Shoot Multiplication Rate Research Articles

In vitro culture of the endangered plant Eryngium viviparum as dual strategy for its ex situ conservation and source of bioactive compounds

Different Eryngium species have been used with ornamental, agricultural and medicinal purposes, as a consequence of their chemical constituents. In the southwest Europe the endemic Eryngium viviparum, presents a high risk of extinction and ex situ strategies are high recommended for efficient conservation and re-introduction program. The objective of this study was to satisfy a dual objective: (i) to develop an ex situ conservation strategy through micropropagation and (ii) taking advantage of the extraordinary potential of plant tissue culture, produce a considerable amount of plant material to carry out a preliminary phytochemical study, based on the accumulation of phenolic compounds and their associated antioxidant activity. First a factorial design was conducted in order to study the effect of two cytokinins (6- benzylaminopurine, BAP, and kinetin, KIN), at three levels (0, 1 and 2 mg L−1), on shoot multiplication. Later another factorial design was applied, by using three levels of MS medium salt strength (full, half and quarter- strength) and four sucrose levels (0, 1, 2, and 3%) for improving shoot elongation and rooting. In parallel, a preliminary quantification of total phenolic and flavonoid contents from E. viviparum aerial parts was determined. The simple micropropagation protocol designed allowed obtaining a high rates of shoot multiplication (5.1–5.8 new shoots), rooting (100%) with healthy long roots (3.1–3.5 cm) and plantlet acclimatization (96%). Moderate antioxidant activity was recorded in hydromethanolic extracts from E. viviparum aerial parts. High correlation between total phenolic content and BAP levels in the culture media was found. In conclusion, the micropropagation procedure described here for the endangered E. viviparum can be used as new and very efficient ex situ conservation strategy, and as potential source of antioxidants, conferring an added-value to this plant.

Sodium azide mutagenesis within temporary immersion bioreactors modifies sugarcane in vitro micropropagation rates and aldehyde, chlorophyll, carotenoid, and phenolic profiles

Chemical mutagens such as sodium azide (NaN3) have been widely used to increase genetic variability in crops, but the undirected mutations induced can have undesirable effects, which need to be characterized. This study investigated the effects of in vitro NaN3 (0–0.45 mM) exposure (30 days) on the micropropagation of sugarcane within temporary immersion bioreactors (TIB). Shoot multiplication rate and cluster fresh weight, and aldehyde, phenolic, carotenoid, and chlorophyll levels were measured on in vitro produced shoots. The soluble phenolic content of the culture medium was also assessed. NaN3 concentration was negatively correlated with sugarcane shoot multiplication rate and fresh weight; at 0.45 mM NaN3, these parameters were only 20% and 39% that of the untreated control, respectively. Shoot multiplication rate and fresh weight, and chlorophyll a and b levels were negatively correlated with NaN3 concentration. In contrast, malondialdehyde, other aldehyde, carotenoid, and exuded phenol levels were positively correlated with NaN3 concentration. Statistical comparisons suggest that shoot multiplication rate and the biochemical parameters that were positively correlated with NaN3 concentration may be the most suitable indicators of stress when optimizing the concentration of NaN3 for sugarcane explants. An interpolated 50% reduction of multiplication rates at 0.23 mM NaN3 suggests that this concentration to be suitable for TIB-based induction of mutagenesis in shoots and eventual production of agriculturally useful mutants.

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

Alpha monofluoro substitution at C5 in homotyphasterol enhances shoot production and multiplication rate of in vitro-grown marubakaido apple rootstock shoots

KEY MESSAGE: 5 fluoro-typhasterol is more effective than the most potent natural brassinosteroid, brassinolide, towards stimulation of shoot production in the marubakaido apple rootstock. Brassinosteroids (BRs) comprise a class of low-abundance plant steroids required for normal plant growth. Here we demonstrate that treatment of in vitro-grown shoots of the marubakaido apple rootstock with 2.5 µg per shoot of 5α-monofluoro homotyphasterol (5F-HTY), a synthetic derivative of the natural BR homotyphasterol (HTY), resulted in significant enhancement in the formation of primary lateral shoots. This treatment also resulted in increased length of primary lateral shoots and main shoots. This growth-stimulatory effect led to a significant increase in the multiplication rate for the rootstock. In contrast to what was found for 5F-HTY, neither HTY nor the synthetic derivative 3α-monofluoro homotyphasterol (3F-HTY) were able to significantly stimulate shoot formation. HTY and 3F-HTY were not able to stimulate primary lateral shoot elongation as well. However, both HTY and 3F-HTY were able to significantly stimulate main shoot elongation. The 5F-HTY-driven enhancement of the multiplication rate described here demonstrates the potential of this compound to improve micropropagation techniques, not only for the marubakaido apple rootstock.

Axillary Shoots Derived from Thin Cell Layer and Adenine Sulphate Application in in vitro Mass Propagation of Gerbera [Gerbera jamesonii (H. Bolus ex Bolus f.)

A new route of in vitro mass propagation protocol of Gerbera jamesonii (H. Bolus ex Bolus f.) derived from application of thin cell layers (TCL) and adenine sulphate (AS) was successfully developed and established. Shoot tip explants and half-strength MS medium containing 0.25 mg/l N6-benzylaminopurine (BAP), 20 g/l sucrose and 7 g/l Swallow agar were used as explant source and basic medium. Different TCL of transversal TCL (tTCL) and longitudinal TCL (lTCL) in four slicing positions of 1, 2, 3 and 4; varieties and clones i.e. G. jamesonii ‘Black Jack’, ‘Carambole’, ‘Nuance’, ‘Violente’, 01.098 and 11.46 clone; AS concentrations viz. 0, 20, 40, 60, 80 and 100 mg/l were tested in the study. Each step of in vitro culture established had unique and specific results. In the initiation stage, first slicing position of ‘Black Jack’ shoot tip tTCL was the most optimal combination treatment to produce 7.0 shoots per explant with 13.5 leaves. The first slicing position on shoot tip explants of 01.098 clone tTCL and 20 mg/l AS in half-strength MS medium containing 0.25 mg/l BAP were the most optimal combination treatment in obtaining the highest number of shoots produced per shoot up to 9.4 shoots per shoot with 34.1 leaves and 2.37 cm length of leaves in the proliferation stage, however the treatment did not give significant effect compared to control. Under periodical subcultures on the basic medium, number of shoots and leaves increased gradually from the initial culture with 3-6 shoots per shoot and 9.4-11.6 leaves till the fourth subculture with 6-11 shoots per shoot and 16.7-28.8 leaves and declined thereafter. Subculturing of shoots in accordance to produce qualified shoots for planting materials could be carried out till sixth to seventh subculture. The highest shoot multiplication rate (SMR) was established on 01.098 clone with as high as 7.3. The well shoots were easily rooted on half-strength MS medium supplemented with 0.1 mg/l BAP, 0.05 mg/l NAA and 1.5 g/l AC. Plantlets were then transferred to ex vitro condition for acclimatization on a mixture of burned-rice husk and organic manure (1:1, v/v) with 85-100% survivability. The ‘Black Jack’ and 11.46 clone were the best genotypes on the acclimatization stage with 100% survivability of plantlets. Results of the study have implication that first slicing position of shoot tip tTCL can be applied in establishing of in vitro propagation protocol for other gerberas.

Shoot multiplication and growth rates of Aquilaria malaccensis Lamk. shoot cultures in temporary immersion system (TIS)-RITA® and bubble column bioreactors

Shoot multiplication and growth rates of Aquilaria malaccensis Lamk. shoot cultures in temporary immersion system (TIS)-RITA® and bubble column bioreactors

A Practical and Efficient Method for the Micropropagation of Japanese Cherry (Prunus sp.)

This study was conducted to establish the procedure for in vitro propagation of Japanese cherry (Prunus sp.) to produce large quantity of plantlets and initial planting materials for climate adaptation research of this plant in Hanoi. Single nodal stems were used as the primary explants and initially produced shoots on MS medium supplemented with 1 mg L-1 BA. The highest shoot multiplication rate (9.57 times) was obtained on MS medium containing 1 mg L-1 BA and 0.25 mg L-1 a-NAA after 8 weeks of culture. 100% of the shoots produced roots with a mean of 10.10 roots per plant within 4 weeks on ½ MSM medium with 4 mg L-1 IBA. The survival rate of in vitro derived plantlets after a 6 to 7-week-period of rooting during acclimatization using a soil: coco peat: smoked rice husks (2:2:1, v/v/v) substrate was 100% and acclimatized plantlets showed good growth and development. This is the first report on a practical and efficient in vitro multiplication protocol for Japanese cherry in Vietnam, starting from shoot initiation to establishment of plants under greenhouse conditions.

Mutagenic effects of sodium azide on pineapple micropropagant growth and biochemical profile within temporary immersion bioreactors

Sodium azide (NaN3) is widely used to induce mutagenesis within in vitro plant systems. However, since this mutagenesis is undirected, its unintended effects demand characterization. This study investigated the mutagenic effects of sodium azide (0-0.45 mM) on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants within temporary immersion bioreactors (TIBs). The content of soluble phenolics in the culture medium was also evaluated. Irrespective of the concentration NaN3 decreased shoot multiplication rate (by 87% relative to the control at 0.45 mM) and fresh weight (by 66% relative to the control at 0.45 mM). Levels of chlorophyll a and b, and soluble phenolics in the culture medium were also negatively correlated with NaN3 concentration. Interestingly, NaN3 application increased shoot carotenoid and soluble phenolic levels but had no significant effect on a range of established plant stress biomarkers: cell wall-linked phenolic levels, malondialdehyde and other aldehydes. Given that 0.19 mM NaN3 decreased shoot multiplication rate by 50% and resulted in propagants that displayed no morphologically abnormalities, increased levels of photoprotective pigments (relative to the control) and no significant increase in lipid peroxidation products, the mutagen can be used at this concentration to induce pineapple mutagenesis in TIB based studies aimed at producing agriculturally-useful mutants.

Shoot multiplication via elongated stem node culture under darkness: a novel method in micropropagation of Paphiopedilum villosum

Paphiopedilum villosum is a beautiful orchid species and has high value in trade; however, this is one of the most difficult to propagate orchids. So far, there has been very little publication on micropropagation. In this study, the effects of plant growth regulators on shoot regeneration from stem node culture of elongated P. villosum shoots in the darkness were investigated. Shoots (1.5 cm) were elongated and produced individual stem nodes under darkness condition for 3 months. Stem nodes were cultured on SH medium and supplemented with individually BA, KIN or TDZ to investigate shoot regeneration. The shoot multiplication rate was also recorded in this study. The highest stem node was observed in the dark with 5.25 cm in the height and the number of stem nodes were 3 stem nodes/shoot. The isolated stem node was cultured on SH medium supplemented with 30 g/L sucrose, 8 g/L agar, 1 g/L charcoal and different concentrations of BA, KIN, TDZ. The results observed after 3 months showed that the best shoot regeneration rate (85%) and highest shoot multiplication coefficient (6.6 shoots/stem node) was obtained when shoots derived from stem node were cultured on SH medium supplemented with 0.5 mg/L TDZ, 30 g/L sucrose, 8 g/L agar and 1 g/L charcoal. Those shoots obtained in the above treatments were subcultured on MS medium supplemented with 0.3 mg/L NAA for rooting and gave the highest number of roots (6.6 roots/shoot) after 1 month; and these plantlets were acclimatized in Taiwan sphagnum moss and transferred into greenhouse with the best survival rate (100%) after 3 months.

SEQUENCING OF MATK GENE FOR TAXONOMIC IDENTIFICATION AND IN VITRO PROPAGATION OF A LOCAL YAM (DIOSCOREA SP.) CULTIVAR GROWN AT MUONG KHUONG, LAO CAI

Khoai mo, local name of Yam species belonging to the Dioscoraceae family, is widely grown at Muong Khuong, Lao Cai province because of their important food and pharmaticeutical valuables. This work aimed to sequence the MatK gene obtained from leaves of a local yam grown in Muong Khuong, Lao Cai. The sequencing result showed a partial sequence of the MatK gene which was submitted to GenBank with accession MF494702 provided. The length of this partial sequence is 916 nucleotides. Phylogeny analysis indicated that local yam cultivar grown at Muong Khuong, Lao Cai belonged to Dioscorea alata species. Nodal segments collected from this local yam were used as initial material for in vitro vegetative multiplication. In the shoot formation step, BAP or BAP and NAA complex were used in shoot formation experience. In vitro shooting effect was higher on MS medium supplemented with BAP at 0.3 mg/l of contrentration in compared to other studied BAP concentrations. The presence of NAA at 0.5 mg/l in medium containing BAP promoted a high rate of shoot multiplication. Highest value of shoot production was obtained on MS medium fortified with BAP (1.5 mg/l) and NAA (0.5 mg/l) from nodal segments. In next step, shoots were successfully rooted in vitro. A high number of roots per shoot and highest length of roots were obtained on MS medium containing 0.2 or 0.3 mg/l of NAA. High acclimatization frequencies were obtained when transferred rooted explants to rich soil, yellow clay soil and soil+sand mixture (1:1). Best survival rate (100%) was achieved on rich soil substrate during ex vitro acclimatization process.

Quercetin and silver nitrate modulate organogenesis in Carissa carandas (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO3), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L−1 6-benzylaminopurine; 0.5 mg L−1 kinetin, and 0.75 mg L−1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L−1 BAP, 0.5 mg L−1 kinetin, and 1.5 mg L−1 AgNO3 was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L−1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.

Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.

In vitro Clonal Multiplication of Two Grape (Vitis spp.) Rootstock Genotypes

Single node segments were used to initiate in vitro cultures in two grape rootstocks namely, Dogridge (Vitis champini) and H-144 (Vitis vinifera × V. labrusca). Culture establishment was enhanced using different growth regulators, while BAP was found essential for culture initiation in both genotypes. Less success (38.31%) was obtained in culture establishment of H-144 but it exhibited better vegetative growth and rooting and ex vitro performance as compared to Dogridge. Higher shoot multiplication rate (12 micro-cuttings per culture) was recorded in H-144 while only 9 micro-cuttings per subculture were registered in Dogridge. Addition of activated charcoal to the rooting medium was found beneficial with enhancement of rooting and reduction in time to root initiation in both genotypes. The results suggested that multiplication of these two grape rootstock genotypes can be carried out efficiently by means of direct in vitro regeneration using nodal segments. In vitro performance of these two genotypes was also compared during different stages of micropropagation.Plant Tissue Cult. & Biotech. 28(1): 1-11, 2018 (June)

Direct Shoot Organogenesis from Seedling Derived Shoot Tip Explants of Endangered Medicinal Plant Saussurea costus (Falc.) Lipsch.

An efficient direct in vitro plant regeneration protocol for Saussurea costus (kuth) was developed using shoot tip explants derived from in vitro grown seedlings to generate genetically uniform plants. Murashige and Skoog (MS) medium supplemented with 1.14 µM thiadiazuron and 2.68 µM naphthalene acetic acid was found to be most optimal for highest for average shoot regeneration (73.33%) with maximum average number of shoots (11.4) and average shoot length (4.17 cm). For in vitro multiplication, shoots cultured on MS medium supplemented with 4.44 µM benzyl adenine, 2.32 µM kinetin and 1.44 µM gibberellic acid were found to grow best with 20.7 average shoot multiplication rate after 5–6 weeks. MS medium supplemented with 2.46 µM indole butyric acid was found to be the best medium for rooting of microshoots (77.78% rooting with 6.0 average number of roots and 3.07 cm average root length). The in vitro raised plantlets were successfully acclimatized under ex vitro conditions. The true to type nature of the in vitro raised plants was confirmed using DNA based ISSR markers for assessment of genetic stability of micropropagated plants. The standardized micropropagation protocol under present study may be tuned towards mass production of roots which are the most economical part of the plant. This would be useful as prerequisite for carrying out any genetic transformation studies while concentrating on any desirable trait in the same herb.

Euclidean distance can identify the mannitol level that produces the most remarkable integral effect on sugarcane micropropagation in temporary immersion bioreactors

Plant scientists usually record several indicators in their abiotic factor experiments. The common statistical management involves univariate analyses. Such analyses generally create a split picture of the effects of experimental treatments since each indicator is addressed independently. The Euclidean distance combined with the information of the control treatment could have potential as an integrating indicator. The Euclidean distance has demonstrated its usefulness in many scientific fields but, as far as we know, it has not yet been employed for plant experimental analyses. To exemplify the use of the Euclidean distance in this field, we performed an experiment focused on the effects of mannitol on sugarcane micropropagation in temporary immersion bioreactors. Five mannitol concentrations were compared: 0, 50, 100, 150 and 200mM. As dependent variables we recorded shoot multiplication rate, fresh weight, and levels of aldehydes, chlorophylls, carotenoids and phenolics. The statistical protocol which we then carried out integrated all dependent variables to easily identify the mannitol concentration that produced the most remarkable integral effect. Results provided by the Euclidean distance demonstrate a gradually increasing distance from the control in function of increasing mannitol concentrations. 200mM mannitol caused the most significant alteration of sugarcane biochemistry and physiology under the experimental conditions described here. This treatment showed the longest statistically significant Euclidean distance to the control treatment (2.38). In contrast, 50 and 100mM mannitol showed the lowest Euclidean distances (0.61 and 0.84, respectively) and thus poor integrated effects of mannitol. The analysis shown here indicates that the use of the Euclidean distance can contribute to establishing a more integrated evaluation of the contrasting mannitol treatments.

Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.)

The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro bud induction and proliferation of a potent medicinal woody shrub R. tetraphylla were investigated. Multiple shoots were induced from nodal explants on Murashige and Skoog medium augmented with different concentrations (0.2–1.0 µM) of TDZ. Continuous exposure beyond 5 weeks of singly supplemented TDZ resulted in hyperhydricity, fasciation and distortion of cultures. To avoid these deleterious effects, benzyl adenine (BA) was supplemented to the media. It not only mitigated the negative effects of prolonged TDZ exposure, but also enhanced shoot multiplication rate. About 30 shoots per explant with an average shoot length of 5.8 cm were obtained after 20 weeks of culture on MS medium fortified with 0.4 µM TDZ and 2.0 µM BA, regeneration frequency was also enhanced by 24% on this media as compared to singly supplemented TDZ. Microshoots (~ 5 cm) were successfully rooted on filter paper bridge in MS liquid medium augmented with different concentrations of indole-3-butyric acid (IBA) after 4 weeks of incubation. Best rooting response (80%) was achieved on MS medium supplemented with 0.3 µM IBA with (9.3 ± 1.45) mean number of roots and (3.3 ± 0.33 cm) root length per shootlet. The in vitro raised healthy plantlets with well developed root and shoot were successfully hardened off and acclimatized in thermocol cups containing sterilized planting substrate, Soilrite® for 8 weeks under culture room conditions prior to field transfer and successfully established in earthen pots with 90% survival rate. The genetic fidelity of in vitro regenerated plantlets was evaluated using random amplified polymorphic DNA markers. No polymorphism was observed in banding pattern among micropropagated and the donor plant, thus, exhibiting their genetic uniformity and clonal fidelity.

Impact of LED light sources on morphogenesis and levels of photosynthetic pigments in Gerbera jamesonii grown in vitro

Gerbera jamesonii cv. Dura is a well-known and economically important ornamental crop that is produced using micropropagation. This study aimed to evaluate the effect of various light wavelengths in six different light emitting diode (LED) treatments on biometric attributes and photosynthetic pigment content in gerbera plants grown in vitro. The LED treatments were B (100% blue), RB1 (50% red and 50% blue), RB2 (70% red and 30% blue), RBW (40% red, 40% blue, and 20% white), RBfR (49% red, 49% blue, and 2% far red), and R (100% red). Light quality was seen to significantly affect plant growth and development. The highest shoot multiplication rate was observed in plants grown under both RB1 and RB2. Plantlets grown under R displayed the greatest shoot elongation, and their petioles were three times longer than those grown under B. Both B and R resulted in reduced leaf blade area. Rooting of shoots was observed in plants grown under all light treatments; however, R stimulated adventitious root formation. The average number of roots produced by plants under all light treatments was calculated and found to match that produced under RB2. The RBfR treatment caused a reduction in leaf dry weight compared to that produced under B, which represented the highest leaf dry weight. Control fluorescent lighting, supplied by Philips TL-D 36W/54 lamps, had a positive effect on root dry weight. Photosynthetic pigment content was higher in the leaves of rooted plants compared to that in plants at the multiplication stage. RB2 resulted in higher concentrations of chlorophyll a and b and carotenoids, whereas lower accumulation of photosynthetic pigments was observed under R. These results demonstrate that light wavelength manipulation through LEDs can be strategically used for the rapid and large-scale propagation of gerbera. The outcomes of this study offer potential to improve micropropagation efficiency and reduce the costs of in vitro plant production.

IN VITRO DEVELOPMENT OF YELLOW LAPACHO (BIGNONIACEAE) USING HIGH-POWER LIGHT EMITTING DIODE

ABSTRACT Handroanthus ochraceus (yellow lapacho) is a medicinal, ornamental and timber tree which can be propagated by in vitro culture. Conventional methods use fluorescent lighting (FL), whereas light emitting diode (LED) has been used for this purpose only recently. The aim of this work was to evaluate the effects of FL and high-power LED (HP-LED) on the in vitro multiplication and rooting of yellow lapacho at different irradiances (15 to 60 µmol m-2s-1). Epicotyls obtained from half-siblings was multiplicated in WPM (Woody Plant Medium) supplemented with 20 µM benzilaminopurine and 1 mM IBA (indolebutiric acid). For rooting, shoots were cultured for 3 days in ½WPM supplemented with 50 µM IBA and for 42 days in auxin-free ½WPM under HP-LED or FL lighting. Under HP-LED, the multiplication rate of shoots increased significantly (61%) from 20 to 40 µmol m-2s-1 respect to FL. Differences in abaxial stomatal density and size were observed between light sources at 20 µmol m-2s-1. High HP-LED irradiance produced the highest rooting percentage. In the rooting stage, the marginal means of treatments without factors interaction showed that HP-LED irradiances significantly increased shoot length by 20%, shoot fresh weight by 77% and shoot dry weight by 30% in comparison to the values under FL. The maximum values calculated from the regression curves were around 50 µmol m-2 s-1 for HP-LED for all parameters except root lenght whereas were around 20 µmol m-2 s-1 for FL for all parameters except fresh and dry weigth of shoot. Here we show that HP-LED lighting improve in vitro culture of H. ochraceus, reduced 81% energy consumption respect to FL and uses only a multispectral LED instead of different single color LEDs. Therefore, HP-LED could be useful for the micropropagation of tree species contributing to sustainable agriculture and ecological restoration of degraded areas.

Salinity induces specific metabolic changes in sugarcane shoot explants in temporary immersion bioreactors

There is a great demand of salt-tolerant sugarcane planting material in Cuba. Temporary immersion bioreactors (TIB) are effective to significantly increase sugarcane in vitro shoot proliferation rate from 1:4 in conventional containers to about 1:35. Sugarcane micropropagation in TIBs under NaCl stress may help screen mutants with salinity tolerance. We developed the experiment shown here to identify a NaCl concentration able to stress shoot in TIBs. At 30 days of culture initiation with different NaCl levels (0 - 200 mM), explant multiplication rate, shoot cluster fresh mass, and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. Content of soluble phenolics in the culture medium was also evaluated. Addition of NaCl decreased shoot multiplication rate and fresh mass. Other statistically significant differences were recorded but the most important were noted in the increased contents of carotenoids, malondialdehyde, other aldehydes and soluble phenolics in the plants, and in the soluble phenolics in the culture medium. This research may be useful for future experiments of in vitro selection of new sugarcane genetic materials with NaCl tolerance. Fifty percent of multiplication rate was reduced with 89 mM NaCl which can be used to stress shoots during micropropagation in TIBs and eventually detect mutants with salt tolerance.

Temporary immersion bioreactors (TIB) provide a versatile, cost-effective and reproducible in vitro analysis of the response of pineapple shoots to salinity and drought

Effects of salinity (NaCl) and the carbon source mannitol (0–200 mM) on micropropagation of pineapple cv. MD2 were analyzed in temporary immersion bioreactors (TIBs). Shoot multiplication rate, shoot cluster fresh weight and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. The content of soluble phenolics in the culture medium was also evaluated. NaCl or mannitol above concentrations of 50 mM decreased pineapple shoot multiplication and fresh weight significantly. Two hundred mM NaCl decreased multiplication rate by 71.5% and cluster fresh weight by 40.0%. NaCl increased 2.4 times the levels of other aldehydes; 1.4 times the soluble phenolics in shoots; and 1.4 times the phenolics excreted to the culture medium. On the other hand, mannitol decreased the multiplication rate and cluster fresh weight by about 60%. Mannitol increased the contents of chlorophyll b 1.4 times and soluble phenolics 2.1 times. Results indicated that pineapple cv. MD2 is more sensitive to NaCl than to mannitol. Multiplication rates indicate that a 50% reduction was obtained with 37.4 mM NaCl and 66.5 mM mannitol. These concentrations can be used to stress shoots during micropropagation in TIBs and screen for/detect somaclonal variants with an increased salinity or drought tolerance.