Non-organogenic Callus Research Articles

Phytochemical Profiling and Assessment of Anticancer Activity of Leptocarpha rivularis Extracts Obtained from In Vitro Cultures.

Plant cell culture is a source of plant material from which bioactive metabolites can be extracted. In this work, the in vitro propagation of Leptocarpha rivularis, an endemic Chilean shrub with anticancer activity, is described. Different media were tested and optimized for the introduction, propagation, and rooting steps of the micropropagation process. At the end of this process, 83% of plants were successfully acclimatized under greenhouse conditions. Callus induction from the internodal stem segment was performed using various combinations of phytohormones. Green-colored, friable, and non-organogenic callus was generated with a callus induction index higher than 90%. The chemical composition of extracts and callus, obtained from clonal plants, was assessed and the results indicate that the phytochemical profiles of extracts from micropropagated plants are like those found for plants collected from natural habitats, leptocarpine (LTC) being the major component. However, no LTC was detected in callus extract. HeLa and CoN cells, treated with LTC or extract of micropropagated plants, exhibit important diminution on cell viability and a drastic decrease in gene expression of IL-6 and mmp2, genes associated with carcinogenic activity. These effects are more important in cancer cells than in normal cells. Thus, micropropagated L. rivularis could be developed as a potential source of efficient antiproliferative agents.

Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta

Key messageDifferences in the composition and the structural organisation of the extracellular matrix correlate with the morphogenic competence of the callus tissue that originated from the isolated endosperm of kiwifruit.The chemical composition and structural organisation of the extracellular matrix, including the cell wall and the layer on its surface, may correspond with the morphogenic competence of a tissue. In the presented study, this relationship was found in the callus tissue that had been differentiated from the isolated endosperm of the kiwiberry, Actinidia arguta. The experimental system was based on callus samples of exactly the same age that had originated from an isolated endosperm but were cultured under controlled conditions promoting either an organogenic or a non-organogenic pathway. The analyses which were performed using bright field, fluorescence and scanning electron microscopy techniques showed significant differences between the two types of calli. The organogenic tissue was compact and the outer walls of the peripheral cells were covered with granular structures. The non-organogenic tissue was composed of loosely attached cells, which were connected via a net-like structure. The extracellular matrices from both the non- and organogenic tissues were abundant in pectic homogalacturonan and extensins (LM19, LM20, JIM11, JIM12 and JIM20 epitopes), but the epitopes that are characteristic for rhamnogalacturonan I (LM5 and LM6), hemicellulose (LM25) and the arabinogalactan protein (LM2) were detected only in the non-organogenic callus. Moreover, we report the epitopes, which presence is characteristic for the Actinidia endosperm (LM21 and LM25, heteromannan and xyloglucan) and for the endosperm-derived cells that undergo dedifferentiation (loss of LM21 and LM25; appearance or increase in the content of LM5, LM6, LM19, JIM11, JIM12, JIM20, JIM8 and JIM16 epitopes).

Callus of American ginseng (Panax quinquefolius) as a source of ginsenosides – medicinal secondary metabolites

Abstract :Asian ginseng (Panax ginseng) and American ginseng (P. quinquefolius) are valuable medicinal herbs whose roots have been used for ages in traditional medicine in China and North America as vitalizing and stimulating agents. The roots are obtained mainly from field cultivation, which is a slow (5-7 years long), laborious, and troublesome process; so in vitro methods started to be used to produce ginseng biomass. In our study, non-organogenic callus of P. quinquefolius synthesized the same active substances like field roots, for more than 6 years. The ginsenosides are derivatives of protopanaxadiol (Rb1, Rb2, Rc, Rd) or protopanaxatriol (Rg1, Re). The synthesis of Rg1 and Re metabolites is preferred in calli cultured in the dark and with ageing of culture.

In Vitro Host-Free Seed Culture, Callus Development and Organogenesis of an Obligatory Root-Parasite Striga hermonthica (Del.) Benth: The Witch-Weed and Medicinal Plant

Striga hermonthica (Del.) Benth a well-known hemi-parasitic weed, it also has been used widely in African folk medicine to remedy broad spectra of diseases. The current contribution is an attempt to establish reproducible in vitro callusing system. In vitro seedling’s stem segments were used as an explant for callus induction, in 1.5% or 3.0% sucrose added into Murashig and Skoog medium (MS) and supplemented with different auxins, α-Naphthalene-3-acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D), Indole-3-acetic acid (IAA), or Indole-3-butryic acid (IBA) at different concentrations each alone or in combination with cytokinin 0.5 mgL−1 6-benzyl aminopurine. The most effective auxin was NAA with maximum 79% callus induction rate. All auxin treatments induced callus in all concentration when used alone or combined with BAP, except 2,4-D, which induced the callus only when combined with BAP. A high sucrose concentration was required for high callus induction rate by all auxin treatments. IAA and IBA auxins induced direct shoot regeneration and had low callus induction rates. NAA, IAA and IBA induced organogenic calli, whereas 2,4-D in combination with BAP induced non-organogenic callus. We further screened preliminarily the phytochemical contents of the callus and intact plant, which was revealed the presence of flavonoids, terpenes, saponins, cardiac glycosides, alkaloids, tannins and coumarins. Experimental data of both seed culture and callus induction could provide a route to further enhance the efficiency of callus initiation of S. hermonthica for medicinal purposes and understanding the infection mechanism of the witch-weed plant.

Involvement of a tobacco leucine-rich repeat-extensin in cell morphogenesis

The unique mutant nolac-K4 (non-organogenic callus with loosely attached cells), which was generated by T- DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, has lost the ability to form adventitious shoots and also shows decreased intercellular attachment. The gene tagged with the T-DNA in this line, named NpLRX1 (LRR-EXTENSIN 1), is a novel tobacco gene that encodes a cell-wall protein containing chimeric leucine-rich repeat and extensin domains. The gene is highly similar to the Arabidopsis LRX genes, and phylogenetic analysis places it in the vegetative LRX clade. NpLRX1 is expressed ubiquitously in vegetative tissues, strongly in the leaf and root vascular bundles, and in emerging lateral roots and root tips. Tobacco leaf disks transformed with an NpLRX1-RNAi construct displayed aberrant adventitious buds and disorganized cell morphogenesis with large intercellular spaces. The shapes of NpLRX1- RNAi-transformed BY-2 cells were irregular, and the cells showed disorganized cortical microtubules. These results suggest that NpLRX1, the lack of which may be responsible for the nolac-K4 mutation, is a new tobacco LRX gene that has regulatory roles in cell morphogenesis that are essential for plant tissue development.

Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells.

When leaf disks from haploid plants of Nicotiana plumbaginifolia Viv. were transformed with T-DNA and cultured on shoot-inducing medium, nonorganogenic callus. designated nolac (for non-organogenic callus with loosely attached cells), appeared on approximately 7% of leaf disks. In contrast, normal callus was generated on T-DNA-transformed leaf disks from diploid plants and on non-transformed leaf disks from haploid and diploid plants. Transmission electron microscopy revealed that the middle lamellae and the cell walls of one line of mutant callus (nolac-H14) were barely stained by ruthenium red. even after demethylesterification with NaOH, whereas the entire cell wall and the middle lamella were strongly stained in normal callus. In cultures of nolac-H14 callus, the level of sugar components of pectic polysaccharides in the hemicellulose fraction was reduced and that in the culture medium was elevated, as compared with cultures of normal callus. These results indicate that pectic polysaccharides are not retained in the cell walls and middle lamellae of nolac-H14 callus. In nolac-H14, the ratio of arabinose to galactose was low in the pectic polysaccharides purified from all cell wall fractions and from the medium, in particular, in the hemicellulose fractions. The low levels of arabinofuranosyl (T-Araf, 5-Araf, 2,5-Araf, and 3,5-Araf) residues in the pectic polysaccharides of the hemicellulosic fraction of nolac-H,14 indicated that no neutral-sugar side chains, composed mainly of linear arabinan. were present in nolac-H14. Arabinose-rich pectins. which are strongly associated with cellulose-hemicellulose complexes, might play an important role in intercellular attachment in the architecture of the cell wall.

Genotype Affects the Morphogenic Response in vitro of Epicotyl Segments of Citrus Rootstocks

The influence of light, hormones and explant orientation on in vitro regeneration in epicotyl cuttings was compared in four Citrus species (C. aurantium, C. macrophylla, C. reshni andC. sinensis ) and the hybrid Troyer citrange (C. sinensis×Poncirus trifoliata). In all cases, explants planted vertically regenerated shoots at the apical end by a process of direct organogenesis without callus formation. When the Troyer citrange explants were incubated horizontally, regeneration at the apical end occurred by an indirect organogenic pathway after callus formation. This change in the pathway of regeneration did not occur in any of the Citrus species, and incubation horizontally resulted in a reduction in the number of buds and shoots formed through the direct organogenic pathway. Shoot formation through the direct organogenic pathway was inhibited by darkness, and this inhibitory effect was counteracted by the cytokinin benzyladenine in Troyer citrange and, partly, in C. sinensis, but not in C. macrophylla. A non-organogenic callus formed at the basal end of most of the cuttings of C. reshni. InC. sinensis and C. aurantium, a non-organogenic callus formed only in a low proportion of explants. Troyer citrange formed an organogenic callus in which buds or roots differentiated depending on the auxin/cytokinin balance. C. macrophylla formed callus in the dark but not in the light. Root formation occurred both in the presence of the auxin naphthaleneacetic acid or low concentrations (2.2 to 4.4μM) of the cytokinin benzyladenine, but no buds were formed. These qualitative and quantitative differences in the organogenic response indicate that the conditions for regeneration must be optimized for each genotype.

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.

Biochemical comparisons of habituated and non-habituated callus lines of Glycine max (L.) cv. Acme

A cytokinin habituated non-organogenic callus line of Glycine max L. cv. Acme, was isolated. Cytokinins were detected in both non- and habituated calli. HPLC analysis indicated that the highest biological activity was associated with compounds that cochromatographed with zeatin and derivatives thereof. Cytokinin-like activity in the habituated callus was higher than in the non-habituated callus. No difference was observed in the ability of the two callus types to metabolise trans-zeatin. The habituated callus however, metabolised N6(△2-isopentenyl)adenine (iP)at a slower rate than the non-habituated line. The regulation of proline and putrescine levels during the growth cycle was similar in both callus types, although less proline was present in the habituated than the non-habituated callus. In both callus types a decrease in putrescine levels was accompanied by an increase in the proline levels on day 21 of the growth cycle. Neither spermine nor spermidine were detected in either the habituated or non-habituated lines. A comparison of the total RNA levels of the habituated and non-habituated material revealed higher RNA levels in the non-habituated than the habituated line. Two-dimensional gel electrophoresis indicated the presence of 3 polypeptides in the habituated callus on day 13 of the growth cycle. These were not detected in the non-habituated callus, of similar age.

Chlorophylls and carotenoids in a fully habituated nonorganogenic callus ofBeta vulgaris

A fully habituated (H) nonorganogenic sugar beet callus, subcultured in the light, did not contain detectable chlorophyll (Chl) nor carotenoid (Car). It accumulated some Car in the dark. Fluorescence spectra indicated that this H callus also accumulated some protochlorophyllide which, however, was not well integrated into the protochlorophyllide-NADPH-photoreductase complex, and therefore not transformed into chlorophyllide in the light. The H callus showed no variable fluorescence which indicated absence of photosynthesis, and therefore it suggested a full heterotrophic behaviour of this peculiar callus line. A green hormone-dependent callus of the same sugar beet had normal fluorescence spectra and kinetics comparable to those of a green leaf.

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H2O2 detoxifying systems.

Plant regeneration in a jute species (C. capsularis) and its possible relationship with glyoxalase-I

The addition of 3 mg/l of nordihydroguaiaretic acid (NDGA) to BAP and tyrosine fortified MS medium was essential to obtain organogenic callus from the hypocotyl segments of two varieties (D-154 and CVL-1) of Corchorus capsularis - one of the two jute species. When the organogenic callus, which is rich in large starch granules, was transferred to MS basal medium, it differentiated into single or multiple shoots usually in the first subculture and sometimes in the second. The activity of glyoxalase-I of the organogenic callus was found to be significantly lower than that observed in the nonorganogenic callus initiated on MS medium supplemented with 2,4-D, tyrosine, BAP or just BAP and tyrosine. This suggests an inverse relationship between differentiation and the level of glyoxalase-I activity in the two varieties of C. capsularis jute.

IN VITRO NITROGEN METABOLISM AND ORGANOGENESIS OF NICOTIANA TABACUM

The role of N metabolism in organogenesis and growth was studied using tobacco pith callus. Callus was cultured on a solid medium containing 10 μM (1.75 mg/l) IAA and 2 μM (0.43 mg/l) kinetin for 56 days. In the growth experiment, ratios of NH4+-N to NO3--N (0:60, 20:40, 30:30, 40:20 and 60:0 mM) were supplied by (NH4)2 SO4 and KNO3. Callus and media were analyzed for inorganic N. Callus supported by 30:30 and 40:20 media removed the highest amounts of NH4+-N and NO3--N from the media and exhibited organogenesis. Final dry weight was greatest in callus supported by the 30:30 medium. In the organogenesis experiment, the transfer history of the inoculum source affected N uptake, organogenesis and growth. Inorganic N was supplied by NH4NO3 and KNO3-. The net uptake of NH4+-N and NO3--N was lower in shoot-forming than in root-forming and non-organogenic callus subculture from 7-day-old stock cultures. The final pH of the medium supporting shoot-forming callus was lowest. Growth, on a dry weight basis, was lowest in shoot-forming callus. Callus subculture from 60-day-old stock cultures formed no shoots.