Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4-6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum.Abbreviations: CTAB -cetyltrimethylammonium bromide; GA 3 -gibberellic acid; GUS -b-glucuronidase; MS -Murashige and Skoog medium

In tissue culture of strawberry, meristem tip culture has been utilized to produce virus-free strawberry plantlets in vitro. Since viruses are scarcely translocated to meristem tissues, in addition to the rare occurrence of somaclonal variations, this culture is useful for maintaining the commercial strawberry cultivars.13) However, stability is not always advantageous, especially when we wish to isolate genetic variation from tissue cultures and apply them to breeding of strawberry. For this purpose, it is necessary that a different culture system with abundant variations is developed.In a series of our works, the authors have clarified the culture conditions for leaf-explants of tomato,711) pepino,9) tobacco,8) and melon,10) and pointed out that somaclonal variations, including disease resistance were frequently induced in leaf-explant-derived callus tissues and successfully propagated to plants regenerated from these callus tissues.4,12) Similar reports have been made by many workers.2,3,5)Also in Japanese cultivars of strawberry, such culture systems have been examined in our laboratory and elaborated as a primary step for obtaining useful variants. In this report, the authors describe an efficient culture system for callus induction and plant regeneration in strawberry leaf explants. Materials and MethodsYoung leaves newly developing from runners of strawberry (Fragaria ananassa cv. Hoko-Wase) were harvested and surface-sterilized with 70% EtOH and 2% sodium hypochlorite, After washing several times with sterilized water, excised leaf segments (leaf-explants) were put onto a MurashigeSkoog (MS) medium6) supplemented with growth regulators, solidified with 0.2% Gellan gum, and adjusted to pH 6.0 with NaOH before autoclaving. The growth regulators used were a-naphthaleneacetic acid (NAA) and 2, 4-dichlorophenoxy-acetic acid (2, 4-D) for auxin, and 6-benzyl-aminopurine (BA) for cytokinin. NAA or 2, 4-D was mixed with BA at various concentrations and added to the MS medium. Culture bottles were tightly sealed with aluminum sheets and Parafilm, and cultured at 26C under a constant illumination of 4,000 lux until some morphological changes in explants or induced calluses were observed (for 30-40 days). Table 1 summarizes the effects of growth regulators on callus induction, growth, and redifferentiation. Induction of actively growing callus tissues was observed in many combinations of NAA and BA and most effective especially when leaf-explants were cultured with 1 mg/l of NAA and BA. When much higher concentrations (2.5-3 mg/l) of NAA were used, both leaf-explants and callus tissues frequently browned and the subsequent growth of callus tissues ceased. With much lower concentrations of the growth regulators, callus tissues induced became brownish, but their growth was vigorous and many adventitious roots were developed from callus tissues 20-30 days after incubation. In any combinations of NAA and BA, however, shoot formation from callus tissues was not observed even when the tissues were excised from explants an...

Four cultivars of Sagagiku group in chrysanthemum were treated with different types of cytokinin to form adventitious shoots from leaf explants. The maximum adventitious shoot formation rate, 82.2%, was observed in the medium with 2.5 X 10-5 M of thidiazuron (TDZ), an urea-type Cytokinin, in •eOgura-nishiki,•f followed by •eSagano-aki•f (56.7%) in the medium with 2.5•~ 10-6 M of N-(2-chloro-4-pyridyl)-N•f-phenylurea (CPPU), an urea-type cytokinin, •eSagano-yuki•f (12.2%) in the medium with 2.5X 10-6 M of 6-benzylamino purine (BA), a purine-type cytokinin, and •eSagano-haru•f (1.3%) in the medium with 5.O X 10-6 M of TDZ. Vigorous roots and significantly higher root formation rates were observed in the medium without 3-indolebutyric acid. In •eSagano-haru,•f however, no roots were differentiated and died during root induction culture. These results suggested that there were large differences in adventitious shoot formation ability among these four cultivars of Sagagiku group, and that TDZ and CPPU were effective for shoot formation in some cultivars of chrysanthemum.

Two cultivars of Zantedeschia aethiopica (group 1) and 21 cultivars in other Zantedeschia spp. (group 2) were hybridized to investigate the cross-compatibility of each combination. From a total of 757 crosses from 37 hybrid combinations that were attempted, 3,144 resulting embryos were cultured. As a result, 175 embryos representing 26 combinations formed shoots, but all hybrids were albino and viable only in vitro. Some hybrids had plastids of both parents, but no relationship between the appearance of albino hybrids and plastid inheritance could be detected.