Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological,cytochemical,and cytogenetical assessment

Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.

Cytochemical localization of H_2O_2 in pigment glands of cotton(Gossypium hirsutum L.)

Programmed cel death （PCD） plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species （ROS） produced by the aerobic metabolism of cels, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands ofGossypium hirsutum L. with 3,3-diaminobenzidine （DAB） staining, 2’,7’-dichlorodihydrolfuorescein diacetate （DCFH2）-DA lfuorescent labeling and CeCl3 cytochemical localization techniques. The results showed that thepigment glandsofG. hirsutum could generate H2O2, and the amount and localization of H2O2 variedat different developmental stages. At the early developmental stage, a smal amount of H2O2 accumulated in the vacuole membrane of pigment gland cels. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cel wals started to accumulate a smal amount of H2O2. When pigment gland cel degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cels. With the degradation of the sheath cels, H2O2was detected in cel wal and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2are consistent with the process of PCD of individual gland cels, which started from the degra-dation of intracelular membrane and ended with the degradation of cel wals. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.

The Synthesis and Storage Sites of Phenolic Compounds in the Root and Rhizome of <i>Echinacea purpurea</i>

Cichoric acid is the main phenolic compound in the root and rhizome of the medicinal part, Echinacea purpurea that is known for possessing immune enhancing characteristics. In this study, we analysis the the synthesis and storage sites of phenolic compound in E. purpurea. We used fluorescent microscopy, transmission electron microscopy, cytochemical and immunocytochemical localization to observe the distribution of phenolic compounds. Our results show that the phenolic compounds were mostly distributed in the cortex parenchyma cells, vascular parenchyma cells and pith parenchyma cells in the root and rhizome, and mainly present in the vacuoles, large intercellular spaces and their surrounding cell walls. No phenolic compounds were observed in the cytoplasm and the organelles. We concluded that the phenolic compounds were synthetized in the cortex parenchyma cells, vascular parenchyma cells and pith parenchyma cells in the root and rhizome, and stored in the vacuoles of parenchyma cells. The above results provided significantly cytological information for further approaching the metabolic regulation and transfer pathways of phenolic compounds in biochemistry and molecular biology.

Induced Differentiation of Epithelioid Carcinoma Cell Lines: Evidence for Tumor Cell Quantal Mitosis

The effects of growth factors and calcium concentrations present in different culture media on induction of terminal differentiation were investigated for four different epidermoid carcinoma cell lines, Hela, KB, A431, and SCC-25, and their responses determined relative to those elicited by normal human keratinocytes subjected to these culture conditions. Differentiation status was determined cyto-chemically by a validated keratin protein staining method, and by autoradiographic analyses. Growth and differentiation promoting factors that influenced the direction of integrated control of growth and differentiation in normal human keratinocytes were found to be effective for some cell lines but not others. The factors examined were 1) high density arrest in serum-free and serum-containing media, 2) media shifts from high density culture in serum-containing media to low density growth factor-depleted or supplemented serum-free medium, and 3) the concentration of calcium in the media. The extent and degree of differentiation achieved varied among different cell lines depend on the presence or absence of serum, EGF and insulin protein growth factors. Certain growth media appear to sponsor keratin protein, cyto-chemically-detected differentiation, and evidence of quantal mitotic division in low density HeLa cell and SCC25 cell cultures. Epidermoid carcinoma cell lines retain limited capacity to commit to early stages of cell differentiation.

Systemic induction of H_(2)O_(2) in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H2O2) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H2O2 could be systemically induced by wounding and exogenous JA. H2O2 increased within 1 h and reached the peak 3―5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H2O2 decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H2O2 burst that was mediated by wounding and exogenous JA. Assay of H2O2 subcellular location showed that H2O2 in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immu- nogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.