BA In Medium Research Articles

Effects of different explant and growth regulator combinations on in vitro propagation of Poinsettia (Euphorbia pulcherrima Willd.)

The aim of the research is to develop a suitable micropropagation protocol for the Poinsettia (Euphorbia pulcherrima Willd.) plant, which is used as an ornamental plant. In the sterilization step, mercuric chloride together with sodium hypochlorite is approved as the best option. Thus, contamination of explants is eliminated. At the shoot propagation stage, BA is absolutely necessary as all doses of BA increased shoot propagation compared to the control. The highest number of shoots per explant (2.62 shoots/explant) and the longest shoots (2.16 cm) were obtained from 1.0 mg.L-1 BA medium. The highest number of leaves (20.41 leaves/explant) was obtained from 2.0 mg.L-1 BA medium. Kinetin was also effective when adding different concentrations to the culture medium compared to the control. In general, BA is more effective than Kinetin in these parameters. Kinetin, on the other hand, performed much better than BA in terms of shoot number. On the other hand, the highest rooting percentage (58.2%) was obtained from the addition of 0.3 mg.L-1 NAA. The highest root number (5.10 roots/explant) was obtained by adding 0.1 mg.L-1 NAA. Good performance was found in the acclimatization phase with plantlets transferred to the soil with a high survival rate reaching 100%. Most of the plantlets started growing well. The plantlets grew well and did not show morphological abnormalities. In addition, a successful plant regeneration was achieved by adding 1.0 mg.L-1 BA and 0.5 mg.L-1 NAA on the callus produced in leaf disc explants and a very good organogenesis was determined in terms of roots and shoots.

The Formula media <i>in vitro</i> Propagation and Conservation of <i>Ludwigia</i> sp.

The aquatic plant "Red Malang” (Ludwigia sp.) has a fairly high economic value as an ornamental aquatic plant, so it has the potential to be developed. The growth of in vitro cultures in culture bottles is high-speed, so it is necessary to find a formula media to inhibit growth so that the frequency of subcultures is reduced. The current research aims to produce a formula media for shoot multiplication and in vitro culture conservation. The research was carried out at the ICABIOGRAD tissue culture laboratory from April 2020 to June 2021. Research activities included plant propagation, conservation, and regeneration after conservation. Plant material was using in the form of a culture collection in the ICABIOGRAD tissue culture laboratory, treatment media for propagation were BA (0; 0.1; 0.3; 0.5; 0.7 and 0.9 mg/L) + thidiazuron (TDZ) (0 and 0.1mg/L). For conservation were MS + BA medium (0 and 0.1 mg/L) + paclobutrazol (0; 0.1; 0.3; 0.5; 0.7 mg/L) and for shoot regeneration after conservation using MS medium without Plant Growth Regulator (PGR). Data analysis using the Anova SAS version 9.0 test program. Further test using DMRT test with alpha level 5%. There was no difference in the mean value between levels of TDZ treatment on the number of shoots and leaves. The difference in the mean value between levels of TDZ treatment was very significant on shoot height, the number of roots, and root length. BA treatment with a concentration of 0.7 mg/L is better because it gives higher results for each observation variable. For conservation, treatment with paclobutrazol 0.5 mg/L inhibited shoot and leaf count, and 0.3 mg/L inhibited shoot formation. Cultures stored for six months grew normally after being regenerated. The highest shoots and the highest number of leaves were obtained from the treatment of paclobutrazol without BA. This study indicated that the propagation media of aquatic plants Ludwigia sp. did not require high concentrations of BA. Cultures could be stored for over six months using paclobutrazol with 0.3-0.6 mg/L.

The effect of 2,4-D, BA and Thidiazuron on somatic embryo induction of liberica coffee of Tungkal Composite from Jambi

The Tungkal Composite of liberica coffee rejuvenation program at Jambi Province is urgently needed due to the latest status of the plants are old and showing diseased attacked. To support the rejuvenation, the provision of seedlings could be done by tissue culture approach through somatic embryogenesis. This research aimed to study the effect of 2,4-D, BA and TDZ on callus formation and embryo induction from liberica coffee leaf explants. Explants used were leaf sections with or without veins. Explants were grown on Murashige Skoog (MS) basal medium with 2,4-D + BA and 2,4-D + TDZ. A completely randomized design was employed in the trial, and each experimental unit was replicated three times. Results showed that callus could be formed on both types of explants, but leaf explants with veins could produce callus faster. The use of BA in medium could induce callus formation faster than TDZ. The higher the concentration of 2,4-D, BA and TDZ, the slower callus initiation. The addition of TDZ in the medium resulted in mostly callus, while callus from medium with BA showed a friable structure. Globular somatic embryos were formed in all treatments with varying amounts. TDZ can induce more globular somatic embryo than BA.

Establishment of an efficient transformation method of garden stock (Matthiola incana) using a callus formation chemical inducer.

Matthiola incana is an important floricultural plant that blooms from winter to spring, and had been desired to be established a transformation system. This study successfully obtained stable transgenic plants from M. incana. We used Agrobacterium tumefaciens harboring a binary vector containing the β-glucuronidase gene (GUS) under the control of cauliflower mosaic virus 35S promoter to evaluate the transformation frequency of M. incana. We observed that cocultivation with the A. tumefaciens strain GV3101 for 5 days effectively enhanced the infection frequency, assessed through a transient GUS expression area in the seedling. Furthermore, the addition of 100 µM acetosyringone was necessary for Agrobacterium infection. However, we could not obtain transgenic plants on a shoot formation medium supplemented with 1 mg l-1 6-benzyladenine (BA). For callus formation from the leaf sections, a medium supplemented with 1-50 µM fipexide (FPX), a novel callus induction chemical, was employed. Then, the callus formation was observed after 2 weeks, and an earlier response was detected than that in the BA medium (4-6 weeks). Results also showed that cultivation in a selection medium supplemented with 12.5 µM FPX obtained hygromycin-resistant calli. Thus, this protocol achieved a 0.7% transformation frequency. Similarly, progenies from one transgenic line were observed on the basis of GUS stains on their leaves, revealing that the transgenes were also inherited stably. Hence, FPX is considered a breakthrough for establishing the transformation protocol of M. incana, and its use is proposed in recalcitrant plants.

Adventitious Shoot Regeneration from Leaf Explants in Sinningia Hybrida 'Isa's Murmur'.

As a valuable ornamental plant, Sinningia hybrida ‘Isa’s Murmur’ (S. hybrida) has genetic flower diversity, which has great potential to develop different flower characters in the horticultural market. The present study focuses on establishing a practical approach for the sustainable propagation of S. hybrida. Compared with aseptic seeding leaves explants, field-grown leaves explants are more suitable for adventitious shoot regeneration. Adding 0.1 mg L−1 NAA and 2.0 mg L−1 TDZ could obtain the highest adventitious shoot proliferation coefficient (24.5), and the induction rate was 91.7%. The shoot proliferation coefficient (20.7) and the greatest shoot length and induction rate (95.3%) were achieved in 0.1 mg L−1 NAA and 2.0 mg L−1 BA medium, accompanied by rooting formation. Adding 0.5 mg L−1 GA3, 1.0 mg L−1 BA, and 0.2 mg L−1 IBA to MS medium can effectively prolong the regenerated buds for rooting. The best for rooting was 1/2 MS medium containing 0.3 mg L−1 IBA, with the maximum number of roots (13.4 per shoot) and survival rate for transplanting (100%). This work aims to build an efficient, definitive, and scalable protocol for S. hybrida regeneration useful for large-scale cultivation and even more protoplast fusion and genetic transformation to develop more colorful or fragrant flowers.

Diagnostics and prevention of aeromonosis Cyprinus sagrio

Relevance. Aeromonosis (carp rubella, hemorrhagic septicemia, infectious abdominal dropsy, Lublin disease) — an infectious disease of fish. Carp, common carp and their hybrids from underyearlings to broodstock are susceptible to the disease. The epizootic reaches its greatest distribution in the spring-summer period, by autumn it fades and the disease takes on a chronic course.Methods. The object of research was the fish Cyprinus carpio. In the experiments, we used the reference strain Aeromonas hydrophila ATCC 7966 of the Global Center for Bioresources, as well as isolates isolated during outbreaks of carp aeromonosis in fish farms in the Moscow region. The diagnosis for the presence of aeromonosis was established on the basis of epizootological, clinical and pathological data and the results of bacteriological studies. The study of biofilms of microorganisms was carried out during cultivation in liquid nutrient media, the CFU of microorganisms was counted and sown on the surface of solid media.Results.The total number of microorganisms (CFU, lg/g) of intestinal microbiocenoses— 54,21 ±0,05 – 66,09 ±0,12. Cultures of microorganisms A. hydrophyla, A. sobria, A. caviae were isolated from blood, heart, spleen, gills, intestine, liver and kidneys of fish. During cultivation at 37 °C for 6–72 h, the adhesion of vegetative forms of bacteria with a typical shape and size of the species and the formation of a bacterial monolayer — a diffuse layer of bacterial cells — was revealed. Viable and non-viable microorganisms were differentiated by fluorescence microscopy. On meat-peptone agar with 5,0% of defibrinated blood, as a rule, the growth of b-hemolytic colonies was observed, less often a-hemolytic colonies. When determining the enzyme DNase, a light zone was found along the inoculation line on the DNA BA medium. Growth of b-hemolytic cultures was observed on meat-peptone agar with 5,0% of defibrinated blood. When determining the DNase enzyme on the surface of the dense nutrient medium “DNA BA”, a light transparent zone was revealed around the colonies of microorganisms. Prevention of aeromonosis is based on systematic monitoring of the etiological structure of pathogens of aeromonosis; feeding in feeding and summer mother ponds begins in the spring when the water temperature rises to 14 °C.

Use of Thidiazuron for High-Frequency Callus Induction and Organogenesis of Wild Strawberry (Fragaria vesca).

Strawberry, belonging to the Fragaria genus, is an important crop that produces popular fruits globally. F. vesca, known as wild strawberry, has great characteristics, such as a robust and powerful aroma, making it an important germplasm resource. The present study aims to establish an efficient regeneration method for the in vitro propagation of F. vesca. Firstly, leaf explants were used to induce callus formation on a Murashige and Skoog medium with combinations of cytokinins (thidiazuron (TDZ) and 6-benzylaminopurine (BA)) and auxin (2,4-dichlorophenoxyacetic acid (2,4-D)). Among them, 0.45–4.54 µM TDZ combined with 0.45–4.53 µM 2.4-D exhibited a high induction rate after 4 weeks of culturing. Different explants were examined for their ability to form a callus, and whole leaves on the medium containing 2.27 µM TDZ and 2.27 µM 2,4-D showed the highest callus induction rate at 100% after 2 weeks of culturing in darkness. The highest shoot regeneration ability through organogenesis from the callus was obtained at 0.44 µM BA. After 2 weeks of culturing, the shoot regeneration rate and shoot number per explant were 96% and 19.4 shoots on an average, respectively. Rooting of shoots was achieved using indole-3-butyric acid (IBA) or an α-naphthaleneacetic acid (NAA)-containing medium, and the resulting plantlets were acclimatized successfully and formed flowers eventually. In this report, we demonstrated that shoot organogenesis was derived from the meristematic cells of calli and by transferring the induced calli to a 0.44 µM BA medium, the regeneration period can be shortened greatly. A protocol for the efficient regeneration of wild strawberry was established, which might be useful for their large-scale propagation or obtaining transgenic plants in the future.

Seed germination, micropropagation from adult and juvenile origin explants and address of hyperhydricity of the Cretan endemic herb Calamintha cretica

The optimum range of temperature for germination (96-100%) of Calamintha cretica, an herb with potential pharmaceutical and horticultural uses, was 15 to 20 °C, with 10 and 30 °C cardinal temperatures. Storage up to one year did not affect germination. The effect of zeatin (ZEA), 6-benzyladenine (BA), kinetin, and 6-γ-γ-(dimethylallylamino)-purine added in MS medium at concentrations from 0.0 to 8.0 mg L-1 was tested for shoot proliferation of both adult- and seedling-origin nodal explants at first- and sub-culture. Both explant types responded similarly during in vitro culture. At cytokinin concentrations up to 1 mg L-1 explant response was high (over 85%) but shoot number per explant was low (1.2-2.2). Increasing cytokinin from 2.0 to 8.0 mg L-1 resulted to an analogous decrease of explant response and shoot length, and an increase of shoot number, particularly when ZEA or BA was used (5.0-6.6 shoots per explant, 0.5-1.0 cm long) with simultaneous though increase of hyperhydricity (up to 50%). The addition of 0.1 mg L-1 naphthaleneacetic acid into the 8.0 mg L-1 BA medium almost eliminated hyperhydricity and increased explant response, while the increase of agar concentration from 8.0 to 12.0 g L-1 eliminated hyperhidricity and induced the highest shoot proliferation (93-95% explant response, 11.2-12.3 shoots per explant, 0.8-1.0 cm long). Microshoots and microshoot clusters rooted (88-96%) on half-strength MS medium either hormone free or supplemented with 1 to 4 mg L-1 indole-3-butyric acid. Plantlets survived at 80% to 100% after ex vitro acclimatization in peat: perlite 1:1 (v/v).

. In vitro propagation of the new orchid Dendrobium trankimianum T. Yukawa

Dendrobium trankimianum T. Yukawa is a beautiful, endemic orchid of Vietnam, a new species with a first - published description in 2004. It is very rare and expected to be added to the IUCN Red List status - CR. In vitro studies of orchid D. trankimianum T. Yukawa were conducted in order to conserve and increase the genetic pool of this precious wild orchid species. The results showed that full-strength MS medium supplemented with 2.0 mg/L BA and 0.5 mg/L NAA (10.24 PLBs/explant; 90.11% explants formed PLBs) or full-strength MS medium supplemented with 1.5 mg/L TDZ and 0.5 mg/L NAA (14.11 PLBs/explant; 92.06% explants formed PLBs) were the most suitable for protocorm formation. For subculture, suitable growth of shoots were obtained on full-strength MS medium supplemented 1.5 mg/L BA (22.35 shoots/explant; shoots length of 1.96 cm) and full-strength MS medium supplemented with 60 g ripe banana per liter (25.11 shoots/explant; shoots length of 2.12 cm). The shoots in vitro were transferred to half-strength MS supplemented with different concentrations of IAA, IBA and NAA to investigate root formation. The best rooting occurred at 0,5 mg/L NAA (7.91 roots/shoot; root length of 4.01 cm; 98.51% root formation). The plantlets with uniform growth were planted on different substrate: Eco clean soil, Coconut fiber, Fern fiber, 50% Rice husk in combination with 50% Eco clean soil for research the most suitable substrate. After 60 days of transplantion and acclimatization, the result showed that Fern fiber was suitable substrate for plantlet growth in a nursery garden (8.0 roots/ explant; root length of 5.5 cm; survival rate of 93.29%).

Evaluation of In Vitro Germination and Callus Induction for Antimicrobial Activities of Jatropha curcas L.

There are many problems related to seed germination and in vitro cultivation of Jatrophacurcas L, this research was to solve this obstacle. This study also aimed to use triphenyl tetrazoliumchloride (TTC) as a new substance for in vitro callus initiation and growth in plant tissue culture fieldsfor the first time. In addition, an efficient regeneration method via seed culture has been developed.For this purpose, different media (MS; B5; WPM with B5 vitamins; wetted cotton and water) were usedfor seed germination. High seed germination percentage for all treatments were achieved after 24days from incubation. WPM was effective medium in increasing the total length of seedlings. Themaximum dry weight (0.24 g) of Jatropha curcas shoots was recorded with B5 medium. In vitro shootformation, MS medium supplanted with 2.0 μM IBA +10 μM BA and MS medium+2.5 μM BA+2.5 μMNAA recorded the highest shoot formation percentage (100%) after one month. Concerning callusinduction, data implied that different growth regulators tested (TDZ, BA, KIN) and TTC as a newsubstance used in plant tissue culture could induce the formation of callus in cotyledons of J. curcasseedlings, furthermore, the combination of BA and Kin in various concentrations was an appropriatemedium for inducing the formation of callus and promoting its growth. Also, callus formation andgrowth were significantly affected by explant types. The balance between auxin and cytokinin is thelimited factor for callus induction. MS nutrient medium supplemented with 2,4-D or NAA or both helpedto form callus at high percentage. regarding antimicrobial activity, the antimicrobial potential ofdifferent plant extracts was screened against four pathogenic microorganisms and reference bacterialstrains, the ethanolic extract of the roots was found to show greater inhibition of B. cereus and E. coliwith zones of inhibition, 21.7 and 10.4 mm respectively, when comparing the extracts from in vivoplant and in vitro callus. Also, ethanol extract from the leaf of the mother plant showed higher inhibitoryzone than that of the leaf-derived callus against S. aureus, B. cereus and E. coli. On the other hand,butanol extract of hypocotyl-derived callus was achieved the highest fraction effect resulted fromdifferent fractionation of crude methanolic extract against antibacterial activity of E. coli, followed byethyl acetate- fraction (15.0 and 13.5 mm) respectively, in contrast, water- fraction came in the last.

Resistance Saffower (Carthamus tinctorius L.) Callus and its branches to salinity Stress

The study was implemented in the year 2013-2014 in the tissue culture laboratory of the field crops department/ faculty of agriculture and forestry for the purpose of producing callus and saline resistant plants , the results showed capability to initiation leaves callus with high levels which was 100% at present 2,4-D alone or its interaction with BA in medium of MS, which supported by sodium chloride salt levels (0.0, 0.05, 0.1 and 0.2 Molar) except a concentration (0.3 molar) which significantly reduced the initiation ratio. The estemation results of proline acomulation in leaves callus as an indicator of salinity resistance showed that the proline content was significantly higher at all levels of salinity used (0.05-0.3 molar) for more than 30 μg/g frish whight compared to 2.3 and 2.8 μg / g After 4 and 8 weeks of treatment any more than 10 times its presence in control tretment. Salinity resistance results showed that branches forming from callus of shoot tip and leaf showed resistance to salinity after four weeks of transfer of branches to growth medium and multiplication which suplimented with sodium chloride salt. Although the number of branches decreased slightly in the fourth week, they were able to grow and showed tolerance to the medium content of 0.2 molar NaCl, while no significant differences were observed in the number of branches during the four weeks on the saline medium (0.1 molar). The number of branches arising from leaf callus did not differ significantly in all medium (0.0-0.2) and showed continuous resistance for four weeks. Results of interaction salt with the plant part showed that increased salinity led to the reduction of branches with a significantly increased concentration of the tip stem and were not significant for the branches of the callus leaves. The effect of the time showed that it was not significant in reducing the number of branches in the two parts of the plant. It was also noticed that there was an increase in the length of the branches of each of the stem tip and leaves callus in the medium containing (0.1 molar NaCl). In high concentrations of salt (0.2 molar) It continued to grow in all saline medium, suggesting that salts may be resistant to saline stress, possibly for genetic mutations that have adapted to growth in these medium,

EFFECT OF SOME FACTORS ON THE MICROPROPAGATION AND MICROGRAFTING OF SOME GRAPE ROOTSTOCKS IN VITRO

This study aimed to investigate the attitude of three nematode and phylloxera resistant grape rootstocks (Freedom, Ramsey and SO4) through the micropropagation stages till be micrografted by Superior scions. During that the effect of paclobutrazol (PP333) on rootstocks stem diameter, transverse sections in graft union regions and acclimatization of resulted micrografts were studied. The results proved that the most effective sterilization durations using 1% NaOCl were 15 min for Freedom and SO4 explants as they achieved 0.00 and 0.00%of contamination and 100% and 90% of survival percentages respectively, while 10 min duration was sufficient for Ramsey explants as it achieved 0.00 % contamination and 100% survival percentages. Significantly, Ramsey achieved the highest number of shoots (1.25), Freedom gave the tallest shoots (4.13 cm)and SO4 got the highest leaf number (7.33) all on MS medium. In multiplication stage; Freedom rootstock proved to be the most reacted rootstock as it significantly accomplished the highest shoot number (2.00) when planted on BA medium, the tallest shoots (3.54 cm) when planted on TDZ medium and the highest leaf number on Kin medium. Whereas, in order to get thicker shoots for easier grafting; using of PP333 at 5 mgl-1 significantly was the best for Freedom (3.55 mm) followed by SO4 (3.03 mm) and Ramsey (2.85 mm) rootstocks. In micrografting stage, Superior micrografts significantly achieved the best results on rooted Freedom rootstocksin scion survival (100.00%) and on un rooted rootstocks in scion bud burst (75.00 %), graft union formation (50.00%), rooting (75.00%). On the same trend, Freedom rootstock was more active in cell dividing activity when grafted by Superior cultivar compared to Ramsey and SO4 rootstocks which were very poor in producing callus cells in graft union. Finally, Freedom rootstock grafted with Superior gained the highest survival (100%) after acclimatization.

Effect of plant hormones on the in vitro culture of breadfruit shoots (Artocarpus altilis (Park.) Fosberg)

Under the influence of plant hormones, after 8 weeks of in vitro culture, the growth of breadfruit shoot (Artocarpus altilis (Park.) Fosberg) was very different. With shoots that were cultured on 1 mg/L BA medium after 10 days of being transferred to ½ MS supplemented with 10 mg/L BA medium, the percentage of shoot development was the highest (86.8 %), and the secretion of phenolic compounds or forming callus were not observed. On the ½ MS supplemented with 12 mg/L BA medium, shoots growed strongly and healthily but the secretion of phenolic compounds and the forming of callus affected the ability of the shoot development. On the ½ MS supplemented with 0.45 mg/L BA, 0.6 mg/L Kinetin (Kin) medium and on the ½ MS supplemented with 0.45 mg/L BA, 0.6 mg/L Kin, 0.35 mg/L GA3 medium, the percentage of shoot developed were lower than those on the ½ MS supplemented with 10 mg/L medium. The addition of 0.35 mg/L GA3 in the culture medium help to appear the lateral more than the remaining experiments. Roles of respiration rate and endogenous hormones were discussed to understand the physiological changes in the in vitro culture shoots breadfruit.

In vitro regeneration and transient expression of recombinant sesquiterpene cyclase (SQC) in Artemisia annua L.

In the present study, we developed an efficient and reliable protocol for in vitro plant regeneration and transient gene expression in medicinal plant Artemisia annua L. For in vitro regeneration, leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.5mg/l NAA plus 2mg/l BA (86.6%) and MS medium supplemented with 1mg/l IAA plus 2mg/l BA (80%). Root induction was obtained on MS medium supplemented with 0.1mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. annua L. in short period via adventitious shoot induction approach. Based on the sequence of a putative sesquiterpene cyclase from A. annua, 1.7kb fragment was amplified by PCR from Iranian variety of this plant after cDNA synthesis. Sesquiterpene cyclase gene was used for sub-cloning into pBI121 between XbaI and SacI sites to allow the expression of the gene driven by CaMV35S promoter. The recombinant plasmids were transferred to competent cells of Agrobacterium tumefaciens strain GV 3850 for vacuum agro-infiltration. HPLC results showed 30.09% increase in atremisinin content (0.15%) in the agroinfiltrated samples (0.67%) in comparison to the control (0.51%).

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05mg/l NAA plus 2mg/l BA (96.3%) and MS medium supplemented with 0.5mg/l IAA plus 2mg/l BA (88.3%). Root induction was obtained on MS medium supplemented with 0.5mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93%) and ATCC15834 (89%) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.

In Vitro Callus Induction and Embryogenesis of Oil Palm (Elaeis guineensis Jacq.) from Leaf Explants

This research was to study in vitro callus induction and somatic embryogenesis in oil palm from leaf explants. Young leaf segments from mature oil palm were cultured on MS medium supplemented with different concentrations of 2,4-D with or without addition of 2 g/l activated charcoal (AC) or 2,4-D and picloram. Embryogenesis induction was done using MS medium containing 2,4-D 450 µM and benziladenine 4.4 µM with 3g/l activated charcoal. The treatment of 2,4-D 15 µM resulted in the highest percentage of callus induction. The treatment of 2,4-D and AC showed that 2,4-D 450 µM and AC led to higher percentage of callus induction than that of 2,4-D 400 µM and 2 g/l AC. Embryogenesis occured in 27 out of 250 clumps of primary callus was occurred after 2-3 times subcultures. Somatic embryo development occurred when the embryogenic callus was transferred on the same basal medium supplemented with casein hydrolysate with 1 µM BA or growth regulator free basal medium with 2 g/l activated charcoal.

Xanthone compounds in shoot cultures of Gentianella bulgarica

Shoot cultures of Gentianella bulgarica established from seedling epicotyls were maintained on MS medium supplemented with BA 0.2 mg l−1 + NAA 0.1 mg l−1. Cultures were prone to precocious flowering requiring the use of small shoot buds for multiplication purposes. The contents of three xanthone compounds identified as DGL, BGL, and DMB, in different plant material were determined by HPLC. The analysis revealed that the production of xanthones was affected by different concentrations of BA in medium. Shoot cultures grown at higher BA concentrations contained more DGL than material grown in nature. The concentrations of other two xanthones were lower in shoot cultures than in plants from nature. The radical scavenging activity of plant extracts and xanthones was investigated by DPPH test. Samples from plants grown in nature showed the highest activity (IC50 = 0.26 mg ml−1), while the extracts of shoot cultures grown in media with higher concentrations of BA showed moderate activities (IC50 from 1.6 to 4.4 mg ml−1).

Encapsulation for in vitro short-term storage and exchange of ginger ( Zingiber officinale Rosc.) germplasm

Synseeds of ginger ( Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100 mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose) + 2.5 mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose) + 2.5 mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25 M sucrose liquid medium for 16 h and stored in cryovials (with out medium) at 25 °C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose) + 2.5 mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories.

Improved clonal propagation of Spilanthes acmella Murr. for production of scopoletin

A reproducible protocol for clonal propagation of Spilanthes acmella has been established. Routinely, the cultures were established in spring (January–April) season because of the highest aseptic culture establishment and high frequency shoot proliferation. Incorporation of 5 μM N6-benzyladenine (BA) to Murashige and Skoog (MS) basal medium showed 100% bud-break and promoted multiple shoot proliferation in cultures. Interestingly, a higher concentration of BA (7–15 μM) promoted stunted shoots with pale leaves while a lower concentration (1–3 μM) resulted in shoots with long internodes and excessive adventitious root proliferation from all over their surface. For recurrent shoot multiplication, single node segments from in vitro-developed shoots were excised and cultured on MS + BA (5 μM) medium where 20.3-fold shoot multiplication was achieved every 5 weeks. Finally, these shoots were successfully rooted on half-strength MS medium (major salts reduced to half-strength) with 50 g l−1 sucrose, with a frequency of 100%. Transplantation survival of micropropagated plants was 88.9%. Additionally, accumulation of scopoletin, a phytoalexin, was revealed for the first time in the uninfected leaves of Spilanthes. Further, the quantitative estimation by HPLC with a fluorescence detector showed that the amounts of scopoletin content (0.10 μg g−1 DW) in the leaves of micropropagated plants are comparable to those of field-grown mother plants. The study thus signifies the effectiveness of in vitro methodology for true-to-type plant regeneration of Spilanthes and their later utility for biosynthesis and constant production of scopoletin throughout the year.

Electrophysiological characterization of store-operated and agonist-induced Ca2+ entry pathways in endothelial cells

In endothelial cells, agonist-induced Ca(2+) entry takes place via the store-operated Ca(2+) entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca(2+) store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca(2+)-release-activated Ca(2+) current (CRAC)-like current when 10 mM Ba(2+)/2 mM Ca(2+) was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca(2+) store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba(2+) medium. On the contrary, in the presence of 10 mM Ca(2+), the ER Ca(2+) content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni(2+) and KB-R7943, two blockers of the Na(+)/Ca(2+) exchanger (NCX). Both blockers also reduced histamine-induced cytosolic Ca(2+) elevation. In addition, removing extracellular Na(+) increased the current amplitude which is in line with a current supported by the NCX. These data are consistent with the involvement of the NCX working in reverse mode (Na(+) out/Ca(2+) in) during agonist-induced Ca(2+) entry in endothelial cells.