Tulip bulbs (Tulipa gesneriana L.), with terminal buds containing a complete flower, require a period of low temperature to prepare the bud for floral stalk elongation and full flower development at subsequent higher temperatures. For the cultivar Apeldoorn, a dry- storage treatment of 12 weeks at 5°C prior to planting at 20°C, win lead to proper floral stalk elongation and full flower development. Shorter periods at 5°C usually result in slower shoot elongation and delayed flowering. Without any cold treatment, the growth of the shoot is strongly reduced and often flower abortion occurs. In these processes, the involvement of gibberellins (GAs) has been implicated, because application of GAs could partly replace the cold treatment. In addition, GA biosynthesis inhibitors could reduce the floral stalk elongation of cold-treated bulbs and this effect was reversed by simultaneous application of GA.
In horticultural practice, there is a need for a practical assay to test whether a particular bulb has received a proper cold treatment. The amount of GAs or of one particular GA, might provide a suitable parameter in a test for properly cold-treated bulbs.
In this study, the role of GAs in the cold requirement of tulip was investigated by analysing the GA levels in cooled and noncooled tulip bulbs, and by studying the effect and metabolism of applied GAs in combination with a' GA biosynthesis inhibitor.
An inventory was made of GAs, in sprouts of cooled (12 weeks 5°C) and noncooled bulbs (12 weeks 17°C) (chapter 3). By combined gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (SIM), GA 1 , GA 4 , GA 9 , GA 12 , GA 24 , GA 34 and three GA-related compounds were detected. They all occurred in sprouts of both cooled and noncooled bulbs. Most of them were found in the conjugated form as well. Among these GAs, GA 4 and/or GA 1 might be the active forms, the others being precursors (GA 9 , GA 12 , and GA 24 ), or an inactivation product (GA 34 ).
Using GC-SIM and deuterated GAs as internal standards, the changes in endogenous GA levels were measured in sprouts and basal plates during cooled and noncooled bulb storage, as well as after planting these bulbs (chapter 4). GA 4 and GA 24 were the major occurring gibberellins, with levels up to ca. 10 ng per sprout or basal plate. GA 1 , GA 9 and GA 34 were present in much lower amounts. The levels of GA 12 and of the GA conjugates and GA-related compounds were not analysed.
During bulb storage, the level of GA 4 per sprout increased, especially in noncooled bulbs. After 12 weeks, these sprouts contained more GA 4 and also more GA 1 than cooled sprouts. However, sprouts in noncooled bulbs did hardly show any development after planting and it is unlikely that the increased level at the end of bulb storage is correlated with floral stalk elongation at subsequent higher temperatures. In the basal plates no significant changes occurred in the GA levels during storage. Therefore, the GA content in sprouts or basal plates at the end of bulb storage, cannot be used as marker in a test for properly cold-treated bulbs.
After planting cooled bulbs, the sprouts started to grow and within the first 11 days the level of GA 4 in the floral sulks increased. In planted noncooled bulbs, sprout growth was negligible and an increase in the level of GA 4 did not occur.
The biological activity of GA 1 , GA 4 and GA 9 , was tested on isolated sprouts, cultivated on a liquid medium in vitro (chapter 5). To compare the sensitivity to exogenous GAs, sprouts from both cooled and noncooled bulbs were used. The growth retardant paclobutrazol was used to study the role of GA biosynthesis. The growth of these isolated sprouts, the response to GAs and the effect of paclobutrazol, appeared to be dependent not only on the pretreatment of the bulbs, but also on the time in the season that the sprouts were isolated and incubated.
At early starting dates of incubation, floral stalks from both cooled and noncooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than the response of sprouts from noncooled bulbs. At later starts, considerable floral stalk elongation already occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and noncooled bulbs was stimulated by GA application. The three tested GAs were not significantly different in stimulating floral stalk elongation. The effect of paclobutrazol. was reversed by simultaneous application of GA. The results of these in vitro experiments demonstrated that, although depending on the time of the year, sprouts from both cooled and noncooled bulbs are responsive to exogenous GAs. Moreover, sprouts from both bulb treatments are capable of GA biosynthesis. The increasing performance of the isolated sprouts when incubated at later starting dates, and the increasing effect of paclobutrazol on these sprouts, suggested an increase in the availability of precursors for the synthesis of GAs. Apparently, low temperatures as well as bulb storage itself enhance GA biosynthesis and GA sensitivity, and consequently floral stalk elongation after planting when conditions are favourable for growth.
The isolated sprouts did not develop a full-grown flower without the addition of GA. GA 4 was more effective than GA 9 in stimulating this flower development. GA 1 could also stimulate flower development, but was no more effective than GA 4 .
The activity of applied GA 9 might be due to its conversion to GA 4 . GA 4 on its turn, might have to be converted to GA 1 before becoming biologically active. The metabolism of applied GA 9 was studied, with the purpose to investigate whether tulip sprouts are able to metabolize GA 9 to biologically active GA 4 or GA 1 , and whether sprouts from cooled and noncooled bulbs show differences in GA metabolism (chapter 6). [ 3H]GA 9 and [ 2H]GA 9 were applied to isolated sprouts by injection into the floral stalk and the metabolites were analysed in the sprouts after 24 h. According to HPLC analyses, [ 3H]GA 9 was converted to GA 4 -like and GA 34 -like compounds. The labelled metabolites of [ 2H]GA 9 were identified by GC-SIM, which demonstrated the conversion of [ 2H]GA 9 to [ 2H]GA 4 and [ 2H]GA 34 . Sprouts from both cooled and noncooled bulbs were able to convert GA 9 to GA 4 and GA 34in vitro . No evidence was found for the production of labelled GA 1 .In the presence of prohexadione (BX-112), known for its inhibiting effect on 2- and 3β-hydroxylations of GAs, the formation of [ 2H] metabolites was less or absent.
In conclusion, there is no direct correlation between the cold-stimulated growth and a change in the endogenous GA status in sprouts or basal plates during cold bulb storage. Further, the sensitivity to GAs increases in cooled sprouts, but also noncooled sprouts are responsive to applied GAs, and GA sensitivity apparently is not limiting for the development of noncooled sprouts in vitro . After cooled bulb storage, GA biosynthesis is essential for floral stalk elongation to proceed. The increase in the level of GA 4 in the growing floral stalks of cooled bulbs, the response of isolated sprouts to GA 4 and the inability of isolated sprouts to produce detectable amounts of GA 1 from applied GA 9 , support the hypothesis that GA 4 is the major intrinsically active GA in the floral stalk elongation of tulip.