Being the third most cultivated crop in South Africa, potatoes are of great economic
importance. As potatoes originated from cooler areas in the world, they do not easily
adapt to South African conditions. The main objective of potato breeding is, therefore,
to extend the crop's limited genetic base. Progress in crop improvement is slow due
to dominance, segregation and other factors caused by the tetraploid character of
cultivated potatoes. A new breeding program for rapid progress has been initiated at
the Vegetable and Ornamental Plant Institute, Roodeplaat, South Africa, which
comprises the combination of conventional and unconventional breeding techniques.
The program is based on the reduction of the ploidy level from the tetraploid to the
dihaploid level to facilitate crossings with diploid wild species. Anther culture is the
preferred technique for the rapid reduction of the ploidy level and has been
successfully applied on different members of the Solanaceae. Cultivated potato,
Solanum tuberosum is, however, an important exception.
In this study various potato genotypes (tetraploid cultivars, dihaploid
breeding lines and a diploid wild species) were used in experiments concerning
microtechniques, alternative culture methods and medium manipulation. The main
objectives were to evaluate and compare the androgenetic ability of the various
genotypes used and to try and identify the factors limiting their in vitro response.
Regarding microtechnique, the study focussed on the investigation of the
frequency of androgenesis - as a function of plant age - and the determination of
defined flower bud lenqths representative of the correct microspore developmental
stage for optimal androgenetic response. Combined with an extensive histological
study on the microspore development within anthers, from the time of flower selection,
after a cold-pretreatment and at various time-intervals during the culture period of 42
days, the following conclusions were reached: In vitro androgenetic response proved
optimal when flowers of responsive genotypes were selected during the first seven to
21 days of the flowering period. Both microspore derived embryoid- and callus
development were visible within responsive anthers after a culture period of only seven
days. The flower bud length required for anthers to be in the optimal stage of
microspore development, e.g. the uninucleate stage, varied between the different
genotypes but could readily be determined with the DAPI (4,6-diamidino-2-
phenylindole) technique. It was also concluded that anthers of the tetraploid cultivar
Atzimba should be selected later, between the late-uninucleate and the early-binucleate
developmental stages. This suggested a limited selection period for Atzimba anthers,
as starch depositioning - which prevent embryogenesis - occurs within anthers during the binucleate stage. Histologically, Atzimba showed limited embryoid development
with no embryoid release, while the diploid wild species, S. canasense, proved
androgenetically unresponsive.
Alternative culture methods were applied to study the effect of different culture
phases (liquid, double layered and agar solidified) and anther orientations (lateral,
dorsal and ventral) on the androgenetic response of the potato genotypes used.
Liquid cultures, based on the so-called shed-pollen technique, enhanced the
androgenetic response of the tetraploid cultivar Atzimba. Optimal embryogenesis was
obtained for responsive breeding line 87.2002/3 with the utilization of agar solidified
media, with maximal response when anthers were cultured in the lateral orientation.
No response was observed from S. canasense.
The effect of medium manipulation on the androgenetic response of the three
genotypes was investigated. The utilization of various combinations of different
concentrations of indole-3-acetic acid (1M) and benzyladenine (BA), the alteration of
the initial time of incubation of anthers on the initiation media and the use of media
without growth regulators compared to that containing gibberellic acid (GA[3]), were
investigated. BA had to be present in the initiation media and had a major, though not
exclusive, effect on embryogenesis compared to 1M. The optimal BA concentration
varied between the two trials. IAA also had an increasing effect on anther response,
both in the absence of BA and, especially, in addition with relatively high BA
concentrations. In this experiment, only breeding line 87.2002/3 responded. The
initial culture of anthers, during the first seven to 21 days of the culture period, on
media containing growth regulators proved essential for microspore derived embryoid
production in the tetraploid cultivar Atzimba. As these growth regulators are
metabolized in the culture media, the regular transfer at shorter, two-weekly intervals
to media containing metabolically active substances, proved important. GA[3] had no
enhancing-effect on embryogenesis in any of the three tetraploid cultivars.
The results obtained in this study suggest that the first 21 days is the critical
stage in the anther culture period in terms of the optimal time for flower selection,
embryoid induction and the increase in embryogenetic response due to growth
regulator influence. It is important to pre-determine the developmental stage when
most microspores were in the uninucleate stage of development and to correlate this
stage with a specific flower bud length. This would assure maximum response of
those genotypes amenable to anther culture. It also implies a more practical and
economical starting pOint to anther culture experiments. Following the determination
of microspore developmental stage and pollen fertility, flowers should be selected from
the donor plants only during the first three weeks of the flowering period. The composition of the nutrient media used for potato anther cultures were sufficient with
respect to growth regulators. The growth regulators SA, IAA and the amines
glutamine and asparagine had to be present in the initiation media, especially during
the first three weeks of the culture period. As microspore development within anyone
anther was found to be asynchronous, the regular transfer of anthers to fresh media
is recommended to assure proper development of all microspores. The use of a
slightly higher IAA concentration could be considered, but care should be taken as
too-high concentrations would induce callus production. Microspore derived embryoid
production is preferred, as the ploidy level of callus derived plantlets normally varies
and somaclonal variation can occur. Liquid media should be considered for anther
culture of tetraploid genotypes, while embryoid production can be increased by
culturing the anthers of responsive genotypes on agar solidified media on the lateral
orientation. Finally, the diploid wild species S. canasense seemed androgenetically
unresponsive, or the media and culture conditions used did not satisfy the specific
requirements of this genotype. Androgenetic amenability should first be transferred
by means of interspecific crossings with a responsive dihaploid genotype, such as the
breeding line 87.2002/3. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10345 |
| Date | January 1995 |
| Creators | Liebenberg, Denise. |
| Contributors | Van Staden, Johannes. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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