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Camellia tuyenquangensis (Theaceae), a new yellow species of camellia from the Chiem Hoa district of the Tuyen Quang province in Vietnam, is described and illustrated. Camellia tuyenquangensis is similar to Camellia luongii but different from the latter species in several morphological features: leaves oblong-ovate to narrow elliptic; flowers 1-2; bracteoles glabrous on both sides; sepal margins ciliate; style cleft one half of the length. The morphological data provisionally support its placement in Camellia sect. Chrysantha Chang.

The Ussurian pear is the most important cultivated pear in the northern part of China. Cultivated Ussurian pears are considered to have derived from Pyrus ussuriensis Maxim. which is native to the northeast of China. In Japan, two varieties of P. ussuriensis, P. ussuriensis var. aromatica and var. hondoensis are native to the northern area and the central area of the main island respectively. In order to reveal the origin of Pyrus ussuriensis var. aromatica distributed in the northern area of main island of Japan, more than 40 explorations have been performed in Japan and in China, and more than 30 natural habitats were recognized. These natural habitats are at risk of extinction because of human development and forest degradation caused by climate change. Population structure and genetic diversity of P. ussuriensis in China and P. ussuriensis var. aromatica in Japan have been investigated using both morphological and molecular markers in order to define appropriate conservation units, and to provide a good focus for conservation management. Distant evolutionary relationships between P. ussuriensis Maxim. in China and P. ussuriensis var. aromatica in Japan inferred from population genetic structure and phylogenetic analysis are also discussed.

Leaf litter respiration (R LL) was directly measured in situ to evaluate relationships with the water content in leaf litter (WC), which is distributed heterogeneously under natural conditions. To do so, we developed a small, closed static chamber system using an infrared gas analyzer, which can measure instantaneous R LL. This study focuses on the measurement of CO2 effluxes from leaf litter using the chamber system in the field and examines the relationship between R LL and WC among seven broadleaf species in a temperate forest. The measurements focused on the position of leaves within the litter layer, finding that both R LL and WC were significantly higher in the lower layer. The value of R LL increased with increasing WC, and the response of R LL to WC was similar among all seven species. Moreover, the temporal variation in WC differed among three species and was associated with leaf litter thickness. The observed heterogeneity in WC induced by the physical environment (e.g., position and thickness of leaf litter) affects the variation in WC and, therefore, both R LL and the decomposition rates of organic matter in the litter layer.

Summary Peace expression determined flower colouration whether pigmented or variegated. In spite of the close relationship between Peace and AtMYB123, a proanthocyanidin regulator, Peace regulates anthocyanin biosynthesis in flowering peach., Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis.

In order to understand the genetic diversity of wild Ussurian pears in China, chloroplast DNA (cpDNA) of 186 wild accessions from 12 populations in Inner Mongolia, Heilongjiang and Jilin Provinces and 51 Chinese and European pear cultivars including Pyrus ussuriensis, Pyrus pyrifolia, Pyrus bretschneideri, Pyrus sinkiangensis and Pyrus communis were investigated. Each accession was classified into one of three types (types A, B and C) based on two large deletions in the hypervariable regions between the accD–psaI and rps16–trnQ genes. Thirty haplotypes were identified by 32 mutations including 17 gaps (in/dels) and 15 base changes. Haplotype network analysis revealed that wild Chinese Ussurian pears could be grouped into subgroup I of type A. A haplotype, Hcp3, in subgroup I detected in Heilongjiang and Jilin Provinces was considered to be a divergent centre in Chinese Ussurian pears. However, the genetic diversity of wild accessions revealed by the two hypervariable regions was quite low. In particular, 98 % of wild Ussurian accessions in Inner Mongolia shared an identical haplotype Hcp1 and are, therefore, monomorphic. In comparison, Chinese pear cultivars were more divergent. These results suggest that the cpDNAs from wild Ussurian pears in Inner Mongolia have specifically differentiated compared to those from pears of other areas. The number of wild Ussurian pears has been decreasing because of desertification and land development, therefore conservation is needed.

By use of Bayesian statistical inference and allelic data for 18 microsatellite loci, we analyzed the genetic structure of Chinese, Korean, and Japanese pear cultivars and of native populations of Pyrus ussuriensis. Although Japanese pear cultivars had a simple genetic structure, Chinese and Korean pear cultivars were admixures of Japanese pear and native P. ussuriensis from the Asian continent. Genetic differentiation between groups of native populations and those of cultivars was high, but cultivars were not well differentiated from each other. Chinese and Korean cultivars, which have traditionally been classified as either P. ussuriensis, P. bretschneideri, or P. pyrifolia, were much closer to Japanese cultivars, which have traditionally been classified as P. pyrifolia, than to native P. ussuriensis. We propose a new classification of cultivars by using the Group concept in accordance with the International Nomenclature for Cultivated Plants, namely, the Pyrus Ussurian pear group, the Pyrus Chinese white pear group, the Pyrus Chinese sand pear group, and the Pyrus Japanese pear group.

The genome structure of pear chloroplast DNA (cpDNA) is extremely highly conserved in comparison with that of other angiosperms, and therefore, relatively few phylogenetic analyses for pear (Pyrus spp.) have been carried out using cpDNA as a marker. In this study, we identified two hypervariable regions in intergenic spacers of cpDNA from 21 species in Pyrus. One of these regions is 857 bp in length and lies between the accD-psaI genes, and the other is a 904-bp region between the rps16-trnQ genes. The mutation rate of gaps for the two regions was 10 and 26 times higher, respectively, than the base change rate. Twenty-five haplotypes were revealed among 21 species in Pyrus by 36 mutations found in the two regions. These included 27 gaps and 9 base changes but excluded cpSSRs. Phylogenetic relationships between the 25 haplotypes were generated by haplotype network analysis. The 25 haplotypes represented three groups (types A–C) with two large deletions, one 228 bp in length between the accD-psaI genes and the other 141 bp between the rps16-trnQ genes. Types A and B consisted mostly of pears native to East and South Asia. Type C contained mainly Pyrus communis and wild relatives native to Europe, West and Central Asia, Russia, and Africa. Type B might have diverged from primitives such as pea pears in type A. Phylogenetic utility of structural alterations (gaps) occurring in the hypervariable regions of Pyrus cpDNA is discussed.

Hybridization between native and cultivated species is a concern in conservation biology. However, detecting such hybridization and distinguishing true natives from prehistorically naturalized species based on phenotypic characteristics is difficult. Here, we report on introgression between native and prehistorically introduced pear (Pyrus) species in Northern Tohoku (northern end of Honshu Island), Japan. We analyzed 20 microsatellites in 226 wild, seemingly wild, or cultivated materials. Phenetic analysis showed that wild Japanese populations of P. ussuriensis var. ussuriensis in Northern Tohoku, previously considered true natives based on morphology and phytogeography, differed from those in continental Asia, confirming their nativeness. However, Bayesian inference of population structures showed that Japanese P. ussuriensis was genetically admixed with two genetic clusters: true native P. ussuriensis var. ussuriensis and prehistorically introduced P. pyrifolia. Even in the Kitakami Mountains, where true native populations of var. ussuriensis are believed to persist, most wild trees were at least somewhat admixed. Prehistorically introduced then naturalized plants are treated as natives in Japan’s conservation management, and some are considered endangered. However, introgression of prehistorically naturalized P. pyrifolia into threatened native P. ussuriensis var. ussuriensis has occurred. This paper examines the implications for conservation management.

Iwateyamanashi (Pyrus ussuriensis Maxim. var. aromatica (Nakai et Kikuchi) Rehd.) is one of the Pyrus species grown wild in Japan. The origin of the Japanese pear (P. pyrifolia) is uncertain but it has been suggested that Iwateyamanashi is the possible progenitor of the modern Japanese cultivar. During the last few decades, the number of Iwateyamanashi trees has been decreasing and therefore, conservation is urgently needed. After 13 explorations in the northern Tohoku region of Japan, 615 pear trees and 30 local names were recognized mainly in Iwate Prefecture. The center of the distribution seemed to be somewhere around Mt. Hayachine to the northern area of Kitakami highlands (from lat. 39 °20′ to 40 °10′N, and from long. 141 °20′ to 141 °50′E). Four morphological characters concerning fruit shape, measured for 85 trees, showed a wide range of continuous variation. For the skin colour of fruit, 51% of trees bore russet type fruits, 22% smooth and 27% intermediate ones. Most of the fruits had five loculi but Sanenashi fruits (seedless pear), one of the old cultivars, had three, and fruits of another two trees had four. More than 80% of trees tended to produce fruits with a calyx but some trees bore fruits without a calyx. These observations indicate a wide range of genetic diversity in Pyrus species which is caused, not only by high heterogeneity in Iwateyamanashi itself, but also by the coexistence of Iwateyamanashi, P. pyrifolia (Burm.) Nakai and hybrid progeny in this area. Already 250 trees have been conserved as genetic resources by grafting at Kobe University.

In order to understand the evolutionary aspects of the chloroplast DNA (cpDNA) structures in Rosaceous plants, a physical map of peach (Prunus persica cv. Hakuhou) cpDNA was constructed. Fourteen lambda phage clones which covered the entire sequence of the peach cpDNA were digested by restriction enzymes (SalI, XhoI, BamHI, SacI, and PstI) used singly or in combination. The molecular size of peach cpDNA was estimated to be about 152 kb. The gene order and contents were revealed to be equivalent to those of standard type of angiosperms by the localization of 31 genes on the physical map. Eighteen accessions from 14 Prunus species (P. persica, P. mira, P. davidiana, P. cerasis, P. cerasifera, P. domestica, P. insititia, P. spinosa, P. salicina, P. maritima, P. armeniaca, P. mume, P. tomentosa, P. zippeliana, and P. salicifolia) and one interspecific hybrid were used for the structural analysis of cpDNAs. Seventeen mutations (16 recognition site changes and one length mutation) were found in the cpDNA of these 18 accessions by RFLP analysis allowing a classification into 11 genome types. Although the base substitution rate in the recognition site (100p = 0.72) of cpDNA in Prunus was similar to that of other plants, i.e., Triticum-Aegilops, Brassica, and Pisum, it differed from Pyrus (100p = 0.15) in Rosaceae. Seven mutations including one length mutation were densely located within a region of about 9.1 kb which includes psbA and atpA in the left border of a large single-copy region of Prunus cpDNAs. The length mutation was detected only in P. persica and consisted of a 277 bp deletion which occurred in a spacer region between the trnS and trnG genes within the 9.1 kb region. Additional fragment length mutations (insertion/deletion), which were not detected by RFLP analysis, were revealed by PCR and sequence analyses in P. zippeliana and P. salicifolia. All of these length mutations occurred within the 9.1 kb region between psbA and atpA. This region could be an intra-molecular recombinational hotspot in Prunus species.

A physical map of chloroplast DNA (cpDNA) of pear [Pyrus ussuriensis var. hondoensis (Nakai et Kikuchi) Rehder] was constructed using five restriction enzymes, SalI, XhoI, BamHI, SacI and PstI. This information will make it possible to investigate the phylogenetic relationships between Pyrus species. Pear cpDNA was found to be a circular molecule with a total size of about 156 kb in which two inverted repeats of 24.8 kb divide the molecule into small (17 kb) and large (90 kb) single-copy regions. The endonuclease recognition sites in the physical map were determined by single and double digestion of 13 lambda phage clones which covered the entire sequence of the pear cpDNA. Twenty nine genes were localized on the physical map of the pear cpDNA. The structure of pear cpDNA was almost the same in terms of genome size and gene order as that of tobacco cpDNA. RFLP analysis was carried out on cpDNAs from five Pyrus species (Pyrus pyrifolia, Pyrus ussuriensis, Pyrus calleryana, Pyrus elaeagrifolia and Pyrus communis). Two mutations, a recognition-site mutation and a length mutation (deletion), were found only in the cpDNA of P. pyrifolia cultivars. These mutations were localized on the physical map of pear cpDNA. The number of mutations of cpDNA in Pyrus species are small in comparison with those of other angiosperms, suggesting a high degree of genome conservatism in Pyrus species.

Eugenia uniflora Linn. is one of the useful trees in tropical areas. In vitro propagation of E. uniflora was achieved by using newly elongating shoots as material. MS medium supplemented with 0.2mgl-1 of 6BA was suitable for shoot regeneration and proliferation. Shoot elongation, root induction and root elongation were successfully carried out by transplanting to 1/2 MSHF medium. By this method more than fifty thousand regenerated plants can be obtained from one stem segment within one year. In vitro propagation of E. uniflora would be applicable not only for nursery production, but also for the conservation of genetic resources.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.

Iwateyamanashi (Pyrus ussuriensis var. aromatica) is one of the Pyrus species which grows wild in Japan. The number of Iwateyamanashi trees has been decreasing, so conservation and evaluation is urgently needed. Over 500 accessions of Pyrus species collected from Iwate in northern Tohoku region are maintained at Kobe University as an Iwateyamanashi germplasm collection. In order to investigate the genetic diversity, five SSR (simple sequence repeat) markers, developed from Japanese and European pear were examined for 86 Pyrus individuals including 58 accessions from Iwate. These SSR loci could discriminate between all the Iwate accessions except for 10 that bear seedless fruit, as well as determine the genetic diversity in Iwateyamanashi germplasms. High levels of variation were detected in 41 alleles and the mean observed heterozygosity across 5 loci was 0.50 for the Iwate accessions. Seedless accessions sharing identical SSR genotype with the local pear variety “Iwatetanenashi” were supposed to have been propagated vegetatively via grafting. In an UPGMA phenogram, Japanese pear varieties (P. pyrifolia) were clustered into two groups with some Iwate accessions including seedless ones. Another 38 Iwate accessions were not clustered clearly, and there was no clear relationship between these accessions and geographical distribution or morphological characters. Allele frequency revealed that the Iwate accessions were genetically more divergent than the Japanese pear varieties. Most Japanese pears possessed a 219 bp deletion at a spacer region between the accD and psaI genes in the chloroplast DNA (cpDNA), but other Pyrus species and two Iwateyamanashi trees did not. In the Iwate accessions, 79.3% had a deletion type cpDNA and others had a standard type cpDNA without deletion. These results are indicative of the wide range of genetic diversity in the Iwate accessions which include Japanese pear varieties. A combination of SSR and cpDNA analyses revealed high heterogeneity in Iwateyamanashi and coexistence of Iwateyamanashi and hybrid progeny with P. pyrifolia. These could be reasons for the wide range of continuous morphological variation described previously.

Pyrus ussriensis Maxim. is native to the northern part of China, but whose habitats are currently being destroyed by environmental changes and human deforestation. An investigation of population structure and genetic diversity of wild Ussurian pear is a priority in order to acquire fundamental knowledge for conservation. A total of 153 individuals of wild Ussurian pear from the main habitats, Heilongjiang, Jilin, and Inner Mongolia in China, possessed low genetic diversity as a result of habitat fragmentation. The genetic diversity of the populations in Inner Mongolia and north east of Heilongjiang was especially low and there was the possibility of inbreeding. Wild Ussurian pears were divided into 5 groups based on the Bayesian clustering method using 20 nuclear SSRs (nSSRs) and 5 groups by haplotype distributions using 16 chloroplast SSRs (cpSSRs), and the populations in Inner Mongolia and north east of Heilongjiang represented unique genotypes. AMOVA indicated there was a 20.05% variation in nSSRs and a 44.40% variation in cpSSRs among populations. These values are relatively high when compared to those of other tree species. Haplotype E, positioned in the center of the cpSSR analysis network and showed the largest number of connections with other haplotypes, represented the most important haplotype. Inner Mongolia and the north east of Heilongjiang are two areas that need urgent conservation because of their genetic vulnerability and peculiarity. We determined 4 conservation units based on the clustering by nSSRs and cpSSRs, and geographic factor. This information is helpful in deciding the conservation strategies for wild Ussurian pear in China.

Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the β-glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25μg/ml kanamycin and 500μg/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.

Shoot tips 0.5mm long were taken from actively growing almond (Prunus amygdalus Batsch cv. hardy) shoots. These were cultured on the modified 1/2 MS medium (UEMATSU and AKIHAMA 1987) supplemented with 6-benzyladenin (6-BA) or N-(2-chloro-4-pyridyl)-N-phenylurea (4PU, Kyowahakko Kogyo K. K.). About 70 to 80% of shoots survived on the paper-wick liquid medium containing 2.0 or 4.0 mg/l 4PU. Growing shoots were transferred to the solid multiplying medium containing 2g/l gerlite and 0.5mg/l 6-BA. These shoots were subcultured on the same fresh medium at forty days intervals. After nine months of culture, the multiplication rate was 6.6-fold per L0rty days. The longest shoot reached to 3.8cm. Within a month after transferring to the rooting medium containing 0.5mg/l naphthalene aceticacid (NAA), root induction occurred.

Shoot tips of eight Prunus cultivars of peach, nectarine, sweetcherry and plum were cultured. Shoot tip of 0.5∼1.0mm taken from each dormant bud was placed on the paper-wick in the medium containing various kind of cytokinins. Survival rates were higher on the medium containing 2.0 mg/l N-(2-chloro-4-pyridyl)-N-phenyl-urea (4 PU, Kyowa Hakko Kogyo K.K.) than other medium containing 6-BA or kinetin. There were varietal differences among eight cultivars in survival rate and shoot growth. Among eight cultivars, three sweetcherry cultivars showed vigorous growth. When shoots were transferred to the multiplying medium with reduced cyto-kinin concentration of 0.5 mg/l 6-BA, 'Ooishiwasesumomo' and 'Taiyou', plum cultivars, multiplied 5.1 and 5.7 times respectively in one month. Roots were induced on the medium containing 0.5 mg/l 4PU. Acclimatized plants have been obtained in plum cultivar 'Taiyou'.