Orchid Mycorrhizal Association of Cultivated Dendrobium Hybrid and Their Role in Seed Germination and Seedling Growth.

Orchids are crucial for the horticulture industry. Mycorrhizal fungi benefit crops by improving nutrition, plant growth, and disease resistance. However, the mycorrhizal association of horticultural hybrid orchids is poorly understood. To address this, we investigated mycorrhizal colonization in the entire root system and assessed the mycorrhizal community using a Dendrobium cultivar, D. Stardust 'Firebird', obtained from three nurseries. Additionally, we isolated and tested mycorrhizal fungi in symbiotic culture to assess their role in the seed germination and growth of Dendrobium species. All plants were colonized by mycorrhizal fungi, with a higher colonization rate in mature than in juvenile plants. Molecular identification of mycorrhizal fungi by Sanger and high-throughput sequencing revealed that the cultivar was associated with a phylogenetically diverse group of fungi, including mycorrhizal fungi from Tulasnellaceae, and several wood-decaying fungi. The Tulasnellaceae isolates significantly enhanced the seed germination of three Dendrobium species and increased the survival rate and growth of asymbiotic seedlings of D. moniliforme. This study is the first comprehensive examination of mycorrhizal associations in horticultural orchid hybrids, providing valuable insights for commercial production.

Two pathways of 2n gamete formation and differences in the frequencies of 2n gametes between wild species and interspecific hybrids.

KEY MESSAGE: Epidendrum produces 2n gametes with high frequency. This paper is the first to report on multiple pathways for forming 2n gametes, meiotic defeats, and pre-meiotic chromosome doubling. Unreduced 2n reproductive cells are predominantly involved in pathways that lead to polyploid plants. Although one of the most common pathways for inducing 2n gametes is through meiotic defects, a small set of isolated species alternatively generates 2n gametes from tetraploid pollen mother cells in the pre-meiotic phase. Hence, determining the mechanisms underlying 2n gamete formation is critical to improving breeding programmes and understanding plant evolution. We investigated sporads to reveal the pathway(s) accounting for the formation and frequencies of 2n gametes in wild species and interspecific hybrids in the genus Epidendrum. We investigated different types of sporads with varying frequencies, sizes, and viability in the wild species and hybrids of the genus Epidendrum. Large tetrad-estimated pre-meiotic chromosome doubling was observed in wild species. The Epidendrum is unique in that it forms 2n pollens via two pathways, namely, meiotic defects and pre-meiotic chromosome doubling. These two pathways of 2n pollen formation could influence the high diversity generation of polyploidy with different degrees of heterozygosity and genetic backgrounds in the genus Epidendrum. Therefore, these findings are proposed to influence polyploid breeding of Epidendrum via 2n pollen, helping us understand evolution and speciation via unreduced 2n gamete formation in Orchidaceae.

Induction of adventitious shoots and tetraploids in Antirrhinum majus L. by treatment of antimitotic agents in vitro without plant growth regulators.

We examined the effects of five antimitotic agents using Antirrhinum majus L. 'Maryland True Pink' on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.

First flowering hybrid between autotrophic and mycoheterotrophic plant species: breakthrough in molecular biology of mycoheterotrophy.

Among land plants, which generally exhibit autotrophy through photosynthesis, about 880 species are mycoheterotrophs, dependent on mycorrhizal fungi for their carbon supply. Shifts in nutritional mode from autotrophy to mycoheterotrophy are usually accompanied by evolution of various combinations of characters related to structure and physiology, e.g., loss of foliage leaves and roots, reduction in seed size, degradation of plastid genome, and changes in mycorrhizal association and pollination strategy. However, the patterns and processes involved in such alterations are generally unknown. Hybrids between autotrophic and mycoheterotrophic plants may provide a breakthrough in molecular studies on the evolution of mycoheterotrophy. We have produced the first hybrid between autotrophic and mycoheterotrophic plant species using the orchid group Cymbidium. The autotrophic Cymbidium ensifolium subsp. haematodes and mycoheterotrophic C. macrorhizon were artificially pollinated, and aseptic germination of the hybrid seeds obtained was promoted by sonication. In vitro flowering was observed five years after seed sowing. Development of foliage leaves, an important character for photosynthesis, segregated in the first generation; that is, some individuals only developed scale leaves on the rhizome and flowering stems. However, all of the flowering plants formed roots, which is identical to the maternal parent.

Shifts in mycorrhizal fungi during the evolution of autotrophy to mycoheterotrophy in Cymbidium (Orchidaceae).

PREMISE OF THE STUDY: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework. METHODS: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences. KEY RESULTS: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales. CONCLUSION: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium. We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants.

N(2)O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies.

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N(2)O) gas to obtain 2n male gametes. N(2)O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N(2)O treatment restores fertility in sterile hybrids. To establish optimal N(2)O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N(2)O gas at different anther developmental stages from fertile and sterile hybrid lilies. N(2)O treatment using the optimal 1-4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N(2)O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC(1) plants. Thus N(2)O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.

The evolutionary history of mycorrhizal specificity among lady's slipper orchids.

Although coevolution is acknowledged to occur in nature, coevolutionary patterns in symbioses not involving species-to-species relationships are poorly understood. Mycorrhizal plants are thought to be too generalist to coevolve with their symbiotic fungi; yet some plants, including some orchids, exhibit strikingly narrow mycorrhizal specificity. Here, we assess the evolutionary history of mycorrhizal specificity in the lady's slipper orchid genus, Cypripedium. We sampled 90 populations of 15 taxa across three continents, using DNA methods to identify fungal symbionts and quantify mycorrhizal specificity. We assessed phylogenetic relationships among sampled Cypripedium taxa, onto which we mapped mycorrhizal specificity. Cypripedium taxa associated almost exclusively with fungi within family Tulasnellaceae. Ancestral specificity appears to have been narrow, followed by a broadening after the divergence of C. debile. Specificity then narrowed, resulting in strikingly narrow specificity in most of the taxa in this study, with no taxon rewidening to the same extant as basal members of the genus. Sympatric taxa generally associated with different sets of fungi, and most clades of Cypripedium-mycorrhizal fungi were found throughout much of the northern hemisphere, suggesting that these evolutionary patterns in specificity are not the result of biogeographic lack of opportunity to associate with potential partners. Mycorrhizal specificity in genus Cypripedium appears to be an evolvable trait, and associations with particular fungi are phylogenetically conserved.

In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae).

BACKGROUND AND AIMS: Many Orchidaceous species are threatened globally by development and over-collection from their natural habitats for horticultural purposes. Artificial propagation from seeds is difficult in most terrestrial orchids native to temperate regions. Seed production is another limiting factor in the artificial propagation for these species because of the lessened probability of pollination and the destruction of fruit by insect larvae. Members of the genus Cephalanthera are distributed across Europe, Asia and North America. C. falcata is a temperate species of East Asia and an endangered species in Japan. As successful propagation from seeds of this species has never been reported, a reproducible method is described here for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro. METHODS: Effects of hand-pollination and bagging treatment of ovaries were examined. Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds obtained from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined within TTC (2,3,5-triphenyl tetrazolium chloride) test solution and histological observations were made on viable seeds by paraffin embedding at each collection stage. KEY RESULTS AND CONCLUSIONS: Hand-pollination followed by bagging treatment of ovaries with aluminium foil was effective for insect control during fruit development, and successfully yielded capsules. Of the capsules, 74.5 % survived to full maturity. The highest frequency (39.8 %) of seed germination was obtained with seeds harvested 70 d after pollination. The frequency declined with progress of seed maturity on the mother plant. Minimal germination was observed with seeds harvested 100 d or later after pollination. Histological observation suggests that accumulation of such substances as lignin in the inner integument surrounding the embryo during seed maturation plays an important role in induction of dormancy.

Diacylated 8-C-glucosylcyanidin 3-glucoside from the flowers of Tricyrtis formosana.

A new diacylated 8-C-glucosylanthocyanin was isolated from the purple flowers of Tricyrtis formosana 'Fujimusume' as one of the major anthocyanins along with four known pigments. The structure of this pigment was determined to be 8-C-(6-O-trans-sinapoyl)-beta-glucopyranosylcyanidin 3-O-(6-O-malonyl-beta-glucopyranoside) by chemical and spectroscopic methods. In addition, four known pigments, 8-C-glucosylcyanidin 3-malonylglucoside, cyanidin 3-glucoside, cyanidin 3-rutinoside and cyanidin 3-malonylglucoside, were identified as the major anthocyanins in the flowers.