Regulation of hyperhydricity in Aloe polyphylla propagated in vitro.
Ivanova, Mariyana Vasileva.
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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.