Combinations Of Auxins Research Articles

Efficient callus induction and indirect plant regeneration from various tissues of Jatropha curcas

The Jatropha curcas is considered as an important energy plant due to the fact that its seed contains high oil content. Nowadays focus is being placed on J. curcas callus induction and plant regeneration. In this study, explants epicotyl, hypocotyl, petiole and cotyledon of 8-day-old seedlings of J. curcas were utilized for callus induction on media supplied with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), naphthyl acetic acid (NAA) or indolebutyric acid (IBA) and 0.1 mg/L kinetin (Kin), and the results demonstrated that the combination of 1 mg/L NAA and 0.1 mg/L Kin was the best medium for callus induction and growth. In addition, induced calli were transferred to regeneration medium containing different combination of auxins and cytokinins, and the data showed that the medium containing 1 mg/L thidiazuron (TDZ) and 1 mg/L Kin combined with 0.1 mg/L IBA was propitious to plant regeneration compared with other combinations. Keywords: Callus induction, indolebutyric acid, Jatropha curcas , kinetin, naphthyl acetic acid, plant regeneration, thidiazuron

Combination of cytokinin and auxin induces apoptosis, cell cycle progression arrest and blockage of the Akt pathway in HeLa cells.

Plant cytokinins and auxins have recently been proposed as novel cancer therapies, which proceed via different mechanisms; however, their combined use has not been investigated. To the best of our knowledge, the present study was the first to show that the cytokinin ortho-methoxytopolin-riboside (MeoTR) strongly inhibited the proliferation of HeLa cells, the effect of which was synergistically enhanced by auxin indole-3-acetic acid (IAA), while IAA demonstrated to have no cytotoxic effects on cells. MeoTR was found to activate intrinsic and extrinsic caspase-dependent pathways, and IAA potentiated this activation. In addition, these effects were blocked by Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), a pan-specific-caspase-inhibitor. IAA increased the MeoTR- induced inhibition of B cell lymphoma 2 (Bcl-2) and survivin, whereas IAA-only decreased Bcl-2 expression. MeoTR downregulated phosphorylated (p)-pyruvate dehydrogenase kinase 1, p-Akt and p-glycogen synthase kinase 3β, the effect of which was more potent in combination with IAA, despite the weak effect of IAA alone. LY294002, an Akt-inhibitor, was able to increase the inhibition of p-Akt through MeoTR and combination treatment. IAA and MeoTR increased the proportion of cells in S phase independently. However, the combination treatment induced a further increase. In addition, IAA and MeoTR treatment downregulated protein levels of cyclin A, cyclin-dependent kinase 2 (CDK2) and p-CDK2, and upregulated protein levels of p21 and p27. Furthermore, the combination treatment enhanced these effects, indicating that IAA potentiated the inhibitory effect of MeoTR on HeLa cells via cell cycle progression arrest and accumulation in S phase, coupled with the negative regulation of Bcl-2. In conclusion, the results of the present study suggested that treatment with these two phytohormones in combination, may offer a novel therapeutic strategy for the treatment of malignant cervical cancer.

In vitrocloning ofPulicaria crispa: An important medicinal plant

A standard protocol for an efficient in vitro micropropagation of Pulicaria crispa, a medicinally important plant, was designed. The in vitro plantlet production was investigated on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. From nodal explants, maximum number of shoots (3 shoots) and beneficial shoot length (3.9 cm) were achieved on MS medium containing 0.5 mg L–1 BAP. MS media with the combination of BAP (0.5–1 mg L−1) and KN (0.5–1 mg L−1) showed maximum response of shoot number and mean shoot length. Rooting of well developed shoot was excellent on half strength MS medium after dipping in 1 mg L−1 IBA for 30 min. The well rooted plantlets were then hardened with high survival rate. This is the first report dealing with in vitro regeneration for P. crispa.

Improving Effect of Salicylic Acid on the Multipurpose Tree <i>Ziziphus spina-christi</i> (L.) Willd Tissue Culture

Salicylic acid (SA) is one of numerous phenolic compounds found in plants with numerous important physiological events. Exogenously application of Salicylic acid (SA) is useful for the growth and development of plants. This investigation aimed to study the improving effect of salicylic acid on the tissue culture of Ziziphus spina-christi. To study its improving effect were used shoot-tip explants cultured on Murashige and Skoog (MS) solid medium supplemented with different concentrations and various combinations of auxins, cytokinins and Salicylic acid. Media without growth regulators enhanced the growth and elongation of shoot-tip explants not its proliferation. Lower concentrations of cytokinin were better for shoot-tip proliferation than higher ones. 6-Benzylaminopurine (BAP) was superior to kinetin (KN) in shoot-tip proliferation. The optimum culture conditions for shoot-tip proliferation were achieved on MS medium supplemented with 0.25 mg/l 6-Benzylaminopurine (BAP) and 25 mg/l Salicylic acid (SA). The optimum culture conditions for callus formation were obtained on MS medium supplemented with 3 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 25 mg/l Salicylic acid (SA), while the optimum culture conditions for somatic callus formation were obtained on MS medium supplemented with 2.0 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 10 mg/l Salicylic acid (SA). The optimum culture conditions for rooting of shoots were obtained on MS medium supplemented with 0.25 mg/l of Indole-3-butyric acid (IBA) and 10 mg/l Salicylic acid (SA). The highest percentage of survival plants was obtained in the soil mixture supplied with 10 mg/l Salicylic acid (SA). Salicylic acid (SA) showed positive effect and good response on callusing, shooting and rooting of Z. spina-christi. Cultures received small amounts of salicylic acid were better than those lack it.

A simple and efficient protocol for the mass propagation of Cymbidium mastersii: an ornamental orchid of Northeast India.

Cymbidium mastersii is an epiphytic orchid distributed mainly in Northeast India. Owing to its high commercial value in the floricultural industry, natural populations are under threat from over-exploitation. Mass propagation provides an alternative means of satisfying the demand. Unfortunately, conventional propagation is slow and difficult, suggesting in vitro methods for mass multiplication may be more appropriate. The objective of this study was to develop an efficient protocol. FOUR NUTRIENT MEDIA WERE EVALUATED FOR SEED GERMINATION AND EARLY PROTOCORM DEVELOPMENT: Murashige and Skoog (MS), half-strength MS, Knudson 'C' (KC), and Vacin and Went (VW). In addition, the effects of plant growth regulators 6-benzylaminopurine (BAP), kinetin (KN), α-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were studied alone and in combination. The maximum percentage seed germination (93.58 ± 0.56) was obtained in MS basal medium after 8-9 weeks of culture. Secondary protocorms (protocorm-like bodies) were developed from primary protocorms on MS medium fortified with different concentrations and combinations of cytokinins (BAP and KN) and auxins (NAA and IBA). The highest numbers of secondary protocorms (20.55 ± 0.62)/primary protocorms were obtained in MS medium supplemented with 5.0 µM BAP and 2.5 µM NAA. The most effective auxin source promoting root production (7.46 ± 0.09 per shoot) was 10.0 µM IBA. The plants were acclimatized effectively (survival percentage 88 %) in a greenhouse using a rooting medium of crushed sterile brick and charcoal (1: 1v/v) and vermicompost (leaf litter + cow dung, 1: 1v/v). An efficient protocol was established for in vitro propagation of C. mastersii using seed as the starting material. The percentage seed germination varied with the composition of the nutrient media and was highest in full-strength MS basal medium. The number of secondary protocorms that developed from primary protocorms was increased by the addition of 5.0 µM BAP and 2.5 µM NAA. In vitro raised plantlets acclimatized in a greenhouse were closely similar to the mother plants in morphology.

Organogênese direta e aclimatização de plantas de patchouli

O patchouli é uma espécie aromática cujo óleo essencial é largamente empregado pela indústria de perfumes. A propagação convencional desta espécie é por estaquia, sendo a micropropagação uma alternativa para propagação clonal de indivíduos livres de patógenos em larga escala. O objetivo deste trabalho foi analisar a influência de combinações de diferentes fontes de auxinas com cinetina sobre a organogênese de patchouli, assim como diferentes tipos de composição de substratos na aclimatização de mudas micropropagadas da espécie. Foram realizados dois ensaios para organogênese. No primeiro, avaliação de cinco concentrações de cinetina (0,0; 1,0; 2,0; 4,0 e 6,0 mg L-1) e quatro concentrações de AIA (0,0; 0,5; 1,0 e 2,0 mg L-1) e no segundo, cinco concentrações de cinetina (0,0; 0,5; 1,0; 2,0 e 4,0 mg L-1) e três concentrações de ANA (0,0; 0,1 e 0,5 mg L-1). Para aclimatização foram analisados os substratos: pó de coco + fertilizante formulado (NPK 3-12-6 + Ca, S, Zn, B, Cu, Fe, Mn e B a 3,0; 2,5; 0,1; 0,025; 0,01; 0,075; 0,05 e 0,0015%, respectivamente) na concentração de 12 g L-1 + calcário (1 g L-1) [PBC]; pó de coco + vermiculita (2:1) + fertilizante formulado na concentração de 12 g L-1 + calcário (1 g L-1) [PBCV (2:1)]; pó de coco + vermiculita (1:1) + fertilizante formulado na concentração de 12 g L-1 + calcário (1 g L-1) [PBCV (1:1)]; pó de coco + 1 g L-1 de calcário + sais do MS [PCMS]; vermiculita acrescido de sais do MS, sendo 15 mL da mistura de sais por planta [VMS]; vermiculita + fertilizante formulado na concentração de 12 g L-1 [VMB]. Neste estudo foi observado que a organogênese direta pode ser promovida em meio MS acrescido de 2,47 mg L-1 de cinetina e 0,1 mg L-1 de ANA, sendo as etapas de alongamento, individualização e enraizamento das brotações promovidas em meio MS básico. Já para aclimatização um dos melhores resultados foi obtido com o substrato PBCV (1:1).

Role of auxins, polyamines and ethylene in root formation and growth in sweet orange

The primary objective of this work was to investigate the role of polyamines (PAs) on root formation and growth in two sweet orange (Citrus sinensis L. Osb.) cultivars Pineapple and Pêra. Adventitious shoots (30-d-old) derived from epicotyl explants were transferred to root induction medium containing Murashige and Skoog salts at different strengths and supplemented with different concentrations and combinations of auxins. Root formation and development decreased in both sweet orange cultivars concomitant with the reduction of medium strength. The α-naphtaleneacetic acid was important during the root differentiation phase, but its combination with indole-3-butyric acid was essential for root elongation. The addition of PAs significantly improved root formation and/or growth, depending on their concentration, whereas the presence of inhibitor of PAs biosynthesis α-difluoromethylornithine (DFMO) inhibited these processes. The rooting impairment caused by DFMO was partially reversed by the supplementation of putrescine. Aminoethoxyvinylglycine AVG and AgNO3 also inhibited in vitro rooting in both sweet orange cultivars, indicating that ethylene was likewise important for rhizogenesis in sweet orange.

Plant Regeneration via Somatic Embryogenesis in Three Iranian Cucumis melo L. genotypes

Efficient genotype-independent plant regeneration via somatic embryogenesis is vital for genetic manipulation and crop improvement of melon (Cucumis melo L.). In this research, the optimal culture media of somatic embryogenesis for three melon genotypes, including two local cultivars (‘Talebi-Isfahan’ and ‘Garmak’) and a drought- and high temperature-tolerant accession (locally named ‘Gargar’), were investigated using cotyledon and zygotic embryo explants. Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D = 0, 1, 2, 3, and 4 mg l−1) and 6-benzyladenine (BA = 0.5 and 1 mg l−1) was used for somatic embryogenesis. The results revealed that 1 mg l−1 2,4-D with 0.5 mg l−1 BA produced the highest embryo induction in both explant types. Talebi-Isfahan and Garmak cultivars did not significantly differ but were superior to the Gargar accession for somatic embryogenesis. No significant effect of BA growth regulator (0, 0.5, 1, and 1.5 mg l−1) was detected in the plant regeneration. It was also found that, under an optimal auxin and cytokenin combination in the growth medium, only melon genotypes with a wider genetic distance could be discriminated for their somatic embryogenesis. The procedure of somatic embryogenesis and plant regeneration of melon reported here is a highly reproducible protocol, which could be well exploited for tissue culture and transformation of every melon genotype.

The effect of in vitro competition on shoot regeneration from hypocotyl explants of Linum usitatissimum

This study was carried out to investigate the relationship between in vitro competition and stress, and their effects on tissue culture response of Linum usitatissimum L. hypocotyl explants. Competition among explants was achieved by varying the spacing among the explants cultured. Four different culture spacing distances were used: 0.5, 1.0, 1.5, and 2.0 cm. Six weeks after culture initiation, hypocotyl fresh and dry weights, shoot regeneration percentage, shoot number per hypocotyl, regenerated shoot length, total shoot number per petri dish, and total chlorophyll content were recorded. The results showed that encouraging competition among explants by decreasing spacing among them from 2.0 cm to 1.0 cm increased shoot number per hypocotyl, regenerated shoot length, and total shoot number per petri dish in both cultivars. When explants were cultured at 0.5 cm spacing, significant stress-initiated decreases were observed in all parameters examined. This study showed that the success of tissue culture studies for related genotype could be increased not only by determination of correct concentrations and combinations of auxins and cytokinins in growth medium but also by evaluating competition among explants cultured.

Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

In vitro rooting of Beilschmiedia berteroana, endemic to the South Central area of Chile

M.E. Uribe, M. Sandoval, A. Mendez, F. Mora, and C. Delaveau. 2011. In vitro rooting of Beilschmiedia berteroana, endemic to the South Central area of Chile. Cien. Inv. Agr. 38(1): 107-115. Beilschmiedia berteroana (“belloto del sur”) is a screrophyllous tree of the family Lauraceae that is endemic to the Central-South region of Chile. It is an endangered species; considered a second priority in the “Extinction Danger” category. This species is known for its timber, fruits and lots of mucilages, also has great potential for its size and ornamental evergreen foliage. This study had as a main objective to develop an in vitro rooting protocol of microshoots, obtained from the proliferation phase, by adding auxin: indole 3-butyric acid (IBA), naphthaleneacetic acid (NAA) and indole 3-acetic acid (IAA) in order to contribute to the recovery of the species. Results are discussed in the rooting phase, where this species is induced or maintained in different trials with exogenous auxin. Rooted microshoots were obtained when 250 µM of IBA were applied for 24 hours, obtaining a 40% rate of rooting, whereas no response was observed with the hormone-free treatment. Therefore, it can be concluded that Beilschmiedia berteroana can be vegetatively propagated in vitro through microcutting induced by IBA or a combination of exogenous auxin (IBA + NAA and IBA + IAA).

Role of exogenous phytohormones on growth and plumbagin accumulation in Plumbago indica hairy roots and conservation of elite root clones via synthetic seeds

The present study describes the role of different exogenous hormones on morphology and plumbagin production in Plumbago indica hairy roots. It was also aimed to conserve elite root clones via synthetic seed technology. Insertion of rolB gene in transformed roots was confirmed by polymerase chain reaction followed by southern blot analysis. Hairy roots were treated with single or in combination of different phytohormones viz. IAA, IBA, 2, 4-D, NAA, BAP, GA3 and ABA. Cultures incubated with GA3 (0.5mgl−1) yielded highest root growth due to formation of profuse lateral branching while NAA (0.5mgl−1) treatment caused highest plumbagin accumulation. Cultures incubated with 2, 4-D exhibited highest inhibitory effect in terms of both root growth and plumbagin production. All phytohormones were found to be effective at lower concentration. In combinatorial study, GA3+NAA (0.5mgl−1, each) was found optimum for root biomass and plumbagin production at earlier stage of culture. Different combinations of auxins and BAP induced different morphologies ranging from reduction of lateral branching to rapid disorganization of root matrix. The combinations of ABA and selected auxins were not found promising at any of selected concentration. Based on the effect of exogenous hormones on hairy root culture, elite root clones were selected and encapsulated with sodium alginate matrix. Uniform shaped alginate coated synthetic seeds were conserved up to 6 months exhibited high regeneration potential without disturbing plumbagin content.

Development of a simple and effective protocol for Agrobacterium tumefaciens mediated leaf disc transformation of commercial tomato cultivars

The transformation of tomato (Solanum lycopersicum) through Agrobacterium tumefaciens is still far from being routine, particularly when it comes to commercial varieties. In the present paper, we present an efficient and simple protocol for leaf disc transformation of three Vietnamese tomato cultivars (DM8, MTS, FM372C) by comparing shoot regeneration media for expanding leaves and examining different parameters of inoculation, co-culture and selection conditions. The present transformation method requires neither feeder layers of cell suspension cultures nor pre-culture. The data clearly show that appropriate cytokinin- and auxin combinations and concentrations provide competent tissues for transformation. Supplementing of 8µM trans-zeatin and 5 µM indoleacetic acid (IAA) into pre-treatment, inoculation and co-culture media resulted in higher frequency of transformation and stronger GUS-expression than that of media supplemented with 4 µM trans-zeatin and 2 µM IAA. The experiments also exhibited that tomato leaf tissues were more sensitive to glufosinate after inoculation with Agrobacteria compared to the untreated controls, so a more sophisticated scheme for the glufosinate selection had to be established.

A Micropropagation, Molecular and Physiochemical Analysis of Canola Brassica napus L. From Seeds

AbstractCanola is important for its oil, anticarcinogenic phytochemicals and low saturated fat content and has wide scope for further improvement through genetic transformations. Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of disease free progeny plants, using modern asceptic plant tissue culture methods and also to conserve germ plasm. A combination of auxin and cytikinin and a shock on growth regulator free medium is used for micropropagation. Molecular and physiochemical analysis done to know variation in seeds protein content and electric conductivity.

Induction of somatic embryogenesis and plant regeneration in the tropical timber tree Spanish red cedar [Cedrela odorata L. (Meliaceae)

Spanish red cedar (Cedrelaodorata L.) is a tropical timber tree native to the Americas from southern Mexico to northern Argentina. Commercial plantations are scarce and, consequently, natural populations are overexploited. Traditional propagation practices for the establishment of large-scale plantations have had limited success in this species due to the relative scarcity of seeds, its broad genetic diversity and the lack of domesticated varieties. In vitro clonal propagation provides an effective method to overcome this situation and increase the yield of commercial plantations through the rapid multiplication of elite materials. Somatic embryogenesis (SE) is one of the most promising strategies for tree propagation due to the possibility of producing artificial seeds, the ability to store and rapidly mobilize germplasm and the opportunity for genetic manipulation. We report here the induction of indirect SE in C. odorata from calli derived from immature zygotic embryos after 12 weeks of culture. Macroscopic, histological, and scanning electron microscopic analyses of the calli revealed the presence of embryogenic cell clusters that formed cotyledonary embryos with clear bipolar structures and no vascular connections with the mother tissue. Different media preparations containing combinations of diverse auxins and cytokinins are known to have different effects on the type and frequency of embryogenic structures. Embryo conversion was achieved using an MS-based medium [Murashige T, Skoog F (1962) Physiol Plant 15:473–497, 1962] supplemented with abscisic acid, and transfer to soil was successful at a rate of 75%. The method described here provides a basis for optimizing the clonal propagation and genetic manipulation of this valuable species.

Thidiazuron-induced shoot organogenesis and production of hepatoprotective lignan phyllanthin and hypophyllanthin in Phyllanthus amarus

Callus cultures from nodal and leaf explants of Phyllanthus amarus were established on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. The leaf-derived callus induced on 2.26 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) + 2.32 μM Kinetin (Kin) upon transfer to medium containing thidiazuron (TDZ) exhibited higher shoot regeneration (32.4 ± 1.3 shoots per culture). Four-week-old shoots rooted readily on 1.5 μM Indol acetic acid (IAA)-containing medium and were successfully acclimatized with 98% survival. The lignans, Phyllanthin (PH) and Hypohyllanthin (HPH), of leaf extracts from naturally grown plants were identified by using TLC, HPLC and H1-NMR. The PH and HPH production in the regenerated shoots was compared to their production in callus cultures, plants under field conditions and in naturally grown plants. The regenerated shoots on MS + 2.27 μM TDZ produced about two times higher PH and HPH than the leaves of naturally grown plant. The present study provides a useful system for further studies on in vitro morphogenesis, elicitor-assisted production of PH and HPH and A. rhizogenes-mediated genetic transformation in Phyllanthus amarus.

Enhancing Sorgoleone Levels in Grain Sorghum Root Exudates

Sorgoleone, found in the root exudates of sorghum [(Sorghum bicolor (L.) Moench], has been a subject of continued research. Sorgoleone production in grain sorghum roots was investigated under different growth conditions. Methanol was the most effective solvent for extracting sorgoleone from grain sorghum roots. Sorgoleone production is high in young developing plants. The maximum concentration (microg mg(-1) root dry weight) was produced in 5-d-old seedlings; beyond this age, production declined. However, considering both root weight and sorgoleone content per seedling, 10-d-old seedlings had the highest total amounts (microg). Compared with the control, sorgoleone content increased 6.1, 8.6, and 14.2 times when sorghum seeds were treated with auxins, Hoagland solution, and a combination of auxins and Hoagland solution, respectively. Among the innate immunity response elicitors, cellulose (an elicitor of plant origin) stimulated higher sorgoleone production than the others, and it produced 6.2 times more sorgoleone than the control. Combined treatment of sorghum seeds with half strength Hoagland solution and 5 microg ml(-1) of IBA significantly increased both root growth and sorgoleone content in sorghum seedlings.

Highly Efficient and Rapid Plant Regeneration in Citrus sinensis

Sweet orange (Citrus sinensis L Osbeck, var Nagpur) was explored for efficient multiple shoot regeneration and rooting in different media. The influence of phytohormones and carbon source on the in vitro morphogenesis of sweet orange epicotyl explants was investigated. Among the various concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP, Kn, Zn, and TDZ) tried, MT (Murashige and Tucker) medium fortified with benzylaminopurine (BAP) at 1 mg l−1 without auxin had a strong promotive effect on shoot regeneration, and elucidated best morphogenic response from one-month-old etiolated epicotyl explants. A 100% regeneration frequency was obtained, and multiple shoots with an average of 8.24 shoots per explant were produced on all of the explants. Root formation was seen in response to all the three auxins viz. IBA, NAA and IAA, but the best response with rapid induction was observed under the influence of indole butyric acid (IBA) at 1 mg l−1. Sucrose was observed to be at par with maltose as carbon source to support shoot regeneration. This study provided promising results, holds potential to be routinely employed for in vitro regeneration of important cultivars of Citrus spp, and can be incorporated for genetic transformation studies in citrus.

MICROPROPAGATION STUDIES ON IMPORTANT FOREST TREES OF HIGH IMPORTANCE: AN EFFORT FOR LARGE-SCALE COMMERCIAL MULTIPLICATION AND THEIR FIELD PLANTATION

The present project is taken up with the aim to develop micropropagation protocols for large and commercial scale multiplication of highly important forest trees, i.e., Eucalyptus hybrids. Two Eucalyptus hybrids namely as FRI-5 (Eucalyptus cainaldulensis Dehn x Eucalytpus tereticornis Sm.) and FRI-14 (Eucalyptus torelliana F.V. Muell × Eucalyptus citriodora Hook) were selected for the present course of investigation with the aim to develop mass scale micropropagation technique using nodal segments as explants from the mature trees (30-32 years old) of both the hybrids. These Eucalyptus hybrids were produced by Forest Research Institute, Dehradun in the early 1960-70s though controlled crossing are available in very limited number and need multiplication true to type for field-testing to ascertain its superiority under varied agro-climatic conditions. These hybrids showed 3-5 superior in terms of total biomass and wood volume when compared to their parents. Cultures were established and multiplied on MS medium supplemented different concentration of BAP either alone or in with combination of auxins i.e. IBA/NAA. MS medium was found to be effective and suitable for all the experiments during plant production. Eighty-five to 92% rooting was achieved on half strength MS medium supplemented with IBA. Ninety-five to 98% plantlets survival was obtained after hardening and acclimatization prior to field transfer. Parameters like height, DBH, clear bole length and self pruning capability of both the hybrid were recorded up to three years to identify the suitability of these hybrids in different varied climatic zones of Uttarakhand.

Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A

Cell suspension cultures of Withania somnifera were established in shake flasks and the effect of different growth regulators (auxins, combination of auxin and cytokinin), inoculum density (2.5–20gL−1), different media (MS, B5, NN and N6), the strength of the MS medium (0.25–2.0×), carbon source (sucrose, glucose, fructose, maltose), concentration of the sucrose (1–8% (w/v)) and the initial pH (4.0–6.5) of the medium were determined for the production of withanolide A. The optimized conditions for biomass accumulation and withanolide A production were found to be 10gL−1 of the inoculum on fresh weight basis, the full strength MS medium, 3% (w/v) sucrose, four weeks culture period and the initial medium pH of 5.8. The results of present study are useful for scale-up process.