Tsutomu Yabuya

Anthocyanin-flavone copigmentation in bluish purple flowers of Japanese garden iris (Iris ensata Thunb.)

The in vitro identification of copigmentation was carried out using anthocyanins, such as malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G and the flavone isovitexin. These are major pigments of Iris ensata. These anthocyanins brought about copigmentation, i.e., the bathochromic shift (bluing effect) of visible λ max due to increased concentrations of isovitexin, and 32 to 35 nm were estimated as the magnitude (Δλ max) of each shift. In addition, the absorption spectrum of 0.1 mM malvidin 3RGac5G, 0.07 mM petunidin 3RGac5G and 0.7 mM isovitexin mixture closely matched those of the fresh outer perianths of the bluish purple cultivars, ‘Suiten-isshoku’, ‘Hekikai’ and ‘Yakonotama’ which belong to the malvidin 3RGac5G - petunidin 3RGac5G type of I. ensata. Therefore, these results indicated that the bluing effect on the flower color of the bluish purple cultivars of this species was caused at least in part by the copigmentation between these anthocyanins and the flavone isovitexin. For the copigment effects of isovitexin among malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G, λ max and Δλ max of delphinidin 3RGac5G was slightly higher than those of malvidin 3RGac5G or petunidin 3RGac5G, and the copigmentation of delphinidin 3RGac5G was characterized by a higher concentration of isovitexin. Finally, the breeding for blue flowers due to copigmentation of delphinidin 3RGac5G with isovitexin in I. ensata was discussed.

Characterization of anthocyanin p-coumaroyltransferase in flowers of Iris ensata

Malvidin and petunidin 3-(p-coumaroyl)rhamnosylglucoside-5-glucosides as well as nonacylated 3-rhamnosylglucoside-5-glucoside of these anthocyanidins were detected as major anthocyanins in cyanic flowers of Iris ensata. Enzyme extracts from flower buds of this plant catalyzed the transfer of the p-coumaroyl moiety from p-coumaroyl-CoA to both the anthocyanidin 3-rhamnosylglucoside and 3-rhamnosylglucoside-5-glucoside to form the anthocyanidin 3-(p-coumaroyl) rhamnosylglucoside and 3-(p-coumaroyl) rhamnosylglucoside-5-glucoside, at a ratio of ca. 1 to 4, respectively. The activities of this enzyme were also examined for various cyanic and acyanic cultivars, in addition to the characterization of the p-coumaroyltransferase. The sequence of acylation and 5-glucosylation in the anthocyanin biosynthesis of this plant is discussed.

Plant regeneration from protoplasts of Iris germanica L.

SummaryProtoplasts were isolated enzymatically from suspension cultures derived from embryogenic calli induced by leaf base culture of Iris germanica. In protoplast culture, the effects of glucose concentration, different sugars and combinations of 2,4-D and KIN on protoplast division and colony formation were examined. N6 medium supplemented with 0.1–1 mg/l 2,4-D, 1 mg/l KIN, 200mg/l casein hydrolysate, 250 mg/l proline, 0.2 M glucose and 20 g/l agarose was suitable for protoplast division and colony formation. When colonies formed were transferred onto hormone-free MS medium, many plantlets were regenerated through somatic embryogenesis. Thus, we could establish a plant regeneration system from protoplasts of I. germanica.

High-performance liquid chromatographic analysis of anthocyanins in Japanese garden iris and its wild forms

SummaryThe anthocyanins of 43 varieties and 4 wild forms of Iris ensata in their outer perianths were analysed by high-performance liquid chromatography procedures. Using the different components of major anthocyanins, each variety and wild form were classified into six types such as malvidin 3RGac5G-petunidin 3RGac5G, petunidin 3RGac5G-malvidin 3RGac5G, malvidin 3RGac5G, petunidin 3RGac5G, delphinidin 3RGac5G and malvidin 3RG5G-petunidin 3RG5G. Among these types, the delphinidin 3RGac5G type was noteworthy because such a type was useful for the breeding of a blue flower. The malvidin 3RGac5G-petunidin 3RGac5G type accounted for ca. 70% of the varieties and all wild forms examined. This type, therefore, was regarded as the basic one for I. ensata, while the other types were variants for major anthocyanins. There was no particular relationship between flower colors such as purple, red-purple, blue-purple, light-purple and pink and the types of major anthocyanins except for one, i.e. all varieties classified into the malvidin 3RGac5G or petunidin 3RGac5G type exhibited pink flowers. Finally, the utility of the relative species for the breeding of new flower varieties of I. ensata was discussed.

Anthocyanin 5-O-glucosyltransferase in flowers of Iris ensata

Abstract Anthocyanin 5-O-glucosyltransferase was characterized in flowers of Iris ensata containing malvidin and petunidin 3-(p-coumaroyl)rhamnosylglucoside-5-glucosides as well as nonacylated 3-rhamnosylglucoside-5-glucoside of these anthocyanidins as major anthocyanins. Enzyme extracts from flower buds catalyzed the transfer of the glucosyl moiety from UDP-glucose to the 5-position of anthocyanidin 3-rhamnosylglucoside to form the anthocyanidin 3-rhamnosylglucoside-5-glucoside, but not to the anthocyanidin 3-glucoside and 3-(p-coumaroyl)rhamnosylglucoside. In addition to the characterization of the 5-O-glucosyltransferase, the activities of this enzyme were also examined for various cyanic and acyanic cultivars. The sequence of 5-O-glucosylation and p-coumaroylation in the anthocyanin biosynthesis in this plant is discussed in detail.

Stability of flower colors due to anthocyanin-flavone copigmentation in Japanese garden iris, Iris ensata Thunb.

The fading of flower color in bluish purple and reddish purple cultivars of Iris ensata and the in vitro stability of malvidin 3RGac5Gand petunidin 3RGac5G due to copigmentation with isovetixin under different pH conditions were examined. The bluish purple cultivars exhibited higher flower color stability than the reddish purple cultivars 2 days after anthesis. In the absence of isovitexin, malvidin 3RGac5G and petunidin 3RGac5G were not able to maintain color stability except at low pH. However, the color stability of malvidin 3RGac5G and petunidin 3RGac5G was increased by copigmentation with isovitexin under all pH conditions tested. Most remarkable was the stabilization of both anthocyanins due to the copigmentation at pH 4.2–6.2. Therefore, it can be concluded that the stability of flower color in the bluish purple cultivars of malvidin 3RGac5Gand petunidin 3RGac5G type of I. ensata is caused at least in part by the copigmentation between these anthocyanins and isovitexin.

Production of somatic hybrid plants between Iris ensata Thunb. and I. germanica

Wide hybridization that cannot be attained through conventional sexual crosses, can now be approached by somatic hybridization. Protoplasts of I. ensata and I. germanica were fused by electrofusion. For the selection of somatic hybrids, protoplasts of I. ensata which did not form colonies in protoplast culture and protoplasts of I. germanica which had regeneration ability for only albino shoot were used in symmetric fusion. On the other hand, the protoplasts of I. ensata and I. germanica protoplasts which were inactivated by iodoacetamide (IOA) treatment were used in asymmetric fusion. Five-six months after cell fusion, green plants were obtained in the symmetric and asymmetric fusion. In the random amplified polymorphic DNA (RAPD) analysis, the green plants had bands specific to both parental species. Therefore, these plants were somatic hybrids between I. ensata and I. germanica.

Chromosome associations and crossability with Iris ensata Thunb. in induced amphidiploids of I. laevigata Fisch. × I. ensata

SummaryThe chromosome associations of amphidiploids of I. laevigata × I. ensata were analysed and compared with those of the parental species and F1 hybrids of I. laevigata × I. ensata. The F1 hybrids showed partial chromosome associations. Their mean chromosome association per cell was 20.73I+3.63II, although the mean chromosome association per cell in the parental species was 0.09I+15.96II for I. laevigata and 0.03I+11.98II for I. ensata, respecively. In contrast, the normal association (28II) was partially restored in the amphidiploids. Their mean chromosome association per cell was 1.93I+26.48II+0.28III+0.03IV+0.03V. In this study, moreover, the crossability between I. ensata (2X and 4X) and the amphidiploids and between I. laevigata and the amphiliploids was examined. No hybrid plants were obtained from both reciprocal crosses between I. ensata (2X) and the amphidiploids and between I. laevigata and the amphidiploids. Only the cross of I. ensata (4X) × the amphidiploids in the reciprocal crosses produced hybrid plants. The observation of their somatic chromosome numbers indicates that these are true hybrid plants between autotetraploid I. ensata and the amphidiploids, and such plants can be called autoallotetraploids between I. ensata and I. laevigata. The interspecific cross-breeding of I. ensata using the autoallotetraploids is discussed.

Plant regeneration from protoplasts of Iris hollandica Hort

The effects of glucose concentrations, different sugars and combinations of 2,4-D and kinetin on cell division and colony formation were examined in cultures of protoplasts isolated enzymatically from suspension cultures of Iris hollandica N6 medium supplemented with 1 mg/l 2,4-D, 1 mg/l kinetin, 200 mg/l casein hydrolysate, 250 mg/l proline, 0.3– 0.5 M glucose and 20 g/l agarose was suitable for cell division and colony formation. When colonies formed were transferred to hormone-free MS medium, many shoots were induced. In addition, when induced shoots were transferred to MS medium with 1 mg/l NAA, root induction was observed. A plant regeneration system from protoplasts of I. hollandica was thus established.

New types of major anthocyanins detected in Japanese garden iris and its wild forms

The anthocyanins of 130 cultivars, 13 lines and 3 wild forms of Iris ensata were analyzed by HPLC, and these plants were classified into 16 types of major anthocyanins. Among these types, 8 types such as petunidin 3RGac5G – delphinidin 3RGac5G, delphinidin 3RGac5G – petunidin 3RGac5G, cyanidin 3RGac5G – peonidin 3RGac5G, delphinidin 3RG – delphinidin 3RGac, petunidin 3RG5G – malvidin 3RG5G, malvidin 3RG5G – peonidin 3RG5G, peonidin 3RG5G – cyanidin 3RG5G and peonidin 3RG – cyanidin 3RG were obtained as new types. In these new types, peonidin 3RG – cyanidin 3RG and peonidin 3RG5G – cyanidin 3RG5G types were noteworthy because cyanidin 3RG and cyanidin 3RG5G are useful for the breeding of red flowers in I. ensata.