<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

High frequency <Emphasis Type="Italic">in vitro</Emphasis> propagation of <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> from nodal buds of mature tree

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm⁻³ N⁶-benzyladenine (BA) and 0.5 mg dm⁻³ α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm⁻³ BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm⁻³ of indole-3-butyric acid solution. Successful field establishment and high (80–90 %) survival of plants was observed.     

Influence of auxins and darkness on in vitro rooting of micropropagated shoots from mature and juvenile Acacia mangium

The influence of total darkness versus a 16/8 photoperiod and of auxins added to the culture medium on the in vitro root formation capacity of Acacia mangium microshoots of juvenile and mature origin was examined. Rooting of the mature clone was significantly increased by exposing the microshoots to auxins (4 and 6 μM IAA or IBA) in darkness, while the promoting effect of darkness combined with 4 μM IAA was more time-restricted for the juvenile-origin microshoots. Overall, the latter rooted in greater proportions than those from the mature source. Maintaining the microshoots of both origins on auxin supplemented medium in darkness resulted in a greater number of adventitious roots formed than under the standard 16/8 lighting conditions. On the other hand, light stimulated root elongation. These results are discussed mainly from the viewpoint of auxin metabolism in relation to adventitious root formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bulgarian golden root <Emphasis Type="Italic">in vitro</Emphasis> cultures for micropropagation and reintroduction

Rhodiola rosea is an endangered medicinal plant used for cancer, cardiovascular, and nervous system diseases therapy. Due to its limited distribution and sustainability alternative methods for production of its valuable substances are under investigation. Using in vitro techniques apical and rhizome buds, leaf nodes, stem and radix segments from wild plants and in vitro seedlings were plated on 24 modified Murashige and Skoog (1962) media. Decontamination of plant material was successful only in 21% of the schemes. The best shoot induction was obtained from seedling explants on media containing 2 mg/l zeatin or N⁶-benzylaminopurine, each. Their reduction stimulated shoot formation in the next passages (multiplication rate up to 5). Efficient rooting was induced on half-strength MS with 2 mg/l Indole-3-butyric acid and stimulated by adding 0.2 mg/l Indolyl-3-acetic acid. Regenerants rooted in perlite, peat, and soil (1:1:2), adapted in greenhouse, and transplanted in the mountains survived (70%) and developed like the wild plants. Salidroside content of these plants after one or two years was high (0.64 and 0.61% in rhizomes and 0.62 and 0.53% in roots, respectively). This is the first established efficient scheme for micropropagation of Bulgarian R. rosea allowing habitats restoration, germplasm conservation, and potential application of biotechnology for production of valuable substances.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Perilla frutescens</Emphasis> from hypocotyl and cotyledon explants

Organogenetic buds were induced from hypocotyl and cotyledon explants of oil crop Perilla frutescens in Murashige and Skoog (MS) medium supplemented with 5.7 M indole-3-acetic acid (IAA) and 8.9 – 13.3 M 6-benzylaminopurine (BA). Shoots were rooted on MS medium with 2.9 M IAA and 1.4 M gibberellic acid (GA3) and the regenerated plants flowered and set seeds normally.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

<Emphasis Type="Italic">In vitro</Emphasis> morphogenesis in plants-recent advances

Summary The capacity of cultured plant tissues and cells to undergo morphogenesis, resulting in the formation of discrete organs or whole plants, has provided opportunities for numerous applications of in vitro plant biology in studies of basic botany, biochemistry, propagation, breeding, and development of transgenic crops. While the fundamental techniques to achieve in vitro plant morphogenesis have been well established for a number of years, innovations in particular aspects of the technology continue to be made. Tremendous progress has been made in recent years regarding the genetic bases underlying both in vitro and in situ plant morphogenesis, stimulated by progress in functional genomics research. Advances in the identification of specific genes that are involved in plant morphogenesis in vitro, as well as some selected technical innovations, will be discussed. REPRINTED FROM: Phillips, G. C. In vitro morphogenesis in plants-recent advances. In: Goodman, R. M., ed. Encyclopedia of plant and crop science, vol. 1. New York: Marcel Dekker, Inc.; 2004: 579–583, http://www.dekker.com/servlet/product/DOI/101081EEPCS120010554; by courtesy of Marcel Dekker, Inc.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Mapping species differences for adventitious rooting in a <Emphasis Type="Italic">Corymbia torelliana</Emphasis> × <Emphasis Type="Italic">Corymbia citriodora</Emphasis> subspecies <Emphasis Type="Italic">variegata</Emphasis> hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana × Corymbia citriodora subspecies variegata hybrid family (n = 186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of strictosidine using<Emphasis Type="Italic">lonicera japonica</Emphasis> leaf extracts and recombinant yeast

Strictosidine is a key intermediate in the biosynthesis of the terpenoid indole alkaloid (T1A) pathway. It results from a condensation reaction, catalyzed by strictosidine synthase (STR), between tryptamine and secologanin. We have now developed a useful method, based on enzyme-assisted synthesis, to produce strictosidine. Our procedure utilizes leaf extracts from Japanese honeysuckleLonicera japonica Thunb. as a secologanin source. In these experiments, an enzyme extract was prepared from transgenic yeastSaccharomyces cerevisiae that expresses theCatharanthus roseus STR (CrSTR) coding region. Strictosidine was then isolated with a 38% yield based on the initial amount of tryptamine in the enzymatic reaction.     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and secondary metabolites production in wild germander (<Emphasis Type="Italic">Teucrium polium</Emphasis> L.)

A protocol for in vitro propagation of the wild germander (Teucrium polium L.) was developed. In vitro plants were developed from ex vitro axillary buds. Then, shoot tips were excised and established on Murashige and Skoog medium. Proliferation of shoots was tested with different levels of 6-furfurylaminopurin, 6-benzyladenine, or thiadiazuron. The highest proliferation of T. polium was obtained when 6-benzyladenine and 6-furfurylaminopurin were used at 2.0 and 1.6 mg l⁻¹, respectively. Thiadiazuron gave the lowest response for shoot proliferation. Rooting was experimented at different levels of Indol-3-butric acid, Indol-3-acetic acid, or 1-naphthaleneacetic acid. 1-Naphthaleneacetic was the only growth regulator which promoted root induction. Rooted plants were acclimatized successfully with 75% survival and grown in the greenhouse. In vitro- and in vivo-grown plants were analyzed for essential oil production. In vitro-grown T. polium on MS medium supplemented with 6-benzyladenine and 1-naphthaleneacetic gave higher oil yield than that grown on hormone-free Murashige and Skoog medium. In vivo (wild)-grown T. polium produced different oil yield when collected in different months (April and October). β-caryophyllene, used as a marker compound in the essential oil, was identified and quantified by gas chromatography (GC) analysis. Gas chromatography/mass (GC-MS) spectrometry analysis was also used to identify other components of in vitro cultures and to compare with in vivo-grown plants.