Low temperature influence on flowering in Citrus. The separation of inductive and bud dormancy releasing effects

Transcription

1 PHYSIOLOGIA PLANTARUM 86: Copenhagen 1992 Low temperature influence on flowering in Citrus. The separation of inductive and bud dormancy releasing effects A. Garcia-Luis, M. Kanduser, P. Santamarina and J. L. Guardiola Garcia-LuIs, A., Kanduser, M., Santamarina, P. and Guardiola. 1. L Low temperature influence on flowering in Citrus. The separation of inductive and bud dormancy releasing effects. - Physio!. Plant. 86: The flowering response of Owari Satsuma mandarin (Citrus unshiu Marc) to low temperature treatments has been determined using potted trees and in vitro bud cultures. In potted trees the chilling treatments released bud dormancy and enhanced both sprouting and flowering, but these two responses could not be separated. However, bud cultures showed no dormancy, and a specific effect of low temperature on flower induction was demonstrated. Low temperature appears to have a dual effect, releasing bud dormancy and inducing flowering. Potential flower buds have a deeper dormancy than vegetative buds, and the first stages of flower initiation seem to occur before the winter rest period. Key words - Citrus (flowering), Citrus unshiu, flower induction, flowering. In vitro flowering, Satsuma mandarin. A. Garcia-Luis (corresponding author) et al., Dept Biologia Vegetal, Univ. Politernica, Valencia, Spain. Introduction When grown In subtropical climates, Citrus species mainly bloom in the spring following the winter rest period, similar to the normal response for temperatezone deciduous fruit trees. In both groups of trees flowering is enhanced by winter chilling, but it is currently believed that the mechanism of this effect is different. In deciduous fruit trees, chilling is required to release differentiated flower buds from dormancy, and flower initiation occurs long before the winter rest period (Gur 1985). In contrast, in Citrus the differentiation of the floral organs does not occur until the first stages of bud sprouting (Randhawa and Dinsa 1947, Ayalon and Monselise 1960, Iwahori and Oohata 1981, Guardiola et a!. 1982), vegetative and reproductive buds being anatomically indistinguishable during the winter rest period. Flower initiation in Citrus is considered to follow an inductive effect of low temperature (Monselise 1985, Davenport 1990). This putative inductive effect of low temperature has not been unequivocally demonstrated. In some experi- ments, it has been shown that chilling enhances bud sprouting (Moss 1969, Southwick and Davenport 1986). The increase in flowering brought about by chilling is paralleled by an increase in the number of developing sprouts, rather than resulting from a reduction in the number of vegetative shoots, which would demonstrate an inductive effect (Guardiola 1981). An alternative interpretation of the inductive low temperature effect could be, therefore, that chilling is required to release initiated but not yet differentiated flower buds from dormancy, much as occurs in deciduous fruit trees. There is biochemical and physiological evidence for early bud initiation during late summer (Iwahori et a!. 1990). To distinguish between these two alternatives, the low temperature effect on flowering needs to be separated experimentally from the effect on bud dormancy. In this study cultured Citrus buds were allowed to form flowers in vitro (Garda-LuIs et a!. 1989, Tisserat et a!. 1990). In this way a specific effect of low temperature on flower induction, independent from the release of bud dormancy, has been demonstrated. Received 3 April. 1992; revised 3 1uly, 1992; in final form 7 September, Physiol. Plant. 86. t<)9~

2 Materials and methods Flowerin~ response of trees In these experiments 3-year-old fruit bearing trees of Owari Satsuma mandarin (Cilrus unshiu Marc) grafted on Carrizo citrange (Poncirus trifoliala x Cilrus sinensis) rootstocks grown in 15-1 pots, were used. These potted trees were kept outdoors for several months alongside with the adult trees used as a source of bud ~xplants (see below), and brought into the growth rooms at different times to determine the effect of chilling on sprouting and flowering. Prior to the experiments the trees were thus subjected to ambient temperatures which, until the end of September, were in the range of C (maximum daily temperature) and C (minimum). Subsequently the temperature decreased gradually, falling to C (maximum) and 6-12 C (minimum) in November, temperatures that induce flowering (Moss 1976). The flowering behaviour of the trees was determined at two times, on September 26 (non-induced trees), and November 20 (partially induced trees). For the experiments, 72 trees uniform with regard to vigour and crop load, were selected from an initial population of 120 trees. At each time, 36 of these selected trees were transferred to growth rooms, and kept under a daily 16-h light and 8-h dark period with a photosynthetic photon flux density of 600!J.mol m- 2 S-I at plant level provided by a mixture of fluorescent (Sylvania cool white F48TI2/CWIVHO) and incandescent lamps. Eighteen of the trees were placed at 22 ± 1 C (day) and 13 ± 1 C (night); the other 18 were kept for 53 days in inductive cold conditions (15 ± 1 C and 10 ± 1 C day and night temperatures, respectively), and then transferred to the high temperature regime. At the beginning of the temperature treatments, half of the number of trees from each set of temperature conditions were defruited, to determine the effect of the fruit on flowering. Bud sprouting and the nature of the developed shoots, either vegetative or flowering, were recorded periodically until the completion of budburst, and the results expressed as a percentage of the total number of nodes of the plant. Only those nodes located on stems less than one year old at the beginning of the experiment were considered, as older buds seldom burst. Since some developing shoots abscised before their developmental stage could be ascertained, they were recorded only as sprouted nodes; so that this number (Tab. 1) is always higher than the sum of vegetative and flowering nodes. The results were subjected to a complete analysis of variance, by which the effect of the 3 main variables [date (experiment), low-temperature treatment and presence of the fruit] and their interactions were determined. Each of the 9 trees of each set of conditions served as a replicate. Since the presence of the fruit had no significant effect on sprouting and flowering, the mean values for the + fruit and - fruit trees are presented in Tab. 1; each number is the average of 18 trees. Flowering of bud cultures in vitro Lateral buds were obtained fr~m shoots developed during the summer flush of growth in 20-year-old, field grown, fruit-bearing Owari Satsuma mandarin trees grafted on sour orange rootstocks (Citrus aurantium L.) grown under identical environmental conditions as those of the potted trees. At the time of sampling the extension growth of the shoots had ceased, and the trees had entered into the winter rest. In one experiment, the behaviour of buds from shoots formed during the spring flush of growth was also determined. The shoots were Tab. 1. Effects of chilling treatments on bud sprouting and flowering of potted trees. Trees were forced to sprout in a growth room Jt 22/13 C, either directly (control trees) or after 53 days at 15/l0 C (chilled trees). Two separjte experiments with the same design were performed starting on September 26 and November 20, respectively. Analysis of variance performed using V x transformed data. NS, not significant;' and.., statistically significant at 5% and 1%, respectively. Experiment and treatments September 26 Control Chilled November 20 Control Chilled Parameter (degress of freedom) Experimental factors Experiment (date) Chilling treatment Remainder Experimental error Nodes Nodes Nodes sprouting, % flowering, % vegetative, % <U Sum of squares Sum of squares Sum of squares (% of total) (% of total) (% of total) (7) 2140 (100) (100) 87.3 (100) (1) 48.3 (23) 61.7 (29) 13.9 (16) (1) (64) (62) 22.8 (26) (5) 28.6 NS (13) 18.3 NS (9) 50.6 (58) (64) Phl'siol. Plant

3 brought into the laboratory and, after surface sterilization for 1 h in a 10% sodium hypochlorite solution, nodal explants consisting of 1-cm-long stem pieces with the unsprouted bud were prepared. This operation was performed either immediately after collecting the shoots or after exposure of the shoots to the appropriate temperature treatment (details given below). Only the 10 most apical nodes of the shoots were used. A minimum of 80 nodal explants per treatment were planted in a solid 1.2% agar (Oxoid, agar technical No. 3) medium including Murashige and Skoog (1962) inorganic constituents supplemented with 90 mm sucrose and 1 mg I-I 6-benzylaminopurine (Garcia-Luis et al. 1989). Cultures were incubated under a 16 h photoperiod in dim light (30 [Lmol m- 2 S-I) provided by Sylvania Gro-Iux lamps, either at a temperature of 22/13 ± 1 C (light and dark respectively), or 15/10 ± 1 C, or an appropriate combination (details given with the experiments). Bud sprouting and flowering were recorded periodically up to day 42; keeping the cultures for a longer time did not result in higher flowering levels. Results and discussion The potted trees had not sprouted after 53 days at IS/10 C, which is in agreement with previous reports (Moss 1969, 1976, Poerwanto and Inoue 1990). Sprouting occurred after placing the trees at 22/13 C. For the control trees budburst was completed after 27 days in the September experiment and 20 days in November. Sprouting was accelerated by the cold treatment of the trees, and took place after 16 (September) and 5 (November) days of transfer of the chilled plants to 22/13 C (data not shown). The percentages of nodes either sprouting or forming flowers were higher in the November than in the September experiment, and further increase was caused by the low temperature treatment of the trees (Tab. 1). These two effects, time of forcing and low temperature treatment, were the only significant ones and accounted, in an additive way, for 90% of the differences in sprouting and flowering as shown by the analysis of variance. Both phenomena were influenced to a similar extent by low temperature. The additive nature of these Tab. 2. The effect of low temperature (IS/lO C) treatment of bud cultures on sprouting and flowering. Buds sampled on November 7 and kept in culture at ls/looe for the time indicated. then transferred to n/l3 e. Bud sprouting and flowering measured after 42 days in culture. Days at Buds Buds lsilooe sprouting. % flowering, % o IS two effects reflect the time dependence of the flowerin effect of low temperatures; it has been shown previousi~ that the low temperature effect on flowering is not vet saturated after 8 weeks of cold treatment (Moss 1969 Southwick and Davenport 1986); hence our :-Iovembe~ trees were not fully induced by the outdoor temperatu_ res. Presence of the fruit had no effect, which seems in contradiction to the inhibitory effect of the fruit On flowering reported by Moss (1971). However, it has been reported that this effect decreases as the fruit matures (Garcia-Luis et al. 1986), and the fruits in our experiment were nearly mature. The number of vegetative shoots was affected in a different way by the experimental parameters as shawn by the analysis of variance (Tab. 1). Their number was increased by the low temperature treatment of the plants in September to a value close to that in the November experiment. At that date, the low temperature treatment had no effect on the numher of vegetative shoots formed. Overall, these results failed to show any transformation of vegetative shoots into generative ones. It is possible to explain them assuming the buds were already differentiated, although initiation was not visible yet, at the start of the experiment, the chilling requirements of the vegetative buds being lower than for floral buds and having been met naturally by the outdoor temperatures during early November. A similar explanation for the low temperature effect on olive flowering recently has been put forward (Rallo and Martin 1991). Previously it has been contended that this evergreen tree is cold-induced to flower (Hackett and Hartmann 1967). Buds in culture behaved differently to those of intact potted trees. All the buds sprouted irrespective of the time of sampling and the temperature of incubation. Differences in the time-course of sprouting were recorded (data not shown), which were probably determined by a weak innate dormancy of the buds (Altman and Goren 1974, Guardiola et al. 1982). However, both the chilling requirements of the potted trees for sprouting and their inability to sprout at C, seem to be imposed by the correlative influence of other tissues of the tree, and the culture of excised buds in vitro enabled us to determine the influence of low temperatures on flower induction without the interfering effect on sprouting. The treatment with low temperature (15/W C) of buds sampled on November 7, before the chilling requirements of the trees had been met, resulted in an increase in flowering (Tab. 2). A significant promotion in flowering was detected after 6 days of cold treatment.. The flowering response was even higher after IS days of cold treatment, and buds treated in this way flowered in a higher proportion than buds on potted trees. Since flowers formed on otherwise vegetative buds, it appears that low temperatures induce flowering; however. an alternative explanation would be that high temperatures Physlol. PI;Jnt. 86. l\)lj~

4 Tab. 3. The effect of temperature of incubation on the flowering response of cultured buds from chilled trees. Buds sampled on December 20 from spring-flush and summer-flush shoots. Bud sprouting and flowering recorded after 42 days in culture. Shoot type and Buds Buds telnperature of incubation sprouting. % flowering. % Spring-flush buds C [5/l0 C Summer-flush buds 22/1 3 C l5/l0 C (22113 C) during the early stages of sprouting inhibit the development of the flower parts of previously induced buds, and allow them to revert to the vegetative condition. This effect has been demonstrated with potted trees (Moss 1969), but using temperatures markedly higher (30/1 SoC) than those used in this study (22/13 C). In an attempt to distinguish between these two possible mechanisms of action, the experiment was repeated with buds sampled on December 20, by which time their chilling requirements for flower induction had been met. The same flowering percentage was obtained at 22/13 C as at IS/10 C, without any evidence of an inhibitory effect of the high temperature on flower development (Tab. 3). The behaviour of the buds from shoots formed during the spring flush of growth was also investigated in this experiment. The spring-formed buds flowered in a lower proportion than those formed during summer, and their flowering was not increased by the in vitro low temperature treatment. Bud sprouting and flower formation took place during the cold temperature treatment, which lasted for 42 days; hence, flowering could not have been increased further by a longer exposure to low temperature. The different buds differ in their flowering response to low temperature. Flowers were also induced when the entire shoots were exposed to low temperatures prior to bud culture. Buds from shoots kept at IS/10 C for 6 days formed more flowers (17% of the cultures) than buds from untreated shoots (1 %), an effect already reported by Southwick and Davenport (1986). Defoliating the shoots before the low temperature treatment greatly reduced the flowering response (S%). Exposure of the shoots to 4 C did not induce flowering; on the contrary, it reduced the flowering of partially induced buds (Tab. 4). In summary, the results of the experiments presented here demonstrate that low temperature treatments can release the buds from dormancy and induce flower formation. The effective temperatures are higher than those reported effective for deciduous fruit trees (Gur 1985). The primary effect is in the bud itself as it occurs in vitro in the absence of expanded leaves. The effect of leaves observed in bud induction in intact shoots may indicate that the leaves normally provide an essential Phl'siol. PI.nt Tab. 4. The effect of cold (4 C) treatment of the shoots on bud sprouting and flowering in culture. The shoots were sampled on November 7 and held in the dark at low temperature for the time indicated before culture. Bud sprouting and flowering measured after 42 days culture at 22/13 C. Days of cold Nodes Nodes treatment sprouting. % flowering. % metabolite, which is a component of our culture medium. We suggest the following interpretation for the regulation of flowering in Citrus. Some early events of flower initiation occur in some buds before the wintl.:r rest period (Guardiola 1981, Iwahori et al. 1990). Vegetative buds have a low chilling requirement and sprout readily in spring. The partially initiated flower buds have a higher chilling requirement, and low temperatures simultaneously release the buds from dormancy and allow the completion of flower initiation. Those potentially flowering buds whose chilling requirements have not been met remain quiescent in spring, and in this way the often reported relationship between sprouting and flowering is established. Acknow/edgemenls - Thanks are due to Mr. M. Sanchez Perales for technical assistance. This research was financed by CtCYT (Grant PB ). References Altman, A. & Goren, R Growth and dormancy cycles in Cilrus bud cultures and their hormonal contro!' - Physio!. Plant. 30: Ayalon, S. & Monselise, S. P flower bud induction and differentiation in the Shamouti orange. - Proc. Am. Soc. Hortic. Sci. 75: Davenport, T. L Cirrus flowering. - Hortic. Rev. [2: Garcia-Luis, A., Almela, Y., Monerri, c., Agusti. M. & Guardiola, J. L Inhibition of flowering in vivo by existing fruits and applied growth regulators in Cilrus unshiu. - Physio!. Plant. 66: , Santamarina, P. & Guardiola, J. L Flower formation from Cilrus unshiu buds cultured in vitro. - Ann. Bot. 64: Guardiola, J. L Flower initiation and development in Cilrus Proc. Int. Soc. Citric.: , Monerri, C. & Agusti, M The inhibitory effect of gibberellic acid on flowering in Cilrus. - Physio!. Plant. 55: Gur, A Rosaceae-Deciduous fruit trees. -In Handbook of Flowering, Vol. I (A. H. Halevy, ed.), pp CRC Inc., Boca Raton, FL. ISBN Hackett, W. P. & Hartmann, H. T The influence of temperature on floral initiation in the olive. - Physio!. Plant. 20: Iwahori, S. & Oohata, J. T Control of flowering of Satsuma mandarin (Cilrus ul1shiu Marc.) with gibberellin Proc. Int. Soc. Citric.: , Garcia-Luis, A., Santamarina, P., Monerri, C. & Guardiola, J. L The influence of ringing on bud develop 6S1

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