Effects Of Basal Media Research Articles

High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba)

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 μM TDZ and 0.27 μM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 μM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N6) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated.

Parametric investigation of static and dynamic cell culture conditions and their impact on hCMEC/D3 barrier properties

In brain research, the hCMEC/D3 cell line is widely used for the establishment of a human in vitro blood-brain barrier (BBB) model. However, its barrier integrity seems to be insufficient for drug permeability studies, represented by rather low transendothelial electrical resistance (TEER) and high permeability of small molecules. Therefore, this study covers a parametric investigation of static and dynamic cell culture conditions to improve barrier functionality of hCMEC/D3. The effect of basal media was investigated by analyzing changes in proliferation rate, barrier integrity and gene expression of cellular junction proteins. The cells were able to grow in different cell culture media, including serum-free media. However, none of these media enhanced strongly the growth rate or barrier integrity compared to the microvascular endothelial cell growth medium-2 (EGM™-2 MV). Furthermore, hCMEC/D3 cells did not respond positively regarding TEER to any tested parameter neither supplements, coating materials nor co-cultures with the human immortalized astrocyte cell line SVGmm. Furthermore, the impact of dynamic conditions was examined by using the Dynamic Micro Tissue Engineering System (DynaMiTES). Cultivation conditions were successfully adapted to the DynaMiTES design and no negative effect was detected by analyzing cell viability and cell count, albeit TEER remained also unchanged. Consequently, the hCMEC/D3 model has considerable limitations and further improvements or alternative cell lines are required.

Optimising protocol for successful development of haploids in marigold (Tagetes spp.) through in vitro androgenesis

For the first time, we have developed a protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. Various factors affecting the in vitro androgenesis response in Tagetes spp. were optimised. At first, a correlation was established between floret size/bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. The floret size of 3.5–4.0 mm (French marigold) and 3.0–3.5 mm (African marigold) contained the highest percent of microspores at early-uninucleate to early-binucleate stage. The frequency of shoot formation from anthers ranged from 0 to 78.6% in T. patula cv. Pusa Arpita and 0 to 63.9% in T. erecta cv. Pusa Basanti Gainda. The French marigold cultivars exhibited higher androgenic response over the African marigold cultivars. The effects of basal media, plant growth regulator combinations and sucrose concentration for direct differentiation of shoot buds and haploid induction were studied. Marigold anthers cultured on newly formulated EMS1 basal media (enriched with coconut water, AgNO3, PVP etc.) exhibited a very high androgenic response over the other commercially available media. We have also optimised the cold temperature pre-treatments and culture condition for high frequency androgenesis in marigold. Ten days cold pre-treated anthers along with 20 days dark incubation of cultures proved highly beneficial for haploid induction. Among a total of 424 anther-regenerants, 56 plants were randomly selected for flow cytometry and cytological analysis. The ploidy analysis revealed 14.3% of anther regenerants as dihaploids, 66.1% as tetraploid (similar to donor mother plant) and 19.6% as polyploid. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells of marigold was also established for rapid screening of haploids. The identified haploids were successfully diploidised and are being utilised in the hybrid breeding programme at our institute. The developed protocol will facilitate doubled haploids based breeding in Tagetes spp. This is the first report of successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. We have studied various factors affecting the in vitro androgenesis response in Tagetes spp. A correlation was established between floret size/ bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. Basal media and cultural conditions were optimised for high frequency androgenesis in marigold. Haploids were successfully induced and their ploidy level was assessed through flowcytometry and cytological analysis.

Effects of media and carbon sources on growth and canthin-6-ones accumulation in hairy root cultures of Eurycoma harmadiana

Eurycoma harmandiana Pierre belongs to Simaroubaceae family which is a Thai traditional medicinal plant used for intermittent fever (Malaria). The major bioactive compounds of the these plants are quassinoids and canthin-6-one alkaloids which possessed cytotoxic, antimalarial, and aphrodiasiac[1 – 2]. In this study, the effects of various basal media and carbon sources on growth and canthinone alkaloids production in hairy root cultures of E. harmadiana was investigated. The effect of basal media on the hairy root biomass was no significantly different, while the highest amount of alkaloids, 9-hydroxycanthin-6-one (5.75 mg/g dry weight) and 9-methoxycanthin-6-one (5.87 mg/g dry weight), could be obtained in B5 liquid medium. At fructose of 3% (w/v), the production of 9-methoxycanthin-6-one (5.74 mg/g dry weight) is promoted to gain the highest yield, while 3% (w/v) mannitol gave the highest amount of 9-hydroxycanthin-6-one (4.94 mg/g dry weight), compared to other carbon sources tested. This study suggests that the successful production of 9-hydroxycanthin-6-one and 9-methoxycanthin-6-one in vitro cultures has a potential in large-scale production using bioreactor technology.

In vitro germination, protocorm formation, and plantlet development of Orchis coriophora (Orchidaceae), a naturally growing orchid species in Turkey

Some species belonging to the genus Orchis Tourn. ex L. (Orchidaceae) are of great economic importance as their tubers or corms are used to produce a hot beverage called salep. Nevertheless, these plants are not cultivated but are rather collected from nature, and due to careless collection many have already been listed as endangered plants. In order to assess the possibility of in vitro propagation, an orchid, Orchis coriophora L., was selected as a model plant, and the effects of basal media and plant growth regulators on in vitro seed germination, protocorm development, and plantlet formation were studied. Mature seeds were cultured in 4 different basal media, each supplemented with various concentrations and/or combinations of auxins and cytokinins/cytokinin-like substances. The highest germination rate (44.2%) was observed in Orchimax medium including activated charcoal plus 1 mg/L indole-3-acetic acid. Protocorms developed plantlets in all the tested media. Orchimax medium including activated charcoal and supplemented with 0.25 mg/L 6-benzyladenine was found to be the most suitable medium for the formation of plantlets from protocorms.

Effect of Basal Media and Sugar Types on in Vitro Regeneration of Grammatophyllum speciosum Blume

Protocorms of Grammatophyllum speciosum Blume. were initiated from immature seeds on solid MS medium containing 15% (v/v) coconut water (CW) and 3% (w/v) sucrose. Protocorms, 2-4 mm in length were used as explants and subcultured to MS and VW (Vacin and Went, 1949) media containing 15% (v/v) CW and 2 or 3% (w/v) sucrose. Protocorms gave the highest formation of PLBs (protocorm-like bodies) at 3.1 PLBs/explant on MS solid medium containing 15% CW and 2% sucrose. For the test with different carbon sources, protocorms were cultured in liquid MS medium supplemented with 4 kinds of sugar, namely sucrose, glucose, sorbitol and mannitol at 2, 4, 6 or 8% (w/v) and cultured for 1, 2, 3 or 4 weeks. They were then transferred to MS solid medium containing 15% CW and 2% sucrose. Results revealed that after 4 weeks in MS liquid medium, sucrose and glucose had an inhibitory effect and 8% glucose gave a high percentage of protocorm browning. In contrast, sorbitol and mannitol were effective for protocorm regeneration and both sugar alcohols had a positive effect on the formation of PLBs and the development of PLBs into plantlets.

Micropropagation of ‘Amethyst’ Purple Raspberry (Rubus occidentalis L. x R. idaeus L. ‘Amethyst’)

Abstract A micropropagation method was developed for a cold hardy purple raspberry cultivar (Rubus occidentalis × R. idaeus ‘Amethyst’). In vitro shoot cultures were initiated from shoot tips of a 30-year old ‘Amethyst’ plant. The effects of basal medium, plant growth regulator, and temperature on shoot proliferation were investigated. Shoots were produced from explants in both Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) media supplemented with different concentrations of thidiazuron (TDZ) and benzyladenine (BA), solely or combined. One micro molar TDZ gave rise to the maximum proliferation rate. Interactions between BA and medium or TDZ were significant. Shoots produced on media with 1.0 μM TDZ had thick stems and small, dark green leaves whether BA was absent or present. Shoots can be rooted both in vitro and ex vitro with or without IBA at 0 to 1.0 μM. However, combination of rooting and shoot multiplication by adding a low level of TDZ to rooting medium produced multi-cane plants resulting in shortening propagation time, increasing survival rate, and lowering the production cost.

Micropropagation of a Cold Hardy Selection of Cercis canadensis L. Through Single-Node Culture

Abstract A cold hardy eastern redbud (Cercis canadensis L., TS 0092) was propagated using single-node micropropagation. In vitro shoot cultures were initiated from rapidly growing shoot tips of a mature tree. The effects of basal media and plant growth regulators on proliferation were investigated. Maximum proliferation rate was obtained from all three tested media, Murashige and Skoog (MS), Woody Plant Medium (WPM), and Driver-Kuniyuki walnut (DKW), supplemented with 0.5 μM thidiazuron (TDZ). Shoots produced on WPM medium appeared stressed showing yellowish and thin leaves compared to those on MS and DKW media. Shoots were in vitro rooted in auxin-free half strength WPM medium after being pulsed for 1 to 16 days in half strength WPM medium with 12.5 to 100 μM indole-3-butyric acid (IBA). In vitro rooting rate was influenced by IBA concentration, pulsing time, and shoot sources (media in which shoots were produced). Shoots produced in DKW medium rooted best after being pulsed in 25 to 100 μM IBA for 8 days although root numbers were similar among treatments. Direct ex vitro rooting was achieved by quick-dipping the microshoots in 10 μM IBA or NAA solutions and inserting them in Jiffy Mix potting medium.

Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes.

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.

병풀 (Centella asiatica L. Urban) 식물체 배양을 이용한 Triterpene Glycoside 생산

기내에서 병풀의 식물체 배양을 확립하였고 기본배지 설정 및 대량원소들, sucrose 농도가 triterpene glycoside (Made-cassoside와 Asiaticoside: M&A) 생산에 대한 영향을 조사하였다. 병풀 식물체가 각각의 배지에서 배양되었을 때, MS와 RCM배지는 각각 식물체 생장과 M&A 생산에 대해 가장 좋은 결과를 나타냈다. 하지만, M&A에 대해 가장 높은 생산성은 B5 배지로 나타내었다. B5 배지의 대량영양소인 KNO<TEX>$_3$</TEX>는 25 mM, NaH<TEX>$_2$</TEX>PO<TEX>$_4$</TEX>는 1 mM, CaC1<TEX>$_2$</TEX>는 1 mM 그리고 MgSO<TEX>$_4$</TEX>는 1~10 mM에서 M&A 생산이 최적을 이루거나 증가시켰다. Sucrose를 농도별로 처리했을 때, 6% sucrose 농도에서 가장 높은 M&A 생산을 얻을 수 있었다. Whole plant cultures of Centella asiatica (L.) Urban in vitro were established and the effects of basal media, some macro elements and sucrose concentration on productivity of triterpene glycosides (madecassoside and asiaticoside: M<TEX>$\varepsilon$</TEX>A) were investigated. Among the media (MS, B5, RCM) tested, MS and 0.5 RCM medium were the best for plant growth and M<TEX>$\varepsilon$</TEX>A production, respectively. However, taking into account the M<TEX>$\varepsilon$</TEX>A productivity, B5 medium was superior (M<TEX>$\varepsilon$</TEX>A: 14.28 mg/g dry wt.). Major macronutrients of B5 medium adjusted with the concentration of 25 mM KNO<TEX>$_3$</TEX>,1 mM NaH<TEX>$_2$</TEX>PO<TEX>$_4$</TEX>, 1 mM CaCl<TEX>$_2$</TEX> and 1~10 mM MgSO<TEX>$_4$</TEX>, caused elevated or optimized levels of M<TEX>$\varepsilon$</TEX>A. On sucrose concentration, the highest yields of M<TEX>$\varepsilon$</TEX>A were obtained from 6% sucrose.

In vitro propagation of the Greek olive cultivar `Chondrolia Chalkidikis'

The effect of basal media (Woody Plant Medium (WPM), Quoirin and Lepoivre (QL) and Olive Medium (OM)) and of various concentrations of cytokinins (6-benzyladenine (BA), zeatin, 6-(γ,γ-dimethylallyl-amino) purine (2iP)), solely or in combinations with each other and with gibberellic acid (GA3), on in vitro shoot proliferation of the greek olive cultivar `Chondrolia Chalkidikis' was investigated. WPM proved to be the most effective one, resulting in better morphological appearance of the microshoots produced. The highest number of new microshoots/explant (1.68), with a 3.0 cm shoot height and a 4.2 proliferation rate, was obtained when this medium was supplemented with 20 μM zeatin. The number of microshoots/explant and proliferation rate increased to 1.85 and 6.8, respectively, by using the combination of 5–20 μM zeatin with 1 μM BA, but shoot height was reduced. 2iP was the least effective of the cytokinins tested. Combination of 20 μM zeatin with 10 μM GA3 affected positively shoot proliferation resulting in 1.80 microshoots/explant, 3.0 cm shoot height and 7.0 proliferation rate. However, the same concentration of GA3 in combination with 1 μM BA reduced the number of new microshoots/explant (0.48) as well as the proliferation rate (2.4), although shoot height remained nearly the same (2.7). The effect of indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and putrescine on root induction was also studied. A rooting percentage of up to 70% and 2.3 roots/microshoot were achieved by the combination of 12 μM IBA + 3 μM NAA, however, abscission of shoot tips and leaves appeared. Both variables were increased, up to 93% and 4.0, respectively, by the addition of 30 μM putrescine in the medium, without any undesired side effect. After acclimatization, survival of rooted microshoots was high (90%) while that of non-rooted ones was low (30%).

Analysis of somaclonal variation through tissue culture and chromosomal localization of rDNA sites by fluorescent in situ hybridization in wild Allium tuberosum and a regenerated variant

The effects of basal media and growth regulators on callus initiation and shoot regeneration have been investigated in wild Allium tuberosum (2n = 4x = 32). Callus initiation was greatest from flower bud explants cultured on MS medium supplemented with 2,4-D and BA at 1 mg l−1 each. Maximum number of shoots was obtained from callus grown on MS medium supplemented with NAA and BA at 0.2 and 2 mg l−1, respectively. The chromosome analysis of regenerants derived from callus revealed variation in ploidy, such as 2n = 28, 29, 30, 31, 33 as well as normal tetraploid. During the culture period for two generations, one aneuploid regenerant with 2n = 30 (named At30) showed better viability and growth than tetraploid plants and other aneuploid variants. In a karyotypic analysis of At30, the chromosomal positions of 5S and 18S-5.8S-26S rDNA were physically mapped by fluorescent in situ hybridization and compared to chromosomes of wild type A. tuberosum. Both wild type A. tuberosum and At30 exhibited two sets of 5S rDNA sites, one on the proximal position of the short arm of chromosome 3, and the other on the intercalary region on the long arm of chromosome 6. There was one 18S-5.8S-26S rDNA site in the secondary constriction including flanking short chromosomal segments of satellite and terminal regions on the short arm of chromosome 8 in wild type A. tuberosum. However, At30 showed only three labelled chromosome 8 indicating that this was one of the lost chromosomes of At30.

Genotype and medium affect shoot regeneration of melon

The effects of basal media, growth regulators and gelling agents on the morphogenetic response of eleven Cucumis melo L. var. reticulatus and inodorus genotypes were examined. Regeneration was achieved from cultured cotyledons of all genotypes and the morphogenic response was affected by the genetic background. Among the combination of factors tested, MS basal medium supplemented with 2.8 μM 6-benzyladenine (BA) and 1.0 μM abscisic acid (ABA) solidified with agar gave the highest frequency of shoot regeneration.

Production of taxol and related taxanes in Taxus brevifolia cell cultures: Effect of sugar

Suspension cells of Taxus brevifolia were cultured as an alternative source of taxol and related taxanes. The effects of basal medium, sugar type, and sugar concentration on growth and taxane production were investigated. To induce taxol production, modification of medium composition was necessary. Fructose supported the best taxol yield and the use of high concentration of sugar was desirable. The maximum level of taxol obtained after 10 days of culture in an optimized condition was 1.43 mg/L, which was 0.013% as a specific content.

Effects of Basal Media on Growth and Polyacetylene Production of Lobelia inflata Hairy Roots.

Effects of Basal Media on Growth and Polyacetylene Production of Lobelia inflata Hairy Roots.

Studies on Ailanthus altissima cell suspension cultures. The effect of basal media on growth and alkaloid production

Time-course experiments have been carried out to investigate the relationship between growth and alkaloid accumulation in A. altissima cell suspension cultures. Results indicate that the type of basal medium, viz. Murashige and Skoog, Linsmaier and Skoog, Schenk and Hildebrandt or Gamborg's B5, has a significant influence on both growth and alkaloid production.

Laboratory identification of Haemophilus influenzae: effects of basal media on the results of the satellitism test and evaluation of the RapID NH system.

The effects of four different basal media, tryptic soy agar, brain heart infusion agar, nutrient agar, and Mueller-Hinton agar, were investigated with respect to the identification of Haemophilus influenzae with a satellitism test in which X and V growth factors were supplied by factor-impregnated filter paper strips. A total of 187 recent clinical isolates of H. influenzae were examined. Of these, 179 strains (95.7%) were correctly identified with tryptic soy agar, 173 (92.5%) with brain heart infusion agar, 105 (56.1%) with nutrient agar, and 133 (71.1%) with Mueller-Hinton agar. Failure to obtain a correct identification was usually the result of satelliting growth around V factor-containing strips, possibly due to the presence of trace amounts of hemin in the basal media, or was because of an absence of growth due to nutritional deficiencies in the basal media. All 187 H. influenzae strains were also examined with a new biochemical and chromogenic substrate micromethod, the RapID NH system (Innovative Diagnostics Systems, Inc., Decatur, Ga.). A total of 168 (89.8%) strains were correctly identified with this system.