Phenolic compounds profiles during ex vitro acclimatization of micropropagated Hypericum polyanthemum.

Accumulation of benzopyrans and total phenolic compounds were assessed in acclimatized field grown plants of Hypericum polyanthemum, an endemic species of southern Brazil, harvested at different developmental stages. The HPLC analysis of bioactive compounds 6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) revealed that the three benzopyrans are accumulated both in the vegetative and reproductive parts with maximum contents observed after 18 weeks (in the former) and 20 weeks (in the later) of plant growth (1.92+/-0.085 g % DW and 2.62+/-0.13 g % DW in the vegetative and reproductive parts, respectively). Highest contents of HP1 (1.56+/-0.12 g % DW) and HP2 (0.19+/-0.01 g % DW) were quantified in the green floral buds of the plants, whereas HP3 reached the highest level (1.02+/-0.08 g % DW) in the overblown flowers. The evaluation of total phenolic compounds showed that the vegetative parts accumulated the highest levels of the metabolites (51.93+/-0.67 mg QE (g DW)(-1)) after 16 weeks of plant growth. Considering the reproductive parts, the open flowers accumulated the greatest levels of the bioactive compounds (75.99+/-0.95 mg QE (g DW)(-1)). The results show that H. polyanthemum can be efficiently propagated and acclimatized to produce benzopyrans and other phenolic compounds.     

Roles of in vitro plantlet age and growing period in the phenolic constituent yields of acclimatized Hypericum polyanthemum

The influences of culture period on growth, plant survival rate and content of phenolic compounds were investigated in vitro and in acclimatized field-grown plants of Hypericum polyanthemum. The growth kinetics of micropropagated plantlets cultured in MS modified medium and the concomitant transference to ex vitro conditions showed that cultures achieved maximum biomass and yield of bioactive compounds after 12 weeks of in vitro growth, with field-grown plants displaying the same survival pattern. Differences in yield among plants cultured in vitro for 8 and 12 weeks that were acclimatized and followed over two years showed that the physiological age of the in vitro cultures influenced biomass production. However, benzopyrans and total phenolic compounds (TPC) contents did not vary significantly, with the exception of the 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) concentration in the reproductive parts, which was higher in the plants grown in vitro for 12 weeks over the two years of the study. All analyzed plant parts from the spring harvest accumulated lower benzopyran levels than plants harvested after 18 weeks of growth in both treatments, except for the levels of 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1) and 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) in the new vegetative parts of the plants, which did not vary. The concentration of TPC, which was detected at low levels in the old vegetative parts in both treatments, was not altered in other plant parts. The information provided by this work will help structure plant growth and collection periods designed to optimize the yield of each required bioactive metabolite.     

Benzopyrans from Hypericum polyanthemum.

From the aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae), three chromenes, 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran; 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran were isolated. Their structures were determined by NMR spectroscopic analyses.     

Supercritical fluid extraction and high performance liquid chromatographic determination of benzopyrans and phloroglucinol derivative in Hypericum polyanthemum

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO₂) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 °C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 °C and for the high molecular-weight compound, uliginosin B, higher pressures were required.     

Influence of fungal elicitation with Nomuraea rileyi (Farlow) Samson in the metabolism of acclimatized plants of Hypericum polyanthemum Klotzsech ex Reichardt (Guttiferae)

Changes in biomass and bioactive metabolites after treatment with the fungus Nomuraea rileyi (Farlow) Samson added as dried culture (DC) or as dried autoclaved cell powder (DACP) for short and long periods of time have been investigated in Hypericum polyanthemum plants grown under controlled conditions and after 18 weeks of field acclimatization. Plants grown under controlled conditions and treated with DACP showed increased concentrations of the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethylbenzopyran), and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethylbenzopyran) while DC affected negatively or did not alter the synthesis of these compounds. Lower levels of the phloroglucinol derivative uliginosin B were detected in all treatments. Long time treatment with DACP of acclimatized plants triggered doubling of biomass as compared to control and chemical analyses demonstrated significant increase on total phenolic compounds in vegetative parts of plants. Moreover, the higher performance liquid chromatography analysis showed different pattern of metabolites accumulation, with higher yields of HP1, HP2, HP3 and uliginosin B in the reproductive parts of the plants treated with DACP during all experiments. The elevation of bioactive metabolites levels in response to the elicitor suggests that these compounds are inducible as part of the defense response of H. polyanthemum.     

Determination of phenolic compounds in flowers of Hypericum species native to South Brazil and Peruvian Páramos

The flowers constitute one of the main sites of accumulation of phenolic compounds in plants of the Hypericum genus. In addition to their important pharmacological activities, some metabolites found in species from the section Brathys and Trigynobrathys appear to have chemotaxonomic significance according to the literature. HPLC analyses were carried out to assess the pattern and accumulation of the dimeric phloroglucinols, benzophenones, benzopyrans, flavonoids and a phenolic acid in flowers of Hypericum species native to southern Brazil and Peruvian Páramos. Qualitative and quantitative differences are reported. Uliginosin B and hyperoside were the main components detected in all species and with maximum concentrations up to 0.188 % in H. caprifoliatum and 5.987 % in H. andinum, respectively. The content of japonicin A varied from 0.003 to 0.087 % in H. myrianthum, while the yield of hyperbrasilol B ranged from 0.006 % in H. laricifolium to 0.011 % in H. caprifoliatum. The major compounds in H. polyanthemum and H. carinatum were the benzopyrans 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1 = 0.200 %), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2 = 0.225 %) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3 = 0.327 %) and benzophenones cariphenone A (0.309 %) and cariphenone B (0.062 %), respectively. Maximum amounts of chlorogenic acid, isoquercitrin, quercitrin and guaijaverin were observed, respectively, in H. campestre (1.458 %), H. andinum (1.161 %), H. carinatum (0.231 %) and H. laricifolium (0.404 %). The results obtained support the taxonomic evidence of the dimeric phloroglucinol derivatives at the section level.     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

Plantlet differentiation in leaf and root cultures of birch (Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl-trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.     

Rosmarinic acid content in basil plants grown <Emphasis Type="Italic">in vitro</Emphasis> and in hydroponics

The accumulation of selected caffeic acid derivatives (CADs), in particular rosmarinic acid (RA), was investigated in different tissues (leaves, roots and plantlet shoots) of sweet basil (Ocimum basilicum L.) plants grown either in vitro or in hydroponic culture (floating system) under greenhouse conditions. Two cultivars with green leaves (Genovese and Superbo) and one with purple leaves (Dark Opal) were tested. The content of CADs in HCl-methanol extracts was determined by HPLC. LC-MS and LC-MS-MS were used to confirm the identification of the metabolites of interest. Apart from rosmarinic acid (RA) and a methylated form of this substance, no other CADs were detected at significant level in any of the analyzed samples. The content of RA ranged approximately from 4 to 63 mg/g DW, depending on the growing system. The highest RA content was found during the in vitro multiplication, in the acclimatized plants and in the roots of hydroponically-grown seedlings at full bloom. In vitro, 6-benzyladenine reduced the accumulation of RA in purple-leaf Dark Opal cultivar, but an opposite effect of this growth regulator was observed in the green-leaf genotypes. Our findings suggest the possibility to scale-up RA production by means of in vitro or hydroponic culture of sweet basil.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Phenolic compounds accumulation in <Emphasis Type="Italic">Hypericum ternum</Emphasis> propagated in vitro and during plant development acclimatization

The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.     

Short-term and long-term time course studies of turmeric (Curcuma longa L.) microrhizome development in vitro

Turmeric (Curcuma longa L.) plantlets were cultured in MS (Murashige and Skoog Physiol Plant 15:473–497, 1962) liquid medium with 6% sucrose. Microrhizome development was observed in the presence of methyl jasmonate (MeJa) (0, 5 and 16 μM) and benzyladenine (BA) (0, 0.32 and 1 μM). Leaf, root, rhizome growth, and sugar use were measured weekly for 6 weeks in small vessels (180 ml) and four times in 23 weeks in larger vessels (2.5 l). MeJa reduced leaf, root, and rhizome biomass. BA had a positive effect on biomass accumulation. Microrhizome mass increased at a linear rate during 6 weeks of culture while roots and leaves accumulated biomass at an exponential rate. Sugar use correlated nearly directly to whole plant dry weight (DW) in the short and long-term experiments. Microrhizomes became a larger fraction of whole plant DW as plantlets aged. After 6 weeks, about 1.8 g of microrhizome DW per liter of media had been produced (in both time courses), and after 23 weeks, about 26 g of microrhizome DW per liter of media had been produced. Secondary rhizomes were first observed at 14 weeks, and most plants had them by 23 weeks. A method for rhizome production in a long-term culture system was described. The linear relationship between DW and sugar use will be useful in the eventual development of a model for sugar use to biomass to secondary metabolite production. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromenes of polyketide origin from Peperomia villipetiola

An extract of leaves and stems of Peperomia villipetiola has been found to contain myristicin (3-methoxy-4,5-methylenedioxy-allylbenzene) and seven chromenes, whose structures are methyl 5-hydroxy-7-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (1), methyl 5-methoxy-7-methyl-2,2-dimethyl-2H-1-chromene-8-carboxylate (2), methyl 7-hydroxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (3), methyl 7-methoxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (4), 5-methanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (5), 5-methanol-7-methoxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (6), and methyl 5-acetoxymethanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate (7). A biosynthetic rationale for 1-7 suggests that orsellinic acid may be a common intermediate. The anti-fungal activities of the chromenes were measured bioautographically against Cladosporium cladosporioides and Cladosporium sphaerospermum: compounds 6 and 7 were found to be the most active.     

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of <Emphasis Type="Italic">Allamanda cathartica</Emphasis> L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00?±?1.30) and shoot length (7.50?±?0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50?±?0.16 mean roots/shoot with 4.05?±?0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.     

Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.)

Summary Somatic embryogenesis in American ginseng (Panax quinquefolium L.) was investigated from three explant sources (root, leaf and epicotyl) with Murashige and Skoog (MS) medium containing different growth regulators. Mature roots and leaves obtained from 3- to 5-yr-old field-grown plants, and seedling leaves and epicotyls from plantlets grownin vitro, were evaluated. From root and epicotyl explants, callus development was optimal with 3,6-dichloro-o-anisic acid (dicamba) (9.0 μM) and kinetin (KN) (5.0 μM) as the growth regulators. When these calluses were transferred after 3 mo. to dicamba alone (9.0 μM), somatic embryo formation was observed at an average frequency of 15.6% in root explants after an additional 3 mo., and 2% in epicotyl explants after an additional 6 mo. No plantlets were recovered because the embryos germinated to form shoots with no roots. From leaf explants, callus growth was optimal with α-naphthaleneacetic acid (NAA) at 10.0 μM and 2,4-dichlorophenoxyacetic acid (2,4-D) at 9.0 μM. Somatic embryos developed on this medium, with the highest frequency (40%) obtained after 3 mo. from seedling-leaf explants. Calluses on mature leaves formed somatic embryos after 7 mo. with NAA/2,4-D at an average frequency of 30%. Transfer of these somatic embryos to 6-benzyladenine/gibberellic acid (4.4/2.9 μM) promoted shoot development but no roots were observed. Up to 100% of germination was observed within 6 wk on half-strength MS salts containing activated charcoal (1%) and on NAA/2,4-D (5.0/4.5 μM) with charcoal (1%). On the latter medium, somatic embryos enlarged and frequently gave rise to new somatic embryos after a brief callusing phase. The embryos germinated through a two-stage process, involving the elongation of the root followed by the formation of a shoot. The highest recovery of ginseng plantlets from germinated embryos was 61.0%. Following transfer to potting medium and maintenance under conditions of high humidity and low light intensity, the plantlets elongated and developed new leaves. A high percentage (50%) of these plants have been acclimatized to soil.     

Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium

Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets. The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in a greenhouse when they were planted in soil. Received: 19 July 1997 / Revision received: 6 October 1997 / Accepted: 3 October 1997     

Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma

A protocol for in vitro propagation of cineraria (Senecio cruentus) was developed. The highest frequency of shoot proliferation was obtained from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0?mg L^?1 6-benzyladenine (BA) and 0.5?mg L^?1 ??-naphthalene acetic acid (NAA), with a mean number of 14 shoots per explant. A high concentration of BA (4.0?mg L^?1) and repeated subcultures resulted in hyperhydric shoots. Decreasing the BA concentration to 1.0?mg L^?1 in the culture medium eliminated hyperhydricity. The concentration of ammonium nitrate (NH₄NO₃) and temperature had marked effects on somaclonal variation. Variation was observed when the cultures were maintained at 15?°C but not at 25?°C. Variants with blue-colored leaves and stems were identified; whereas, normal plants maintained their green-colored leaves and stems. The highest frequency of variation (67.5?%), with a mean number of 3.0 variant shoots per explants, was obtained on shoot proliferation medium (MS?+?2.0?mg L^?1 BA and 0.5?mg L^?1 NAA) devoid of NH₄NO₃. The best rooting (100?%), with the highest number of roots per shoot (10.8) and the greatest root length (6.8?cm) was obtained on medium supplemented with 0.1?mg L^?1 NAA. In vitro-grown plantlets were successfully acclimatized in a greenhouse, and transferred to the field.     

珠美海棠试管苗的土壤支撑生根培养和带坨移栽

以粘壤土做培养基支撑物,选用只含有0．5mg·L^-1IBA和15g·L^-1蔗糖的液体培养基,珠关海棠试管苗茎段生根培养的结果显示,试管苗生根率可达到100．0％,明显高于琼脂支撑培养,根长也显著提高,并且长出正常的根毛。土支撑培养生根的珠美海棠试管苗开瓶炼苗21d后带坨移入营养钵中,移栽后不喷雾、并毋需覆盖塑膜、午后空气相对湿度控制在50％-65％,其成活率达到92．2％;在开瓶炼苗21d后,黑暗中叶片气孔的关闭率从26．7％增加到88．5％。