In vitro cultures of Salvia officinalis L. as a source of antioxidant compounds

The concentrations of carnosic acid, carnosol and rosmarinic acid in different materials from differentiated (multiple shoot cultures and regenerated plants) and undifferentiated (callus and cell suspension) in vitro cultures of <em>Salvia officinalis</em> were determined by HPLC. The results suggested that diterpenoid (carnosic acid and carnosol) production is closely related to shoot differentiation. The highest diterpenoid yield (11.4 mg g^-1 for carnosic acid and 1.1 mg g^-1 for carnosol) was achieved in shoots of 10-week-old micropropagated plants. The levels were comparable to those found in shoots of naturally growing plants. Undifferentiated callus and cell suspension cultures produced only very low amounts of carnosol (ca. 0.05 mg g^-1 of dry weight). In contrast, content of rosmarinic acid in callus and suspension cultures as well as shoots growing in vitro and in vivo was similar and ranged between 11.2 and 18.6 mg g^-1 of dry weight.

In vitro Organogenesis and Alkaloid Accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3α-tigloyloxytropine and 3-tigloyloxy- 6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l-1) and indole-3-acetic acid (IAA, 0.5 mg l-1) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.

Black stem galls on aspen: anatomy and histochemistry

Large black stem galls occur sporadically on trembling aspen (Populus tremuloides) in western Canada. Although little is known about their cause or structure, trees having these galls are less likely than surrounding aspen to have advanced decay caused by the fungus Phellinus tremulae. The anatomy and histochemistry of black galls and associated branch galls were studied and compared with normal wood and bark. Light microscopy showed that the cambium of black galls produces greater numbers of cells per growth ring and that growth rings are two to three times wider than normal. Vessel elements and fibers are unusually small and misshapen. Gall xylem has characteristics associated with wounding or infection: ray cells filled with phenolic deposits, and vessel elements occluded by tyloses and granular material. Frequent radial strands of undifferentiated callus tissue surrounded by necrophylactic periderms indicate sites of cambial damage of unknown cause. White areas within dark-colored gall xylem of some samples were free of most of these abnormalities, suggesting that a persistent agent is required for continuing tumor growth. Thickened outer bark harbored a variety of saprophytic fungi, especially hyphomycetes. Surface and internal morphology of black galls was also compared with similar stem galls caused by poplar budgall mites (Aceria parapopuli) and was found to be different. Bacteria, fungi, or mites were not obvious within living tissue, and further studies are necessary to determine the etiology of black galls. Key words: Populus tremuloides, poplar, black gall, wood anatomy.

Resistance to Phytophthora cinnamomi in callus tissue derived from three avocado cultivars

Calli induced from various plant parts of avocado cultivars Topa Topa (susceptible), Duke 7 (moderately resistant), and Martin Grande (resistant) were inoculated with the root and collar rotting pathogen Phytophthora cinnamomi and examined for expression of resistance. Resistance was assessed quantitatively by measuring the rate of hyphal extension across the callus surface from the point of inoculation. Resistance in the callus tissue was associated with sparse, limited mycelial growth, in contrast to prolific, dense growth on and within susceptible callus. Rates of hyphal extension on calli of the resistant ‘Duke 7’ and ‘Martin Grande’ ranged from 1–3 mm day−1 compared with 4–7 mm day−1 on calli of ‘Topa Topa’. This difference in growth was significant (P = 0.05). This is the first report of avocado callus exhibiting resistance to P. cinnamomi and it mirrors that recorded for the whole plant for the three avocado cultivars tested. Thus, the level of resistance expressed by each cultivar appears to be innate whether the host tissue remains in an organized state or is in the form of an undifferentiated callus mass. The basis of the resistance expressed appeared to be largely physiological and (or) biochemical rather than anatomical. Assessing rates of hyphal colonization on undifferentiated callus masses in vitro may provide a new and useful assay for screening and selecting host lines in avocado resistant to P. cinnamomi. Key words: callus, avocado, in vitro, resistance, Phytophthora cinnamomi, Persea.

Patterns of variability in DNA content and nuclear volume in regenerating cultures of Nicotiana tabacum

The frequency distributions of DNA content per nucleus were examined in five isonicotinic acid hydrazide (INH) resistant callus lines of tobacco and in developing buds and shoots regenerating from the lines. They were compared with the distribution for a diploid plant, with an estimated 2C value of 7.8 pg. The total range of all the callus cultures was between 5 and 40 pg, with modes between 7 and 16 pg. In the oldest (6 years) callus culture, the mean and standard deviation were lower in the buds developing from the culture than in the undifferentiated callus: however, older shoots again showed a higher mean and more disperse distribution. In contrast, for 4-year-old secondary callus cultures from plants simultaneously regenerated from a second INH-resistant (I 24) line, the mean DNA content per nucleus of the developing buds was higher than that of the undifferentiated callus. Less variation and a more nearly diploid distribution were observed in a 2-year-old callus culture of a fertile plant chosen from the progeny of one of these I 24 plants and in the buds and older shoots regenerated from this culture. Thus, in these five moderately heteroploid cultures a consistent pattern of selection for euploid levels of DNA did not occur during the observed stages of regeneration. Examination of volume as well as DNA content of nuclei in the different tissues showed that variability of nuclear volume measurements, as indicated by coefficients of variation, was correlated with variability of DNA content per nucleus, although measurements of volume and DNA content per individual nucleus were not always highly correlated. It is suggested that high coefficient of variation of nuclear volume, in conjunction with large nuclear size, could serve as a rapid preliminary indicator of highly heterogeneous DNA levels in the nuclear population of Nicotiana cultures.

Enrichment of Solanum khasianum Callus Generating Rootlets with Steroidal Glycoalkaloids

Undifferentiated callus cultures of Solanum khasianum contain only traces of steroidal glycoalkaloids, whereas cultures which have just started to generate rootlets contain these com pounds at a level of up to 5.2% of the dry weight. Only 0.34% steroidal glycoalkaloids occur in the seeds; they consist predominantly of solasonine and solanidine as well as traces of an unidentified com pound. Conversely, steroidal glycoalkaloids from tissue cultures starting organogenesis contain the unidentified com pound as a major fraction, in addition to small amounts of solasonine and solanidine.

In vitro Preservation of Globe Artichoke Germplasm

Aseptically, shoot bud and undifferentiated cultures of globe artichoke were successfully stored for 12 months at 5ºC in the dark. Under these conditions, high percentage of cultures remained viable without perceptable serious signs of senenscence. Although the rates of regrowth slightly decreased, the recovery percentages were enough to obtain high frequencies of healthy globe artichoke (= Cynara scolymus L.). The storage at cold and dark conditions was strongly effective compared with storage in the medium under osmotic stress. Also, the differentiated cultures registered a higher viability compared to undifferentiated cultures. RAPD analysis suggests that plantlets derived from the preserved cultures were identical to those derived from nonstored differentiated tissues culrtures. However, undifferentiated (callus) cultures showed relatively less genetic variation.Key words: Globe artichoke, Germplasm, In vitro preservationDOI = 10.3329/ptcb.v17i1.1115Plant Tissue Cult. & Biotech. 17(1): 1-9, 2007 (June)