Callus Explants Research Articles

A Comparison between Synthetic and Organic Iron Chelators on in Vitro Growth of Nicotiana tabacum Callus Cultures

Nicotiana tabacum callus growth (fresh weight) was measured after culture in the light (16-hour photoperiod) or in darkness for four different culture media, differing in iron chelate type or concentration. All media contained MS basal medium supplemented with 30 g·L–1 sucrose, 2 mg·L–1 IAA, 0.2 mg·L–1 KIN, and 7 g·L–1 agar, pH 5.8. Three of the media contained iron-metalosate (Albion Laboratories), an organic iron chelate, at 100, 200, and 400 micromolar concentrations, and the fourth medium contained 100 μm Fe-EDTA. Twenty-five culture tubes were prepared for each of the 4 different media concentrations and 2 light treatments (8 treatments total). A 1-cm3 callus explant was used for each treatment and cultured for 56 days at 20°C. About 20-fold increases in callus fresh weight were observed for cultures incubated in light or in darkness. In addition, callus growth was not significantly affected by iron chelate type, suggesting the potential utility of this organic chelator in tissue culture media to alleviate potential problems of light-induced EDTA instability and subsequent IAA inactivation. These cultures are being maintained to examine the influence of iron chelate type on organogenesis.

The role of insulin-like growth factor II in magnetic field regulation of bone formation

Musculoskeletal tissue is uniquely sensitive to biophysical input, as demonstrated by both mechanical and electrical stimulation experiments. However, the mechanism by which biophysical input couples to cellular processes is not well understood. The results presented in these studies suggest that these stimuli are bioactive due to stimulation of growth factor biosynthesis by musculoskeletal target cells. We chose insulin-like growth factors (IGFs) as the model growth factor, as the IGFs are capable of stimulating chemotaxis, proliferation, and differentiation of osteoprogenitor cells. These specific studies addressed whether short-term exposure to combined a.c. and d.c. magnetic fields (CMF) would increase production of IGF-II by both osteoblast-like cell cultures as well as rat fracture callus cultures. In vitro studies on human osteoblast-like cell cultures demonstrated statistically significant increases in IGF-II levels and DNA synthesis after only 30 min CMF exposure. In rat fracture callus explant cultures, IGF-II levels were increased at least two-fold dependent on the callus differentiation stage, and these results were comparable with the effect of the osteotropic agent, parathyroid hormone. In summary, these results suggest that the mechanism by which CMF, and other biophysical stimuli, regulate musculoskeletal repair is by modulation of endogenous growth factor (IGF-II) synthesis and secretion.

True-to-the type in vitro propagation of Aechmea fasciata Baker

In vitro propagation of Aechmea fasciata Baker was studied with the of decreasing the frequency of aberrant plant formation. Shoots differentiated from leaf explant callus were cultured on MS based agar solidified media. The frequency of aberrant plant formation was lower on kinetin (KIN)/indolyl-3-acetic acid (IAA) than on 6-benzyladenine/naphthyl acetic acid (NAA) supplemented media. A stable dark-green shoot culture clone was established by persistent use of media supplemented with 1.0 mg 1 −1 KIN and 1.0 mg 1 −1 IAA and by elimination of shoot clusters which contained shoots with pale green, light yellow and variegated leaves. The frequency of aberrant shoot formation in this clone was less than 1% prior to rooting and after potting and acclimatization aberrant plants could not be detected. On media supplemented with 0–2.0 mg 1 −1 NAA or indolyl-3-butyric acid adventitious roots were short. Their length significantly increased upon addition of 1% activated charcoal or if sucrose was replaced by equimolar glucose and fructose. The dark-green clone has been so far maintained for more than 40 subcultures throughout a period exceeding 5 years without significant changes in vigor or frequency of aberrant plant formation. Since summer 1992 it is in use for large scale commercial propagation.

Effect of Elm Selection, Explant Source and Medium Composition on Growth of Ophiostoma ulmi on Callus Cultures

Abstract We examined the effects of elm selection, explant source and media composition on growth of the Dutch elm disease (DED) fungus Ophiostoma ulmi on callus cultures. Calluses were generated from leaf and stem tissue of an American elm (Ulmus Americana L.) seedling (A), susceptible to the disease; an American elm selection 8630, resistant to the disease; and a Siberian elm (U. pumila L.) seedling, also resistant to DED. Calluses were generated on modified Murashige-Skoog (MMS) medium, either with (MMSC) or without coconut milk. Explant source did not affect the fungal growth rate on the callus. Rate of O. ulmi growth on American elm A callus was similar on both media; on Siberian and 8630, fungal growth rate was more rapid on callus cultured on MMS than on MMSC. However, in the absence of callus tissue, O. ulmi growth on MMSC medium was more than five times as rapid as it was on MMS. We observed significant interactions between explant source and selection, and between medium and selection. Fungal growth was always more rapid on American A, and American 8630 then on Siberian. Scanning electron microscopy revealed heavy fungal sporulation on American A, slight on Siberian and none on American 8630. High performance liquid chromatography analysis showed that the secondary metabolic profiles were distinguishable for callus tissue versus explant tissue, but were similar for calli generated from different explant sources.

Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains

For optimization of the transformation procedure with Pisum sativum L. stern explant callus was used to test the effect of disarmed Agrobacterium tumefaciens strains, cocultivation procedures (preconditioning of explants; use of Nicotiana tabacum L. nurse cultures), duration of cocultivation (2, 3 or 4 days), and agents for selection (kanamycin or hygromycin). The succinamopine strain EHA101(pBI1042) produced the highest percentage of transformed calli (77%) when used in conjunction with tobacco nurse culture during four days of cocultivation. Using this strain, kanamycin (76%) and hygromycin (77%) were equally effective selective agents, but for strain LBA4404(pBI1042) percentage of transformed calli was higher for hygromycin (63%) than for kanamycin (17%). The procedures and strains shown to be optimal for transformation of pea callus will now be complemented by a pea regeneration system.

Protoplast culture and plantlet regeneration in stock (Matthiola incana R. Br.).

Protoplasts were isolated from callus derived from excised cotyledons of stock (Matthiola incana R. Br.), by using an enzyme mixture of 1.5% Meicelase and 0.05% Macerozyme R-10. Modified Murashige-Skoog major elements, Ringe and Nitsch minor elements and modified vitamins supplemented with 0.2mg/l 2, 4-D, 1mg/l NAA, 1mg/l BAP, 8.1% mannitol, 1% glucose and 1% sucrose supported protoplast division at 25°C. The frequency of colony formation 10 days after culture was the highest (17.9% of isolated protoplasts) at protoplast density of 0.5×105/ml. Colonies developed to the callus-stage 2 months after initial culture. When callus pieces were cultured in full strength MS medium supplemented with 1mg/l BAP or 1mg/l zeatin, adventitious buds were induced in 9%-16% of callus explants. The buds elongated with leaves open at 20°C and rooted in medium supplemented with 0.1mg/l or 5mg/l IBA.

Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum ait

Callus and suspension cultures, established from seedling and leaf explants of Solanum laciniatum Ait were analysed for solasodine using a spectrophotometric assay. Solasodine concentration in both types of culture ranged from 0.5 - 1.0 mg/g dry wt., with a small number of callus explants containing higher concentrations. There was no overall fall in concentration as a result of serial subculture, and in suspension cultures the level remained constant throughout a single passage. Solasodine concentration was enhanced by the induction of organogenesis in both primary leaf explants and callus. ABA, added at 0.04 mg 1(-1), increased solasodine yield in callus cultures whilst CEPA, at concentrations of 10 mg 1(-1) and higher, inhibited production. Dark grown callus contained significantly more solasodine than light grown.

Embryogenesis and Plantlet Regeneration from Callus of Hibiscus acetosella1

Abstract Plantlets have been produced by germination of somatic embryos derived from callus of Hibiscus acetosella Welw. ex. Hiern. Five of the media used were based on Nitsch and Nitsch's Medium H (purchased formulated without IAA or sucrose). To this base were added, per liter: 40 g glucose for NH; 10 g sucrose for NH-1; 40 g sucrose, 1 mg 2,4-dichlorophenoxyacetic acid (2,4-D), and 1 mg 6-furfurylaminopurine (kinetin) for RM-1; 40 g glucose, 250 mg NaH2PO4·H2O, 28 mg FeC6H5O7·nH2O, 100 mg i-inositol, 30 mg adenine, 0.1 mg (2-chloroethyl)trimethylammonium chloride (chlormequat), and 4 mg 2,4-D for SEM-1; and 40 g glucose, 0.1 mg chlormequat, 0.05 mg B-napthoxyacetic acid (NOA), and 10 mg 2-isopentyladenine (2iP) for HC. The B5 medium was Gamborg's B5 without 2,4-D. All media contained 8 g agar and had the pH adjusted to 5.7 prior to autoclaving. Primary explants placed on HC produced adventitious shoots and callus. When callus explants from HC or primary explants of roots were placed on RM-1, a callus containing embryoid-like structures was produced. Torpedo stage embryos were induced by subculturing this callus from RM-1 on SEM-1 and could be proliferated by sequential transfer from SEM-1 to RM-1, then back to SEM-1. When callus containing torpedo-stage embryos was transferred to B5, the embryos germinated and produced rudimentary plantlets with elongated hypocotyls, short roots, and small cotyledons. These developed into plants when transferred to NH-1.

Isozyme Patterns in Tobacco Plant Parts and Their Derived Calli1

This study was initiated to determine if different tissues of the same plant maintain persistent tissue specific patterns of gene expression during in vitro culture or if in vitro cultures are characterized by identical patterns of gene expression no matter what their tissue of origin. Two‐millimeter sections were excised from the root, lower leaf, upper leaf, pith, sepal, ovary, and anthers of a mature ‘Wisconsin 38’ tobacco (Nicotiana tobacum L.) plant and were grown on a common medium for callus formation. Transfer to fresh medium was made when the calli reached about 8 mm in diameter. One to five such transfers were made depending upon the growth rate of different explants. Extracts from the original explants and each callus transfer were assayed for several isozyme systems using horizontal starch‐gel electrophoresis. Malate dehydrogenase and peroxidase isozymes were found to have good xesointion and to be suitable for the study. The patterns of the isozymes displayed tissue specific differences at the explant level; however, substantial differences between the isozyme patterns of the calii and that of the original explant were displayed. Initially there were differences in isozyme patterns among the calli from different origins and within the different transfers of the same explant callus. However, after the second or third callus transfer, isozyme patterns became more uniform for calli of the same explant.

Shoots from Douglas-fir cultures

Whole Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seed embryos were placed on defined medium containing 0.05 or 0.1 mg/ℓ BAP (benzylaminopurine). One vigorous shoot usually grew from the tip of each swollen cotyledon on the 0.05 BAP medium, but small multiple shoots were produced on the 0.1 BAP medium. After transfer of embryos or excised cotyledons to medium without hormones, the vigorous shoots developed normally but the small multiple shoots did not. However, when cotyledons were grown on medium containing auxin and cytokinin, callus was produced that was isolated and subcultured monthly to fresh medium. Shoots were produced from subcultured cotyledon callus in 11 of 35 cultures, for a frequency of 31%. Two of 30–50 excised shoots rooted after treatment with 10 mg/ℓ IBA (indolebutyric acid). A few shoots were also produced from subcultured needle callus and from stem explant callus from young seedling material.

HYDROXYPROLINE AS AN INHIBITOR OF AUXIN-INDUCED CELL ELONGATION.

AUXIN-INDUCED cell elongation can be inhibited by a variety of substances1. Among these inhibitors are the ammo-acid antagonists canavanine2, β-2-thienylalanine3 and ethionine4,5. This has been taken as evidence that protein synthesis is necessary for cell elongation6. Recently, Steward et al.7 have presented evidence that hydroxy-L-proline can also be an amino-acid antagonist. The growth of carrot callus explants could be blocked by this compound, and the inhibition could be completely reversed with L-proline. Since grdwth in this tissue is largely due to cell division, an investigation has been undertaken into the effects of hydroxyproline on the elongation of Avena coleoptile sections in order to assess the effect of this compound on auxin-induced cell elongation.