Magenta Boxes Research Articles

Improvement of ex vitro transfer of tobacco plantlets by addition of abscisic acid to the last subculture

Tobacco (Nicotiana tabacum L.) plantlets were grown on Murashige and Skoog medium in ventilated Magenta boxes and for the last subculture 10 µM ABA was added to the medium. After three weeks plantlets were transferred into pots with Perlite moistened with water and grown in controlled conditions (16-h photoperiod, day/night temperature 25/20 °C, air humidity about 45 %) either under low or high irradiance of 150 (LI) and 700 (HI) µmol m-2 s-1, respectively. Content of endogenous ABA was 271.7 pmol g-1(f.m.) in ABA treated plantlets, while in control plantlets it was only 53.3 pmol g-1(f.m.). After ex vitro transfer, stomatal conductance and transpiration rate decreased considerably in comparison with in vitro grown plantlets and remained lower also 7 d after ex vitro transfer, especially in ABA-treated plants and so wilting of plants was practically eliminated. Net photosynthetic rate also decreased 1 d after ex vitro transfer but after 7 d it was mostly higher than that of in vitro grown plantlets. Water use efficiency significantly increased in ABA-treated plants. Chlorophyll a+b content did not change immediately after ex vitro transfer, nevertheless, after 7 d chlorophyll content was higher in ABA-treated plants. Pool of xanthophyll cycle pigments (XCP) and the degree of their deepoxidation (DEPS), which are connected with harmless dissipation of light energy, increased under high irradiance. Contents of XCP and ABA precursors (neoxanthin and violaxanthin) were lower in ABA-treated plants than in control plants indicating less stress in these plants. Most chlorophyll a fluorescence parameters did not change considerably after ex vitro transfer and so the photoinhibition was not observed even under HI. Slight increase in non-photochemical quenching under HI in ABA-treated plants suggested their better photoprotection. Thus application of ABA to the last subculture can improve acclimatization of in vitro grown plants to ex vitro conditions

Sustained growth promotion in Arabidopsis with long-term exposure to the beneficial soil bacterium Bacillus subtilis (GB03)

Volatile emissions from the commercial growth promoting soil bacterium Bacillus subtilis (GB03) are effective in augmenting short-term growth, photosynthetic capacity, and salt tolerance in Petri-dish grown Arabidopsis seedlings. In contrast, the impact sustained GB03 volatile exposure on plant growth and development has yet to be examined. Here is provided physical and physiological data establishing that bacterial volatiles induce long-term growth promotion, elevated photosynthetic capacity and iron accumulation, as well as delayed albeit higher seed count compared with water-treated control plants. Plants were grown unrestricted in double Magenta boxes containing solid MS media for up to twelve weeks with GB03 volatiles introduced in separate containers within the chamber so that plant bacterial interactions are only by air-borne transmission. These results establish that GB03 volatiles induce sustained beneficial effects on Arabidopsis growth including robust and extended vegetative growth followed by elevated seed set.

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

MASS PROPAGATION OF COFFEE TRANSPLANTS UNDER SCALED-UP PHOTOAUTOTROPHIC MICROPROPAGATION SYSTEM

Somatic embryos were obtained from leaf discs of coffee plants cultured in vitro. The main objective of our study was to develop a scaled-up micropropagation system under photoautotrophic conditions (PA) using Coffea arabusta somatic embryos as a model plant. At different developmental stages of somatic embryos (torpedo, precotyledonary, cotyledonary and germinated), the physiological variables in relation to the photosynthetic ability were investigated. Results revealed that cotyledonary and germinated stage embryos were physiologically capable to grow photoautotrophically. However, high photosynthetic photon flux (PPF: 100 μmol m 2 s -1 ) treatment for 14 days increased the photosynthetic efficiency of the somatic embryos and possibly make them more suitable to grow under PA conditions. To confirm these findings the somatic embryos were cultured under PA conditions (sugar-free medium with CO 2 enrichment in the culture vessel environment and high PPF). From the results it was concluded that cotyledonary stage is the earliest stage somatic embryo, which can be successfully grown photoautotrophically for conversion into plantlets. To scale up the plantlet conversion process of coffee somatic embryos under PA conditions a bioreactor was especially designed. The growth and conversion percentage of cotyledonary stage embryos cultured in the newly developed bioreactor were compared with those of commercially available systems such as RITA temporary immersion bioreactor and Magenta box. Results revealed that the growth, percent conversion and ex vitro survival of plants were highest in the newly developed bioreactor.

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolytica A1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.

SCALE-UP MICROPROPAGATION OF VEGETABLE SPECIES: FROM LABORATORY TO COMMERCIAL REALITY

The efficient, commercial-level use of bioreactor systems for the scale-up micropropagation of vegetable species is demonstrated for cucumber (Cucumis sativus L.). Nodal explants were derived from aseptically grown seedlings of the cucumber F 1 hybrid 'Stella' and bearing four leaves. Explants were inoculated on gelled Murashige and Skoog (MS) basal medium supplemented either with no growth regulators or the antigibberellin flurprimidol at 0.5 mg l -1 . Explants were incubated in Magenta® boxes under a photosynthetic photon flux density of 150 pmol m -2 s -1 at 24°C. After one month, nodal clusters were transferred into liquid MS medium with the same composition as above and incubated twice in either: (1) 2.5 1 airlift bioreactors (Osmotek Lifereactors®) or (2) 250 ml flasks on an orbital shaker (80 rpm) for 20-30 days. Regenerated plantlets were transferred to soil and acclimatized in the greenhouse. Significantly more plants were regenerated in bioreactors than in Magenta boxes® or in flasks. In addition, bioreactor-derived plants had an increased fresh and dry weight, number and length of roots, number of nodes and leaf surface. These traits were associated with patterns of macronutrient and sugar accumulation, as well as ascorbic acid and free antioxidant phenolics concentration. The percentage of plants that were successfully acclimatized increased to 95% after grafting them on squash. The prospects for applying this technique to other cucumber varieties, as well as other commercially important vegetable species, are discussed.

PRODUCTION OF SOMATIC EMBRYOS OF AMERICAN GINSENG IN SUSPENSION CULTURE AND REGENERATION OF PLANTLETS

ISHS XXVI International Horticultural Congress: Biotechnology in Horticultural Crop Improvement: Achievements, Opportunities and Limitations PRODUCTION OF SOMATIC EMBRYOS OF AMERICAN GINSENG IN SUSPENSION CULTURE AND REGENERATION OF PLANTLETS

Responses of Tobacco Plantlets to Change of Irradiance During Transfer from in vitro to ex vitro Conditions

Chlorophyll a fluorescence kinetics, net photosynthetic rate (PN), water relations, and photosynthetic pigment contents were studied during acclimation of in vitro grown tobacco to higher irradiance (HL; 700 μmol m−2 s−1). Plantlets were grown on medium containing sucrose in glass vessels (G-plants) or in Magenta boxes (M-plants) with better CO2 supply in the latter ones. The effect of HL was studied either (1) in plantlets grown under original in vitro conditions (closed vessels), (2) in in vitro plantlets exposed to ambient CO2 concentration (covers removed), or (3) in plantlets transplanted to ex vitro into pots with sand and nutrient solution. Higher PN, and fraction of closed photosystem 2 (PS2) centres (1 − qP), and lower content of xanthophyll cycle pigments were found in M-plants compared to G-plants. HL treatment caused photoinhibition particularly in plants kept in closed vessels. This was indicated by the decrease in the ratio of Fv/Fm and by the increase in non-photochemical quenching, 1 − qp, and content of xanthophyll cycle pigments. Better CO2 supply ensured by the removal of closure lead to the moderate reduction of symptoms of photoinhibition, although stomatal conductance (gs), transpiration rate (E), and PN were negatively affected. The main reason was the decrease in relative air humidity, which caused similar reduction of PN, E, and gs after the transfer of plantlets to ex vitro. Nevertheless, plant response to HL seemed not to be affected by any possible root injury caused by transfer to ex vitro. The differences in contents of xanthophyll cycle pigments, degree of de-epoxidation, PN, and quenching parameters between M- and G-plantlets were still significant 7 d after ex vitro transfer and HL acclimation.

Development of a Rapid Technique for the Evaluation of the Response of Brassica napus to Verticillium wilt.

Extensive efforts are being made to develop oilseed rape cultivars with resistance to Verticillium wilt. Evaluation of these cultivars involves either dipping roots of young plants in a conidial suspension followed by replanting in soil, or sowing the seed directly in artificially infested soil. These methods for screening for resistance take a long time and are expensive. The objective of this research was to develop a rapid and reliable method of screening cultivars for resistance to Verticillium wilt. A box test method in which 4-day-old seedlings were inoculated and grown under sterile conditions in double magenta boxes was developed. The Brassica napus cultivars Accord, Express, Hanna, and Libraska were inoculated with isolates of Verticillium longisporum or V. dahliae. The results from the box test method were compared with root-dip inoculation of seedlings grown in a soil mixture under greenhouse conditions. In addition, plants from the box test were randomly selected and transferred to pots containing sterile soil to permit comparison of plants from the box test at similar developmental stage with plants in the root-dip method. Disease evaluation was based on the number of days after transferring seedlings into the boxes that root browning, root and shoot blackening, and wilting of cotyledons developed. Additional disease evaluation was based on a 0 to 5 disease severity scale based on the percentage of wilted leaves (number) and/or the percentage of stunting (mm) of inoculated plants compared with noninoculated plants. Disease severity was higher on Hanna than on Express, and isolates of V. longisporum were more virulent than isolates of V. dahliae. There was a significant correlation (r = 0.93) of disease rating between the traditional root-dip method and the new box test method. Analysis of variance revealed that the interaction between cultivars and isolates was highly significant (P ≤ 0.01), whereas method of inoculation did not affect the results. In the box test, plants were rated after 25 days, which is at least 3.5 times more rapid than the root-dip method.

In vitro precultivation of tobacco affects the response of antioxidative enzymes to ex vitro acclimation

Both growth under elevated CO2 (CE) conditions and/or treatment with abscisic acid (ABA) during acclimation to ex vitro conditions and precultivation in vitro in tightly closed glass vessels (G-plants) or ventilated Magenta boxes (M-plants) affected activities of antioxidative enzymes of tobacco 28 days after the transfer. Single treatment with 5 μmol/L ABA immediately after transplantation caused an increase in net photosynthetic rate and in the content of chlorophyll a, and decreases in the activities of glutathione reductase, Mn-SOD, peroxidases, malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G-6P-DH) in both types of plantlets. Contrary to this, CE effects were dependent on the plant origin and promoted activities of peroxidase, ME, and G6PDH were observed more in M-plants than in G-plants. Effects of a single ABA treatment lasted throughout the whole acclimation and alleviated transplantation shock more efficiently than CE, irrespective of plant precultivation. However, under combined treatment (CE+ABA) CE effects prevailed over ABA.

Growth of In vitro banana (Musa SPP.) shoots under photomixotrophic and photoautotrophic conditions

In vitro banana (Musa spp.) shoots were cultured under photomixotrophic (30 gl−1 sucrose and 0.2 h−1 number of air exchanges of culture vessels) and photoautotrophic (0 gl−1 sucrose and 3.9 h−1 number of air exchanges) conditions for 28 d in 370 cm3 Magenta boxes (GA7-type) containing 70 ml of half-strength Murashige and Skoog (MS) medium with 22.2 μM N6-benzyladenine (BA). The effects of varying CO2 concentration (475 or 1340 μmol mol−1) and light intensity (photosynthetic photon flux (PPF) of 100 or 200 μmol m−2 s−1) were investigated. Fresh and dry weights of banana shoots grown photomixotrophically were significantly greater on day 28 than those grown photoautotrophically. Photoautorophic shoots had a larger number of unfolded leaves and greater leaf area than photomixotrophic plants by days 14 and 28, regardless of CO2 concentration. The shoot fresh and dry weights on day 14 in photoautotrophic conditions were significantly greater at PPF of 200 μmol m−2 s−1 than at 100 μmol m−2 s−1. The increase in net photosynthetic rate of photoautotrophic banana shoots was significant compared with photomixotrophic shoots. The multiplication ratio of in vitro banana shoots grown photoautotrophically in a 28-d culture period was the greatest at 100 μmol m−2 s−1 PPF and 475 μmol mol−1 CO2.

Effects of light, CO2 and humidity on carnation growth, hyperhydration and cuticular wax development in a mist reactor

Plant survival ex vitro requires functioning stomata, adequate cuticular wax composition and deposition, and normal morphological development. Light intensity, CO2 and relative humidity were altered inside an acoustic window mist reactor to study their effects on carnation (Dianthus caryophyllus) growth, stomata development, hyperhydration and epicuticular wax content. Increasing the light intensity from 65 to 145 μmol m−2 s−1 and enrichment of the gas phase with CO2 (1350 ppm) reduced the number of hyperhydrated plants from 75 to 25% and increased the percentage dry weight of normal (healthy) plants from 17 to 25%. Lowering the relative humidity (≈70% RH) surrounding the plants during the mist-off phase for the last 2 wk of culture reduced the number of hyperhydrated plants from 70 to 9% and also increased the percentage of dry weight of normal plants from 16 to 25%. The stomata on plants grown in conditions of high light or low humidity had smaller apertures and appeared sunken when compared to stomata from plants grown in low light and high relative humidity. The epicuticular wax profiles of plants from the greenhouse or Magenta boxes showed a distinct shift in wax compounds with developmental age, plant type (hyperhydrated or normal), and type of box that was used (vented or not). In addition, very different wax profiles were observed from plants grown in reactors with altered CO2 and light intensities.

Survival of Chlorophyllous and Nonchlorophyllous Fern Spores through Exposure to Liquid Nitrogen

Survival of Chlorophyllous and Nonchlorophyllous Fern Spores through Exposure to Liquid Nitrogen

Photosynthetic pigments and gas exchange during ex vitro acclimation of tobacco plants as affected by CO2 supply and abscisic acid

Nicotiana tabacum L. plantlets were grown in glass vessels or in Magenta boxes with better CO2 supply. To improve the ex vitro transfer we tested application of abscisic acid and elevated CO2 concentration. In the first two weeks after transfer, net photosynthetic rate, chlorophyll a+b content, and Chl a/b ratio were higher, and content of xanthophyll cycle pigments lower in M-plants than in G-plants, but during further growth the differences almost disappeared. ABA application alleviated the risk of wilting because it decreased stomatal conductance. The effect of ABA was enhanced under CE (28 days after transfer). In situ, PN was always higher at CE than at CA, but when measured under CA, positive effect of CE was found 2 and 16 days after transfer in M-plants and only 16 days after transfer in G-plants. Slightly increased Chl a content was found in all ABA-treated plants, and in M-plants grown under CE. The content of xanthophyll cycle pigments was lower under CE compared to CA, and the lowest one was found in ABA-treated M-plants grown under CE. On the contrary, the degree of their deepoxidation (DEPS) was slightly higher in plants grown under CE. No significant effects of ABA-treatment or growth under CE on fluorescence kinetic parameters were found and inconsistent effects on photochemical activities. The photochemical efficiency of PS2 (variable to maximum fluorescence ratio, Fv/Fm) after ex vitro transfer was similar to that in in vitro grown plants. This together with the values of DEPS indicated that no photodamage during ex vitro transfer occurred.

In vitro regeneration in chile pepper (Capsicum annuum L.) from ?half-seed explants?

An in vitro regeneration protocol has been developed from ‘half-seed explants’ of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.

The Role of Sucrose Alone and Combined with Different Cytokinin-like Compounds on in Vitro Tuberization of Potato

The role of sucrose alone and in combination with different cytokinin-like compounds on the microtuberization of potato, Solanum tuberosum `Atlantic', was investigated. Single nodal segments were placed in Magenta boxes containing Murashige & Skoog medium supplemented with one of 15 treatments in a 3 × 5 factorial. Treatment factors were sucrose at 3%, 6%, or 9%, and cytokinin-like compounds at five levels [cytokinin-free; 2 mg kinetin/L; 0.1 mg thidiazuron (TDZ)/L; 1.0 mg AC 243,654/L; 0.1 mg AC 239,604/L]. Except in a few cases in kinetin and TDZ treatments, nearly all cytokinin treatments failed to induce tuberization at the 3% sucrose, noninductive level. However, all cytokinin treatments induced tuberization in the presence of 6% sucrose. By raising the sucrose level from 6% to 9%, more and larger microtubers were obtained in the cytokinin-free medium. At the 9% sucrose level, even though more tubers per box were produced by TDZ and AC 243,654 treatments, less total fresh weight of tubers per box resulted from kinetin, TDZ and AC 243,654 treatments because tubers formed were smaller. Higher sucrose concentrations (9%) favored tuberization in the cytokinin-free medium, whereas 6% sucrose was optimum for the medium containing cytokinins. Sucrose might produce a strong tuberization signal that might either change endogenous hormone levels affecting tuberization or activate a number of genes coding tuber proteins and enzymes related to starch synthesis.

The Effects of Different Concentrations of Gibberellic Acid (GA3) and Kinetin on in Vitro Tuberization of Potato (Solanum tuberosum L.)

To determine the influence of gibberellic acid (GA3) and 6-furfuryl aminopurine (kinetin) concentrations alone and in combinations on in vitro tuberization of potato, nine treatments consisting of combinations of gibberellic acid and kinetin at three levels of concentration (0, 2, and 5 mg·liter–1) were included in Murashige and Skoog medium supplemented with 6% sucrose. Four single nodes of in vitro plantlets from Solanum tuberosum L. cultivar Atlantic were placed into each magenta box. All magenta boxes were arranged in a randomized complete box design with five replications and cultured under a short photoperiod condition (8 h light/16 h dark). Gibberellic acid strongly inhibited tuberization when used alone or with kinetin, whereas kinetin induced tuberization at both 2 and 5 mg·liter–1. Although tuberization was initiated in the absence of kinetin because of the high concentration of sucrose and short photoperiod, the presence of kinetin accelerated the in vitro tuberization process of potato.

UPDATE ON MICROPROPAGATION OF PACHYSANDRA PROCUMBENS MICHX.

Pachysandra procumbens, the Allegheny spurge, is a native herbaceous perennial. Microcuttings were maintained in liquid MS medium in Magenta boxes with membrane rafts. A preliminary study looked at 1,2.5,5, and 10 mg BA/liter and 0 and 1 mg NAA/liter. Shootscultured with 5 or 10 mg BA/liter and no NAA produced significantly more axillary shoots. When shoots were cultured on media containing 5,10, 15, 20, or 25 mg BA/liter, trend analysis indicated significant linear and quadratic trends. Shoots proliferated similarly when cultured with 5 to 20 mg BA/liter. Non-fully expanded shoots proliferated significantly better compared to fully expanded shoots. After 6 weeks, microcuttings dipped in 500 or 1000 ppm IBA (50% ethanol) had produced significantly more roots of greater length compared to the control. Sixteen weeks after potting, rooted microcuttings potted in MetroMix 500, Promix BX, or a nursery mix (60% pine bark, 20% peat, 20% stone dust) had initiated significantly more shoots.

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40μM), 0.5 μM of either BA or TDZ, and 7 g·1(-1) TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 μM 2,4-D and 0.5 μM TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.