Development of an optimum proliferation medium via the graph kernel statistical analysis method for genetically stable in vitro propagation of endemic Thymus cilicicus (Turkey)

Thymus cilicicus is an endemic Eastern Mediterranean element that has aromatic-medicinal properties. Its natural population spreads across gravelly ground and open rocky areas of South and Southwest Anatolia. The current study on in vitro propagation of T. cilicicus focused deeply on environmental applications such as the development of an optimum medium composition for efficient and genetically stable micropropagation and improved preservation procedures for long-time conservation of elite germplasms for further studies. For this purpose, MS and OM media were used individually and in combination with cytokinins, charcoal, AgNO3, Fe-EDDHA, and H3BO3. The raw data were statistically analyzed via the graph kernel method to optimize the nonlinear relationship between all parameters. The optimal proliferation medium for T. cilicicus was OM supplemented with a combination of 10 g L-1 charcoal and 1 mg L-1 KIN and the calculated averages of the best regeneration rate, the best shoot number and the best shoot length were 96.89%, 3 and 1.24 respectively on this medium. The determination of genetic stability of in vitro grown plants on the optimum medium compositions obtained by the graph kernel method was carried out with the use of the ISSR-PCR technique. All the ISSR primers produced a total of 192 reproductive band profiles, none of which were polymorphic. Furthermore, the micropropagated plants were successfully rooted and acclimatized to greenhouse conditions. In this study, we present a graph kernel multiple propagation index which considers all the possible parameters needing to be analyzed. Such an index is used for the first time for the determination of the optimum proliferation medium.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

Mutasi Induksi Dendrobium sylvanum var. flava Menggunakan Kolkisin secara In Vitro

ABSTRACTIn Vitro mutation using colchicine on 2 month of self-pollinated protocorm like bodies of Dendrobium sylvanum var. flava was conducted to determine the effects of concentration and immersion duration in colchicine on proliferation of PLBs, and to identify of ploidy variants based on stomatal variable. Research was arranged using factorial completely randomized design with three factors in three replications. The first factor was concentration of colchicine, consisted of five concentrations (0.02; 0.04, 0.06; 0.08; dan 0.1%). The second factor was duration of immersion in the colchicine, consisted of four durations (1; 24; 48; 72 hours). The third factor was proliferation medium consisted of two concentrations of BAP (1; 0.5 mg L-1). Analysis of variance showed the significant effect of colchicine treatment on percentage of survived explants. LD50 in media 1 mg L-1 BAP was obtained at a colchicine concentration of 0.069% with duration immersion of 58.19 hours. On Media 0.5 mg L-1 BAP, LD50 was obtained at colchicine concentration of 0.054% with duration immersion of 47.63 hours. Percentage of solid polyploid mutant of Dendrobium sylvanum var. flava can not be determined on MV2 generation because the stomata leaf showed chimeras based on the chloroplast number in cell guard and stomata size.Keywords: colchicines, Dendrobium sylvanum, mutation, number of chloroplast stomatal density.ABSTRAKMutasi dengan kolkisin pada PLBs hasil selfing Dendrobium sylvanum var. flava umur 2 bulan dilakukan secara In Vitro dengan tujuan mempelajari pengaruh konsentrasi kolkisin, durasi perendaman dalam kolkisin, media proliferasi terhadap pertumbuhan PLBs Dendrobium sylvanum var. flava serta mengidentifikasi variasi ploidi berdasarkan variabel stomata. Penelitian disusun dalam Rancangan Acak Lengkap Faktorial 3 Faktor dengan 3 ulangan. Faktor pertama ialah konsentrasi kolkisin yang terdiri atas 5 taraf (0.02; 0.04, 0.06; 0.08; dan 0.1%). Faktor kedua ialah durasi perendaman yang terdiri atas 4 taraf (1; 24; 48; 72 jam). Faktor ketiga ialah media proliferasi media V&W yang ditambah BAP terdiri atas 2 taraf (1; 0.5 mg L-1). Hasil analisis ragam menunjukkan pengaruh kolkisin yang nyata terhadap variabel persentase hidup. LD50 pada media 1 mg L-1 BAP diperoleh pada konsentrasi 0.069 % dengan durasi perendaman 58.19 jam. Pada media 0.5 mg L-1 BAP, LD50 diperoleh pada konsentrasi 0.054 % dengan durasi perendaman 47.63 jam. Persentase mutan poliploid pada MV2 Dendrobium sylvanum var. flava ini belum dapat ditentukan karena stomata daunnya masih kimera berdasarkan karakter jumlah kloroplas sel penjaga dan ukuran stomata.Kata kunci: Dendrobium sylvanum, jumlah kloroplas, kerapatan stomata, kolkisin, mutasi.

Neem Oil Used as a “Complex Mixture” to Improve In Vitro Shoot Proliferation in Olive

Shoots of the olive cultivar Moraiolo were previously cultured in aseptic conditions on Olive Medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. Then, 1-cm long uninodal explants with two leaves and two axillary buds were excised from the proliferated masses and placed on the same proliferation medium enriched with four concentrations of neem oil (0—control, 0.1, 0.5, and 1.0 mL·L−1), added before sterilization. The addition of 0.1 mL·L−1 of neem oil to the medium gave an improvement in shoot regeneration. More vigorous shoots (longer proliferated shoots) were obtained along with a higher number of nodes (multiplication rate). Overall, there was a significant increase in the total fresh and dry proliferated weights. To our knowledge, this is the first report showing a strong and beneficial effect of neem oil, used as a “complex mixture,” on in vitro plant regeneration.

Effect of Epoxomicin on 3D Neurosphere

Developmental neurotoxicity (DNT) entails the toxic effects imparted by many chemicals on brain during early childhood. Various chemicals would have their maximum effects on brains during early childhood. Some toxicants have confirmed to induce developmental toxic effects on CNS. Most of agents cannot be identified with certainty due to the defective nature of predictive toxicology models used. A novel method that can overcome most of the limitations of conventional techniques is the use of 3D neurospheres system (in-vitro system can recapitulate most of the changes during the period of brain development making it an ideal model for predicting neurotoxic effects). In the present study we verified the possible DNT of epoxomicin which is a potent anti-tumor agent isolated from Actinomycetes that is used as a selective and irreversible inhibitor of the 20S proteasome with anti-inflammatory activity. Methods Rat neural progenitor cells were isolated from rat embryos (E14) extracted from placental tissue. Cortices were aseptically dissected from brains of the fetuses and the tissues were triturated by repeated passage through a fire-polished constricted Pasteur pipette. The dispersed tissues were allowed to settle for 3 min. The supernatant was then transferred to a fresh tube and centrifuged at 1,000 g for 5 min. The pellet was placed in Hank’s balanced salt solution cultured as free-floating neurospheres in proliferation medium. Two doses of epoxomicin (1µM and 10µM) were used in cultured neurospheres for a period of 14 days. For proliferation analysis, spheres were cultured in proliferation medium. After 0,4,5,11 and 14 days, sphere size was determined by software analyses (CellProfiler, Broad Institute, www.cellprofiler.org) Diameter of each neurosphere was measured and exported as excel file to statistical analysis. For viability analysis, trypsin-EDTA solution were added to neurospheres for 3 min to dissociate them into single cells suspension, then viability was evaluated by the Trypan Blue exclusion test. Epoxomicin found to affect differentiation and viability of neurospheres, these effects were positively correlated to doses and progress of time. Results Epoxomicin found to affect proliferation and viability of neuropsheres. Conclusion The study confirms DNT of epoxomicin and suggests possible gross morphologic changes and the decrease in viability, it proposes possible focal lesions on exposure to epoxomicin in early childhood.

Residual effect of growth regulators in etiolation and regeneration of in vitro pineapple plants

This work aimed to evaluate the influence of naphthaleneacetic acid (NAA) and gibberellic acid (GA3) plant regulators in in vitro etiolation and subsequent regeneration of the PE x SC-60 pineapple hybrid. Nodal segments of in vitro plants with approximately 5-7 cm height were incubated in basic MS culture medium supplemented with 0.0; 0.5 and 1.0 mg L-1 of naphthaleneacetic acid (NAA) in combination with gibberellic acid (GA3) in concentrations of 0.0; 0.5 and 1.0 mg L-1, and maintained at 27 ºC under dark condition. Evaluations were carried out at 90 and 180 days after incubation period. The best results for length of etiolated stems were obtained with 1.0 mg L-1 of NAA. In the experiment followed by the regeneration, stems with 3 cm from the etiolation treatment, were cultivated in proliferation medium and the number of regenerated plants per treatment was evaluated at 60 days of cultivation. The treatment that promoted the best etiolation of plants also promoted the worst regeneration rates, demonstrating the residual effect of the auxin used in the previous step in the regeneration of plants of the pineapple hybrid evaluated.