Norihiko Kaizuma

Effect of genotype on shoot regeneration from cotyledonary explants of rapeseed (Brassica napus L.)

SummaryAbility of shoot regeneration from cotyledonary explants of rapeseed (B. napus) was surveyed for 100 cultivars. Effects of explant age and plant growth regulators were determined before screening the genotypes. The optimal condition required 4-day-old cotyledons as explant and 4.0 mg/l benzylaminopurine as plant growth regulator. Gas-permeable tape as sealing material was more effective for shoot regeneration than Parafilm. When testing cultivars, shoot regeneration response was strongly influenced by genotype with a range of variation from 97% (percentage of explants regenerating shoots) in ‘Arabella’ and ‘Norin 26’ to 0% in ‘Norin 18’ and ‘Norin 30’. The response was not dependent on origin and cropping type (spring vs. winter type). The ability of shoot regeneration was not related to that of microspore embryogenesis. The regenerants rooted on medium containing 2.0 mg/l indole-3-butyric acid and after planting in soil flowered and set seeds. Histological studies showed that shoot meristems developed in callus which had grown from the vascular bundle tissue within 8 days.

Scanning electron microscopy of microspore embryogenesis in Brassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.

Microspore culture of radish (Raphanus sativus L.): influence of genotype and culture conditions on embryogenesis

A number of factors influencing embryogenesis from isolated microspores of radish (Raphanus sativus) were examined. Of 11 genotypes evaluated, six produced embryos ranging from 8.3 embryos per 105 microspores for ‘Chugoku-ao’ to 0.2 for ‘Tenshun’, but five genotypes were not responsive. An initial culture period at elevated temperature before incubation at 25°C was essential for induction of microspore embryogenesis. However, the optimum period of the treatment varied among genotypes and/or experiments. Bud size also influenced microspore embryogenesis. Though optimum bud size was different between genotypes, the microspore populations represented in these buds contained uninucleate and binucleate microspores. Selection of embryogenic microspores using percoll density gradient resulted in up to 1.3-fold increase of embryo yield. Though almost all embryos failed to develop directly into plantlets, plants were obtained by multiple subcultures. The regenerated plants had hyperploid chromosome numbers.

Scanning electron microscopy of desiccation-tolerant and -sensitive microspore-derived embryos of Brassica napus L.

Abstract Desiccation tolerance can be induced in microspore-derived embryos of Brassica napus L. by application of abscisic acid (ABA). Scanning electron microscopy was employed to study and compare desiccation-tolerant and -sensitive microspore-derived embryos. The external surface of those desiccated embryos in which desiccation tolerance had been induced was uniformly shriveled due to severe dehydration, and their internal tissue system was well preserved. In contrast, in desiccation-sensitive embryos, dehydration caused tearing of the epidermis and collapse of the internal tissue system. After the desiccated embryos had been rehydrated, the size and external morphology of the desiccation-tolerant ones recovered to the pre-desiccation state within 1 day, whereas the sensitive ones did not recover or remained shriveled. The effect of ABA on the induction of desiccation tolerance is discussed.

Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor : detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F2 seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Tibi5 for this novel variant. The sequence analysis of Tibi5 revealed that six nucleotides were different between Tibi5 and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr→Phe), 74 (Ser→Arg), 114 (Met→Val), 120 (Leu→Ile) and 137 (Pro→Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tibi5 is an intermediate between Tia and Tib types. Tibi5 type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tibi5 type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.