Tetsushi H et al, Japan. J. Breed., 32(3):247-252(1982)
Summary
An anther culture technique was employed in Citrus aurantiuln L.; 'Sour Orange', 'Bouquet', and two varieties grown in Japan, 'Kabusu (Shu-to)' and 'Choshu-to'. Anthers collected from immature flower buds were cultured on the MURASHIGE and SKOOG's (19692) medium supplemented with or without indole-3-acetic acid (IAA) and kinetin at 28℃ in darkness. Embryoid formation vvas generally evident 14 weeks after inoculation of anthers of 'Sour Orange' and 'Choshu-to'. Anthers containing pollen grains at the late uninucleate stage formed embryoids effectively on media with 0.02mg/l of kinetin. Nuclear divisions of pollen grains and multinucleate pollen grains were observed in the 'Sour Orange' an-thers, while embryoids from pollen grains were not observed microscopically so far. After the incubation in light with a 16-hour photoperiod, some of these embryoids differentiated into shoots, developing into plantlets, while some of the other embryoids produced more embryoids mainly from the hypocotyl region. Induction of roots and shoots was stimulated by transfer from the embryoid induction media containing growth regulators to a medium eliminated growth regulators. Root tip cells of some of the differentiated plantlets had the diploid chromosome number.
Objectives
In this paper, the formation of plantlets from anthers of Citrus aurantium L. is described and the result are discussed in comparison with that of the poncirus.
Materials
‘Sour Orange’(Citrus aurantium L.) was used for most of this study and for histological experiments. ‘Kabusu(Shu-to)’ and ‘Choshu-to’, both of which are now grown in Japan, and ‘Bouquet’ were used for a varietal comparison with two different media.
Explant
anthers
Initial culture
The media were based on that of MURASHIGE and SKOOG (1962) supplemented with 50 g/L of sucrose. Priorto autoclaving, they were adjusted to pH 5.8 and solidified with 8 g/L of agar. Various tested media including indole-3-acetic acid (IAA) and kinetin are listed in Table 1 and 2. The cultures were incubated at 28±1℃ in darkness for embryoid induction. Once embryoids were differentiated they were placed in light (white fluorescent light, 500lux) with a 16-hour photoperiod. For ‘Sour Orange’ the media with 0.02 or 0.2 mg/l of kinetin were effective to form the embryoid. The media with 2.0 mg/l of kinetin were not effective. An embryoid was also formed on the medium with 0.002 mg/l kinetin and 2.0 mg/l of IAA, however, the medium with 0.2 mg/l kinetin and 2.0 mg/l of IAA was not effective. The medium supplemented with 0.2 mg/l of kinetin and 0.02 mg/l of IAA was effective for both ‘Sour Orange’ and ‘Choshu-to’. Any organ was not induced from anthers of ‘Kabusu’and‘Bouquet’.
Shoot multiplication
Rooting
For the induction of roots and shoots, embryoids produced from the anthers were transplanted to a medium with 20 g/l of sucrose and without any growth regulators. After transplanting, some embryoids formed shoots and/or roots. Other embryoids, however, did not grow, did not turn green, neither developed into pseudobulbils. Some of them eventually became brown.