LS Medium Research Articles

Melatonin Mitigates Lead-Induced Oxidative Stress and Modifies Phospholipid Profile in Tobacco BY-2 Suspension Cells.

Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin's potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium-control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth-just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.

Studies on the Accumulation of Secondary Metabolites and Evaluation of Biological Activity of In Vitro Cultures of Ruta montana L. in Temporary Immersion Bioreactors.

The present work focuses on in vitro cultures of Ruta montana L. in temporary immersion PlantformTM bioreactors. The main aim of the study was to evaluate the effects of cultivation time (5 and 6 weeks) and different concentrations (0.1-1.0 mg/L) of plant growth and development regulators (NAA and BAP) on the increase in biomass and the accumulation of secondary metabolites. Consequently, the antioxidant, antibacterial, and antibiofilm potentials of methanol extracts obtained from the in vitro-cultured biomass of R. montana were evaluated. High-performance liquid chromatography analysis was performed to characterize furanocoumarins, furoquinoline alkaloids, phenolic acids, and catechins. The major secondary metabolites in R. montana cultures were coumarins (maximum total content of 1824.3 mg/100 g DM), and the dominant compounds among them were xanthotoxin and bergapten. The maximum content of alkaloids was 561.7 mg/100 g DM. Concerning the antioxidant activity, the extract obtained from the biomass grown on the 0.1/0.1 LS medium variant, with an IC50 0.90 ± 0.03 mg/mL, showed the best chelating ability among the extracts, while the 0.1/0.1 and 0.5/1.0 LS media variants showed the best antibacterial (MIC range 125-500 µg/mL) and antibiofilm activity against resistant Staphylococcus aureus strains.

Effects of basic media and plant growth regulators on Direct-somatic embryogenesis induction in vegetative strains obtained from the tissue culture of digitalis purpurea

This study aimed to investigate the effects of certain nutrient medium types enhanced with plant-growth regulators on the direct inductionof somatic embryogenesis in vegetative strains produced and propagated from a tissue culture of Digitalis purpurea. The results showthat MS medium was superior to other tested nutrient media-B5, LS, and 5C01—in terms of the days required to initiate direct somaticembryogenesis induction and induction rate. In contrast, the LS medium resulted in the highest number of embryos. Moreover, 2 mg/LBA (benzyl adenine) was superior to similar concentrations of BAP (benzyl aminopurine) and Kin (kinetin) in the MS medium, whereasBAP performed better in LS and B5. On the other hand, auxin 2.4D (dichlorophenoxy acetic acid) inhibited somatic embryogenesis, as didNAA (naphthalene acetic acid) and IAA (indole acetic acid) when added individually. Regarding the type of explant, the stems showedthe induction rates and numbers of direct-somatic embryos produced in all the basic media.Keywords: Digitalis purpurea; Vegetative strains; Direct-somatic embryogenesis; Explant; Basic media

IN VITRO PROPAGATION OF PLUM ROOTSTOCK DOCERA 6

This paper describes research on the application of tissue culture techniques to the micropropagation of interspecific rootstock ʹDocera 6ʹ. The experimental work was carried out in the period 2017-2018, in the in vitro propagation laboratory of the Fruit Growing Institute Plovdiv. Аxillary buds were employed as initial explantеs in two different seasons (March-May; September-October). The action of the mineral medium was studied in the multiplication stage. The best result was obtained on LS medium included BAP 0.5 mg/l and IAA 0.05 mg/l. Тhe obtained average multiplication rate is 3.08. The concentration of auxin applied to the basal medium influence the quality of the root system Treatment with high concentrations of IBA added to the rooting medium gives the best results (V5). Тhe influence of the season on growth and development of micropropagated ʹDocera 6ʹ rootstock during ex vitro acclimatization is also part of our research. The spring acclimatization gives better results than the autumn.

Farklı Besi Ortamlarının Tatlı Patateste in Vitro Çoğaltım Üzerine Etkil

This study was carried out in Tokat Gaziosmanpaşa University, Faculty of Agriculture, Field Crops Department, Industrial Plants Tissue Culture Laboratory in 2020. In the study, effects of different media (LS media, LS+1 mg/L Gibberellic acid (GA3), 0.1 mg/L kinetin (KIN) and LS+0.5 mg/L indole-3-acetic acid (IAA) on sweet potato genotypes (Havuc and Hatay Yerlisi) were investigated. Plant and root weights, plant and root lengths, and number of nodes and roots were determined. In this study, plants grown in cytokinin and gibberellin media had higher root number, root weight, root length, plant height, number of nodes and plant weight compared to the ones grown in auxin media. Havuc local genotype had higher values in terms of the investigated traits in all of the media studied.

Health literacy and associated factors in patients with chronic diseases, Brazil

Abstract Chronic diseases of the population have been a global challenge for public health. Considered a new field of study, health literacy (LS) is the ability to obtain and understand basic health information and services needed to make health decisions. Worse outcomes, such as less control of chronic diseases, have been associated with LS. Thus, the objective of the study was to investigate the association of LS, clinical and behavioral conditions in patients with chronic non-communicable diseases (NCDs) users of the public health service in Brazil. It was a cross-sectional study, conducted from July to December 2019, with 238 adult users of the public health service, in a random sample, from 10 basic health units, in Piracicaba-SP, Brazil. A questionnaire was applied to obtain socioeconomic, behavioral, and LS data (Suka et al, 2013). The outcome was LS (high, medium and low) and the independent variables: CNCDs (diabetes, systemic blood pressure), clinical conditions (pressure, tooth loss, pain, visible plaque and insertion loss), behaviors (brushing, use of dental floss), determinants (type and frequency of service use) and self-perception (oral health and quality of life) in health. Descriptive statistical analysis was performed, and the Chi-Square test, based on a conceptual model adapted for LS (5% significance level). They had low levels of literacy 35.3% (n = 84), average 35.7% (n = 85), and high 29.0% (n = 69). Low LS levels were associated with older individuals, low education, less brushing and flossing, irregular use of dental services, edentulism, pain, loss of insertion, negative self-perception of oral health and quality of life, and better systolic blood pressure index. The results of this study show the association of LS with behavioral and clinical factors, showing an important role in the health-disease process in this population. LS should be considered in health promotion policies in future studies. Key messages Our study shows the importance of health literacy, and its association with health outcomes in individuals with chronic non-communicable diseases. Within the important global epidemiological panorama of chronic diseases, better understanding the role of LS is important, and it can collaborate with health promotion strategies for this population.

Mass production of Pinellia ternata multiple egg-shaped micro-tubers (MESMT) through optimized growth conditions for use in ethnomedicine

The root tuber of Pinellia ternata (Thunb.) Briet has been used over the years in traditional medicine to treat myriad of diseases. In this study, a standard in vitro mass production of P. ternata multiple egg-shaped micro-tubers (MESMT) protocol was developed to meet the increasing demand for P. ternata tubers. Ten different types of media, as well as different sucrose and plant growth regulators (PGRs) concentrations were evaluated for their suitability to enhance optimum P. ternata MESMT production. At 6 weeks of culture, LS medium supplemented with 6% sucrose, 1.0 mg/L BA cytokinin and 0.5 mg/L IAA auxin was the most suitable condition for the mass production of P. ternata MESMT. Liquid chromatography-mass spectrometry and Fourier transform infrared spectroscopy analyses of the P. ternata MESMT harvested after 25 weeks of growth in soil showed a close similarity and drug equivalence to the commercialised P. ternata sample. Although these results provide a basis for future mass production of P. ternata MESMT, further testing should be conducted to validate this patented P. ternata MESMT (No. 10-1955009) for use in ethnomedicine. This is the first study to report on the optimal conditions for P. ternata MESMT increased mass production for traditional medicine purpose. This is first study on in vitro propagation of Pinellia ternata Multiple Egg-Shaped Micro-Tubers and provides an invaluable basis for its large-scale production to meet its demand in traditional medicine.

Effect of light quality on regeneration and naphthoquinones accumulation of Arnebia euchroma

The influence of light quality on the regeneration (i.e., formation, proliferation, growth, development, and rooting) and naphthoquinones’ accumulation of Arnebia euchroma was investigated in vitro. The cotyledons of sterile seedings were placed on modified LS medium supplemented with 0.8 mg/L thidiazuron to obtain callus, which was incubated on the corresponding medium at different culture stages under varying light conditions, including dark, fluorescent lamp (Fl), white light-emitting diodes (LEDs), blue LEDs, red LEDs, and a combination of 25% red, 25% blue, and 50% white LEDs [RBW (1:1:2)]. During the establishment of the A. euchroma regeneration system, we obtained the maximum fresh weight, dry weight, germination rate, shoot number, and proliferation rate under RBW (1:1:2) LEDs in the formation and multiplication phases; the growth index, shoot height, leaf number, and photosynthetic pigment concentration were promoted under red LEDs in the growth and development phase; and the maximum rooting rate (89.29% ± 2.06%) was observed under red LEDs in the rooting phase. To study the effect of light quality on naphthoquinones’ accumulation of A. euchroma, we used the euphylla to achieve adventitious roots, and the highest shikonin (121.77 ± 6.68 μg/g DW) and β′β-dimethylacrylalkannin (619.18 ± 15.71 μg/g DW) contents were detected in the dark culture. The lowest naphthoquinone contents were recorded under Fl in the adventitious roots. Overall, RBW (1:1:2) LEDs enhanced A. enchroma formation and proliferation, while red LEDs promoted the plant’s growth, development, and rooting. The dark condition was beneficial to naphthoquinones’ accumulation, whereas the light condition inhibited the naphthoquinones production of A. enchroma adventitious roots. Determining the suitable light quality for the regeneration and accumulation of naphthoquinones that is conducive to the protection and promotes resource utilization is essential.

Micropropagation of traditional deep water rice (Oryza sativa L.) cv. TNR1 for viable seed production and germplasm conservation

Abstract Extinction of traditional crop varieties is becoming a common scenario in the world due to adoption of hybrid/improved varieties. Conservationists are very busy with protection of those verities for novel genes to develop agriculture through molecular breeding. However conservation feasibility is always questionable for traditional seeds because of recalcitrance and low viability in nature. This is the first report on the protection of traditional deep water rice (Oryza sativa L.) cultivar, TNR1 from possible extinction using callus induction and regeneration system. TNR1 is known for its traditional (antidiabetic) and genetic (salinity and submergence resistance genes) importance. The matured embryos were inoculated on LS medium prepared with 2.5 mg L−1 2, 4-D and 0.1 mg L−1 KN showed 74.5% of embryogenic callus induction. The calli was regenerated on MS medium fortified with 1.5 mg L−1 NAA and 3.0 mg L−1 BAP and obtained 83.4% of regeneration. Regenerated plants were successfully acclimatized in the field with 100% survival. The germination percentage of tissue culture derived seeds was found to be high (84.2%) compared to traditional seeds (36.4%). The outcome of this study revealed that, tissue culture enhanced the viability of recalcitrant seeds. In future, this protocol will be an invaluable tool for both transgenic plant production or conservation of threatened traditional rice germplasms.

Regulators, culture media and types of lights in vitro lavender culture

ABSTRACT: Lavender is an aromatic ornamental plant that is used widely in the perfume, pharmaceutical, cosmetical and food industries. That is why it is important to study ways to promote a production of lavender raw material. For this, four experiments were carried out in order to study the germination and initial in vitro development of lavender. In the first part the efficiency of the use of hydrogen peroxide in the pre-treatment of seeds inoculated in culture media with different concentrations of gibberellic acid was evaluated. Besides that, the influence of the use of Growlux lamps and white fluorescent lamps on plantlets cultivated in MS and LS media at salts concentrations of 50 and 100% was evaluated. Finally, the effect of the gibberellic acid, putrescine, spermine and spermidine in different concentrations was evaluated. Better results were estimated when hydrogen peroxide pretreatment was applied to the seeds with subsequent inoculation in a medium containing 2,5 mg L-1 of gibberellic acid. The use of Growlux lamps did not influence the characteristics observed when compared to the use of white fluorescent lamps, however it was concluded that the species develops more in a LS medium. Concerning the use of polyamines was found that germination, aerial part and number of leaves of the seedlings were generally favored at the concentration of 0,5 mg L-1 except when spermine was applied, which resulted in a lower number of leaves under this condition.

Modeling and Optimizing a New Culture Medium for In Vitro Rooting of G\xd7N15 Prunus Rootstock using Artificial Neural Network-Genetic Algorithm

The main aim of the present investigation is modeling and optimization of a new culture medium for in vitro rooting of G×N15 rootstock using an artificial neural network-genetic algorithm (ANN-GA). Six experiments for assessing different media culture, various concentrations of Indole – 3- butyric acid, different concentrations of Thiamine and Fe-EDDHA were designed. The effects of five ionic macronutrients (NH4+, NO3−, Ca2+, K+ and Cl−) on five growth parameters [root number (RN), root length (RL), root percentage (R%), fresh (FW) and dry weight (DW)] were evaluated using the ANN-GA method. The R2 correlation values of 0.88, 0.88, 0.98, 0.94 and 0.87 between observed and predicted values were acquired for all five growth parameters, respectively. The ANN-GA results indicated that among the input variables, K+ (7.6) and NH4+ (4.4), K+ (7.7) and Ca2+ (2.8), K+ (36.7) and NH4+ (4.3), K+ (14.7) and NH4+ (4.4) and K+ (7.6) and NH4+ (4.3) had the highest values of variable sensitivity ratio (VSR) in the data set, for RN, RL, R%, FW and DW, respectively. ANN-GA optimized LS medium for G×N15 rooting contained optimized amounts of 1 mg L−1 IBA, 100, 150, or 200 mg L−1 Fe-EDDHA and 1.6 mg L−1 Thiamine. The efficiency of the optimized culture media was compared to other standard media for Prunus rooting and the results indicated that the optimized medium is more efficient than the others.

Exploring Efficient Callusing and Rapid Regeneration System in Upland Rice (<i>Oryza sativa</i> L.)

An efficient callusing and rapid regeneration system is all time needed for successful genetic transformation in rice. Therefore, mature healthy dehulled kernels of a popular upland rice variety Khandagiri was used for in vitro culture using four callus induction media and various hormone recipes singly or in combination. MS medium was equivalent to LS medium for callusing response while, BM and B5 media had shown delayed and poor callusing response without inducing somatic embryos. MS with 2.5mg/l 2, 4-D + 0.5mg/l Kn induced fast growing creamy, embryogenic and nodular calli with minimum necrosis. MS with 2.0mg/l BAP + 0.5mg/l NAA revealed excellent somatic embryogenic regeneration response with on an average 8.8 shoots per responsive callus as compared to any other combinations of BAP/Kn with NAA. The regenerated shoots were rooted on MS medium with NAA(1.0mg/l) + BAP (0.2mg/l) with highest rhizogenetic response within a week. The present reproducible in vitro culture system is amenable for rapid plantlet regeneration in upland rice.

Mature Embryo-Based in vitro Regeneration of Indica Rice Cultivars for High Frequency Plantlets Production

The study was conducted at Biotechnology Division of Bangladesh Rice Research Institute (BRRI) to investigate the effects of plant growing medium and plant growth regulator (PGR) for the callus induction and high frequency plantlets regeneration of indica rice. Ten indica rice varieties viz. BR5, BR11, BRRI dhan28, BRRI dhan29, BRRI dhan33, BRRI dhan41, BRRI dhan47, BRRI dhan48, BRRI dhan49 and BRRI dhan50 were cultured on MS, N6 and LS media. The MS medium was found better for callus induction as compared to N6 and LS media. Among the tested varieties BRRI dhan48 induced the highest percent and best quality callus. Interaction effects of BRRI dhan48 to MS medium yielded 71.55% callus induction. The regeneration efficiency of BRRI dhan48 was tested on MS medium supplemented with different combination of NAA plus BAP and NAA plus kinetin. MS medium supplemented with 2.0 mg L-1 NAA and 2.0 mg L-1 Kn was found the best in respect of percent regenerated (76.67%) plantlet as well as for the growth of plantlets in vitro.Bangladesh Rice j. 2016, 20(2): 81-87

Chemical inhibition of the histone acetyltransferase activity in Arabidopsis thaliana

Chemical inhibition of chromatin regulators provides an effective approach to investigate the roles of chromatin modifications in plant and animals. In this work, chemical inhibition of the Arabidopsis histone acetyltransferase activity by γ-butyrolactone (MB-3), the inhibitor of the catalytic activity of mammalian GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is evaluated. Arabidopsis seedlings were germinated in LS medium supplemented with different concentrations of MB-3, and inhibition in the root length and yellowed leaves were observed. The yellowed leaves phenotype of the plants grown in 100 μM of MB-3 was reverted when plants were additionally treated with 1 μM of TSA, a histone deacetylase inhibitor. Using an immunoblot assay with specific antibodies revealed a reduction of H3K14 acetylation levels at 3 and 24 h post-treatment. At 24 h post-treatment a reduction of H3K9 acetylation levels was observed. Targets of GCN5 related to stress were downregulated at 3 h post-treatment but no change was observed in target genes related to developmental transition. Our results indicate that MB-3 is a chemical inhibitor of the histone acetyltransferase in Arabidopsis and suggest that this inhibitor could function in other plants species.

High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L.) using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.

Clonal propagation and synthetic seed production from nodal segments ofCape gooseberry (Physalis peruviana L.), a tropical fruit plant

Physalis peruviana L. contains polyphenols and carotenoids with antiinflammatory and antioxidant activities used against diabetes. To establish an efficient regeneration system using nodal segments excised from 4-week-old germinated seedlings, direct plant regeneration, without additional rooting stage, was achieved on LS medium containing 0.5 mg/L 6-benzylaminopurine (BAP), kinetin (KIN), thidiazuron (TDZ), or gibberellic acid (GA3), alone or in combination with 0.25 mg/L indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA), after 2 weeks of incubation. The highest mean numbers of shoots and well-developed roots were obtained on LS medium containing solely 0.5 mg/L TDZ, producing 5.3 shoots and 3.3 roots per explant after 2 weeks of incubation. Direct shoot and root formation were also recorded on LS medium containing no plant growth regulators. Due to the high regeneration capacity of nodal segments, synthetic seed production was also investigated using the sodium alginate (NaAlg) encapsulation technique. Four different matrix compositions, including NaAlg with or without LS medium containing 3% (w/v) sucrose alone or in combination with 0.5 mg/L abscisic acid (ABA) as a growth retardant were tested for the regrowth performance of synthetic seeds after storage at 4 °C up to 70 days. The highest regrowth (100%) was observed at 28 days of storage for all matrix compositions. All plantlets were acclimatized to the soil and then progressively transferred to the field. The fruits were harvested after 5 months. This study might provide a new insight through protocol development for micropropagation and synthetic seed production of many solanaceous species with economical relevance.

Somatic embryogenesis and encapsulation of immature bulblets of an ornamental species, grape hyacinths (Muscari armeniacum Leichtlin ex Baker)

An efficient in vitro regeneration system was developed for the ornamental Muscari armeniacum on Linsmaier and Skoog basal medium (LS) supplemented with benzyladenine (BA) at varying concentrations (0.5, 1.0, or 2.0 mg/L) alone or in combination with 0.5 mg/L alpha-naphthalene acetic acid (NAA). The highest mean number of direct somatic embryo formations was observed on LS medium containing 2.0 mg/L BA and 0.5 mg/L NAA, with a mean of 7.9 somatic embryos per explant after 10 weeks of culture. Green nodular calli induced on LS medium containing 5.0 mg/L BA alone or in combination with 0.5 mg/L NAA were transferred to LS medium supplemented with or without 0.5 mg/L gibberellic acid (GA3) for 8 weeks, producing 23.3 immature bulblets. Immature bulblets produced in vitro were either embedded in a sodium alginate matrix for the encapsulation process or were transferred directly to LS medium supplemented with or without GA3 at 0.5 or 1.0 mg/L for growth and development for 6 weeks. Encapsulated bulblets were then stored at 4 °C in darkness for 10 weeks and almost all encapsulated bulblets retained their viability and resumed their growth under nonaxenic greenhouse conditions.

Genotype Independent Regeneration and Agrobacterium?mediated Genetic Transformation of Sweet Potato (Ipomoea batatas L.)

Development of an efficient genotype independent regeneration and genetic transformation system in sweet potato continues to be of great interest. Agrobacterium?mediated genetic transformation protocol was established in two different cultivars of sweet potato using Agrobacterium strain EHA105 harbouring binary plasmid pBI121 containing GUS and nptII genes. The internodal stem segments from 30?day?old micropropogated plants were used as explant with different combinations of media and hormones. MS and LS media with various concentrations of growth regulators proved to be non?responsive and the infecundity was severe with the addition of cytokinins. Nonetheless, MS with 2,4?D and TDZ gave a good percentage of callusing but with low differentiation. In different concentrations of NAA, significant amount of callusing was observed but percentage of rooting remained low in both the genotypes. Gamborg’s B5 supplemented with NAA proved to be the most suitable media and hormone combination, which yielded shoot formation after 8 ? 10 weeks with a regenera? tion efficiency of 40 ? 70%. Stable integration of transgene was confirmed by PCR analysis. Furthermore, qRT?PCR analysis was performed to assess the transcript accumulation in addition to the GUS enzymatic assay in the transgenic lines.Plant Tissue Cult. & Biotech. 23(1): 87?100, 2013 (June)DOI: http://dx.doi.org/10.3329/ptcb.v23i1.15565D.O.I. 10.3329/ptcb.v23i1.15565

Somatic Embryogenesis in Pearl Millet (Pennisetum Glaucum (L) R.Br.) Using Light and Electron Microscopy

The morphology and histology of somatic embryogenesis were examined using embryogenic calli derived from immature inflorescences of pearl millet using light and electron microscopic techniques. Young immature inflorescences, 30-35 days old (2.5 cm long) were cultured on LS medium supplemented with 2.0 mgL - 1 of 2, 4-D and 0.5 mgL -1 of kinetin. The primary callus initiated was non-embryogenic, loose, white but differentiated rapidly into pale yellow, nodular and friable embryogenic callus within 2 weeks. Histological studies revealed the formation of somatic embryos and their maturation. Initial stages of somatic embryo induction showed calli with internal segmenting divisions, which gave rise to discrete groups of cells. Continued divisions and organization in some discrete groups formed globular structures, presumably proembryoids, which showed a distinct epidermis. Further these structures developed to form somatic embryos with a well- organized bipolar structure showing embryonic axis, scutellum, coleoptile and coleorhiza. Multiple shoots (5-6) were produced from each embryo when transferred to MS regeneration medium supplemented with hormonal combinations, viz., BAP + IBA, 2.0 + 0.5 mg L -1 which further developed into complete plantlets.

Plant Regeneration from Young Shoot of Nilegrass (Acroceras macrum)

To optimize the method for embryogenic callus induction and plant regeneration from young shoot explants of Nilegrass (a warm-season forage grass species), young shoots were cultured onto LS medium supplemented with 0.5, 1, 3 and 5 mg/l 2,4-D, 0.1 mg/l BA, 4 mg/l Thiamine-HCl,100 mg/l, α-ketoglutaric acid, 30 g/l sucrose and 2.6 g/l phytagel for 8 weeks. The highest callus formation and embryogenic callus induction was obtained on LS medium supplemented with 1 mg/l 2,4-D (27.50% and 13.75%, respectively). The embryogenic nature of the compact structure was confirmed by scanning electron microscopy. The embryogenic calli were cultured on LS media supplemented with 0.5, 1, 3 and 5 mg/l BA, 3% sucrose and 2.6 g/l phytagel. After 8 weeks of culture, the highest regeneration percentages (100%) was obtained on 5 mg/l BA. The plantlets were successfully transferred to grow in the soil.