Nodal Segment Explants Research Articles

Effect of interaction between different plant growth regulators on in vitro shoot multiplication of Citrus latifolia Tan. (persian lime)

In this paper a shoot multiplication is described for Citrus latifolia Tan. (persian lime) using nodal segment explants of young one – old – year trees by two different pathways contain with and without callusing phase. The best result for multiple shoot formation and regenerated shoot formation was 3.2 and 2.6 shoots per explants with 4.44 µM BA plus 0.053 µM NAA and 4.44 µM BA plus 0.049 µM IBA respectively. Alike shoot regeneration, shoot elongation was occurred in medium with 4.44 µM BA and 0.049 µM IBA. Micropropagated and regenerated plants are under other experiments.

Factors influencing large-scale micropropagation of Sphagneticola calendulacea (L.) Pruski and clonality assessment using RAPD and ISSR markers

Sphagneticola calendulacea (L.) Pruski (synonym Wedelia chinensis (Osbeck) Merrill) has great significance in traditional systems of medicine and is popularly known for its hepatoprotective properties. A comprehensive micropropagation technique for its conservation and season-independent, large-scale production was established. Initial culture was established in different basal media [Murashige and Skoog (MS), Schenk and Hildebrandt (SH), Gamborg’s (B5), and Woody Plant medium (WPM)] following a non-toxic, simple, and effective sterilization of ex vitro shoot tip (ST) and nodal segment (NS) explants. MS medium favored the highest rate (∼87.27%) of multiple (∼2.03) shoot bud initiation from NS explants that was further multiplied with supplementation of thidiazuron (TDZ), kinetin, N6-(Δ2-isopentenyl) adenine, or zeatin in combination with indole-3-acetic acid (IAA) or α-naphthaleneacetic acid (NAA). MS medium containing TDZ (0.2 mg L−1) and NAA (0.05 mg L−1) resulted in the maximum (∼98.82%) response with the highest number (∼33.12) and length (∼6.04 cm) of multiple shoots. The maximum number (∼12.18) and length (∼6.24 cm) of healthy roots per plant was achieved on ½ MS medium plus 1 mg L−1 IAA. In a simple acclimatization process of 6 wk, the survival rate of plantlets cultured in a combination of soil, sand, and vermicompost (1:1:1; v/v/v) was 82.00%. DNA fingerprinting of the in vitro-regenerated plantlets via analysis of random amplification of polymorphic DNA and intersimple sequence repeat markers showed clonal fidelity of the plantlets with the mother plant.

Effect of growth regulators on in vitro multiplication of potato (Solanum tuberosum L.) cv. diamant

Shoot tip and nodal segment explants from field grown plants were used as experimental materials in this investigation. All explants were cultured on MS medium supplemented with various plant growth regulators. For surface sterilization of explants, HgCl2 (0.1%) for 2 minutes was found to be most effective for complete killing of surface pathogens and getting healthy tissues. Shoot regeneration was observed from both shoot tips and nodal explants for the studied plant. Various concentrations of BAP (0.1, 0.2, 0.3mg/l) and GA3 (0.1, 0.2, 0.3mg/l) were used for shoot multiplication. In case of BAP, the highest length of shoot was recorded 4 cm and the highest percentage of shoot multiplication (73%) was noticed in MS+0.2mg/l BAP. And in case of GA3, the highest response for shoot multiplication (82%) was noticed in MS+ 0.1 mg/l GA3.But among all of the media formulations used in this experiment, the highest response for shoot multiplication (95%) within7-10 days was noticed in hormone free MS media. In case of root regeneration, the highest percentage (96%) of root induction was recorded in MS medium supplemented with no hormone. Asian Australas. J. Biosci. Biotechnol. 2017, 2 (1), 81-88

Effect of different factors on in vitro growth and shoot proliferation of guava (Psidium guajava L.) cv. Allahabad Safeda

In the present experiment an attempt has been made to optimize the effect of different medium, levels of sucrose, pH, adenine sulphate and light intensity for culture establishment and shoot proliferation in guava using nodal segment explants. Culture establishment was greatly influenced by media types. Maximum establishment ofexplants (74.57%) was recorded on MS medium supplemented with 2.0 mg/l BAP + 0.2 mg/l IBA. In proliferation study, the maximum shoot proliferation was observed in MS + 1 mg/l BAP + 0.25 mg/l GA3. Sucrose 3% was found to be more favorable for maximum proliferation and growth of shoots moreover, it was reduced gradually as increased or decreased levels of sucrose from 30 g/l. among the various pH levels tested, pH 5.5 recorded maximum number of shoots (8.08) and maximum length of shoots (3.75 cm).In proliferation medium the length of shoot, numbers of shoots and growth rate were increased as increased the adenine sulphate level in the medium. Maximum proliferation was observed on 160 mg/l adenine sulphate in the medium. High light intensity 3000 lux was found to be most suitable for proper growth of regenerated shoots. Low light intensity (1000 lux) resulted in stunted growth. All the above factors significantly influenced shoot multiplication and growth. Thus, optimization of these factors showed significantly increased number of shoots and rapid multiplication. This could be useful for the in vitro production of cost effective healthy planting material of guava

Effects of the bendazol fungicide on in vitro development of the nim (Azadirachta indica A. JUSS)

Two experiments were conducted to evaluate Bendazol fungicidal effects in neem micropropagation. In these experiments, the nodal segment explants from <em>in vitro</em> plants were used. In the first experiment, the explants remained in DKW culture medium for a period of 30 days containing different concentrations of Bendazol (M1 -50, M2 - 100, M3 - 200, and M4 - 400 mg.L<sup>-1</sup>). The control treatment (M0) was prepared with DKW medium + BAP (0.225 mg.L<sup>-1</sup>). In the second experiment, the explants were maintained for only one week in media supplemented with Bendazol or BAP, and then they were transferred and kept in free Bendazol/BAP media for three weeks. In each experiment, the design was completely randomized with five treatments, 10 replicates per treatment, and one explant/cultivation flask. The variables analyzed included the formation of calluses and roots, lateral bud development, shoot height, contamination and plant death. There was no significant difference in tree variables (shoot, callus formation and shoot height) between treatments in both experiments. There was no death, plant contamination and rooting during the experiment. The results indicate that Bendazol can be used at low doses for <em>in vitro</em> neem cloning thereby replacing BAP and ultimately reducing production costs.

In vitro propagation and ex vitro rooting of Aerva lanata (L.) Juss. ex Schult.: a rare medicinal plant

The present study describes an improved and reproducible in vitro regeneration system for Aerva lanata using nodal segment explants obtained from a mature 2 years old plant. Direct organogenesis of shoots from nodal segments was achieved by culturing on Murashige and Skoog (MS) medium supplemented with 1.5 mg l−1 6-benzylaminopurine (BAP). On this media combination, 8.7 ± 0.21 shoots per explant were induced. Shoots multiplication was extensively influenced by the type and the concentrations of plant growth regulators used, repeated transfer of mother explants and the sub-culturing of in vitro regenerated shoot clumps on fresh medium. Maximum numbers of shoots (49.39 ± 1.24) was observed on MS medium augmented with 1.0 mg l−1 BAP and 0.2 mg l−1 α-naphthalene acetic acid. Full, half and one-fourth strength of MS medium with different concentrations of auxins was tested for in vitro root formation. One fourth strength MS medium with 2.0 mg l−1 indole-3 butyric acid (IBA) was optimum for in vitro root induction (36.9 ± 0.72 roots per shoot). Experiments on ex vitro rooting were conducted to shorten the regeneration pathway and observed highest percentage (100 %) of rooting from the cut ends of the shoots with 200 mg l−1 IBA. About 12.6 ± 0.30 roots were induced from the shoots using this method. The well rooted plantlets were acclimatized successfully in Soilrite under the greenhouse conditions with 92 % survival rate.

In vitro propagation, DNA content and essential oil composition of Teucrium scorodonia L. ssp. scorodonia

Teucrium scorodonia ssp. scorodonia is a valuable medicinal plant. It is rare in Poland, therefore availability of raw material is limited and an effective method of producing in vitro-derived plants for pharmaceutical reasons would be desirable. However, the protocol for micropropagation of this species has not been developed yet. Here, its regeneration ability on media supplemented with different growth regulators was evaluated using shoot tip and nodal segment explants. Murashige and Skoog (MS; Physiol Plant 15:473–497, 1962. doi: 10.1111/j.1399-3054.1962.tb08052.x ) medium supplemented with indole-3-acetic acid (IAA) (0.1 mg L−1) and 6-benzyladenine (BA) (2 mg L−1) was the best for multiple shoot induction, resulting on average in about 7 and 8 axillary shoots per shoot tip and nodal explant, respectively, within 5 weeks. Flow cytometric analysis revealed that most of the in vitro-developed shoots of T. scorodonia possessed similar nuclear DNA content as seedlings (about 2.1 pg/2C). In vitro rooting of shoots was achieved at 100 % efficiency on MS medium without growth regulators or containing auxin. The addition of auxin, IAA, indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA) led to an increase in the number of roots. Rooted plantlets were transferred into ex vitro conditions and further grown in the field, where they matured and flowered. The essential oils from aerial parts of the in vitro-originated plants and their seed-derived counterpart were isolated and analyzed by gas chromatography-mass spectrometry (GC–MS). In the oils, 84 components were identified. Although the essential oils from the micropropagated plants and the seed-derived plants were qualitatively similar, quantitative differences were found.

In vitro propagation of Andrographis paniculata Nees.-A threatened medicinal plant of Bangladesh

An efficient protocol was developed for in vitro regeneration of plantlets from shoot tip and nodal segment explants of Andrographis paniculata Nees. Nodal segment explants produced the highest number of shoots (18±1.24) when they were cultured on MS supplemented with 11.10 ?M/l BAP. Addition of 10% coconut water and 2.0 g/l activated charcoal to the above mentioned medium increased the number of shoots (30) per culture. Shoot tip explant also showed better performance in the same medium. Addition of 100 mg/l urea and 2.0 g/l activated charcoal to the medium showed proper shoot elongation. The isolated shoots rooted well (90%) on half-strength MS fortified with 9.80 ?M/l IBA, where average number of roots per shoot was 28-30. The plantlets were acclimatized successfully in poly bags containing a mixture of soil, sand and compost in 2:1:1 ratio. Finally acclimatized plantlets were transferred to experimental field.Jahangirnagar University J. Biol. Sci. 3(1): 67-73, 2014 (June)

BREAKING UP DORMANCY OF ADANSONIA DIGITATA L. SEEDS AND REGENERATION OF PLANTLETS FROM STEM NODAL SEGMENTS IN VITRO

In Yemen, there are only three trees of Adansonia digitata L. which is adopted in this study. Natural regeneration and independent germination of Adansonia digitata is poor although it is one of the tallest trees in the world that was recorded to live about 4000 years. Unfortunately, the tree is a subject of extinction. In the present study, the seeds of Adansonia digitata L. were pretreated with full and half strength of hydrochloric, sulfuric and nitric acids for one and/or two hour as a mean of scarificationtobreakthedormancyoftheseeds.Seedswerethensurfacesterilizedandallowedtogerminateonhormone–freeMS basal salts culture medium in vitro. The obtained seedlings were used as a source of nodal segments explants for propagation in vitro. Results of the present study have shown that all of the acid treatments overcame seed dormancy and enhanced seed germination in various degrees. The percentage of seed germination increased from 18.6% in the control group to 86.6% using a pretreatment of half strength sulfuric acid for one hour. The germinated seeds grown into seedlings of a mean of 11.1 centimeter after 8 weeks of growth. Stem nodal segments were cultured on agar solidifiedMSbasalsaltssupplementedwithvarious concentrations of benzyle adenine (BA) and naphthalene acetic acid (NAA). The maximum regenerant shoot length (15.1±03 cm) was obtained with 8 mg/l BA plus 0.5 mg/l NAA while The maximum number of leaves per regenernt reached up (7.2±0.2) using 10 mg/l BA + 1.0 mg/l NAA.The obtained regenerants developed roots on the same culture media. They were acclimatized for one week and then transferred to soil.

In vitro plant regeneration of potato (Solanum tuberosum L.) at the rate of different hormonal concentration

One of the goals of the experiment is to standardization of HgCl2 treatment for explants sterilization. The objectives also include developing a reproducible cost effective protocol for large scale production of Solanum tuberosum of Cardinal variety plantlets from selectively better clones through plant in vitro propagation methods. Selection of growth regulators for proper multiple shoots regeneration, elongation and root induction. To produce genetically uniform plantlets within a short time capable surviving in natural condition raised in in vitro environment. Shoot tip and nodal segment explants from field grown plants were used as experimental materials in this investigation. All explants were cultured on Murashige and Skoog medium supplemented with various plant growth regulators. For surface sterilization of explants, HgCl2 (0.1%) for 2 minutes was found to be most effective for complete destroying of surface pathogens and getting healthy tissues. Shoot regeneration was observed from both shoot tips and nodal explants for the studied plant. Maximum number of shoot per culture (17) was recorded and it also obtained the highest average length of the shoot (5cm) in Murashige and Skoog medium containing no hormone. On the other hand 6-benzyl amino purine (0.2mg/l) in 3 media showed the highest rate of shoot multiplication (73%) and the highest average length (4cm). In case of Gibberellic acid (0.1mg/l) in Murashige and Skoog media showed its highest rate of shoot regeneration (82%) and the highest average length (4.5cm). From the overall experiment it was observed that shoot tips are more responsive for micro propagation. In root induction Murashige and Skoog medium supplemented with different concentration (0.5, 1, 1.5 and 2mg/l) of indol-3-acetic acid and kinetin. Indol-3-acetic acid and kinetin (1.5+1.5 mg/l) showed its lowest rate of root regeneration (40%) and the average length of the root (1.5 cm). On the contrary Murashige and Skoog medium with no hormone showed the rate of root regeneration (96%) and the highest average length of the root (2.5 cm). The supplemented Murashige and Skoog media with no hormone showed the best performance for root regeneration.Asian J. Med. Biol. Res. June 2015, 1(2): 297-303

Thidiazuron induced efficient in vitro multiplication and ex vitro conservation of Rauvolfia serpentina – a potent antihypertensive drug producing plant

An efficient system for in vitro propagation of the endangered medicinal plant Rauvolfia serpentina has been developed. Proliferation of shoots was achieved from nodal segment explants, excised from a field grown plants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–2.5 μmol/L) although with low regeneration response and few number of shoots per explant. Greater number of shoots was achieved from nodal explants pretreated with higher concentrations of TDZ (5–100 μmol/L) in liquid MS medium for different time periods (4, 8, 12 and 16 days), followed by their transfer on a growth regulator-free medium. The highest response in terms of percent regeneration (90%), average number of shoots/explant (23.17 ± 2.15) and maximum shoot length (5.3 cm ± 0.83 cm) were achieved by pretreating the nodal explants with 50 μmol/L TDZ for 8 days. Rooting was achieved in MS medium supplemented with 0.5 μmol/L indole-3-butyric acid with an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transferred to soil, where 90% of them grew well and exhibited a normal development. Chlorophyll a and b, carotenoids, net photosynthetic rate and antioxidative enzymes, viz., superoxide dismutase and catalase activities were measured in leaf tissues during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Most of the activities showed a decreasing trend but subsequently increased after 7 days of acclimatization. These changes suggested the adjustment of the in vitro propagated plantlets to ex vitro conditions.

In vitroplantlet formation in Carrizo citrange: A promising citrus rootstock

The shoot tip and nodal segment explants from three-year-old plants of Carrizo citrange were exposed to mercuric chloride (0.1%) for different durations. In case of nodal segments, maximum survival (39.00%) and minimum microbial contamination (60.80%) was observed with mercuric chloride treatment for 10 min., whereas, shoot tips failed to establish. The explants were established on Murashige and Skoog (1962) medium supplemented with different concentrations of BAP, NAA and IBA. Maximum culture establishment (97.20%) was observed highest on MS medium supplemented with BAP (2 μM) + NAA (1 μM). Season of culturing also effected the establishment of shoot cultures as maximum establishment (96.60%) was observed during April-May followed by August-September. The highest shoot proliferation (3.64 per explant) and shoot length (3.71 cm) were also recorded on MS medium supplemented with BAP (2 μM) + NAA (1 μM). The micro-shoots were rooted on half-and full-strength media supplemented with different concentrations of IBA and NAA. Earliest root induction (10.60 days) and maximum rooting (53.89%) were observed on MS medium supplemented with IBA (10 μM) + malt extract (500 mg/l). Maximum number of roots per shoot (1.37) and root length (5.0 cm) were observed on half-strength MS medium containing IBA (2.5 μM) + NAA (2.5 μM). The maximum ex vitro plantlet survival (81.4%) was observed in potting mixture consisting of cocopeat + vermiculite + perlite (2:1:1).

INFLUENCE OF CULTURE MEDIUM, EXPLANT LENGTH AND GENOTYPE ON MICROPROPAGATION OF Pinus taeda L.

This work aimed to establish a micropropagation protocol for Pinus taeda L. Apical shoots from 5-day seedlings, of different genotypes (F27, B05 and PC), were cultured on WV5 medium supplemented with 44 µM 6-benzylaminopurine (BAP) and 0.05 µM α-naphtaleneacetic acid (NAA), for 14 days, followed by subcultures on growth regulator-free medium. Explants were sectioned into apical shoots and nodal segments for multiplication. Different lengths of explants, BAP concentrations and cultivation periods were tested. Rooting was induced on WV5/2, WV3/2, GDm/2 and agar-water culture media supplemented with 2.68 µM NAA and 0.44 µM BAP for nine days, followed by a 4-week subculture on growth regulator-free medium. It was verified that genotype influenced shoot formation and rooting. The length of 1.0 cm is recommended for nodal segment explants to obtain a high number of axillary shoots. For apical shoots, 0.5 cm explant and WV5 medium formulation allowed the best results for elongation. The best period of subculture was eight weeks, both for nodal segments and for apical shoots. The higher percentage of nodal segments with shoots (99.2%) and the higher average number of shoots per explant (4.0) were obtained with F27 genotype in medium culture containing 2.5 µM BAP. For apical shoots, the best result for elongation was observed for the shorter explants (0.5 cm) on WV5 culture medium (218.3%). The maintenance of in vitro clonal micro garden of vigorous shoots was obtained for two years of 8-week subcultures on WV5 culture medium. The best rooting rate (55.6%) was obtained when shoots were inoculated in agar-water medium with 2.68 μM NAA and 0.44 μM BAP for nine days. Plantlets were successfully acclimatized (85% of survival), so a micropropagation protocol was established.

In Vitro Propagation, Encapsulation, and Genetic Fidelity Analysis of Terminalia arjuna: a Cardioprotective Medicinal Tree

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100mM CaCl2[Symbol: see text]2H2O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50 ± 3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5mgl(-1) of benzylaminopurine (BAP) and 0.1mgl(-1) of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250mgl(-1)) + 2-naphthoxy acetic acid (NOA, 250mgl(-1)) for 5min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80% survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.

Development of a Successful Protocol for <i>in vitro</i> Mass Propagation of <i>Celastrus paniculatus</i> Willd. – A Valuable Medicinal Plant

<em>Celastrus paniculatus </em>Willd. belongs to family <em>Celastraceae</em>. Seeds provide an extremely important medicinal oil which is reported to sharpen the memory and also used to cure a number of diseases. The rate of seed germination is low and other vegetative propagation methods also cannot be used for cultivation. This was recorded as a highly threatened plant in the 1999 list of threatened fauna and flora of Sri Lanka. The objective of this study was to develop a successful protocol for in vitro mass propagation of <em>C. paniculatus</em> and to study the growth and physiology of greenhouse established tissue cultured plants. Nodal segments, shoot tips and leaf discs were used as explants. They were cultured in Murashige and Skoog (MS) medium supplemented with different combinations of growth regulators in order to induce callus. It was observed that MS medium supplemented with 5.0 μM 6-benzylaminopurine (BAP) and 7.0 μM indole-3-acetic acids (IAA) was the best medium for callus induction from tested explants. Both shoot tips and nodal segment explants produced multiple shoots in the MS medium in the presence of 10.0 μM BAP and 14.0 μM IAA. Multiple shoots were induced in the MS media with 5.0 μM BAP and 0.5 μM IAA. Elongated shoots developed roots and the highest rooting percentage (73.3%) was obtained in the MS medium supplemented with 5.6 μM IAA and 9.6 μM Indole-3-butyric acid (IBA). Rooted plants were acclimatized in different potting mixtures and a mixture of river sand: top soil: compost (1:1:1) gave the highest survival rate (80.0%). Rate of photosynthesis and the stomatal resistance of in vitro produced plants increased with time indicating that tissue cultured plants adapted to natural environment. Growth of the acclimatized plants had a sigmoid pattern of normal growth in the greenhouse. DOI: <a href="http://dx.doi.org/10.4038/tar.v21i1.2583">http://dx.doi.org/10.4038/tar.v21i1.2583</a><br /><br /><em>Tropical Agricultural Research</em> Vol. 21(1): 21-29 (2009)

Endophytic bacteria from Ilex paraguariensis shoot cultures: localization, characterization, and response to isothiazolone biocides

Nodal segment explants of Ilex paraguariensis, collected from greenhouse-grown plants, were found to contain endophytic bacteria. After culturing in bioreactors, 16 rRNA gene analyses and analytical profile index biochemical tests were used to identify these bacteria as Stenotrophomonas malthophilia. The presence of bacterial cells in the intercellular spaces of stem cortical parenchyma was detected in histological sections by scanning electron microscopy. A range of commercial isothiazolone biocides were tested for their ability to repress the growth of Gram-negative bacteria grown in liquid media during the micropropagation phase. The addition of 0.75 ml l−1 Delcide™ TG (5-chloro-2-methyl-4-isothiazolin-3-one + 2-methyl-4-isothiazolin-3-one, 1.05% and 0.45%, respectively) to the culture media resulted in 100% visibly clean cultures, with no suppression of shoot growth.

Effect of 2, 4-D, BAP, KN, IAA and IBA onin vitroRegeneration ofOcimum canumSims – An Important Hoary Basil Plant

The effect of 2, 4-dichlorophenoxyacetic acid (2, 4-D), 6-benzylaminopurine (BAP), kinetin (KN), IAA (indole-3-acetic acid) and indole-3-butyric acid (IBA) was evaluated for in vitro shoot regeneration of Ocimum canum Sims. This important medicinal plant species belongs to the family Lamiaceae and it is also known as hoary basil. Nodal segment explants were cultured on Murashige and Skoog (MS) medium containing different phytohormones. 2, 4-D resulted in callus induction from nodal segments when supplemented with MS medium. The highest callus induction (90.17%) was recorded on MS medium containing 2, 4-D (2 mg/l). When nodal segments were cultured on MS medium supplemented with BAP (0.5–2.0 mg/l), KN (0.5–2.0 mg/l) as sole and with combination of BAP (0.5 mg/l) and IAA (0.10–1.00 mg/l), multiple shoots proliferation was initiated. The maximum number of shoots (12.75±0.64), with average length 6.04±0.08 cm, was recorded with MS medium containing BAP (1.5 mg/l) as sole. Repeated subculture of in vitro proliferated shoots yielded 35–40 shoots from a single nodal segment explant after 4–5 months. Developed single shoots were transferred for rooting to ½ MS medium containing IBA (1.5 mg/l) for best rooting of each shoot (3.99±0.23). The regenerated plantlets with well-developed roots were transferred to plastic pots containing a mixture of garden soil, farm yard manure and sand (1:1:1) in controlled environment for achieving high survival percentage (80 – 85%). Thus, a comprehensive micropropagation protocol was developed for O. canum Sims first time using nodal segment explants. This protocol can be used for commercial propagation and for future genetic improvement studies.

Estabelecimento &lt;i&gt;in vitro&lt;/i&gt; de clones híbridos de &lt;i&gt;Eucalyptus globulus&lt;/i&gt;

http://dx.doi.org/10.5902/198050986626O presente estudo teve como objetivos avaliar o comportamento de clones de Eucalyptus grandis W.Hill ex Maiden x Eucalyptus globulus Labill e Eucalyptus urophylla S.T.Blake x Eucalyptus globulus Labill e o efeito de diferentes introduções in vitro (aos 30, 90 e 150 dias após a poda do ápice das minicepas) na fase de estabelecimento in vitro. As minicepas, fornecedoras dos explantes para a introdução in vitro, foram conduzidas em minijardim clonal semi-hidropônico. Segmentos nodais de 21 clones de Eucalyptus urophylla x Eucalyptus globulus e 8 clones de Eucalyptus grandis x Eucalyptus globulus foram coletados, desinfestados e inoculados em meio de cultura MS, suplementado com 0,5 mg L-1 de BAP e 0,1 mg L-1 de ANA. Após 30 dias em cultura, foram feitas as avaliações e concluiu-se que houve variação entre clones, bem como entre introduções in vitro, para as variáveis estudadas, sendo possível o estabelecimento in vitro da maioria dos clones.

High frequency shoot regeneration and plant establishment of Rauvolfia serpentina: An endangered medicinal plant

An efficient method was developed for high frequency shoot regeneration and plant establishment from nodal segment explants of Rauvolfia serpentina. Among the three different cytokinins (6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP)) tested as supplements to Murashige and Skoog (MS) medium, BA at 5.0 μM concentration was found effective in inducing multiple shoots. Effects of different medium (Murashige and Skoog (MS), Gamborg medium (B5), Schenk and Hildebrandt (SH) and Woody Plant Medium (WPM)) on in vitro regeneration were also evaluated. The highest frequency of shoot regeneration (90%) and maximum number of shoot (45.4 ± 3.2 per explants) with an average shoot length of 4.7 ± 0.21 cm were standardized on WPM supplemented with 5.0 μM BA along with 1.0 μM α-naphthalene acetic acid (NAA). In vitro regenerated micro-shoots rooted best on WPM supplemented with 1.0 μM NAA. Plantlets with well-developed shoot and roots, following acclimation in the greenhouse, were successfully established in field condition, and 90% of plants were survived. The regenerated plants were morphologically normal and did not show any immediate detectable phenotypic variation. The method developed can be successfully employed for large-scale multiplication and long-term in vitro conservation of R. serpentina. Key words: Nodal explants, plant growth regulators, in vitro, plant regeneration, micro-propagation.

In vitroculture and plant regeneration from nodal explants ofVerbesina encelioides(Cav.) Benth &amp; Hook

A protocol for complete regeneration of Verbesina enceloides, a medicinally important plant, was accomplished through in vitro culturing. The nodal segments excised from aseptically grown seven day old seedlings were employed for multiple shoot induction using BAP in combination with mesoinositol. Different phytohormonal concentrations were tried but maximum percentage of response was elicited in 3 mg L−1 of BAP. From nodal segments explants, high percentage of multiple shoot formation was induced between Jan to June on MS medium supplemented with BAP (3 mg L−1). In vitro regeneration seemed to be more successful by axillary bud regeneration than by indirect organogenesis. As such shoot regeneration from axillary buds was obtained on MS media with low levels of BAP as well though higher concentration (3 mg L−1) in media supplemented with mesoinositol gave efficient shooting. Chlorophyll contents varied between tissue cultured plants (TC) and field grown (FG) plants markedly. The TC plants exhibited higher contents of chlorophyll pigments especially chlorophyll a. Rooting of the excised shoots from secondary cultures was excellent on half-strength MS medium having 1 mg L−1 IBA. Micro propagated rooted plants were transplanted on garden soil for further evaluation. The survival percentage was nearly 65%.