Seiichi Fukai

CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums

Chrysanthemum is a typical short-day (SD) plant that responds to shortening daylength during the transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT)/Heading date 3a (Hd3a) plays a pivotal role in the induction of phase transition and is proposed to encode a florigen. Three FT-like genes were isolated from Chrysanthemum seticuspe (Maxim.) Hand.-Mazz. f. boreale (Makino) H. Ohashi & Yonek, a wild diploid chrysanthemum: CsFTL1, CsFTL2, and CsFTL3. The organ-specific expression patterns of the three genes were similar: they were all expressed mainly in the leaves. However, their response to daylength differed in that under SD (floral-inductive) conditions, the expression of CsFTL1 and CsFTL2 was down-regulated, whereas that of CsFTL3 was up-regulated. CsFTL3 had the potential to induce early flowering since its overexpression in chrysanthemum could induce flowering under non-inductive conditions. CsFTL3-dependent graft-transmissible signals partially substituted for SD stimuli in chrysanthemum. The CsFTL3 expression levels in the two C. seticuspe accessions that differed in their critical daylengths for flowering closely coincided with the flowering response. The CsFTL3 expression levels in the leaves were higher under floral-inductive photoperiods than under non-inductive conditions in both the accessions, with the induction of floral integrator and/or floral meristem identity genes occurring in the shoot apexes. Taken together, these results indicate that the gene product of CsFTL3 is a key regulator of photoperiodic flowering in chrysanthemums.

Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture

An efficient system for the in vitro plant and shootregeneration of Lilium longiflorum was developed andaccomplished using transverse thin cell layers (tTCL) of young stems.tTCLs were cut transversely along young stems from which the shoot-tipshad been removed. Sections were measured accurately using a graded gridand were cut in 4 mm × 4 mm × 1 mm cubes, eliminatingepidermal tissue, and were cultured on one-half MS medium containing 8 gl−1 agar, different sucrose concentrations (10, 20, 30 or 40g l−1), and with or without 1 mg l−1 activatedcharcoal (AC). Plants formed on the surface of tTCLs within 60 days onone-half MS medium containing 8 g l−1 agar and 20 gl−1 sucrose. Sections of 1 mm taken just below the apicalarea developed buds within 15 days, whereas the sections closer to thebase required about 45 days. Shoot regeneration was enhanced whensucrose concentration was used at 30 or 40 g l−1 after 60days of culture. No root formation occurred. Both shooting and rootingoccurred when sucrose was used at 20 g l−1. The plantletswere transferred to soil and grew well under greenhouseconditions.

The gated induction system of a systemic floral inhibitor, antiflorigen, determines obligate short-day flowering in chrysanthemums

Significance Photoperiodic floral initiation is thought to be regulated by a systemic flowering inducer (florigen) and inhibitor (antiflorigen) produced in the leaves. Here, we show the discovery of an antiflorigen (CsAFT) from chrysanthemum, which is produced in the leaves under a noninductive photoperiod to systemically inhibit flowering. This antiflorigen production system prevents precocious flowering and enables the year-round supply of marketable flowers by manipulation of day length. Photoperiodic floral induction has had a significant impact on the agricultural and horticultural industries. Changes in day length are perceived in leaves, which synthesize systemic flowering inducers (florigens) and inhibitors (antiflorigens) that determine floral initiation at the shoot apex. Recently, FLOWERING LOCUS T (FT) was found to be a florigen; however, the identity of the corresponding antiflorigen remains to be elucidated. Here, we report the identification of an antiflorigen gene, Anti-florigenic FT/TFL1 family protein (AFT), from a wild chrysanthemum (Chrysanthemum seticuspe) whose expression is mainly induced in leaves under noninductive conditions. Gain- and loss-of-function analyses demonstrated that CsAFT acts systemically to inhibit flowering and plays a predominant role in the obligate photoperiodic response. A transient gene expression assay indicated that CsAFT inhibits flowering by directly antagonizing the flower-inductive activity of CsFTL3, a C. seticuspe ortholog of FT, through interaction with CsFDL1, a basic leucine zipper (bZIP) transcription factor FD homolog of Arabidopsis. Induction of CsAFT was triggered by the coincidence of phytochrome signals with the photosensitive phase set by the dusk signal; flowering occurred only when night length exceeded the photosensitive phase for CsAFT induction. Thus, the gated antiflorigen production system, a phytochrome-mediated response to light, determines obligate photoperiodic flowering response in chrysanthemums, which enables their year-round commercial production by artificial lighting.

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum

Somatic embryogenesis was achieved directly from pseudo-bulblettransverse thin cell layers (tTCL) of Lilium longiflorum.Embryo-like structures (globular embryos) were obtained from different sizeexplants of pseudo-bulblet tTCLs after 45 days culture on Murashige and Skoog,1962 (MS) medium containing 5.4 μM naphthalene acetic acid(NAA)and 1.1 μM thidiazuron (TDZ). The embryo-like structures werethen isolated and mass proliferated on MS medium, containing 5.4μM NAA and 0.4 μM TDZ, every 45 days. A0.8–1.0 mm thick explant was shown to be optimal forobtaining the highest number of embryo-like structures. For plant regenerationthese structures were transferred to hormone-free MS medium with 30g/l sucrose. All of these structures formedplantlets after 90 days culture.

The chimeric structure of the apical dome of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam.) is affected by cryopreservation

Abstract The shoot tips of a periclinally chimeric cultivar of chrysanthemum ( Dendranthema grandiflorum (Ramat.) Kitam. cultivar ‘Apricot Marble’) were excised, loaded with cryoprotectants and cryopreserved. Amongst plants derived from the conventional shoot tip culture, 99% had normal flower color. In contrast, 70% of plants derived from cryopreserved shoot tips had a pink flower color instead of the original apricot. The pink petals contained anthocyanins in the epidermis but lacked carotenoids in the epidermis and mesophyll. These observations indicate that many of the shoots regenerated from thawed shoot tips were adventitious in origin and were derived from the epidermis of the shoot tip.

The effect of antibiotics on the in vitro growth response of chrysanthemum and tobacco stem transverse thin cell layers (tTCLs)

Abstract Plant tissue culture systems are always under threat of microbial contamination from the air, the tissue culturer, and from the tissue cultured plant itself, and antibiotics can be utilized to eliminate unwanted contaminants from tissue culture. In addition, the genetic transformation of plants requires selection and regeneration of transformed tissues through the use of an antibiotic-degrading gene. This study utilizes a wide range of antibiotic agents to examine their effect on chrysanthemum and tobacco thin cell layer (TCL) tissue culture systems, with an increasing gradient of phytotoxicity being observed: bialaphos®>chloramphenicol>rifampicin>streptomycin>minomycin>ampicillin>penicillin G=penicillin V, demonstrating that the limiting factor to the use of these agents is in fact the plant. Following the establishment of threshold survival levels, a decrease in explant survival and reduced biomass, malformation of roots and inhibition of shoot formation in chrysanthemum was observed, and further, in the case of tobacco endoreduplication.

Cryopreservation of shoot tips of Chrysanthemum morifolium and related species native to Japan

SummaryThe shoot tips of Chrysanthemum morifolium (syn. Dendranthema grandiflorum) and related species native to Japan were cryopreserved using preculture for 2 days, slow cooling (0.2°C/min) until −40°C with 10% dimethyl sulphoxide and 3% glucose prior to immersion into LN2 and rapid thawing. High survival rates were observed in 3 cultivars of chrysanthemum, 12 species and 2 interspecific hybrids, and slightly low survival rates in 3 species. The shoot regeneration rates of the frozen shoot tips varied from 9.4 to 100% depending on species. Shoot tips of chrysanthemum showed high viability even after a storage of 8 months in LN2. The thawed chrysanthemum shoot tips grew and flowered normally in a greenhouse under natural conditions.

Cryopreservation of chrysanthemum shoot tips

Abstract Shoot tips of chrysanthemum (Dendranthema grandiflorum (Ramt.) Kitam.) were precultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg 1−1 6-benzylaminopurine (BA), 1 mg 1−1 1-naphthaleneacetic acid (NAA), 2% sucrose and 5% dimethyl sulphoxide (DMSO) for 2 days, slowly cooled with a cryoprotectant solution (10% DMSO and 3% glucose) at a rate of 0.2°C min−1 from 0 to −40°C and then immersed and stored in liquid nitrogen. After thawing in warm water, more than 87% of the shoot tips survived and 47% of them regenerated shoots.

The changes in shoot regeneration potential of protocorm-like bodies derived from Lilium longiflorum young stem explants exposed to medium volume, pH, light intensity and sucrose concentration pretreatment

Summary An efficient system has been developed for the in vitro shoot regeneration of Lilium longiflorum Thunb. by culturing pretreated protocorm-like bodies (PLBs) derived from young stem transverse thin-cell layers (tTCLs) exposed to various treatments of different medium volumes, pH, light intensity and sucrose concentrations. After 30.d culture in 50 ml plastic petri dishes containing Murashige and Skoog medium supplemented with 0.2 mg l–1 thidiazuron (TDZ), the explants formed PLBs in all treatments. PLBs with the highest fresh weight were obtained after 30.d of culture when explants were cultured on MS medium containing 0.2 g l–1 TDZ with either 25 ml volume, pH 6.1, 20 μmol m–2 s–1 light intensity, or with 30 g l–1 sucrose supplement. However, most shoot formation from PLBs was found using MS hormone-free medium with either 30 ml volume, pH 5.0, 40 μmol m–2 s–1 light intensity, or with 20.g l–1 sucrose after 30.d of culture. The highest shoot regeneration of L. longiflorum was derived from PLBs when explants were exposed to various media, indicating that the choice of explants and pretreatment in the medium with optimum volume, pH, light intensity and sucrose concentreation are needed.

Effects of high temperature on flower colour and anthocyanin content in pink flower genotypes of greenhouse chrysanthemum (Chrysanthemum morifolium Ramat.)

Summary The relationship between the intensity of flower colour and changes in the content of the main anthocyanins under various controlled temperatures was examined in order to clarify the effects of high temperature on flower colouration in six pink flower genotypes of greenhouse chrysanthemum (Chrysanthemum morifolium Ramat.). Poor colouration of flowers was observed at 30°C in all genotypes except ‘Chatoo’. This genotype showed little difference in flower colour between different temperature treatments. The degree of change in flower colour differed depending on the genotype, whereas no clear differences in flower colouring were observed between Summer – Autumn flowering and Autumn-flowering genotypes. All genotypes showed lower contents of the two anthocyanins tested [cyanidin 3-O-(6’’-O-monomalonyl- -glucopyranoside) and cyanidin 3-O-(3’’,6’’-O-dimalonyl- -glucopyranoside)] at higher temperatures. Therefore, flower colour changes were attributable to changes in these two main anthocyanins. Differences in colouration between genotypes and temperature conditions were also detectable in values that were measured using a colorimeter. Changed parameters that were visually verifiable were the a* value, representing the degree of red colour, and the C* value, representing chroma. For ‘Sei-Monako’, which showed visually greater differences between temperature treatments, the a* and C* values were low under high temperature conditions. On the other hand, in ‘Chatoo’, the differences detected by eye and those in a* and C* values between temperature treatments were small. In addition, the present results indicate that mean temperature is more important than either day or night temperature in determining the degree of flower colouration.