DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA: THE COCONUT‐MILK PROBLEM

In an attempt to define a medium and to investigate the effects of glycine, floral buds of Aquilegia were grown in culture on coconut milk and defined media containing either White's minerals or a new mineral formula, with and without glycine. Development obtained on the best defined medium was 66 to 76% of that achieved on the equivalent coconut-milk medium. The inability of buds to achieve development equal to that on the coconut-milk medium may be partially explained by the existence of a barrier to carpel formation and development. The effect of glycine on floral buds grown on a defined medium was dependent not only upon the minerals used but also on the stage of development of the bud. With White's minerals, glycine inhibited the development of buds in early and intermediate stages but was slightly promotive with buds in later stages. With the new minerals, glycine was slightly promotive with early and intermediate stages of development but was inhibitory with later stages. Further work is necessary before development on a defined medium will equal or exceed development on the cocontut-milk medium. THIS COMMUNICATION is intended to report on preliminary attempts to achieve a completely defined medium without coconut milk which would support the development of floral buds in vitro. Since coconut milk is highly variable in measurable constituents and may contain important unknown growth factors, the removal of coconut milk from the medium is absolutely essential before any definitive work can be done with the effects of amino acids, nucleic acids or hormones. Subsequent to the report of Tepfer, Karpoff, and Greyson (1966), Filner (1966) stated that glycine in the presence of White's minerals severely inhibited growth of tobacco callus tissues. Since glycine and White's minerals were important constituents of Medium H2 (Tepfer et al., 1966), it was essential to investigate the effects of glycine on the development of buds not only on the coconut milk medium but on defined medium containing either White's minerals or a new mineral formulation. MATERIALS AND METHODS-Floral buds of Aquilegia formosa Fisch. were sterilized and prepared for dissection according to methods described previously (Tepfer et al., 1963; Tepfer, 1965; Tepfer and Karpoff, 1965; Tepfer et al., 1966). Inflorescences were dissected and the individual buds were explanted onto agar media in plastic petri dishes. A diagram of a fllora bud that has just formed its last whorl of floral organs is shown in Fig. 1. The five sepals (Se) I Received for publication 1 March 1968. Supported by NSF Grants GB1268 and GB5663 to S. S. Tepfer. The authors thank Dr. Howard T. Bonnett for reviewing the manuscriDt. are initiated singly and are not exactly in a true whorl. A true whorl of five petals (P) alternates with the sepals. There are eight whorls of stamens with four whorls each in vertical alignment above the sepals and petals. Each stamen whorl is numbered according to its position above the sepals and petals. Above the stamens, there are two whorls of staminodia (Std) and a terminal whorl of carpels (Ca). The initiation of each individual sepal and of each whorl of primordia is considered to represent a developmental stage. The average number of stages initiated is then used as a measure of the extent of development. The last whorl of primordia initiated is the single carpel whorl. After the initiation of the 17 whorls of primordia, further differentiation of the flower was indicated by defining specific stages in carpel development as star carpels, dimpled carpels, grooved carpels, elongating carpels, and erect carpels. For example, a floral bud placed in culture at the S + 3 stage that reaches elongating carpels, has advanced through 10 developmental stages. The entire series of developmental stages was divided arbitrarily into five phases: (I) buds before sepal formation, during sepal formation, or with sepal and petal primordia; (II) buds from stage S+ 1 through stage P+2 (1-4 stamen whorls); (III) buds from stage S+3 through P+4 (5-8 stamen whorls); (IV) buds at Std (staminodium) stages; (V) buds in various stages of carpel development. Equal numbers of buds of each phase, except phases IV and V, were explanted onto each medium. Phase IV buds were grown for qualitative observation only and were not included in the statistical analysis. Phase V buds were not used.