Two-step Culture Method Research Articles

Efficient isolation of mesenchymal stem cells from amniotic fluid via a two-step culture method

Efficient isolation of mesenchymal stem cells from amniotic fluid via a two-step culture method

Recreating Tissue Structures Representative of Teratomas In Vitro Using a Combination of 3D Cell Culture Technology and Human Embryonic Stem Cells.

In vitro studies using human embryonic stem cells (hESCs) are a valuable method to study aspects of embryogenesis, avoiding ethical issues when using embryonic materials and species dissimilarities. The xenograft teratoma assay is often traditionally used to establish pluripotency in putative PSC populations, but also has additional applications, including the study of tissue differentiation. The stem cell field has long sought an alternative due to various well-established issues with the in vivo technique, including significant protocol variability and animal usage. We have established a two-step culture method which combines PSC-derived embryoid bodies (EBs) with porous scaffolds to enhance their viability, prolonging the time these structures can be maintained, and therefore, permitting more complex, mature differentiation. Here, we have utilised human embryonic stem cell-derived EBs, demonstrating the formation of tissue rudiments of increasing complexity over time and the ability to manipulate their differentiation through the application of exogenous morphogens to achieve specific lineages. Crucially, these EB-derived tissues are highly reminiscent of xenograft teratoma samples derived from the same cell line. We believe this in vitro approach represents a reproducible, animal-free alternative to the teratoma assay, which can be used to study human tissue development.

Comparative analysis of mouse embryonic palatal mesenchymal cells isolated by two primary culture methods

Cell culture plays a vital role in mechanism research, as a supplement to experiments in vivo. At present, there are two main methods to obtain mouse embryonic palatal mesenchymal (MEPM) cells, while no systematic investigation about characteristics of cells using these two methods to get a clear application. In this study, using the traditional two-step primary culture method as the control group, we found that the MEPM cells of the simplified one-step method and the control group were both consistent with the surface markers of mesenchymal stem cells. The vimentin (mesenchymal marker) was positive, cytokeratin 14 (CK14, epithelium marker) was negative. In addition, along with the increasing passages, the cells cultured by two methods had similar changes with a decreasing proliferation activity, increasing apoptosis rate, declining migration, diminishing stemness, and obvious senescence. Moreover, the senescence of MEPM cells in the one-step group was lower than that in the control group. Our findings indicate that there are no significant differences between the two methods and one-step method can be a good method in investigating palatal development mechanisms by shortening the processing of the MEPM cell culture and may reduce the damage to the cells done by more enzymes and processing.

A Novel Approach to Enhance the Urease Activity of Sporosarcina pasteurii and its Application on Microbial-Induced Calcium Carbonate Precipitation for Sand

Urease is involved in the formation of carbonate sediments by microbial-induced calcium carbonate precipitation (MICP), and Sporosarcina pasteurii used extensively in this technique owing to its high urease production. In this study, a simple two-step culture method with the appropriate medium was developed to enhance the urease activity of S. pasteurii. Urea played an important role in the culture process, particularly during the pre-cultivation step and the newly developed method improved both urease activity and specific urease activity. Furthermore, the increase in urease activity by MICP resulted in increased production of calcium carbonate and better strength of bio-cemented sand.

Coexpression of WUSCHEL related homeobox (WOX) 2 with WOX8 or WOX9 promotes regeneration from leaf segments and free cells in Nicotiana tabacum L.

To examine the effect of the ectopic expression of three Arabidopsis genes, including WOX2, WOX8 and WOX9, on the regenerative competency of tissues and cells cultured in vitro, we developed a transgenic variety of Nicotiana tabacum, in which these genes were under the transcriptional control of a chemical-inducible expression system. We designed a two-step culture method to feasibly demonstrate the effect as follows. Leaf segments of approximately 10 mm2 were prepared from transgenic plants and their hybrids and cultured in a liquid medium based on modified Murashige and Skoog medium supplemented with an auxin, 2,4-dichrorophenoxyacetic acid and/or an expression inducer β-estradiol for 10 days in dark. The segments were subsequently cultured on a solidified medium in the absence of both the auxin and inducer in light for 3 weeks. We observed remarkable regeneration of plantlets only in segments derived from the hybrids possessing two transgenes, WOX2 combined with WOX8 or WOX9, but no regeneration in the segments derived from their parental lines. We also observed that free cells released from the hybrid explants in the liquid medium developed into embryo-like structures due to the transient application of the inducer. In a wide range of species including recalcitrants, the effect of the coexpression of these genes may be useful for developing an alternative to conventional protocols that requires cytokinin.

Comparison of the Efficiency to One-Step and Two-Step Anther Culture in Rice (Oryza sativa L)

Comparison of the Efficiency to One-Step and Two-Step Anther Culture in Rice (Oryza sativa L) Jeong Hoon Min, Jin Soo Sim, Hyun-Suk Lee, Young-Hie Park, Gyu-Hwan Park, Kyung-Min Kim Abstract Anther culture method has commonly been performed two-step culture of callus induction during the plant regeneration stage. Two-step culture was generally required to a much longer duration of culture for regeneration. There are still needed to be solved the disadvantage of the two-step culture technique. Anther of F1 plants derived from Drimi2*3/AR1 of new plant type and improved rice cultivars were compared with method efficiency using one-step and two-step culture method. The results showed that one-step culture was more effective than two-step culture. Callus induction averaged 8.2% for one-step culture and 6.1% for two-step culture. The proportion of plants that survived after colchicine treatment was similar as 17.5% for one-step culture and 16.7% for two-step culture. However, there was a highly significant difference when results of plant regeneration were compared. Frequency of regenerated plant averaged 13.6% for one-step culture, which was 7.1% higher than two-step culture (6.5%). These results provide valuable data that may be used to establish an efficient plant regeneration system for rice cultures. Full Text: PDF DOI: 10.15640/jaes.v4n1a10

Effective two-step culture method for development of early preantral mouse follicles

Effective two-step culture method for development of early preantral mouse follicles

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

Human Embryonic Stem Cells Differentiate into Functional Natural Killer Cells with the Capacity To Mediate Anti-Tumor Activity.

Hematopoiesis from human embryonic stem cells (hESCs) follows developmental kinetics similar to what is observed during normal human ontogeny. Myeloid, erythroid and megakaryocytic progenitors can be routinely generated from hESCs. However, little is known about the ability of hESCs to differentiate into the lymphoid lineage. Natural killer (NK) cells are important mediators of donor anti-host alloreactivity seen after allogeneic transplant for myeloid leukemias. Our studies use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. CD34+ and CD34+CD45+ hESC-derived hematopoietic progenitor cells were co-cultured with inactivated AFT024 stromal cells in medium supplemented with IL-7, IL-15, SCF and FL. Generation of NK cells was established by phenotypic and functional analysis. CD34+ umbilical cord blood (UCB) cells were utilized as a positive control. After 14 days of culture of CD34+ hESC-derived cells, more than 90% of the cells express CD45, a pan-hematopoietic cell marker, but few CD56+ cells are observed. At 21 days of culture a distinct CD56+CD45+ cell population develops (14.9%), which increases to 37.5% of cells after 28 days of culture. Similar results are observed for CD34+CD45+ hESC-derived cells, characterizing that both CD34+ and CD34+CD45+ cell populations contain hematopoietic progenitors with NK cell developmental potential. Limiting dilution analysis of hESC-derived progenitor cells demonstrates CD34+ hESC-derived cells have a low NK cell progenitor frequency. However, sorting for CD34+CD45+ hESC-derived cells significantly increased the NK cell cloning frequency (1.92% ± 1.20%) to a level comparable to the frequency observed for CD34+ UCB cells cultured in the same manner (3.57% ± 1.68%). The hESC-derived NK cells also express receptors known to regulate NK cell cytolytic activity, including killer-Ig-like receptors (KIRs), C-type lectin-like receptors (CD94 and NKG2A) and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). Furthermore, hESC-derived NK cells also express CD16, an Fc-receptor typically expressed on more mature NK cells. The expression of KIRs is significantly higher for the hESC-derived NK cells compared to the UCB-derived NK cells. This may lead to future strategies to generate selective alloreactive NK cell populations for therapy. To investigate the functional properties of the hESC-derived NK cells, cytolytic activity was tested against K562 erythroleukemia cells and Raji B-lymphoblastoid cells. hESC-derived NK cells effectively killed K562 cells, with activity similar to that seen with UCB-derived NK cells. As expected, Raji cells were resistant to direct cytotoxicity by both hESC and UCB-derived NK cells. However, treatment of Raji cells with anti-CD20 antibody results in effective antibody-dependent cell-mediated cytoxicity by the hESC-derived NK cells. The hESC-derived NK cells also demonstrate ability to upregulate production of cytokines such as IFN-γ upon stimulation. Furthermore, we also find that hESC-derived progenitors also have T cell and/or B cell potential based on cells that express Ikaros, Rag1, and IL7Rα. These results demonstrate that the CD34+ and CD34+CD45+ hESC-derived cell populations contain lymphoid progenitor cells that can develop into both innate and adaptive immune cells. The ability to generate functional NK cells that can target and lyse human tumor cells via two distinct mechanisms suggests potentially novel anti-cancer therapy applications of hESCs.

Human Embryonic Stem Cell-Derived NK Cells Acquire Functional Receptors and Cytolytic Activity

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Bicarbonate enhances synchronous division of the giant nuclei of sporophytes in Bryopsis plumosa.

A mature sporophyte of Bryopsis plumosa (Hudson) C. Agardh forms a huge number of zoospores in its cell continuum. Zoospore formation starts with the division of a single giant nucleus and subsequent repeated mitosis. We found that an elevation of photosynthetic activity triggered the division of a mature giant nucleus. Transfer to short-day conditions was not necessary. Giant nuclei did not divide in darkness or in the presence of 1 micro M DCMU. Giant nuclei of as many as 90% of sporophtyes started to divide following the addition of 5 microM NaHCO3 to the growth medium under continuous white light (6-12 W m(-2)). Frequency of nuclear division increased with increased light intensity. By combining those parameters that promoted the division of giant nuclei, we developed the "two-step culture method" which is composed of preliminary and main cultures. This new method guarantees that giant nuclei of more than 90% of all sporophytes synchronously divide between 72 and 96 h after the transfer to the main culture (continuous white light of 12 W m(-2) in PES medium supplemented with 5 mM NaHCO3).

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-α (TNF-α), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.

작약의 약 및 소포자 배양에서 Phenylacetic Acid ［PAA］가 배형성에 미치는 영향

작약의 약 및 소포자 배양 효율을 향상시키기 위하여 배지 내에 첨가되는 PAA의 농도별 배형성 정도와 배의 형태적 변이 등에 대한 실험을 수행하여 얻어진 결과를 요약하면 다음 과 같다. 작약의 약배양에서 배지 내에 첨가되는 PAA의 농도에 따라 소포자 유래의 배형성률은 다르게 나타났는데 2 mg/L의 PAA가 첨가된 배지에서 배형성률이 가장 높게 나타났으며, PAA의 농도가 증가됨에 따라 배형성를은 낮아진 반면 캘러스 형성률은 증가하는 경향을 나타내었다. 소포자에서 유래된 배의 형태는 PAA의 농도에 따라 큰 변이를 나타내었는데 2개의 자엽을 가진 정상배의 출현빈도는 2 mg/L의 PAA가 첨가된 배지에서 가장 높게 나타났으며 PAA의 농도가 그 이상으로 증가하면 오히려 비정상배의 출현빈도가 높아지는 경향이었다. 작약의 소포자 배양에서도 2 mg/L의 PAA 첨가 배지에서 10일간 전배양된 약을 PAA (2 mg/L)를 함유한 MS액체 배지에 배양했을 때 누출된 소포자 (shed microspore)의 수도 많았고 분열률도 높았으며, 이들 소포자로부터 형성된 배의 수도 가장 많았다. The objective of this study was to determine the effects of phenylacetic acid (PAA) on embryo production in anther and microspore culture of herbaceous peony (Paeonia lactiflora Pall.). The anthers of herbaceous peony were cultured on MS medium with 0 to 100 mg/L PAA according to two-step culture method. The ruptured anthers were transferred onto embryo formation medium without growth regulators. The MS medium with 2 mg/L PAA was effective in enhancing of direct embryogenesis and producing of normal embryo with two cotyledons from the cultured anthers. However, the increase of PAA concentration more than 5 mg/L PAA inhibited the embryo formation and promoted to callus formation from the anthers. The PAA affects significantly on the division of microspore and embryo formation in shed pollen culture and the best result was obtained from a medium supplement with 2 mg/L PAA. The preculture of anther for 10 days on solid medium with 2 mg/L PAA was effective for embryo formation from shed microspore of herbaceous peony.

In Vitro Mass Production of Human Erythroid Cells from the Blood of Normal Donors and of Thalassemic Patients

ABSTRACTWe describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (105 cells/mL) or light-density (106 cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10−6 M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated ≅1–2 × 107 erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45low/glycophorin (GPA)neg/CD711ow cells at day 7, 50–60% of which became CD45neg/GPA+/CD71high by days 15–20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (>90% benzidinepos and CD45neg/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the β-glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34 + cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34 + hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34 + progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34 + hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7–11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-α. By the two-step culture, total nucleated cells were increased 208±66 (mean±SD, n=13), or 94±29 ( n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34 + cells at the time of starting culture. Out of the total nucleated cells, 23±10.4% of cells in CB cell culture and 25±5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a + cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34 + cells using SCF, GM-CSF and TNF-α. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a + DC generated from CD34 + cells, and the phenotypes and functions of these CD1a + DC could be enhanced efficiently by treatment with a calcium ionophore agent.

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or α-naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA3), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.

Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis-derived plants of allotetraploid Coffea arabica L. (Rubiaceae).

The most important commercial species of coffee, Coffea arabica, which produces 73% of the world's coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid, 2n=4x=44) species known in the genus. High-frequency somatic embryogenesis, plant regeneration and plant recovery were achieved from leaf explants of a mature, elite plant of C. arabica cv. Cauvery (S-4347) using a two-step culture method. To assess the genetic integrity of the nuclear, mitochondrial and chloroplast genomes among the hardened regenerants, we employed multiple DNA markers (RFLP, RAPD, ISSR) for sampling various regions of the genome. Although the nuclear and mitochondrial genomes of the mother plant and five ramets derived from the mother ortet were similar in organization, this was not so in the somatic embryo-derived plants where both nuclear and mitochondrial genomes changed in different, characteristic ways and produced novel genome organizations. A total of 480 genetic loci, based on the data obtained from a total of 16 nuclear, mitochondrial and chloroplast gene probes, in combination with nine restriction enzyme digests, 38 RAPD and 17 SSR primers, were scored in 27 somatic embryo-derived plants and the single control. Among these, 44 loci were observed to be polymorphic. A relatively low level of polymorphism (4.36%) was found in the nuclear genome, while polymorphism in the mitochondrial genome (41%) was much higher. No polymorphism was detected in the chloroplast genome. The polymorphism in the mitochondrial genome was found in only 4 plants. Such selective polymorphism was not true for the nuclear genome. Thus, this in-depth and comprehensive study demonstrates, for the first time, the presence of subtle genetic variability and novel genome organizations in the commercially well-established somatic embryogenesis-derived plants of this important coffee species.

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l−1. As the cell concentration increased from 0.42 to 1.68 g l−1 in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l−1. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell−1 h−1 irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l−1 under nitrogen-limiting conditions, as high as 65 g l−1 of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183.