Plant regeneration from shoot tips and callus of papaya

Summary Two methods of in vitro culture were employed to regenerate papaya plants. One involved regeneration of plants from callus and the other, production of multiple plants from single shoot-tip explants. Callus was induced from stem sections of papaya seedlings in a medium containing 1 mg per 1 NAA and 0.1 mg per 1 kinetin. The callus regenerated shoots and/or embryoids when transferred to a medium of lower auxin, 0 to 0.05 mg per 1 IAA, and higher cytokinin, 1 to 2 mg per 1 kinetin Multiple shoots were produced when the excised shoot-tip explants were cultured in a medium supplemented with 0.05 mg per 1 IAA and either 5 mg per 1 kinetin or 0.5 to 1.0 mg per 1 benzyladenine. Root formation of the shoots or embryoids that derived from callus or shoot tips occurred in a medium containing 5 mg per 1 IAA and in a light intensity of 3000 to 4000 Ix. The rooted plants could be established in soil and under standard greenhouse conditions after they had been acclimated by initially growing them in moist vermiculite contained in polyethylene-covered pots. This research was supported by the National Science Council, Republic of China.     

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS?+?TDZ, and ½ MS?+?IAA?+?TDZ?+?PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS?+?IAA?+?TDZ?+?PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS?+?IAA?+?TDZ and ½ MS?+?IAA?+?TDZ?+?PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

INVESTIGATIONS ON THE ROOT FORMATION IN THE TISSUES OF HELIANTHUS TUBEROSUS CULTURED IN VITRO

Excised rhizome fragments of Helianthus tuberosus ‘Violet de Rennes’ produce roots in vitro; the sequence of morphogenetic phenomena is as follows: proliferation, differentiation of phloem and tracheids, organization of cambiums, and formation of root primordia from cambiums. This rhizogenesis is conditioned by five limiting factors: mineral salts, sugar, auxin, temperature, and light. The optimum level of each of these factors was determined and in certain cases also the threshold of action. For white light the beginning was about 1/1000 lux. These five limiting factors do not represent the same site of action. The mineral salts, especially nitrogen and calcium, have their effect when the cambiums have been organized. The sugars act before the formation of cambiums. Light operates principally upon cambium and this action is independent of photosynthesis. It was not possible to determine whether the auxin has a preferential site of action. A temperature higher than 15 C is essential to obtain the formation of cambiums but further production of roots is possible at 15 C or even less. The variations in temperature more or less around the optimum stimulate rhizogenesis. The supra-optimal temperatures mainly have their effect before the formation of cambiums and infra-optimal temperatures after. The effect of light can be exhibited at low temperature. Gibberellic acid in association with auxin favors root formation in the dark whereas in light it exhibits inhibiting properties. Rhizogenetic potentialities of tissues are reduced when these are cultured in the dark in presence of auxin. On the other hand, if auxin is applied after the isolation of explants, rhizogenesis is stimulated and the tissues are able to produce roots even in the dark. The action of light and of auxin can be dissociated; when the explants having absorbed a small quantity of auxin are transferred to a medium without auxin for 60 days in the dark, they conserve the rhizogenetic potential which is expressible on transferring them to light. Finally, grafting experiments suggested that light could induce the formation of a rhizogenetic factor in the tissues which is transmittable from cell to cell. These experiments have definitely established that the induction of root formation is governed by several factors which have complex interactions.     

Micropropagation with 3-methyleneoxindole: Its obligatory role in indole-3-acetic acid-mediated auxin action

Summary 3-Methyleneoxindole (MO), a metabolite of the plant auxin indole-3-acetic acid (IAA), was more active than IAA in supporting Stage II and III micropropagation of several plant species. In Stage II micropropagation, characterized by the rapid numerical increase of shoots, the optimal IAA concentration was 0.01 mM compared to 0.1 mM MO for most plants. In Stage III micropropagation where auxin is required for the rhizogenic response, 0.1 μM MO was more effective than 0.01 mM IAA. Inhibition analysis of plant growth with chlorogenic acid (CGA) suggested an obligatory role for MO in IAA-mediated auxin reactions: CGA, which blocks the enzymatic oxidation of IAA to MO, in vivo, completely abolished IAA's ability to support the growth of explants during micropropagation. In contrast, CGA did not inhibit the auxin activity of MO, the product of the blocked reaction. The growth rate and rooting efficiency of tobacco propagules in Stage III medium was improved substantially if these were first exposed to a high concentrations of MO and subsequently transferred to media containing low or no MO.     

In vitro Organogenesis in Explants from Different Cultivars of Begonia X hiemalis

Petiole explants from 17 cultivars of Begonia X hiemalis were grown on a basal agar medium with different combinations of NAA and BA as well as on media lacking microelements or vitamins. The stock plants were kept either under short days (7–8 h of light perday) at 15°C or under long days (15–16 h of light) at 18–21°C. The day length during the in vitro culture was 20 h of light and the temperature 21°C. Explants from short-day treated stock plants did not show any differentiation. In explants from long-day treated stock plants, the percentage of explants with shoot, root and with both shoot and root initiation were recorded after 55 days. Explants forming both shoots and roots were transferred to soil, and plantlet formation was observed after another 55 days. The percentage of explants with organ and plantlet formation differed between cultivars. With increasing NAA and decreasing BA concentrations, the percentage of explants forming only roots increased, whereas the percentage of explants with only shoots decreased. Plantlet formation was most frequent in explants from NAA: BA ratios of 2: 1 and 10: 1, and a variation was found between different cultivars. When the vitamin fraction was not added to the medium, this did not influence formation of shoots, roots and plantlets. When the microelements were omitted. shoots, roots and callus were formed, but no plantlets.     

Induction of embryogenic callus in Cucurbita pepo hypocotyl explants by indole-3-ethanol and its sugar conjugates

Hypocotyl explants of pumpkin ( Cucurbita pepo L.) were inoculated aseptically on solid media containing Murashige and Skoog (MS) macro- and micronutrients, organic supplements, glucose as a carbon source, and indoleacetic acid (IAA) or one of its possible metabolic precursors, i.e. indole-3-ethanol [3-(2-hydroxyethyl)-indole, tryptophol], 2-(indol-3-yl)-ethyl α-L-arabinopyranoside (tryptophol arabinoside), and 2-(indol-3-yl)-ethyl β-D-glucopyranoside (tryptophol glucoside). Embryogenic callus and adventitious roots developed on the explants, the response depending on the source of auxin, its concentration, and endogenous, possibly genetic, factors. Hypocotyl sections did not form embryogenic tissues and did not survive on media which did not contain an auxin or auxin precursors. Fewer explants responded to IAA than to indole-3-ethanol. Its arabinopyranoside was even more effective, while only a few hypocotyl sections proliferated in the presence of the corresponding glucopyranoside. Embryogenic callus induced by any of the four above compounds could be subcultured on media containing the same source of auxin. Selected callus lines have been maintained for more than three years and have continued to form embryoids. Indole-3-ethanol and IAA were suitable for clonal propagation of regenerated plantlets by axillary and apical bud culture.     

Morphogenesis and Auxin Sensitivity of Transgenic Tobacco with Different Complements of Ri T-DNA

Leaf explants of hairy root tobacco (Nicotiana tabacum) regenerants characteristically differentiate roots from the wound margins on hormonefree medium. The same response can be elicited on normal tobacco by culturing the explants in the presence of auxin. We show here that the spontaneous rooting of transformed plants is neither due to the activity of right T-DNA-borne auxin genes nor to a substantially altered balance of endogenous hormones. Rather, an increased sensitivity to auxin is conferred to transformed cells by the left T-DNA (TL-DNA). Analysis of the morphogenetic behavior of transgenic tobacco plants obtained by transferring segments of TL-DNA cloned in a binary vector system allowed us to pinpoint TL-DNA genes responsible for this increased auxin sensitivity of hairy root tissues. Three genes (open reading frames 10, 11, 12) are responsible for the spontaneous rooting of leaf explants and confer to transgenic plants an exaggerated response to auxin.     

In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.     

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Effects of Auxin and Kinetin on the Development and Differentiation of Potato Tissue Cultured in vitro

The present investigation deals with the influence of auxin and kinetin on cultures of potato tuber tissue, and the effect of the size of the explants on the organ formation. Exogenous application of auxin is indispensable for initiating callus formation of the tissue, but kinetin is not necessarily required. Kinetin serves to maintain the callus development, indicating that the stimulation of callus growth due to exogenous auxin would presumably be mediated by the addition of kinetin to the medium. A relatively low concentration of auxin in the medium is required for root formation on the culture. In this case, IAA is markedly superior to any other auxin in root forming activity. NAA is somewhat less effective than IAA, and 2,4-D shows no stimulation on root formation. The growth and organ formation of potato tissue are remarkably affected By the variation in sizes of the explants. Spontaneous formation of a sprout was observed on the large explant in the medium without auxin. Small explants form only callus or roots in the presence of auxin and kinetin irrespective of combination or concentration. It seems likely that a sufficient amount of stimulus for initiation of bud formation may be contained in the large sized explant itself.     

Potential of bacterial indoleacetic acid to induce adventitious shoots in plant tissue culture

AIMS: The main aim of this study was to investigate the possible role of indoleacetic acid (IAA) from bacteria to induce in vitro adventitious shoots in internodal explants of Brassica oleracea L. METHODS AND RESULTS: Culture supernatant of Halomonas sp. RE1 and Halomonas sp. HT1 that contain 21 and 40 microg ml(-1) IAA, respectively, was used to supplement Murashige and Skoog (MS) medium. Two combinations that were supplemented with bacterial supernatant (BS) are MS + BS and MS + BS + 10%CW (coconut water) while basal MS medium was used as control. The amounts of BS used in this experiment were 50, 100, 150 and 200 microl in 5 ml MS medium in each combination. In vitro-grown internodal explants of B. oleracea were inoculated on these media combinations and incubated in a growth chamber at 25 +/- 1 degrees C and exposed to 16-h cool fluorescent light. After 5-6 weeks of incubation adventitious shoot induction was observed in all treatments that were supplemented with BS as compared with the controls where very low response was observed. The frequency of shoot induction was high in media that were supplemented with 10%CW in the presence of bacterial auxin. CONCLUSIONS: It was concluded that IAA of microbial origin has the potential to induce adventitious shoots in internodal explants. SIGNIFICANCE AND IMPACT OF THE STUDY: IAA from bacteria can be effectively used in plant tissue culture; especially a combination of MS + BS + 10%CW is very cost-effective as compared with synthetic phytohormones for in vitro studies.     

A Requirement for DNA Synthesis during Auxin Induction of Cell Enlargement in Tobacco Pith Tissue

IAA (indoleacetic acid) is known to induce cell enlargement without cell division in tobacco pith explants grown on an agar medium without added cytokinin. The very long lag period before IAA (2 × 10^?5M) stimulates growth, about 3 days, can be useful to study the metabolic changes which lead to the promotion of growth. When the disks are transferred to a medium without IAA after 2 days or less of treatment with IAA, the IAA does not stimulate growth. Disks transferred after 3 days, subsequently show an auxin response, almost as great as those given IAA continuously. At 5 × 10^?4M, 5-fluorodeoxyuridine (FUDR), which inhibits DNA synthesis by blocking formation of thymidylate, completely suppresses the lAA-induced growth if it is added together with the IAA or 1 day later. When the FUDR is given 2 days after the IAA, there is a small increment of auxin-induced growth, and an even greater amount if added after 3 days. The period when exogenous auxin must be present to stimulate growth corresponds to the period of FUDR sensitivity. The FUDR inhibition is prevented by thymidine but not by uridine. Other inhibitors of DNA synthesis, hydroxyurea and fluorouracil, also inhibit auxin-induced growth. Thus DNA synthesis seems to be required for auxin induction of cell enlargement in tobacco pith explants. In contrast, FUDR does not inhibit auxin-induced growth in corn coleoptile and artichoke tuber sections.     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.     

Effect of Auxins and Light on Rooting Stem Cuttings of Populus nigra Salix tetrasperma, Ipomea fistulosa and Hibiscus notodus in Relation to Polarity

The apical and basal ends of stem cuttings of Populus nigra, Salix tetrasperma, Ipomoea fistulosa and Hibiscus notodus were treated with 10 mg/l solutions of IAA and IBA for 24 hours and were planted either erect or inverted both in light and dark. Observations for the number of cuttings that rooted and the roots produced on them were recorded at weekly intervals. In Salix, Ipomoea and Hibiscus rooting was more on cuttings planted erect, while in populus it did not differ much with the manner of planting. The reduced rooting in inverted cuttings may be ascribed to the low level of endogenous auxin at the apex due to polar transport. An exogenous application of auxins enhanced rooting on inverted cuttings. In dark, roots on Populus and Salix cuttings were produced both above and within the rooting medium. The weak polarity of these two plants may be due to the potential root primordia reported in their stem. The formation of callus occurred on the top of Populus cuttings whether planted erect or inverted but it differentiated into branches on erect cuttings only. In those planted in an inverted position the callus failed to differentiate in spite of the application of kinetin, auxins, TIBA, coumarin and sucrose, and dried ultimately.     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.