Direct Organogenesis of Citrus Cultivars from Shoot Tip Nodal Segments.

BACKGROUND: Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. OBJECTIVE: The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. METHODS: The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. RESULTS: The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. CONCLUSION: A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

The Metabolite Content of the Post-Culture Medium of the Tree Fern Cyathea delgadii Sternb. Cell Suspension Cultured in the Presence of 2,4-D and BAP.

The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern's cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L-1 and 0.2 mg·L-1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.

Limonoid detection and profile in callus culture of sweet orange

Plant tissue culture has emerged as an important tool to produce bioactive compounds from various plant species, including the sustainable production of limonoids that are receiving considerable attention due to the benefits associated with human health such as anticancer activities. The purpose of the present study was to evaluate the capacity of limonoids aglycone production from callus culture from sweet orange cv. Pera (Citrus sinensis) seeds and identify the compounds produced in this cell line. Callus induction occurred in Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic (2,4-D), malt extract, agar and coconut water. For the analysis and identification of the limonoids, CG-MS-EI ion-positive mode and UPLC-QTOF-ESI were used operating in positive and negative mode. An intense peak corresponding to limonin appeared in the callus extracts at a retention time of 58.1 min. in CG-MS-EI and four major limonoids aglycone by positive ion mode UPLC-QTOF-ESI: limonin, nomilin, deacetylnomilin, and nomilinic acid. The culture medium was efficient at the bioproduction of limonoids aglycone in callus cultures of C. sinensis seeds. Therefore, data obtained from UPLC-QTOF-ESI proved its importance at identifying new compounds that benefit human health, and may assist future work in the identification of known or new limonoids in Citrus species and related genera.

Germination of Mansonia altissima (A. Chev.) A. Chev. var. altissima: an endangered valuable timber species in Africa

The in vitro seed germination which results in the production of disease-free seedlings and greenhouse germination of the seeds of Mansonia altissima was investigated in order to establish a better way of germination of the timber species. Five levels of GA3 treatment were used in in vitro germination with three replicate and two seeds were inoculated in each of the jam bottle. Whereas, in greenhouse germination, five levels of different treatments were used, replicated three times and each Petri plate contained 15 seeds. The experiment was repeated twice and the data from each experiment was put together and used for the statistical analysis. The results showed that seeds germination occurred eight days after inoculation in in vitro but in the case of greenhouse germination, it took only five days. For in vitro rapid germination of Mansonia altissima, the MS medium should be supplemented with 1.0 μm of GA3. Equally, in greenhouse germination, the seeds need to be soaked in 1.0 mM of GA3 for 24 hours. Alternatively, in the absence of GA3, the seeds can be soaked in water for 24 hours before broadcasting the seeds on the seedbed for germination, as this will help to identify nonviable seeds.

The nitric oxide suppressed Arabidopsis mutants- Atnoa1 and Atnia1nia2noa1-2 produce nitric oxide in MS growth medium and on uranium exposure.

The mutants Atnoa1 and Atnia1nia2noa1-2 having a defective chloroplast developmental process, showed enhanced chlorophyll levels when they were grown on Murashige and Skoog (MS) medium and on exposure with uranium (U) on Hoagland medium. Thus we hypothesized that these mutants probably produced NO in MS medium and on exposure with U. Wild-type Col-0, Atnoa1, Atnia1nia2noa1-2 plants were cultured on modified Hoagland and 1/10 MS media and NO generation in the roots of these mutants was monitored using NO selective fluorescent dyes, DAF-2DA and Fl2E. Both Atnoa1 and Atnia1nia2noa1-2 triple mutants produced NO as observed by increases in DAF-2T and Fl2E fluorescence when these mutants were grown on MS medium but not on Hoagland medium. In presence of NO scavenger, methylene blue (MB, 200 µM), DAF-2T and Fl2E fluorescence was completely abolished. On the other hand treatment of the plants with 25 µM U triggered NO generation. U-treated Atnoa1 and Atnia1nia2noa1-2 plants upregulated genes (POR B, POR D, CHL D) involved in the chlorophyll biosynthesis. From these results it was concluded that Atnoa1 and Atnia1nia2noa1-2 are conditional NO producers and it appears that NO generation in plants substantially depends on growth medium and NIA1, NIA2 or NOA1 does not appear to be really involved in NO generation in MS medium or after U exposure.

In vitro multiplication of cassava varieties / Multiplicação in vitro de variedades de mandioca / Multiplicación in vitro de variedades de mandioca

The implantation of a model of sustainable development for agriculture can happen with the contribution of the mandiocultura. But for this, culture needs to be strengthened. In vitro propagation is an instrument for this purpose. Micropropagation can provide growers with large quantities of vigorous and healthy cassava seedlings in a short time. The objective of this study was to evaluate the in vitro establishment of four varieties of cassava cultivated in the municipality of Colorado do Oeste, State of Rondônia, popularly known as Arara, Caturra, Cacau Vermelha and Roxinha. For that, an experiment was carried out in the Laboratory of In Vitro Cultivation at the Pole of Technological Innovation of the University of Cruz Alta (UNICRUZ), with a completely randomized design in a 4 x 2 factorial scheme, with 6 replications. The treatments consisted of explants grown in Murashige and Skoog (MS) medium without the presence of growth regulator and MS medium supplemented with of 6-benzylaminopurine (BAP). The results indicate that the mean contamination percentage of the explants was 47.19%, differing among the varieties. The best growth response in culture media, in the multiple comparison of means (Scott-Knott's test, 5%), was obtained with MS medium without BAP addition, with significant difference between varieties. Under the conditions of this experiment, it was evidenced that micropropagation is a viable tool for obtaining varieties of interest, with desired phytosanitary qualities, with varietal and large-scale authenticity.(AU)

A implantação de um modelo de desenvolvimento sustentável para a agricultura pode acontecer com a contribuição da mandiocultura. Mas para isso, a cultura precisa ser fortalecida. A propagação in vitro é um instrumento para este fim. A micropropagação pode proporcionar aos produtores grande quantidade de mudas de mandioca vigorosas e sadias em um curto espaço de tempo. O objetivo deste trabalho foi avaliar o estabelecimento in vitro de quatro variedades de mandioca cultivadas no município de Colorado do Oeste, Rondônia, popularmente conhecidas como Arara, Caturra, Cacau Vermelha e Roxinha. Para isso, foi realizado um experimento no Laboratório de Cultivo In Vitro no Polo de Inovação Tecnológica da Universidade de Cruz Alta (UNICRUZ), com delineamento inteiramente casualizado em esquema fatorial 4 x 2, com 6 repetições. Os tratamentos consistiram em explantes cultivados em meio Murashige e Skoog (MS) sem a presença de regulador de crescimento e meio MS suplementado com 6-benzilaminopurina (BAP). Os resultados indicam que a porcentagem média de contaminação dos explantes foi de 47,19%, diferindo entre as variedades. A melhor resposta de crescimento em meios de cultura, na comparação múltipla de médias (teste de Scott-Knott, 5%), foi obtida com meio MS sem adição de BAP, com diferença significativa entre as variedades. Nas condições deste experimento, ficou evidenciado que a micropropagação é uma ferramenta viável para obtenção de variedades de interesse, com qualidades fitossanitárias desejadas, com autenticidade varietal e em larga escala.(AU)

La implantación de un modelo de desarrollo sostenible para la agricultura puede suceder con el aporte de la mandiocultura. Pero, para eso, la cultura necesita ser fortalecida. La propagación in vitro es un instrumento para ese fin. La micropropagación puede proporcionar a los cultivadores grandes cantidades de plántulas de mandioca vigorosas y saludables en poco tiempo. El objetivo de esta investigación ha sido evaluar el establecimiento in vitro de cuatro variedades de mandiocas cultivadas en el municipio de Colorado del Oeste, Rondônia, popularmente conocidas como Arara, Caturra, Cacau Vermelha y Roxinha. Para eso, se realizó un experimento en el Laboratorio de cultivo in vitro en el Polo de Innovación Tecnológica de la Universidad de Cruz Alta (UNICRUZ), con delineamiento completamente casualizado en esquema factorial 4 x 2, con 6 repeticiones. Los tratamientos consistieron en explantes cultivados en medio Murashige y Skoog (MS) sin la presencia de regulador de crecimiento y medio MS suplementado con 6-bencilaminopurina (BAP). Los resultados indican que el porcentaje de contaminación promedio de los explantes fue de 47.19%, diferenciándose entre las variedades. La mejor respuesta de crecimiento en medios de cultivo, en la comparación múltiple de medias (prueba de Scott-Knott, 5%), se obtuvo con medio MS sin adición de BAP, con una diferencia significativa entre las variedades. Bajo las condiciones de este experimento, se evidenció que la micropropagación es una herramienta viable para obtener variedades de interés, con cualidades fitosanitarias deseadas, con autenticidad varietal y de gran escala.(AU)

An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.).

An improved micropropagation protocol has been developed for a cosmetically important, dye yielding crop, henna (Lawsonia inermis). Quality of henna product is governed by naphthoquinone based pigment lawsone, thus in vitro multiplication of superior healthy plant to achieve enhanced productivity in terms of dye content and biomass deserve due attention. In the present study, nodal explants collected from an elite plant screened on the basis of superiority in lawsone content was cultured on MS medium supplemented with 0.5 µM benzyl adenine (BA) gave significantly (p < 0.05) high number of shoots (24.33). The explants placed on MS medium augmented with 0.5 µM BA and 2-isopentenyladenine (2-iP) resulted in the formation of maximum number of shoots (43.67) and was elongated (12.57 cm) within 4 weeks of culture period. Enhanced axillary bud proliferation and production of mass number of micro shoots was achieved by the continuous subculture in MS medium containing 0.5 µM BA and 2-iP. In vitro raised micro shoots were dipped in 0.44 mM NAA for 5 min followed by planting in polyethylene pots containing a soil: vermiculite (1:1 v/v) mixture produced rooted plantlets (100%). Different auxin types and its concentrations had significant role rooting of L. inermis. Rooting response of various size shoots of L. inermis treated with 0.44 mM NAA showed 100% rooting in 4.1-5 cm size class shoots. After two months of potting, survived (95%) plants were successfully transferred to medicinal plant garden of the Department. The lawsone content of one-year-old micropropagated plants (23.04 mg/g dw) growing in normal environmental conditions and elite mother plant (22.84 mg/g dw) was almost similar. Through the present study, efficient cloning of superior germplasm of L. inermis was established.

Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals.

Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4 min aeration every 8 h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium). A critical aeration rate of 440 ml min-1 was identified, corresponding to a shear rate of 96.7 s-1, pneumatic power input of 8.8 mW kg-1 and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0 mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25 mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1 mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500 mg l(-1) of IBA (indole-3 butyric acid) for 3 min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85 % survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale.

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 µM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 µM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 µM BAP plus 10.74 µM NAA and 17.76 µM BAP plus 2.685 µM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 µM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 µM BAP plus 10.74 µM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP-NAA, where they grew 2-3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 µM BAP plus 5.37 µM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.

In vitro regeneration of shoots and ex vitro rooting of an important medicinal plant Passiflora foetida L. through nodal segment cultures.

Methods were developed in the present investigation for cloning and large scale plant production of Passiflora foetida L. germplasm selected from the East-Coast region of South India. Nodal shoot segments were used as explants. The explants were dressed and surface sterilized with 0.1% (w/v) HgCl2. Multiple shoots were induced (6.13 ± 0.22 shoots per explant) by proliferation of nodal shoot meristems on Murashige and Skoog (MS) semi-solid medium + 2.0 mg l-1 6-benzylaminopurine (BAP). The shoots of P. foetida were further multiplied (16.45 ± 0.44 shoots per explant) on MS medium + 0.5 mg l-1 each of BAP and Kinetin (Kin). The in vitro generated shoots were rooted on half-strength MS medium containing 2.5 mg l-1 indole-3 butyric acid (IBA). By this method 67% shoots were rooted. About 97% shoots were rooted ex vitro (8.33 ± 0.29 roots per shoot) when the cut ends of the shoots were treated with 300 mg l-1 IBA for 5 min. The in vitro rooted plants were hardened and acclimatized in the greenhouse and successfully (100%) transplanted to the field.

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert.

Ephedra foliata Boiss. & Kotschy ex Boiss., (family - Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l(-1) of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR's) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l(-1) ammonium sulphate {(NH4) 2SO4} (MMS3) + BA (0.25 mg l(-1)), Kinetin (Kin; 0.25 mg l(-1)), Indole-3-acetic acid (IAA; 0.1 mg l(-1)) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l(-1) of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.

In vitro micropropagation of Stevia rebaudiana Bertoni in Malaysia

Stevia rebaudiana Bertoni is a medicinal plants and commercially use as non-caloric sweetener for diabetic patient. In the present study, a protocol was developed for in vitro micropropagation using 6-benzylamino purine (BAP) and Kinetin (Kn) for the formation of multiple shoot proliferation and Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA) for the induction of roots. Maximum shoot formation (7.82 ± 0.7 shoots per explants) was observed on a Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BAP and 0.25 mg L-1 Kn. The maximum number of roots (30.12 ± 2.1 roots per explants) was obtained on a MS medium containing 1.0 mg L-1 IBA. The well rooted plantlets were successfully weaned and acclimatized in plant soil with survival rate of 83.3 %.

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (<90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced>80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (<90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²âº chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.

Involvement of peroxidase activity in developing somatic embryos of Medicago arborea L. Identification of an isozyme peroxidase as biochemical marker of somatic embryogenesis.

The legume Medicago arborea L. is very interesting as regards the regeneration of marginal arid soils. The problem is that it does not have a good germinative yield. It was therefore decided to regenerate via somatic embryogenesis and find a marker of embryogenic potential. In this study, peroxidase activity was evaluated in non-embryogenic and embryogenic calli from M. arborea L. A decrease in soluble peroxidase activity is observed in its embryonic calli at the time at which the somatic embryos begin to appear. This activity is always lower in embryonic calli than in non-embryonic ones (unlike what happens in the case of wall-bound peroxidases). These results suggest that peroxidases can be considered to be enzymes involved in somatic embryogenesis in M. arborea. In addition, isozyme analyses were carried out on protein extracts using polyacrylamide gel electrophoresis. The band called P5 was detected only in embryogenic cultures at very early stages of development. This band was digested with trypsin and analyzed using linear ion trap (LTQ) mass spectrometer. In P5 isoform a peroxidase-L-ascorbate peroxidase was identified. It can be used as a marker that allows the identification of embryological potential.

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni).

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL(-1) agar and 2.0 mgL(-1) 2,4-D. The addition of 7.0 gL(-1) agar and BA (1.0, 2.0 and 4.0 mgL(-1)) significantly (P<0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL(-1) BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL(-1)) and Kin (1.0 mgL(-1)). Lower agar (3.5 gL(-1)), IAA (2.0 mgL(-1)), and NAA (2.0 mgL(-1)) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL(-1)) in combination with Kin (3.0 mgL(-1)) and 3.5 gL(-1) agar increases dulcoside-A content (Dul-A; 71.8 µg/g-DW) in shoots compared to control (50.81 µg/g-DW). Similar PGRs with 7.0 gL(-1) significantly increases the production of steviosides (Stev. 82.48 µg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 µg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL(-1)) and 7.0 gL(-1) agar than in control (07.39 µg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.

CLAVATA3-like genes are differentially expressed in grape vine (Vitis vinifera) tissues.

The CLAVATA3 (CLV3)/endosperm surrounding region [(ESR) CLE] peptides function as intercellular signaling molecules that regulate various physiological and developmental processes in diverse plant species. We identified five CLV3-like genes from grape vine (Vitis vinifera var. Pinot Noir): VvCLE 6, VvCLE 25-1, VvCLE 25-2, VvCLE 43 and VvCLE TDIF. These CLV3-like genes encode short proteins containing 43-128 amino acids. Except VvCLE TDIF, grape vine CLV3-like proteins possess a consensus amino acid sequence known as the CLE domain. Phylogenic analysis suggests that the VvCLE 6, VvCLE25-1, VvCLE25-2 and VvCLE43 genes have evolved from a single common ancestor to the Arabidopsis CLV3 gene. Expression analyses showed that the five grape CLV3-like genes are expressed in leaves, stems, roots and axillary buds with significant differences in their levels of expression. For example, while all of them were strongly expressed in axillary buds, VvCLE6 and VvCLE43 expression prevailed in roots, and VvCLE25-1, VvCLE25-2 and VvCLE TDIF expression in stems. The differential expression of the five grape CLV3-like peptides suggests that they play different roles in different organs and developmental stages.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants.

OBJECTIVE: To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants. METHODS: Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3-4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration. RESULTS: Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field. CONCLUSIONS: The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.

Induction of Biologically Active Flavonoids in Cell Cultures of Morus nigra and Testing their Hypoglycemic Efficacy.

The antidiabetic activity of both leaves and MJ-treated cell cultures of Morus nigra was evaluated after their oral administration to streptozotocin-induced diabetic rats. The antidiabetic activity of extracts from leaves given to streptozotocin (STZ)-diabetic rats for 10 days increased with increasing doses of leaves extract up to 500 mg/kg/day. The administration of 500 mg/kg/day of leaves extract reduced the concentration of glucose from 370 ± 7.31 mg/dl (control) to 154 ± 6.27 mg/dl, and a significant increase in the insulin level from 11.3 ± 0.31 µU/ml (control) to 14.6 ± 0.43 µU/ml was recorded. Cell suspension cultures were established from the young leaves of Morus nigra cultivated on modified MS medium supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA), 0.2 mg/l 6-(furfurylamino)purine (kinetin). The changes in cell weight and flavonoid content were monitored between day zero and 12. The linear increase in fresh weight was found to be parallel to flavonoids production. Cell cultures treated with 100 µM methyl jasmonate for 24 hours showed a noticeable increase in level of flavonoids and significant and more effective hypoglycemic activity than that for extract from leaves. The major flavonoids were isolated by TLC and HPLC and identified as rutin, quercetin, Morusin and cyclomorusin by co-chromatography and mass spectrometry in comparison to samples of authentic reference compounds.