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Micropropagation and acclimatization of Aloe polyphylla and Platycerium bifurcatum.Chukwujekwu, Jude Chinedu. 11 December 2013 (has links)
Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in
vitro grown plants. The basal medium was MS medium (MURASHIGE and
SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol, and 30 gl ¯¹ sucrose.
Agar (0.8 %) was used as the gelling agent. Different cytokinins, singly or in
combination with auxins (IBA and NAA), were tested for shoot proliferation
activity. All the cytokinins tested (kinetin, zeatin, iP, and BA) gave a good shoot
proliferation response. The optimal concentrations for shoot proliferation of each
of the cytokinins tested were: zeatin (0.5 mgl ¯¹), kinetin (1.5 mgl ¯¹), iP (1.0 mg ¯¹)
and BA (1.5 mgl ¯¹). In combination with auxins, the optimal combinations were
kinetin/NAA (2.0/0.1 mgl ¯¹), kinetin/lBA (1.5/1.0 mgl ¯¹), zeatin/lBA (1.0/0.5 mgl ¯¹),
zeatin/NAA (1.0/1.0 mgl ¯¹), BA/IBA (1.0/1.0 mgl ¯¹), BA/NAA (1.5/0.1 mgl ¯¹).
Although it gave the highest number of shoots per explant, BA was responsible for hyperhydricity.
Temperature and sucrose also influenced shoot proliferation. The optimal
temperature was 25°C, while 30 gl ¯¹ was the optimal concentration of sucrose for
shoot proliferation. Plants rooted well in plant growth regulator-free MS medium.
Amongst the potting mixtures tested, soil: sand: vermiculite (1:1:1 v/v) was the best with 98 % plantlet survival.
In the second part of this project, Platycerium bifurcatum cultures were
established using leaf explants. The basal medium was MS medium
(MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol
and 30 g l ¯¹ sucrose. For bud initiation, 1.0 mgl ¯¹ BA was used, while 0.8 % agar
was used as the gelling agent. Three different strengths of MS medium (full, half,
and one-quarter strength) without plant growth regulators were tested for further
bud growth and development. Half-strength MS proved to be the best for further bud growth and development. Rooting was best achieved in one-quarter strength
MS medium without plant growth regulators. In vitro grown plantlets were
successfully acclimatized using peat as the potting medium. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.
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Characterization and control of micropropagation problems in aloe, devil's claw and banana.Bairu, Michael Wolday. January 2008 (has links)
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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.
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Regulation of hyperhydricity in Aloe polyphylla propagated in vitro.Ivanova, Mariyana Vasileva. January 2009 (has links)
Micropropagation of Aloe polyphylla, an endangered species with a high ornamental and medicinal value, is an important part of its conservation. However, the in vitro culture was hindered by the phenomenon of hyperhydricity. The research reported in this thesis was undertaken for two reasons. Firstly, to understand the role of various culture factors involved in the process of hyperhydricity in A. polyphylla and to identify the in vitro conditions, under which this disorder can be prevented. Secondly, we conducted an investigation into the underlying mechanisms of this phenomenon by probing if it was mediated through internal cytokinins. Ammonium (NH4 +) ions, applied cytokinins (CKs) and CK concentrations were tested in multifactorial combinations and significantly influenced the regeneration rate and occurrence of hyperhydricity. Shoots were grown on media with different NH4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with BA, zeatin or TDZ at 0, 5 or 15 ìM. Elevating the levels of NH4 +, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4 + (10.3 mM). However, in the presence of higher NH4 + concentrations, CKs increased hyperhydricity in a concentrationdependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4 + and low BA or zeatin (5 ìM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. In view of the fact that NH4 + was supplied in the form of NH4NO3, it was difficult to determine if NH4 + or nitrate (NO3 -) ions were associated with the increase in hyperhydricity. We further examined the role of nitrogen (N) supplied as inorganic NH4 + or NO3 -, or organic glutamine. The omission of total N from the culture medium resulted in low multiplication and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 - was used as the sole N source, shoots of fine quality were produced and hyperhydricity was completely eliminated. Overall, the MS N mix was superior to any single N source for multiplication and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 - on shoot regeneration. Furthermore, not only the absolute amount of N, but also the relative amounts of NH4 + and NO3 - influenced the multiplication rate, frequency of hyperhydricity and shoot quality. The highest regeneration was obtained with NH4 + : NO3 - ratios (mM) of 20 : 40, 30 : 30 and 40 : 20. Decreasing the ratio of NH4 + : NO3 - lowered the occurrence of hyperhydricity. The potential of glutamine as the sole source of N was also demonstrated, since its application resulted in the production of good quality shoots and almost no hyperhydricity. Shoot explants grown in static liquid media became hyperhydric and lost their ability to regenerate. The type of gelling agent used to solidify the medium affected greatly hyperhydricity and shoot multiplication. Gelrite resulted in a significantly lower multiplication rate and four times higher hyperhydricity (64.7%) compared to when agar was used. Gelrite was further selected to test the hypothesis if hyperhydricity can be overcome by decreasing the relative matric potential of the media, and respectively the availability of water, as represented by increasing gelrite concentrations. Satisfactory reduction in hyperhydricity was achieved only at 16 g l-1 gelrite, however the regeneration also decreased. The nature of the gelling agent is therefore essential for the successful control of this phenomenon. It appears that a crucial prerequisite for the reduction of hyperhydricity in tissue cultures of A. polyphylla is the gaseous exchange between the in vitro atmosphere and the outside environment. In ventilated cultures, achieved by using a modified lid with a hole (d = 7 mm) covered with polyester or cotton mesh, hyperhydricity was completely eliminated, irrespectively of the type of gelling agent. Ventilation was further advantageous for the in vitro regenerants by increasing their leaf chlorophyll content as well as epicuticular wax deposition, the last one being indicative of the development of the water loss regulation mechanisms of explants. The increased culture ventilation, however, was negatively correlated with the regeneration rate and shoot growth. Endogenous CKs were measured in in vitro regenerants after an eight-week cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of hyperhydric shoots was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.
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