Tissue culture studies on citrus and Welwitschia.
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Date
1972
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Abstract
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[1]), and White (BM[2]) , 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[2] 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[1] + 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[1] + GA[3] (1 mg/l) , embryoids developed
roots and later, shoots. It was necessary to remove plantlets from
the GA[3]-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[3] to BM[1] 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.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1972.
Keywords
Plant tissue culture., Theses--Botany.