Nodal Culture Research Articles

Genome-wide identification of NAC transcription factors and regulation of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs.

Modulation in the enzymatic antioxidants, MDA level and elicitation in conessine biomolecule in Holarrhena pubescens (medicinal tree) cultures exposed to different heavy metals: Ni, Co, Cr and As.

Nodal explants of Holarrhena pubescens, an important medicinal tree, were cultured on Murashige and Skoog's medium (MS) containing 15µM BA (control) alone and on medium supplemented with different concentrations (0, 1, 5, 25, 50, 100 and 200mg/L) of heavy metals such as NiCl2, CoCl2, As2O3 and CrO3 to study their toxic effect. After 28days of treatments, the nodal segments were harvested to assess the average number of shoots per explants, average shoot length, malondialdehyde content, proline content, conessine accumulation and antioxidant enzymatic activity. Among all the metals tried, best morphogenic response was achieved at 5mg/L CrO3 where 80% culture differentiated an average of 3.21 ± 0.08 shoots per explant having 0.95 ± 0.018cm average shoot length. Highest concentration (200mg/L) of all the heavy metals proved lethal for morphogenesis. Maximum inhibition in average shoot number and average shoot length was observed in nodal explants treated with 25mg/L As2O3 where an average of 0.49 ± 0.047 shoots having an average shoot length of 0.3 ± 0.02cm. Contrarily, addition of heavy metals in culture medium proved strong elicitors, exhibiting significant enhancement in the biosynthesis of conessine, an important bioactive compound. HPLC analysis of the crude extract of in vitro grown untreated nodal cultures revealed an average of 117.06 ± 2.59µg/g d. w. of conessine, whereas those treated with 100mg/L of CoCl2 accounted for 297.1 ± 7.76µg/g d. w. (an increase of 156% over control). Among the heavy metals tried, CoCl2 proved to be the best for conessine enhancement which was in the order of CoCl2 > Cr2O3 > NiCl2 > As2O3 in the nodal explants. Concomitantly, MDA content, the antioxidant enzymes activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GR) and ascorbate peroxidase (APX) were also observed to be differentially expressed with the increase in the heavy metals concentration from 1 to 200mg/L. Free proline, too, increased up to 3.5-fold over control. The results obtained during the present investigation revealed that the overall response of the nodal explants in terms of morphogenesis, conessine content and antioxidant enzyme activities was metal specific as well as dose dependent.

A Review on In Vitro Regeneration of Ethnomedicinal Plant Turkey Berry (Solanum torvum Swartz)

In this review, we report on the different plant growth regulators (PGRs) affecting the callus induction, callus-mediated regeneration, organogenesis, somatic embryogenesis from different explants and micropropagation through shoot tip and nodal cultures in Turkey Berry (Solanum torvumSW). The fruits of this plant guard against liver and kidney damage, stop certain cancers, and regulate blood sugar levels. They benefit digestion, the management of gout and menstruation, the treatment of anaemia and diabetes, the prevention of intestinal worms, cardiovascular disorders, and strokes. The protocol developed for the induction of callus can be utilized to isolate pharmaceutically important secondary metabolites in S. torvum, and the regeneration protocols optimized in this study can be used for genetic transformation and conservation of S. torvum, not only as a medicinal plant but also a model system.

Study on in vitro propagation of \(\textit{Vaccinium myrtillus}\) Linn. via nodal culture method

Vaccinium myrtillus Linn. is an imported fruit tree with high economic value. Currently, in Vietnam there has not been any publication on in vitro propagation of the blueberry plant. This study is focused on in vitro propagation of V. myrtillus by nodal culture. The results showed that in vitro shoot growth and proliferation were the best when nodes were cultured on WPM medium supplemented with 0.5 mg/L zeatin, 30 g/L sucrose, 10 g/L agar, pH 5.8 (shoot height 4.24 cm, 1 shoot/explant, 8.60 nodes/shoot). The third nodes to sixth nodes proved to be suitable materials for in vitro propagation of V. myrtillus. The proliferation and growth of shoots on the medium without activated charcoal (plant height 3.92 cm, 1 shoot/explant, 8.60 nodes/shoot) were better than those grown on the medium supplemented with 1 g/L of activated charcoal (plant height 3.64 cm, 1 shoot/explant, 7.90 nodes/shoot). For the in vitro rooting stage, the WPM medium containing 1.0 mg/L IBA, 30 g/L sucrose, 10 g/L agar, pH 5.8 resulted in plant height 5.49 cm, root length 1.42 cm, root formation rate 100%. After transferring the cultivated-tissue Vaccinium myrtillus to the greenhouse, substrate with coconut fibre powder, the results were observed to be plant height 6.94 cm, root length 3.18 cm, survival rate 95%. The plantlet growth increased overtime after 4 months in the greenhouse.

CO2 supplementation eliminates sugar-rich media requirement for plant propagation using a simple inexpensive temporary immersion photobioreactor

In vitro plant propagation systems such as temporary immersion bioreactors (TIBs) are valuable tools that enable production of disease-free plants with improved traits. However, TIB systems can be expensive, difficult to implement, and prone to contamination due to sugar rich propagation media. Using rapidly growing chicory root cultures to expedite design-build-test cycles, we report here an improved, low-cost version of a previously reported Hydrostatically-driven TIB (Hy-TIB) that facilitates economical use of gas mixtures. Bioreactor improvements include decreased material costs, expanded modes of operation, and a horizontal orientation of a plastic film plant growth chambers that increase propagule light exposure. To take advantage of these improvements, we describe here experiments that evaluate the impacts of elevated CO2 on propagation of cacao (Theobroma cacao) secondary embryos and nodal cultures of yam (Dioscorea spp.) during both phototrophic and photomixotrophic growth. Our experiments show that elevated CO2 during plant propagation significantly improved both cacao and yam propagule development and eliminated the need for supplemental sugars in tissue culture growth media. Thus, our improved Hy-TIB shows potential as a simple, low-cost, and scalable propagation platform with cost-effective gas composition control and reduced risk of contamination overgrowth. We provide detailed instructions for assembly of this Hy-TIB design and discuss the implications of its adoption in food-insecure regions of the world.

Study on in vitro propagation of Sophora tonkinensis Gagnep through stem node culture

In the present study, authors propagated Sophora tonkinensisGagnep plants using stem nodal culture. The results indicated that on MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 0.75 mg/l TDZ shoots proliferated from stem segments have the best count and height of shoots. The most appropriate medium for multiplication of shoots was the MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 0.75 mg/l TDZ, 0.5 mg/l IBA, 2.0 g/l peptone, 30 g/l carrot puree, pH 5.5 with the results of 20.60 shoots/explant, shoot height of 3.75 cm and 4.6 leaves/shoot after 8 weeks of culture. Root formation of shoots carried out on the MS medium supplemented with 30 g/l sucrose, 5.5 g/l agar, 200 ml/l coconut water, 1 g/l activated charcoal, 1.0 mg/l αNAA gave the best result. In the nursery, a mixture of humus + coconut fiber powder (70:30 ratio) was regarded as the best substrate due to the high survival rate of plantlets (92%) and healthy plantlets (10.30 cm high with 7.2 leaves and 4.3 new roots/a plantlet) at 10 weeks after planting

Optimisation of gentiopicroside production in Gentiana kurroo Royle from adventitious root cultures in a liquid culture system

Gentiana kurroo Royle is a critically endangered medicinal herb of the Indian Himalaya. It has bioactive seco-iridoid glycosides, notably gentiopicroside, in the rhizome and roots. In this study, we report on the in vitro production of gentiopicroside (GPD) from adventitious (AD) roots induced directly from the leaf, nodal, and suspension cell cultures of G. kurroo. Murashige and Skoog (MS) media fortified with Indole-3-butyric acid (IBA) 2 mg L−1 + Naphthaleneacetic acid (NAA) 0.5 mg L−1 and IBA 2 mg L−1 + Indole-3-acetic acid (IAA) 1 mg L−1 produced the maximum number of roots. Suspension cell cultures derived AD roots showed a 1.41-fold higher biomass production than adventitious roots induced from leaf and nodal explants. Among the various concentrations of MS media salts evaluated, half-strength MS suspension media favored the higher biomass and GPD accumulation. The maximum accumulation of GPD (2.58 mg g –l dry weight (DW)) and AD root biomass (18.96 g L−1 DW) was observed on the 48th day. Furthermore, GPD produced from AD root cultures were separated by using TLC, characterized using Nuclear Magnetic Resonance (NMR) and High-Resolution Liquid Chromatograph-Mass Spectrometer (HR LC-MS) and quantified with High-Performance Liquid Chromatography (HPLC).

Selection and Micropropagation of an Elite Melatonin Rich Tulsi (Ocimum sanctum L.) Germplasm Line

Tulsi (Ocimum sanctum L.) is a sacred plant of medicinal and spiritual significance in many cultures. Medicinal properties of Tulsi are ascribed to its phytochemicals with antioxidant capabilities. The current study was undertaken to screen a large seed population of Tulsi to select germplasm lines with high antioxidant potential and to standardize protocols for micropropagation and biomass production to produce a phytochemically consistent crop. A total of 80 germplasm lines were established under in vitro conditions and screened for their antioxidant potential determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioassay. The micropropagation of a selected line, named Vrinda, was established using nodal cultures grown on Murashige and Skoog medium containing benzylaminopurine (1.1 µM), gibberellic acid (0.3 µM), and activated charcoal (0.6%). The antioxidant phytohormones melatonin and serotonin were quantified in the field and greenhouse grown tissues of Vrinda and melatonin levels were found to be consistent in both conditions with higher serotonin levels under field conditions. This integrated approach combining the in vitro selection and propagation offers potential applications in the development of safe, effective, and novel natural health products of Tulsi, and many other medicinal plant species.

Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

Rapid in vitro adventitious rooting and proliferation by leaf and nodal cultures of Momordica cymbalaria Fenzl.

Rapid in vitro adventitious rooting and proliferation by leaf and nodal cultures of Momordica cymbalaria Fenzl.

Antioxidant activity of „Micromeria croatica“ (Pers.) Schott grown in plant tissue culture „in vitro“ versus ones from the natural habitats

Micromeria croatica, like many other species belonging to the Lamiaceae family, is characterized by good antioxidant activity. To avoid the exploitation of natural plant populations, it is recommended to grow them in vitro culture. The present study aimed to examine and compare the antioxidant potential of M. croatica obtain through nodal culture in vitro and collected from natural habitats. Different antioxidant methods were used: DPPH, ABTS, total reducing power, total phenol content, and flavonoid content. The obtained results indicate that the cultivation of plants by the in vitro culture technique stimulates the synthesis of secondary metabolites that promote antioxidant activity. It is increased in micropropagated plants primarily due to the increased phenol content by 136%. The possibility to test and then apply in practice the biological activity of the herb M. croatica is limited by the fact that the species is a local endemic.

Micropropagation, genetic fidelity assessment and phytochemical studies of Clerodendrum thomsoniae Balf. f. with special reference to its anti-stress properties

Clerodendrum thomsoniae commonly known as bleeding heart vine or bag flower which is a good candidate for a new crop for the floriculture industry. In this study, in-vitro callus regeneration of C. thomsoniae through nodal culture has been attempted. Murashige and Skoog’s medium supplemented with 2 mg/l BAP and 0.5 mg/l

PERTUMBUHAN KULTUR TUNAS AKSILAR KENTANG (Solanum tuberosum L.) DENGAN PENAMBAHAN SUPER FOSFAT DAN KNO3 PADA MEDIA AB MIX SECARA IN VITRO

Murashige&Skoog-based medium Potatoes are one commodity that has the potential to be developed as a resource in the context of food diversification, farmers' income riser, non fossil export commodities and raw materials for processing industry. The objective of this research was to find out the effect of SP-36 fertilizer, KNO3 fertilizer, as well as the interaction between the two fertilizers on the growth of potato nodal culture on AB Mix media in vitro. The results of this study are expected to provide economical potato tissue culture media development. This research used factorial complete randomized design. The treatment were SP-36 concentration: 0 ppm; 50 ppm; 100 ppm; and 200 ppm, in combination with KNO3 concentration: 0 ppm; 100 ppm; 200 ppm; 400 ppm; and 600 ppm, The variables observed included number of leaves, leaf color, length of plantlets, fresh weight of plantlets and percentage of plantlets growth. Based on the results of the analysis of variance (ANOVA) F. Calculate < F. Table with the average success of plantlet growth between 87.5-100%. In addition, there are four types of leaf color produced, namely the color of yellowish green, pale green leaves, green, and dark green. Research showed that the interaction between SP-36 fertilizer and KNO3 fertilizer on AB Mix media had no significant effect on all observed variables.

English

English

Enhanced anthraquinone production in presence of silver nitrate in suspension culture of Gynochthodes umbellata (L.) Razafim. & B. Bremer (Rubiaceae), a traditional medicinal plant mentioned in Hortus Malabaricus

Silver nitrate (AgNO3) enhanced production of anthraquinone was standardized in cell suspension cultures of Gynochthodes umbellata, a plant mentioned in the Hortus Malabaricus. The present research investigates the effect of silver nitrate, an abiotic elicitor on production of anthraquinone in in vitro cell suspension cultures of G. umbellata. Friable callus culture was established using in vitro derived leaf segment obtained from the nodal explant culture maintained in Murashige and Skoog (MS) medium containing 2 mg/l benzyl amino purine (BAP) and 3% sucrose. The in vitro derived leaf segments (0.5cm2) were cultured on MS medium containing 1 mg/l 2,4-D and 2% glucose for the production of friable callus. After 30 days of culture, uniform yellow friable callus was inoculated into MS liquid medium containing 1 mg/l 2,4-D and 2 % glucose for raising suspension culture. Uniform cell suspension was transferred to same media constituents and treated with different concentrations of AgNO3 on 25th day of culture. Fresh weight, dry weight and accumulation of anthraquinone content was studied and found that AgNO3 caused a marginal increase in biomass and anthraquinone based on the concentration and duration of AgNO3 treatment. A maximum fresh weight (19.48 g/fwt) dry weight (1.92g/dwt) and highest amount of anthraquinone content (48.62 mg/gdwt) were recorded in MS medium supplemented with 1 mg/l 2,4-D, 2%glucose and 3.5µM AgNO3 after 72 hrs of incubation.

Clonal Propagation and Karyomorphological Study of Solanum torvum Swartz: An Ethnobotanical Species of Tripura, India

An efficient method of clonal propagation through nodal culture of Solanum torvum Swartz. is described. Different concentrations of BAP/Kn alone or in combination with IAA were tested for direct shoot bud induction and proliferation. Lower concentration of BAP/Kn alone produced better shoot proliferation and elongation. Maximum number of shoot proliferation was achieved from MS supplemented with Kn 0.5 mg/l with an average 4.0 ± 1.41 shoots during 28 days of culture. Addition of IAA to the culture media in combination with BAP/ Kn significantly reduced the number of shoot formation. Regenerated plants also produced roots during subsequent culture in the same media supplemented with BAP/Kn alone or in combination with IAA. The easy nature of in vitro rooting of S. torvum was recorded without any separate root induction phase. Regenerated plants were successfully transferred to the field condition. Clonal feature was cytologically confirmed through the study of mitotic metaphase chromosomes of regenerated plants which reveals 2n = 24 somatic chromosomes. Comparative karyomorphological details between the mother and regenerated plants of S. torvum revealed close similarity in their chromosomal complements and falls under the category of "1B" Stebbin’s symmetric index suggesting true to type nature of the regenerated plant.Plant Tissue Cult. & Biotech. 28(1): 69-76, 2018 (June)

Development of an effective propagation protocol for <i>Osbeckia octandra</i> (L.) DC. (Heen Bovitiya)

Effective multiplication technique for mass propagation of Osbeckia octandra (L.) DC, an endemic species to Sri Lanka with potential values as an ornamental flowering plant was investigated. Production of planting materials was evaluated using seeds, conventional vegetative propagation and tissue culture techniques. Effect of substrate on in-vivo seed germination and effect of Gibberellic acid (GA3) on seed germination under in-vivo and in- vitro conditions were investigated. Survival rates of in-vitro germinated seedlings were tested with three different acclimatization environments. Rooting performances of cuttings were evaluated by using both softwood and semi-hardwood cuttings treated with Indole-3-butyric acid (IBA). Effect of shoot multiplication of plant under in-vitro conditions was investigated by establishing nodal cultures. The substrate containing sand: soil at 1:1 ratio was found as the best for seed germination. There was no effect of GA3 on seed germination and a higher (P<0.05) number of seedlings (61.27%) could be obtained by the seeds under in-vitro conditions. Use of non-sterilized growing medium (sand : soil - 1:1) in a polythene sleeve arranged as a single propagator was the most cost effective way of acclimatizing in-vitro grown plants, and use of semi-hardwood cuttings with an initial dip in 500 mg L-1 IBA solution for 10 minutes was the most effective method for rapid plant multiplication. A successful shoot production was achieved by culturing single nodes on full strength Murashige and Skoog medium supplemented with 0.1 mg L-1 Benzyl-aminopurine under in-vitro conditions.

In vitro conservation of Cape gooseberry through slow-growth nodal segment cultures

Abstract: The objective of this work was to evaluate the effects of temperature and osmotic agents on the in vitro conservation of Cape gooseberry (Physalis peruviana). Temperatures at 18 and 25°C, as well as the osmotic agents sucrose, mannitol, and sorbitol were tested. A short-term in vitro conservation of Cape gooseberry can be achieved at 18°C, using 30 g L-1 sucrose.

Elite Screening and In Vitro Propagation of Tinospora cordifolia (Willd.) Miers ex Hook F. &amp; Thoms.

A protocol was established for the high frequency in vitro propagation of Tinospora cordifolia through nodal explant culture. Plants were collected from different geographical areas for screening superior germplasm based on berberine content, the prominent alkaloid present in the plant. Of the different accessions estimated for berberine, candidate elite plant (CEP) 8 recorded highest quantity (757.3 µg g dw) of berberine, therefore selected as elite mother plant for in vitro culture. MS medium supplemented with different concentrations of benzyl adenine (BA) and kinetin were used for in vitro culture of nodal explants derived from elite plant. The best result was obtained in 2 µM BA supplemented medium with 75% response, 2.95 shoots per culture reaching 4.89 cm shoot length. Initial cultures were subsequently used for the subculture with the multiplication rate of 3.83 shoots at the fourth subculture. Use of phloroglucinol at varying concentrations did not improve shoot number. The in vitro raised shoots after 4 weeks of culture were excised for rooting at ex vitro conditions. Two auxins, IBA and IAA at different concentrations were used to induce rooting of which IBA (30 µM) treated microshoots produced significant (p < 0.001) rooting (87.77%) and root number (7.29) in sand and soil (1:1) mixture. The ex vitro rooted plants showed 80% survival. Ex vitro rooting effectively synchronized rooting and acclimatization steps thereby reduced time, labour and cost of production. An estimate of propagation efficiency revealed a total of 1044 hardened plants were produced from a single node within a 6 months culture period.

In Vitro Propagation of Rubber Tree (Hevea Brasiliensis) Using Shoot-Tip and Nodal Cutting Explants

Hevea brasiliensis which belongs to the Euphorbiaceae family is the primary source of natural rubber. Propagation of rubber tree by grafting on to unselected seedlings sustains intraclonal heterogeneity for vigour and productivity which could be improved via in vitro techniques. Micropropagation from rubber nodal and shoot tip explants is possible. In vitro technique is needful to mass propagate disease-free and genetically similar rubber plantlets. In vitro results in increased growth and vigour of rubber tree, However, in vitro techniques of rubber tree have not been given much critical research attention in Ghana. Propagation of H. brasiliensis by in vitro techniques was used to study alternative procedures for mass production of rubber planting materials. Murashige and Skoog (MS) basal medium amended with 30.0g/L sucrose, 100.0mg/L myo-inositol, 2.0g/L activated charcoal, 1.0mg/L silver nitrate AgNO3, 2.0mg/L GA3 and control, 2.5, 5.0, 7.5 or 10.0mg/L kinetin was used to culture both H. brasiliensis shoottip and nodal explants. The MS medium with 5.0mg/L kinetin significantly (P<0.05) enhanced higher shoot development (84.00%), number of shoots (3.60) and leaves (23.40) of the shoot-tip explants compared to other treatments. In nodal explants, the control medium developed higher shoots (94.00%), the height of shoot (4.80cm), number of leaves (19.20), number of shoots (6.00) and number of roots (7.00) than those with kinetin treatments. Conversely, 7.5mg/L kinetin of the nodal culture also performed significantly after the controls. Successful in vitro regeneration of plantlets was achieved using Hevea brasiliensis shoot-tip and nodal cutting explants cultured on an MS medium supplemented with kinetin.