An extensive hairy root production precedes shoot regeneration in protoplast-derived calli of cauliflower (Brassica oleracea var. botrytis)

Summary Protoplasts isolated from mesophyll tissues of cauliflower (Brassica oleraceavar. botrytis) were induced to divide in culture, with 2% of them producing calli. Upon transfer to a regeneration medium containing a low auxin/cytokinin balance (0.02mg/l 1-naphthaleneacetic and 2mg/l 6-benzylaminopurine), they displayed an extensive production of hairy roots before the regeneration of shoots. Negative effects of root differentiation on the subsequent caulogenesis by such calli were not observed, since 97% of the calli regenerated hairy roots and 93% gave shoots.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - MES 2-N-morpholinoethane sulfonate     

Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.)

Summary Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l^-1 6-benzyl-aminopurine and 0.1 mg.l^-1 l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - IBA 4-indole-3yl-butyric acid - IPE initial plating efficiency - f wt fresh weight - KM Kao and Michayluk (1975) - MES 2-N-morpholino ethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA l-naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000)     

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

High cyclin E staining index in blastemal, stromal or epithelial cells is correlated with tumor aggressiveness in patients with nephroblastoma

Purpose

Identifying among nephroblastoma those with a high propensity for distant metastases using cell cycle markers: cyclin E as a regulator of progression through the cell cycle and Ki-67 as a tumor proliferation marker, since both are often deregulated in many human malignancies.

Methodology/Principal Findings

A staining index (SI) was obtained by immunohistochemistry using anti-cyclin E and anti-Ki-67 antibodies in paraffin sections of 54 postchemotherapy nephroblastoma including 42 nephroblastoma without metastasis and 12 with metastases. Median cyclin E and Ki-67 SI were 46% and 33% in blastemal cells, 30% and 10% in stromal cells, 37% and 29.5% in epithelial cells. The highest values were found for anaplastic nephroblastoma. A correlation between cyclin E and Ki-67 SI was found for the blastemal component and for the epithelial component. Univariate analysis showed prognostic significance for metastases with cyclin E SI in stromal cells, epithelial cells and blastemal cells (p = 0.03, p = 0.01 and p = 0.002, respectively) as well as with Ki-67 SI in blastema (p<10⁻⁴). The most striking data were that both cyclin E SI and blastemal Ki-67 SI discriminated between patients with metastases and patients without metastasis among intermediate-risk nephroblastoma.

Conclusions

Our findings show that a high cyclin E SI in all components of nephroblastoma is correlated with tumor aggressiveness and metastases, and that assessment of its expression may have prognostic value in the categorization of nephroblastoma.     

miRNA Profiles as a Predictor of Chemoresponsiveness in Wilms’ Tumor Blastema

The current SIOP treatment protocol for Wilms’ tumor involves pre-operative chemotherapy followed by nephrectomy. Not all patients benefit equally from such chemotherapy. The aim of this study was to generate a miRNA profile of chemo resistant blastemal cells in high risk Wilms’ tumors which might serve as predictive markers of therapeutic response at the pre-treatment biopsy stage. We have shown here that unsupervised hierarchical clustering of genome-wide miRNA expression profiles can clearly separate intermediate risk tumors from high risk tumors. A total of 29 miRNAs were significantly differentially expressed between post-treatment intermediate risk and high risk groups, including miRNAs that have been previously linked to chemo resistance in other cancer types. Furthermore, 7 of these 29 miRNAs were already at the pre-treatment biopsy stage differentially expressed between cases ultimately deemed intermediate risk compared to high risk. These miRNA alterations include down-regulation in high risk cases of miR-193a.5p, miR-27a and the up-regulation of miR-483.5p, miR-628.5p, miR-590.5p, miR-302a and miR-367. The demonstration of such miRNA markers at the pre-treatment biopsy stage could permit stratification of patients to more tailored treatment regimens.     

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

A protoplast-to-plant system in chrysanthemum: differential responses among several commercial clones

Summary Plant regeneration has been obtained from Chrysanthemum mesophyll protoplasts. Of the twenty-nine clones studied, division was observed for eighteen clones, and coupled with colony formation for sixteen of them. Elimination of NH₄NO₃ from the culture medium greatly improved colony survival in culture. Calli were obtained from colonies of five clones, and for clone n^o 42 buds were produced. Regeneration ability, for such protoplast-derived calli of clone n^o 42, was retained over a prolonged culture period. The regenerated plants were successfully transferred to the glasshouse (4 to 5 months from protoplast isolation).Abbreviations BAP 6-benzylaminopurine - F.P.E. final plating efficiency - f.wt. fresh weight - GA₃ gibberellic acid - IAA indole-3-acetic acid - I.P.E. initial plating efficiency - MS Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichloro-phenoxyacetic acid