Micropropagation Conditions Research Articles

Metabolic profiling of Lomatozona artemisiifolia Baker plants grown in vitro and collected from nature using molecular networking and chemometric analysis

Brazilian Cerrado is recognised as a biodiversity hotspot due to the presence of endemic species with great biological potential. Particularly, Lomatozona artemisiifolia, is a rare species found in the Cerrado region in midwestern Brazil. Efforts have been made for its conservation in the Cerrado, such as the use of in vitro micropropagation, demanding a comparative analysis between grown plants and those collected from nature. For this purpose, we performed the chemical study of L. artemisiifolia by LC-ESI-MS/MS and molecular networking analysis in the Global Natural Products Social Molecular Networking (GNPS) with in silico annotation using Network Annotation Propagation (NAP), which led to the observation of labdane diterpenes and flavonoid subclasses as the most representative specialised metabolites of this plant. In addition, molecular networking and chemometric analysis were correlated, allowing the metabolite profile emerging from field growth and micropropagation conditions to be observed.

Effective Contamination Agent For Tissue Culture Applications of Bacopa monnieri

Biocides and plant protection products have been used in plant tissue culture sterilization procedures since they have broad spectrum, are inexpensive and resistant to autoclave process. This study was conducted to determine the effects of contamination agents on tissue culture applications. MS medium was supplied with 0.5mg/l 6-benzylaminopurine and 0.1 mg/L indole-3-butyric acid. PPM and Contaminacide and their three dosages (0, 3 and 6 ml/L) were used for maintaining tissue culture aseptic conditions. Contamination rate and plant growth parameters were subjected. after lids remained open for three days in Bacopa monnieri micropropagation conditions. Although preservation-free and 3mlL-1 PPM-added media were contaminated, contamination did not occur in all dosages of Contaminacide and 6 ml/L PPM. In conclusion 3ml/L Contaminacide added media were superior in terms of contamination rate and plant height, fresh and dry weight (LSD;p

Volatile profile and micropropagation conditions of Bauhinia forficata Link.

The online version contains supplementary material available at 10.1007/s13205-023-03634-8.

Micropropagation of Paulownia elongata tree through Plant Tissue Culture Technology

The study was carried out at biotechnology research center laboratories for the purpose of Micropropagation of Paulownia elongata tree by using plant tissue culture technique for the purpose of identifying the best plant Micropropagation conditions. The plants were sterilized superficially by immersing them in the Clorox solution, then the sterilized plants were cultured in MS media supplemented with several concentrations of (BA and Kinetin) for the purpose of obtaining the best vegetative growths. Obtained plants were also cultured in MS media supplemented with different concentrations of (IBA and NAA) for the purpose of obtaining the best root growth. Finally plants were moved for the adaptation stage. The results indicated that single-nods cultured in (MS) media supplemented with a concentration of 2 and 2.5 mg / L of (BA) growth regulator resulted in good vegetative growth represented in the number of leaves, while the best treatment of branches growth was using 2 mg / L concentration of the Kinetin (K) growth regulator but For the length of the plant, the treatment of the control achieved the best results. For the root site, the results showed that the culture media (MS) plus 0.2 mg / L of the growth regulator (NAA) resulted in the best root growth. As for the adaptation, the results showed that 85% of the plants could be adapted to the sterile environment of soil with 1: 1 size / volume before transferring them to the greenhouse.

ADVANCE STUDIES ON MICROPROPAGATION OF DATE PALM.

Date palm is one of the most important fruit crops in the present and past, as the cultivated areas increase due to its high economics and grown in most lands and its tolerance to difficult conditions and therefore the demand for its offshoots was abundant. This study aimed to micropropagate date palm cultivar Amri through direct organogenesis, improving the growth of plantlets and establish a protocol for photoautotrophic and photomixotrophic as a new micropropagation system in order to obtain highest success percentage and lowest the loss percentage in the acclimatized plantlets during the acclimatization stage, this was done by performing two main experimental groups In-vitro and Ex-vitro Where the effect of different concentrations of auxins, cytokinins and AC. during the direct organogenesis stage was studied, as well as the study of some substances affecting the growth and improvement of plants during the elongation and rooting stages, such as the use of different concentrations of CNTs, spirulina platensis algae extract and Kafom-Cu, PEG and SiO2 and Kafom-Mg. The effect of ventilation rates in cultivation vessels with different concentrations of sucrose in the rooting medium and under CO2 enriched conditions (photoautotrophic and photomixtrophic) or under normal conditions for micro-propagation in the in vitro during the rooting and pre-acclimatization stages in addition to the acclimatization stage were studied in which different systems were studied. For greenhouse cultivation, it also affects different alcohol sugar concentrations of sorbitol and salicylic acid concentrations. Anatomical and chemical studies were also conducted on the best treatments during the rooting and pre-acclimatization stages. The following results were obtained: Adding 0.2 mg l-1 2,4-D + 0.12 mg l-1 NAA + 0.12 mg l-1 IAA + 0.1 mg l-1 2iP + 0.15 mg l-1 BA + 0.5 g l-1 AC to the cultivation medium encouraged the formation of direct shoots. While planting the plants on a medium containing MS added to 20 mg l-1 of spirulina extract increased the length of the shoots, the addition of K-Mg at 1.2 mg l-1 to the rooting medium achieved the highest values ​​of vegetative growth parameters and root system, with the plantlets and media free from contamination. Addition of SiO2 at 20 mg l-1 improved the trunk thickness. It also showed the use of the rate of ventilation 4 holes in the planting pot + 15 g l-1 of sucrose added to the rooting medium under the conditions of enriched with CO2 at 1500 ppm achieved the highest growth vigor and the highest percentage of survival and increased the content of the leaves of amino acids and reducing sugars and their content of Chlorophyll a + b. cultivation plantlets in mixture of Peat moss with Perlite (1: 1 v / v) plus 3 g/pot Barbary-Plante with foliar spray and watered by sorbitol at 30 g l-1 plus salicylic acid at 40 mg l-1 gave the highest values ​​for survival percentage, leaf width and growth vigor (91%, 2.73 cm, 4 respectively).

The Optimal <i>In Vitro</i> Culture Condition for Shoot Multiplication of Evergreen Oak Tree, <i>Quercus myrsinaefolia</i> Blume

The evergreen oak tree, Quercus myrsinaefolia Blume, is not only economically important for wood, medicine, landscape trees, etc., but also becoming more important in terms of ecology due to climate change. However, asexual reproduction was difficult, so this study was conducted to establish the optimum conditions for micropropagation by shoot multiplication. The surface sterilized seeds of Q. myrsinaefolia were successfully germinated in WPM basal medium. BAP (1.0 mg/L) treatment was most effective for inducing multiple shoots. The highest induction rates of adventitious roots from the multiple shoots was shown in the treatment of 1.0 mg/L NAA. Both MS and WPM medium were most effective for growth of multiplied plantlets. For ex vitro acclimatization, the survival rates of multiplied plantlets were 100% in vermiculite and commercial soil. The results of this study can be used for proliferation and supply, and establishment of ex situ conservation of Q. myrsinaefolia elite trees.

Some important aspects in Moringa oleifera Lam. micropropagation

Type and source of explant as well as the type of cytokinin were important factors for successful moringa micropropagation. Explants obtained from in vitro grown plant materials were better than others obtained from soil growing seedlings. In addition, nodal segments were better than shoot cuttings in terms of number of shoots/ explants, frequency of shoot formation and number of nodes/shoot. While callus formation on the base of nodal segment on BAP containing media were higher than those of KIN, especially under the influence of high concentration as an aspect of vitrification, BAP was better than KIN in moringa multiplication. Low nutrient medium (half strength MS) supplemented with 0.5 mg l-1 IAA was essential for successful root formation. The suitable conditions for moringa micropropagation on full strength MS or SH may exert low stress and low need to raise the expression of SOD and POX. On the other side, stress due to over increase of chemical components of double MS medium or low nutrient content of half strength MS, B5 or WPM expressed the highest number and staining intensity of SOD and POX bands, vice versa was detected in case of CAT.

Pomegranate Micropropagation, Callogenesis and Genetic Integrity Assessment Using Simple Sequence Repeat Markers

In recent years, the awareness of pomegranate health benefits has grown exponentially; nonetheless the existing propagation methods remain a challenge to supply adequate suitable planting materials needed for commercial production. Micropropagation can lead to mass production of plantlets and callus-mediated in vitro regeneration can open avenues for the use of genetic engineering to improve this crop. The aim of this study was to evaluate appropriate conditions for pomegranate micropropagation, callogenesis and use Simple Sequence Repeat markers to screen for somaclonal variation. Cytokinins (Benzylaminopurine, Kinetin and Thiadiazol-5ylurea) were tested for shoot induction from nodal explants while auxins (1-Naphthaleneacetic acid, Indole-3-butyric acid and Indole-3-acetic acid) were tested for root induction of in vitro regenerated shoots. 1-Naphthaleneacetic acid combined with Benzylaminopurine was assessed for their ability to induce callus from cotyledon and leaf explants. Genetic integrity between mother plant, callus and in vitro regenerated shoots were assessed using eight Simple Sequence Repeat markers. Maximum number of shoots and leaves were obtained on full strength Murashige and Skoog media with 6.9 µM kinetin. The highest number of roots was achieved on half strength Murashige and Skoog media with 4.9 µM Indole-3-butyric acid and the longest root was got on half strength Murashige and Skoog media with 5.3 µM Indole-3-acetic acid. Leaves and cotyledons demonstrated to be potential explants for callus formation at all hormonal combination levels tested. Eight out of 13 amplified alleles were polymorphic. A wider genetic variation was found with similarity coefficient range of 0.46-0.92. More somaclonal variation was in regenerated shoots compared to callus.

Comparison of two different micropropagation systems of Saccharum officinarum L. and expression analysis of PIP2;1 and EIN3 genes as efficiency system indicators

In sugarcane commercial plantations, seedlings are obtained by vegetative propagation from stem segments. However, this form of seedling production has a downside as it is responsible for spreading various pathogens that accumulate in plants during the cultivation cycle. The development of a disease-free and pathogen-free large-scale production protocol is useful to help minimize the spread of such diseases in commercial plantations. For this reason, a plant tissue culture methodology such as in vitro micropropagation offers the best alternative. In vitro micropropagation protocols using bioreactors based on the temporary immersion system such as BioMINT II™ is a cost-effective design. Our aim was to evaluate the efficiency of two different systems of in vitro micropropagation for sugarcane seedlings; semi-solid and BioMINT II™ in terms of biomass production, morphological and physiological parameters. Beside this, we tested by RT-qPCR two genes (PIP2;1 and EIN3) involved in plant development, as possible molecular markers for quality analysis of development during the tested in vitro micropropagation conditions. At 28 days we obtained more shoots and better quality physiological parameters in the BioMINT II than in the semi-solid system. On the other hand, we characterized and correlated the expression of PIP2;1 and EIN3 with the morphological parameters of the two systems. Our results give a better option for biomass production with the BioMINT II™ and suggest additional experiments of the PIP2;1 and EIN3 genes to be considered as molecular markers for quality analysis of physiological parameters during in vitro micropropagation.

Establishment of optimal conditions for micropropagation by node culture and multiple shoots formation from sucker explants of thornless Blackberry (Rubus fruticosus L. cv. BB21)

This study was conducted to develop a simple, rapid, and reliable method for in vitro propagation of disease-free and true-to-type clones from sucker explants of thornless blackberry (Rubus fruticosus L. × R. parvifolius L.). To induce multiple shoots, the sucker explants were sterilized in 1% NaOCl solution, and then were aseptically cultured on the full and 1/2 MS solid medium supplemented with BAP (0.1, 0.5, 1.0, 2.0 mg/L). After six weeks of culture, the highest frequency (85.4%) of shoot formation from sucker explants was obtained on the full-strength MS medium with 1.0 mg/L BAP. Node explants obtained from multiple shoots were cultured on the various media of full- or half-strength of AD, B5, MS, SH, QL, WPM media, respectively. After 30 days of culture, plant growth was good on the half-AD, half-QL medium. After 90 days of culture, plant growth was good on the full MS and full SH medium. The survival rate of the plantlets after transfer to plastic pots containing soil mixture (sand: soil: vermiculite was 1:1:1, vol.) in the greenhouse was 98%. The results indicate that a multiple-shoot procedure can be applied for an efficient mass propagation of Rubus fruticosus L. × R. parvifolius L.

A comparative study on growth and morphology of wasabi plantlets under the influence of the micro-environment in shoot and root zones during photoautotrophic and photomixotrophic micropropagation

The growth of wasabi (Wasabia japonica Matsumura) plantlets under different micro-environments inside culture vessels in photoautotrophic micropropagation (PA) and photomixotrophic micropropagation (PM) conditions were compared. After 28 days of culture, dry weight, relative growth rate, leaf area, and leaf chlorophyll contents of plantlets in PA were greater than those in PM. The number of leaves did not differ significantly between PA and PM conditions. PA promoted root growth and development with a greater number of roots, root length, root diameter, root fresh weight, root dry weight, and root xylem vessel system. Dissolved oxygen concentration in PA culture medium sharply decreased after 7 days of culture and then recovered. In PM culture medium, no significant fluctuation of dissolved oxygen concentration was apparent. The net photosynthetic rates of plantlets in PA were much higher than those in PM and increased with culture time. In contrast, the net photosynthetic rates of wasabi plantlets in PM kept a low and constant value during the culture period. With the presence of gas exchange membranes attached to the vessel lids, the detected vapor pressure deficit was higher in PA than in PM conditions. Higher stomatal density and larger stomatal aperture on the abaxial and adaxial surfaces of the leaves in PM medium promoted leaf water loss following ex vitro conditions. Thus, PA is applicable for producing healthy wasabi transplants.

Growth media and mycorrhizal species effect on acclimatization and nutrient uptake of banana plantlets

The objective of this study was to investigate the acclimatization and performance of banana (Dwarf Cavendish) in two substrates inoculated with different AM fungi (Glomus. caledonium and G. macrocarpun) and assess the plantlets dependency on inoculation for phosphorus (P) and zinc (Zn) uptake along with biomass development. In addition, to investigate the role of mycorrhizal fungi in supporting acclimatization phase, a plant growth promotion study was set-up in greenhouse using micropropagated plantlets. Two growth media, as GM-I and GM-II were used accompanied by G. caledonium and G. macrocarpum. In first phase, 9 weeks acclimatization study was conducted and in second phase, acclimatized plants were propagated for 16 weeks in both inoculated and non-inoculated conditions. Plantlets acclimatization and nutrient uptake were recorded along with other parameters. Mycorrhizal inoculation significantly increased banana plantlets growth, root infection and P uptake. Plantlets inoculated with G. caledonium exhibited increase in shoot and root dry mass, P and Zn concentration, and root infection in the GM-I. The shoot and root dry mass, P and Zn concentration, and root infection were higher in GM-I than Konaktas soil series. Banana plantlets are mycorrhizal dependent (MD) and soil-grown banana plantlets are more MD than plants grown in the GM-I. Mycorrhizal inoculation seems to be a significant factor in decreasing mortality and increasing production of high-quality banana plantlets under micropropagation conditions.

Direct formation of axes in new plantlets of Kappaphycus alvarezii (Doty) Doty, as influenced by the use of AMPEP K+, spindle inhibitors, and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K+ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L−1 AMPEP K+ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L−1 AMPEP K+ + 1.0 mg L−1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L−1 AMPEP K+ + 0.5 mg L−1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K+ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L−1 AMPEP K+ with PGR, 6.0 ± 0.58 mm with 0.5 mg L−1 AMPEP K+ + 0.1 mg L−1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L−1 AMPEP K+ + 0.1 mg L−1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L−1 AMPEP K+ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K+ with PGR and AMPEP K+ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K+ + colchicine with PGR and AMPEP K+ + oryzalin with PGR, respectively. The use of AMPEP K+ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K+ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.

Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets

Cattleya intermediais an Atlantic Forest species endemic to Brazil that is classed as vulnerable on the list of threatened species. In this study, C. intermedia plantlets were micropropagated in an asymbiotic culture and the influence of different concentrations of sucrose (15, 30, 45 and 60 g L-1, plus a zero sucrose medium) and macronutrient salts (complete Murashige and Skoog (MS) medium and half MS medium (with half-strength macronutrients)) on survival and development of the plantlets was evaluated. In all treatments 100% plantlet survival was achieved. The integrated analysis of height of aerial part, number of leaves per plantlet, fresh mass, number of roots per plantlet and length of the longest root showed that the plantlets exhibited greatest development at the half-strength macronutrient concentrations with 45 or 60 g L-1 of sucrose, as well as at the complete macronutrient concentration with 60 g L-1 of sucrose. Plantlets acclimatized and reintroduced to an environment in which the species occurs naturally exhibited 98.6% survival. The results obtained in this study allowed the establishment of optimal conditions for asymbiotic micropropagation, which is a requisite for future studies focused on conservation of C. intermedia.

The significance of non-controlled natural light, temperature and humidity in the commercial micropropagation of Solanum tuberosum L. Cultivar Diamant

The aim of our study was to reduce the unit cost of in vitro micropropagation of the Solanum tuberosum cultivar Diamant widely cultivated in Bangladesh with the guarantee that the quality and quantity of the in vitro plants produced was not jeopardized. This was done by entirely replacing the conventional micropropagation conditions of maintaining the in vitro plants in a controlled room whose temperature varies between 25 and 30°C, its humidity between 60 to 70% and its light intensity of 20,238 to 20,409 for 19 hours; with a room whose roof was made of corrugated plastic sheets that allow a partial passage of natural light. Under this conditions the amount of light, temperature and humidity were not controlled. During the time work the temperatures in this non-conventional room fluctuate between 14 and 40°C, the light intensities were between 20,017 to 20,687 Lux and the humidity between 40 to 90%. Experiments were initiated in May, 2009 through March, 2014 covering summer, rainy and winter seasons. After two years of laboratory research and two years of field studies, we have not found differences between yield production of the micropropagated plants grown under control and non-controlled conditions, very often the latter plants were robust and adapted faster when transfer to field conditions. All plants used in the field experiments were no more than seven in vitro passages. A RBCD yield trail of the plants was done during two seasons and it was no found any difference in yield between them. Moreover, a yield trail of the minitubers to produce breeder seed (second generation) was done during the season 2013-2014 and no differences were found between the controls and the tubers derived from the low cost.
 Plant Tissue Cult. & Biotech. 24(2): 131-139, 2014 (December)

CALLUS INDUCTION AND PLANT REGENERATION IN ECHINOCACTUS PLATYACANTHUS

The aim of this research was to establish the optimal conditions for micropropagation of E. platyacanthus, a cactus that is used as a source of water and fodder for livestock during the dry season. To this end, two experiments were carried out. In the first, seed germination on Murashige and Skoog (MS) medium with different concentrations of mineral salts (50, 75 and 100%) and sucrose (10, 20, 30 and 40 g L-1) was evaluated. From the first experiment, we selected three-month-old seedlings, approximately 1.0 cm in length, and from each one of them three explants were obtained; the explants, in turn, were placed on MS medium with different concentrations of benzyladenine (BA) (0.0, 0.5, 1.0 and 2.0 mg L-1) and naphthaleneacetic acid (NAA) (0.0, 0.2, 0.5 and 1.0 mg L-1). In both experiments the factors and levels were combined in a factorial arrangement and established in a completely randomized design. In all cases, the culture medium was supplemented with myo-inositol (100 mg L-1), thiamine (0.4 mg L-1) and agar (6 g L-1), and its pH was adjusted to 5.7. In the first experiment, we began recording the number of germinated seeds 15 days after sowing (DAS); in the second, 60 DAS the variables callus fresh weight, callus diameter, shoot number and length and root number and length were recorded. The data were subjected to analysis of variance and mean separation tests (Tukey, p≤0.05) using the SAS Version 9 statistical package (SAS, 2003). Seed germination began at 16 DAS and ended 37 days later (53 DAS). Salt proportions under or equal to 75% were associated with germination rates of 70%, statistically higher than those obtained with MS medium with 100% mineral salts. The highest callus fresh weight was obtained in BA concentrations at or above 1.0 mg L-1. NAA concentrations at or above 0.5 generated callus diameters two to three times greater than without the hormone (0.59 cm). The NAAA—BA interaction was significant in shoot height, with the highest value (0.68 mm) being obtained with 0.2 mg L-1 NAA and 0.5 mg L-1 BA. Root number and length were negatively affected by the BA concentration.

МИКРОРАЗМНОЖЕНИЕ in vitro СУБТРОПИЧЕСКИХ, ДЕКОРАТИВНЫХ КУЛЬТУР И ЭНДЕМИКОВ ЗАПАДНОГО КАВКАЗА: ОРИГИНАЛЬНЫЕ И ОПТИМИЗИРОВАННЫЕ ПРОТОКОЛЫ

To obtain good planting material in floriculture and horticulture, the effective procedures are required for testing and sanitation. Micropropagation is also commonly known as an approach to intensifying plant breeding due to accelerated reproduction of valuable forms and as a way to save biodiversity of rare species. Nevertheless, for many flowering plants, and subtropical fruit crops and endemic species especially, the reliable protocols for in vitro cultivation, regeneration and propagation are not yet reported. This paper presents the results of a revised protocol application and the improvement of clonal micropropagation methods for tea Camellia sinensis (L.) Kuntze, lemon Citrus limon (L.) Burm, blossoming shrub Camellia japonica L., and Lilium caucasicum Miscz. ex Grossh., an endangered endemic species in the Western Caucasus. Some peculiarities in obtaining sterile culture are revealed, and the types of explants and seasonal terms of their introduction to in vitro culture are identified. Nutrient mediums and conditions for clonal micropropagation are optimized. Thus, cultivation of tea explants on WPM supplemented with phytohormones (i.e. 6-BAP at 3.0 mg/l and adenine at 0.9 mg/l) facilitated the induction of numerous adventitious microshoots. Aactive growth of C. japonica microshoots was also observed on the WPM medium with 6-BAP at 2 mg/l and indole-3-acetiс acid at 0.5 mg/l. In lemon cultivars, microengrafting is approved as a reliable propagation method. It was revealed that after the third cycle of microengrafting the multiplication index for microstalstalks was 2 times higher comparing to that for microshoots from auxiliary buds of donor plants, and made up 4.6 pc. per shoot. The results of in vitro cultivation of the various parts of Lilium caucasicum bulb scales showed that the induction of morphogenesis was not the same. After 48 days in vitro cultivation under sterile conditions, from the basal and medial scale parts the adventitious shoots were mainly developing, whereas callusand rhizogenesis occurred rarely, while in the apical scale parts the callus formation dominated. In special publications there are no data on L. caucasicum cell and tissue in vitro cultivation. So, the protocol for in vitro cultivation and propagation of this endangered endemic species of the Western Caucasus is reported here for the first time.

Role of Achromobacter xylosoxidans AUM54 in Micropropagation of Endangered Medicinal Plant Naravelia zeylanica (L.) DC

This study was carried out to evaluate the inoculation effects of Achromobacterxylosoxidans AUM54 and Indole-3-butyric acid (IBA) on the growth of the medicinal plant Naraveliazeylanica (L.) DC under micropropagation conditions. Results revealed that the micropropagated shoots treated with the combination of endophytic bacterium and IBA promoted shoot growth, root length, number of roots, chlorophyll content, nitrogen content, antioxidant enzymes, and stress tolerance compared with the control plants. A significant increase in shoot fresh and dry weights (64.65 and 8.85 %), root fresh and dry weights (61.65 and 3.91 %), shoot length (30.17 %), root length (28.57 %) and number of roots (276.9 %) was observed in treated plants over controls. Total chlorophyll and nitrogen content of bacterized plants also treated with IBA showed a 48.39 and 116.66 % increase, respectively, compared with controls. A significant increase in peroxidase (22.52 %) and superoxide dismutase levels (48.38 %) and fewer changes in the polyphenol oxidase level were observed in plants treated with A. xylosoxidans AUM54 and IBA. Moreover, stress ethylene levels were reduced by 21.4 and 14.5 % due to bacterization with A. xylosoxidans AUM54 and IBA treatment during postacclimatization and acclimatization stages, respectively. The shoot primordial with application of A. xylosoxidans AUM54 and IBA (1 mg l−1) had increased survivability of N. zeylanica plants by 30 % during the acclimatization stage under greenhouse conditions. From the present study it could be inferred that the association of endophytic bacterium A. xylosoxidans AUM54 and IBA with in vitro shoots of N. zeylanica improved root initiation, promoted plant growth and development under micropropagation conditions, reduced stress ethylene levels, and increased survivability during the postacclimatization stage. Therefore, A. xylosoxidans AUM54 along with IBA treatment can be used as a valuable tool for micropropagation of N. zeylanica and other endangered plants.

Bromelain Enzyme from Pineapple: In Vitro Activity Study under Different Micropropagation Conditions

The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.

ANTIBACTERIAL ACTIVITY OF SECONDARY METABOLITES FROM IN VITRO CULTURE OF DROSERA GIGANTEA AGAINST THE PLANT PATHOGENIC BACTERIA PSEUDOMONAS SYRINGAE pv. SYRINGAE AND P. SYRINGAE pv. MORSPRUNORUM

The carnivorous plant Drosera gigantea contains naphthoquinones among which plumbagin is known to have an antibacterial activity. The aims of this study were (i) to optimize conditions for micropropagation of D. gigantea and (ii) to evaluate the antibacterial activity of extracts of D. gigantea tissues and plumbagin against the bacterial fruit tree pathogens Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum. A number of media with different growth regulators [1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), thidiazuron (TDZ) and 6-benzylaminopurine (BAP)] or activated charcoal (CA) were tested for D. gigantea micropropagation. The best medium proved to be a halfstrenght Murashige and Skoog medium (1/2 MS) with 0.4% CA. All of the tested growth regulators resulted in changed phenotype of daughter plants and RAPD analysis showed that some of the regulators caused somaclonal variation. The minimal inhibitory concentrations and the minimal bactericidal concentrations (MBCs) of methanol and chloroform extracts of D. gigantea as well as pure plumbagin were determined and were in the range of 375-1875 μg dry weight (dw)/ml for methanol extract, 240-1425 μg dw/ml for chloroform extract and 5-150 μg dw/ml for plumbagin. The lowest MBCs (750-1875 μg dw/ml) were obtained with methanol extract of D. gigantea. These results suggest that in vitro-grown D. gigantea could be a potential source of antibacterial compounds, useful for the control of bacterial fruit tree pathogens but it should be further tested under in vivo conditions.