Mist trickling bioreactor for <Emphasis Type="Italic">Centaurium erythraea</Emphasis> Rafn growth of shoots and production of secoiridoids

Shoots of Centaurium erythraea Rafn were cultivated in 5 l mist trickling bioreactor for 21 and 28 days increasing their dry weight from 0.54 g to 13.7 g and 18.3 g, respectively. About 6880 shoots from 223 initial shoot-tips in 21-day bioreactor producing cycle were produced. The shoots could be successfully rooted and transferred to soil. Secoiridoid accumulation (expressed as a sum of gentiopicroside, sweroside and swertiamarin) in shoots after 21 days of culture reached about 303 mg l⁻¹.     

Regeneration of plants using nutrient mist culture

Summary A nutrient mist was used forin vitro culture of plant tissue in a novel bioreactor, wherein the tissues were grown on a biologically inert screen within a sterile chamber which allows excess media to drain away from the tissue. Plants tested includedDaucus, Lycopersicon, Ficus, Cinchona, andBrassica. The latter 4 genera were fully regenerated within the bioreactor. Tissue inocula included callus, anthers, and shoot meristems. All plants grew at least as well in nutrient mists as in agar and always produced a greater quantity of shoots of a higher quality and often faster than agar cultures. Cost analysis estimates showed up to a 65% savings in production costs (labor and materials) could be realized using nutrient mist culture. Nutrient mist culture offers significant improvements in the micropropagation of plants.     

Multiple shoot culture of Dianthus caryophyllus using mist culture system

Summary A mist bioreactor system for the plant tissue cultures was developed. Using this system, the growth of Dianthus caryophyllus multiple shoots was directly measured. Tissue growth in mist bioreactor system was far better than that on agar medium and almost comparable to that in liquid medium. The mass increase (final dry weight/initial dry weight) in the mist culture was 2.85 while 3.28 in the liquid flask culture. Shoots were seriously vitrified in flask culture but these vitrifications could be considerably cured by using the mist culture system.     

Flowering ofin vitro grown spinach shoots in the presence of the Herbicide Sandoz 9789

Stem tips ofSpinacia oleracea were isolated and grown in sterile culture, with Sandoz 9789 added to the medium. Although the herbicide provoked a complete loss of all chloroplast pigments, developed shoots were able to flower, in response to long days; in short days flowering was inducible by adding gibberellins. Sandoz 9789 decreased the elongation of stems by up to 30%; the percentage of flowering plants in culture was also decreased. However, among those plants that flowered, the majority were male, so that Sandoz 9789 produced a shift of the sex ratio toward maleness.     

Uptake and transport of lead by perennial ryegrass from flowing solution culture with a controlled concentration of lead

Summary Perennial ryegrass was grown in flowing solution culture in a glasshouse, and during February lead was added to the nutrient solution and held at a constant concentration; uptake and transport of lead were followed in conditions of low intensity daylight or higher intensity artificial light. Uptake of lead by the roots was most rapid during the first 4 days after addition to the nutrient solution. After this time there was a steady increase in uptake per g dry weight of root with plants grown in artificial light having a much higher rate of uptake than plants grown in daylight. Roots always contained more lead than the corresponding shoots and concentration was always greater in the roots than in the shoots. The concentration in both roots and shoots increased with time but that in plants grown in artificial light was higher than that in plants grown in daylight. Two phases of uptake were identified, an initial rapid phase which is probably an exchange phenomenon, and a slow sustained phase which may be under metabolic control. A lower proportion of the total lead taken up remained in the roots of plants grown in artificial light than in those grown in daylight. This difference may have resulted from differences in (i) the production of organic carriers and/or (ii) transpiration. re]19750930     

Morphogenesis induction and organogenic nodule differentiation in <Emphasis Type="Italic">Populus euphratica</Emphasis> Oliv. leaf explants

Populus euphratica Oliv. is a deciduous poplar species, occurring mainly in riparian areas of China and Middle Eastern countries, and presenting high tolerance to extreme temperatures and high soil salinity. In this study, an optimized protocol for development and propagation of P. euphratica from leaf explants is reported, based on a morphogenic process that involves organogenic nodule differentiation. Adventitious shoot regeneration of P. euphratica from organogenic nodules of leaf explants was achieved within a range of concentrations of α-naphtalenacetic acid and 6-benzylaminopurine, at a fixed 2:1 ratio. Cambial cells started to divide 5 days after inoculation on culture medium and, after 12 days, several organizing centres were already formed. Non-friable callus tissue, together with organization centres, formed structures that evolved to nodules after about 40 days which were, then, able to regenerate new shoots after 50–60 days. The nodules did not separate from the mother explants and were able to successfully give rise to new adventitious shoots. These were rescued and successfully grown and rooted in different culture media, and fully developed plants were obtained. The regeneration system here described for P. euphratica is innovative, reproducible and data from histological studies of the morphogenic process support the classification of the regenerative structures as organogenic nodules.     

Micropropagation of Sterculia urens Roxb. — an endangered tree species

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl^–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.     

Secondary metabolism in micropropagated <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. grown in non-aerated liquid medium

Hypericum perforatum L. is a medicinal plant that has been extensively studied because of its bioactive properties. The objective of this study was to establish a system that could lower the cost of in vitro propagation by using liquid medium, as well as to evaluate the secondary metabolism in the systems tested. Nodal segments of H. perforatum were obtained from in vitro shoots and grown in three liquid culture systems: total immersion (TI), partial immersion (PI), and paper bridge support (PB). Semi-solid medium (3 g L⁻¹ Phytagel™) was used as control (SS). The organogenic responses were evaluated, and phenolic compounds, hypericin, and the activity of polyphenol oxidases (PPO) and peroxidases (POX) were quantified. After 80 days of culture, induction and proliferation of adventitious shoots were similar in the PI and SS systems (65.3 and 71.3 shoots, respectively), whereas PB resulted in the fewest shoots per explant (29.5 shoots). Longer shoots were obtained under the PI conditions. Hyperhydricity was observed in the shoots from the TI system. Browning was visible in shoots from the TI and PB systems. The highest concentrations of phenolic compounds and hypericin were observed in shoots derived from PI and PB, at 80 days of culture. POX activity was higher in shoots cultured in PI at 40 days, whereas PPO was significantly more active at 80 days of culture. Likely, POX was more related to shoot growth, whereas PPO played a later role in response to the culture environment and medium stress.     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

High frequency in vitro production of microrhizomes of Curcuma amada

Microrhizomes were induced at the base of the in vitro derived shoots of Curcuma amada grown in the liquid MS medium supplemented with BA (5 mg/l), sucrose (80 g/l) and incubated under 16 hr photoperiod. They were produced year round in culture, harvested after 90 days of growth in medium, and could be used as seeds and germinated to plantlets showing normal growth.     

Responses of Nicotiana tabacum to CO2 enrichment at low-photon flux density

Bean plants ( Phaseolus vulgaris ) grown in phosphate-deficient (–P) medium display deficiency symptoms after about 2 weeks of culture. A decrease in inorganic phosphate level in roots was observed after 10 days of culture, and after 17 days it was more than 30 times less than control. The dry weight of the shoots of phosphate-deficient plants was lower whereas the dry weight of the roots was higher as compared to the control roots. After 2 weeks of culture, the sucrose level in –P roots almost doubled compared to control roots. An increase in glucose and fructose was observed earlier than that of sucrose, and after 10 days of culture in phosphate-deficient medium the glucose level in –P roots was about 3 times higher than that in +P roots. At the same time, a decrease in hexose-phosphate level was observed. This decrease may reflect a higher drain from the hexose phosphate pool, or it may be due to the lower capacity for hexose phosphorylation of phosphate-deficient plants, as judged by the lower hexokinase and fructokinase activities. The ratio of non-phosphorylated to phosphorylated sugars in –P roots was about 5 times higher as compared to control roots. We propose that glucose and fructose accumulation in phosphate-deficient roots represents a non-metabolic, probably vacuolar pool which is not utilized for growth and metabolism of the roots.     

Controlling hyperhydration of carnations (Dianthus caryophyllus L.) grown in a mist reactor

Carnations (Dianthus caryophyllus L.) grown in vitro often develop physiological abnormalities such as hyperhydration. The amount of hyperhydration and growth was compared between carnations grown in mist reactors and conventional semisolid micropropagation systems (vented or unvented GA7 culture boxes). Plants grown in the mist reactor with long misting times (10 min h(-1)) produced more dry mass than those grown with <10 min h(-1); however, more misting also produced more hyperhydrated plants (70% hyperhydration). Control of hyperhydration in the mist reactor involved either reducing the overall nutrient mist supply or altering the mist supply throughout the culturing period. Stepped decreases in the mist supply throughout the 3-week period or an overall decrease in the duration of misting reduced hyperhydration to 13% and 5%, respectively. However, for both misting regimes, the biomass of normal (healthy) plants (fresh and dry weights) was limited. Further analysis suggested that, although normal plant biomass increased with longer mist exposure, hyperhydration levels also increased while the water content, based on percent dry weight, approached that of hyperhydrated plants. Sufficient normal plant development (fresh weight, leaf and shoot numbers, height, and rooting) with < 50% hyperhydration was obtained by weekly, stepped increases in the nutrient mist supply.     

In vitro plantlet regeneration from seedling nodal explants of Acacia catechu

Multiple shoots were initiated after 20 days in stem nodes excised from in vitro grown seedlings of Acacia catechu, on Murashige and Skoog's medium adjuvanted with 1 to 100 microM of N6-benzyladenine (BA). Explants were subcultured on the same medium augmented with 1.5 g l(-1) of polyvinylpyrrolidone (PVP) after 30 days. In the second subculture, after 30 days, the explants were transferred to a medium lacking PVP, but containing 10 microM of BA, where nine or ten shoots differentiated per explant within next 30 days. If individual shoots along with some callus were subcultured on BA (10 microM), nearly 15 shoots per explant regenerated in 90 days. Thus, the average number of shoots obtained from each node was 142 after 180 days. Since a seedling develops four nodes after 20 days, theoretically an average of 568 shoots can be obtained from a single seed. If shoots were individually subcultured on 1/2-strength MS medium with 14.7 microM of indole-3-butyric acid (IBA), roots developed in 20 days. Addition of 40 mg l(-1) of glutamic acid to the rooting medium prevented leaf senescence. These plantlets thrived well in garden soil, sand and silica (1:1:1).     

Effect of Photoperiod and Nitrogen Supply on Basal Shoots Development in Rhododendron Catawbiense

In order to control young plant form by modifying culture conditions, plants of Rhododendron catawbiense from in vitro culture were grown in a greenhouse under different photoperiodic treatments (long or short days) combined or not with a several-week nitrogen starvation. After 12 weeks of culture under long days (16 h) with nitrogen supply, plants showed a rhythmic acrotonous development. When long days were combined with a six-week nitrogen starvation, the apical growth pause was extended leading to an increase of the number of acrotonous lateral ramifications. Short-day (8 h) treatment affected distal burst potential and moreover when a concomitant nitrogen starvation was applied. This lack of distal development allowed basal buds swelling, leading to basitonous plants. When plants were returned back to long days after 2, 4 or 6 weeks under short days, distal buds resumption competed with basal shoots development. Durable basitonous plants were obtained by a 12-week short days treatment combined with a 6-week nitrogen starvation.     

Plant Regeneration from Petiole Protoplasts of Brassica napus

Two cultivars of Brassica napus, Altex and Canadian twins, were used as materials. Protoplasts isolated from petioles of plants grown in vitro were cultured in Nitsch medium supplemented with 0.5mg/L BA, 0.5mg/L NAA, lmg/L 2,4-D, 100mg/L serine, 800mg/L glutamine, 4% sucrose and 0.4mol/L mannitol. After 2 days of culture, the first division was observed. The division frequency estimated after 10 days of culture was 30-60%. One week after transferring onto MS medium containing 6mg/L GA3. and 3mg/L BA, protoplast-derived calli regenerated into shoots. The regeneration frequency of the two cultivars was 24% and 31% respectively. It was found that the protoplasts isolated from petioles could float on the surface of the 3% sucrose contained solution which was very favourable both to purification, and culture of the protoplasts.     

The effect of replacing potassium by sodium on cation uptake and transport to the shoots in four legumes and Italian ryegrass

White clover, red clover, lucerne, sainfoin and Italian ryegrass were grown in a series of culture solutions in which 0, 25, 50, 75 and 100% of the potassium was replaced by sodium. In the shoots, the potassium: sodium ratios indicated selectivity for potassium which decreased in the order: sainfoin>lucerne>red clover>white clover > Italian ryegrass. In the roots only sainfoin showed selectivity for potassium: with the other species the ratio of potassium to sodium in the roots was similar to that in the other species the ratio of potassium to sodium in the roots was similar to that in the culture solution. All species showed selective transport of potassium to the shoots in preference to sodium: this was least in Italian ryegrass and white clover and greatest in sainfoin, which in contrast to other species also showed restricted transport of potassium to the shoots. Replacement of potassium by sodium slightly increased the magnesium concentration in the shoots but had no effect on the calcium concentration.     

Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L) via organogenesis

Green bean (Phaseolus vulgaris L.) plants were regenerated from 3-day old seedling explants via organogenesis. The explants contained a cotyledon and a small portion (2–3 mm) of embryonic axis split in half. Explants were cultured on a defined medium containing glutamine as the sole nitrogen source. A ring of meristematic tissue was produced at the base of the axillary bud located at the cotyledonary node. The meristematic tissue was produced only if the axillary bud was present together with the cotyledon in the explant. Buds and shoots developed from the meristematic ring. Selected shoots produced roots when excised from the cluster of buds and transferred to root induction medium. Rooted shoots (plantlets) grew well and produced viable seeds when grown in the greenhouse. Histological studies revealed the origin of buds from the peripheral layers of the meristematic ring.Production of buds and shoots was a continuous process, so that new shoots could be removed from the explant for plantlet production every 10–14 days. With the cultivar Dark Red Kidney, an average of 49 buds and 8 shoots were regenerated per explant by 30 days after culture initiation. Sixty-seven percent of the shoots produced roots, and 90–95% of the plantlets survived greenhouse acclimatization to produce healthy plants.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L.

Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to be highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminopurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation.Abbreviations MS Murashige and Skoog - IAA 3-indoleacetic acid - BA 6-benzylaminopurine