Tissue culture studies on citrus and Welwitschia.
Part I. IN VITRO CULTURE OF CITRUS EMBRYOS AND NUCELLAR ISOLATES Zygotic embryos of the Ellendale mandarin, a monoembryonic variety of citrus, were cultured on modified basal media of Murashige and Skoog (BM), and White (BM) , supplemented with various growth regulators and nutrient additives. The growth of immature embryos was greatly enhanced by the addition of 400 mg/l casein hydrolysate (CH) to the basal media. Coconut milk (CM) and malt extract (ME) enhanced growth to a lesser extent, while the addition of indoleacetic acid (IAA) and kinetin (KIN) at the concentrations used, was in no way beneficial. Nucellar isolates excised from abortive and normal Ellendale mandarin ovules eight to 20 weeks after anthesis, were cultured on BM[I] and BM in the presence of various concentrations and combinations of IAA, indolebutyric acid, naph~haleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), KIN, CM, benzyl adenine, 6-dimethylallylamino purine, yeast extract (YE), ME, CR, adenine (AD), adenine sulphate (AS), ascorbic acid (AA), and benzylthiazole-2-oxyacetic acid. Some of the isolates which remained alive for four months did develop callus but n~ differentiation of embryoids or other structures occurred. Unfertilized ovules from 8-12-week-old Washington Navel orange fruits provided nucellar isolates which were cultured on media similar to those upon which mandarin nucelli were unsuccessfully cultured. In the case of Navel orange nucelli however, BM + 400 mg/l filter-sterilized ME, and BMl + 40 mg/l AD yielded numerous pseudobulbils which later developed embryoids. Adenine (10 mg/l) was more effective than 20 mg/l which in turn was more effective than 30 mg/l. Adenine was more effective than its equivalent amount supplemented in the sulphate form except at 10 mg/l where the two forms were equally effective. Zeatin (ZE) at 0,2 mg/l did induce some pseudobulbils and embryoids, but all these treatments were less effective than 400 mg/l ME. When transferred to BM + GA (1 mg/l) , embryoids developed roots and later, shoots. It was necessary to remove plantlets from the GA-supplemented medium shortly after the first foliage leaves developed in order to prevent the development of weak, spindly plants . Plantlets were transferred from BM[1 ]+ GA to BM only, and then after careful conditioning they were planted out in soil. This appears to be the first successful attempt at inducing adventive embryogenesis 1n the nucellus of unpollinated, unfertilized citrus ovules in vitro. Part II. EMBRYO AND FREE-CELL CULTURE OF WELWITSCHIA MIRABILIS Welwitschia embryos, cultured on BMI supplemented with CR, and low levels of IAA and KIN, germinated and developed leaves but not roots. Embryos cultured on BM[I] with 5,0 and 10,0 mg/l NAA produced an abundance of friable callus from the hypocotyl root axis. This callus was used for starting suspension cultures aimed at inducing vegetative embryogenesis. A number of nutritional additives and hormones were used alone and in combination at various concentrations. Cells of numerous shapes and sizes were observed but no organogenesis was apparent in either suspension cultures or in cell colonies plated out on semi-solid agar media. A closer study of cell aggregates formed in suspensions supplemented with CM + 2,4-D revealed that internal division occurred in approximately 40 per cent of the larger cells. It is suggested that this internal division may constitute the first step in embryogenesis of Welwitsahia cells in suspension culture. It is also tempting to speculate that this process, which has been reported by other researchers, is the first step 1n embryogenesis of free cells in general. Although this attempt at inducing adventive embryogenesis in cell cultures of Welwitschia was unsuccessful, some encouraging results were obtained on potentially suitable media and possible initial stages in the organization of embryoids.