Gamborg B5 Research Articles

Efficient production and enhanced accumulation of Valerenic acid in Valeriana officinalis: Early identification of high-performing hairy root clones and Pioneering use of hydrogen peroxide as an elicitor

Valeriana officinalis (valerian) roots and rhizomes possess a long history of medicinal use due to their sedative, antiepileptic, and anticonvulsant properties. Valerenic acid, a bioactive sesquiterpene with therapeutic potential, is present in limited quantities within these tissues. This study explores the application of hairy root cultures for enhanced valerenic acid production. Hairy root induction was attempted on valerian leaves and petioles using three Rhizobium rhizogenes strains (ATCC15834, A4, and MSU440) across three culture media (Murashige and Skoog (MS), Gamborg's B5 (B5), and Schenk and Hildebrandt (SH)). A non-destructive imaging system and periodic analyses were employed to identify superior hairy root clones exhibiting increased branching frequency. Leaf explants co-cultured with R. rhizogenes strain ATCC15834 yielded the most promising clones, characterized by the highest dry weight (1.03 mg) and valerenic acid content (0.384 mg/g dry weight) when grown in a half-strength SH liquid medium. Following strain and media optimization, the impact of 50 mM hydrogen peroxide as an elicitor on valerenic acid production was investigated. This treatment resulted in a significant 1.76-fold increase in valerenic acid accumulation compared to the control group at the first day post-treatment. This approach presents a valuable strategy for the early identification of high-yielding hairy root lines. Moreover, the utilization of hydrogen peroxide, a safe and cost-effective elicitor, offers a rapid method for enhancing valerenic acid production in the selected superior clone. This study establishes a promising platform for the sustainable production of valuable plant compounds within both research and industrial settings.

Ölmez Otu (Helichrysum italicum (ROTH) G. DON) Bitkisinin in vitro Mikroçoğaltımı

Immortelle grass (Helichrysum italicum (Roth) G. Don), which spreads in the Southern Marmara and Aegean regions, can be grown in arid and semi-arid regions. In addition, due to its rich essential oil and secondary metabolite content, it has an important place in modern medicine and cosmetics, including traditional treatment methods. Although the propagation of plants by shoot regeneration in vitro has been achieved in many plant species, studies on tissue culture in immortelle grass are limited. This study aims to optimize the tissue culture study in immortelle grass and provide a basis for the next in vitro, molecular, and secondary metabolite studies. In addition, it promotes the plant by optimizing the healthy and disease-free seedling production method for cultural agriculture in the region. Three different (15%, 25%, and 35%) NaOCl concentrations were tested for 10 and 20 minutes during the sterilization phase of the explants. The most successful result was obtained in the medium containing 35% NaOCl for 10 minutes. Sterilized explants were transferred to MS and Gamborg B5 nutrient media containing BAP, GA, and NAA plant growth regulators for shoot regeneration. The best regeneration in explants was obtained in MS medium containing 0.5 mg L-1 BAP, 1 mg L-1 GA, and 0.2 mg L-1 NAA. No growth was observed in trials containing Gamborg B5, and vitrification and darkening occurred in the explants. After four weeks, the shoots reaching a length of 3 cm were taken into MS and ½MS medium containing 0 MS, 0.5 mg L-1 IBA, 1 mg L-1 IBA, 1.5 mg L-1, and 2 mg L-1 IBA as a rooting medium. 100% rooting was observed in all prepared media within four weeks. As a result of micropropagation studies, the rooted plants were transferred to the acclimatization stage within three months and then moved to the pots in the greenhouse and to the field one month later.

Influence of Explants Type, Nodal Positions and Two Cytokinins on Cassava (Manihot esculenta, Crantz) In vitro Organogenesis

Cassava In vitro regeneration is essentially influenced by exogen factors as well as explants nature. This work aimed to evaluate explants type and positions influence, phytohormone on cassava plantlets regeneration in modified MS media. The apex and nodes of the position 3, 5, and 7 were initiated on Murashige and Skoog (MS) based media supplemented with Gamborg B5 vitamins and growth regulators such as naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (ZEAT) in different concentrations. The results indicated a significant difference (p=0.0001) among explants sprouted regarding the nodes position and the cultivar genotype during the time of culture. The highest rate of regenerated plantlets was found with nodes of position 5 (95.33%) followed respectively by nodes of the position 7 (93.33%), nodes of the position 3 (89.33%), and the apex (76%). Regarding the growth regulators, NAA (0.5 mg/l) + BAP (0.5 mg/l) combination was favorable for height growth (26.2cm), and leaves development (7.84 cm) in most of cultivars, whereas NAA (0.5 mg/l) + ZEAT (0.5 mg/l) combination was more favorable to rhizogenesis in overall cultivars. Among the cultivars, the best performance in stem height (26.2 cm), in leaf length (7.84 cm), and roots number (4.64) were observed with INA 1er cultivar. The cultivars RB89509, BEN 86052, and DOKU were less grown on MS supplemented with 0.5 mg/l of BAP or ZEAT. These findings could contribute of using nodes of the positions 5, 7 as potential explants and Zeatin for cassava plantlets rhizogenesis.

Use of nurse endosperm for the culture of haploid embryos produced by durum wheat x maize crosses

The use of doubled-haploids in plant breeding programs enables accelerating the release of new varieties adapted to climate change. The durum wheat x maize crosses technique is a method of choice for producing durum wheat haploid plants. The haploid embryos produced by this method develop without albumen and their survival is ensured by post-pollination hormonal treatments. In this study, nine post-pollination treatments with 2,4-Dichlorophenoxyacetic acid (2,4-D), Picloram and Dicamba at the concentrations of 10, 50 and 100 mg.L-1 were applied to 7 durum wheat genotypes. The effects of genotype and post-pollination treatment on durum wheat haploid embryos produced by durum wheat x maize crosses and the use of the endosperm nursing technique for haploid plantlets regeneration were investigated. The haploid induction parameters varied with the durum wheat genotypes as well as the post-pollination treatments. The phenomenon of polyembryony resulting from durum wheat x maize crosses is reported for the first time in this article. The durum wheat genotypes showed different abilities to produce monoembryo and polyembryos. The post-pollination treatments with 2,4-D (10 mg.L-1) and Picloram (10 and 100 mg.L-1) gave a higher embryo formation frequency than the treatments with Dicamba. The embryo conversion to plantlet was greatly improved, especially in recalcitrant genotypes using the durum wheat endosperm as supplemental nourishment in combination with the Gamborg B5 regeneration medium.
 Keywords: Durum wheat; Haploid embryo; Maize; Nurse Endosperm Technique; Polyembryony

“In Vitro” Ovule Culture to Improve Genetic Variability in Hydrangea macrophylla

In flowering plants, such as Hydrangea macrophylla, the main breeding objective is to increase genetic variability in ornamental traits. This study investigates in vitro techniques, through ovule culture, to overcome the hybridization barriers and increase the efficiency of crossing in Hydrangea macrophylla in which breeding has been hampered by a fairly long breeding cycle and lack of information about its genetic resources. Two different types of media were compared, Gamborg B5 and Murashige and Skoog basal salts, to verify the germination rate of immature ovules in different intraspecific crosses. The germination rate and viability of the seedlings were influenced by the parental genotypes in the different combinations of crossing, highlighting, in some cases, the poor compatibility between some of them. The crossing combination “Parental A × Parental B”, showed the highest germinated ovules percentage (78.3%). The media used seem to less affect the ovule germination while mainly influencing the development and growth of the young seedlings and in particular the number of leaves, the branching attitude, and root length, with the Gamborg medium determining up to a 30% increase, compared to MS medium. In addition, we tested the effectiveness of using SSR markers to assess the parentage of the putative hybrids even though only three out of twelve SSR markers showed allelism. Although the number of SSR markers was low, they were allowed to profile the parentage according to Mendelian laws.

Study on induction of Allium sativum L. hairy roots with antibacterial activity against Vibrio

Allium sativum L. has long been used to improve the health of humans and other species due to its antibacterial activity. Applying biotechnology to create a stable and large-scale production process for Allium sativum L., medicinal herbs by hairy root culture technology are essential. However, Allium sativum L. is a monocotyledonous plant, so its ability to induce hairy roots is lower than that of dicotyledonous plants, which requires more research investment. In this study, we succeeded in inducing hairy roots in monocotyledonous plants Allium sativum L, by evaluating parameters impacting hairy root induction such as plant organs, acetosyringone concentration, infection time and co-culture time. The results showed that the optimal procedure for induction of Allium sativum L. hairy roots by Agrobacterium rhizogenes ATCC 15834 was as follows: 10 day old in vitro Allium sativum L. sprouts were wound and soaked in 100 μM acetosyringone solution for 10 min, infection time was 5 minutes, co-culture time was 48 hours cultured on B5 medium (Gamborg B5 medium) having the induction rate of hairy roots reaching 64.4% after 14 days of culture. Allium sativum L. hairy roots had good antibacterial effect against all three strains of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus because the MBC/MIC ratios are all less than or equal to 2. Especially, for V. cholerae (MIC = 1.84 mg/ml, MBC = 2.11 mg/ml), hairy roots have antibacterial activity equivalent to tetracycline antibiotics and better than non-infectious roots.

Sugar beet cells cellular and extracellular events taking place in response to drought and salinity

Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To understand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expression of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liquid medium (PG0). Severe stress altered the intracellular concentrations of the most measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence categories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcription factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracellular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and the affected expression of cellular and extracellular proteins in sugar beet cells.

Plant regeneration from protoplasts of Pastinaca sativa L. via somatic embryogenesis

In the present study we report the development of an effective and relatively efficient protocol for protoplast-to-plant regeneration of parsnip (Pastinaca sativa L.) via indirect somatic embryogenesis. The regenerative potential of three open-pollinated and four hybrid cultivars was assessed. The protoplast isolation efficiency after digestion of source material in an enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3.6 × 106 of cells per g of fresh mass. Protoplasts embedded in an alginate matrix and cultured in parsnip protoplast culture medium with phytosulfokine-α and putrescine reconstructed their cell wall and re-entered mitotic divisions. After the release from alginate, microcallus proliferated continuously on Gamborg B5 medium with vitamins supplemented with 100 nM of phytosulfokine-α. Indirect somatic embryogenesis occurred during the callus culture of cultivar ‘Półdługi biały’. The regenerated and acclimatized plants were morphologically similar to their donors and displayed no variation in the ploidy level.

Cretan Dittany (Origanum dictamnus L.), a Valuable Local Endemic Plant: In Vitro Regeneration Potential of Different Type of Explants for Conservation and Sustainable Exploitation

Origanum dictamnus L. is a medicinal local endemic to the Island of Crete, Greece. Its propagation through biotechnological tissue culture techniques is essential due to its augmented multi-industrial sector demand. For direct organogenesis, among different culture media variants (MS, Gamborg B5), and cytokinins [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyl adenine (2-iP)], the MS + added with BA (2.2 μM) was the most effective treatment for shoots and roots formation. For indirect organogenesis, all explant types (leaves, petioles, roots) showed a 100% callusing rate after 2 months in all media variants tested; ODK1: 20 μM thidiazuron (TDZ) + 5 μM indole-3-butyric acid (IBA) or ODK2: 0.5 μM kinetin + 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D). The leaves and petiole explants assured a low rate of shoot regeneration (20%) in ODK1. Afterwards, leaf-, petiole-and root-callus derived from both media were transferred to four new media plant growth regulators-free or with BA + IBA + gibberellic acid (GA3). After 10 months from callus transferring, the petiole callus gave rise to roots (20-75%) while the leaf callus exhibited 10-30% shoot or 30% root regeneration. In this study, indirect organogenesis of O. dictamnus was carried out for the first time, thus various organs can be used for plant regeneration, and the developed protocol may be applicable in the horticulture industry.

Prediction of In vitro organogenesis of Bacopa monnieri using artificial neural networks and regression models

This study was conducted to determine if artificial neural networks (ANN) can be used to accurately predict in vitro organogenesis of Bacopa monnieri compared with statistical regression. Prediction models were developed for shoot and root organogenesis (outputs) on two culture media (Murashige and Skoog and Gamborg B5) affected by two explant types (leaf and node) and two cytokinins (6-Benzylaminopurine and Thidiazuron at 1.0, 5.0, and 10.0 μM levels) with and without the addition of auxin (1-Naphthaleneacetic acid 0.1 μM) (inputs). Categorical data were encoded in numeric form using one-hot encoding technique. Backpropagation (BP) and Kalman filter (KF) learning algorithms were used to develop nonparametric models between inputs and outputs. Correlations between predicted and observed outputs (validation dataset) were similar in both ANN-BP (R values = 0.77, 0.71, 0.68, and 0.48), and ANN-KF (R values = 0.79, 0.68, 0.75, and 0.49), and were higher than regression (R values = 0.13, 0.48, 0.39, and 0.37) models for shoots and roots from leaf and node explants, respectively. Because ANN models have the ability to interpolate from unseen data, they could be used as an effective tool in accurately predicting the in vitro growth kinetics of Bacopa cultures.

Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha‐ l‐iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4‐ d + 20.5 g/l KNO3) resulting in 3.4 fold eGFP secretion when compared to the non‐induced control. These optimized conditions were applied to the IDUA‐expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150‐fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non‐induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

In vitro direct organogenesis of the Cretan dittany (Origanum dictamnus L.), an important threatened Greek endemic species

Dittany of Crete (Origanum dictamnus L.) is a threatened medicinal-aromatic plant of Lamiaceae family which is a local endemic to the Island of Crete, Greece. Its high culinary use and increasing demand in the pharma, perfumery, cosmetic and food industry along with its overexploitation from its natural habitat has threatened this species and has necessitated its large-scale production for industrial exploitations using advanced technologies. Micropropagation is considered a good tool for ex situ conservation of endangered species with reduced populations in the wild, low germination rates and low seed production. In this study, moderate germination percentages (40-41.38%) were exhibited for seeds after 40 days of culture at 21-23 oC in MS medium regardless of photoperiod regime (16h light/ 8h dark, 24h darkness), without significance difference. In the proliferation and rooting stage, three basal culture media (MS, WPM, Gamborg B5) were tested in combination with two concentrations of 6-benzyladenine (BA) (1.1, 2.2 μM) and indole-3-butyric acid (IBA) (0.125, 0.25 μM), all supplemented with 0.3 μM gibberellic acid (GA3), 20 g L-1 sucrose and 6 g L-1 Plant Agar (pH: 5.8). The results showed that the MS medium + 2.2 μM BA + 0.25 μM IBA was the most effective treatment for micropropagation of shoot nodal explants in a single stage within a 30-day culture period, exhibiting 85% shoot formation, 1.8 new shoots/ explant 2.8 cm long with a 3.2 proliferation rate, 100% rooting, 16.5 roots/ rooted explant 2.1 cm long. Rooted plants obtained in vitro from MS medium enriched with 2.2 μM BA + 0.25 μM IBA gave 100% ex vitro survival rate on a peat: perlite (1:1 v/v) substrate mixture after 2 months in the greenhouse mist. In this study, an efficient in vitro propagation system of O. dictamnus is described for the first time through optimization of direct organogenesis stages (seed germination, proliferation, rooting, ex vitro acclimatization), as a means to facilitate domestication procedure, ex-situ conservation and future sustainable exploitation strategies, thus promoting wider usability of this local endemic with significant commercial potential.

Особенности индуцированного морфогенеза и регенерации растений из соматических тканей люцерны сортов отечественной селекции в культуре in vitro

The authors have studied Morphogenesis and regeneration features of domestic breeding cultivars of alfalfa and conditions for obtaining regenerant plants from the morphogenic callus tissues. As a result of the study, the efficient sterilization protocol of alfalfa seeds was established with the use of sterilizing agents as 70% ethanol and 10% sodium hypochlorite which resulted in the production of 100% aseptically viable seedlings for the further in vitro studies. Callus initiation from cotyledons and hypocotyls was implemented on a Gamborg B5 nutrient medium containing 2,4-dichlorophenoacetic acid (2,4-D), kinetin in a concentration of 5,0 mg/l, and naphthaleneacetic acid (NAA) of 0,1 mg/l. Induction of morphogenic callus was achieved on a Gamborg B5 medium with 0,2 mg/l 6-benzylaminopurine (BAP). Further shoot initiation occurred on a hormone-free Gamborg B5 medium with the addition of Gamborg B5 vitamins, and on a half-strength medium with the addition of 0,2mg/l kinetin. It was found that established method of obtaining regenerated plants from the callus cultures of Medicago Varia proved itself effective.

Breaking seed dormancy and regeneration in Cannabis sativa L.

Cannabis sativa L. is an important medicinal plant species that grow under natural conditions and has been legalized in 20 out of 81 Turkish provinces. The female inflorescence is a highly branched compound raceme with indeterminate habit of growth. This results in different maturing of seeds on the inflorescence and induce physiological dormancy on seeds. The study aimed to improve seed germination percentage using various concentrations of GA₃, GA₃ + BAP, germination on water and water solidified with agar, MS or Gamborg B5 medium. The results showed that the best seed germination was noted on Gamborg B5 medium. Different explants were used to regenerated plantlets on Gamborg B5 medium. All explants were suitable for callus regeneration variably. Only the stem nodes of Samsun Vezirköprü were suitable to induce shoots and plantlets. These plantlets were acclimatized on clay loam soils and transferred to field condition during October 2020, where they acclimatized successfully. These studies provide an effective insight into the mechanism seed dormancy in C. sativa. Further studies using other plant growth regulator concentrations will improve shoot regeneration and aid in utilizing the methods for breeding purpose.

Study of the effects of Lemna minor extracts on human immune cell populations.

Lemna minor is a plant with a huge repertoire of secondary metabolites. The literature indicates that extracts of Lemna minor have antioxidant, antiradical, immunomodulatory and anti-inflammatory properties. The objective of the present study was to find a suitable technique to extract active compounds from this plant and verify whether these extracts have immunomodulatory activity. We grew L. minor on a standard medium with Gamborg B5 and vitamins. We extracted compounds from the plant by maceration and decoction. The phytochemical profile of the extracts was characterized by chromatography, spectrophotometry, and spectroscopy. The extracts were tested on cultures of mononuclear cells from four human subjects. These cells were pulsed with carboxyfluorescein succinimidyl ester, grown in triplicate in standard culture medium without (control) and with increasing concentrations of Lemna extracts. Flow cytometry was used to evaluate cell death and proliferation of the total mononuclear cell population and of CD4+, CD8+, B cell and monocyte populations. The Lemna extracts were not cytotoxic and did not cause cell necrosis or apoptosis in immune cells. At low concentrations, they induced very limited proliferation of CD4+ cells within 48 hours. At high concentrations, they induced proliferation of CD8+ cells and B lymphocytes within 48 hours. Unfortunately, we failed to confirm any immunomodulatory activity of Lemna extracts. Growth and death rates of human immune cells were not significantly affected by adding Lemna extracts to the culture medium.

Repression of Carotenoid Accumulation by Nitrogen and NH4+ Supply in Carrot Callus Cells In Vitro.

The effect of mineral nutrition on the accumulation of the main health beneficial compounds in carrots, the carotenoid pigments, remains ambiguous; here, a model-based approach was applied to reveal which compounds are responsible for the variation in carotenoid content in carrot cells in vitro. For this purpose, carotenoid-rich callus was cultured on either BI (modified Gamborg B5) or R (modified Murashige and Skoog MS) mineral media or on modified media obtained by exchanging compounds between BI and R. Callus growing on the BI medium had abundant carotene crystals in the cells and a dark orange color in contrast to pale orange callus with sparse crystals on the R medium. The carotenoid content, determined by HPLC and spectrophotometrically after two months of culture, was 5.3 higher on the BI medium. The replacement of media components revealed that only the N concentration and the NO3:NH4 ratio affected carotenoid accumulation. Either the increase of N amount above 27 mM or decrease of NO3:NH4 ratio below 12 resulted in the repression of carotenoid accumulation. An adverse effect of the increased NH4+ level on callus growth was additionally found. Somatic embryos were formed regardless of the level of N supplied. Changes to other media components, i.e., macroelements other than N, microelements, vitamins, growth regulators, and sucrose had no effect on callus growth and carotenoid accumulation. The results obtained from this model system expand the range of factors, such as N availability, composition of N salts, and ratio of nitrate to ammonium N form, that may affect the regulation of carotenoid metabolism.

Metabolic profiling, in vitro propagation, and genetic assessment of the endangered rare plant Anarrhinum pubescens

BackgroundAnarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. ResultsTwenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard’s similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. ConclusionsThis study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.

The effect of medium formulation and BAP concentration of growth and development of Aquilaria malaccensis Lam. shoot in vitro

Tissue culture of agarwood tree (Aquilaria malaccensis Lam) was done to produce a large number of transplants in the laboratory. The compositions of medium culture are commonly modified to obtain the best growth of explants to regenerate into plantlets. Our study aimed to evaluate and compare different medium formulations supplemented with BAP on agarwood shoot culture growth. In this study we used MS, NN, B5, AR and WP medium containing 0 (control treatment), 0.25, 0.5, 1.0 and 2.0 mg/L BAP. The shoot tips were cultured for 16 weeks in the treatment media. Height of shoots, number of leaves, number of adventive shoots, and number of roots was recorded every week to determine the growth. The results showed that after 16 weeks of culture, the best medium for shoot height was NN medium with BAP fortification (3.54cm) for number of leaves and adventive shoots proliferation was Gamborg B5 medium fortified with 0.5 mg/L BAP (21.63 leaves and 8,38 shoots), while NN medium without BAP was best for rooting.

Preculture in an enriched nutrient medium greatly enhances the Agrobacterium-mediated transformation efficiency in Arabidopsis T87 cultured cells.

The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg's B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5µM benzyl adenine (BA) and 0.1µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5µM) and NAA (0.1µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2mM indole-3-butyric acid (IBA) for 5min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.