Subculture Process Research Articles

Clostridium beijerinckii strain degeneration is driven by the loss of Spo0A activity.

Solventogenic clostridia represent a diverse group of anaerobic, spore-forming bacteria capable of producing acetone, butanol and ethanol through their unique biphasic metabolism. An intrinsic problem with these organisms however is their tendency to degenerate when repeatedly subcultured or when grown continuously. This phenomenon sees cells lose their ability to produce solvents and spores, posing a significant problem for industrial applications. To investigate the mechanistic and evolutionary basis of degeneration we combined comparative genomics, ultra-deep sequencing, and concepts of sociomicrobiology using Clostridium beijerinckii NCIMB 8052 as our model organism. These approaches revealed spo0A, the master regulator gene involved in spore and solvent formation, to be key to the degeneration process in this strain. Comparative genomics of 71 degenerate variants revealed four distinct hotspot regions that contained considerably more mutations than the rest of the genome. These included spo0A as well as genes suspected to regulate its expression and activity. Ultra-deep sequencing of populations during the subculturing process showed transient increases in mutations we believe linked to the spo0A network, however, these were ultimately dominated by mutations in the master regulator itself. Through frequency-dependent fitness assays, we found that spo0A mutants gained a fitness advantage, relative to the wild type, presumably allowing for propagation throughout the culture. Combined, our data provides new insights into the phenomenon of clostridial strain degeneration and the C. beijerinckii NCIMB 8052 solvent and spore regulation network.

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes.

Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1–S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13–S20, obtained after 13–20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Growth of Kaffir Lime (<i>Citrus hystrix</i> DC) Cell Line Derived from Seed Explant After Yeast Elicitation Using Pure and Technical Grade Yeast

The addition of elicitors in kaffir lime (Citrus hystrix DC.) culture is one of strategies for obtaining and increasing the production of secondary metabolites. Saccharomyces cerevisiae is one of the elicitors that can be used to increase secondary metabolites such as terpenoids. However, in its use, the pure cultures of S. cerevisiae are expensive. Therefore, the first objective of this study was to analyze the ability of technical grade (commercial baker’s yeast) to be used as an elicitor and measure the growth of kaffir lime cell line after being elicited by pure and technical grade (commercial baker’s yeast). The second objective is to determine the best time to subculture kaffir lime cell line after elicitation. We observed the morphology and measured the growth curve of pure and technical grade yeast until the 4th subculture generation. Furthermore, we used both grades of yeast for elicitation. Kaffir lime cell suspension was treated with 10 ppm pure grade or 5 ppm and 10 ppm technical grade yeast for 4 days. After elicitation, kaffir lime cell lines were subcultured and their growth was analyzed. The result showed that the morphology and growth curve of technical grade until 4th subculture generations was similar to the pure grade. On the other hand, after elicitation using pure and technical grade yeast and being subcultured, the growth of the elicitated kaffir lime cell line had the same pattern as the control group, but the cell density of the control group was higher than the elicitated group. The initial stationary phase of kaffir lime cell line was on the 17th day which is the best time to subculture. The subculturing process is important to maintain the viability of the kaffir lime cell line.

POMEGRANATE WONDERFUL CULTIVAR MICROCLONAL PROPAGATION TECHNOLOGY IN VITRO

Introduced in the article, the Wonderful variety of pomegranate was studied in vitro for microclonal propagation, sterilization at 1.0 and 3.0% solution of sodium hypochlorite (NaOCl) at different times, swelling of the variety in different nutrient media, the percentage of germination. The subculture process under the influence of nutrient media hormones BAP, Kin, IBA and GA3 and the results of pomegranate rooting are covered.

The Effect Hormone BAP (6-Benzyl Amino Purine) on the Growth of Potato Axillary Shoots (Solanum Tuberosum L.) in Vitro

Potatoes are one of the foodstuffs that contain lots of carbohydrates, minerals, and vitamins. To develop potato cultivation, superior varieties are needed that produce high yields, are resistant to pests, and have good tuber quality. The growth of potato plantlets in the in vitro subculture process is determined by many factors, one of which is the administration of growth regulators and the concentration of growth regulators. The type of media or nutrients used also affects the growth and development of the explants and seedlings produced. This study aims to trigger the growth of potato plantlets so that their growth is optimal. The method used is a true experimental quantitative method with a one-factor Completely Randomized Design (CRD) design, namely variations in BAP concentration consisting of 5 treatments with 5 replications. Data collection techniques through observation or observation. Parameters observed were number of shoots, number of leaves, number of roots, and plantlet height. The data analysis technique used is the one way ANOVA test and for the average difference between treatments, the HSD (Honestly Significant Difference) test at 5% level is used. Based on the results of the study, it was known that there was an effect of giving the hormone BAP (6-Benzyl Amino Purine) on the growth of potato axillary shoots (Solanum tuberosum L.) in vitro. The results showed that there was a significant effect on the number of shoots (P = 0.007) with an average of 1.64 pieces, the number of leaves (P = 0.002) with an average of 1.42 pieces, the number of roots (P = 0.000) with an average of 1. an average of 1.70 pieces, and plantlet height (P=0.000) with an average of 2.65 cm. It was concluded that treatment D (1.5 ppm BAP) was the best treatment in increasing the growth of potato plantlets.

Cytochrome Oxidase 2 (COX2), β-Tubulin (TUB) and Chitin Synthase Subunit 2 (CHS2) Expression in Pythium insidiosum Thai Strains

This study aimed to investigate the real-time fluctuation of temperature-sensitive gene expression in a Pythium insidiosum growing at human body temperature (37 ºC) when it can cause life-threatening disease, whereas its natural habitat is in agricultural water sources with lower temperature. Genes encoding cytochrome oxidase 2 (COX2), β-tubulin (TUB), and chitin synthase subunit 2 (CHS2) were tested for their expression at the infection temperature (37 ºC) and natural habitat temperature (27 ºC) using real-time RT-PCR. Sixteen strains of P. insidiosum, representing 3 phylogeographic preferences, were analyzed. The subculturing process at 27 ºC was repeated at least 2 - 3 cycles. The results showed that at 37 ºC, the COX2 was expressed 2.5-fold higher than that of 27 ºC (p = 0.0347) while the level of TUB and CHS2 mRNA disclosed indistinguishable profiles. These results suggested that these genes were suitable to be used as housekeeping genes for temperature-susceptible gene expression studies. This work was likely to be the 1st study examining the gene expression levels of the Thai strain of P. insidiosum under thermal stress conditions. Follow-up studies for the COX2 genes may useful to provide valuable insight into the pathogenesis, diagnostic, or therapeutic targets for further investigation.

Autogenic successions of bacteria and fungi in kefir grains from different origins when sub-cultured in goat milk

Kefir grains are a unique symbiotic association of different microbiota, including a variety of bacterial and fungal species. The microbiota in kefir grains is strongly influenced by the geographical origin and sub-culturing environment. After sub-culturing in goat milk for 2 to 4 months, amplicon sequencing (16S rRNA and ITS1 region) was applied for the identification of bacterial and fungal autogenic succession of three kefir grains collected from China (CN, Asia), Germany (DE, Europe) and United States of America (USA, America). Taxonomic analysis displayed three main bacterial and fungal species in kefir grains from different origins during sub-culturing process (Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus kefiri for bacteria, Kazachstania unispora, Kluyveromyces marxianus and Saccharomyces cerevisiae for fungi). Based on the results of beta diversity analysis, microbiota in kefir grains from CN and DE would be stable when sub-cultured in goat milk for more than three months. Differently, a highly microbial stability has been found for the sample from USA during the whole sub-culturing process. These results helped to understand the composition and stability of microbiota in kefir grains when sub-cultured in goat milk.

ОСОБЛИВОСТІ СОЦІАЛЬНО-ПСИХОЛОГІЧНОЇ АДАПТАЦІЇ ОСОБИСТОСТІ ЮНАЦЬКОГО І ДОРОСЛОГО ВІКУ В НЕФОРМАЛЬНІЙ СУБКУЛЬТУРІ

У статті подано результати дослідження, які дозволяють розширити наукове розуміння адаптації та розглядати її не лише, як процес пристосування до нових обставин, а й як важливий процес особистісного зростання і розвитку. Презентоване дослідження мало на меті виявлення особливостей соціально-психологічної адаптації особистості юнацького та дорослого віку чоловічої й жіночої статі в неформальній субкультурі, порівняно з респондентами, які до неї не належать. Метод дослідження – опитувальник соціально-психологічної адаптації (СПА) К. Роджерса та Р. Даймонд. Вибірка складалася з юнацького і дорослого віку чоловічої й жіночої статі. За допомогою процедур рандомізації було створено дві групи респондентів, кожна з яких складалася зі 160 осіб, які були представниками, відповідно, неформальних і формальних груп.Розкрито результати вивчення інтегральної соціально-психологічної адаптації учасників неформальних і формальних груп та її окремих складників. За переважною більшістю складових соціально-психологічної адаптації, активні учасники неформальних угруповань мають вищі показники, порівняно з пересічними респондентами, зокрема, за шкалами «адаптивність», «прийняття себе», «емоційний комфорт», «внутрішній контроль», «прагнення до домінування», «конформність», «ескапізм», проте нижчі показники за шкалою «прийняття інших». Емпірично визначено вікові особливості соціально-психологічної адаптації, а, саме, труднощі й переваги перебігу адаптаційних процесів представників юнацького та дорослого віку, що належать/не належать до неформальних субкультур. Достовірно найбільш дезадаптивними є пересічні юнаки, порівняно з пересічними дорослими чоловіками (р≤0,05) і хлопцями-неформалами (р≤0,01). В юнацькому віці кращі адаптаційні властивості в неформальному субкультурному середовищі виявили хлопці, порівняно з дорослими чоловіками, а в дорослому – жінки, порівняно з юними дівчатами. Здійснено статеву диференціацію особливостей перебігу адаптації в юнацькому та дорослому віці, з урахуванням впливу на цей процес неформальної субкультури. Виявлено негативну вікову динаміку інтегральної адаптації у представників чоловічої статі та позитивну – у представниць жіночої статі, які належать до неформальної субкультури. Загалом юнаки й дорослі жінки є більш адаптивними, порівняно з юнками і дорослими чоловіками. Доведено, що активні учасники неформальних груп соціально і психологічно краще адаптовані до цих груп, порівняно з адаптацією учасників формальних груп.

Impact of pH on succession of sourdough lactic acid bacteria communities and their fermentation properties.

Sourdough, a traditional fermented dough, is made via natural fermentation by lactic acid bacteria (LAB). Its pH changes from near neutral to acid during the subculture process. However, the product quality of subcultured sourdough depends on the unpredictable succession of LAB communities, the influential factors of which are still unclear. To elucidate one end of the LAB community succession mechanism, we evaluated the effect of pH by designing four subculture experiments using a model medium adjusted to pH 6.7, 5.5, and 4.5, as well as a natural sourdough subculture. All experiments began by inoculating a sourdough LAB mixture, and both bacterial successions and fermentative properties were monitored until ten subculture steps. In media subcultures, lactic acid production was higher in higher pH media. Three LAB genera, Weissella, Pediococcus, and Lactobacillus, each represented by one operational taxonomic unit (OTU), were successively detected in all subcultures. In later steps with lower pH media, an OTU closely related to Lactobacillus brevis dominated, replacing an OTU closely related to the Weissella cibaria-confusa group that was more dominant than the L. brevis OTU in the near-neutral pH medium. In the sourdough subculture, the three genera were also detected, while Lactobacillus was dominant in earlier steps due to the emergence of an OTU closely related to Lactobacillus sanfranciscensis. These results suggest that a lower pH is favorable for the sequence of sourdough bacterial community evolution finalizing with Lactobacillus domination. Further research is needed to elucidate additional factors other than pH that influence the pattern of LAB community shift.

Vertical capacitance aptasensors for real-time monitoring of bacterial growth and antibiotic susceptibility in blood

For the treatment of bacteremia, early diagnosis and rapid antibiotic susceptibility tests (ASTs) are necessary because survival chances decrease significantly if the proper antibiotic administration is delayed. However, conventional methods require several days from blood collection to AST as it requires three overnight cultures, including blood culture, subculture, and AST culture. Herein, we report a more rapid method of sensing bacterial growth and AST in blood based on a vertical capacitance sensor functionalized with aptamers. Owing to their vertical structure, the influence of blood cells sunk by gravity on capacitance measurements were minimized. Thus, bacterial growth in blood at 100–103 CFU/mL was monitored in real-time by measuring changes in capacitance at f = 10 kHz. Moreover, real-time capacitance measurements at f = 0.5 kHz provided information on biofilm formation induced during blood cultures. Bacterial growth and biofilm formation are inhibited above the minimal inhibitory concentration of antibiotics; therefore, we also demonstrated that vertical capacitance aptasensors could be applied to rapid AST from positive blood cultures without a need for the subculture process.

Quantitative analysis of operators' flow line in the cell culture for controlled manual operation.

IntroductionAlthough cell culture has been widely used in the life sciences, there are still many aspects of this technique that are unclear. In this study, we have focused on the manual operations in the cell culture process and try to analyze the operators’ flow line.MethodsDuring a course of approximately 6 years, we obtained the operators’ flow line data from two places (three layouts) and 38 operators (93 subcultures) using two network cameras and a motion detection software (Vitracom SiteView).ResultsOur investigation succeeded in quantifying the flow line of the subculture process and analyzed the time taken to carry out the process, to travel around the workplace. For the subculture process, the total time of the process being rerated the time of the operation in the place where the main operation is performed; the total distance of travel and the counts of travel not being related to the total time of the process. Based on these results, we propose a new way of evaluating the efficiency of cell culture process in terms of time and traveling. We believe that the results of this study can guide cell culture operators in handling cells more efficiently in cell manufacturing processes.ConclusionsThe flow line analysis method suggested by us can record the operators involved and improve the efficiency and consistency of the process; it can, therefore, be introduced in cell manufacturing processes. In addition, this method only requires network cameras and motion detection software, which are inexpensive and can be set up easily.

Optimization of Alginate-Based Encapsulation Utilization For Viability and Stability of The Mesenchymal Stem Cell

In the past few decades, attention and research in the field of stem cell are progressing very rapidly. Hospitals in Indonesia have been using stem cells as an alternative to cure some illnesses like diabetes, heart disease, fractures and joints, dental implants, etc. Currently, adult stem cells can be obtained not only from the spinal cord and peripheral vessels, but also from fat tissues of the human body, where it can be isolated as adherent stem cells (mesenchymal stem cells). Consideration of fat tissue as the source of mesenchymal stem cells (MSCs) for autologous tissue engineering is because they are readily available in abundant quantities through minimal invasive procedures, as well as easily cultured and propagated. It is possible to proliferate and differentiate into the desired direction of the network. Stem cell growth requires conditions to grow such as requiring optimum growing conditions such as an environmental temperature of 37°C and a concentration of 5% CO2. Maintenance of MSCs also requires a subculture process, i.e. the process of moving MSCs from a full culture medium to new media; continuous subculture process can cause changes in MSCs. The viability of stem cells may be disrupted by micro-conditions in wounds such as hypoxia, oxidative stress, and inflammation. Therefore, the purpose of this research was to investigate whether alginate-based encapsulation can increase and maintenance stem cell growth at different temperature by using some concentration of alginate and CaCl2 as the formula. Results shown that alginat with low concentration and CaCl2 100mM is suitable for MSCs growth (as in MTT result shown) at 25°C temperature. This can be due to the MSCs encapsulated can adapt and grow within the alginate microcapsule with low concentration. In addition, the media may also easier to get into the microcapsule alginate.

Selective elimination, clump production and patterning of hiPSCs by light on PAG-polymer-functionalized substrates

Event Abstract Back to Event Selective elimination, clump production and patterning of hiPSCs by light on PAG-polymer-functionalized substrates Kimio Sumaru1, Kana Morishita1, Toshiyuki Takagi1, Taku Satoh1 and Toshiyuki Kanamori1 1 National Institute of Advanced Industrial Science and Technology, Biotechnology Research Institute for Drug Discovery, Japan Introduction: Utility of human stem cells has been increasingly examined for drug assay or tissue engineering. For these purposes, automatable cell culture method is required to implement the pure and efficient propagation of those cells. In the culture of human pluripotent stem cells to maintain their undifferenciated state and viability stably, the cell should be treated in cell clumps avoiding discrete state which induces apoptosis in the subculture process, and be purified by removing the differenciated portion timely. To provide powerful tool to process cell culture systems on a culture substrate in such circumstances, we developed a method to kill the cultured cells selectively by using the combination of photo-acid-generating (PAG)-polymer-functionalized culture substrates and micro-patterned light irradiation[1]. Recently we found that the cell monolayer culture of the substrate can be cut by irradiation of line pattern. In this study, we examine the application of this photo-manipulation to the maintenance culture of human iPS cells (hiPSCs) (Fig.1). Experimental and Results: We synthesized a poly(methylacrylate) functionalized with visible-light-responsive PAG group[2], and fabricated a photo-responsive culture substrate by spin-coating the polymer solution on polystyrene culture surface[1]. We cultured the colonies of hiPS cell line (201B7) on the substrate and irradiated light locally in the areas, where the cells were undifferentiated, using a PC-controlled micropattern irradiation system. After 2 hours, we observed that the cell viability was diminished only in the irradiated area (Fig.2). Also we demonstrated to produce cell clumps with te similar size from the grown colonies by the photo-induced cutting without critical damage. Together with the above purification method, this photo-manipulation was expected to provide powerful tools in automatizing the propagation of the human pluripotent stem cells. This study was financially supported by JSPS KAKENHI (25282148).

Efficient Subculture Process for Adherent Cells by Selective Collection Using Cultivation Substrate Vibration.

Cell detachment and reseeding are typical operations in cell culturing, often using trypsin exposure and pipetting, even though this process is known to damage the cells. Reducing the number of detachment and reseeding steps might consequently improve the overall quality of the culture, but to date this has not been an option. This study proposes the use of resonant vibration in the cell cultivation substrate to selectively release adherent calf chondrocyte cells: Some were released from the substrate and collected while others were left upon the substrate to grow to confluence as a subculture-without requiring reseeding. An out-of-plane vibration mode with a single nodal circle was used in the custom culture substrate. At a maximum vibration amplitude of 0.6 µm, 84.9% of the cells adhering to the substrate were released after 3 min exposure, leaving a sufficient number of cells for passage and long-term cell culture, with the greatest cell concentration along the nodal circle where the vibration was relatively quiescent. The 72-h proliferation of the unreleased cells was 20% greater in number than cells handled using the traditional method of trypsin-EDTA (0.050%) release, pipette collection, and reseeding. Due to the vibration, it was possible to reduce the trypsin-EDTA used for selective release to only 0.025%, and in doing so the cell number after 72 h of proliferation was 42% greater in number than the traditional technique.

Transition from somatic embryo to friable embryogenic callus in cassava: dynamic changes in cellular structure, physiological status, and gene expression profiles.

Friable embryogenic callus (FEC) is considered as the most suitable material for efficient genetic transformation of cassava. Heavy genotype dependence of FEC induction and amenability to somaclonal variation limits the production and maintenance of reliable FEC. Identifying key elements involved in biological processes from somatic embryos (SEs) to FEC at different stages provides critical insights for FEC improvement. Cytological observation showed a dramatic change of subcellular structures among SEs, fresh FEC (FFEC), and old FEC (OFEC). Decrease of sucrose and increase of fructose and glucose were detected in OFEC. A total of 6871 differentially expressed genes (DEGs) were identified from SEs, FFEC, and OFEC by RNA-seq. Analysis of the DEGs showed that FEC induction was accompanied by the process of dedifferentiation, whereas the epigenetics modification occurred during the continuous subculturing process. The cell structure was reconstructed, mainly including the GO terms of “cell periphery” and “external encapsulating structure”; in parallel, the internal mechanisms changed correspondingly, including the biological process of glycolysis and metabolisms of alanine, aspartate, and glutamate. The significant reduction of genomic DNA methylation in OFEC indicated altered gene expression via chromatin modification. These results indicate that the induction and long-term subculture of FEC is a complicated biological process involving changes of genome modification, gene expression, and subcellular reconstruction. The findings will be useful for improving FEC induction and maintenance from farmer-preferred cassava cultivars recalcitrant to genetic transformation, hence improving cassava through genetic engineering.

장기간 계대배양 된 장미 배발생 캘러스로부터 식물체 재분화 및 비정형체로부터 새로운 배발생캘러스 재생

장미 형질전환체를 개발하기 위한 유전자 도입 재료로써 5 ~ 6년 이상 장기간 유지 증식된 장미 배발생캘러스의 이용 가능성이 확인되었다. 2007년 및 2009년에 각각 처음으로 기내 뿌리유래 캘러스로부터 유도된 후 유지 증식배지에서 계대배양 해 온 장미 '스위트 옐로우' 품종 및 KR056002와 KR056006 2계통 유래 배발생캘러스로부터 식물체의 재분화 능력을 확인하였다. 장기간 계대배양 된 배발생캘러스로부터 신초의 모습을 갖춘 식물체로 재분화 되기까지 소요기간 및 재분화율은 '스위트옐로우' 품종은 3 ~ 4개월, KR056002 및 KR056006은 4 ~ 5개월로, 이들 배발생캘러스가 기내뿌리로부터 처음으로 배발생 된 후 최초 신초 재분화 때와 동일한 양상이었다. 또한 체세포배발생캘러스의 유지 증식을 위한 계대배양과정에서 발생되는 비정형체 위에 새로운 배 및 배발생캘러스가 유도되었다. 이 비정형체는 새로운 배발생캘러스를 유도할 수 있는 재료로서 이용될 수 있을 것이다. Long-term subcultured rose embryogenic calluses, which had been maintained for more than 5 to 6 years since the first embryogenesis from calluses induced from in vitro roots of rose, were identified as potential material for the development of transgenic plants. The first embryogenic calluses from 'Sweet Yellow' and two breeding lines (KR056002 and KR056006) were obtained in 2007 and 2009, respectively. Subsequently, we found that plants regenerated from long-term embryogenic calluses (LEC). Whereas the LEC from 'Sweet Yellow' takes 3 to 4 months to regenerate plants, those of the two breeding lines take 4 to 5 months. This period of time is the same as that taken for plants to regenerate from the first embryogenic callus. New embryogenesis was observed from atypical bodies (ABs) that appeared during the process of long-term subculture. We found that it is possible to use the AB as a material for new embryogenesis.

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.

Establishment of cell culture system of rough-legged buzzard and biological characteristic analysis on different tissue cells cultured in vitro

In total, three different tissues from the rough-legged buzzard were obtained by culture and successfully cryopreserved and then recovered. During the subculture process, biological characteristics including as cell morphology, growth curve, cell adhesion rate, and karyotype were analyzed and compared, and overall all three kinds of tissue cells exhibited fibroblast-like growth. Oviduct-derived cells had the strongest adherent ability, followed by lung-derived cells and trachea-derived cells. The doubling times of lung-derived cells, trachea-derived cells, and oviduct-derived cells were 29.91±0.39 h, 33.18±0.21 h, and 30.67±0.28 h, respectively, with population doubling times 3.54±0.01, 4.52±0.02, and 4.38±0.03, respectively. Likewise, we noted the chromosome number of the rough-legged buzzard was 68, within the typical type of ZW. These results may potentially provide material and a basis for further research in the field, with the successful preservation of genetic information of rough-legged buzzard.

Genetic Stability of In vitro Multiplied Phalaenopsis gigantea Protocorm-like Bodies as Affected by Chitosan

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant.