μM Indole-3-butyric Acid Research Articles

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper).

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95% of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0μM 6-benzylaminopurine. Elongated (2-3cm) shoots were rooted on MSB medium with 2.5μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3% was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.

Management of cytokinin–auxin interactions for in vitro shoot proliferation of Althaea officinalis L.: a valuable medicinal plant

An effective, rapid and improved protocol for in vitro regeneration has been established for Althaea officinalis using nodal explants, excised from 4-week-old aseptic seedlings. Different cytokinins and auxins, alone or in combination were investigated with respect to shoot induction and development. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kn) and 2-isopentenyl adenine (2-iP) tested as supplement to Murashige and Skoog (MS) medium, BA proved to be the most effective for induction and proliferation of shoots from nodal explants. Proliferation capacity of the induced explants was differentially influenced by a combination of cytokinins [6-benzyladenine (BA), Kinetin (Kn) and 2-isopentenyl adenine (2-iP)] and auxin [indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), α-naphthalene acetic acid (NAA)]. The optimum response was observed with (7.5 μM) BA and (0.5 μM) NAA, which induced shoot regeneration in 95 % of the cultured nodal explants, with an average of 26 shoots per explant. High frequency of rooting (89 %) was obtained when shoot were transferred to ½ MS medium supplemented with (20 μM) IBA. In addition, a histological study was undertaken to better understand the origin of the regenerated shoot buds.

Anatomical changes induced by Azospirillum brasilense in in vitro rooting of pink lapacho

The pink lapacho Handroanthus impetiginosus, a South American native deciduous tree, is widely grown as an ornamental, medicinal, and timber resource. In vitro culturing allows ex situ conservation but may cause anatomical changes that decrease plant survival. This study evaluated the ability of Azospirillum brasilense to reverse such changes in H. impetiginosus in in vitro culture under different conditions. Handroanthus impetiginosus shoots were induced with different indole-3-butyric acid (IBA) pulse treatments (3 days), transferred to two half-strength media [Murashige–Skoog salts with Gamborg’s vitamins (½MSG) and woody-plant medium (½WPM)], and were inoculated or not inoculated with A. brasilense Cd or Az39 strains. Rooted plant samples were processed for observation under light and scanning electron microscopy. Factorial analysis revealed significant triple interaction between medium type, IBA concentration, and bacteria inoculation (p ≤ 0.01) for all anatomical parameters. A. brasilense Cd inoculation significantly increased (p ≤ 0.05) mesophyll thickness in ½WPM for 10–50 μM IBA inductions and in ½MSG for 0, 10, and 40 μM IBA inductions compared to uninoculated plants. The Cd strain decreased stomatal density by 29 % in plants grown in ½MSG compared to uninoculated plants, whereas the Az39 strain increased the same by 30 %. Bacterial colonization improved periclinal epidermal cell division, bundle fiber development, secondary stem growth, and root hair density compared to that in uninoculated plants. The H. impetiginosus rooting by A. brasilense inoculation, mainly with the Cd strain, decreased undesirable anatomical modifications generated in vitro, enhancing adaptation to ex vitro conditions after transplanting.

Establishment of an efficient micropropagation and callus regeneration system from the axillary buds of Bambusa ventricosa

Based on the screening of various hormone combinations, we have established an efficient micropropagation and callus regeneration system from the axillary buds of B. ventricosa. We found that 6-benzyladenine (6-BA) had a dominant role in promoting bud sprouting, multiple bud induction and proliferation in B. ventricosa. Meanwhile α-naphthaleneacetic acid (NAA) was found to be an effective factor of inducing rooting in proliferated buds. The Murashige and Skoog medium containing 22.2 µM 6-BA was optimal for bud initiation, and the MS medium containing 26.6 µM 6-BA provided a good result for multiple bud induction. However, for buds proliferation MS medium containing 22.2 µM 6-BA, 0.23 µM Thidiazuron (TDZ: N-phenyl-N-[(1, 2, 3-thidiazol-5-yl) urea]) and 0.27 µM NAA was found to be very effective. The optimal medium for rooting of proliferated bud was MS medium containing 2.7 µM NAA, 4.9 µM indole butyric acid and 4.4 µM 6-BA. Based on the establishment of an efficient micropropagation system, we investigated the frequencies of callus formation from buds of in virto raised plantlets under different culture conditions. We showed that media containing 27 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2.7 µM NAA and 0.0045 µM TDZ effectively produced callus. The callus induction rate varied between it to 60 percent. TDZ was found to be a main factor influencing the callogenesis of B. ventricosa. Medium containing 22.6 µM 2, 4-D, 2.2 µM 6-BA and 5.4 µM NAA was preferred for callus amplification among the four tested subculture media. Plants were successfully regenerated on a MS medium containing 13.3 µM 6-BA and 2.7 µM NAA, subsequently acclimatized and transplanted to an experimental pod.

In vitro propagation of Picconia azorica (Tutin) Knobl. (Oleaceae) an Azorean endangered endemic plant species

Picconia azorica (Tutin) Knobl. (Oleaceae) is a woody plant endemic from the Azores. It is considered as a part of a true relict taxon, reflecting its phylogeny, genetics and geography. Given the fragile status of this species, effective management and conservation are necessary for its survival and repopulation, initially for ecological reasons, but ultimately for forestry purposes as a valuable source of timber. Micropropagation is a useful tool for conservation programs, and in vitro culture techniques have, therefore, been developed for P. azorica by testing different combinations of media and growth regulators at each micropropagation stage. Adult plants from the field were successfully established in vitro on Rugini olive medium supplemented with 24.6 µM 2-isopentenyl adenine (2iP), resulting in 91.2 % sprouting of the nodal explants that survived the disinfection stage, although similar results were achieved using 18.2 µM zeatin. At the multiplication stage, the best shoot quality and the best compromise between shoot length and new axillary shoot development were achieved using the modified macronutrients Driver and Kuniyuki walnut medium supplemented with 24.6 µM 2iP and 0.6 µM indole-3 acetic acid. Rooting was most successful with half-strength macronutrients combined with 10 µM indole-3 butyric acid. Using this micropropagation protocol, a wide range of accessions were successfully established in vitro and are now being propagated on a larger scale for ecosystem restoration.

Optimizing rooting and survival of oil palm (Elaeis guineensis) plantlets derived from somatic embryos

In species of the Arecaceae, initial conversion of somatic embryos to non-rooted plantlets (those with only shoot development and no roots) is common. A consistent methodology for improving rooting and survival of oil palm plantlets derived from somatic embryos was developed. Two experiments were carried out. In the first, non-rooted shoots regenerated via somatic embryogenesis were inoculated in nutrient medium supplemented with 0, 53.7, or 107.4 μM indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA). In the second, the optimum treatment as determined in the first experiment was evaluated in three culture systems: semi-solid medium (SS), stationary liquid (SL), and double-phase system (DP), with and without the addition of activated charcoal. Next, the plantlets were acclimatized in a greenhouse, where the influence of the presence or absence of roots, the number of roots, the length of the main root, and the height of the shoot on survival were assessed. Supplementation with 53.7 μM IBA and the DP system without activated charcoal provided the highest percentage of rooted plantlets (92.9%) and number of roots (6.3) and improved the length of the aerial parts (11.0 cm) and the survival of the plantlets during the acclimatization (82.1%). The best survival of plantlets was obtained when they were acclimatized with at least one root and with a height greater than 7.5 cm.

In vitro root induction improvement by culture in darkness for different globe artichoke cultivars

A propagation protocol was developed and optimized for globe artichoke (Cynara scolymus L.) plants of cvs “Blanca de Tudela,” “Calico,” and “Salambo.” For culture initiation and shoot proliferation, the effect of different kinetin (K) concentrations in the culture medium on shoot induction was tested. A study was also conducted to evaluate different in vitro rooting strategies. The results indicate that a low level of kinetin (1.4 μM) was optimal for the three cultivars tested for both shoot induction and multiplication. The best results for root induction (73.3% in “Blanca de Tudela,” 80% in “Calico,” and 93.3% in “Salambo”) were achieved with a 5-d culture in darkness, in the presence of 29.5 μM indolebutyric acid (IBA). The survival percentage of plants rooted was higher after acclimatization, 75–83%. The results presented here show that a low level of kinetin and darkness appear to be key factors in a plant propagation protocol for artichoke.

In vitro regeneration of Citrus hystrix DC

An efficient plantlet regeneration system for Citrus hystrix DC has been developed in this study. N6-benzyladenine (BA) was most commonly used plant growth regulator in micropropagation of Citrusspp. However, our study demonstrated that BA alone was insufficient to induce normal in vitro shoot regeneration of C. hystrix due to the occurrence of leaf senescence on the regenerated shoots. The inclusion of silver nitrate (AgNO3) at 10.0 µM into Murashige and Skoog (MS) medium containing 2.22 µM BA could effectively overcome the leaf senescence and improved shoot regeneration from different explants (cotyledonary node, cotyledon, hypocotyl and primary root) of C. hystrix. In the rooting experiment, the application of continuous indole-3-butyric acid (IBA) and naphthaleneacetic acid (NAA) treatment of 4.92, 9.84, 14.76, and 19.69 µM IBA or 1.34, 2.69, and 5.37 µM NAA in combination with 20.0 µM AgNO3 resulted in poor rooting of C. hystrix shoots. Rooting was significantly improved upon exposure of shoots to IBA pulse treatment of 9,840 µM for 16 h prior to their transfer on MS medium supplemented with 20.0 µM AgNO3. Assessment of various potting mixtures for acclimatization showed medium containing loamy soil: Peatgro: sand (1:1:1; v/v/v) produced high percentage (83.3 %) of plantlet survival.

Clonal Fidelity of Micropropagated Psidium guajava L. Plants Using Microsatellite Markers

Micropropagation of Psidium guajava L. (guava) is a viable alternative to currently adopted techniques for large-scale plant propagation of commercial cultivars. Assessment of clonal fidelity in micropropagated plants is the first step towards ensuring genetic uniformity in mass production of planting material. In the present study, 31 plants of guava cultivar “Lucknow 49” regenerated by micropropagation were tested for genetic fidelity by comparing them to the mother plant from which explant material was obtained. Efficient rooting of in vitro proliferated shoots was obtained by culture on 1/2 strength MS medium supplemented with either 9.8 μM indole butyric acid (IBA) or 11.4 μM indole acetic acid (IAA). Leaf samples of 31 regenerated plants were compared to the mother plant using 17 simple sequence repeat (SSR) markers. While 16 SSRs detected the same allele, locus mPgCIR07 detected slight differences, where six micropropagated plants were 1 bp smaller (152 bp) than the parental genotype (153 bp). Differences in leaf tissues for anthocyanin pigmentation were also noted among micropropagated plants. Results of the study indicated efficient rooting of “Lucknow-49” cultivar for rapid propagation of planting material, and revealed that micropropagated plants were identical for 16 of the 17 loci examined. Although most mutations induced by tissue culture may not have an effect on phenotype, the possibility that novel phenotypes can be generated in a commercial setting exists.

Adenine sulphate and AgNO3PromoteIn vitroShoot Proliferation inMillettia pinnata(L.) Panigrahi-a Biodiesel Producing Tree

A complete protocol is presented for in vitro regeneration of Milletia pinnata (L.) Panigra-hi, a biodiesel producing medicinal tree, using cotyledonary nodes derived from axenic seed-lings. Shoot development was induced from cot-yledonary nodes on Murashige and Skoog (1962) medium supplemented with 8.88 and 11.1 μM benzyladenine (BA) with or without ad-ditives (silver nitrate (AgNO3) and/or adenine sulphate (Ads)). The maximum number of shoot bud (24.33 buds/explant) and elongated shoots (8.33 shoots/explants) was developed on a medi-um containing 8.88 μM BA with 11.84 μM AgNO3 and 108.6 μM Ads. Upper 2.0 cm portion of elon-gated shoot was harvested for rooting and re-maining portion was cut into nodal segments and cultured on a fresh medium for further mul-tiplication. Thus, from a single cotyledonary node about 40–45 shoots were obtained within eight weeks of culture. Sufficiently long shoots were excised and rooted on half-strength MS supple-mented with 9.84 μM indole butyric acid (IBA). Plantlets were successfully acclimated and estab-lished in soil.

Effect of Nutrient Media and KNO3 on <i>in Vitro</i> Plant Regeneration in <i>Saraca asoca</i> (Roxb.) Willd

Forest trees in general and those belonging to family Fabaceae in particular, have proved to be recalcitrant for propagation through tissue culture. Saraca asoca (Roxb.) Willd. (Family-Caesalpinaceae) is one such tree which has become vulnerable in nature due to over exploitation of its bark. Four nutrient media [MS (Murashige and Skoog Medium), WPM (Woody Plant Medium), B5 (Gamborg’s Medium) and NN (Nitsch and Nitsch Medium, 1969)] and five doses of BA (N6-Benzyladenine) (0, 2.2, 4.4, 8.8 and 17.8 μM) and their all possible interactions were tested for shoot induction and proliferation from nodal segments of 3-year-old plants. B5 medium supplemented with 2.2 μM BA was screened out as the most suitable medium shoot induction, proliferation and elongation of regenerated shoots. In order to enhance shoot number, the nitrogen source in B5 medium was modified and five strengths of KNO3 (0.25×, 0.5×, 1.0×, 1.25× and 1.5×) were tested. The different strengths of KNO3 (Potassium nitrate) had statistically significant effect on number of shoots and on 0.25× strength of KNO3, maximum number of shoots (1.92) were obtained. The modified strengths of KNO3 did not significantly affect the elongation of shoots. Effect of 5 durations (quick dip, transfer of shoots after 1 day, after 3 days, after 5 days and after 7 days) of pulse treatment with 200 μM IBA (Indole-3-butyric acid) in 1/2 strength MS liquid medium was tested. Thereafter, the shoots were transferred to semi-solid half strength MS medium supplemented with 0.2 μM IBA and 3.96 μM phloroglucinol. Pulse treatment of 5 days duration resulted in 37.5% in vitro rooting of shoots. Plantlets were hardened in soilrite soaked with half strength MS medium in culture room and later shifted to a soil mixture in shade house.

Establishment of an efficient in vitro plantlet regeneration system from leaf explants of ornamental crabapple (Malus spp.)

SUMMARYCrabapple (Malus spp.) trees are wild relatives of apple trees and have become increasingly important as an ornamental plant germplasm resource. To establish an efficient regeneration system from leaves would be valuable for the rapid propagation of ornamental crabapple. We therefore evaluated the effects of the type and concentration of various plant growth regulators (PGRs) and carbohydrates used during the regeneration of adventitious buds, shoot multiplication, and rooting in culture. Leaf explants from three cultivars of crabapple (‘Flame’, ‘Strawberry Parfait’, and ‘Royalty’) were used. The most successful types and concentrations of PGRs for the induction of adventitious buds were 4.6 μM thidiazuron (TDZ) plus 1.0 μM α-naphthaleneacetic acid (NAA) for ‘Flame’ and ‘Strawberry Parfait’, and 4.6 μM TDZ plus 2.5 μM NAA for ‘Royalty’. Sucrose at 116 mM was the most effective carbon source to induce buds in ‘Flame’ and ‘Royalty’, while 88 mM sorbitol was the optimum sugar for ‘Strawberry Parfait’. Furthermore, using young leaves placed abaxial side-up on the medium and 10 d of dark treatment promoted leaf regeneration in all three cultivars. Moreover, 6.6 μM 6-benzyladenine (6-BA) and 176 mM sucrose promoted shoot multiplication, while the optimum concentrations for shoot growth were 2.2 μM 6-BA and 58 mM sucrose. In contrast, the best PGRs to induce adventitious rooting were 5.0 μM indole-3-butyric acid (IBA) for ‘Flame’, 4.0 μM IBA for ‘Strawberry Parfait’, and 4.0 μM IBA plus 0.5 μM NAA for ‘Royalty’. Thus, the type and concentration of PGR and carbohydrate source are critical factors in establishing an efficient in vitro regeneration system from leaf explants in crabapple. This information will be valuable when developing efficient methods to genetically transform crabapple or other fruit tree species

Callus induction and proliferation fromRosa hybridaleaf explants

To establish a resourceful tissue culture system to induce anthocyanin pigments, research was conducted to obtain high quality callus from leaf explant of Rosa hybrida cv. Pusa Ajay. Of the different treatments employed for callus induction using Murashige and Skoog (MS) medium, a treatment combination of 19.60 μM indole3-butyric acid (IBA) + 4.65 μM kinetin (Kin) + 108.6 mM adenine sulphate (AdS) gave the maximum induction coefficient (97.75 ± 1.11%) along with maximum gain in callus biomass accumulation. This treatment also resulted in the lowest fresh-and dry-cell weight ratio (26.52) suggesting actual gain in callus biomass accumulation. Correlation analysis indicated a significant positive correlation between callus induction coefficient and cell fresh-(r = 0.939) and dry-(r = 0.951) weight.

An optimized method for in vitro propagation of African baobab (Adansonia digitata L.) using two-node segments

Adansonia digitata L. (African baobab), is an important multi-purpose tree, whose distribution is at present limited to wild or semi-domesticated individuals widespread in Africa. Its distribution is threatened by seedling clearance for other land use and potentially by overharvesting induced by growing commercial use of baobab fruit. Recently, efforts have been made to establish baobab domestication and conservation strategies, with mixed results due to the low germinability of baobab seeds, a factor that hinders the possibility of developing commercial A. digitata plantations. Here, micropropagation was tested as a method for clonal propagation of explants from in vivo-grown seedlings. In vitro shoot multiplication was achieved by enhanced axillary bud proliferation of sterilized two-node segments. Bud break was dependent on cytokinin supply, but the combination of 1.0 or 10.0 μM zeatin riboside and 10.0 μM indole-3-butyric acid (IBA) increased the formation of microshoots after 8 weeks of culture. Regenerated microshoots rooted successfully in in vitro nutrient medium containing 10.0 μM IBA and normally grew in a greenhouse after acclimatization.

GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA3) followed by incubation at dark, and (iv) immersion in 400 ppm GA3 followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0×MS medium containing 4.44, 8.88, 17.76μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0×MS medium containing 17.76μM BAP plus 10.74μM NAA and 17.76μM BAP plus 2.685μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0×MS medium containing 4.9μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0×MS medium containing 17.76μM BAP plus 10.74μM NAA) were transferred to 1.0×MS medium containing 30g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0×MS medium containing 4.44μM BAP plus 5.37μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.

Azospirillum brasilense enhances in vitro rhizogenesis of Handroanthus impetiginosus (pink lapacho) in different culture media

Handroanthus impetiginosus is an ornamental tree used in urban forestry programs and landscaping, produces good timber, and has medicinal qualities. Clearing of natural forest has been increased and motivates to find techniques that optimize rhizogenesis to ex situ conservation and reforestation. This study evaluated Azospirillum brasilense as an enhancer of in vitro rooting of H. impetiginosus under different indole-3-butyric acid (IBA) pulse inductions and media formulations. In vitro shoots were induced with 0 to 50-μM IBA pulse for 3 days, transferred to two half-strength media (Murashige and Skoog salts with Gamborg’s vitamins [MSG] and woody plant medium), and inoculated or not with A. brasilense Cd or Az39 strains. The statistic response surface methodology determined that both A. brasilense strains decreased auxin requirement for rooting up to 49 % in half-strength MSG. In this medium, Cd strain with 30-μM IBA pulse produced the 98 % of rooting shoots 21 days before uninoculated plants induced with the same IBA pulse. Also, the biometric parameter index increased from 127 to 286 % in shoots inoculated and induced with 0 to 30 μM IBA, relative to uninoculated. Our results show that biofertilization with A. brasilense significantly promotes in vitro rooting of H. impetiginosus decreasing auxin requirements and micropropagation costs.

Thidiazuron induced in vitro multiplication of Mentha arvensis and evaluation of genetic stability by flow cytometry and molecular markers

An efficient and reproducible system for the development of genetically uniform clones of a highly valuable essential oil producing crop Mentha arvensis was developed by pre-conditioning of nodal explants with thidiazuron (TDZ) supplemented liquid Murashige and Skoog (MS) media. Multiple shoot formation was achieved by pretreating of nodal explants with 5, 15, 25 or 50μM TDZ for 24h, followed by their transfer on TDZ free semi-solid MS media. The maximum response in terms of percent regeneration (92.6±3.65), average number of shoots per explants (23.7±1.45), and greatest shoot length (5.2±0.72) were obtained from nodal explants treated with 15μM TDZ for 24h followed by culture of explants on TDZ free MS medium for 8 weeks. Elongated shoots when transferred to half-strength MS medium supplemented with 0.5μM indole-3 butyric acid (IBA) resulted in 91±2.91% rooting. In vitro regenerated plantlets with well-developed shoot and roots were successfully established in pots containing garden soil with a 90% survival rate. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using flow cytometry and molecular markers. No changes in plant genome size/ploidy level, and in directed amplification of minisatellite-region (DAMD) and inter simple sequence repeat (ISSR) profiles were found among the micropropagated plants, which were similar to that of the donor plant, illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite.

Micropropagação de Aechmea setigera Mart. ex Schult. & Schult. f.: Uma Bromélia Endêmica da Amazônia Ocidental

As bromélias da Amazônia são em geral pouco conhecidas. Aechmea setigera é uma bromélia endêmica da Amazônia com potencial ornamental. O objetivo do trabalho foi avaliar as respostas fisiológicas sob efeito de reguladores de crescimento nas etapas da micropropagação, bem como, estabelecer um protocolo como subsídio para a conservação.

Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation.

In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ.