Frequency Of Colony Formation Research Articles

Efficient plant regeneration from protoplasts isolated from long-term, shoot primordia-derived calluses of garlic ( Allium sativum)

We developed an efficient method for the regeneration of plants from garlic (Allium sativum) protoplasts. Protoplasts were isolated from six-year-old cultures of shoot primordia-derived calluses. At the isolation condition applied, high yield of protoplasts was obtained from the calluses compared with that from the initial shoot primordia. Throughout long-term culturing, highly regenerative calluses that propagated rapidly and appeared genetically stable were selected. The frequency of colony formation from protoplasts was clearly enhanced by increasing cell density. Adventitious buds were induced from protoplast-derived calluses at ca. 90 % efficiency, and the regeneration efficiency from adventitious buds averaged nearly 70 %. Plants that regenerated from adventitious shoots showed normal phenotypes, suggesting that long-term culturing of these garlic calluses does not induce dramatic genetic alterations.

Suppression of tumorigenicity of human lung carcinoma cells after transfection with connexin43.

The human lung carcinoma cell line PG is defective in gap junctional intercellular communication (GJIC). Connexin43 (Cx43) mRNA, which is expressed in normal human lung cells, is undetectable in these tumor cells. To explore if up-regulation of Cx43 gene expression will suppress malignancy of PG cells, Cx43 cDNA was co-transfected with pSV2neo cDNA into PG cells. Control cells were transfected with the blank vector plus neo cDNA. Several stable Cx43 transfectant clones, which acquired high levels of Cx43 expression and the capacity of GJIC, were compared with control clones and the parental cell line, both of which lacked Cx43 expression and GJIC. The control clones resembled the parental cells in exhibiting high cell growth rate, weak attachment to the substratum and a high frequency of colony formation in soft agar. In contrast to the control cells, Cx43 transfected clones showed reduced growth rate, enhanced attachment to the substratum and inhibition of colony formation in soft agar. In vivo results from nude mice experiments showed high tumorigenicity with control clones and inhibition of tumorigenicity in Cx43 transfected clones. The consistency between in vitro and in vivo results strongly suggests a tumor suppressing effect of the Cx43 gene in human lung carcinoma cells.

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts.

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0-5.0×106 were obtained following 4-6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 105 protoplasts/ml was optimal for subsequent division. An initial division frequency of 12-15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μM), and zeatin (2.3 μM). Colonies consisting of 100-200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1-2 mm in diameter) were formed after an additional 4-6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μM) and benzylaminopurine (2.2 μM). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μM gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.

Absence of the microsatellite mutator phenotype in human bronchial epithelial cells transformed by alpha particles

The immortalized human bronchial epithelial cell line, BEP2D, can become malignantly transformed following its irradiation with alpha particles. Exposed cells progress through a latent period of eight to ten weeks prior to exhibiting malignant properties. The molecular basis for this delay is unclear, however it is thought to involve a manifestation of genomic instability brought on by ionizing radiation. In this study we addressed whether the microsatellite mutator phenotype which arises in cells that lack mismatch repair function could have played a role in the radiation-induced transformation of BEP2D cells. Three cell lines, including BEP2D and two transformed clonal derivatives, H2BT2L and R30T1L, were examined for the presence of the microsatellite mutator phenotype using a selectable reporter system developed in our laboratory. This reporter system is based on the ability of stably transduced cells to restore the reading frame of a hygromycin B phosphotransferase transferase gene rendered out of frame by the insertion of a (CA)(13) repeat tract immediately downstream of the ATG start codon. The parental BEP2D cell line had a relatively low hygromycin B resistant colony formation frequency of 2.36 x 10(-4). This value was similar to the frequencies of two malignantly transformed derivatives H2BT2L (0.996 x 10(-4)) and R30T1L (1.87 x 10(-4)). We therefore conclude that the H2BT2L and R30T1L cell lines are not deficient in their hMSH2, hMLH1, hPMS2 or hMSH3 mismatch repair gene functions, and that other factors orchestrated the alpha particle induced malignant transformation of H2BT2L and R30T1L cells.

Plantlet regeneration from protoplast-derived haploid embryogenic calli of wheat

To search for an alternative method for protoplast culture, regenerable embryogenic calli were obtained from anther culture of three wheat cultivars, Karl 92, Jinghua #1, and Pavon 76. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in modified PMI and LM8P media without going through cell suspension culture. After 8–11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3–4 × 105 protoplasts ml−1. Frequency of colony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, while Karl 92 failed to produce colonies, even though its embryogenic calli were friable and fast-growing on the maintenance medium. Green haploid plantlets of Jinghua #1 and Pavon 76 have been regenerated from protoplasts, which were cultured on a differentiation medium first and then on a rooting medium.

Plant regeneration from patchouli protoplasts encapsulated in alginate beads

Mesophyll protoplasts were isolated from leaves of in vitro grown patchouli (Pogostemon cablin Benth.). The protoplasts were encapsulated in alginate beads, approximately 2–3×103 protoplasts per 25 μl bead. Successful colony formation was induced when the protoplast beads were inoculated into a liquid medium supplemented with 10-6 M NAA and 10-5 M BA. The frequency of colony formation was improved greatly by the inclusion of several beads per ml medium. To induce high colony formation for a single bead, it was essential to culture protoplasts in the presence of nurse beads containing actively-growing cells of the same species. Rapid regeneration of plants from protoplast-derived calluses was accomplished by a two-step culture procedure with liquid and then solid media. Gas-chromatographic analyses showed that regenerated plants produced an essential oil comprising a full-set of patchouli sesquiterpenes.

Plant Regeneration through Direct Culture of Isolated Pollen Grains in Rice.

Immature pollen grains of an Indica type rice cultivar IR24 were isolated and cultured to regenerate green plants without preculture of anthers. Either low-temperature pretreatment of panicles at 10'C in the dark or exposure to sugar starvation in the beginning of culture were necessary for induction of cell division of the grains. Low-temperature pretreatment was more effective treatuent than exposure to sugar starvation. It was found that there was a complementary relationship between the effects of low-temperature pretreatuent and of exposure to sugar starvation the pollen grains required shorter period of exposure to sugar starvation to start cell division as low-temperature pretreatnent was prolonged. The optimal condition for induction of cell division of the pollen grains was 21 days of the pretreatuent with I day of sugar starvation or 28 days of the pretreatment without sugar starvation. Though 2, 4-D was not necessary for induction of cell division, the development of the derived colonies followed by shoot regeneration was observed only in the presence of 2, 4-D. Reduced nitrogen source in the induction medium also affected the frequency of both colony formation and plant regeneration. The medjum containing 20mM KN03 and 5mM glutamine as nitrogen sources gave the highest frequency of colony formation at 0.20/0 and its frequency reached 2.0 times higher than the control R2 medium containing 40mM KN03 and 2.5mM (NH4)2S04. On the other hand, the calli which were formed in the medium containing 20mM KN03 and 5mM alanine had the highest regenerating ability. In addition, the calli which were formed in the medium containing 20mM KN03 and 5mM alanine showed marked higher frequency of green plant regeneration than ones in any other media tested, when 21 days of low-temperature pretreatment without exposure to sugar starvation was applied as induction condition.

Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1 alpha.

We recently developed a two-step methyl cellulose culture system for murine lymphohemopoietic progenitors that are capable of differentiation along the myeloid and B-lymphoid lineages. In this system, two-factor combinations, which include steel factor plus interleukin (IL) 6, IL-11, or granulocyte colony-stimulating factor effectively supported the lymphomyeloid potential of primary colonies. Interestingly, IL-3 could neither replace nor act synergistically with steel factor in maintaining the B-lymphoid potential of the primary colonies although the frequency of colony formation was the same with IL-3 and steel factor. We now report that addition of IL-3 or IL-1 alpha to a permissive system suppresses the B-lymphoid potential of primitive progenitor cells in primary culture in dose-dependent fashion. In vivo transfer of the primary colonies to scid mice confirmed the suppressive effects of IL-3 and IL-1 alpha. In addition, IL-1 alpha inhibited pre-B-cell colony formation in the secondary culture. Once pre-B-cell colonies had formed in secondary culture, neither factor affected the proliferation of the pre-B cells. These results suggest negative regulatory roles for IL-3 and IL-1 alpha in early stages of B lymphopoiesis.

Hypersensitivity to oxygen is a uniform and secondary defect in Fanconi anemia cells

Cells from patients with Fanconi anemia (FA) frequently show an increased sensitivity to DNA crosslinking agents such as mitomycin C (MMC). FA cells also show abnormal sensitivity to oxygen tension. In order to examine the correlation between the two cellular defects in FA, several FA fibroblast lines were tested for their sensitivity to MMC and oxygen by colony-formation frequency. The sensitivity to MMC in different FA lines varied in a broad range from normal level to extreme hypersensitivity, whereas all of the FA lines showed similar hypersensitivity to oxygen. When FA fibroblasts were transformed by SV40 large T-antigen, the hypersensitivity to oxygen was normalized while the MMC sensitivity still remained. These results suggest that the cellular sensitivity to oxygen is a secondary defect rather than a primary effect of mutations in FA. However, it is a more uniform phenotype that the MMC sensitivity, and therefore, it may be closely related to the common clinical symptoms of FA. Since 1% oxygen showed the highest colony-formation frequency for FA cells, establishment of FA primary fibroblasts was attempted at the low oxygen condition. FA fibroblast cells showed greatly enhanced growth and migration at 1% oxygen resulting in fast establishment of FA primary fibroblasts.

Expression of a swine class II gene in murine bone marrow hematopoietic cells by retroviral-mediated gene transfer.

As a first step in assessing the efficacy of a gene transfer approach to the induction of transplantation tolerance in our miniature swine model, double-copy retroviral vectors engineered to express a drug-resistance marker (neomycin) and a swine class II DRB cDNA were constructed. Infectious particles containing these vectors were produced at a titer of greater than 1 x 10(6) G418-resistant colony-forming units/ml using both ecotropic and amphotropic packaging cell lines. Flow cytometric analysis of DRA-transfected murine fibroblasts subsequently transduced with virus-containing supernatants demonstrated that the transferred sequences were sufficient to produce DR surface expression. Cocultivation of murine bone marrow with high-titer producer lines leads to the transduction of 40% of granulocyte/macrophage colony-forming units (CFU-GM) as determined by the frequency of colony formation under G418 selection. After nearly 5 weeks in long-term bone marrow culture, virus-exposed marrow still contained G418-resistant CFU-GM at a frequency of 25%. In addition, virtually all of the transduced and selected colonies contained DRB-specific transcripts. These results suggest that a significant proportion of very primitive myelopoietic precursor cells can be transduced with the DRB recombinant vector and that vector sequences are expressed in the differentiated progeny of these cells.

Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

Autocrine transforming growth factor-β controls G0 phase of early human hematopoietic progenitors in serum- or serum-free culture

In a previous paper [8] we have shown that, with human CD 34+ cells grown in culture with serum, antisense TGF-β oligonucleotides significantly enhanced the frequency of colony formation by multilineage, early erythroid and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-β is produced by a subpopulation of early progenitors. We now show that this effect is observed as well in serum-free culture which supports the development of mixed colonies. Therefore, the G0 phase control of some early progenitors, by an autocrine production of TGF-β, can be observed in vitro independently of serum or accessory cells.

Thymic tumors induced by 3-methylcholanthrene treatment of rat Peyer's patches

Studies were performed to characterize thymic tumors which were induced after a single injection of 500 μg or 3-methylcholanthrene (3-MC) into surgically exposed Peyer's patches (PP) of Copenhagen rats. Detailed gross, histological, and morphological analyses revealed thymic tumors differing in size and weight (1 to > 8 g) with distorted architecture and infiltration by lymphocytes and epithelial cells in varying proportions. Approximately 25% of the rats with thymic tumors exhibited abnormal spleens, whereas 66% developed low grade leukemias. A majority of the thymic tumors contained cells which exhibited (1) phenotypic markers characteristic of normal thymocytes, (2) abnormal DNA, and (3) increased percentages in S + G 2 phases of the cell cycle. Further studies of tumor cell isolates demonstrated an increased frequency of colony formation on soft agar, as well as the ability to elicit thymic tumors upon transplantation. Collectively these studies describe chemically induced thymic lymphomas.

Role of DT diaphorase the cytotoxicity of menadione and 4-nitroquinoline-1-oxide in cultured mammalian fibroblastic cells

The role of DT diaphorase on the cytotoxicity (reduction in colony formation frequency) of menadione and 4-nitroquinoline-1-oxide (4NQO) was examined in two fibroblastic cell lines (Chinese hamster V79H3 cells and NG2 Syrian hamster cells). The addition of dicoumarol (10 −4 M − 3 × 10 −4 M), a specific inhibitor of DT diaphorase, resulted in an intensification of the cytotoxicity of menadione, supporting the hypothesis that DT diaphorase protects cells against the oxidative stress induced by quinones. On the other hand, the toxicity of 4NQO was greatly reduced by the addition of dicoumarol (10 −5 M − 3 × 10 −4 M), showing that DT diaphorase is the key (or the sole) enzyme involved in the activation of 4NQO in the above cells.

Protoplast culture and plantlet regeneration in stock (Matthiola incana R. Br.).

Protoplasts were isolated from callus derived from excised cotyledons of stock (Matthiola incana R. Br.), by using an enzyme mixture of 1.5% Meicelase and 0.05% Macerozyme R-10. Modified Murashige-Skoog major elements, Ringe and Nitsch minor elements and modified vitamins supplemented with 0.2mg/l 2, 4-D, 1mg/l NAA, 1mg/l BAP, 8.1% mannitol, 1% glucose and 1% sucrose supported protoplast division at 25°C. The frequency of colony formation 10 days after culture was the highest (17.9% of isolated protoplasts) at protoplast density of 0.5×105/ml. Colonies developed to the callus-stage 2 months after initial culture. When callus pieces were cultured in full strength MS medium supplemented with 1mg/l BAP or 1mg/l zeatin, adventitious buds were induced in 9%-16% of callus explants. The buds elongated with leaves open at 20°C and rooted in medium supplemented with 0.1mg/l or 5mg/l IBA.

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C(1), xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO(3), an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O(2)-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A(2), which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki- ras or c-Ha- ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki- ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.

Characterization of Conditioning Factors that Increase Colony Formation from «Black Mexican Sweet Corn» Protoplasts

Summary Colony-formation frequency of protoplasts isolated from suspension cultures of «Black Mexican Sweet (BMS) corn» ( Zea mays L.) is increased about ten-fold when conditioned medium (CM) isolated from BMS suspension cultures is supplied to protoplasts plated at 50,000 protoplasts/ml. The objective of this study was to characterize the factor(s) (CMF) from conditioned medium responsible for the increased colony formation. Colony-formation frequency was a linear function of the amount of CM, indicating that either a single CMF is present in CM or that several CMF are present but each acts independently. Removal of polyvalent cations stabilized CMF at elevated temperatures. CMF was most stable at pH 5, losing activity rapidly at pH = 8 or pH = 2. Based on its partitioning between water and ethyl acetate or butanol, and its solubilities in mixtures of water and isopropanol, CMF is very hydrophilic. Neither anion-exchange (NO 3 - form, in H 2 O) nor cation-exchange (Na + form, in H 2 O) resins at pH 5 retained CMF, suggesting that CMF is uncharged. Ultrafiltration showed that the molecular weight of CMF is slightly less than 1350. Subsequent gel filtration with Biogel P-2 and Biogel P-6 resolved CMF into a major peak with an apparent molecular weight of ca. 1200. Thus, CM appears to contain a single major CMF, which has characteristics similar to that of an oligosaccharide. Although the final structure of CMF is unresolved, CMF is chemically unlike the reported conditioning factor(s) of either Hordeum vulgare anther cultures or of dicot suspension cultures, or the cell-division-inducing phytohormones typically used in the culture of protoplasts.

The effect of conditioned medium on colony formation from ‘black mexican sweet’ corn protoplasts

Protoplasts from suspension cultures of ‘Black Mexican Sweet’ corn ( Zea mays L.) exhibited increased colony formation frequency when cultured in the presence of conditioned medium isolated from Black Mexican Sweet (BMS) cultures. The increase in colony formation frequency of protoplasts plated at 5 × 10 4 protoplasts/ml was used as a bioassay for detecting conditioning factors and to investigate the response of protoplasts to conditioned medium. Conditioned medium was stable when stored frozen at −14°C, retained 80% activity when stored for 10 days at 24°C and was inactivated by heating to 100°C for 10 min. The most active isolates of conditioned medium were recovered during the exponential growth phase of the suspension cultures. Colony formation frequencies were greatest when conditioned medium was added to protoplasts within 12 h of plating and was not removed for 9 days thereafter. Addition of conditioned medium to protoplasts increased cell division frequency and did not increase protoplast viability or cell wall formation above the control cultures. Most of the conditioning activity was due to factors that were rejected by a 500 molecular weight (MW) cutoff ultrafiltration membrane. A mixture of threonine, glutamate, cysteine and methionine (0.25 μM each), which were the only amino acids detected in conditioned medium, and proline (1 mM) increased colony formation but to lower levels than 25% conditioned medium. Our results indicate that BMS culture cells produce conditioning factors that increase and sustain cell division from BMS protoplasts and that free amino acids are not the active factors in conditioned medium.

Requirement of conditioned medium or a high concentration of serum for anchorage-independent growth of adenovirus type 12 E1a-transformed rat cells (HY1).

Adenovirus type 12 (Ad12) Ela-transformed rat cells (HY1) grew in methocel medium containing 9% fetal calf serum (FCS), but the frequency of colony formation was very low (the order of 10(-4)). The addition of conditioned medium or a high concentration of serum (20% FCS) to the methocel medium accelerated colony formation, and plating efficiency increased 10- to 100-fold. In contrast, in stationary culture, HY1 cells grew well even in 1%-FCS medium. These results indicate that HY1 cells require high concentrations of growth factors for anchorage-independent growth. The effects of conditioned medium or FCS also were demonstrated in several transformed cell lines induced by transfection of combined sets of Ad12-transforming genes (E1a, E1b and E4). These growth behaviors suggest that the first step in cell transformation with adenovirus 12 is the acquisition of responsiveness to growth factors in methocel culture, which must be the function of the Ad12-E1a gene products. The function of the other two Ad12-transforming genes was discussed.