Presence Of Naphthaleneacetic Acid Research Articles

In vitro cultivation of Mouriri elliptica (Mart.) a species with alimentary and medicinal potential using alternative to agar media

Alternative supports can be successfully used in place of agar for 'in vitro' culture to increase seedling vigor. The objective of the present study was to evaluate the efficiency of alternative support materials compared to agar in the 'in vitro' cultivation of 'Mouriri elliptica' (Mart.) in the absence or presence of naphthalene acetic acid (NAA). The alternative support materials used were medium-grain vermiculite, sugarcane ('Saccharum spp.' L.) bagasse and queen palm fiber ['Syagrus romanzoffiana' (Chamisso) Glassman]. For this, young plants with at least two axillary buds under in vivo conditions were transferred to in vitro condition considering four replications with three young plants. After 45 days was evaluated the growth and anatomical explant characteristics. Autoclaving these alternative supports without the media and proceeding the explant desinfestation was an efficient method to contamination control and plant growth in a in vitro condition. No differences were observed between agar, vermiculite and sugarcane bagasse cultures for most growth characteristics evaluated. Greater numbers of adventitious and secondary roots and greater root length were significantly observed in plantlets grown in the presence of vermiculite and the absence of naphthalene acetic acid. In the agar culture, roots had weak points and poorly differentiated tissues, with parenchymal tissue predominating. The addition of 2.0 mg L-1 naphthalene acetic acid used did not stimulate rooting in this specie. So, other auxin source and concentrations should be evaluated in future studies. Both support materials used, vermiculite and sugarcane bagasse, representing promising agar substitutes to obtain seedlings with roots.

Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra.

BackgroundGentiana scabra is commonly known as ‘Longdan’ is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials.ResultsAn efficient hairy root culture system of Gentiana scabra and influence of different plant growth regulators (PGRs) on the production of gentiopicroside, swertiamarin and loganic acid constituents were described. Leaf explants were infected with Agrobacterium rhizogenes, which induced hairy roots up to 21%. The transformed hairy root lines were confirmed by PCR using rolB and rolC gene-specific primers. Among various solid and liquid media, B5 liquid medium resulted maximum root biomass (36- fold higher) in 4-weeks. Quantitative analysis showed loganic acid was 6.6- fold higher in the presence of zeatin (1 mg/l) and gentiopicroside accumulation was 1.8- fold higher in the presence of naphthaleneacetic acid (NAA, 1 mg/l), as compared to the roots of plants grown in greenhouse. On the other hand, 1.4- and 2.5- fold higher gentiopicroside and swertiamarin were observed in the presence of 1.0 mg/l NAA as compared to commercial Gentiana herb No. 2. The result also showed iridoid and secoiridoid contents affected greatly by age, physiology and growing environment of the plant.ConclusionsThe use of hairy root cultures is an excellent alternative to harvesting natural or in vitro grown plants to produce pharmaceutically important metabolites in less time with ensured quality.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-17) contains supplementary material, which is available to authorized users.

Multiple shoot regeneration of plumular apices of chickpea

Chickpea (Cicer arietinum L.) is an important grain legume used almost all over the world. Development of new repeatable and reliable tissue culture protocols is very important for improvement of breeding and genetic transformation studies. It is a highly recalcitrant plant and there is an urgent need to develop a regeneration protocol that can ensure easy multiple and qualitative superior shoots that could be rooted and yield fertile plants. Unconditioned plumular apices and plumular apices preconditioned with 10 mg L^{-1} benzylaminopurine (BA) for 10 days were cultured on Murashige and Skoog (MS) medium supplemented with 0.25-2.00 mg L^{-1} BA with or without 0.25 mg L^{-1} naphthalene acetic acid (NAA). Comparing the 2 culture conditions, the preconditioned explants had 2-fold to 5-fold more regeneration and shoots per explant compared to the unconditioned plumular apices. The presence of NAA in the culture medium positively increased the number of shoots per preconditioned explant at the lower concentrations of BA. In contrast, NAA inhibited the number of shoots per explants and the mean shoot length of the unconditioned explants at all concentrations of BA. An increased sucrose concentration of 45 and 60 g L^{-1} (R1) with 1.00 mg L^{-1} indole-3 butyric acid resulted in 50% rooting with multiple and earlier hardening of shoots. Subculturing of multiple shoots on MS medium containing 45-60 g L^{-1} sucrose (R2) enhanced the rooting frequency by 60%-100%. The rooted plantlets were successfully acclimatized.

Clonal propagation of <i>Wattakaka volubilis</i> through nodal explant culture

A protocol was developed for microporpagation of Wattakaka volubils (L.f) Stapf, an important medicinal plant in India. In vitro shoot multiplication nodal explants of W. volubilis was achieved on solid MS basal medium supplemented with BAP (6-Benzylaminopurine) and NAA (Naphthalene acetic acid). Presence of NAA in the culture medium along with BAP promoted multiplication of shoots than BAP alone. The highest shoot multiplication rate (23.4±0.48) was observed after 28 days of culture on MS basal medium supplemented with 0.6 mg/l BAP and 0.2 mg/l NAA. Elongated shoots produced roots within 6 weeks in half strength MS basal medium supplemented with 0.6 mg/l IBA (Indole-3-butyric acid) and 3% (w/v) sucrose. Rooted plantlets were acclimatized with a survival rate of 95% and transferred to the green-house and thereafter successfully transferred to the field with 90% survival rate. Keywords: medicinal plants, micropropagation, nodal culture, survival rate DOI: http://dx.doi.org/10.4038/cjsbs.v40i1.3406 CJSBS 2011; 40(1): 53-58

In vitro shoot regeneration from preconditioned explants of chickpea (Cicer arietinum L. ) cv. Gokce

The present study reports the successful shoot regeneration of preconditioned mature embryo and embryonic axis explants of chickpea cv. Gokce . Explants were preconditioned with 10 mgl benzylaminopurine (BA) for 7 days followed by culture on Murashige and Skoog (MS) medium containing 0.25, 0.50, 1.00 and 2.00 mg/l BA with or without 0.25 mg/l naphthalene acetic acid (NAA) supplemented with 4 mg/l activated charcoal and 1 mg/l polyvinylpyrrolidon (PVP). Shoot regeneration was recorded on all explants. Maximum number of (14.75) shoots per explants on mature embryo were recorded on MS medium containing 2.0 mg/l BA with 0.25 mg/l NAA. Whereas, 16.83 shoots per explants were recorded on MS medium containing 2.0 mg/l BA. Presence of NAA in the culture medium decreased the mean shoot length of both explants compared to medium devoid of 0.25 mg/l NAA. Regenerated shoots were rooted on MS medium containing 1.0 mg/l indole-butyric acid (IBA) after 4 weeks of culture. Rooted plantlets were transferred to pots for acclimatization under green house conditions. Key words : In vitro, chickpea, pulse treatment, shoot regeneration.

Histologia da embriogênese somática induzida em embriões de sementes maduras de Urochloa brizantha apomítica

O objetivo deste trabalho foi descrever o processo de embriogênese somática em Urochloa brizantha cv. Marandu (Syn. Brachiaria brizantha cv. Marandu) e fornecer subsídios para o aprimoramento dos métodos de cultura de tecidos e transformação genética. Calos embriogênicos foram obtidos por indução em embriões isolados de sementes maduras, e cultivados in vitro, em meio de cultura que continha ácido 2,4-diclorofenoxiacético, 6-benzilaminopurina e caseína hidrolisada. Plântulas foram regeneradas a partir dos calos embriogênicos, na presença de ácido naftalenoacético e cinetina. Esse processo foi descrito morfologicamente por observações em microscopia de luz de secções seriadas semifinas de tecidos fixados, ao longo do processo de regeneração, em FAA [formaldeído (40%): ácido acético glacial: etanol (50%), a 5:5:90 v/v/v]. Os embriões das sementes de U. brizantha cv. Marandu não têm epiblasto e são classificados como do tipo panicoide. Nas condições estabelecidas de cultura in vitro, calos embriogênicos e embriões somáticos de U. brizantha cv. Marandu, desenvolvem-se a partir de células meristemáticas do escutelo.

Bulblet regeneration from ex vitro root explant in lily hybrids

The influence of growth regulators on <i>in vitro</i> bulblet formation from <i>ex vitro</i> roots was studied in asiatic and oriental hybrids of <i>Lilium</i> The root segments (3–4 mm) isolated from the middle zone of 2–3 cm <i>ex vitro</i> root were cultured on Murashige and Skoog (MS) medium containing 1 or 1.5 mg/dm<sup>3</sup> naphthalene acetic acid (NAA) and/or benzyladenine (BA). Bulblets were not produced in the presence of NAA and BA alone. A significant increase in the per cent explants producing bulblets was observed with 1.5 mg/dm<sup>3</sup> NAA and 1 mg/dm<sup>3</sup> BA. Maximum number of bulblets and average fresh weight per bulblet was observed with 2 mg/dm<sup>3</sup> NAA and 1.5 mg/dm<sup>3</sup> BA after 90 days of culture. No differences were found among cultivars in bulblet regeneration of explant or bulblet number although more weighty bulblets occurred in cv. Apeldoorn. About 82% bulblet survival was recorded in coco peat after 30 days of transfer to pots.

Influence of growth regulators and nitrogenous compounds on in vitro bulblet formation and growth in oriental lily

The influence of growth regulators and nitrogenous compounds on in vitro bulblet formation and growth was studied in two hybrids of <i>Lilium</i>. Bulbscales isolated from pre-cooled bulbs of hybrids Rosato and Marco Polo were used. The basal portion with plate (5 × 6 mm) of inner bulbscales was cultured on Murashige and Skoog (MS) medium containing 0.5 or 1 mg/dm<sup>3</sup> naphthaleneacetic acid (NAA) and/or benzyladenine (BA). The presence of NAA (0.5 mg per dm<sup>3</sup>) showed higher explant regeneration, producing about three bulblets per explant as compared to control. About four bulblets per explant were produced at both concentrations of BA. The bulblets with significantly higher fresh weight were obtained on medium containing NAA. Approximately a three-fold increase of bulblet fresh weight was observed with all the concentrations of TDZ in both cultivars. The bulblets cultured with nitrogenous compounds after attaining the size of 14−16 cm flowered during the second year of the growing period without any phenotypic variations.

Plant regeneration from callus of Cuscuta reflexa – an angiospermic parasite – and modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid

Callus induction and plant regeneration system for an obligate parasite, Cuscuta reflexa (family Cuscutaceae), has been established. Callusing was induced from explants of basal portion of seedlings as well as from shoots (vines) in a modified Murashige and Skoog (MMS) media supplemented with 1 mg·L –1 benzyl adenine (BA) and 3 mg·L –1 naphthalene acetic acid (NAA) (MMS-1). The MMS media containing 1 mg·L –1 BA (MMS-2) was found to be better than the MMS-1 media for callus proliferation and long term maintenance. Use of glucose in place of sucrose enhanced callus induction and growth. The browning of callus was substantially reduced by supplementing 0.1 mg·L –1 gibberellin to the MMS-1 media. Shoot regeneration from the green callus was achieved in the MMS media containing 3 mg·L –1 NAA and 1 mg·L –1 kinetin. Cuscuta callus was characterised by having several folds higher peroxidase activity than those of culture-raised and field-grown vines. Catalase activity also revealed a pattern similar to that of peroxidase except for the fact that it could not be detected in field-grown vines. Developmental profiles of peroxidase and catalase, during the growth of the callus in the absence and presence of NAA, revealed inhibition of both the enzymes by NAA, though inhibition of peroxidase was stronger as compared to that of catalase. The inhibitory effect of NAA was also evident when in-gel activity staining of the two enzymes were performed. In callus, three acidic and four basic isoperoxidases were present, while in field-grown vines, two acidic and one basic isoperoxidases were present. Investigation of the effect of salicylic acid on catalase and peroxidase revealed inhibition of both enzymes in a concentration dependent manner during seedling growth. The inhibitory effect of salicylic acid was also evident when in-gel enzyme activity staining of the two enzymes was done. The results are discussed in light of the proposed mode of action of salicylic acid.

Bud regeneration from inflorescence explants for rapid propagation of geophytes in vitro.

Bulbs, corms and other subterranean storage organs are commonly used as explant source material for the establishment of geophytes in vitro. The inflorescence stalk was found to be a good alternative source of explants to overcome explant contamination originating from underground storage organs. Inflorescence explants of Allium, Dichelostemma, Eucrosia, Gladiolus, Haemanthus, Hyacinthus, Narcissus, Nerine and Ornithogalum were used to establish cultures in vitro. The regeneration potential of the inflorescence was compared with regeneration from bulb twin scales or from apical buds isolated from corms. Gladiolus (Iridaceae) explants isolated from the floral stem just below the expanding florets, still enclosed in the bracts, were highly regenerative in the presence of naphthalene acetic acid (NAA) and kinetin. In the presence of 2,4-dichlorophenoxyacetic acid and benzyl aminopurine (BA) in the medium, explants isolated from the tissue at the junction between the peduncle and the pedicels of a young Nerine (Amaryllidaceae) inflorescence regenerated several buds. The scapes of young unemerged inflorescences taken from sprouting bulbs of Narcissus (Amaryllidaceae), following a 15 °C storage treatment, regenerated buds in the presence of NAA, BA, elevated phosphate and adenine sulfate in the medium. The number of buds regenerated depended on the location on the scape from which the explant was isolated, and on the duration of the 15°C treatment. In Allium (Alliaceae), capitulum tissue between the flower pedicels regenerated buds. Explants excised from the peduncle, as well as the pedicel-peduncle junction of Dichelostemma (Alliaceae), Ornithogalum, Hyacinthus (Hyacinthaceae) and Eucrosia (Amaryllidaceae) regenerated several buds in each type of explant. In the case of Haemanthus (Amaryllidaceae), pedicel-peduncle junction explants regenerated buds only when excised from inner whorl florets. Propagation protocols and the potential use of expediently isolated inflorescence explants for efficient micropropagation of geophytes are discussed.

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested.

Enhancement of Naphthaleneacetic Acid-Induced Rhizogenesis in TL-DNA-Transformed Brassica napus without Significant Modification of Auxin Levels and Auxin Sensitivity

Determination of the abscisic acid and indoleacetic acid (IAA) contents of floral stem segments of nontransformed and pRi A(4) T(L)-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor) using a high performance liquid chromatography-enzyme-linked immunosorbent assay procedure and mass spectrometry controls showed that IAA levels were not modified. The regeneration abilities of the in vitro cultured explants were compared on media supplemented with several plant growth regulator combinations. No regeneration occurred on hormone-free media, and shoot production was similar in both genotypes when supplemented with benzyladenine. In the presence of naphthaleneacetic acid (NAA), transformed explants were characterized by faster root regeneration and reduced shoot organogenesis. The optimum for root formation was the same in nontransformed and transformed plants, but the sensitivity threshold was slightly lower in the latter. The NAA inductive period was shorter (14 versus 22 h) with transformed tissue. Root neoformation occurred about 72 h earlier on transformed explants. Our results suggest mainly that there is an acceleration of the auxinic signal transduction and/or that the events preliminary to the formation of roots occur faster in the transformed tissues than in the normal ones.

Advances in haploid plant induction in onion ( Allium cepa L.) through in vitro gynogenesis

The aim of this research was to improved the efficiency of previous techniques for the production of haploid onion plants through in vitro gynogenesis. Taking into account the last published results, two experiments were carried out: (1) unpollinated ovule culture of the long-day cv ‘Erso 1’; (2) ovary and whole flower culture of the long-day cvs ‘Cosmic’, ‘Morgana’ and of the short day cvs ‘KLR’, ‘Senshyu Yellow’. The yield of gynogenic embryos from ovule culture significantly increased when: (1) 100 g/l of sucrose was supplied in both preculture and culture media and (2) 2,4-dichlorophenoxyacetic acid (2,4D) and 6-benzyladenine (6BA) were both used at 2 mg/l in the preculture medium. Embryo yield from ovary culture was significantly improved in the four cvs by using a modified BDS medium containing 100 g/l of sucrose, 1 mg/l of naphthaleneacetic acid (NAA) and 2 mg/l of 6BA; a high percentage of embryos were induced from direct flower culture by using a modified BDS medium containing 100 g/l of sucrose, 2 mg/l of 2,4D and 2 mg/l of 6BA. Statistical analysis of the data revealed a close relationship between the kind of cultured organ (ovary or flower) and the type of auxin in the medium. Ovary culture gave the best yield in the presence of NAA while 2,4D gave good results in both ovary and flower culture. Thirtysix per cent of the embryos obtained could regenerate whole plantlets and 88.3% of the regenerants were haploid. The capacity of each type of organ to produce embryos was also influenced by the genotype used and, perhaps, by the environmental conditions of donor plant growth. For this reason, gynogenesis induction should always make use of both ovary and flower culture.

The formation of adventitious buds in vitro on micro-cross sections of hybrid Populus leaf midveins

Micro-cross sections (MCS) of midveins from hybrid Populus leaves were1used to study adventitious bud formation in vitro. MCS of 100 μm, 200 μm, 300 μm, 400 μm, and 500 μm in length were made with a Lancer Vibratome and cultured in Woody Plant Medium (WPM). In the presence of naphthaleneacetic acid (NAA), 400 and 500-μm sections had equal bud forming capacity and produced as many adventitious buds (average four shoots/MCS) as a 1-cm explant under the same culture conditions. This means that MCS of midveins are approximately 25 times more efficient in shoot regeneration than a 1-cm explant. The conditions used were suboptimal for the 200-μm and 100-μm sections. Shoot numbers from the 200-μm MCS were considerably reduced and the 100-μm MCS did not form buds at all. The size of lamina remnants was critical with 1–2 mm on each side of the midvein being optimal for bud formation. The MCS (300 and 400 μm) were not only an effective system for production of adventitious shoots, but also allowed direct microscopic observations of early morphogenetic events in vitro. These direct observations indicate that the callus and buds are initiated from a group of small chlorophyllous cells located in areas of the midvein that are adjacent to the mesophyll cells.

Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro

Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained from 22.0 μM cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.

Regeneration of Plantlets By Embryogenesis from Callus Cultures of Carica Candamarcensis

Summary Somatic embryogenesis was induced on callus derived from hypocotyl sections of Carica candamarcensis growing on several nutrient media in the presence of naphthaleneacetic acid (NAA) (1-5 mg/l) and kinetin (1 mg/l) within 3 months culture in the light. Embryoids developed into plantlets when subcultured on a half-strength Murashige and Skoog medium (MS) supplemented with indoleacetic acid (IAA) (0.5 mg/l) and casein hydrolysate (200 mg/l), Callus derived from immature pericarp tissue or peduncles did not show morphogenetic responses.

IN VITRO PROPAGATION OF OTTAWA 3 APPLE ROOTSTOCK

Methods were developed for obtaining normal shoot cultures and for rapid in vitro proliferation and rooting of Ottawa 3 apple rootstock from meristem tips. The presence of naphthalene acetic acid (NAA) and benzyladenine (BA), both at concentrations of either 0.5 or 1.0 mg/L in the culture medium, was most effective for in vitro shoot proliferation, but growth was abnormal. Normal growth was achieved when shoots were cultured with a combination of 0.5 mg/L NAA, 0.5 mg/L BA and 5.0 mg/L gibberellic acid. One hundred percent rooting was achieved after 2 wk on agar medium supplemented with 6.25 mg/L indole butyric acid.Key words: Tissue culture, Malus, meristem cloning, growth regulators

Root and Shoot Development from Sugarcane Callus Tissue1

Growth of shoots differentiated from sugarcane (Saccharum sp.) callus was enhanced by 5 mg naphthalene acetic acid (NAA)/I in the absence of coconut water, while 0.2 mg 2,4‐dichlorophenoxyacetic acid (2,4‐D)/1 stunted their growth. In the presence of NAA, a crown developed at the base of the shoot on which new shoots and roots developed, grew, and formed a clump of plantlets.Root growth could also be induced by trimming the leaves or by incubation at 15C for 4 to 6 weeks. Leaf trimming resulted in more vigorous root growth than cold treatment, although clonal differences were observed.Results indicate that a medium which stimulates differentiation does not necessarily stimulate shoot growth, and the hormonal requirements for shoot differentiation differ from requirements for root induction in sugarcane callus.