In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.     

Isolation and characterization of four somatic embryogenesis receptor-like kinase (<Emphasis Type="Italic">RhSERK</Emphasis>) genes from miniature potted rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> cv. Linda)

The isolation and expression analysis of four partial gene sequences from rose (Rosa hybrida cv. Linda) belonging to the receptor-like kinase gene superfamily are reported. These genes have been designated RhSERK1 to RhSERK4 (Accession No. EF631967 to EF631970) as they exhibit high sequence identities with genes from the somatic embryogenesis receptor-like kinase (SERK) family in other plant species. The RhSERK genes are differentially expressed in non-embryogenic callus, embryogenic callus, mature somatic embryos and a range of tissues from intact plants, indicating a broad role in plant growth and development. However, the expressions of RhSERK3 and RhSERK4 were approximately fivefold higher in embryogenic callus than in non-embryogenic callus, and they are even higher when compared to tissues from intact plants. In addition, RhSERK4 expression was approximately eightfold higher in somatic embryos than in embryogenic callus. These results suggest that the expression pattern of RhSERK3 and RhSERK4 may be used as a marker of somatic embryogenesis.     

Characterisation of the <Emphasis Type="Italic">Vitis vinifera</Emphasis>PR10 multigene family

Background  

Genes belonging to the pathogenesis related 10 (PR10) group have been studied in several plant species, where they form multigene families. Until now, such an analysis has not been performed in Vitis vinifera, although three different PR10 genes were found to be expressed under pathogen attack or abiotic stress, and during somatic embryogenesis induction. We used the complete genome sequence for characterising the whole V. vinifera PR10 gene family. The expression of candidate genes was studied in various non-treated tissues and following somatic embryogenesis induction by the auxin 2,4-D.     

Protocorm-like body (PLB) formation and plant regeneration from the callus culture of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall <Emphasis Type="Italic">ex</Emphasis> Lindl.

An efficient system was established for a higher frequency of protocorm-like body (PLB) formation from the callus of Dendrobium candidum Wall ex Lindl. The calluses were induced from longitudinally bisected segments of protocorms and subcultured two times every 40d on Murashige and Skoog medium with macronutrients at half strength, micronutrients at full strength, 2% sucrose, and with 8.8μM 6-Benzylaminopurine. PLB formation was achieved when calluses were transferred onto the same basal medium without any plant growth regulators. PLBs developed into intact plantlets about 2cm in height and with four roots when on basal medium with 2.7μM 1-naphthaleneacetic acid. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Histological observations proved that globular somatic embryos could be produced from the inside and surface of the embryogenic callus during PLB formation.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the wild carrot <Emphasis Type="Italic">Daucus carota</Emphasis> L. subsp. <Emphasis Type="Italic">halophilus</Emphasis> (Brot.) A. Pujadas for conservation purposes

Daucus carota subsp. halophilus, is a wild crop relative of domestic carrot. It is an aromatic plant widely used in folk medicine due to recognized therapeutic properties of its essential oils. Experiments were carried out to evaluate the potential of in vitro propagation techniques to the conservation of this endemic and endangered taxon. The results showed that shoot tips of in vitro germinated seeds were able to proliferate in the presence of benzyladenine, with the best results being achieved using 4.4 μM, both in the first and second cultures. Shoots rooted after being transferred to 1/2-Murashige and Skoog basal medium. The results indicated that the concentration of benzyladenine used during the multiplication phase did not interfere with the rate of root formation. The obtained plantlets were morphologically and anatomically identical to those obtained by seeds. Some of the in vitro produced shoots developed flowers that produced viable pollen. Plant regeneration was also achieved by somatic embryogenesis induction in cotyledons and root segments cultured in the presence of 4.5 μM 2,4-dichlorophenoxyacetic acid. Somatic embryos converted into plantlets in a medium without growth regulators. Plants obtained either by shoot proliferation or somatic embryogenesis were acclimatized and are now growing at the Coimbra Botanical Garden. The first attempts to reintroduce these plants in the original habitat were successful. It can be concluded that the protocols developed are a useful approach to the conservation of this endemic species.     

Expression pattern of two paralogs of the <Emphasis Type="Italic">PI</Emphasis>/<Emphasis Type="Italic">GLO</Emphasis>-like locus during <Emphasis Type="Italic">Orchis italica</Emphasis> (Orchidaceae,Orchidinae) flower development

The class B MADS-box genes belong to two distinct functional groups: the AP3/DEF-like and the PI/GLO-like sub-families. In orchids, AP3/DEF-like genes are present in four copies, each with a different role in floral organ formation, which is described in the “orchid code” model. Interestingly, the orchid PI/GLO-like genes are present in two copies in Orchidinae, whereas they are described as single copy in the other orchid lineages. The two PI/GLO-like paralogs have site-specific different selective constraints; in addition, they show relaxation of purifying selection when compared to the single-copy lineages. In this study, we present a comparative analysis of the expression patterns of the two PI/GLO-like paralogs, OrcPI and OrcPI2, in floral tissues of Orchis italica in different developmental stages using real-time PCR. The two genes show similar expression profiles in the tissue examined, with differences detectable between immature and mature inflorescence. In all cases, OrcPI2 is expressed at a higher level than OrcPI. Real-time PCR results reveal that the co-expression of the two duplicated loci could have a fully or partially redundant function. The possible evolutionary fate of OrcPI and OrcPI2 is discussed as well as their involvement in ovary development.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Somatic embryogenesis in <Emphasis Type="Italic">Buchanania lanzan</Emphasis> spreng

Summary We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Somatic embryogenesis in clonal neem, <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. and analysis for <Emphasis Type="Italic">in vitro</Emphasis> azadirachtin production

Summary Efficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately 15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic embryos.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Indirect somatic embryogenesis and morphohistological analysis in <Emphasis Type="Italic">Capsicum chinense</Emphasis>

Capsicum chinense is recalcitrant in in vitro morphogenesis. No efficient, reproducible somatic embryogenesis regeneration system exists for this species, impeding regeneration from transformed cells. An indirect somatic embryogenesis protocol is developed using mature C. chinense zygotic embryo segments (ZES). The ZES cultured in semi-solid Murashige-Skoog (MS) medium supplemented with 8.9 μM naphthaleneacetic acid, 11.4 μM indoleacetic acid and 8.9 μM 6-benzylaminopurine, developed an embryogenic callus and 8% of the calli developed somatic embryos. Torpedo-stage somatic embryos were detached from the callus and subcultured in semi-solid MS medium without growth regulators, producing a 75% conversion rate to plantlets with well-formed root tissue. Histological analysis showed the developed structures to have no vascular connection with the callus and to be bipolar, confirming that this protocol induced formation of viable somatic embryos from mature C. chinense ZES. All acclimated plantlets survived under greenhouse conditions. This protocol will facilitate regeneration of genetically transformed plants using either biolistics or Agrobacterium tumefaciens approach.