Evergreen Azaleas Research Articles

Evaluation of root-associated fungi of evergreen azalea Rhododendron ripense Makino in its Japanese native habitat

Evaluation of root-associated fungi of evergreen azalea <i>Rhododendron ripense</i> Makino in its Japanese native habitat

High-quality evergreen azalea genome reveals tandem duplication-facilitated low-altitude adaptability and floral scent evolution.

SummaryAzalea belongs to Rhododendron, which is one of the largest genera of flowering plants and is well known for the diversity and beauty in its more than 1000 woody species. Rhododendron contains two distinct groups: the most high‐altitude and a few low‐altitude species; however, the former group is difficult to be domesticated for urban landscaping, and their evolution and adaptation are little known. Rhododendron ovatum has broad adaptation in low‐altitude regions but possesses evergreen characteristics like high‐altitude species, and it has floral fragrance that is deficient in most cultivars. Here we report the chromosome‐level genome assembly of R. ovatum, which has a total length of 549 Mb with scaffold N50 of 41 Mb and contains 41 264 predicted genes. Genomic micro‐evolutionary analysis of R. ovatum in comparison with two high‐altitude Rhododendron species indicated that the expansion genes in R. ovatum were significantly enriched in defence responses, which may account for its adaptability in low altitudes. The R. ovatum genome contains much more terpene synthase genes (TPSs) compared with the species that lost floral fragrance. The subfamily b members of TPS are involved in the synthesis of sesquiterpenes as well as monoterpenes and play a major role in flora scent biosynthesis and defence responses. Tandem duplication is the primary force driving expansion of defence‐responsive genes for extensive adaptability to the low‐altitude environments. The R. ovatum genome provides insights into low‐altitude adaptation and gain or loss of floral fragrance for Rhododendron species, which are valuable for alpine plant domestication and floral scent breeding.

Whole-genome sequencing and analysis of two azaleas, Rhododendron ripense and Rhododendron kiyosumense.

To enhance the genomics and genetics of azalea, the whole-genome sequences of two species of Rhododendron were determined and analysed in this study: Rhododendron ripense, the cytoplasmic donor and ancestral species of large-flowered and evergreen azalea cultivars; and Rhododendron kiyosumense, a native of Chiba prefecture (Japan) seldomly bred and cultivated. A chromosome-level genome sequence assembly of R. ripense was constructed by single-molecule real-time sequencing and genetic mapping, while the genome sequence of R. kiyosumense was assembled using the single-tube long fragment read sequencing technology. The R. ripense genome assembly contained 319 contigs (506.7 Mb; N50 length: 2.5 Mb) and was assigned to the genetic map to establish 13 pseudomolecule sequences. On the other hand, the genome of R. kiyosumense was assembled into 32,308 contigs (601.9 Mb; N50 length: 245.7 kb). A total of 34,606 genes were predicted in the R. ripense genome, while 35,785 flower and 48,041 leaf transcript isoforms were identified in R. kiyosumense through Iso-Seq analysis. Overall, the genome sequence information generated in this study enhances our understanding of genome evolution in the Ericales and reveals the phylogenetic relationship of closely related species. This information will also facilitate the development of phenotypically attractive azalea cultivars.

Application of Mutant &lt;i&gt;ap3&lt;/i&gt; Allele-based Markers for the Selection of the Long-lasting Flower Phenotype (Misome-shō) in Evergreen Azalea Cultivars

Application of Mutant &lt;i&gt;ap3&lt;/i&gt; Allele-based Markers for the Selection of the Long-lasting Flower Phenotype (Misome-shō) in Evergreen Azalea Cultivars

Humic Acid and Auxins Induced Metabolic Changes and Differential Gene Expression during Adventitious Root Development in Azalea Microshoots

Auxins and humic acid (HA) were investigated for their roles in adventitious root induction in azalea microshoots in our previous study. To reveal the regulatory mechanisms of auxins and HA in this process, measurements of the levels of reactive oxygen species (ROS), carbohydrates, and phenolic compounds and gene expression during in vitro root development were performed. During the adventitious root induction process, ROS levels in shoots treated with auxins and HA increased compared to untreated shoots, especially during the earliest period after transfer to the media. Media supplemented with NAA experienced increases in H2O2 contents by 480%and 250%, respectively, after 7 and 14 days of culture. The phenolic compound levels were also enhanced in the shoots treated with auxins and HA, reflecting the different rooting-promoting abilities of both auxins and HA. The highest levels of total phenolic [68.6 mg·g−1 fresh weight (FW)], polyphenolic acids (121.72 μg caffic acid/g FW), and total flavonols (162.42 μg quercetin/g FW) were recorded after 21 days for NAA media, but the maximum levels of anthocyanins (49.76 μg cyanindin/g FW) were recorded after 21 days for IBA medium. Soluble carbohydrate, starch, and soluble protein levels were increased in the shoots treated with all treatments; however, the influence of NAA treatments was stronger than that of other treatments for most investigated parameters. The NAA significantly enhanced soluble carbohydrates by 30%, 37%, and 25%, respectively, at 14, 21, and 28 days compared with untreated microshoots. Expression of the POD1 gene increased in the shoots submitted to HA treatment media. Expression levels of auxin response factors (ARFs) increased with IBA- and NAA-treated explants, suggesting that ARFs may have diverse regulatory roles in adventitious root induction in evergreen azalea. Moreover, the profiles of the IAA1, IAA9, IAA14, and IAA27 transcripts were analyzed to reveal their roles in the adventitious rooting of evergreen azalea microshoots. These results indicate that auxins and HA promote adventitious root induction in Rhododendron plants through their impact on ROS, carbohydrate contents, phenolic compound levels, and expression levels of different genes related to root development in evergreen azalea plants.

Correction to: Inheritance of the narrow leaf mutation in traditional Japanese evergreen azaleas

This article has been published with an erroneous version of Table 2. Please find the correct Table 2 in this document.

Long-lasting Corolla Cultivars in Japanese Azaleas: A Mutant AP3/DEF Homolog Identified in Traditional Azalea Cultivars from More Than 300 Years Ago.

Floral shape in higher plants typically requires genetic regulation through MADS transcription factors. In Japan, hundreds of azalea cultivars including flower shape mutations have been selected from the diversity of endogenous species and natural hybrids since the early 17th century, the Edo era (1603–1867). The long-lasting trait, known as “Misome-shō” in Japanese, has been identified in several species and cultivar groups of evergreen azaleas (Rhododendron L.) from three hundred years ago in Japan. However, the natural mutation conferring the long-lasting trait in azalea remains unknown. Here, we showed MADS-box gene mutations in long-lasting flowers, R. kaempferi ‘Nikkō-misome,’ R. macrosepalum ‘Kochō-zoroi,’ R. indicum ‘Chōjyu-hō,’ and R. × hannoense ‘Amagi-beni-chōjyu.’ All of the long-lasting flowers exhibited small-sized corollas with stomata during long blooming. In the long-lasting flowers, transcript of the APETALA3 (AP3)/DEFICIENS (DEF) homolog was reduced, and an LTR-retrotransposon was independently inserted into exons 1, 2, and 7 or an unknown sequence in exon 1 in gDNA of each cultivar. This insertion apparently abolished the normal mRNA sequence of the AP3/DEF homolog in long-lasting flowers. Also, long-lasting flowers were shown from F2 hybrids that had homozygous ap3/def alleles. Therefore, we concluded that the loss of function of the AP3/DEF homolog through a transposable element insertion may confer a stable long-lasting mutation in evergreen azaleas.

The effect of humic acid on endogenous hormone levels and antioxidant enzyme activity during in vitro rooting of evergreen azalea

Abstract It is important to produce roots in vitro for woody plants, such as azaleas. The influence of humic acid (HA) on histological development, antioxidant enzyme changes and endogenous hormone levels was evaluated during adventitious root formation in evergreen azalea microshoots. Explants (microshoots) were transferred to Anderson rooting medium supplemented with HA at 0, 0.5, 1, 2 and 5 mg L−1 for 56 days. HA at 1 and 2 mg L−1 improved the morphological root character of microshoots, such as root length, root number and rooting percentage compared with other treatments. The data collected during anatomical evaluation indicated that the cell division occurred on the third day of culture in the phloem adjacent to the cambium, which led to differentiation of the root primordium after ten days. Both treatments of HA (1 and 2 mg L−1) increased the endogenous hormone levels of indole-3-acetic acid (IAA) and gibberellic acid (GA) in rooted shoots, especially at the first period of root development. However, the increase of zeatin riboside (ZR) and isopentenyl adenosine (iPA) levels was shown during the in vitro rooting process. Moreover, HA contributed to higher activities in peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total soluble protein compared with other treatments at the same concentration. The results demonstrated that HA is effective for rooting in evergreen azaleas, and this effect was related to physiological and metabolic changes during adventitious root formation. Therefore, this information could help in developing a new type of rooting stimulator to reduce the high cost of plant growth hormones that are used for micropropagation.

Adaptation to iron deficiency and high pH in evergreen azaleas (Rhododendron spp.): potential resources for breeding

The growth of evergreen azaleas (Rhododendron spp.) can be altered by iron (Fe) chlorosis when plants are cultivated in a neutral-alkaline substrate. In this study, morphological and physiological responses to alkalinity and Fe deficiency were evaluated in five diploid Japanese azaleas to assess their potential as resources for breeding. R. obtusum ‘Kirin’, R. indicum ‘Shinsen’, R. × pulchrum ‘Sen-e-oomurasaki’, R. indicum ‘Osakazuki’, and R. ripense were pot cultivated in a peat-based substrate for 10 weeks, in acid and alkaline growing media with both adequate and inadequate Fe nutrition. Plant performance was generally affected by high pH of the substrate, while Fe deficiency by itself influenced few of the evaluated parameters, possibly due to the complex adaptive response mechanisms of these slow growing ornamental shrubs. According to the biochemical and physiological variations recorded on a long period of cultivation, R. indicum ‘Osakazuki’ reported the best performance. This azalea could be a valuable resource for breeders.

Floral Morphology and MADS Gene Expression in Double-flowered Japanese Evergreen Azalea

Floral Morphology and MADS Gene Expression in Double-flowered Japanese Evergreen Azalea

Effect of anthocyanin profile and petal pH on flower coloration in evergreen azalea

Effect of anthocyanin profile and petal pH on flower coloration in evergreen azalea

Contribution made by the carotenoid cleavage dioxygenase 4 gene to yellow colour fade in azalea petals

Carotenoid content, composition, and the expression patterns of carotenoid biosynthesis and cleavage genes during petal development were compared among a yellow-flowered deciduous azalea (Rhododendron japonicum f. flavum), a white-flowered evergreen azalea (‘Miyamasatsuki’), and their progeny, to determine the factors that cause reduction in carotenoid content as the petals of the progeny develop. During the early, green petal flowering stage, total carotenoid contents were 31.27 μg g−1 Fresh Weight (F. W.) in R. japonicum f. flavum, 17.84 μg g−1 F. W. in ‘Miyamasatsuki’, and 42.18 μg g−1 F. W. in their progeny. During subsequent flower development, total carotenoid contents remained similar to the green petal stage for R. japonicum f. flavum. However, the content decreased in ‘Miyamasatsuki’ and their progeny at one day before anthesis, and became less than 3 μg g−1 F. W. during the later stages. The expression levels of PSY and PDS increased significantly in R. japonicum f. flavum than in ‘Miyamasatsuki’ as the flowers developed. Their expressions in the progeny were mid-way between both parents. The expression level of CCD4 was significantly higher in ‘Miyamasatsuki’ and the progeny than in R. japonicum f. flavum for all development stages. This result suggested that the high expression level of CCD4, which was inherited from ‘Miyamasatsuki’, was the main factor controlling the reduction in carotenoid content in the progeny.

Hydroponic Screening for Iron Deficiency Tolerance in Evergreen Azaleas

Evergreen azaleas grow in acid soil and suffer from iron deficiency when cultivated in substrate with pH higher than 6.0. In order to select tolerant plants, 11 azalea genotypes were tested for 21 days in alkaline solution (pH 9), buffered with sodium hydrogen carbonate (1 g·l-1). Leaf damage, root length and mortality rate were recorded. While leaf damage and mortality rate allowed to discriminate genotypes, root development appeared not directly linked to iron deficiency tolerance. Rhododendron ‘Juko’, R. scabrum, R. macrosepalum ‘Hanaguruma’, R. x pulchrum ‘Oomurasaki’, and R. x pulchrum ‘Sen-e-oomurasaki’ resulted iron efficient genetic resources, useful for azalea cultivation and gardening in calcareous soils. On the contrary, R. obtusum ‘Kirin’, R. tosaense, R.x mucronatum ‘Fujimanyo’ and R. obtusum ‘Susogo-no-ito’ resulted iron deficiency sensitive genotypes. R. x mucronatum ‘Ryukyushibori’ and R. indicum ‘Kinsai’ showed intermediate responses.

Hydroponic Screening for Iron Deficiency Tolerance in Evergreen Azaleas

Evergreen azaleas grow in acid soil and suffer from iron deficiency when cultivated in substrate with pH higher than 6.0. In order to select tolerant plants, 11 azalea genotypes were tested for 21 days in alkaline solution (pH 9), buffered with sodium hydrogen carbonate (1 g·l-1). Leaf damage, root length and mortality rate were recorded. While leaf damage and mortality rate allowed to discriminate genotypes, root development appeared not directly linked to iron deficiency tolerance. Rhododendron ‘Juko’, R. scabrum, R. macrosepalum ‘Hanaguruma’, R. x pulchrum ‘Oomurasaki’, and R. x pulchrum ‘Sen-e-oomurasaki’ resulted iron efficient genetic resources, useful for azalea cultivation and gardening in calcareous soils. On the contrary, R. obtusum ‘Kirin’, R. tosaense, R.x mucronatum ‘Fujimanyo’ and R. obtusum ‘Susogo-no-ito’ resulted iron deficiency sensitive genotypes. R. x mucronatum ‘Ryukyushibori’ and R. indicum ‘Kinsai’ showed intermediate responses.

Inheritance of the narrow leaf mutation in traditional Japanese evergreen azaleas

Morphological mutations have been admired and preserved in the classical cultivars of the Japanese evergreen azalea since the Edo era (1603–1867). One such mutation is the presence of narrow leaves and petals. In this study, we investigated the characteristics and inheritance patterns of the narrow leaf-type mutational form of Rhododendron macrosepalum ‘Seigaiha’, R. kaempferi ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant. The leaves of all cultivars showed weak development in the transverse plane and thickened margins. Subsequently, based on this qualitative character, the inheritance of the narrow leaf mutation was investigated. F1 seedlings of cross combinations between normal and narrow leaf-type plants had normal leaf-type. All F1 progenies of cross combinations between narrow leaf-type plants had narrow leaves like their parents. In addition, seedlings obtained from backcross and sib-cross tests using narrow leaf mutants segregated into normal and narrow leaf-types at a 1:1 and 3:1 ratio, respectively. These results indicate that the narrow leaf trait of three different azalea cultivars, ‘Seigaiha’, ‘Kin-kujyaku’, and R. kiusianum Yanagi-ba mutant, is probably a recessive mutation caused by a single, common gene.

常緑性ツツジ野生種とトキワバイカツツジとの種間交雑における受精前および受精後障害

常緑性ツツジ野生種とトキワバイカツツジとの種間交雑における受精前および受精後障害

EVERGREEN AZALEAS TOLERANT TO NEUTRAL AND BASIC SOILS: BREEDING POTENTIAL OF WILD GENETIC RESOURCES

Evergreen azaleas (Rhododendron subg. Tsutsusi) are ornamental plants which usually grow in acidic soils. They perform best when the pH ranges between 4.5 and 6.0. On calcareous soils, they often show strong iron-deficiency chlorosis symptoms and their growth is inhibited. Therefore their cultivation area is limited. However, wild azaleas were seen to show different adaptability to soil pH, according to species and habitat environment. With a long-term goal of selecting commercial genotypes tolerant to pH higher than optimum, we collected plant materials in the wild and in historical gardens and we performed specific crosses. To evaluate their neutral-basic pH tolerance, seeds were placed to germinate at nine different pH regimes ranging from 2 to 8.5. Ultra acid pH inhibited seed germination especially in R. macrosepalum, R. ripense and R. japonicum. However a clear correlation between germination capacity and pH was not observed. Genotype dependent differences were better observed by a screening, in which rooted cuttings were grown in nutritive solutions at three pH regimes (6.0, 7.5, and 9.0). R. indicum showed serious chlorosis symptoms in all conditions. By contrast, R. macrosepalum var. hanaguruma and R. scabrum were tolerant to neutral-basic pH, showing very limited leaf damage. As confirmed by molecular analyses, these two latter species belong to the subsection Macrosepala and are close related to R. ripense, whose elevated pH tolerance was already known. These wild genetic resources appear promising for pH tolerance breeding efforts.

Determination of genetic relationships between evergreen azalea cultivars in China using AFLP markers

Evergreen azaleas are among the most important ornamental shrubs in China. Today, there are probably over 300 cultivars preserved in different nurseries, but with little information available on the cultivar itself or relationships between cultivars. Amplified fragment length polymorphism (AFLP) markers were employed to determine the genetic relationships between evergreen azalea cultivars in China. One hundred and thirty genotypes collected from gardens and nurseries, including cultivars classified in the groups East, West, Hairy, and Summer, unknown cultivars, and close species, were analyzed using three primer pairs. A total of 408 polymorphic fragments were generated by AFLP reactions with an average of 136 fragments per primer pair. The average values of expected heterozygosity and Shannon's information index were 0.3395 and 0.5153, respectively. Genetic similarities were generated based on Dice coefficients, used to construct a neighbor joining tree, and bootstrapped for 100 replicates in Treecon V1.3b. Principal coordinate analysis (PCO) was performed based on Dice distances using NTSYS-pc software. The AFLP technique was useful for analyzing genetic diversity in evergreen azaleas. Cluster analysis revealed that cultivars in the West and Summer groups were quite distinct from other groups in the four-group classification system and that the East and Hairy groups should be redefined.

Chimeric inheritance of organelle DNA in variegated leaf seedlings from inter-subgeneric crossing of azalea

The inheritance of organelle DNA was investigated using PCR-RFLP markers in reciprocal cross combinations of inter-subgeneric azalea hybrids between evergreen azaleas (Rhododendron nakaharai and its hybrids) and fragrant deciduous azaleas (R. arborescens and R. viscosum) for the purpose of breeding fragrant evergreen azaleas. The hybrid progenies included green leaf, pale green leaf, varie- gated leaf and albino seedlings. Most viable green leaf seedlings had inherited ptDNA from the deciduous parent and non-viable albino and pale green leaf seedlings had inherited ptDNA from the evergreen parent. On the other hand, variegated leaf seedlings had chimerically inherited ptDNA from both parents. Their green leaf segments had ptDNA from the deciduous parent, and the pale green and white segments had biparental or maternal ptDNA depend- ing on the progeny. In this study, we obtained interesting inter-subgeneric azalea hybrid progenies that had chimerically inherited organelle DNA and had different colored leaf segments corresponding to the composition of ptDNA from each parent. These results suggest that variegated leaf progenies with chimeric ptDNA from both parents can be subsistent, whereas albino seedlings resulting from plastome- genome incompatibility between the plastid genome from evergreen azalea and the nuclear genome from deciduous azalea are non-viable.

Study on Effects of Different Plant Growth Regulators Types in Shoot Regeneration and Node Formation of Sutsuki Azalea (Rhododendron indicum): A Commercially Important Bonsai

Sutsuki azalea with botanical name Rhododendron indicum is one of the oldest evergreen azaleas, not only considered as a famous ornamental shrub, but also as a medicine to treat disease. Sutsuki azaleas are popular bonsai plants for many reasons. The overall goals of this research are to establish the most suitable cytokinin for in vitro shoot regeneration of Sutsuki azalea. The influence of Plant Growth Regulators was studied on shoot numbers, shoot length and node numbers after 10 weeks. In current experiment, after sterilization the apical shoots contain 4 developing leaf primordia separated and were cultured on ½ Anderson medium with different hormonal treatments. In this stage included three levels of isopentil adenin (2ip) (0, 2, 10) mg/l, Zeatin (0,0/1,0/5) mg/l and Thidiazuron (TDZ) (0, 0/04, 0/2) mg/l that were studied two concentration of hormones with each other. The results showed that 10mg/l 2ip together with 0/2mg/l TDZ was the best treatment for increasing the shoot number and 10mg/l 2ip together with 0/1mg Zeatin were the best treatment for increasing shoot length and node number.On the basis of available literature, this is the first and significant study regarding the comparative effect of different PGRs on in vitro propagation study of R. indicum. This significant study could be useful for large scale propagation and regeneration of this valuable bonsai.