Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Somatic embryogenesis and plantlet regeneration in semul (<Emphasis Type="Italic">Bombax ceiba</Emphasis>)

Somatic embryogenesis in semul, Bombax ceiba L. (Bombacaceae) was achieved from immature zygotic embryo explants on MS medium. The cytokinin BA induced somatic embryogenesis at higher frequencies than the auxin 2,4-d. The rate of somatic embryogenesis was inversely related to the concentration of BA. A constant supply of 0.44 M BA was necessary for a high multiplication rate. Conversion of somatic embryos into plantlets is reported.     

Somatic embryogenesis from zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

We describe the multi-step regeneration system of medicinal plant Schisandra chinensis (Turcz.) Baill. The seeds were pre-treated with 0.005 μM thidiazuron. Subsequently the zygotic embryos of the early heart stage were cultured on medium with 50 μM of 2,4-dichlorophenoxyacetic acid (2,4-D) and after three weeks the embryogenic calli were transferred to a medium with 10 μM of 2,4-D and 4 μM of 6-benzyladenine and were sub-cultured at the 4-week intervals. Abscisic acid (30 μM) and polyethyleneglycol (3 %) significantly influenced the synchronization of development of the somatic embryos (SEs) to the globular stage. The following culture on a medium without growth regulators resulted in full developed cotyledonary stage SEs. Indole-3-butyric acid (0.05 μ) contributed to their rapid conversion to plantlets.This experimental work was supported by project No. 521/02/1129 of the Grant Agency of the Czech Republic.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis>: influence of cytokinins

The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA₃) was applied post-flower-emergence.     

<Emphasis Type="Italic">In vitro</Emphasis> fruiting and seed set of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. CV. Sweet banana

Summary Plantlets of Capsicum annuum L. ev. Sweet Banana regenerated via somatic embryogenesis from immature zygotic embryos were capable of producing flower, fruit, and seed when cultured in small tissue culture containers. In vitro floral buds were first formed on plantlets that grew on plantlet development medium [agar-gelled Murashige and Skoog (MS) basal medium containing 1 mgl⁻¹ (5.3 μM) α-naphthaleneacetic acid (NAA)] in a growth room at 22°C and continuous illumination. However, floral buds rarely developed further into mature flowers. This problem was overcome using the vented autoclavable plant tissue culture containers. In vitro fruit formation and ripening was observed when liquid half-strength MS basal medium supplemented with 5 μg ml⁻¹ silver thiosulfate, 1 mg l⁻¹ (5.3 μM) NAA, and 3% sucrose was added to the surface of the plantlet development medium. Hand-pollination improved fruit set. Further research in needed to determine why the pepper seeds formed in vitro failed to germinate.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

Micropropagation of the endangered <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis> Baijnath

Summary The species, Kniphofia leucocephala is extant at only one location, Langepan, KwaZulu-Natal in South Africa, where the population is threatened by afforestation and possibly grazing. Consequently, a continuous culture system was established as part of a program for the propagation and re-introduction of plants into the wild. The efficiency of the system in terms of shoot multiplication and, particularly, the frequency and rate of root initiation was strongly influenced by the concentration of benzyladenine in the shoot multiplication medium. The optimum shoot multiplication medium for subsequent root initiation contained 2 mgl⁻¹ (8.9 μM) benzyladenine alone. The shoots were successfully rooted and acclimatized. Approximately 200 shoots can be produced from one shoot after five 4-wk cycles. Thus, large numbers of plantlets can be propagated in this continuous culture system, serving conservation interests.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.