Role of physiological factors in tulip bulb scale micropropagation

The propagation of tulip is severely hampered by the slow traditional propagation methods. With the increased need for new varieties with e.g. an improved resistance against pests, next to the continuous interest in new varieties with e.g different colors, the 25-30 years needed before a new cultivar can be commercially introduced is a problem. Therefore, alternative, faster methods to propagate tulip are wanted and the application of tissue culture techniques seems very promising. However, still a number of problems exist; especially tissue culture starting from tulip bulb scales has been proven cumbersome, because the explants are only vital and able to regenerate shoots (from which new bulbs can be produced) in a very limited period of the season. Our project was aimed at the elucidation of the physiological factors responsible for this loss of vitality and regeneration potential.

Two hypotheses were tested. In one hypothesis the role of oxidative stress in these problems was evaluated. If tissues are not able to process the reactive oxygen species produced during the cutting of an explant or to cope with the stress during the incubation, the tissue can be severely damaged by oxidative stress phenomena like membrane damage, production of toxic substances (TBArs or phenolics) and damage to DNA or structural proteins, leading to loss of viability and death of the explants. In our second hypothesis the role of tulipaline A (α-methylene-γ-butyrolactone) in the loss of vitality was evaluated: since tulipaline A has been implicated in fungal defence of tulip bulb scale tissue as being a toxic substance to the fungus which is released upon wounding, we hypothesized that release or production of tulipaline A might cause toxic reactions in tulip tissue, leading to loss of vitality and death of the explants upon cutting.

To test our hypothesis of extreme sensitivity to oxidative stress we used different approaches in the first place: we compared physiological parameters of the stress response in regenerating tulip explants (bulb scales and stalk explants) with non-regenerating tulip bulb scale explants. In a second approach we applied different tissue culture conditions (differing oxygen concentrations and anti-oxidative additives to the nutrient medium) and measured physiological parameters implicated in the oxygen stress response (i.e. changes in enzyme activities (LOX, SOD, peroxidase, catalase, PAL and polyphenoloxidase), membrane composition and production of TBArs and phenolics). To obtain an indication of the role of the changed oxygen conditions a further comparison was made with other tissue culture systems like lily and apple stem segments. This also enabled us to determine in which phase of the regeneration process the changed conditions have the most effect.

The physiological response of regenerating and non-regenerating scale explants did not differ significantly. The response was furthermore comparable to the pattern of changes observed in well regenerating tulip stalk explants. Some differences were observed after 2-3 weeks of incubation; these differences are probably caused by the poor viability of non- regenerating explants and not the cause of this poor viability since most damaging events and the start of regeneration are supposed to occur before the stage in which the differences are found. The experiments with modified oxygen conditions in which we applied 100 % oxygen to increase or 2 % oxygen to reduce oxygen stress also yielded no indications of an extreme sensitivity of tulip bulb scale tissue to oxidative stress. Regeneration was not improved by the application of low oxygen conditions and vitality was not negatively influenced by high oxygen conditions; high oxygen conditions seemed favourable for faster growth of the regenerating structures but slightly negative to the formation of primordia. No improvement of vitality could be observed after addition of anti-oxidative substances, this also suggesting no extreme sensitivity of the tulip bulb scale explants for oxidative stress.

Tulipaline content was determined in stalk explants and in regenerating and non- regenerating bulb scale explants. Surprisingly, tulipaline A and not its precursor tuliposide A was the most abundant compound in scale explants, although tulipaline was expected to be too reactive to occur in the free form in living cells. Therefore, it is likely that in bulb scales the tulipaline is present either in specialised. vesicles or in an environment which reduces its reactivity. The amounts of tulipaline we found did differ strongly between stalks and bulb scales: the higher amounts of tulipaline in bulb scales might suggest a role in the lower regeneration potential but the comparison between regenerating and non-regenerating bulb scale explants did not confirm this suggestion. The tulipaline content changed during incubation; a clear decrease upon cutting the explants is seen, resembling a wounding effect. The content did increase later on during incubation of the explants indicating production of tulipaline A but still no clear relation with vitality could be established. Furthermore, a comparison between initial tulipaline content and browning response for different tulip cultivars, indicated a negative instead of a positive correlation between the two parameters. All these observations suggest that the low viability of tulip bulb scale explants is not directly related to a toxic effect of tulipaline A.

Since both tested hypotheses do not seem to be related to the poor viability of tulip bulb scale explants outside the period in which regeneration is possible, the cause of this problem has to be found elsewhere. Probably the gradual progress of senescence leads to a stage where no redifferentiation is possible. Therefore, the development of a method to arrest bulb material in a physiological state where regeneration is possible might be necessary for the of micropropagation of tulip via bulb scale explants.