μM GA3 Research Articles

Gibberellins and light synergistically promote somatic embryogenesis from the in vitro apical root sections of spinach

Gibberellins (GAs) play a pivotal role in the induction of somatic embryogenesis from in vitro root apices of spinach plants. With the aim to understand the role of GAs in this process and to improve somatic embryo (SE) regeneration efficiency, the impact of light and GAs on SE initiation from the in vitro root apices was studied. The root sections were isolated from in vitro-grown SE-derived plants and placed on medium containing 20 µM α-naphthaleneacetic acid (NAA) and 0–10 µM GA3 or GA1, and cultivated under light conditions or in darkness. The most efficient SE regeneration response (100% regenerating SEs and 40.73 SEs per root apices) was achieved only in the presence of both light and GAs, with GA3 always exhibiting much stronger effect than GA1. Considering that light enhances GAs biosynthesis and the necessity of GAs for SE initiation, the expression levels of genes encoding the key enzymes involved in the final steps of GAs synthesis (SoGA20-ox1 and SoGA3-ox1) and deactivation (SoGA2-ox1, SoGA2-ox2 and SoGA2-ox3) were analyzed. Light enhanced the expression of all five GA-ox genes, while exogenously supplied NAA + GA3 provoked downregulation of SoGA20-ox1 and SoGA3-ox1 and upregulation of SoGA2ox-2 and SoGA2ox-3 expression. The expression of SoGA2ox-1 only slightly decreased. The results indicated the capability of isolated spinach roots to perceive the light and autonomously produce GAs. The expression levels of genes encoding key enzymes involved in GA biosynthesis suggest that lower levels of GAs favor SE initiation. Light and gibberellins (GA) synergistically promote somatic embryogenesis in spinach. Expression levels of genes encoding key enzymes for GA metabolism suggest that lower levels of GAs may favor somatic embryogenesis.

Induction of direct somatic embryogenesis and shoot organogenesis and histological study in tree peony (Paeonia sect. Moutan)

Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species, but it is recalcitrant to in vitro regeneration. Here, we present a protocol for induction of direct somatic embryogenesis (SE) and direct shoot organogenesis (SO) from zygotic embryos (ZEs) of Paeonia rockii and P. ostii. The results showed that explant genotypes, ZEs stages, sucrose and plant growth regulators (PGRs) have greatly influences on in vitro morphogenesis. The highest frequency of SE (48%) and mean number of somatic embryos (5) was obtained from mature ZE of P. rockii ‘Jing Hong’ when cultured on modified Murashige and Skoog (mMS) medium supplemented with 2.22 µM BA and 0.23 M sucrose.The proliferation can be achieved by recurrent production of embryogenic callus and somatic embryos on mMS medium supplemented with 0.89 µM BA. The germinated rate of somatic embryos was 45% on WPM medium containing 2.22 µM BA and 1.44 µM GA3 after dormancy release at 4 °C under dark for 20 days. In addition, direct SO was obtained firstly in tree peony by using a variety of cytokinins. Morpho-histological analysis confirmed the initiation, development and reserve accumulation of somatic embryos and shoots. The study will be benificial to the propagation and breeding of tree peony. Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species which is recalcitrant to in vitro regeneration. Direct somatic embryogenesis protocol of P. rockii ‘Jing Hong’ was developed and morpho-histological analysis confirmed the initiation, development and reserve accumulation during in vitro morphogenesis. The study will provide valuable reference for propagation and breeding of tree peony.

In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid.

Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed germination (93.3%) was achieved on ½ MS medium without vitamins plus 5% CW, 1 µM NAA, and 1.5 µM GA3. Secondary protocorm formation was best achieved on ½ MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid.

Silver compounds regulate leaf drop and improve in vitro regeneration from mature tissues of Australian finger lime (Citrus australasica)

Finger lime is a small-leaved citrus native to Australia that is becoming increasingly popular worldwide. Finger lime is monoembryonic and cannot be propagated true to type from seeds. Initial studies to develop a tissue culture protocol for the mass production of elite lines being developed in our breeding program were unsuccessful due to complete leaf abscission during the culture establishment phase. In this study, silver-containing compounds such as silver thiosulfate (STS), silver nitrate (AgNO3), and silver nanoparticles (AgNPs) were applied at three concentrations (20, 40, and 60 µM) to manage ethylene biosynthesis in finger lime tissue cultures. The Murashige and Skoog (MS) medium supplemented with 2.2 µM BAP and 60 µM silver thiosulfate was the best medium for culture establishment. The same level of STS with 0.70 µM GA3 produced the largest number of shoots and prevented leaf abscission. Micropropagated shoots were successfully rooted in half-strength MS medium supplemented with 0.10 µM NAA. The addition of 60 µM silver thiosulfate (STS) to the MS medium resulted in efficient micropropagation of the finger lime explants and controlled leaf abscission. The study evaluates the efficacy of various silver-containing compounds on their ability to effectively control ethylene production and allow for the development of an efficient tissue culture protocol for Finger lime.

Effect of exogenous application of IAA and GA3 on growth, protein content, and antioxidant enzymes of Solanum tuberosum L. grown in vitro under salt stress

Indole-3-acetic acid (IAA) and gibberellic acid (GA3) are essential for the growth and development of plants. In the present study, the ameliorative potential of these phytohormones on growth, protein content, and antioxidant enzymes was investigated in in vitro-grown Solanum tuberosum L. cultivars ‘Cardinal’ and ‘Desiree’ under salt stress. A 4 × 3 factorial combination of 0, 40, 60, or 80 mM NaCl with 0, 7, or 14 μM IAA, or 0, 14, or 21 μM GA3, were added to Murashige and Skoog (MS) basal medium, followed by inoculation of nodal explants or callus cultures. The data for root and shoot number and length, number of nodes and leaves, fresh weight of plants, increase or decrease in fresh weight of callus cultures, total soluble protein, and superoxide dismutase (SOD) and peroxidase (POD) activities were recorded after 30 d. The growth of both callus cultures and nodal explants subjected to NaCl stress was substantially reduced compared with the control. Both IAA and GA3 successfully alleviated the harmful effects of salt stress on all of the growth parameters studied. Salt stress resulted in decreased protein content, which increased when the media also contained phytohormones. The activities of SOD and POD were increased with either IAA or GA3 under NaCl stress. Therefore, the exogenous application of both IAA and GA3 not only played a positive role in terms of in vitro potato growth but also significantly affected the biochemical parameters tested.

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar 'Kufri Chipsona 1'. It was observed that explants pre-cultured for 2days on MS2 medium (MS medium containing 10µM silver nitrate, 10µM BA, 15µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar 'Kufri Chipsona 1'.

Biochemical Characterization on Photosynthetic Activities during Dark Incubated Senescence of Wheat Primary Leaves by Gibberellic Acid

In annual crop plants like maize, rice and wheat etc. Senescence limits crop yields of annual crops like maize rice and wheat. Delayed leaf senescence is a desirable agronomic trait to improve crop yield. In this study 10 µM GA reduced the loss of wheat primary leaves under incubated dark conditions. GA reduced the loss of pigments, proteins, electron transport activities, spectral properties. The restoration of WCE activity by GA was closely associated with the restoration of PS II activity compared to that of PS I. GA treated leaf thylakoid membranes showed an increase in absorption at 680 nm moderate increase at 480 nm and 440 nm at 72 h during dark incubation. GA protected the degradation of water oxidation complex polypeptides (33, 23, 17 KDa) of PS II and slightly protected the PS I polypeptides.

Expression Patterns of Key Hormones Related to Pea (Pisum sativum L.) Embryo Physiological Maturity Shift in Response to Accelerated Growth Conditions.

Protocols have been proposed for rapid generation turnover of temperate legumes under conditions optimized for day-length, temperature, and light spectra. These conditions act to compress time to flowering and seed development across genotypes. In pea, we have previously demonstrated that embryos do not efficiently germinate without exogenous hormones until physiological maturity is reached at 18 days after pollination (DAP). Sugar metabolism and moisture content have been implicated in the modulation of embryo maturity. However, the role of hormones in regulating seed development is poorly described in legumes. To address this gap, we characterized hormonal profiles (IAA, chlorinated auxin [4-Cl-IAA], GA20, GA1, and abscisic acid [ABA]) of developing seeds (10–22 DAP) from diverse pea genotypes grown under intensive conditions optimized for rapid generation turnover and compared them to profiles of equivalent samples from glasshouse conditions. Growing plants under intensive conditions altered the seed hormone content by advancing the auxin, gibberellins (GAs) and ABA profiles by 4 to 8 days, compared with the glasshouse control. Additionally, we observed a synchronization of the auxin profiles across genotypes. Under intensive conditions, auxin peaks were observed at 10 to 12 DAP and GA20 peaks at 10 to 16 DAP, indicative of the end of embryo morphogenesis and initiation of seed desiccation. GA1 was detected only in seeds harvested in the glasshouse. These results were associated with an acceleration of embryo physiological maturity by up to 4 days in the intensive environment. We propose auxin and GA profiles as reliable indicators of seed maturation. The biological relevance of these hormonal fluctuations to the attainment of physiological maturity, in particular the role of ABA and GA, was investigated through the study of precocious in vitro germination of seeds 12 to 22 DAP, with and without exogenous hormones. The extent of sensitivity of developing seeds to exogenous ABA was strongly genotype-dependent. Concentrations between 5 and 10 µM inhibited germination of seeds 18 DAP. Germination of seeds 12 DAP was enhanced 2.5- to 3-fold with the addition of 125 µM GA3. This study provides further insights into the hormonal regulation of seed development and in vitro precocious germination in legumes and contributes to the design of efficient and reproducible biotechnological tools for rapid genetic gain.

Meta-Topolin (mT) enhances the in vitro regeneration frequency of Sesamum indicum (L.)

An efficient micropropagation protocol developed for sesame using cotyledonary node explants derived from 7-day-old in vitro grown seedlings. The efficacy of meta-Topolin (mT) was evaluated during different stages of regeneration. Multiple shoots were initiated and proliferated on Murashige and Skoog (MS) medium supplemented with various concentrations of mT and N6–Benzyladenine (BA), individually. Multiple shoots induced in the medium containing mT, responded well for shoot elongation, rooting and in vitro acclimatization. mT at a concentration of 6.21 μM in MS medium induced maximum number of shoots (23.36 shoots per explant) from 90.66% of cotyledonary node explants. About 73.33% of shoots induced on medium containing mT were elongated (5.93 cm per shoot) in liquid MS medium fortified with 5.77 μM GA3, and 95.66% of them developed profuse roots (8.62 roots per shoot; 13.82 cm in length) in liquid MS medium containing 2.46 μM IBA. All the plantlets with roots were hardened and successfully acclimatized in the greenhouse with a survival rate of 94.33%. Multiple shoots obtained from the cotyledonary node in the medium containing BA showed significantly higher H2O2 content, enhanced antioxidant enzyme (SOD, APX, and CAT) activities, and lower chlorophyll than for the shoots derived from the medium containing mT. SCoT and RAPD markers revealed that all the regenerated plants from the medium amended with either BA or mT showed similar banding pattern to that of the in vivo grown plant, thus confirming the genetic stability of regenerated sesame plants.

The Technique of Water Extract Preparation from Goat Willow Leaves with Allowance for Circadian Rhythm of Their Biological Activity to Stimulate Scots Pine Seed Germination

The preparative technique for water extraction of goat willow (Salix caprea L.) growing leaves was elaborated in order to stimulate Scots pine (Pinus sylvestris L.) seed germination by the extracts (patent no. 2662999). It was shown that the effects of the extract were accounted for by its biological activity, which considerably varied depending on the time of day for extract sampling. The maximal increase in gibberellin-like activity (equivalent to 3.4–3.6 µM GA3) and marked decrease in ABA-like activity (equivalent to 0.15‒0.30 µM ABA) were recorded in the willow leaf extracts harvested in the morning (8:00 a.m.) or evening (8:00 p.m.) in South Karelia (60° N) in mid-May. Application of the extracts to pine seeds afforded the maximal effects on their germination. Thus, the second-class seeds displayed qualitative characteristics of the first-class seeds (germination exceeding 80%) after the treatment. It is concluded that the introduction of the method into practice will allow utilization of the accessible, widespread plant raw material for production of the effective natural preparation, which is capable of improving sowing qualities of Scots pine seeds.

Evaluation on Stevia rebaudiana Bertoni seed germination and seedling development under phytohormones treatment

Poor germination and the long period required for the development of seedlings suitable for planting in the field are among the major problems that affect the cultivation of Stevia rebaudiana. We investigated the abilities of different phytohormone concentrations to improve stevia seed germination and seedling growth. Seeds were germinated on agar gel (AG), supplemented with various concentrations (0, 1, 10 and 100 μM) of gibberellic acid (GA3), thidiazuron (TDZ), kinetin (KN) and N6-benzyladenine (BA) under in vitro conditions. The resultant seedlings were transferred onto Murashige and Skoog (MS) medium for further growth. In most cases, a phytohormone concentration of 1 μM was most favorable for germination. The highest germination was achieved in cases of AG enrichment at a concentration of 1 μM GA3. Regarding the seedlings, the optimal effect on fresh weight, stem, and root length, as well as leaf number was observed when KN was added at a concentration of 10 μM during the germination stage. Generally, a high negative correlation was observed between the phytohormone concentration and the plantlets’ appearance. Conversely, higher chlorophyll and carotenoid contents were observed when 100 μM of GA3, KN and BA was applied at the germination stage. In most cases, antioxidant enzyme activity was also higher under phytohormone treatments. We concluded that the addition of a certain phytohormone concentration at the germination stage enhanced stevia germination and improved seedling development.

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36 h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0 μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0 μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5 μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15 μM TDZ for 24 h) explants was achieved on WPM augmented with 6.0 μM TDZ and 0.5 μM NAA after 8 weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (∼0.5 g) and were transferred onto WPM fortified with different concentrations (2.0–9.0 μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0 μM) and gibberellic acid (GA3, 0.5–3.0 μM). Maximum shoot number (10.4 ± 0.74) and average shoot length (4.75 ± 0.71 cm) were observed in WPM enriched with 2.0 μM BAP, 1.0 μM IAA and 1.5 μM GA3 after 8 weeks of incubation. The developed shoots (4 cm) were excised, pulse-treated for 24 h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0 μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60 days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.

Gallium(III) Nitrate Inhibits Pathogenic Vibrio splendidus Vs by Interfering with the Iron Uptake Pathway.

It is well known that iron is critical for bacterial growth and pathogenic virulence. Due to chemical similarity, Ga3+ competes with Fe3+ for binding to compounds that usually bind Fe3+, thereby interfering with various essential biological reactions. In our present study, gallium(III) nitrate [Ga(NO3)3] could repress the growth of V. splendidus Vs without complete inhibition. In the presence of Ga(NO3)3, the secretion of homogentisic acid-melanin (HGAmelanin) in V. splendidus Vs cells could be increased by 4.8-fold, compared to that in the absence of Ga(NO3)3. HGA-melanin possessed the ability to reduce Fe3+ to Fe2+. In addition, HGA-melanin increased the mRNA levels of feoA and feoB, genes coding Fe2+ transport system proteins to 1.86- and 6.1-fold, respectively, and promoted bacterial growth to 139.2%. Similarly, the mRNA expression of feoA and feoB was upregulated 4.11-fold and 2.71-fold in the presence of 640 μM Ga(NO3)3, respectively. In conclusion, our study suggested that although Ga(NO3)3 could interfere with the growth of V. splendidus Vs, it could also stimulate both the production of Fe3+-reducing HGA-melanin and the expression of feoA and feoB , which facilitate Fe2+ transport in V. splendidus Vs.

Responses of Foreign GA3 Application on Seedling Growth of Castor Bean (Ricinus communis L.) under Salinity Stress Conditions

Castor bean (Ricinus communis L.), a promising bioenergy crop, is readily planted in marginal lands like saline soils. A controlled experiment was conducted to explore the possibility of using gibberellic acid (GA3) as a promoter for caster bean grown under NaCl conditions and to try to determine the most appropriate concentration of GA3 for seedling growth. The seeds of salt-tolerant cultivar Zibi 5 were firstly soaked with 0, 200, 250, and 300 µM GA3 for 12 h and then cultured with 1/2 Hoagland solution containing 0, 50, and 100 mM NaCl in pots filled with sand. Plant height, stem diameter, leaf area, dry mater of each organ, activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), soluble protein, and proline content in the leaves were examined. Plant height and stem diameter, SOD, and POD activity was significantly highest in the treatment of 250 µM GA3 under salt concentration of 50 mM NaCl among all the testing days; protein content was highest when GA3 concentration was 250 µM under 100 mM NaCl treatment. This indicated that caster bean seed soaking with 250 µM GA3 could be the most suitable concentration for promoting seedling growth of caster bean, improving their stress resistance.

High-frequency somatic embryogenesis, nuclear DNA estimation of milkweed species (Asclepias latifolia, A. speciosa, and A. subverticillata), and genome size stability of regenerants

A high-frequency somatic embryogenesis was developed for three Asclepias species, A. latifolia (broadleaf milkweed), A. speciosa (showy milkweed), and A. subverticillata (horsetail milkweed) using gibberellic (GA3) and the amino acid l-proline. A somatic embryo initiation medium consisting of MS salts (Murashige and Skoog, in Physiol Plant 15:473–497, 1962) with Gamborg’s (1968) vitamins, 1.5 µM 2,4-D, 2.3 µM kinetin, and 2% (w/v) sucrose supplemented with various concentrations of l-proline (0, 8.7, or 17.4 mM) combined with various of concentrations of GA3 (0, 2.9, or 5.8 µM), resulting in nine different media (MWM0–MWM8). All media produced callus, but no embryos were obtained on the control medium which contained no l-proline or GA3. Once calli produced somatic embryos, they were transferred to a medium referred to as somatic embryo conversion medium or SECM, which contained MS salts with Gamborg’s vitamins (Gamborg et al., Exp Cell Res 50:151–158, 1968), 2.3 µM kinetin, 2.9 mM GA3, 1.5% (w/v) sucrose, 8 g/L. The conversion percentage of somatic embryos into plants was high for all media, in particular for MWM2 (17.4 mM l-proline + 0 µM GA3) with conversion rates of 90.2, 93.4, and 97% for A. latifolia, A. speciosa, and A. subverticillata, respectively. Flow cytometry was used to estimate the nuclear DNA content of both seed-derived and in vitro grown plants. The 2C-DNA values of all three species were 0.92 pg, which did not differ from the values of in vitro grown plants, thus verifying that the regeneration system produces genetically stable plants.

In vitro propagation of Rosa ‘Konstancin’ (R. rugosa × R. beggeriana), a plant with high nutritional and pro-health value

Abstract The aim of the study was to develop an efficient micropropagation system for Rosa ‘Konstancin’, an interspecific hybrid between R. rugosa and R. beggeriana, whose fruits have high pro-health value. Shoot cultures were initiated from shoot buds collected in May and August from 15-year-old field-grown Rosa ‘Konstancin’ shrubs. The effect and interaction of different concentrations of phytohormones, sucrose and iron sources on in vitro initiation, multiplication and rooting of shoots were studied. The time of collecting explants from donor plants significantly affected the initiation of shoot culture of Rosa ‘Konstancin’. Considerably higher frequency of bud break (100%) was obtained in explants isolated in August as compared to those collected at the end of May (30%). All buds developed into single shoots after 2-4 weeks of growing on the basal Murashige and Skoog medium containing 2.2 µM BAP, 0.3 µM GA3 and 88 mM of sucrose. The highest multiplication rate (4.8 shoots/explant) in a 5-week period was obtained on MS medium containing 50% of nitrogen salts, 3.1 µM BAP, 0.9 µM GA3 and 58 mM sucrose. High rooting frequency (100%) and quality of rooted plantlets was obtained on a medium containing 0.5 µM IBA, 138 µM Fe-EDDHA and 88 mM sucrose. Fe-EDDHA had a beneficial effect on the growth and photosynthetic activity of Rosa ‘Konstancin’ plantlets, which were successfully acclimatized ex vitro, with a more than 90% survival rate.

Embryo rescue of Spondias tuberosa and exogenous application of gibberellic acid

The umbuzeiro (Spondias tuberosa Arruda) is a native fruit tree of Caatinga and has great economic importance. Its fruits are collected extractivistically due to lack of commercial plantations. Furthermore, the seeds have physical dormancy, which reduces germination rates. However, tissue culture can be used to overcome such problems by excision and in vitro culture of embryos with gibberellin. The objective of this study was to evaluate different disinfection times of zygotic embryos in sodium hypochlorite (NaOCl) and the effect of gibberellic acid (GA(3)) in the in vitro establishment of excised embryos. For aseptic test, zygotic embryos (15 replicates/treatment) were excised and immersed in NaOCl (1.25% active chlorine) for different times (5, 10, and 15 min) and rinsed three times with distilled and sterilized water. The explants were cultured on Murashige and Skoog medium (MS) supplemented with 2.88 µM GA(3). After 30 days we evaluated the contamination and germination percentages. The influence of different concentrations of GA(3) was also tested. Zygotic embryos (24 replicates/treatment) were inoculated onto MS medium with different concentrations of GA(3) (0.0, 0.72, 1.44, and 2.88 µM). After 30 days the germination and normal seedlings percentage, height, number of leaves, fresh and dry weight of shoot and root were evaluated. The experimental design of both trials was completely randomized. The immersion in NaOCl for more than 10 min reduced contamination as well as the germination percentage (p=0.0281). The effect of GA(3) was significant (p=0.0346) in stem elongation in a concentration of 2.88 µM compared to control without gibberellin. The 10-min time was effective in disinfection and gibberellic acid promotes increased height of seedlings.

Effects of gibberellic acid on Tifton 85 bermudagrass (Cynodon spp.) in constructed wetland systems.

This study aimed to evaluate 1) the influence of gibberellic acid (GA3) in the development of Tifton 85 bermudagrass grown in constructed wetland systems (CWs) and 2) the plant's capacity to remove nutrients and sodium from synthetic municipal wastewater (SMW). The experiment was carried out in Viçosa, Minas Gerais, Brazil, and consisted of foliar applications of GA3 set in randomized blocks design, with four replicates and 6 treatments as following: NC (control with plants); 0 μM GA3; N1: 5 μM GA3; N2: 25 μM GA3; N3: 50 and N4: 100 μM GA3 per CWs, NC* (control with no plants): 0 μM GA3. The study was conducted over two crop cycles in the spring 2016. The parameters used to evaluate the performance of the Tifton 85 bermudagrass were its plant height, productivity, chlorophyll measurement, number of internodes, nutrients and Na removals. Chemical analyses of the effluents were conducted. In response to the application of GA3, the increase in height of Tifton 85 bermudagrass in the first crop cycle was higher than the increase in height in the second crop cycle. The decrease in plant growth in response to GA3 in the second crop cycle may be linked to the age of the plant tissue and climatic conditions. The greater growth of the plants cultivated in the CWs allows a more efficient removal of pollutants, using simple management and low cost. The results suggest that applying 50 μM of GA3 to the development of Tifton 85 bermudagrass provides higher dry matter yield and removal of nitrogen, phosphorus, and sodium for the first crop cycle in CWs. However, in the second crop cycle, the application of GA3 had no effect on dry matter production and nutrient removal by Tifton 85 bermudagrass in CWs.

In vitro propagation of Campomanesia rufa: An endangered fruit species

ABSTRACT The Campomanesia rufa is a fruitful species native from Brazil considered as endangered by the IUC (International Union for Conservation of Nature) and low information regarding its propagation is available. In this context, the aim of the present study was to develop in vitro germination, micropropagation and callogenesis protocols for the species. For in vitro germination, seeds were inoculated in MS medium supplemented with GA3 (gibberellic acid) and for shoot induction, the medium was supplemented with three different cytokinins BA (benzyladenine), BAP (6-Benzylaminopurine) or TDZ (Thidiazuron). For shoot growth, culture medium containing BA, BAP, TDZ were maintained under white fluorescent lamps and blue:red light-emitting diodes (LED). GA3 was also tested on in vitro shoot elongation. For oxidation control, the medium was supplemented with PVP (polyvinylpyrrolidone) and for callus induction, 2.4-D (dichlorophenoxyacetic acid). The results showed 68% germination regardless of GA3 concentration. In the propagation stage, BAP at 4.5 µM induced a higher number of shoots (4.53) and LED lamps as the light source combined with the culture medium with 1 μM BAP induced a higher number of shoots (4.08). The highest average of C. rufa length (31.9 mm) was obtained using 8.8 µM GA3. The use of PVP (584.3 uM) controls up to 27.3% oxidation in young leaf explants. The use of 10 µM 2.4-D leads to a higher callus formation (58.7 %). Therefore, it can be concluded that the use of BAP is efficient in the induction of shoots, PVP controls oxidation leaf segments, and 2.4-D induces callus in C. rufa.

An efficient in vitro regeneration protocol to generate stable transgenic lines of Black gram (Vigna mungo)

An efficient and quick in vitro regeneration protocol was developed for black gram (Vigna mungo) using wounded embryonic axis with cotyledon as explant. Murashige and Skoog (MS) medium supplemented with 4.44 μM BAP and 2.32 μM Kinetin was found to be effective in producing maximum number (mean 7.80) of multiple shoots. The individual shoots elongated to 4.5 cm when MS medium was supplemented with 2.89 μM GA3 along with 0.44 μM BAP and 0.46 μM KIN. A novel in vitro rooting technique was also optimized for black gram using half-strength liquid MS medium supplemented with 1.34 μM NAA. The shoots in this medium produced the highest number (mean 7.50) of roots with root length of 6.02 cm. The plantlets were transferred to soil mixture and placed in greenhouse where more than 80% successfully grew to maturity. The same protocol was successfully used to generate transgenic black gram lines carrying Bt-Cry2Aa gene through Agrobacteriummediated transformation with a transformation efficiency of 0.42%. The rooted T0 plants grew to maturity and produced T1 seeds with the presence and expression of transgene in T1 plants. Thus, we have standardized an in vitro regeneration protocol suitable for generation of stable transgenic black gram plants.