Kenji Takayanagi

Phenotypic variation and the relationships among mustard (Brassica juncea L.) germplasm from Pakistan

An investigation was conducted to determine the extent of diversity and relationships among the Brassica juncea germplasm from Pakistan using morphological characters. A total of 52 accessions, including the collected germplasm as well as commercial cultivars/improved lines, were studied under field conditions at Tsukuba, Japan during 1995 and 1996. All the accessions were characterized for 35 agro-morphological characters from seedling emergence to crop maturity. Morphological data were analyzed by numerical taxonomic techniques using two complementary procedures: cluster and principal component analyses. Phenograms based on Euclidean distance placed the accessions into six groups during both years. Landrace groups were primarily associated with morphological differences among the accessions and secondarily with the breeding objectives and horticultural uses. The mustard germplasm collected from Pakistan showed a comparatively low level of phenotypic variation amongst themselves and were genetically similar to the oilseed cultivars. However, the oilseed forms and vegetable cultivars were genetically distinct. This study revealed that the evaluated germplasm appears to have a narrow genetic base which undergoes a high level of genetic erosion. This is perhaps due to the use of the same ancestors in the selection of new lines for similar horticultural traits, replacement by major crops and changes in agricultural land uses.

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

Abstract Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis.

Transformation of Antirrhinum majus L. by a rol-type multi-auto-transformation (MAT) vector system

A total of 11 independent beta-glucuronidase (GUS) positive hairy roots were induced following co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing rol-type multi-auto-transformation (MAT) vector pNPI702. A total of 326 adventitious shoots were regenerated from the hairy root lines on 1/2 MS medium without plant growth regulators at 25 degrees C under a 16 h/day photoperiod condition 4 months after infection of the A. tumefaciens GV2260. The absence of the rol genes in five plants was verified by polymerase chain reaction (PCR) and Southern blot analysis. Acclimatized transformants exhibited normal phenotypes in height and in the morphology of leaves and flowers. Furthermore, the GUS gene was strongly expressed in the leaves, inflorescence of the transformed plant, and the progeny. This result demonstrates that the rol-type MAT vector can be used to study gene functions controlling the morphogenesis of Antirrhinum majus plants.

Genetic variation in radish (Raphanus sativus L.) germplasm from Pakistan using morphological traits and RAPDs

Genetic diversity of 30 radish (Raphanus sativus L.) accessions was investigated at the phenotypic level with morphological characters and at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Thirty-six morpho-physiological traits were recorded from seedling stage to harvest. The 31 primers used generated 202 RAPD bands, of which 158 (78.2%) were polymorphic. Multivariate procedures were used to classify the germplasm on the basis of phenotypic traits and RAPD fragments. Dendrograms were generated for the Euclidean distance from the morphological data and the Neis genetic distance from the RAPD markers. Phenotypically, all the accessions were classified into four major groups corresponding to the different forms of cultivated radish. The morphological diversity existing within each of these groups suggested that they should be discriminated into the three botanical convarieties, sativusT (large-rooted), caudatus (pod-type) and oleifer (oilseed-type). Clustering of the accessions did not show any pattern of association between the morphological characters and the collection sites. Instead, landrace groups were associated with their morphological similarities and horticultural uses. On the other hand, the intra-specific genetic relationships of several accessions based on RAPD analysis were related primarily to their collection sites rather than to their phenotypic affinities. The level of polymorphism exhibited by the various convarieties could be exploited in genetic mapping populations to tag economically important traits. These genotypes also could serve as a useful germplasm source for root, leaf, pod and seed. This preliminary study of traditional radish landraces from Pakistan provides useful information regarding their horticultural potential.

Introgressive hybridization between Rhododendron kiusianum and R. kaempferi (Ericaceae) in Kyushu, Japan based on chloroplast DNA markers

Wild evergreen azalea populations of Rhododendron kiusianum and R. kaempferi ( Ericaceae ) were analysed using a chloroplast DNA (cpDNA) PCR-RFLP marker that was used to detect introgressive hybridization in our previous study of the Kirishima Mts populations. The populations of the intermediate region in the Unzen Mts, which show phenotypic variation, were demonstrated to result from interspecific hybridization between Rhododendron kiusianum and R. kaempferi , possessing cpDNA from either R. kiusianum (1030/420 bp) or R. kaempferi (950/420/80 bp). Most individuals of Rhododendron kiusianum in the Kujyu Mts, the Aso Mts and the surrounding mountains exhibited the PCR-RFLP pattern of R. kaempferi . These results from the Kujyu Mts and the Aso Mts indicate that natural hybridization and cytoplasmic introgression from Rhododendron kaempferi to R. kiusianum have occurred in the relatively distant past. In the case of Mt Yufudake and Mt Haneyama, the Rhododendron kiusianum population retains the effects of natural hybridization with R. kaempferi in the cpDNA as well as in the variation in flower characteristics. All individuals of Rhododendron kiusianum on Mt Onogaradake in the Takakuma Mts exhibit R. kiusianum cpDNA (1030/420 bp), in spite of variation in flower colour.

High frequency of shoot regeneration from hypocotyls and stem segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l−1 NAA + 2 mg l−1 Z and 0.5 mg l−1 IAA + 2 mg l−1 Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l−1 NAA + 2 mg l−1 Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.

Regeneration of Plants from Protoplasts of Butterbur (Petasites japonicus Fr. Schmidt)

Japanese butterbur (Fuki) is a perennial composite and distributed throughout Japan, Korea and China. Several species related to this plant, e.g. Petasites fragrans Presl, are distributed in Europe (Shibata and Shimizu 1978). In Japan, this plants is widely distributed from Hokkaido to Kyushu, and has been cultivated since the tenth century for its succulent petioles as vegetables (Fig. 1). The flower stalk of the butterbur (Fukino-tou) is highly valued as one of the seasonal foods which heralds the arrival of early spring.