三吉 一光

We studied the effects of various treatments given before or at inoculation with Agrobacterium tumefaciens strain EHA105 (pIG121Hm) on the transient GUS gene expression in leaf segments of Saintpaulia ionantha Wendl. Sonication and vacuum infiltration treatments in the presence of Agrobacterium had no positive effect on GUS expression. In contrast, the explants cultured for 3 to 5 weeks on shoot induction medium containing 0. 5 mgr1 NAA and 0.5 mg1-1 BA, prior to Agrobacterium inoculation markedly increased transient GUS expression. The addition of acetosy-ringone to the co-cultivation medium enhanced the GUS expression. A similar enhancing effect was observed in all of the 4 cultivars examined, suggesting the wide applicability of the pre-culture treatment. © 2002, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.

An albino mutant designated ali was isolated from an M2 population derived from a tobacco (Nicotiana tabacum L. 'BY-4') plant which had been irradiated with a 14N-ion beam at an early stage of embryonic development. Leaf mesophyll cells of ali were devoid of developed chloroplasts, and no stacked thylakoid membranes could be detected in the plastids. The reduction of pigment and the arrest of chloroplast development in ali were associated with distinct alterations in gene expression. Expression of plastid-encoded genes for the photosynthesis system (rbcL, psbA) was greatly reduced in ali, but at the transcript level plastid-encoded genes for 23S rDNA and 16S rDNA expression were only slightly reduced. Transcription of the nuclear-encoded Lhc and rbcS genes in the mutant was similar to that in the wild-type. There was no accumulation of Rubisco-L, Rubisco-S, and PSII-D1 polypeptides in the ali mutant. The number of chloroplasts per cell was similar in both ali and wild-type plants. These results suggest that ali is a novel albino mutant in which chloroplast gene expression is affected. © 2001 Annals of Botany Company.

Cypripedium macranthos is a wild orchid that is becoming endangered. Efficient methods for its propagation from seed, which is indispensable for conservation, production and breeding, have not been reported. The effects of sodium and calcium hypochlorite, pre-chilling and cytokinins on the germination of seeds of Cypripedium macranthos Swartz were examined. The duration of treatment with a solution of hypochlorine prior to sowing was one of the critical factors that affected germination. Approximately 70% of seeds that had been treated with either a solution of NaClO that contained 0.5% available chlorine for 60 min or with one of Ca(ClO)2 with 3.2% available chlorine for 7 h. germinated after months of culture at 20°c subsequent for 2 months chilling at 4°C. Chilling seeds at 4°C prior to culture at 20°C was another factor that stimulated germination. Even chilling for 2 weeks had a promotive effect on germination, and chilling for 2 months enhanced it most effectively: the frequency of germination was 67 after 3 months of culture at 20°C. However, the promotive effect of chilling on germination were reduced by holding seeds at 20°C for 3 and 6 weeks prior to chilling treatment. Germination of 58-70% was achieved by the addition of 1 μM cytokinin to the medium, while the frequency was only 17% in cytokinin free medium. We report a reproducible and efficient method for enhancing seed germination of C. macranthos which involves treatment with hypochlorite prior to sowing, and the combination of chilling at 4°C prior to germination and exposure to a cytokinin.

The effects of kinetin and gibberellin were examined under anaerobic conditions (0 % oxygen) and aerobic conditions (20 % oxygen) on the germination of dehusked seeds of indica and japonica rice cultivars that had been harvested at different times during the formation of seeds. Surjamkhi was used as a representative of deep dormant indica cultivars and Assam IV as a less dormant indica cultivar. Sasanishiki was used as the japonica rice cultivar. Both phytohormones were applied at a concentration of 10-3 M which proved to have the greatest stimulatory effect in preliminary work at concentrations of 10-3-10-5 M. Under aerobic conditions, inhibition of germination by dehusking of Sasanishiki seeds that had been harvested either 30 or 60 d after anthesis was overcome by kinetin and all seeds germinated. Complete germination induced by kinetin under aerobic conditions was also achieved with the dehusked seeds of the indica rice cultivar Assam IV that had been harvested on two occasions and of Surjamkhi that had been harvested 28 d after anthesis. In contrast, germination of dehusked japonica seeds stimulated by anaerobiosis was inhibited by kinetin. The stimulatory effects of gibberellin on the germination of indica and japonica rice seeds were observed under aerobic and anaerobic conditions. Under anaerobic conditions, the responses of dehusked indica and japonica rice seeds to kinetin and gibberellin differed, being negative with kinetin and positive with gibberellin. Under aerobic conditions, the stimulatory effects of kinetin on germination of dehusked seeds were greater than those of gibberellin. Thus, treatment with kinetin appears to be useful for breaking the considerable dormancy commonly observed in the dehusked seeds of indica rice. Mechanisms are proposed to explain the stimulatory effects of these phytohormones on the germination of dehusked seeds of indica and japonica rice under aerobic and anaerobic conditions.

An examination was made of the effects of ethanol at 0.2-6.0% (v/v) on the germination, under aerobic conditions, of intact and dehusked seeds of indica rice (cv. Assam IV), which had been harvested 14, 21 and 28 d after anthesis, and of the japonica rice (cv. Sasanishiki), which had been harvested 30 and 60d after anthesis. The inhibition of germination caused by dehusking japonica rice was overcome by 0.5-5% ethanol, with maximum germination (frequently 100%) achieved at 3-5% (30d after anthesis) or 1-4.5% (60d after anthesis) ethanol. Further increases in the ethanol concentration reduced germination. The germination of dehusked indica rice was slightly inhibited at 0.5 and 1% ethanol, whilst the promotion of germination by 2% ethanol increased as the seeds matured. At all harvests germination was greatest at 3% ethanol, and at 5-6% ethanol germination fell to 0%. Inhibition, no effect, or minimal stimulation of the germination of intact seeds of both japonica and indica rice by ethanol was observed at the concentrations examined. The absence of oxygen stimulated germination of dehusked japonica rice, but this germination was inhibited by ethanol. In contrast ethanol had little or no effect on the failure of dehusked indica seeds to germinate in anaerobic conditions. Thus ethanol treatment may help break the strong dormancy of dehusked seeds of indica and japonica rice. The possible role of ethanol in stimulating germination in rice is discussed.

The germination of intact, dehusked, and peeled seeds (caryopses) of the japonica rice cultivar Sasanishiki, harvested 30, 40, 47 and 60 days after anthesis, and of the indica rice cultivar Assam IV, harvested 14 and 28 days after anthesis, was examined. Dehusking strongly inhibited germination of Sasanishiki seeds, with the exception that seeds harvested 30 days after anthesis gave minimal germination percentages even when left intact. Peeling (removal of the pericarp and testa) restored or enhanced germination, and 60-100% of seeds germinated after 10 days. By contrast, the rank order of germination of Assam IV seeds was intact, dehusked, and peeled seeds, with peeled seeds yielding germination percentages of 100%. In Sasanishiki, inhibition of germination of peeled seeds was observed at reduced oxygen concentrations (1-4% oxygen). This inhibition might explain the inhibitory effects of dehusking on germination of seeds from the japonica cultivar. It is possible that the testa and pericarp, which cover the embryos of dehusked seeds, acted as a bather to the diffusion of oxygen to the embryo.

Differences in germination of intact and dehusked seeds of two japonica and two indica cultivars of rice (Oryza sativa L.) were examined during the development and maturation of seeds both under high-temperature (30/23 °C) and low-temperature (20/13 °C) regimes and a 14-h photoperiod in growth cabinets. As described previously for seeds grown in the paddy fields, germination of freshly harvested japonica rice seeds that developed and matured in growth cabinets was inhibited by dehusking. We observed a roughly consistent triphasic pattern with respect to the germination of intact and dehusked seeds during the development and maturation of seeds of indica and japonica rice. The triphasic pattern consisted of: (a) an initial phase, during which germination was stimulated by dehusking both in indica and japonica rice; (b) a second phase during which almost no dehusked seeds of indica or japonica rice germinated; and (c) a third phase during which intrinsic differences between indica and japonica rice were observed, with dehusking stimulating germination of indica rice but inhibiting that of japonica. Temperature regimes did not affect this triphasic pattern, but the time from the day of anthesis to mass maturity was affected by temperature regimes, and the effect was more pronounced in indica than in japonica rice.

Morphogenic calli were obtained efficiently from ab initio cultures of isolated microspores in eggplant. Initial culture of freshly isolated microspores in sucrose-free medium at high temperature (35°C) for 3 d was a prerequisite for callus induction. The microspores were re-cultured in modified NLN medium containing 2% sucrose and phytohormones (NAA 0.5 mg 1-1, BA 0.5 mg 1-1) in the dark. After 4 weeks of re-culture, small calli derived from microspores were transferred to MS medium containing 4 mg 1-1 zeatin and 0.2 mg 1-1 IAA for shoot regeneration. The ploidy of 12 randomly selected regenerants was assessed by chromosome counts in root tips. Only one of the regenerants was haploid, 7 were diploid, 3 were triploid and one was tetraploid. The diploids set seeds after self-pollination and showed no segregation for morphological traits in the progeny, suggesting that they were spontaneously doubled haploids.

Gynogenetic plants of pot gerbera (Gerbera jamesonii) were successfully produced from cultures of unpollinated ovules in vitro. Genotypic variations in the number of ovules that formed callus were found among the lines tested. One particularly responsive genotype was found among 17 genotypes tested where the frequency of callus-forming ovules was 17.5%. Four genotypes formed no callus at all. The frequency of shoot formation from the callus varied from 0-19.6% in nine genotypes. Ploidy was determined by flow cytometry, and 37 (80.4%) regenerants were haploid, seven (15.2%) were diploid, and two (4.3%) were mixoploid with both haploid and diploid cells. The doubling of chromosomes was achieved by treatment with 0.05% colchicine for 2-6 d in vitro, and 24.2-34.1% of treated haploid plants were found to have been diploidized.

Pre-soaking the seeds of Calanthe discolor Lindl. for 7 days in solutions containing α-naphthaleneacetic acid (NAA) at 1-100 mg l-1, 2-chloroethane phosphonic acid (Ethephon) at 1000 mg l-1 or 6-benzylaminopurine (BA) at 10, and 100 mg l-1 prior to germination in a growth-regulator-free medium resulted in overall 1.5-2.9 fold increases in protocorm formation compared with control cultures. Gibberellic acid (GA3) exerted no stimulatory effect on seed germination and protocorm formation. The possible mechanisms of seed germination and protocorm formation of this species are discussed. © 1995.

Mature seeds of Calanthe discolor were sterilized with 1% sodium hypochlorite solution and sown in liquid media after ultrasonic treatment. The seed coat was removed from almost all the seeds after 4 min of sonication treatment. However, prolonged treatment of more than 8 min increased the percentage of destroyed embryos. The rate of seed germination was significantly increased by the sonication treatment. Less than 10% of the seeds germinated in the control cultures, whereas 60% germinated in the cultures with sonication treatment for 4-16 min. © 1988.