants. Chromatographically homogeneous tomatine-4-¹⁴C in 96% ethanol, when incubated in whole, ripe tomatoes was rapidly converted to 3β-hydroxy-5α-pregn-16-en-20-one in combined form. The identity of this steroid and its acetyl derivative was established by comparing their TCL mobilities with reference materials and by recrystallizing them to constant specific activity.     

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.     

Chemical components of callus tissues of rice

Chemical components of callus and other tissues (leaf, root, and seed) of rice were investigated to find the variation in callus metabolism. The isolated compounds were squalene, three sterols, three triterpenes and a fatty alcohol; the last being present only in the callus. Cylindrin, isoarborinol and chlorophyll were not found in the callus. Fatty acids were detected in all tissues in varying amounts, the callus containing lauric acid in greater quantity than the leaves, roots, and seeds.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Effects of IAA and four IAA conjugates on morphogenesis and callus growth from tomato leaf discs

The effects of indole-3-acetic acid (IAA) and four IAA conjugates, indoleacetylalanine (IAAla), indoleacetylaspartic acid (IAAsp), indoleacetylglycine (IAGly), and indoleacetylphenylalanine (IAPhe), on growth and morphogenesis in tomato leaf discs in vitro were examined. Free IAA stimulated root initiation in the absence of cytokinin and stimulated callus growth in the presence of 0.89 M benzylaminopurine (BAP). Free IAA also inhibited shoot initiation obtained with 8.9 M BAP. The activities of the IAA conjugates depended on the conjugating amino acid, the concentration of the conjugate, and the response being measured. IAAsp had little or no activity in promoting root initiation or callus growth or in inhibiting shoots, while IAPhe was similarly inactive except at the highest concentration tested (100 M). IAAla and IAGly were both very active in inhibiting shoots and promoting callus growth, but were much less active in stimulating rooting, except at 100 M, at which concentration they were as effective as free IAA. Thin-layer chromatography of the IAA conjugates revealed that IAAla, IAGly and IAPhe were largely stable to autoclaving, but that IAAsp underwent some hydrolysis to products identical with free IAA and aspartic acid. Pretreatment of seedlings with IAA, IAAla or IAGly altered the subsequent shoot initiation response from leaf discs on media with and without IAA.     

Withanolide production by <Emphasis Type="Italic">in vitro</Emphasis> cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its association with differentiation

Withanolides-steroidal lactones, isolated from various Solanaceous plants have received considerable attention due to their potential biological activities. Five selected withanolides (withanone, withaferin A, withanolide A, withanolide B, withanolide E) were identified by HPLC-UV (DAD) — positive ion electrospray ionization mass spectroscopy in Withania somnifera (L.) Dunal cv. WSR plants and tissues cultured in vitro at different developmental phases. Cultures were established from five explants on Murashige and Skoog’s medium supplemented with different plant growth regulators. Results suggest that production of withanolides is closely associated with morphological differentiation.     

Distribution of littorine and other alkaloids in the roots of datura species

Littorine, various tropoyl and tigloyl esters and cuscohygrine are components of the roots of all nine Datura species studied. The genus exhibits a uniform alkaloid spectrum and its inclusion, with Solandra, in the tribe Datureae is supported on phytochemical grounds.     

Dechlorodauricumine from cultured roots of Menispermum dauricum

Dechlorodauricumine, a possible organic substrate for biochlorination, was isolated from cultured roots of Menispermum dauricum, a rich source of chlorinated alkaloids. Its structure was established by spectroscopic and chemical methods.     

Role of nano-SiO2 in germination of tomato (Lycopersicum esculentum seeds Mill.)

Agricultural biotechnology is very familiar with the properties of nanomaterial and their potential uses. Therefore, the present experiment was conducted to test the beneficial effects of nanosilicon dioxide (nSiO₂: size- 12 nm) on the seed germination of tomato (Lycopersicum esculentum Mill. cv Super Strain B). Application of nSiO₂ significantly enhanced the characteristics of seed germination. Among the treatments, 8 g L⁻¹ of nSiO₂ improved percent seed germination, mean germination time, seed germination index, seed vigour index, seedling fresh weight and dry weight. Therefore, it is very clear that nSiO₂ has a significant impact on the seed germination potential. These findings could provide that alternative source for fertilizer that may improve sustainable agriculture.     

Metabolite profiling and quantification of phenolic compounds in methanol extracts of tomato fruit

The consumption of tomatoes and tomato products has been associated with a reduction in the risk of contracting some types of cancer and other chronic diseases. These beneficial properties may be attributed to the presence of key metabolites and the interactions among them. We have developed and validated an analytical method for the comprehensive profiling of semi-polar metabolites in the methanol extract of three cultivars of raw tomatoes (Daniela, Raf and Rambo) grown in Almería, in south-east Spain. Diode-array and time-of-flight/ion-trap mass spectrometry detectors were used to ensure the wide detection of metabolites with highly divergent properties. The masses thus detected were assigned by matching their accurate mass-signals with tomato compounds reported in the literature, and supplemented by UV and MS/MS information, reference compounds and existing metabolite databases. In this way we were able to identify tentatively 135 compounds belonging to various structural classes, 21 of which are to our knowledge reported for the first time in the tomato fruit. Among the metabolites identified, the most abundant were phenolic compounds. This class of secondary metabolites is attracting considerable attention from producers and consumers due to their antioxidant activity and nutritional properties. Their quantitative analysis was achieved by using closely related derivatives for each family.     

Pn-AMPs,the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato,Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.     

The position and growth of lateral roots on cultured root axes of tomato,Lycopersicon esculentum (Solanaceae)

Root axes of tomato (Lycopersicon esculentum) were cultured in vitro in three different concentrations of sucrose in order to vary their growth rate. Lateral root growth and the initiation of lateral root primordia were studied on each group of axes. Various aspects of primordium initiation, positioning, and emergence were quantified with a view to discovering variable and constant features of these processes. Variable parameters were the rate and frequency of root primordium emergence. Constant parameters, at least under the prevailing conditions, were the spacing between successive laterals and primordia, and the position of the primordia in relation to the vascular system. A model of primordium initiation is presented which combines controls determined by the divisional history of the potential primordium cell and by the vascular pattern.Dedicated with great respect to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday in recognition of her distinguished contributions to cytology.     

Enzymes of starch metabolism in Nicotiana tabacum callus

Activities of enzymes of starch metabolism were determined in tobacco callus grown in light or darkness and in the presence or absence of gibberellic acid. There was a higher rate of starch turnover in light-grown cultures, as judged by the activities of synthetic and degradative enzymes. Gibberellic acid-treated tissues contained a lower level of starch than the corresponding control tissue. This decrease could be correlated with the activity of phosphorylase. Cultured tissue and seedling material were found to have comparable levels of activity for the starch metabolizing enzymes.     

Formation of acetyltropine in Datura callus cultures

The production of tropoylesters in suspension cultures of Datura innoxia stem callus was significantly stimulated by dl-tropic acid, phenylpyruvate or tropine, but was little affected by l-phenylalanine or l-ornithine. Analyses have shown that acetyltropine is synthesized in large quantities by cultured cells when tropine has been supplied to various cultures of D. innoxia and D. tatula. Acetyltropine has been isolated from either the culture medium or cells supplied with tropine. These results indicate that tropine absorbed by the cultured cells of Datura is esterified predominantly by acetic acid to form acetyltropine, instead of other tropane alkaloids.     

Effects of water deficit stress on the developmental growth of excised tomato roots cultured in vitro

Summary Excised tomato roots (Lycopersicon esculentum Mill. cv Bonny Best) were cultured in the presence of mannitol to determine the effects of varying degrees of mild water deficit on their developmental growth. It was found that over the 7-d culture period, the cultured roots could regulate their own developmental responses to the water deficit such that elongation of the primary root axis was favored over that of the lateral roots. Higher degrees of water deficit proportionately decreased lateral root number and density, but lateral root primordia (visualized by clearing roots in chromium trioxide) continued to be formed in water-stressed roots. Measurements of water and osmotic (solute) potentials of the root tips showed that the cultured roots osmoregulated and did not suffer a loss in turgor pressure as a result of the mannitol treatments. However, reciprocal transfer experiments showed that root cultures were unable to resume growth after removal from water deficit conditions, thus indicating a probable requirement for the shoot for complete recovery.     

C6-Green leaf volatiles trigger local and systemic VOC emissions in tomato

In response to insect feeding, tomato plants (Lycopersicon esculentum) release elevated levels of volatile organic compounds; that is, monoterpenes and sesquiterpenes are released both locally and systemically with wounding while C(6) green leaf volatiles are released only from damaged leaves. With the exogenous application (100 nmol) of the C(6)-tomato-volatile (E)-2-hexenal, an increase in the release of local and systemic terpenes was observed, while an equimolar amount of methyl jasmonate triggered only local emissions of terpenes. Labeling studies with 13CO(2) showed that de novo synthesis was not required for monoterpene or sesquiterpene release immediately following chemical treatment or insect feeding. Comparative measurements were made between aldehyde doses applied to the plant and levels naturally released from leaves with insect damage.     

Biscoclaurine alkaloids in callus tissues of Stephania cepharantha

Although callus tissues derived from tubers of Stephania cepharantha cannot synthesize the main alkaloids of the original plant, cepharanthine and isotetrandrine, they are able to synthesize biscoclaurine alkaloids, berbamine and aromorine, the latter not being found in the original plant. These results suggest that enzymes controlling specific methylation and methylenedioxy group formation are absent from the callus. The maximum content of total alkaloid in the callus tissues subcultured for 9 months was more than 3 times that of original plant. Alkaloid content was affected by addition of various auxins, IAA being most effective.     

Media and gelling agent effect on cotton callus initiation from excised seed hypocotyls

A factorial experiment was performed to develop a medium which would support initiation and proliferation of callus in a diverse group of exotic lines of Gossypium hirsutum. Seed hypocotyls of T1, T25 and T133 were cultured on Linsmaier and Skoog (LS) basal medium (1965) with NAA or 2,4-D tested in combination with BA or kinetin. The best medium from this study was then compared to five published media for support of callus initiation and growth of the varieties Acala 1517-75, Coker 500, Dunn 120, Paymaster 303 and TM1. Furthermore, the effects of two gelling agents, Difco-Bacto agar and Kelco Gelrite, were investigated with each of the six media. Significantly more callus was initiated on media solidified with Gelrite than with agar. The best callus production occurred on LS medium supplemented with 30gl^-1 glucose, 0.1 mgl^-1 BA and 0.1 mgl^-1 2,4-D.     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.