Introduction of asymbiotically propagated seedlings of <Emphasis Type="Italic">Cephalanthera falcata</Emphasis> (Orchidaceae) into natural habitat and investigation of colonized mycorrhizal fungi

Asymbiotic seedling propagation and introduction of seedlings into a natural habitat were achieved for Cephalanthera falcata. For immature seeds collected 65 days after pollination, high germination rate (av. 50%) was achieved on Hyponex agar medium plates. Root development occurred in about 10% of the protocorms 5 months after seed sowing. Rooted protocorms were transferred to a culture bottle containing 100 ml of the Hyponex agar medium and incubated continually. In about 30% of the transferred individuals, shoot height reached 1.5–2 cm 8 months after the transfer. After acclimatization in wet vermiculite at 4°C for 6 months, 135 individuals were planted in a natural stand of C. falcata in mid February 2001. Shoot appearance rate was 44.4% at the first year and flowering was noted in some plants. At the fifth year, shoots with an average height of 21.6 cm still appeared in four plants, and flowering was noted in three of them. Colonization of mycorrhizal fungi was examined in two of them as well as one wild plant, in which the mycorrhizal fungi were identified to be in Thelephoraceae or Russulaceae. These fungi are known to form ectomycorrhiza with trees, and thus a tripartnership symbiosis consisting of C. falcata, mycorrhizal fungi and trees was suggested. The involvement of ectomycorrhizal fungi might be the reason for the low survival rate in the field experiment, because the distribution of ectomycorrhizal fungi relevant to this orchid is assumed to be uneven. The possibility of introducing artificially propagated orchids into natural habitats was discussed.     

Adventitious rooting of conifers: influence of physical and chemical factors

In conifers, vegetative propagation of superior genotypes is the most direct means for making large genetic gains, because it allows a large proportion of genetic diversity to be captured in a single cycle of selection. There are two aims of vegetative propagation, namely large-scale multiplication of select genotypes and production of large numbers of plants from scarce and costly seed that originates from controlled seed orchard pollinations. This can be achieved, in some species, either through rooted cuttings or rooted microshoots, the latter regenerated through tissue culture in vitro. Thus far, both strategies have been used but often achieved limited success mainly because of difficult and inefficient rooting process. In this overview of technology, we focus on the progress in defining the physical and chemical factors that help the conifer cuttings and microshoots to develop adventitious roots. These factors include plant growth regulators, carbohydrates, light quality, temperature and rooting substrates/media as major variables for development of reliable adventitious rooting protocols for different conifer species.     

In vitro conservation of <Emphasis Type="Italic">Alectra chitrakutensis</Emphasis>: a critically endangered root parasitic plant of high medicinal importance

The present study provides an effective in vitro propagation method for critically endangered parasitic plant Alectra chitrakutensis (M.A. Rau) R. Prasad & R.D. Dixit (Scrophulariaceae). The plant comprises tremendous medicinal properties to treat many diseases such as leprosy, leucoderma, tuberculosis and paralysis. The protocol has been developed using rhizome explant on the modified Murashige and Skoog medium containing various phytohormones to raise complete plantlets without any host interaction. 0.5 mg/l kinetin and 1.0 mg/l NAA was found to be the most excellent medium combination to obtain plantlets with an average shoot length of 11 ± 1.04 cm; and 100% response for rooting. The same medium combination also supported flowering in vitro. Further, comparative molecular and chemical characterization of in vitro and in vivo grown plants were carried out by performing RAPD and HPTLC analysis, respectively, to make sure the genetic as well as chemical stability among the plants. RAPD analysis with MAP (01-20) and OPJ primers (01-20) exhibited a monomorphic band pattern which revealed the genetic similarities among the tested plants. Similarly, presence of azafrin, the most important pharmaceutically active compound in in vitro generated plants indicates the efficacy of protocol to fulfill the objective of in vitro propagation of this critically endangered plant species. Moreover, the present study is the first report towards in vitro conservation of parasitic plant A. chitrakutensis without host from the rhizome explant.     

Field performance of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. plants propagated via somatic embryogenesis

Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4–6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.     

The flowering pattern of the perennial herb Lobularia maritima: an unusual case in the Mediterranean basin

In plant communities of the Mediterranean Basin most plant species reach their blooming peak in spring and have characteristically short flowering periods of two-three months. The perennial herb Lobularia maritima represents an exception to these characteristics, because it flowers for almost 10 months, and has its flowering peak in autumn. In this five-year study, we describe the flowering pattern of L. maritima at the population and community levels. Despite the unusually extended flowering period of L. maritima, the species showed characteristic low among-year variability in the length of the flowering period but large interannual variation in the distribution of flowers throughout the flowering period. The flowering pattern (unimodal or bimodal) of L. maritima individuals differed among the five years, suggesting that L. maritima plants are plastic enough to tailor their flowering to variable environmental conditions. We conclude that flowering phenology of L. maritima represents a very particular case in the plant community studied, and the influence of abiotic and biotic factors on the phenology of this species is discussed.     

Clonal identification by microsatellite loci in sporadic flowering of a dwarf bamboo species, <Emphasis Type="Italic">Sasa cernua</Emphasis>

Dwarf bamboo species are monocarpic. They flower simultaneously and die after several decades. The type of flowering in the genus Sasa ranges from sporadic to gregarious. In order to determine whether or not the sporadic flowering of dwarf bamboo is fixed genetically, we investigated the distribution of clones using eight microsatellite (SSR) loci in a sporadic flowering patch of Sasa cernua Makino, a major dwarf bamboo species found in central Hokkaido. In May 2006, flowering occurred on 60.5% of living culms in a 1600 m² patch. We established a 50 × 10 m study plot in this patch and noted 1267 clumps consisting of 2529 living culms. We investigated all 1267 clumps and identified six multilocus genotypes as clones using five variable SSR loci. All flowering clumps belonged to the same clone. On the other hand, non-flowering vegetative clumps were also discovered to be of the same clone. These data suggest that all flowering culms originated from a single clone of a sporadic flowering patch of S. cernua. Clonal analysis for investigation of sporadic flowering of S. cernua revealed that only a portion of a clone flowers and dies instead of the whole clone.     

Rare nationwide synchronized massive flowering and decline event of <Emphasis Type="Italic">Sasa borealis</Emphasis> (Hack.) Makino in South Korea

Sasa borealis, a monocarpic species of dwarf bamboo, is widely distributed throughout Korea. It dominates forest floors, thereby inhibiting mainly the biodiversity. Although it flowers very rarely, examples have recently been observed in multiple locations, providing a good opportunity to study reproduction phenomena, and to aid in biodiversity restoration. Therefore, we investigated the nationwide timing of flowering events by using data collected from a social network service (SNS). We also more closely examined flowering and decline event, focusing at the patch and culm levels on Mt. Jeombong. We then analyzed the main factors affecting flowering. Our SNS and survey results showed that S. borealis is in a current flowering cycle that started in 2013 and continues to the present (83% of all events happening within this period) with a peak in 2015 (48% of the cases occurring in that year). This clearly demonstrated nationwide, synchronized, and massive flowering. Although the culm density in patches was not related to flowering, patches with large culms tended to flower (F = 8.241, p = 0.01). We suspected that this nationwide flowering event was triggered by prolonged drought during the spring months of 2014 and 2015 (F = 5.207, p < 0.05), which led to concurrent, massive flowering in patches mature enough to do so. Because this species prefers a wet habitat, we concluded that severe, prolonged drought induced environmental stress for those plants. After flowering, culms in those particular patches tended to die off within one year. This large-scale synchronized decline should have an enormous effect on the vegetation dynamics of a forest dominated and suppressed by Sasa. Future investigations might incorporate methods of ecological control and manipulation to increase biodiversity there.     

Correction to: Shoot regeneration process and optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Sinningia speciosa</Emphasis>

The objective of this research was to induce mitotic chromosome doubling in Anemone sylvestris L. The mitosis inhibitor oryzalin was directly added to the induction medium at 1, 2, 5, 10 and 15 μM for 8, 10 or 12 weeks of cultivation. Three tetraploid plants (2n?=?4x?=?32), 0.8% (polyploidization efficiency), were obtained from diploid plants (2n?=?2x?=?16) in three treatments (1 μM for 10 weeks, 5 μM for 8 weeks and 8 μM for 10 weeks). Ploidy level was confirmed by flow cytometry. Morphological characteristics (e.g. flower diameter, total plant height, leaf area) and chlorophyll content differences between diploid and tetraploid A. sylvestris were observed together with polyphenol content and antioxidant activity. The inter primer binding sites markers were used for evaluation of polymorphism. New genotypes with different morphological and biological characteristics were obtained through somatic polyploidization. The tetraploid plants were stronger, more vigorous and had an early flowering, which is essential for its use as an ornamental plant. The iPBS analysis showed unique amplicons that can be used for the purposes of molecular identification of tetraploid plants of A. sylvestris in the future. The results demonstrate the first report of in vitro induction of tetraploids of A. sylvestris.     

Transformation of<Emphasis Type="Italic"> Kalanchoe blossfeldiana</Emphasis> with <Emphasis Type="Italic">rol-</Emphasis>genes is useful in molecular breeding towards compact growth

Dwarf genotypes of the economically important flowering potted plant Kalanchoe blossfeldiana were developed by molecular breeding. Root inducing (Ri)-lines were regenerated by applying CPPU to the hairy roots, which were produced by inoculating leaf explants with a wild-type Agrobacterium rhizogenes strain ATCC15834. Amplification by polymerase chain reaction (PCR) and Southern blot analysis confirmed the presence of T-DNA in the Ri-lines. Six Ri-lines were characterised in a greenhouse trial revealing that several morphological traits changed with respect to ornamental value such as plant height, number of lateral shoots, leaf size, leaf number, flower size and number of flowers. The Ri-lines differed in their degree of Ri-phenotype, and the internodes of the Ri-lines were clearly shorter, giving a compact growth habit compared to control plants. Time to anthesis was the same in Ri-line 331 as in control plants and delayed by only 3 days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.     

Molecular and Functional Characterization of <Emphasis Type="Italic">ZmNF-YC14</Emphasis> in Transgenic Arabidopsis

The optimum transition stage from vegetative to reproductive development is important for flowering plants to obtain the desired plant architecture to maximize yield. In this study, we overexpressed the maize NUCLEAR FACTOR Y, SUBUNIT C 14 (ZmNF-YC14) gene in Arabidopsis to investigate its potential functions in the regulation of plant transition. Overexpression of ZmNF-YC14 in Arabidopsis inhibited plant flowering and retarded the duration of the branch-producing inflorescence phase 1 (I₁). These plants exhibited increased length of I₁ and higher ratio of inflorescence phase 1 to inflorescence phase (I) (I₁:I) under long-day conditions. As a consequence, these transgenic plants exhibited dramatic changes in their overall inflorescence morphology. In addition, the phenotypes of inflorescence morphology caused by ZmNF-YC14 overexpression were enhanced by exogenous gibberellin (GA) treatment, which obtained a significant increase in I₁:I, inflorescence phase 1 to inflorescence phase 2 (I₂) (I₁:I₂), and remarkable decrease in I₂ and I₂:I in transgenic plants compared to those under normal conditions. Taken together, the results of this study suggest that ZmNFYC14 negatively regulates flowering and controls flower formation under long-day conditions in Arabidopsis and may be involved in a GA regulation pathway.     

KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

KT/HAK/KUP potassium transporter protein-encoding genes constitute a large family in the plant kingdom. The KT/HAK/KUP family is important for various physiological processes of plant life. In this study, we identified 27 potential KT/HAK/KUP family genes in rice (Oryza sativa) by database searching. Analysis of these KT/HAK/KUP family members identified three conserved motifs with unknown functions, and 11-15 trans-membrane segments, most of which are conserved. A total of 144 putative cis-elements were found in the 2 kb upstream region of these genes, of which a Ca2+-responsive cis-element, two light-responsive cis-elements, and a circadian-regulated cis-element were identified in the majority of the members, suggesting regulation of these genes by these signals. A comprehensive expression analysis of these genes was performed using data from microarrays hybridized with RNA samples of 27 tissues covering the entire life cycle from three rice genotypes, Minghui 63, Zhenshan 97, and Shanyou 63. We identified preferential expression of two OsHAK genes in stamen at 1 day before flowering compared with all the other tissues. OsHAK genes were also found to be differentially upregulated or downregulated in rice seedlings subjected to treatments with three hormones. These results would be very useful for elucidating the roles of these genes in growth, development, and stress response of the rice plant.     

Expression of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> induces precocious flowering in soybean irrespective of maturity group and stem growth habit

The flowering integrator gene FLOWERING LOCUS T (FT) in Arabidopsis thaliana is conserved between diverse plant species and is thought to be the flowering signal ‘‘florigen’’, a universal long-distance mobile signal. In soybean, two FT homologs having a function to induce flowering in Arabidopsis have been identified. In this study, we showed that the expression of FT from Arabidopsis by the Apple latent spherical virus (ALSV) vector promoted precocious flowering and terminated vegetative growth in a wide range of genotypes of soybean, without using a short-day treatment. Four determinate and two indeterminate cultivars, infected with ALSV expressing FT (FT-ALSV), set flower buds on shoot apices and terminated vegetative growth at the fourth- to seventh-node stages under long-day conditions. In contrast, non-infected, healthy plants did not set flower buds on shoot apices at the same stage under the same conditions. After flowering, soybean cultivars infected with FT-ALSV, belonging to different maturity groups and stem growth habits, matured and produced seeds. The results suggest that the basic flowering pathway controlled by FT in A. thaliana might also be conserved in soybean. A system for precocious flowering and shortening of generation time using FT-ALSV would be a useful and novel technology for efficient soybean breeding.     

Somatic embryogenesis from mature <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxburgh as basis for mass production of elite forestry bamboos

A reliable protocol for mass propagation via somatic embryogenesis in mature bamboos has been established using pseudospikelets of Bambusa balcooa. Fourty percent of the explants gave rise to multiple regenerants within 4 months. This conversion rate is sufficiently high to use the process in commercial mass production. Further, shoot apical meristems can also be used as primary explants without lost of efficiency. Regenerated plants were uniform and identical to the mother plant and to plants obtained by axillary branching with respect to growth characteristics and morphology. Furthermore, epigenetic changes could not be detected by Methylation Sensitive AFLP (MSAP). During the complete process no changes in ploidy level could be observed. The process allows for a cost reduction for this tropical bamboo for forestry of up to 57% compared to micropropagation via axillary branching. For the first time, a reliable process based on somatic embryogenesis has been developed that is well suited for commercial micropropagation of elite mature bamboos.     

孝顺竹开花过程中DNA甲基化水平动态研究

以孝顺竹为材料,利用HPLC和MSAP技术在其未开花至开花的生长过程中进行甲基化水平的检测,分析竹子开花过程中基因组DNA甲基化的动态,以揭示DNA甲基化水平与竹子开花现象的相关性。结果显示:(1)孝顺竹基因组DNA甲基化率在不同的时间处于动态变化中,进入开花状态的孝顺竹植株其甲基化水平极显著低于开花竹丛中未开花植株和未开花竹丛;开花和未开花植株的总甲基化率分别为9.00%和12.42%,全甲基化率分别为5.06%和7.53%。(2)MSAP位点中有66.83%的位点在开花和未开花材料中甲基化状态保持一致,33.17%的位点在开花和未开花植株中发生甲基化变化:其中22.28%的位点在开花植株中发生完全的去甲基化,1.98%的谱带在开花植株中发生甲基化,8.91%的位点在开花和未开花材料中甲基化水平呈现上升或降低的趋势。研究表明,开花的孝顺竹同时发生甲基化和去甲基化,但发生去甲基化的概率明显大于发生甲基化的概率,最终导致其甲基化水平极显著降低。     

The role of abscisic acid and plant growth regulators in tissue culture-induced rejuvenation of strawberry ex vitro

Plant growth regulators applied in vitro affected strawberry plant performance ex vitro for a period of up to 4 months. Benzyl-adenine and gibberellin enhanced juvenile characteristics; in general, more runners and monofoliolate leaves were produced, net photosynthesis was reduced, leaf diffusive resistance was increased and, on occasion, flowering was delayed. In contrast, abscisic acid and a gibberellin biosynthesis inhibitor, paclobutrazol, resulted in a more adult phenotype; specifically, flowering was earlier, net photosynthesis and leaf diffusive resistance rates were equal to those of adult plants, and fewer leaves were monofoliolate. Tissue culture-produced plants reacquire the adult phenotype earlier than seedlings mature. Abscisic acid application to seedlings also resulted in earlier and increased flowering.Endogenous abscisic acid concentrations were greater in adult plants and less in tissue culture and seedling plants at 3 and 7 weeks ex vitro, after germination or after adult runner propagation. No flowering occurred at 3 weeks in any propagation type and only runner-produced plants flowered at 7 weeks. At 15 weeks, no statistical difference in abscisic acid concentrations existed among propagation treatments and all propagation types flowered. The endogenous concentrations of abscisic acid in plants propagated by all three methods were much higher at three weeks compared to concentrations at 7 or 15 weeks.Abbreviations ABA +cis, trans-abscisic acid - BA benzyl-adenine - GC-SIM-MS combined gas chromatography-selected ion monitoring mass spectrometry - GA gibberellin A₃ - RS leaf diffusive resistance in sec cm^-1 - MS Murashige & Skoog medium devoid of plant growth regulators - Pn net photosynthesis in mol CO₂ fixed m^-2 sec^-1 - paclo paclobutrazol as 50WP - TC-plants plants produced from tissue culture - Tween20 polyoxyethylene-sorbitan monolaurate - RP runner plants - SDLG selfed seedlings     

Gender-specific flowering responses to day length in the dioecious plant <Emphasis Type="Italic">Silene latifolia </Emphasis>at different temperatures

Silene latifolia Poiret is a reliable model system for studying many of the classical problems in biology because little difference in its physiological response to environmental stimuli has been found between genders. In this experiment, we studied flowering responses to day length in male and female S. latifolia plants grown at different temperatures. The primary objective was to examine whether flowering in male and female plants was differentially influenced by day length and temperature. We hypothesized that temperature would interact with day length in affecting flowering, because both genders have been found to flower in a wide range of day lengths and temperatures. Plants were grown in environmental chambers at a target temperature of 15/10 or 30/25°C (day/night). Day length was increased by half-hour intervals until all plants had flowered. Results showed that S. latifolia is a qualitative long-day plant with a critical day length of 11 h. Flowering accelerated with increasing day length until 16.5 h, when all plants flowered. Males and females differed in relative flowering at different day lengths, but to a greater extent at high than at low temperature. This study demonstrated that flowering of male and female S. latifolia respond differently to day length at different temperatures. A more uniform experimental protocol, especially in controlled environments, is called for to make this model system a more useful tool in biological studies.     

Experimental alteration of DNA methylation affects the phenotypic plasticity of ecologically relevant traits in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Heritable phenotypic variation in plants can be caused not only by underlying genetic differences, but also by variation in epigenetic modifications such as DNA methylation. However, we still know very little about how relevant such epigenetic variation is to the ecology and evolution of natural populations. We conducted a greenhouse experiment in which we treated a set of natural genotypes of Arabidopsis thaliana with the demethylating agent 5-azacytidine and examined the consequences of this treatment for plant traits and their phenotypic plasticity. Experimental demethylation strongly reduced the growth and fitness of plants and delayed their flowering, but the degree of this response varied significantly among genotypes. Differences in genotypes’ responses to demethylation were only weakly related to their genetic relatedness, which is consistent with the idea that natural epigenetic variation is independent of genetic variation. Demethylation also altered patterns of phenotypic plasticity, as well as the amount of phenotypic variation observed among plant individuals and genotype means. We have demonstrated that epigenetic variation can have a dramatic impact on ecologically important plant traits and their variability, as well as on the fitness of plants and their ecological interactions. Epigenetic variation may thus be an overlooked factor in the evolutionary ecology of plant populations.     

Doubled haploids in production of male sterility maintaining triticale (<Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) lines

The androgenetic response of several selected male sterility-maintainer genotypes of triticale was investigated. Androgenesis induction was obtained in all cultivars, but a large genotypic variation in green plant regeneration was observed. The number of regenerated triticale plants varied from 0.1 to 4.7 green plants per spike, depending on genotype. Spontaneous doubling of chromosomes varied from 14 to 60% for particular genotypes and, on average, reached the value of 34% for all genotypes. Fertile DH lines obtained in this study will find practical application in the development of triticale male sterile lines that are desirable in hybrid breeding.