Characterization and control of micropropagation problems in aloe, devil's claw and banana.
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Date
2008
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Abstract
The development of the science of micropropagation from the very initial concept
of totipotency to the modern day advancement and sophistication has been
affected by a wide range of problems such as hyperhydricity, shoot-tip necrosis
and somaclonal variation. These problems are largely the result of the obvious fact
of trying to grow plants in an environment that is different from the one plants are
used to naturally. The extent of these problems ranges from minor technical
inconvenience to significant economic loss. Characterization and control of
micropropagation problems has been one of the priorities of plant tissue culture
research due to the enormous contribution of this discipline for plant production,
improvement and conservation.
The prevalence and severity of these tissue culture problems varies widely among
plant species. The rationale of this research project was therefore, to identify plant
species most affected by the problems studied, characterize the problem and find
mechanism(s) to control or minimize the damage caused by the problem. The
literatures reviewed provide sufficient background information for the experimental
chapters. Due to the different nature of the problems and variation in the plant
species they affect, the model plant, the methodologies used and parameters
analysed were also different. The findings of these investigations, in their own
different way, addressed certain problems that individually and collectively pose
difficulties to the micropropagation industry. The difference in the content of the
experimental chapters is therefore the result of the broader objective of the
research project to tackle such difficulties.
The success and failure of tissue culture system greatly depends on the choice of
PGR’s. This choice can be made based on comparative study of their biological
activity. Some promising reports on the role of topolins in micropropagation led to
the idea of testing these cytokinins for their potential in tissue culture. As a
prerequisite to subsequent investigations, the biological activity of some selected
topolins and BA derivatives was tested using the soybean callus bioassay. The
activity of the cytokinins tested varied significantly. The results demonstrated that
the structure of a cytokinin dictates its activity. Modifications of side-chain
improved the activity of oT but had no effect on pT. The presence of the methyl
group had an enhancing effect on cytokinin activity of topolins or at least it did not
reduce it. BA derivatives BA9THP (conjugated at N9 position), 3FBA and
2Cl6(3OHBA)R (halogenated derivatives) also showed good cytokinin activity and
hold good promise for future research.
In an attempt to alleviate hyperhydricity in Aloe polyphylla and optimize the
micropropagation protocol, meta-topolin and its derivatives were tested at various
concentrations together with BA and zeatin. Of all the cytokinins tested mT
produced the best results in terms of shoot and root growth. Five μM was found to
be the optimum concentration at which complete control of hyperhydricity was
achieved without compromising shoot and root growth. Plantlets rooted in a
multiplication media. BA generally had a negative effect on growth and
development both in vitro and ex vitro. Acclimatization of plantlets was achieved
easily by initially transferring plantlets to a mist house (for three weeks) followed
by transfer to the greenhouse. The type of cytokinin also had an effect on ex vitro
growth with BA-treated plants producing the lowest shoot and root biomass.
Various experiments were conducted to characterize and control factors affecting
STN in Harpagophytum procumbens. Media type and strength, PGR, carbon
sources, sub-culturing, calcium and boron were tested. Results indicated that all of
the tissue culture components tested affected STN. From the different media types
tested, half strength was MS found to be the preferred medium. Increasing
cytokinin concentration increased the incidence of STN and the problem was
aggravated by the addition of auxin to the multiplication medium. Optimum shoot
multiplication was achieved by omitting auxin and using the cytokinin mTR.
Plantlets produced basal callus which interfered with rooting. The quantity of this
basal callus was minimum when mTR was used.
Sub-culturing plantlets onto fresh medium every two weeks helped minimize STN.
Off all the sugars tested 3% sucrose was optimum. Other sugars either
aggravated STN or inhibited growth when compared at equi-molar concentration.
Increasing the concentration of either Ca or B prevented the development of
necrotic shoots. When the concentration of both elements is increased
simultaneously negative effects on both growth and STN were observed. Using 6
mM Ca in half strength MS medium was optimum. B was toxic at higher
concentrations. Plantlets rooted readily in half strength cytokinin-free MS media
supplemented with 2.5 μM IAA. Rooted plantlets produced using the optimized
protocol were acclimatized successfully by transferring directly to a greenhouse in
a 1:1 ratio of sand and soil mixture.
The effect of meta-toplins on micropropagation and somaclonal variation of
banana was investigated. Tissue cultured explants of cultivars ‘Williams’ and
‘Grand Naine’ were cultured in MS media containing the cytokinins BA, mT,
MemT, MemTR and mTR at various concentrations. Results of the investigation
revealed that superior multiplication and lower abnormality index was recorded
from the mTR and mT treatments at 22.2 μM concentration. These treatments,
however, had an inhibitory effect on rooting. The effect of these treatments (22.2
μM mT and mTR) in comparison with equi-molar concentration of BA on
somaclonal variation of ‘Williams’ banana was tested using RAPD-PCR at the 7th
multiplication cycle. No significant difference was found between the treatments. It
should however be highlighted that cultures were initially maintained for three
multiplication cycles in media containing BA. The inherent stability and initial effect
of BA could have influenced the results.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.
Keywords
Plant micropropagation--Methodology--Research., Aloe polyphylla--Micropropagation., Grapple plant--Micropropagation., Bananas--Micropropagation., Cytokinins., Necrosis., Theses--Botany.