PEMANFAATAN LIMBAH BUAH PISANG DAN AIR KELAPA SEBAGAI BAHAN MEDIA KULTUR JARINGAN ANGGREK BULAN (Phalaenopsis amabilis) TIPE 229

Coconut water and rotten banana fruits are commonly found in traditional markets as organic wastes. One way to overcome the problems caused by these organic wastes is to convert these unuseful matter into an important and economically useful matter by using them as components of tissue culture media. One important commodity that is usually propagated by tissue culture is Phalaenopsis orchid type 229 (Phalaenopsis amabilis). Therefore, it would be more benefit to substitute the expensive chemicals with organic wastes such as coconut water and banana puree.In this experiment, addition of coconut water and banana pure to the minimum media containing commercial fertilizer red Polyhyponex, sucrose and commercial agar did not show any inhibition of Phalaenopsis orchid plantlet growth. This probably caused by sufficient macro and micro nutrients provided by those organic matter and Polyhyponexfertilizer. Moreover, addition of 100 mL/L of coconut water and 100 mg/L banana puree gave the optimum leaf and adventitious shoot formation. On the other hand, addition of 150 mL/L coconut water gave the optimum height and root formation. In this case,growing Phalaenopsis orchid plantlet should be done in 2 subculture period. The first subculture is to obtain maximum amount of leaf and shhot formation while the second subculture is to obtain optimum height and root formation.Key words : Waste, coconut water, banana puree, tissue culture, Phalaenopsis orchi

Sucrose and Cytokinin Interactions in Relation to Ethylene and Abscisic Acid Production in the Regulation of Morphogenesis in Pelargonium × hortorum L.H. Bailey In Vitro

The aim of the study was to determine the effect of exogenous sucrose and cytokinin on ethylene production and responsiveness in relation to the shoot formation of Pelargonium × hortorum ‘Bergpalais’ in vitro. Increasing the concentration of sucrose from 15 to 40 g L−1 in medium containing meta-topolin (mT) resulted in a two-fold decrease in the number of shoots and leaves as well as a reduction in ethylene production. The addition of ethylene synthesis inhibitor (AVG) to mT-medium significantly reduced the ethylene production and the shoot growth, but it had no significant influence on the shoot formation. The mT-induced shoot formation was, however, significantly reduced in the presence of ethylene action inhibitor (AgNO3), in a manner dependent on sucrose levels. At the end of the subculture period, increased sucrose concentrations (15–40 g L−1) in the presence of mT and AgNO3 resulted in a 3.7-fold increase in ethylene emission. At the same time, the supply of sucrose caused a 2.8-fold increase in the level of endogenous abscisic acid (ABA). Our results may suggest that the inhibitory effect of high sucrose concentration (30 and 40 g L−1) may depend on its influence on ethylene sensitivity. It also suggests that sucrose-regulation of the shoot formation of Pelargonium in vitro is mediated by ABA.

Effects of cytokinins on antioxidant enzymes in in vitro grown shoots of Pelargonium hortorum L. H. Bayley

<p>The aim of this study was to determine the influence of <em>meta</em>-topolin (<em>m</em>T) and 6-benzyl-aminopurine (BAP) on the hydrogen peroxide (H₂O₂) level and antioxidant enzymes activities in relation to the shoot formation and senescence process in <em>Pelargonium hortorum</em> cultivars, which differ in their susceptibility to leaf yellowing under <em>in vitro</em> conditions.</p><p>In an early senescing cultivar ‘Grand Prix’, the addition of an aromatic cytokinin <em>m</em>T to abscisic acid (ABA)-enriched Murashige and Skoog (MS) basal medium more efficiently inhibited leaf yellowing than BAP. In both genotypes, <em>meta</em>-topolin was also the most effective in shoot formation. It was found that <em>Pelargonium</em> species varying in their susceptibility to senescence differ in H₂O₂ production and antioxidant enzymes activities. Generally, <em>meta</em>-topolin more effectively enhanced H₂O₂production and POD activity than BAP and control medium, but its effect depended on genotype. The highest H₂O₂ production stimulated by <em>m</em>T was observed on day 5 of subculture in late senescing cv. ‘Bergpalais’. In both geranium genotypes, superoxide dismutase (SOD) and catalase (CAT) levels were highest at the beginning of the subculture period, during the initiation of shoot formation. SOD showed the highest activity on day 5 of subculture on the medium without cytokinin and generally being higher in cv. ‘Bergpalais’ than in cv. ‘Grand Prix’. CAT activity was positively regulated by both cytokinins. POD activity was most effectively enhanced by <em>m</em>T, but on different days of subculture - on the 2^nd day of subculture in cv. ‘Bergpalais’ and on the 22^nd day of subculture in cv. ‘Grand Prix’. The enhanced activity of POD in the presence of <em>m</em>T, 4-fold higher than on control medium, at the end of subculture in <em>P. hortorum </em>‘Grand Prix’ coincided with the inhibition of leaf senescence.</p>

Cultured stellate cells in human vocal fold mucosa

Objectives:Stellate cells in the maculae flavae, located at both ends of the human vocal fold mucosa, have been considered an independent category of cells. We aimed to isolate and subculture these stellate cells, and to observe their morphological characteristics.Methods:Stellate cells from the maculae flavae and fibroblasts from Reinke's space were cultured in three normal, adult human vocal fold mucosa preparations.Results:The subcultured cells from Reinke's space were conventional fibroblasts. The subcultured cells from the maculae flavae were stellate in shape and had cytoplasmic processes. They were larger than conventional fibroblasts, and lipid droplets in the cytoplasm disappeared in the second culture. These stellate cells proliferated by attaching their cytoplasmic processes to each other. During the seven to 10 month subculture period, each cell type continued to exhibit its own morphological characteristics.Conclusion:This study demonstrated that such stellate cells form an independent cell category, which should be considered as a new category of cells within the human vocal fold.

Study of culture conditions for improved micropropagation of hybrid rose

An efficient protocol was developed for micropropagation of hybrid roses by manipulating growth regulators, photoperiods, gelling agent and subculture period. Multiple shoots were achieved from nodal explants of <I>Rosa hybrida</I> cvs. Cri Cri, Pariser Charme and First Red on the Murashige and Skoog (MS) medium supplemented with 1.5–2.0 mg/l BA (6- benzylaminopurine), 50 mg/l Ads (adenine sulfate) with 3% (w/v) sucrose. Inclusion of indole-3-acetic acid (IAA; 0.1–0.25 mg/l) into the cytokinin-rich medium promoted high frequency of shoot multiplication. The induction of multiple shoots was also affected by photoperiod and subculture period. Higher multiplication was achieved under 16 h photoperiod in all tested cultivars. The rate of multiplication was low when photoperiod both increased or decreased. The frequency of shoot multiplication was best up to the 6^th to 7^th subculture and thereafter it declined. Rooting was readily achieved upon transferring the microshoots onto half-strength MS medium supplemented with 0.25 mg/l IBA (indole-3-butyric acid) and 2% (w/v) sucrose. The percentage of rooting was less on MS medium containing NAA (1-naphthalene acetic acid) or IAA as compared with IBA. More than 60% of rooted plantlets were established in the greenhouse. The<I> in vitro</I> raised plantlets were grown normally and flowered within one month after their transfer to open field.

Development of Agrobacterium-mediated Transformation System in Pear Cultivars with Low-regeneration Frequency

The transformation of pears such as `Conference', `Doyenne du Comice' and `Passe-Crassane' has been attributed to the high regeneration frequency from leaf discs (71% to 97%; Leblay et al. 1991). However, it has been difficult to transfer desirable genes into cultivars with low-regeneration frequency such as `Silver bell' (35.4%) and `La France' (10.7%), which are the two major pear cultivars in Japan. In this study, we developed an Agrobacterium-mediated transformation system for `Silver bell' and `La France'. For `Silver bell', leaf discs derived from in vitro shoots were used as explants. The antibiotics for selection of transformants and elimination of Agrobacteria were investigated. In the most optimum condition, which is 30 mg·L-1 Kanamycin and 500 mg·L-1 Sulbenicillin, a 3.2 % transformation efficiency was obtained. However, no success was recorded in an effort to transform `La France' using leaf disc explants because of very poor regeneration frequency. Therefore, axillary shoot meristems were used as explants for transformation of `La France'. The conditions for antibiotic selection and elimination of Agrobacteria were also investigated. In 5 mg·L-1 Kanamycin and 375 mg·L-1 Carbenicillin, transformed shoots were produced at 4.8% efficiency. No chimera was observed in the transgenic shoots during a 2-year subculture period. Since the inoculated explants developed into multiple shoots during selection, it was thought that the problem of chimera might have been overcome. Therefore, this transformation method using axillary shoot meristem may be applicable to pear cultivars recalcitrant to regeneration from leaf disc. To the best of our knowledge, this is the first report of a transformation system in pear cultivars with low regeneration efficiency.

The role of rejuvenated and adult forms of English oak (Quercus robur) in in vitro cultures

In vitro plant material of clones (Q. robur) NL 100 A (adult) and NL 100 R (rejuvenated) received from Germany (A. Meier-Dinkel, 1995) were used in these experiments. WPM medium was used for the multiplication phase. Plantlets were subcultured monthly. Differences in quality and colour of the adult and rejuvenated cultures induced us to follow and compare the changes of mineral- and chlorophyll content and dry weight during the propagation phase. Mineral and chlorophyll content as well as dry weight were measured weekly on three samples during the subculture period. In the case of propagation rates we stated, they were similar around the year, but both clones had a high peak in April. Examining the cation-content, we detected that, the plantlets had a highest quantity of several elements during the 2nd and 3rd week of subculture. The iron content was the highest in the 1st week and after that it decreased continuously. It is supposed, that the content of iron is not enough in the media. The chlorophyll content of the rejuvenated clone was higher than that of the adult one. In the rooting experiments it was stated that, after one-week cold treatment the rooting ability was the best.

Sugar Use in Relation to Shoot Induction by Sorbitol and Cytokinin in Apple

Microshoots of `McIntosh' apple (Malus domestica Borkh.) were grown on Murashige-Skoog (MS) nutrient medium supplemented with either sucrose or sorbitol or with sucrose and an elevated level of cytokinin. Shoot growth was recorded and concentrations of fructose, glucose, sorbitol, and sucrose were analyzed in nutrient media and shoots during a 6-week subculture period. Axillary branching was stimulated by high cytokinin and sorbitol media, with increased biomass production and carbohydrate use on the high-BA medium only. The sucrose in the nutrient medium was hydrolyzed to fructose and glucose, which were equally taken up by shoots. Sorbitol was taken up somewhat less effectively. The elevated level of BA decreased sucrose hydrolysis in the nutrient medium. There were high concentrations of sorbitol in shoots grown on the sorbitol medium, and sorbitol also accumulated at the end of the culture period in shoots grown on sucrose. The amount of sucrose was low, and glucose was more abundant than fructose in microshoots. The starch content of leaves was not affected by treatments or sampling time. Chemical names used: N6-benzyladenine (BA).

Micropropagation of Agarita (Berberis trifoliata Moric.)

Experiments were conducted to develop a clonal propagation system for agarita (Berberis trifoliata Moric.). Actively growing agarita shoots were collected from a mature plant at the Texas A&M Univ. Research and Extension Center in El Paso and successfully established on a basal medium consisting of woody plant medium (WPM) salts and Murashige and Skoog vitamins, sucrose at 30 g·L–1, and 0.8% Phytagar supplemented with 11.1 μm BA. Cytokinins (benzyladenine, kinetin, and thidiazuron), subculture period, and age of cultures were tested. The optimal shoot proliferation conditions were WPM basal medium supplemented with 5.5 μm BA and a subculture period of 4 weeks. Culture age did not affect shoot proliferation but did affect rooting. Preliminary experiments with 1.0 μm NAA resulted in nearly 100% rooting of microshoots <6 months old. Shoots from 21-month-old cultures had to be placed on a cytokinin-free medium before successful rooting. On basal medium supplemented with NAA (5.4 μm), 68% of the microshoots rooted with an average of 1.2 secondary roots per microshoot. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); 1-naphthaleneacetic acid (NAA); N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ); 6-furfurlaminopurine (kinetin).