Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O(2)(*-)), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial 'burst' in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O(2)(*-) production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

Shoot regeneration and occurrence of hyperhydricity in Aloe polyphylla were greatly affected by the type of gelling agent. The use of gelrite resulted in a significantly lower multiplication and almost four times higher hyperhydricity (65%) compared to agar-solidified medium. Gelrite was further selected to evaluate if hyperhydricity can be overcome by altering the physical properties of the gel, as represented by increasing gelrite concentrations. Four concentrations of gelrite (0, 2.4, 6 and 16 g l -1 ) were tested in combination with zeatin, N 6 -benzyladenine (BA) or thidiazuron (TDZ). Almost all explants grown in liquid media in the presence of cytokinins became hyperhydric and lost their ability to regenerate. The greatest shoot formation was obtained on media with 2.4 g l -1 gelrite and 5 lM zeatin or BA, however hyperhydricity was very high. Satisfactory reduction in hyperhydricity was achieved only at 16 g l -1 gelrite, under which conditions the multiplication also decreased. The use of TDZ resulted in very low shoot regeneration and high hyperhydricity irrespective of the gelrite concentration.

The role of nitrogen (N) supplied as inorganic ammonium (NH 4 ? ) and nitrate (NO 3 -), and organic glutamine on shoot regeneration, incidence of hyperhydricity and production of good quality shoots in Aloe polyphylla explants was investigated. The omission of total N from the culture medium resulted in low proliferation 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 NO 3 -was used as the sole N source, shoots of good quality were produced and hyperhydricity was completely eliminated. Overall, the MS N combination was superior to any single N source for proliferation and growth of shoots, suggesting a synergistic effect between NH 4 ? and NO 3 -on shoot regeneration. Moreover, the relative amounts of NH 4 ? and NO 3-also influenced the parameters tested. The highest regeneration was obtained with NH 4 ? :NO 3 -ratios (mM) of 20:40, 30:30 and 40:20. Decreasing the ratio of NH 4 ? :NO 3 -lowered the incidence of hyperhydricity. This study also demonstrates the potential of glutamine as the sole source of N, since its application resulted in the production of good quality shoots and almost no hyperhydricity.

In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH 4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N 6 -benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 lM. Elevating the levels of NH 4 + , in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH 4 + (10.3 mM). However, in the presence of higher NH 4 + concentrations, CKs increased hyperhydricity in a concentration-dependant 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 NH 4 + and low BA or zeatin (5 lM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.

An investigation into the role of ventilation to reduce hyperhydricity in tissue cultures of Aloe polyphylla Schönland ex Pillans revealed that gaseous exchange between the in-vitro atmosphere and the outside environment is an essential prerequisite for controlling this disorder. In closed culture vessels, hyperhydricity affected as much as 84% of the newly-formed shoots on media gelled with gelrite. The leaves of hyperhydric shoots had a bright green colour, smooth epidermis and large, open stomata. Gaseous exchange was promoted by using modified lids with a hole covered with polyester or cotton mesh. In ventilated cultures, hyperhydricity was completely eliminated irrespective of the type of gelling agent used. Natural ventilation was further advantageous for the microplants in terms of leaf chlorophyll content as well as the deposition of epicuticular wax, indicating the onset of mechanisms that regulate water loss from the explants. Although culture ventilation was negatively correlated to the regeneration rate and shoot growth, it has the potential to control the appearance of abnormal phenotypes and can be easily adopted for routine A. polyphylla propagation in vitro.

Increasing fructokinase (FRK) activity in cotton (Gossypium hirsutum L.) plants may reduce fructose inhibition of sucrose synthase (Sus) and lead to improved fibre yield and quality. Cotton was transformed with a tomato (Solanum lycopersicum L.) fructokinase gene (LeFRK1) under the control of the CMV 35S promoter. In a greenhouse, the LeFRK1 plants had increased fibre and leaf FRK activity over nonexpressing nulls, but not improved fibre length and strength. Compared with the nulls, LeFRK1 plants yielded 13–100% more seed-cotton mass per boll and more bolls per plant, and therefore more seed cotton and fibre yield per plant. The enhanced yield was related to a greater seed number per boll for LeFRK1 plants. Photosynthetic rates were not appreciably different among genotypes. However, more area per leaf and leaf number (in some instances) for LeFRK1 plants than for nulls enhanced the capacity for C gain. Larger leaf areas for LeFRK1 plants were associated with larger stem diameters. Lower sucrose levels in developing leaves of LeFRK1 plants suggest that LeFRK1 overexpression leads to improved in vivo Sus activity in developing leaves and possibly in developing seeds. The improvement in yield for LeFRK1 plants may also be the result of improvements in photosynthate supply as a consequence of greater leaf area.

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N 6 -benzyladenine (BA) or zeatin (0, 5 and 15 lM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). 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 M. Ivanova AE J. Van Staden (

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