In vitro regeneration of adult Pinus sylvestris L. trees

The propagation of adult conifer trees by tissue culture has been studied for the last twenty years, but problems related to the juvenile to adult phase change of trees have limited the practical applications of these tissue culture procedures. This paper describes a micropropagation protocol for the in vitro propagation of mature Scots pine trees. In this study, dormant shoot buds, which had not started to elongate, were collected from twenty-one adult Pinus sylvestris trees (> 15 years old) during the winter. The sampled buds were cut transversely into slices of 0.5 to 1 cm in thickness and were cultured on three types of culture media (DCR, WP and LPm) supplemented with four cytokinins (BA, mT, Tdz and Z), at two different concentrations (25 and 50 µM), except for Tdz, whose concentrations were diluted to 5 µM and 2.5 µM. The evaluated culture media did not show significant differences in the bud organogenesis capacity. In fact, the highest organogenic response was obtained with buds cultured on DCR and WP media and by explants cultured on medium supplemented with 25 µM meta-topolin. This protocol is a successful and efficient biotechnological approach to the micropropagation of adult P. sylvestris trees.     

The effect of Stratiotes aloides L. and nutrients on the growth rate of Lemna minor L.

We tried to find the reasons for frequently observed low biomass of Lemna minor among floating rosettes of Stratiotes aloides as opposed to the abundance of Spirodela polyrhiza in these stands. The effect of S. aloides on L. minor was analysed in outdoor culture experiments. The duckweed collected from among dense stands of the floating form of S. aloides (test sites) showed a significantly lower growth rate than that from stands free of the water soldier (control sites). Water from the latter stands was significantly richer in nitrate-nitrogen and ammonium-nitrogen, but not in soluble reactive phosphorus suggesting possible nutrient depletion as a reason for observed differences. Growth rates of L. minor were indeed significantly correlated with in situ concentrations of all dissolved nutrients and showed saturation at around 0.18 mg N-NH₄ dm⁻³ and 0.05 mg SRP dm⁻³ above which the growth rates did not increase significantly. We used stepwise regression to test the combined effect of nutrients and the presence/absence of S. aloides on the growth rate of L. minor. The regression showed a negligible effect of ammonium ions and nitrates on the growth rate of the duckweed. At low concentrations of soluble reactive phosphorus the presence of both the S. aloides and SRP concentrations significantly affected the growth of L. minor. Above saturating SRP concentrations none of the two effects was significant. Results of our experiments seem to suggest that the presence of S. aloides exerts a negative effect on the growth of L. minor only at low concentrations of soluble phosphorus. Since the depletion of nutrients in water by the floating form of S. aloides is unlikely, we point to S. polyrhiza as a superior competitor for nutrients and suggest a possible allelopathic effect of the water soldier on L. minor.     

S-allele diversity in Prunus L. Cerasus subgenus from Iran

In this study, S-allele diversity of eight wild and two commercial species of the Cerasus subgenus in Iran was investigated using two primer pairs. A high level of S-allele polymorphism was detected among and within the species evaluated. Furthermore, most of wild species showed 2–4 alleles based on S-allele primers and may be considered as tetraploid. Sweet cherry cultivars, Siah-Mashhad, Siah-Shabestar, Takdaneh-Mashhad, Siah-Daneshkadeh and Protiva showed S₃S₁₂, S₃S₁₂, S₃S₁₂, S₃S₅ and S₃S₄ combinations, respectively, allele S₃ showing the highest frequency. Three Iranian sweet cherry cultivars had the same allelic combination (S₃S₁₂) that the same ancestor in genealogy of these cultivars may explain the loss of diversity observed at the S-locus. Wild cherry (mazzard) accessions showed wide range of alleles such as S₁, S₂, S₇, S₁₄ and S₂₀ and unknown alleles, while sour cherries showed S₆, S₉, S₁₃ and S₂₇ alleles. In conclusion, the conservation of these highly diverse native species of Iranian wild Cerasus germplasm is recommended for future breeding activity.     

Micropropagation and bioreactor studies of the medicinally important plant Lessertia (Sutherlandia) frutescens L.

This study describes a protocol for rapid and efficient in vitro propagation of Lessertia frutescens (cancer bush), a medicinally important plant species native to southern Africa. Single node explants were grown in various culture regimes of MS medium containing 30 g/l sucrose supplemented with various concentrations of cytokinins and auxins and solidified with 8 g/l agar. These were (a) 2.22, 4.44, 13.32 and 22.19 µM BA; 2.32, 4.65, 13.95 and 23.23 µM K and 0.45, 2.27, 4.54 and 13.62 µM TDZ (b) a combination of 2.22 µM BA with 0.57, 2.85, 5.71 and 11.42 µM IAA, 0.49, 2.46, 4.9 and 9.8 µM IBA or 0.54, 2.69, 5.37 and 10.74 µM NAA and (c) different media types viz. MS, SH basal salt medium and WPM at 1, ½ and ¼ salt strength which were each supplemented with 2.22 µM BA and 0.54 µM NAA. Single node explants were also grown in MS liquid medium supplemented with 2.22 µM BA and 0.54 µM NAA in temporary and continuous immersion bioreactors. Maximum number of shoots (12.9) per single node explant was obtained in the temporary immersion bioreactor but 50% of these shoots showed symptoms of hyperhydricity. In solid culture the best shoot multiplication response (10 shoots) was obtained in full strength MS. Roots were induced using shoot tips cultured in ½ MS solid medium supplemented with various concentrations of IBA or NAA. The highest rooting percentage (78%) was achieved in 19.6 µM IBA. Rooted plantlets were cultured in a mixture of perlite and vermiculite (1:1; v/v) and successfully acclimatized in a growth chamber with an 85% survival rate.     

Combined effect of salinity and temperature on Spirorbis spirorbis L. and Circeus spirillum L. larvae from the White Sea

The combined effect of salinity and temperature on Spirorbis spirorbis L. and Circeus spirillum L. larvae from the White Sea was studied in the laboratory experiments. In the White Sea, S. spirorbis is distributed through the depth of 1-20 m and is affected by all varieties of fluctuations in salinity and temperature. C. spirillum lives in more wide range of depths 1-55 m and is more stenohaline. S. spirorbis larvae are sufficiently more resistant to the low salinity (10‰) than C. spirillum larvae. Both species are stenothermic. Highest survivorship of S. spirorbis larvae was marked under 5 °C in all experimental salinities. Under temperature treatments of 10-15 °C, the larval survivorship was sufficiently restricted in all salinities. Highest survivorship of C. spirillum larvae was also marked under 5 °C but in more narrow salinity range.The number of larvae undergoing metamorphosis in both species was very low, only about 10% of the total number. Highest number of successful attachments in both species was marked in high salinities (25-30‰) and does not exceed 25% of survivors. Experimental data suggests that salinity and temperature affect directly general survivorship of the larvae and secondary-attachment and metamorphosis processes.     

Mutagenesis by radiowaves in Antirrhinum majus L.

Pollen of Antirrhinum majus was irradiated with radiowaves (γ = 1.5 m; field strength 1.5 V/m) in 3 series for 4, 12 and 43 $\text{3}\text{4}$ h, then crossed on styles of emasculated untreated flowers. After selfing of the M₁ generation an increase in embryonic lethality and in mutations for characters of the seedlings and young plants was observed. These observations confirmed the mutagenic action of the treatment that was described earlier in Vicia faba and Oenothera hookeri.     

Introgressive hybridization between Plantago major L. taxa

Occurrence and direction of introgressive hybridization between Plantago major taxa were tested. Plantago major plants were collected from 10 Egyptian locations. Four populations of two European taxa were used for comparison. These are ecologically and geographically separated and were identified as the subspecies Plantago major ssp. major and Plantago major ssp. intermedia. In the Egyptian populations, most individuals fall within the variation range of P. major ssp. intermedia. Only one population, from Burg El-Arab, morphologically resembled clearly P. major ssp. major and showed some ISSR fragments that characterize pure populations of this taxon. All individuals of population 2 (Alexandria) and some individuals of populations 1, 3, 5, 6 and 10 (Alexandria, Aswan) morphologically corresponded to P. major ssp. intermedia. All individuals collected from Egypt had ISSR fragments characterizing both pure P. major ssp. major and pure P. major ssp. intermedia. Most of these individuals had a higher percentage of intermedia-type fragments than major-type fragments, suggesting that P. major ssp. intermedia in Egypt shows some introgression towards P. major ssp. major. The pure populations were distinct from each other, while the Egyptian populations were intermediate according to principal coordinate analysis (PCoA) of ISSR data. In the populations collected from Egypt, major and intermedia cannot be seen as separate species. The study suggests that the dominant taxa introgressed to the minority population(s). Taxon frequency may be a key component in determining the direction of introgression.     

Furostanol saponins from Yucca gloriosa L. rhizomes

Two new and one known furostanol saponins were isolated from the rhizomes of Yucca gloriosa. The structures of the isolated compounds were established by detailed spectroscopic analysis (1D-, 2D NMR, ESI-MS, HR-MALDI-MS). The glycosidic profile of Y. gloriosa in comparison to that of Yucca schidigera, the well known commercial source of saponins, is briefly discussed. The similarity between the two species regarding the high steroidal content suggests that it might be possible to use Y. gloriosa for the same applications as Y. schidigera.     

Anti-allergic inflammatory components from Sanguisorba officinalis L.

Sanguisorba officinalis L. was well known as a traditional herbal medicine to treat inflammation and allergic skin diseases. The aim of this research was to indentify compounds with anti-allergic inflammatory property. Twenty-five compounds (1–25) were isolated from S. officinalis including two new compounds (1 and 8), and their chemical structures were identified by NMR and ESIMS analysis. Consequently, the anti-allergic inflammatory activities of these isolates were investigated by inhibiting β-hexosaminidase and IL-4 production in PMA/A23187-stimulated RBL-2H3 cells. Compounds 6, 8, 13, 17–18 and 25 significantly inhibited β-hexosaminidase release and IL-4 production. Additionally, compounds 8, 17 and 25 effectively suppressed the activation of NF-κB and NF-κB p65 translocation into the nucleus. Anti-inflammatory effects of isolated compounds were evaluated in LPS-stimulated RAW264.7 macrophages, and they showed dramatic inhibition on LPS-induced overproduction of nitric oxide (NO) and TNF-α. Consistently, the protein levels of iNOS and COX-2 were remarkably decreased by the single compounds 8, 13 and 25. These results showed that compounds 8, 13 and 25 from S. officinalis may have a therapeutic potential for allergic inflammatory diseases.     

A Simple in Vitro Method to Study Tuber Growth of Solanum tuberosum L.

A simple in vitro method is described which allows the study of effects of mineral nutrient supply on growth rate of the tuber. Single node cuttings carrying axillary tubers are cultured under semi-sterile conditions in cooled (+ 1°C) nutrient solutions. Tuber growth is monitored volumetrically and mineral nutrient uptake calculated from the nutrient solution taken up by the cuttings.     

Endogenous isoflavone methylation correlates with the in vitro rooting phases of Spartium junceum L. (Leguminosae)

Spartium junceum L. (Leguminosae) is a perennial shrub, native to the Mediterranean region in southern Europe, widespread in all the Italian regions and, as a leguminous species, it has a high isoflavone content. An in vitro culture protocol was developed for this species starting from stem nodal sections of in vivo plants, and isoflavone components of the in vitro cultured tissues were studied by means of High Performance Liquid Chromatography (HPLC) analytical techniques. Two main isoflavones were detected in the S. junceum tissues during the in vitro propagation phases: Genistein (4′,5,7-Trihydroxyisoflavone), already reported in this species, and its methylated form 4′,5,7-Trimethoxyisoflavone, detected for the first time in this plant species (0.750 ± 0.02 mg g⁻¹ dry tissue). The presence of both of these compounds in S. junceum tissues was consistently detected during the in vitro multiplication phase. The absence of the methylated form within plant tissues in the early phases of the in vitro adventitious root formation was correlated with its negative effect displayed on root induction and initiation phases, while its presence in the final “root manifestation” phase influenced positively the rooting process. The unmethylated form, although detectable in tissues in the precocious rooting phases, was no longer present in the final rooting phase. Its effect on rooting, however, proved always to be beneficial.     

Production of a hybrid of Nicotiana repanda willd.× N. tabacum L. by ovule culture

Nicotiana repanda (n = 24) × N. tabacum (n = 24) was obtained by ovule culture following regeneration from the callus. The hybrid showed an aneuploid chromosome number (44) and morphological characteristics intermediate between those of N. repanda and N. tabacum. The resistance of N. repanda to tobacco mosaic virus was incorporated into the hybrid.