Yoshiji Niimi

In vitro bulblet formation from leaf segments of lilies, especially Lilium rubellum Baker

Abstract Bulblets were induced from 5-mm lily leaf segments, cultured on Murashige and Skoogs mineral salts medium (1962) together with α-naphthalene acetic acid (NAA) 1 mg/l, 6-benzylaminopurine (BA) 0.1 mg/l and several organic elements. Bulblet formation was affected by several factors, especially the time of excision and the origin of the explant; bulblets developed well in leaf explants excised before flowering, and mainly in those excised within 15 mm of the leaf base. However, bulblets were seldom induced in explants excised after flowering, regardless of their origin. NAA was almost indispensable for bulblet formation, but BA had a slight stimulatory effect only when NAA was also present. Sucrose concentration and light also affected bulblet regeneration.

Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily ‘Connecticut King’

A system for producing haploid plants from anther cultures was developed for the Asiatic hybrid lily ‘Connecticut King’. Anthers containing microspores at the mid- to late-uninucleate stages were cultured on MS media supplemented with various plant growth regulators. Microspores containing 3 or 4 vegetative-like nuclei were observed 2 to 3 weeks later, and yellowish nodular calluses appeared within dehisced anthers 2 to 3 months after culture. Picloram was superior to 2,4-d for inducing nodular calluses. Anthers from greenhouse-grown plants required higher concentrations of both picloram and cytokinins than those from field-grown plants and most frequently produced nodular calluses (17.6%) on MS medium containing 2 mg 1−1 picloram and 2 mg 1−1 zeatin. The nodular calluses regenerated many bulblets following transfer to MS medium supplemented with 0.1 or 0.5 mg 1−1 picloram and 0.01 mg 1−1 BA, and the bulblets developed into plantlets (bulblets with scaly leaves and roots) after transfer to MS medium containing 0.1 mg 1−1 NAA. Chromosome counts of root-tip cells of 11 plantlets revealed that five were haploids (2n = 12), two diploids (2n = 24), and four mixoploid. This result suggests that at least some plantlets were of gametophytic origin.

Interspecific hybrids between Lilium nobilissimum and L. regale produced via ovules-with-placental-tissue culture

Reciprocal pollination was made between Lilium nobilissimum and L. regale. Pollen tubes reached the base of style within 144 h after pollination, but no mature seeds were obtained in either cross combination. Explants, or ovules-with-placental-tissue excised from each carpel 30 and 40 days after pollination (DAP), were cultured on a medium composed of major salts of B-5 macronutrient (Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Exp. Cell Res. 50, 151‐158), micronutrient, Fe-EDTA and vitamins of MS (Murashige, T., Skoog, F., 1962. Physiol. Plant. 15, 473‐497), 5% sucrose and 0.2% gellan gum. In L. regale Lilium nobilissimum, 3% of ovules excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none of the ovules excised 30 DAP produced any seedlings. Bulbs of L. regale and hybrids transplanted to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on leaves, but those of Lilium nobilissimum were sensitive to these diseases. Flowering individuals were nearly intermediate between parents in their morphological characteristics. All flowering individuals (2na 24 chromosomes) were identified as hybrids based on karyotype, isozyme and random amplified polymorphic DNA analyses. # 2000 Elsevier Science B.V. All rights reserved.

Bulblet-productivity of explants from scales, leaves, stems and teplas of Lilium rubellum Baker

Abstract The number of bulblets per cultured expiant was greatest in the expiants of leaves, i.e. 2.2 bulblets, followed in decreasing order by scale-, stem- and tepal-expiants. Bulblets formed in the expiants of stems were heaviest, 135 mg on average. The expiants of leaves were excellent for regenerating many bulblets per expiant, while those of stems produced the heaviest bulblets.

Detection of cucumber mosaic virus, lily symptomless virus and lily mottle virus in Lilium species by RT-PCR technique

Cucumber mosaic virus (CMV), lily symptomless virus (LSV) and lily mottle virus (LMoV) were detected in three lily cultivars by reverse transcription-polymerase chain reaction (RT-PCR). Results were compared with those obtained in an enzyme-linked immunosorbent assay (ELISA). The amplified DNA fragments with expected size and specific bands for each virus were obtained by RT-PCR using degenerate primers. In 18 out of 22 plants tested at least one virus was detected with RT-PCR in the 18 (81.8%) plants compared to 4 (18.2%) plants with ELISA. The present study showed that the RT-PCR is a more sensitive method than ELISA to determine the presence of viruses in Lilium plants of various species.

Production of triploid plants of Japanese pear (Pyrus pyrifolia Nakai) by anther culture

Triploid plants were grown from the diploid pear scion cultivar (Pyrus pyrifolia Nakai) by anther culture. Androgenic callogenesis occurred at about 10 days after anther excision, and most calli formed within four weeks and stopped growing at less than 1–3 mm in diameter. Androgenic embryogenesis occurred within 8–12 weeks after anther culture. The formed embryos were generally white, globular or heart-shaped. To increase callogenesis and embryogenesis, the effects of cold pre-treatment, light and activated charcoal (AC) on anthers collected from buds were investigated. Cold pre-treatment, dark and lack of AC increased callogenesis more than no cold pre-treatment, light and adding AC. A decisive condition was not found for embryogenesis. The embryogenesis frequency was extremely low and only six embryos were grown. Two embryos expanded greenish cotyledons and developed foliage, and they proliferated when transferred monthly to fresh proliferation medium. The proliferated shoots were rooted and acclimated. Flow cytometric analysis and investigation of the number of chromosomes of regenerants showed that all cells were triploid.

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35 °C for a few days and by then incubating them in the dark at 25 °C. Pre-culturing anthers at 35 °C for 4 days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15 °C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.

Influence of low and high temperatures on the initiation and the development of a bulb primordium in isolated tulip embryos

Abstract The influence of 4°C and 24°C on the germination and the subsequent development of isolated tulip embryos was studied, using embryo culture techniques. A low temperature not only influenced the breaking of the embryo dormancy, but also the initiation of a bulb primordium. High-temperature treatment after chilling accelerated the development of an initiated bulb primordium.

Distribution of indole-acetic acid, gibberellin and cytokinins in apoplast and symplast of parthenocarpic tomato fruits

The levels of indole-acetic acid (IAA), gibberellic acid1 (GA1), trans-zeatin (Z) and trans-zeatin riboside (ZR) in seedless fruits of parthenocarpic tomato (Lycopersicon esculentum Mill. cv. Rarkuna First) were analysed using 13C6-IAA, 2H2-GA1, 2H5-Z and 2H5-ZR, as internal standards by liquid chromatography–mass spectrometry. Fruits were sampled at 6 cm in diameter (referred to as 6-cm-fruit) and 8 cm (8-cm-fruit, mature green stage) and separated into pericarps, partitions and locule tissues. The pericarps and partitions were centrifuged for the collection of apoplast (AP) solution (sap outside a cell) and symplast (SP) solution (sap within a cell). IAA concentrations of the pericarps and partitions were higher in 8-cm-fruit than in 6-cm-fruit. In the partitions, IAA concentrations of SP solution were higher than those of AP solution in both 6- and 8-cm-fruit. The SP solution of the partitions in 6-cm-fruit had the highest concentration of Z (4.6 pmol/g fresh weight) and was 2.7 times than the AP solution, while in the pericarps Z concentrations were the same level in AP and SP solution. The ZR concentration in locule tissues in 6-cm-fruit (55 pmol/g fresh weight ) was the highest of all parts. The results suggest that the sites of synthesis may be the SP of partitions for IAA and Z, and locules for ZR.

Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures

Abstract The tulip embryo grows upward within the seed under low temperature at first and becomes nearly the same length as the seed after about 40 days of inoculation on an inorganic salt medium, but the distinct cell divisions of the undifferentiated vegetative apex can scarcely be ascertained during this period. After germination, the seedlings grow downward and the real divisions of the apex begin: at first, anticlinal or periclinal divisions take place actively in the deeper layers of the apex, followed by frequent anticlinal divisions in the peripheral layers as well as in the deeper layers. As a result, the vegetative apex can be detected distinctly within 70 days of inoculation as a mass of cells which becomes a slightly domed apex.