Use Of MS Medium Research Articles

In vitro regeneration of Senna alata (L.) Robx. (Fabaceae): An ancient medicinal plant

Senna alata, a legume of medicinal importance, presents antifungal and antibacterial antioxidant activity. The main limitation is the presence of seeds with integument dormancy within natural populations. This study aimed to establish an efficient protocol for the in vitro propagation and acclimatization of seedlings. The seeds were disinfected with different concentrations of NaOCl; for the regeneration of the shoots, cotyledon segments were inoculated in MS and WPM medium, supplemented with plant growth regulators, auxins (1-NAA and 2,4-D), interaction with cytokinins (TDZ and BAP), and activated carbon. The seedlings were acclimated in nursery conditions using vermiculite, sand, and soil substrates. Using NaOCl (0.5%) for 30 min resulted in 98.0% disinfected seeds while 95.4% of scarified seeds germinated. High node and shoots numbers (8.3 ± 0.4; 12.6 ± 0.8, respectively) were obtained when using the MS medium containing 1-NAA + TDZ (0.1 + 1.0 mg L-1), while the use of MS medium supplemented with 2,4-D + TDZ (0.1 + 1.0 mg L-1) resulted in longer shoots (6.0 ± 0.5 cm). Using WPM medium with IBA + BAP (1.0 + 0.1 mg L-1) improved rooting (14.0 cm ± 1.3) and the number of roots (5.0 ± 0.0) in the treated plants. The vermiculite substrate with sand promoted 81.5% of acclimatized plants. This study allowed obtaining high levels of germination, regeneration in vitro and acclimatization of S. alata.

Micropropagation of lavender: a protocol for production of plantlets

ABSTRACT Lavender (Lavandula dentata) has several medicinal applications, both in the fresh form and after industrial processing. Productivity increase could be obtained through the mass propagation, expanding the possibility of obtaining raw material for production of essential oil. The objective of this work was to develop a micropropagation protocol from explants obtained from seeds. Aspects of in vitro germination, multiplication, elongation and rooting were evaluated and, in sequence, the acclimatization process as well. The in vitro germination of lavender seeds can be obtained using a medium composed of agar and water. For multiplication, the use of MS medium (Murashige & Skoog, 1962) added with 5.0 µM 6-benzylaminopurine to the nutrient medium is the most appropriate. For elongation and rooting, the use of growth regulators is unnecessary. Acclimatization can be obtained directly by transferring the explant to plastic bags with substrate, kept in a greenhouse. It was possible to develop a protocol for the in vitro cultivation (establishment, multiplication, elongation, rooting and acclimatization) of lavender from explants of seed origin.

Plantlet Formation and Acclimatization of Sugarcane cv. PS 881 with Different Types and Concentration of Auxin

This research has been carried out with a view to induce rooting and plantlet formation, followed by acclimatization. Micro shoots of sugarcane cv. PS 881 were cultured on Murashige and Skoog medium supplemented with different types and concentration of auxins for root induction. This research conducted experimentally using a split-plot design. The main plots were three types of auxins, which consisted of IAA, IBA, and NAA. The subplots were auxin concentrations with four levels, i.e. 0 I¼M, 5 I¼M, 10 I¼M, and 15 I¼M. Significantly faster root emergence time and higher number of roots observed in the Murashige and Skoog basal medium supplemented with 10 I¼M NAA. The best root length obtained in the Murashige and Skoog basal medium supplemented with NAA 0 I¼M. Plantlets derived from NAA 10 I¼M treatment showed the best performance during acclimation with a 100% survival rate. NAA at a concentration of 10 µM considered to be the best treatment in plantlet formation and acclimatization of sugarcane cv. PS 881. This study showed that the use of MS medium with 10 uM NAA is able to increase the growth of PS 881 sugarcane plantlets. The results of this study can increase the availability of high quality seedlings and increase national sugar production.

Temporary Immersion Bioreactors: Establishment of Cassava

Cassava (Manihot esculenta Crantz-Euphorbiaceae) is one of the most important crops for world agriculture. It is the main source of many family’s calorie intake and contributes toward food security. Its cultivation is necessitated sterile vegetative propagules with essential physiological and genetic quality, which can be obtained by plant tissue culture techniques. However, the maintenance of in vitro propagation processes requires automation, making large-scale production feasible. This work aimed at developing protocols for the establishment of cassava cultivation in Temporary Immersion Bioreactors (TIBs). Two nutrient media (MS medium reduced to half-strength of the salt concentration and the same formulation plus 4,43µM of 6-Benzylaminopurine [BAP]) and different culture cycles were evaluated in Bioreactors (15 minutes of immersion and 4 hours of stationary phase [15M4H]; 15 minutes of immersion and 8 hours of stationary phase [15M8H]). The establishment of culture in bioreactors was promising. Based on our results, the use of MS medium without growth regulators was more effective for shoot formation; there were differences in the 15M4H cycle when growth regulators were used. However, the 15M8H cycle had better performance in both treatments: with or without growth regulators. The use of culture medium without BAP led to better rooting; calli formation increased when BAP was used. The present research investigation indicated the use of MS medium reduced to half- strength the salt concentration without the presence of growth regulator and the use of the culture cycle constituted by 15 minutes immersion and 8-hour intervals in stationary phase was effective for Cassava cultivation in TIBs.

Effect of utilization of tomato extract and foliar fertilizer as media on shoots multiplication of banana cv Ambon in vitro

The good quality banana seeds are still difficult to obtain. There are two ways to provide seeds, namely conventional and tissue culture (in vitro). Tomato extract contains natural ZPT or phytohormone which can be utilized in modification of banana tissue culture media. The aim of this study was to determine the influence of media types and tomato extracts in various concentrations for multiplication of banana cv. Ambon in vitro. The study was conducted from October - December 2016 at the Tissue Culture Laboratory of Horticulture Seed Center, Salaman, Magelang. The experimental design used was completely randomized design with two treatment factors. The firs factor was media type with the addition of foliar fertilizer, the second factor was modification of tomato extract with 4 levels. The results showed that the different of the treated media treatment did not affect the emerge of leaf and leaf length, the number of roots and root length. The emerge of the leaves of all treatments occurred at 6 days after planting with the highest average length was obtained in MS treatment with a combination of tomato extract 50 ml/l (10.3 cm). The use of MS medium with a combination of tomato extract 50 ml/l generated the average root number 15.5 with a root lengths 7.5 cm. Substitution of MS medium with tomato extract and foliar fertilizer did not show better results compared to the use of MS media in the multiplication of banana shoots in tissue culture.

Jasmonic acid changes the composition of essential oil isolated from narrow-leaved lavender propagated in in vitro cultures

The aim of the present study was to determine the effect of jasmonic acid added to the culture medium on composition of Lavandula angustifolia essential oils. The chemical composition was determined by gas chromatography coupled to mass detector (GC/MS). The experiment was conducted with the use of MS medium supplemented with increasing concentration of JA (0.2, 0.5, 1, 1.5 mg∙dm−3). It was found that the analysed essential oils varied in terms of chemical composition depending on the content of JA in the medium. All obtained essential oils were characterised by a high content of σ-cadinene (17.06–29.64%), borneol (6.66–17.47%), caryophyllene oxide (8.30–14.01%), τ-cadinol (4.87–9.16%), beta-caryophyllene (3.54–6.57%), 1.8-cineole (1.94–5.87%), β-pinene (1.48–3.05%), geranyl acetate (0.56–2.14%) and myrtenal (0.65–2.14%).

In vitro establishment of blackberry (Rubus sp.) cultivar 'Xavante'

ABSTRACT: The objective of this study was to evaluate the in vitro establishment of 'Xavante' blackberry under different conditions of plant luminosity and concentrations of salts of MS medium. The maintenance of 'Xavante' blackberry plants in the absence of light, with the use of MS medium with salt concentrations of 75% and 100% reduce bacterial contamination and the percentage of explants oxidized and provide the highest percentage of surviving and established explants. The 50% salt dilution in MS medium is not recommended when plants are subjected to the absence of light due to the high percentage of oxidized explants and consequently lower survival and establishment.

Germinação in vitro e criopreservação de sementes de Zinnia elegans

Zinnia elegans Jacquim is a species of big cutting and ornamental potential, but its seeds have low germination percentage. This study aimed to establish a protocol for in vitro germination and water content for the cryopreservation of seeds. The length, width, thickness, and weight of one thousand seeds were determined. MS and WPM culture mediums, as well as the concentrations of gibberellic acid (GA3) were tested regarding in vitro culture. For cryopreservation, the seeds were subjected to drying on silica gel or laminar flow for different times. Then, the seeds were stored in liquid nitrogen (-196° C) for 24 hours. After this period, the seeds were thawed and inoculated into culture medium. The biometric results of seeds showed 8.6 mm average length, 4 mm width, and 0.9 mm thickness. The weight of one thousand seeds was 851 mg, characterizing them as small, lightweight, and easy to disperse. The use of MS medium with no addition of GA3 enhanced germination (67%). The initial moisture content of Z. elegans seeds was 9%. Seeds subjected up to 2 hours of drying in both treatments obtained 23% germination in silica gel and 19% in laminar flow. Z. elegans seeds may be desiccated by 4% moisture content and cryopreserved with no loss of germination potential.

IN VITRO GERMINATION AND CHEMICAL COMPOSITION OF ZANTEDESCHIA AETHIOPICA CALLUS

Zantedeschia aethiopica is often used for cut flowers. However, seedling production is very limited, mainly due to diseases. The objective of this work was to develop an in vitro propagation protocol for the species and to analyse its callus chemical composition. Seeds obtained from mature and immature fruits were inoculated in half-strength MS and WPM culture media supplemented with 15 g L-1 sucrose. Different GA3 concentrations (0.0, 0.2, 0.4, 0.6 and 0.8 mg L-1) were added to the MS medium in the presence of 15.0 g L-1 sucrose. For callus induction, nodal segments were inoculated on MS medium supplemented with 2,4-D (0.0, 1.0, 2.0 and 3.0 mg L-1) and 15 g L-1 sucrose. For callus chemical analysis, total phenols, starch, total soluble sugars, reducing and non-reducing sugars, lipids and protein were quantified. Seeds from immature fruits presented 33% germination in WPM medium while seeds from mature fruit presented 24% germination on MS medium. Regarding the use of MS medium plus 15 g L-1 sucrose with different GA3 concentrations, the higher germinated rate, using immature (20%) or mature fruit seeds (10%), were obtained with 0.8 or 0.2 mg L-1 GA3, respectively. The callus induction occurred in media supplemented with 1.0 mg L-1 (53%) and 2.0 mg L-1 (47%) of 2,4-D. The callus chemical composition was as follows: total phenols (802.47 mg 100 g-1), starch (19.8 g 100 g-1), total soluble sugar (3.1 g 100 g-1), reducing sugar (1.2 g 100 g-1), non-reducing sugar (1.8 g 100 g-1), ether extract (1.0%) and protein (20%). These compounds may help the understanding of cellular dedifferentiation processes that take place in Z. aethiopica species.

IN VITRO REGENERATION OF ANNATTO (BIXA ORELLANA L.) PLANTLETS FROM NODAL AND INTERNODAL ADULT STEM SEGMENTS

This work aimed to evaluate the in vitro morphogenic response of adult explants of Bixa orellana and to establish a regeneration protocol from adult nodal and internodal explants from 16-year-old adult plants propagated by air-layering. For internodal segments the following treatments were used: T1, MS medium lacking growth regulators (MS0); T2, MS + 4.92 µM 6-I³,I³-dimetylalylamino-purine (2-iP); T3, MS + 4.56 µM Zeatin (ZEA); T4, MS + 4.44 µM 6-benzyladenine (BA); T5, MS + 4.54 µM Thidiazuron (TDZ), with ten repetitions for each treatment. Thirty days after establishment of INS, better regeneration rates were achieved with ZEA and TDZ, when compared to BA, 2-iP and MS0. For nodal segments the best results were obtained using the combination of 2iP and BA. In conclusion, the promotive effect of direct adventitious organogenesis from adult internodal segments or axillary shoot proliferation from adult nodal segments, in B. Orellana, is dependent on the type and concentration of cytokinin used. Histological analyses showed the presence of meristemoids in the regenerated tissue. This region was characterized by the typical organization of the vascular system into a cylindrical structure, with central xylem, and with fast proliferating and radially distributed cells. Our analyses also revealed that all budding activity had a vascular system connected to the original explant, confirming the organogenic pathway-related ontogeny. We suggest the use of MS medium supplemented with cytokinins ZEA or TDZ and 2iP or BA for regeneration of annatto from INS and NS, respectively, which will reduce the regeneration time of this species in vitro. The protocol could be further used in transformation systems applied to adult elite genotypes to improve bixin production. This is the first report on adventitious regeneration from adult explants of annatto.

In vitro behaviour of Aspasia variegata, an epiphytic orchid from the Brazilian Cerrado

Aspasia variegata occurs naturally in the savanna of the Mato Grosso do Sul State, Brazil and it has been widely collected for its beautiful flowers. Additionally, its habitat has been greatly reduced and little or no investigation of its spread has been performed. Aiming to establish a protocol to obtain seedlings of the orchid A. variegata, different compositions of culture medium were tested to identify which one provided better in vitro growth and development and to assess the influence of these media in seedling acclimatisation. Thus, seeds obtained from mature capsules were inoculated in Knudson culture medium for 120 days until the protocorm stage. They were transferred to different culture media formulations, including MS and Knudson with half or full formulation, and 3.0 and 6.0g L-1 activated charcoal were added to them or not. After 180 days of protocorm inoculation, seedlings were evaluated for length of roots and shoots, number of roots and leaves, and chlorophyll contents. After that, seedlings were transferred to trays containing a mixture of Plantmax® and coconut fibre (1:1) for acclimatisation. Best results for the in vitro growth of A. variegata were obtained with the use of MS medium supplemented with 6.0g L-1 activated charcoal. Higher levels of chlorophyll were obtained, however, in treatments containing MS salts without activated charcoal presence, and lower levels in media containing Knudson salts with the presence of activated charcoal. The seedlings originated by higher chlorophyll levels during in vitro cultivation presented the highest survival rates and better development in the acclimatisation phase.

Development of genotype-independent regeneration system for transformation of rice (Oryza sativa ssp. indica)

Rice (Oryza sativa ssp. indica) is an important economic crop in many countries. Although a variety of conventional methods have been developed to improve this plant, manipulation by genetic engineering is still complicated. We have established a system of multiple shoot regeneration from rice shoot apical meristem. By use of MS medium containing 4 mg L(-1) thidiazuron (TDZ) multiple shoots were successfully developed directly from the meristem without an intervening callus stage. All rice cultivars tested responded well on the medium and regenerated to plantlets that were readily transferred to soil within 5-8 weeks. The tissue culture system was suitable for Agrobacterium-mediated transformation and different factors affecting transformation efficiency were investigated. Agrobacterium strain EHA105 containing the plasmid pCAMBIA1301 was used. The lowest concentration of hygromycin B in combined with either 250 mg L(-1) carbenicillin or 250 mg L(-1) cefotaxime to kill the rice shoot apical meristem was 50 mg L(-1) and carbenicillin was more effective than cefotaxime. Two-hundred micromolar acetosyringone had no effect on the efficiency of transient expression. Sonication of rice shoot apical meristem for 10 s during bacterial immersion increased transient GUS expression in three-day co-cultivated seedlings. The gus gene was found to be integrated into the genome of the T(0) transformant plantlets.

An efficient micropropagation system for Celastrus paniculatus Willd.: a vulnerable medicinal plant

A micropropagation protocol was developed for Celastrus paniculatus, a vulnerable medicinal plant. Cultures were initiated from nodal explants collected from young shoots of a 12-year-old plant in MS basal medium. An average of five shoots were produced in MS medium supplemented with 1.5 mg l−1 benzyl adenine (BA) and 0.1 mg l−1 naphthalene acetic acid (NAA) after two subculture cycles with a 30-day interval. Continuous subculture in the same medium for three more cycles resulted in reduction of the number of multiple shoots (2 or 3 shoots), vitrification of the shoots, and callus formation. Vitrification of cultures could be overcome by the use of MS medium supplemented with lower concentrations of BA (0.05mg l−1) and NAA (0.01mg l−1). Among the various rooting trials, ex vitro rooting of shoots with simultaneous hardening was most efficient. The method standardized in the present study is simple, as it eliminated separate steps for in vitro rooting and hardening. Qualitative chemical similarity of the tissue culture regenerants with the mother plant was confirmed using high performance thin-layer chromatographic (HPTLC) profiling.

<i>In vitro</i> propagation of pointed gourd (<i>Trichosanthes dioica</i> Roxb.) through encapsulated shoot tips

Plants were regenerated from encapsulated shoot tips of pointed gourd. Shoot tips isolated from multiple shoot cultures of AM-8 and AM-15 cultivars of pointed gourd were encapsulated in sodium alginate beads. For germination and shoot proliferation, encapsulated shoot tips (artificial seed) were cultured in MS basal medium containing different concentrations and combinations of BAP and NAA. Use of MS medium resulted in 90% conversion of encapsulated shoot tips into plantlets. The results exhibited that BAP and combinations of BAP and NAA play an important role in germination of artificial seed being encapsulated by sodium alginate beads. The plantlets were successfully established in earthen pot. Under the present study, limited experimental efforts have been made to establish the protocol for encapsulating the shoot tips for the production of artificial seed and their subsequent regeneration. It is the first report in Bangladesh in developing artificial seed production technique using vegetative tissue of pointed gourd. Key Words: In vitro propagation; pointed gourd; shoot tips. DOI: 10.3329/bjar.v34i4.5832Bangladesh J. Agril. Res. 34(4) : 555-563, December 2009