Tissue Culture Protocols Research Articles

THE ROLE OF NAA, GA3, ACTIVATED CHARCOAL, AND SUCROSE ON ZYGOTIC EMBRYOS CULTURE OF HYBRID OG OPEN POLLINATED CLONE (Elaeis guineensis JACQ. X Elaeis oleifera)

Oil palm OG hybrid genetic potentials are high in term of vegetative and generative traits, such as: low height increment, compact palm, high free fatty acid and high carotenes. However, OG hybrid reproduction is hindered due to its low germination rate. So, alternative method for germination is needed, which is in vitro embryo zygotic culture. This study objective was to reveal the best medium composition for OG hybrid embryo culture by modificated several oil palm basic medium from oil palm tissue culture protocol and to understand the role of plant growth regulators, activated charcoal, and sucrose on in vitro growth and development of OG hybrid embryo. The design used was Completely Randomized Design (CRD) with 16 replication and 4 embryos for each replicates, while the treatments were: A1 = M129 (control); A2 = M129 + activated charcoal; A3 = M129+ activated charcoal + sucrose; A4 = ½ M129 + activated charcoal + sucrose; A5 = M129 + GA3 ; and A6 = M129 + GA3 + NAA. From the study we found that the best medium for OG hybrid open pollinated clone's embryo culture was M129 with GA3 and NAA (A6). This medium gave percentage of in vitro germination up to 14.06%, and later on the plantlets from this medium gave the best vegetative growth performance such as high number of leaves and primer roots.

Estabelecimento de protocolo para micropropagação de b. Portulacoides (amaranthaceae)

Objetiva-se neste trabalho o estabelecimento de protocolo para cultura de tecidos vegetais (importante ferramenta para conservação de germoplasma e para manutenção da espécie ameaçada) com a espécie Blutaparon portulacoides (A. St.-Hil.) Mears, esta espécie ocorre entre o limite superior da maré alta e as dunas e está ameaçada pela ação antrópica. Para tanto proceder-se-á adequação de metodologia dos ensaios biológicos e de procedimentos bioquímicos e da biotecnologia vegetal. O material vegetal foi coletado em seu habitat natural na faixa litorânea em duas localidades: Maricá (RJ) e Ubatuba (SP), em seguida foi cultivado em casa de vegetação, sendo assim uma fonte de material vegetal para todo o trabalho. Explantes (segmentos do caule e as porções mais apicais) foram coletados e submetidos a experimentos in vitro, eles foram distribuídos em 6 variações do meio de cultura M&S (MURASHIGE; SKOOG, 1962), para cada variação o experimento contou com 30 repetições, foram avaliados o desenvolvimento da zona pilífera e o comprimento médio das raízes principais. Sendo que o tratamento com 40g/L de Sacarose e metade dos macro-nutrientes do meio padrão M&S esteve presente entre todos os grupos que apresentaram melhor resultado dentro do experimento, sendo assim uma alternativa para a micropropagação desta espécie.

In vitro mass multiplication of screw pines (Pandanus spp.) - an important costal bio- resource

An efficient in vitro mass multiplication protocol was developed for selected species of screw pine (Pandanus fascicularis Lam., P. furcatus Roxb.). The protocol could be successfully used for large scale production of planting materials leading to cultivation and making bio-fences or green belts in coastal areas, wetlands for protection and preventing soil erosion. This will facilitate the replenishment of the bio resource and also provide good breeding space for various fauna associated with it. For developing the tissue culture protocol, shoot tips and tillers from the mature plants were used as the explants. Surface sterilization with 0.1 % mercuric chloride for 1 min. Yielded good fraction (65 %) of contamination free explants. Explants inoculated in MS solid medium supplemented with 6-benzylaminopurine (BAP, 2.5 mg/l) and indole-3-acetic acid (IAA, 0.5 mg/l), induced 2–3 shoot buds in 5 weeks. These buds either individually or along with the explant portion, sub-cultured to fresh media containing the same hormonal combinations, resulted induction of 5–6 shoot buds in 4 weeks. Repeated subcultures of shoot buds in the same medium produced 8–10 shoot buds in every 4 weeks. Shoot elongation was achieved (~ 3 cm) by transferring the shoot clusters or individual shoots to basal medium, and the elongated shoots were rooted in vitro or ex vitro. Root induction in shoots (87.4 %) was achieved in MS medium supplemented with 0.1 mg/l indole-3-butyric acid (IBA). Rooted shoots were established in paper/polythene cups filled with fine sand, which subsequently recorded 85 % establishment under the shade-net house with proper irrigation. Plants grown in poly-bags were later successfully established in different field conditions and recorded 100 % survival.

In vitro Culture and Biochemical Attributes of Bergenia ciliata (Haw.) Sternb.

Bergenia ciliata is one of the medicinally important and vulnerable plants of Kashmir Himalaya. A highly reproducible tissue culture protocol of B. ciliata vis-a-vis evaluating its biochemical attributes was studied. The best response for callus formation was obtained on Gamborg’s half strength medium with thidiazuron (2 mg/l) and 1-napthlene acetic acid (2 mg/l) after 6 weeks. Maximum number of 54 shoots were obtained on B5 ½ strength medium + 6-benzylaminopurine 2 mg/l + indole-3-acetic acid 1 mg/l. The complete plants were acclimatised in lab and then transferred to green house conditions with survival rate of 65 %. The in vitro raised, wild plant leaves and callus of B. ciliata were evaluated for biochemical attributes (chlorophyll, carotenoids, total carbohydrates and total soluble protein). Total carbohydrate content was higher in wild plant leaves (15 mg/g) as compared to in vitro plant leaves (11.7 mg/g). Chlorophyll a, b, total chlorophyll and carotenoid content was found to be 0.15, 0.05, 0.24, 0.10 mg/g respectively in wild grown leaves and 0.11, 0.05, 0.15 and 0.06 mg/g respectively in leaves of in vitro raised plants. Carotenoid content, however, was observed significantly (P < 0.005) higher (0.22 mg/g) in callus. Soluble protein content was found to be 142.5 mg/g fresh weight in wild plant leaves but it was 157.5 mg/g fresh weight in in vitro plant leaves. In current study, reproducible in vitro regeneration protocol was developed. It was also observed that in vitro culture had significant influence on expression of primary metabolites vis a vis plant growth.

English

The first objective of this study was to assess and optimize somatic embryo production in a genetically diverse range of cacao genotypes. The primary and secondary somatic embryogenesis response of eight promising cacao clones and a positive control was evaluated using modified versions of standard protocols. The second objective was to optimize the efficiency of primary somatic embryogenesis for a commercially important cacao clone, CCN 51, which has proven to be quite recalcitrant to standard protocols, relative to CCN 10, a clone also included in our analysis. The efficiency of the overall process was assessed by determining the number of somatic embryos produced per starting somatic tissue explant, as well as the quality of embryos (normal vs. abnormal) produced. Donor floral explants were subjected to five tissue culture steps, each 15-25 days in duration. Although all studied genotypes produced primary somatic embryos, most of them originated only from brown or brown-white callus. Overall, flower petals performed better than staminodes, and our best performing genotype yielded an average of 7-10 embryos produced in brown callus explants with embryogenic response during primary somatic embryogenesis procedures. In conclusions our analysis from a pilot in a small-scale are: 1) it is possible to achieve a high production of plants by somatic embryogenesis, although the efficiency is highly genotype-dependent; it is therefore necessary to optimize hormone balance and hormone type, as well as the explant type for each genotype, 2) through the use of secondary somatic embryogenesis, it is possible to increase somatic embryogenesis production at least ten-fold, and 3) the observed response variation between genotypes may reflect differences in endogenous and exogenously-supplied hormones. The importance of adapting the tissue culture protocol to the genotype is discussed. Key words: Somatic embryogenesis, 2,4-D, cellular competence, propagation, abnormalities, ethylene, cacao.

Optimization of in vitro regeneration protocol for a popular Indica rice (Oryza sativa L. cv Swarna)

Though regeneration system in rice has been very well established compare to other crop plants, the fact remains that, most of the indica rice varieties are still recalcitrant for regeneration and genetic transformation. Therefore, refinement of tissue culture protocol for generation of embryogenic calli and regeneration of the fertile plants from a single cell should be a pre requisite event for development of transgenic plants. Here, in this study we reported high frequency robust regeneration protocols for a popular Indica cultivar Swarna.Mature seeds were used as initial material as explants. Highest callus induction % was observed in MSCIMP medium containing 2.0 mg-1 2,4, D + 0.5 mg-1 Kn as phytohormonal combinations. In addition, maximum regeneration was observed in 2.0 mg-l KN + 0.5 mg-l NAA. Regenerated plants were shifted to rooting medium followed by polyhouse for hardening. The callus induction and regeneration reported in this study were well suited for transformation agronomical important genes or functional genomics studies.

Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors.

Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or 137Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p < .001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin-cyclin dependent kinase inhibitors p21Waf1 and p27Kip1, and perturbations in cell cycle progression (p < .001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p < .001) and caspase 8 activation (p < .05) and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Exposure of the tumor cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells.

In vitro Shoot Micro Propagation of Medicinal Applications and Ornamental Value of Cestrum nocturnum

Background/Objectives: Cestrum nocturnum L. a night Blooming jasmine belongs to Solanaceae family widely circulated all over tropical as well as subtropical areas of the World. It is mainly popular for ornamental fragrant flowering and hedge plant but also sometimes for traditional medicinal purpose. Due to strongest smelling characteristics of the plants, it is used in many industries for making Perfumes, essential Oils, Soaps, Candles, Body Oils, etc. The existence of natural plants of economic importance are threatened due to rapid urban development, including industrialization, residential development, educational, commercial etc., reduce the land for cultivation. Hence plant tissue culture protocol may be adapted for production and utilization of economically popular plants, including C. nocturnum involving limited space and short period of time. Methods: Shoot tip explants of naturally grown C. nocturnum were excised sterilized and endued on ‘Murashige and Skoog’ (MS) medium enriched changed concentration of BA, NAA, as well as GA3 singly or in combination. Excised micro shoots were examined for root development on 0.5 MS using IBA, NAA as well as IAA separately. Findings: The highest amount of multiple bud were observed in low concentration of BA (01.50 milligram × l-1), resulted no. of shoot 4.40 as well as 4.20/explant, no. of leaves 15.40 as well as 4.20/explant as well as size of different shoot 5.360 as well as 4.860 cm. The concentration of IBA and IAA were found to be best for root formation in micro shoots (13.20, 6.80 roots/micro shoots) as well as root size (8.39, 5.73 cm) individually. Application: There are many opportunity of plant tissue culture which offer marvelous chances in plant propagation, plant development as well as creation of plants with necessary agronomical features. Finally often hardening plantlets were gradually adjusted to natural condition and acclimatized with 90% success. This established protocol could help plant cell biotechnology, horticulture, medical and industrial sector of the country.

Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties

ABSTRACTCost-effective tissue culture protocols have been established for the commercial multiplication of three banana varieties, ‘Rasthali’ (AAB – Silk), ‘Grand Naine’ (AAA – Cavendish), and ‘Udhayam’ (ABB – Pisang Awak). Reverse osmosis water and 3% (w/v) table sugar were used as the low-cost water and carbon source, respectively. Six different gelling agent treatments were tested: sago alone (T1), Isabgol alone (T2), sago + agar (T3), Isabgol + agar (T4), sago + Isabgol (T5), and agar alone as a control (T6). Full-strength Murashige and Skoog (MS) medium supplemented with 3 mg l–1 6-benzylaminopurine (BAP) and 1 mg l–1 indole-3-acetic acid (IAA) were used for culture initiation and subculturing. Rooting was accomplished on low-cost MS medium containing 1.0 mg l–1 α-napthaleneacetic acid (NAA), 1.0 mg l–1 indole-3-butyric acid (IBA), and 250 mg l–1 activated charcoal. Statistical analysis indicated that sago + Isabgol (T5) produced the maximum number of shoots (10 per explant) in ‘Udhayam’ and ‘Rasthali’, while sago alone (T1) produced the maximum number of shoots (6 per explant) in ‘Grand Naine’. The genetic stability of tissue-cultured banana plantlets produced using these low-cost substitutes was assessed using inter-simple sequence repeat (ISSR) markers. The results indicated that the ISSR profiles of the five treatments and the control (T6) were similar, indicating genetic stability using these cost-effective tissue culture protocols. Reductions in cost over the control (l–1 of MS medium) ranged from 65% to 86%, while the per plant production cost was reduced by 12.5%–20.0%. Adoption of these treatments (T1–T5) as low-cost tissue culture protocols for in vitro propagation would reduce production costs significantly, leading to an expansion of the area planted with tissue-cultured banana, thereby increasing productivity.

Micropropagation, Micromorphological Studies, and In Vitro Flowering in Rungia pectinata L.

A tissue culture protocol was developed for an important medicinal plant Rungia pectinata L. in the present study. Nodal shoots were used as explants and surface-sterilized with 0.1% HgCl2 solution. Murashige and Skoog (MS) medium was used to establish the cultures of R. pectinata. The bud break was reported on MS medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BAP). About 98% response was observed with this media combination and maximum 3.2 shoots per explant with 4.3 cm length were recorded. The shoots were further multiplied using MS medium augmented with 0.5 mg L−1 each of BAP and kinetin (Kin) + 0.1 mg L−1 indole-3 acetic acid (IAA). Maximum 13.2 shoots per explant with 5.2 cm length were observed. All the shoots were rooted (4.9 roots per shoot with 3.5 cm length) on half strength MS medium fortified with 2.0 mg L−1 indole-3 butyric acid (IBA). In vitro flowering was induced from the shoots on half strength MS medium supplemented with same concentrations and combinations of growth regulators used for shoot multiplication under 12/12 hr light/dark photoperiod. The plantlets were hardened in the greenhouse for two months and finally transferred to the field. The foliar micromorphological studies revealed the developmental changes in stomata, vein density, and trichomes during the culture of shoots under in vitro conditions.

The usefulness of apricot gum as an organic additive in grapevine tissue culture media

The growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. In addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. Hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. The proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. The growth parameters of treated samples were measured and compared to gum-free medium. Earlier callus initiations with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. The usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot multiplication and rooting parameters. Due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. Overall, the gum at the rate of 4.0 g/L was found to be a logical concentration with respect to encouraging response in all three species. Owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. However, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media.

A Reliable Homemade Tissue Culture Protocol for &lt;i&gt;Dendrobium&lt;/i&gt; Orchid Cultivation

Murashige and Skoog (MS) medium, a common tissue culture media for orchids which is mostly supplemented with various natural organic substances with high nutritional values can accelerate the growth of plant tissues. Nevertheless, the knowledge of actual compositions of these natural added substances is limited, causing various culture results. In this study, we have investigated the effects of added natural organic nutrient ratios on 2 orchid tissue cultures, Dendrobium, Dendrobium farmeri Paxt. and Dendrobium griffithianum Lindl. Additionally, simple, affordable medium recipes, MS media supplemented with banana were thus economically sterilized by either a simple type steaming vapor boiler or an autoclave for reliability improvement. The results showed that the orchids grew better in medium supplemented with Namwa banana especially at 150 g/L for Dendrobiumfarmeri Paxt. and 75 g/L for Dendrobium griffithianum Lindl. The suitable steaming procedure at 100°C for 60 minutes was adequate for eliminating most pathogens from the media and helped both orchid seedlings cultivated very well. Moreover, the optimized homemade tissue culture protocol provides fast effectiveness, simplicity, consistently high nutritional values, and practically being suitable technique for ready transferring to the community.

High-frequency plant regeneration from transgenic rice expressing Arabidopsis thaliana Bax Inhibitor (AtBI-1) tissue cultures

Abstract Genetic transformation was affected by material of explant, age of callus, and medium of regeneration. Two rice seed cultivars (Ilpum and Baekjinju) and mediums were investigated in this study for enhancing regeneration of transgenic rice expressed AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor. Regeneration rate of Ilpum rice transformant in gelrite of 5 and 8 g were 27.4% and 18.0%, respectively. In Baekjinju, regeneration rate of transformant was 5.4% and 4.3% in 5 and 8 g gelrite, respectively. The highest number of transformant plant in this study was regenerated from Ilpum cultivar on MS medium (30.4%) and was applied for the subsequent experiment. The callus regeneration rate of transformant were 40.7% in callus infection of up-side, it was higher regeneration then in the down-side (3.9%). The regeneration rate of callus of 25 days and 35 days were 14.7% and 38.6%, respectively. The most important application of this work is in genetic transformation of rice, particularly for improvement transgenic plant tissue culture protocol with high frequency of plant regeneration.Keywords Agrobacterium -mediated, Transformation, Rice, Regeneration, AtBI-1

Disinfection procedures for in vitro propagation of Anthurium

Abstract Disinfection of plant material is the most important step of the tissue culture protocol. In this process, an attempt is made to eliminate microbial contaminants from the surface and interior of plant material, thus giving the explant a fighting chance at survival in vitro. Initial cultures of Anthurium species and cultivars, which are usually established from ex vitro material grown in a greenhouse, pots or in the field, easily contaminate the in vitro milieu. This review highlights the differences in disinfection protocols that exist for different species or cultivars of Anthurium. The protocol needs to be adjusted based on the material used: spadices, spathes, seeds, leaves, or roots. Regrettably, most of the currently published protocols, derived from a literature that spans over 100 published papers, have numerous weaknesses and flaws in the information provided pertaining to disinfection and infection levels. Advice for future Anthurium researchers should thus be followed cautiously.

A reproducible method for the expansion of mouse CD8 + T lymphocytes

Murine adoptive CD8+ T-cell immunotherapy studies require the generation of large numbers of high viability CD8+ cells. Here we report a tissue culture protocol for the reliable expansion of CD8+ T-cells derived from murine spleen to give a 20-fold expansion after 4days in culture. The cells were transfected with an mRNA GFP construct and transferred into NOD mice. GFP positive cells could be detected 7days after transfer thus confirming that the cells survive and are functional for up to 1week.

Immunomodulatory potential of shatavarins produced from Asparagus racemosus tissue cultures.

Medicinal properties of Asparagus racemosus (vernacular name: Shatavari) are attributed to its steroidal saponins called shatavarins. This plant is facing the threat of being endangered due to several developmental, seasonal constrains and malpractices involved in its collection and storage. To support its conservation, a tissue culture protocol is standardized which produces 20 fold higher levels of shatavarin. Here we evaluate the bioactivity and immunomodulatory potential of in vitro produced shatavarins from cell cultures of AR using human peripheral blood lymphocytes. In vitro produced shatavarin stimulated immune cell proliferation and IgG secretion in a dose dependent manner. It stimulated interleukin (IL)-12 production and inhibited production of IL-6. It also had strong modulatory effects on Th1/Th2 cytokine profile, indicating its potential application for immunotherapies where Th1/Th2 balance is envisaged. Our study demonstrating the bioactivity of tissue cultured AR extracts supports further in vivo evaluation of its immunomodulatory efficacy.

Production of virus-free garlic (Allium sativum L.) through meristem tip culture after solar or hot air treatment of cloves

SummaryGarlic (Allium sativum L.) is a clonally propagated crop that can be infected by many virus species which tend to accumulate year-after-year, leading to yield reductions and varietal degeneration. Therefore, the production and use of virus-free planting material is vital to sustain productivity. In this study, Onion yellow dwarf virus (OYDV; Potyvirus), Garlic common latent virus (GarCLV; Carlavirus), Shallot latent virus (SLV; Carlavirus), and Garlic virus X (GarV-X; Allexivirus) were detected in cloves of the commercial garlic cultivar, ‘G-1’ (‘Jamuna Safed’) by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). DAC-ELISA was performed using specific antisera against each virus species, and confirmed through reverse transcription-polymerase chain reactions (RT-PCR) using virus or genus-specific oligonucleotide primers. To obtain virus-free plants of ‘G-1’, cloves were subjected to solar heat treatment for 5, 10, or 15 d, or to hot air treatment (at 37°C, 40°C, or 42°C for 7, 14, or 21 d) combined with meristem isolation and in vitro culture, then indexed for all viruses using DAC-ELISA and RT-PCR. Meristems from solar heat-treated or hot air-treated cloves regenerated after 90 d and 45 d in culture, respectively. Solar heat treatment for 15 d resulted in 17 - 18% regeneration, and all were free from GarCLV, SLV, and OYDV. However, when solar heat-treated for 10 d, only 66% of the 17 - 18% regenerated plants were free from all viruses, including GarV-X. Hot air treatment at 37°C or 40°C eliminated GarCLV, SLV, and OYDV in 20 - 38% and 42 - 62% of regenerated plants, respectively, for the different durations of exposure. However, GarV-X could not be eliminated from any of the regenerated plants by hot air treatment, irrespective of the duration of exposure. Meristems from cloves that had been hot air-treated at 42°C resulted in 50 - 66% regeneration, of which 53 - 57% were free from GarCLV, SLV, and OYDV when treated for 7 d or 14 d, and 29% were free from all viruses when treated for 21 d. For most treatments, an increase in the duration of exposure to heat resulted in reduced regeneration, but increased the rate of recovery of virus-free plants. To our knowledge, this is the first study to report the use of solar treatment for virus elimination in garlic and the development of an efficient tissue culture protocol for the production of virus-free garlic plants using different thermotherapy treatments coupled with meristem tip culture.

MICROPROPAGATION OF SOME NEW PEACH ROOTSTOCKS

Two experiments were conducted in 2011 and 2012 seasons on two newly introduced peach rootstocks, namely Garnem and Cadaman (which are widely used in Europe) in addition to the wellknown Nemaguard peach rootstock for comparison. The aim of this study is to optimize tissue culture protocol for propagation of the rootstocks under study. Results indicated that Nodal cuttings were better than shoot-tips in regard to aseptic culture (without contamination after 10 days) during establishment stage. However after one month, the three rootstocks gave rise to 100% survival percentage. Meanwhile number of leaves on Nemaguard rootstock was significantly the greatest. During proliferation stage, Garnem produced the highest shoot number, while Cadaman rootstock produced significantly the lowest shoot number. Regarding the effect of BA supplementation, it was found that BA at 2.0 ml/l resulted in the longest shoots. During rooting stage, 2.0 mg/l GA3 significantly gave the longest shoots. At root formation, there were no significant differences between the 3 rootstocks concerning root number , rooting percentage and root length. 0.1 mg/l IBA incorporated in half MS medium strength gave the highest significant values for the same traits. During acclimatization stage, Garnem rootstock achieved the highest significant survival ratio, while half strength Murashige and skoog (MS) medium resulted in better survival ratio, plant height and number of leaves. Sand + peat moss potting mixture achieved better survival ratio, plant height and number of leaves than Sand + Vermiculite. Protein banding patterns profile data proved the presence of band with molecular weight 20.094 k Da in Garnem rootstock, which may be responsible for high number of shoots and survival ratio. This band was absent in other rootstocks.

Tissue culture and aromatic profile in Salvia dolomitica Codd

In this study, we developed tissue culture protocol of Salvia dolomitica, a particularly fragrant sage and we evaluated the yield and the profile of essential oil (EO) produced from plants grown in vivo compared to in vitro plant material cultured in two light intensity growth conditions. Plant extracts secreted from glandular trichomes are, in Lamiacea, a valuable source of biologically active volatile compounds. Microscopical observations permitted to describe, for the first time, secretory structures from S. dolomitica leaves developed in vitro showing morphological identity to in vivo glands. The yield of EO obtained from light-stressed in vitro plants was higher than which obtained from the non-stressed in vitro plants and threefold higher that in vivo plants. Phytochemical analysis of EOs and volatile compounds showed quali-quantitative differences between S. dolomitica in vitro grown plantlets and those from open field. α-Pinene, β-phellandrene and borneol accumulated predominantly in micropropagated plants while β-caryophyllene, α-guaiene, δ-cadinene and valencene in in vivo material. Both EO and SPME analysis had the same trend: the percentage of total sesquiterpenes decreased strongly in vitro, on the contrary the total monoterpenes are abundant. The effect of the high light treatment on aromatic profile demonstrating that this stress can affect the EO composition and this can be exploited by researchers to address the accumulation of valuable amounts of EO.

Plant biotechnology: use of tissue culture techniques in species Boerhavia paniculata Rich and Crinum americanum L as alternative for the production of new drugs in vitro

Background Crinum americanum L. popularly known as lily is an ornamental plant that belongs to the family Amaryllidaceae alkaloids presenting with anti-inflammatory, antimicrobial and inhibition acetilcolinisterase. The species Boerhavia paniculata Rich is a plant from the family Nyctaginaceae popularly known as “Pega Pinto” that produces isoflavones that have action antiinflmatorias urinary tract and with its concentration in the roots. Aimed at the production and accumulation of these substances in vitro, this work aimed to study the induction of roots of the species Boerhavia paniculata Rich and callus induction of the species Crinum americanum L through the techniques of tissue culture protocols developed cell culture for production of these metabolites and evaluate the qualitative HPTLC by these cells [1,2,5].