redox status evaluation in dogs affected by mast cell tumour

Oxidative stress status has been evaluated in depth in human medicine and its role in carcinogenesis has been clearly established. The purpose of this prospective study was to evaluate antioxidant concentrations and oxidative stress in dogs with mast cell tumours (MCTs) that had received no previous treatments, and to compare them to healthy controls. In 23 dogs with mast cell tumour and 10 healthy controls, oxidative status was assessed using the Reactive Oxygen Metabolites‐derived compounds (d‐ROMs) test, antioxidant activity was measured by the Biological Antioxidant Potential (BAP) test, and α‐tocopherol levels were evaluated using high‐performance liquid chromatography and ultraviolet analysis. At baseline, dogs with MCT had significantly higher d‐ROMs (P < 0.00001) and lower BAP (P < 0.0002) compared with healthy controls. However, no significant difference was observed for α‐tocopherol (P = 0.95). Results suggest that oxidative stress pattern and oxidative defence barrier are altered in dogs with newly diagnosed MCT compared with control dogs. Future studies are needed in order to assess the prognostic role of oxidative stress and to evaluate the impact of different therapeutic approaches.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

Interspecific hybridization in vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers

Vanilla, Vanilla planifolia Andrews, is native to Mexico and Central America, but is now cultivated in other parts of the tropics. Continuous clonal propagation has resulted in very little variability for crop improvement programmes in vanilla. In this study, an attempt has been made to increase the spectrum of variation by interspecific hybridization with Vanilla aphylla, an Indian species which is tolerant to Fusarium. Interspecific hybrids were successfully produced and morphological characters and molecular profiles revealed the true hybridity of the progenies. Ten seedling progenies of V. planifolia, and four interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) using a number of different loci as markers were evaluated and 319 amplified fragment length polymorphisms (AFLPs) and 83 random amplified polymorphic DNAs (RAPDs) loci were marked. The profiles indicate similarity between the parents, selfed progenies and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla. This is the first report in vanilla, indicating that RAPD and AFLP profiles coupled with morphological characters can be utilized to assess the variability and hybrid nature of genotypes and of successful interspecific hybridization and production of hybrids between V. planifolia and V. aphylla.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

Promotion of lateral female strobili production inPinus densiflora by cytokinin application at a specific stage

BAP (N ⁶-benzylaminopurine) was sprayed 5, 10 and 15 times at four different periods to promote flowering of a grafted Japanese red pine (Pinus densiflora) clone. Four stages were I, August 4 to September 1; II, September 6 to October 4; III, October 5 to November 14 and IV, November 30 to December 28, in 1994. Concentration of BAP was 250 mg/l. In May, 1995, no apical female strobili were observed at the top of the new shoots in any treatment. However, BAP application at stage III (floral differentiation period) produced lateral female and bisexual strobili at the lower part of new shoots. The numbers of lateral female and bisexual strobili were 87 out of 147 to 234 out of 162 new shoots. Frequency of BAP application did not affect the numbers. Marked decrease in number of male strobili induced by BAP application at stages I and II was observed. In contrast, the number of new shoots was increased by the BAP application at the above stages. Treatment at stage III and IV did not affect significantly the number of male strobili and new shoots. These results indicate that BAP application at stage III specifically induces the development of lateral female and bisexual strobili in Japanese red pine.     

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

Adventitious shoot regeneration and protoplast isolation and culture were examined from leaf explants of in vitro shoot cultures of several cauliflower (Brassica oleracea var. botrytis) cultivars, sourced from Europe and Australia, was investigated with the aim to develop improved nuclear and plastid transformation protocols for this vegetable crop. Eight out of 10 cultivars regenerated shoots from at least 79% of leaf explants. Mesophyll protoplasts from leaves gave high yields and division frequencies. Growth of shoot cultures in large glass vessels with vented lids was the key factor in obtaining high protoplast division frequencies of up to 71% and at least 70% of protoplast calluses regenerating shoots.     

Development of a transformation protocol for Tecomella undulata (Smith) Seem from cotyledonary node explants

A protocol was optimized for genetic transformation of Rohida (Tecomella undulata) from cotyledonary node tissues using Agrobacterium tumefaciens strain GV2260 harboring binary vector pBinAR containing osmotin and nptII gene under control of CaMV35S promoter. This is the first report on the transformation of T. undulata. The effective concentration of selectable marker antibiotic used for screening of transformants was 85.97 μM in shooting media and 42.98 μM in rooting media. PCR and Southern blot confirmed integration of osmotin gene into genome of Rohida.     

Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities

Taking into account the recalcitrance of date palm (Phoenix dactylifera L.) to tissue culture, establishment and proliferation of embryogenic suspension cultures in two date palm cultivars, namely Boufeggouss (BFG) and Bouskri (BSK) was implemented using liquid medium with different concentrations of the 6-benzylaminopurine (BAP) (0.3, 0.4, 0.5 mg/l) and various subculturing times (7 days, 15 days and 20 days). Total phenols and peroxidase activities have been determined and oxidative browning process checked in different cultures. The liquid medium with 0.3 mg/l BAP allowed cell suspension cultures to produce the greater globular embryos and the highest number of somatic embryos in comparison with the other treatment for BFG and BSK date palm cultivars. The low rate of culture transfers (every 15 or 20 days) tended to increase the phenolic contents and peroxidase activities in plant tissue leading to an enhancement of tissues/cells browning and then a decrease in the proliferation of embryogenic cells. The transfer of cultures on fresh medium every 7 days allowed a substantial reduction of tissue/cell oxidative browning, which is related to reduction of phenolic compounds and decrease in peroxidase activities with a resultant increase in the proliferation of embryogenic cells. This result suggests that oxidative browning is mainly peroxidase-based in date palm suspension cultures. Relationships between low concentrations of BAP as cytokinin, high growth of embryogenic cells, low contents of phenolics, decrease of peroxidase activities and oxidative browning process, are discussed, in order to optimize date palm multiplication through somatic embryogenesis and solve recalcitrance of many date palm cultivars to tissue culture.