Ability of plant callus cultures to synthesize and accumulate lower terpenoids

Callus cultures derived from seven oil-producing plants have been maintained under different regimes of media, temperature and illumination, and have been assayed for ability both to synthesize and to accumulate mono- and sesqui-terpenes. Callus of Pinus radiata (the sole gymnosperm) accumulated (α- and β-pinenes at levels comparable with those in the parent stem and needles; and that of Jasminum officinale accumulated traces of several monoterpenes (< 0.1 % the amount in petals) but cultures of Rosmarinus officinalis, Lavandula angustifolia, Anethum graveolens, Ocimum basilicum and Tanacetum vulgare did not detectably accumulate the lower terpenoids or secrete them into the medium. However, all seven culture lines yielded cell-free extracts containing prenyltransferase and an isomerizing system (as assayed by conversion of IPP into GPP, NPP and FPP) with activities some fold greater than those extracted from the parent mature plants, or up to 90-fold the levels extractable from young seedlings of the various species. Callus of five of the species (A. graveolens and O. basilicum were not assayed) also contained MVA-kinase, MVAP-kinase, MVAPP-decarboxylase and IPP-DMAPP isomerase at levels comparable with those in the parent tissue. Hence the angiosperms yielded cultures that presumably contained the crucial enzyniic machinery necessary for the synthesis of the lower terpenoids, although accumulation of those compounds did not occur. Reasons for this unexpected situation are discussed. These results imply that callus cultures may be a convenient source of biomass for studies on the enzymes of terpenoid biosynthesis.     

Reduction of ferric leghemoglobin in soybean root nodules

Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions.

     

Light-dependent monoterpene synthesis in Pinus radiata cultures

Callus cultures of Pinus radiata that synthesized monoterpenes de novo and which were stable for at least 1 year have been established. The products differed from those of parent plants in that α-pinene (87–100%) rather than β-pinene was the main component. The best lines accumulated monoterpenes (ca 2 × 10^?3% wt/wet wt)in yields 40–20% of that in the parent stem and needles. The composition of the extractable oil depended on the light regime. After culture in total darkness toluene and acetone accumulated. These compounds also occurred (at low levels) in dark-grown seedlings and in seeds of P. radiata and a route for their formation from α-pinene is suggested. Cell-free extracts of the culture lines converted [¹⁴C] IPP into geraniol, nerol and α- and β-pinenes in up to 46% total yield. These are the most active crude extracts for monoterpene biosynthesis that have been reported from either tissue cultures or higher plants.     

The toxicity of monoterpenes to plant cell cultures

The effects of exogenous monoterpenes on cellular viability and growth of fine suspension cultures of Pelargonium fragrans have been investigated in order to establish whether product toxicity might play a role in determining the level of monoterpene accumulation in morphologically undifferentiated plant cell cultures. Producing lines of P. fragrans accumulate monoterpenes in the medium at concentrations which we now demonstrate to be toxic to fine suspension cultures. The problems associated with end-product toxicity must be solved before the potential of plant tissue culture techniques for the commercial production of monoterpenes be fully realised.     

Rearing Migratory Endoparasitic Nematodes in Citrus Callus and Roots Produced from Citrus Leaves

Radopholus similis and Pratylenchus coffeae were reared on callus and roots developed from citrus leaves. Callus formed best when leaf petioles were immersed in Astatula fine sand and the leaves were sprayed daily with 4 ppm 2,4-D solution and maintained at 25 or 30 C. The nematodes completed one generation in 20 days at 25 C. Highest populations of R. similis (1,127) occurred after 50 days, and the highest for P. coffeae (619) after 70 days. Leaf-callus cultures from R. similis-resistant citrus rootstocks showed the same degree of infection as susceptible rough lemon callus after 30 days.     

Cometabolic isoterpinolene formation from isolimonene by denitrifying Alcaligenes defragrans

Alcaligenes defragrans strains denitrify on monoterpenes with an unsaturated hydrocarbon structure. A new cometabolic reaction, the formation of isoterpinolene from isolimonene, was detected in cultures that grew on a monoterpene. The biotransformation of isolimonene, a monocyclic monoterpene with a sp³-hybridized C1 atom of the menthane skeleton, contrasts with the complete mineralization of monoterpenes with a sp²-hybridized C1 atom. This selectivity indicates a demand for a sp²-hybridized C1 atom as structural property for monoterpenes that can be oxidized by A. defragrans.     

Bisbenzylisoquinoline alkaloids in Berberis cell cultures

Callus cultures of 34 Berberis cell lines representing 33 species were screened for alkaloids using TLC and HPLC. Jatrorrhizine was always found to be a major constituent; two B. stolonifera lines were the richest in bisbenzylisoquinolines. The new alkaloid 2-norberbamunine was isolated from cultures of B. stolonifera V29 together with berbamunine, aromoline, berbamine, isotetrandrine and jatrorrhizine. The time course of bisbenzylisoquinoline accumulation was studied in B. stolonifera V29 suspension cultures and it was found that the concentrations of these alkaloids rose throughout the cell growth phase.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Callus Induction,Plant Regeneration and an Assessment of Cytological Variation in Regenerated Plants of Lolium multiflorum L.

Callus cultures were initiated from roots, apical meristem tips and leaf explants of several genotypes of Lolium multiflorum L. (Italian Ryegrass). Genotypes were selected which showed a high frequency of callus initiation and from which plants could be regenerated. Plants could be routinely produced from root-derived callus of only one of the genotypes tested. The selected genotypes were still amenable if the temperature and concentration of 2,4-D in the medium were altered. Increase in temperature caused callus from one genotype to give rise to more albino regenerants. Callus formation and plant regeneration occurred at a higher frequency from diploid than tetraploid explants. All regenerants from the diploid cultures had the 2n = 2x = 14 chromosome number whereas plants regenerated from callus derived from tetraploid cultures lost up to 3 chromosomes.     

Morphogenesis and plant regeneration from cotyledonary cultures of Eucalyptus

Callus cultures were established from hypocotyls and cotyledons derived from young seedlings of Eucalyptus citriodora. Successful plantlet production from cotyledonary callus was achieved within 6 weeks on Murashige and Skoog's basal medium supplemented with zeatin (1 mg/l) and indoleacetic acid (0.2 mg/l). Leaf and shoot callus obtained from one-year-old plants did not differentiate. Results reported contribute to defining optimal conditions for callus growth and plantlet formation.     

Monoterpene synthesis in shoots regenerated from callus cultures

-Shoots regenerated from two-year old callus cultures of Lavandula angustifolia and Rosmarinus officinalis accumulated monoterpenes characteristic of the parent tissue. No such compounds could be detected in undifferentiated callus maintained under a variety of conditions.     

In vitro organogenesis in two dioecious species,Rumex acetosella L. and R. acetosa L. (Polygonaceae)

Callus cultures were established from dioecious plant species Rumex acetosella and R. acetosa, using cotyledons, hypocotyls and stem tips of aseptically germinated seedlings as primary explants. Cultures were also established from male and female R. acetosella adult plants, starting from vegetative lateral buds. Cell division was induced using a high 2,4-D concentration, while bud induction and multiplication were stimulated on a medium with high BAP/IAA ratio. Cotyledon fragments of both species produced only rhizogenic calli. Hypocotyl-derived calli of R. acetosella produced buds, while those of R. acetosa showed no bud forming response under these conditions. Bud multiplication occurred in stem tip cultures of both species and in lateral bud cultures of R. acetosella. Calli derived from male plants produced more buds than those from female. Shoots were easily rooted using IBA, and plantlets were effectively transferred to soil. Flowering was not induced in culture. The sex of regenerated male and female plants was not altered by the culture conditions.     

Secondary metabolites produced by callus cultures of various Ruta species

Callus cultures were established from hypocotyl explants of R. bracteosa, R. chalepensis and R. macrophylla. Calli were maintained for more than three years on MS-medium supplemented with 1 mg l^-1 of each 2,4-D and kinetin. Acridone and furoquinoline alkaloids and coumarins have been isolated from four week old calli grown on a hormone containing and hormone-free medium. A new chlorinated acridone alkaloid has been detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog [6]     

Production of β-β,Dimethylacrylshikonin in Callus Cultures of <Emphasis Type="Italic">Onosma</Emphasis> echioides Var <Emphasis Type="Italic">hispidum</Emphasis> Clarke

Callus tissues derived from leaf segments of Onosma echioides var hispidum on three basal media viz. Murashige & Skoog(MS), Gamborg et al (B₅) and White’s containing 3% sucrose produce napthaquinone pigments in presence of and NAA.However, β-β,dimethylacrylshikonin synthesis was triggered in dark in undifferentiated parenchyma cells on B₅ agar mediumcontaining 1 x10^?5 M Kn and 2×10^?6 M IBA when proliferated calli after 16 weeks (4^th generation) were transferred to it andincubated at 23 ± 1 °C. The pigment biosynthesis increased linearly from 4^th to 6^th week after a lag of first 3 weeks. Callus grewexponentially after a lag of 2 weeks and diminished from 6^th week onward. During 8 weeks of growth, callus grew from 0.8 to8.2 g and the β-β, dimethylacrylshikonin showed the highest level of 25.41 μg g^?1 of fresh tissue. Light microscopic examinationof semi-thin sections of pigmented tissue revealed pigment accumlation between the plasma membrane and cell wall andalso in the intercellular spaces. Exposure of cultures to white fluorescent light for more than 2 h resulted in completerepression of pigment bosynthesis. The investigations suggest that the regulatory mechanism for the biosynthesis andaccumulation of napthaquinone pigment(s) may be similar to that of Lithospermum erythrorhizon. Pigment producingcapability of callus cultures of O. echioides var hispidum can be exploited as an alternative raw source for the production ofshikonin derivatives.     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Rubidium chloride tolerant callus cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accumulate more potassium and cross tolerate to other salts

Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog’s (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95–100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K⁺ in comparison with their corresponding controls. Suspension cultures were established and uptake of ⁸⁶Rb⁺ was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K⁺ and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35–40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K⁺ and also an increased influx of it.     

Plant regeneration from somatic embryos in black pepper

Embryogenic callus derived from zygotic embryos of black pepper (Piper nigrum Linn.) were induced to form somatic embryos on solid and liquid Schenk and Hildebrandt basal medium. Callus proliferation, somatic embryo-genesis and germination of embryos were achieved in about 8 months in static cultures while it took only 8 weeks in liquid suspension cultures. The highest number of embryos and plantlets was produced from cells grown as suspension cultures raised in half-strength medium without growth regulators and sucrose level reduced from 3% to 1.5%. Regenerated plants were established in soil.     

Genotype Dependent Somatic Embryogenesis and Regeneration from Leaf Base Cultures of Sorghum bicolor

A simple and reproducible protocol for callus induction and regeneration of plantlets from leaf base cultures of agronomically important Indian cultivars of Sorghum bicolor(L.) Moench (296 Band RS 585) has been developed. A strong genotype dependent response was observed and the genotype 296 B was found to be the most responsive as compared to the other genotypes tested. Cultures were raised from the III, IV and V leaf bases, excised from 12-day-old in vitro raised plantlets and cultured on Callus Induction Medium (CM). Callus initiation took place after 10-14 days of culture. The explants were maintained on this medium for 30- 35 days, after which they were transferred to Regeneration Medium (RM). Histological examination indicated that somatic embryogenesis was prevalent in the leaf base cultures and the embryos started to germinate after 15-20 days of transfer to RM. Plantlets with complete shoot and root system have been raised with as many as 30 plantlets regenerating from a single explant. These plantlets could be easily separated from one another and transferred to culture tubes for faster growth and development. Later, individual plants numbering more than 50 were transferred to pots containing soil: soilrite (1:1) for hardening. A high regeneration frequency of up to 40 % could be obtained in the genotype 296 B followed by 10.8 % in the genotype RS 585 and 7.8 % in C 43.     

Cellular and whole plant responses ofVigna radiata to NaCl stress

The effect of different NaCl regimes was examined on the growth and ion accumulation in whole plants and callus cultures ofVigna radiata. Whole plants grown in sand culture were watered with Hoagland's solution supplemented with 0–350 mol m⁻³ of NaCl. Callus cultures were initiated from leaves of 7-d old seedlings of the same seed stock and grown in modified PC-L2 medium containing the same levels of NaCl as in Hoagland's solution. Callus showed the same tolerance to salt as did the whole plant suggesting thatV. radiata appears to have a mechanism(s) for salt tolerance which operates at the cellular level. Ion analysis of whole plant showed that root sodium concentrations of the tolerant cultivar G-65 was much higher while shoot sodium was much less than those of salt sensitive cultivar ML-1. Callus cultures of cv. G-65 also accumulated higher Na⁺ levels. Thus, the greater salt tolerance of cv. G-65 was associated with the control of sodium accumulation at the shoot or cellular level. Communicated by J. POSPíŠILOVá     

ACC deaminase producing Pseudomonas putida strain PSE3 and Rhizobium leguminosarum strain RP2 in synergism improves growth, nodulation and yield of pea grown in alluvial soils

Plant growth promoting rhizobacteria affects the overall performance of plants by one or combination of mechanisms. However, little information is available on how ACC deaminase secreting bacteria enhance crop production. The present study aimed at identifying ACC deaminase producing and phosphate solubilizing bacterial strains and to assess their plant growth promoting activities. Additionally, the effect of two ACC deaminase positive bacterial strains Pseudomonas putida and Rhizobium leguminosarum on pea plants was determined to find a novel and compatible bacterial pairing for developing efficient inoculants for enhancing legume production and reducing dependence on chemical fertilizers. The isolated bacterial cultures were characterized biochemically and by 16S rRNA sequence analysis. The plant growth promoting activities was determined using standard microbiological methods. The impact of P. putida and R. leguminosarum, on pea plants was determined both in pots and in field environments. Of the total 40 bacterial strains, strain PSE3 isolated from Mentha arvenss rhizosphere and RP2 strain from pea nodules produced ACC deaminase, solubilized insoluble phosphate, synthesized indole acetic acid, ammonia, cyanogenic compounds, exopolysaccharides and had antifungal activity. The dual inoculation of P. putida strain PSE3 and R. leguminosarum strain RP2 had largest positive effect and markedly increased the growth, symbiotic characteristics, nutrient pool and quantity and quality of pea seeds. The measured parameters were further augmented when inoculated pea plants were grown in soils treated with urea or DAP. A significant variation in the measured parameters of pea plants was observed under both pot and field trials following microbial inoculation but the bacterial cultures did not differ significantly in growth promoting activities. The results suggest that ACC deaminase positive bacterial cultures endowed with multiple potential can be targeted to develop mixed inoculants for enhancing pea production and hence, to reduce dependence on synthetic fertilizers.