Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system

Background

Freezing tolerance is an important factor in the geographical distribution of plants and strongly influences crop yield. Many plants increase their freezing tolerance during exposure to low, nonfreezing temperatures in a process termed cold acclimation. There is considerable natural variation in the cold acclimation capacity of Arabidopsis that has been used to study the molecular basis of this trait. Accurate methods for the quantitation of freezing damage in leaves that include spatial information about the distribution of damage and the possibility to screen large populations of plants are necessary, but currently not available. In addition, currently used standard methods such as electrolyte leakage assays are very laborious and therefore not easily applicable for large-scale screening purposes.

Results

We have performed freezing experiments with the Arabidopsis accessions C24 and Tenela, which differ strongly in their freezing tolerance, both before and after cold acclimation. Freezing tolerance of detached leaves was investigated using the well established electrolyte leakage assay as a reference. Chlorophyll fluorescence imaging was used as an alternative method that provides spatial resolution of freezing damage over the leaf area. With both methods, LT₅₀ values (i.e. temperature where 50% damage occurred) could be derived as quantitative measures of leaf freezing tolerance. Both methods revealed the expected differences between acclimated and nonacclimated plants and between the two accessions and LT₅₀ values were tightly correlated. However, electrolyte leakage assays consistently yielded higher LT₅₀ values than chlorophyll fluorescence imaging. This was to a large part due to the incubation of leaves for electrolyte leakage measurements in distilled water, which apparently led to secondary damage, while this pre-incubation was not necessary for the chlorophyll fluorescence measurements.

Conclusion

Chlorophyll fluorescence imaging is an alternative method to accurately determine the freezing tolerance of leaves. It is quick and inexpensive and the system could potentially be used for large scale screening, allowing new approaches to elucidate the molecular basis of plant freezing tolerance.     

Evaluation of three methods to assess winter hardiness of strawberry genotypes

Summary

In northern regions, winter injury is a frequent problem in strawberry (Fragaria X ananassa Duch.) production. Controlled freezing tests provide an option for screening of winter survival potential in different genotypes. In this study, winter hardiness of strawberry genotypes was evaluated by three low-temperature procedures: an artificial hardening-dehardening-rehardening programme accompanied by freezing tests, prolonged freezing test, and laboratory cold hardiness measurements of field-grown plants. Rooted runner cuttings of five June-bearing strawberry cultivars and three new selections were tested in the course of two years. Relative winter hardiness of the genotypes could not be predicted by the hardening-dehardening-rehardening programme employed in the present study. Prolonged freezing at –6?C yielded promising results, whereas storage at _4?C and –8?C failed in discrimination between genotypes. Field-grown plants were sampled for cold hardiness measurements in January, March and April. The genotypes were best differentiated in January, at a fully acclimated state. The results indicate that strawberry winter hardiness can be evaluated by testing differences in the ability to survive lengthy exposure to sub-lethal low temperatures, or by applying conventional freezing tests to measure cold hardiness in different genotypes after field acclimation.     

Comparative analysis of laboratory freezing methods to establish cold tolerance of detached rhizomes and intact crowns in garden chrysanthemums (Dendranthema × grandiflora Tzvelv.)

Since 1926, the University of Minnesota herbaceous perennial breeding program has released N = 84 garden chrysanthemum cultivars (Dendranthema × grandiflora) with important traits for northern temperate climates, such as winter hardiness. Recent breeding objectives have identified the need for development of non-destructive phenotypic markers and destructive laboratory freezing tests for co-selection of cold tolerance in Dendranthema, Gaura, and other herbaceous perennial flower crops. Such methods have become critical to flower breeding programs in northern temperate regions during periods of above-average winter temperatures and minimal snow cover due to the ‘el Niño’ effect. Two different, destructive laboratory freezing tests were evaluated for their effectiveness in determining cold tolerance. Acclimated crowns of n = 6 garden chrysanthemum genotypes, ranging from hardy to non-hardy in USDA Z3-4, were used in Omega Block (using detached, emergent rhizomes) and chamber (using entire, intact crowns with emergent, non-emergent rhizomes) freezing test methods. Comparative winter survival in the field was monitored over locations and years. Cold tolerance was assessed at 0 to −12 °C with varying ramp and soak time periods. LT₅₀ temperatures and number of living emergent rhizomes were also determined. Rhizome quality at 1, 3, and 5 cm depths was rated for regrowth on a 0 (dead) to 5 (undamaged) scale. The chamber freezing method was the most powerful to discern accurate LT₅₀ values. Cold tolerant genotypes included ‘Duluth’ and Mn. Selection 98-89-7 (LT₅₀ = −12 °C). Four genotypes were rated as non-hardy (LT₅₀ = ≤−10 °C). Cold-tolerant genotypes also had significantly higher regrowth ratings for rhizomes at 1 and 3 cm depths. Future research will implement the chamber freezing method to assay the inheritance of winter hardiness in intact crowns of segregating populations.     

核桃属植物休眠期的抗寒性鉴定

 以核桃属（Juglans）中的普通核桃、核桃楸、河北核桃和黑核桃4个种的18份种质休眠期的1年生枝条为试验材料,采用电解质渗出率法、组织褐变法及氯化三苯基四氮唑（TTC）染色法并配合Logistic方程确定枝条的低温半致死温度（LT₅₀）;分析了LT₅₀与枝条解剖结构之间的相关性。结果表明：随着处理温度的降低,休眠期各核桃种质1年生枝条的相对电导率（REC）均呈上升趋势;供试种质间抗寒性差异大,LT₅₀在–38 ~–22 ℃之间;种间抗寒性差异明显,依次为黑核桃 > 核桃楸、河北核桃 > 普通核桃。电解质渗出率法、组织褐变法及TTC染色法均可用于休眠期核桃属植物的抗寒性鉴定,尤其以电解质渗出率法准确度高。用略高于大部分核桃种质LT₅₀的温度（–24 ℃）处理枝条,其相对电导率与LT₅₀之间达到极显著正相关,此温度处理下的相对电导率以及枝条的LT₅₀均可作为休眠期核桃属抗寒性鉴定的理化指标。核桃属4树种1年生茎粗大致相同的枝条中各组织的厚度差异显著,其中木栓层厚度及其在茎结构中所占的比率与枝条的LT₅₀之间达到极显著负相关,可作为休眠期核桃抗寒性鉴定的形态结构指标。     

Some physiological changes in strawberry (Fragaria × ananassa ‘Camarosa’) plants under heat stress

Summary

The effects of heat injury induced by long exposures were evaluated in strawberry (Fragaria × ananassa ‘plants’) Camarosa in this study. Seedlings were grown in 14 × 12 cm pots using perlite for three weeks at 25/10°C day/night temperature, and watered daily by modified 1/3 Hoagland nutrient solution. Half of the plants were transferred to a growth chamber with a constant 25°C, 16/8 h (light/dark) photoperiod regime and 1200 lux light intensity for a week to acclimate the plants. Temperature was increased stepwise (5 K per 48 h) to 30, 35, 40°C and finally to 45°C. In addition to others, plants were transferred from the outside to the growth chamber, at each temperature step to impose a heat shock. Leaf relative water content (RWC, %), loss of turgidity, chlorophyll content (Spad value) and heat-stress tolerance (HTS; LT₅₀) were measured in control and stressed plants. Total soluble proteins and total DNA were extracted from the leaves following the above treatments using standard procedures and total protein contents were determined using a Bradford assay. In general, effects of gradual heat stress (GHS) and shock heat stress (SHS) on the variables studied were mostly significant, except for chlorophyll content, while the effect of temperatures was significant for all the variables. Interaction between the heat stress type and temperature treatments was not significant for leaf RWC, loss of turgidity and chlorophyll content. Data also indicated that total protein and DNA contents were changed significantly by heat stress types (GHS and SHS) and/or temperature treatments. The plants exposed to GHS exhibited a significant increase in HST compared with the plants exposed to SHS (LT₅₀ of 41.5°C and 39°C, respectively). Consequently, gradual heat stress increased HST in strawberry leaves. Increased HST may be associated with the accumulation of several heat-stable proteins in GHS plants.     

苹果砧木枝条电阻抗参数与其抗寒性的关系

 以18株2年生苹果砧木西府海棠（Malus micromalus Makino）实生后代单株为试材,研究了不经冷冻处理的枝条的电阻抗（EIS）参数与其抗寒性（电导法测定枝条冷冻半致死温度）的关系。结果表明,3个时期（3月、7月、11月）枝条的EIS参数高频电阻率r和低频电阻率r1、胞外电阻率re、弛豫时间τ与抗寒性呈显著负相关（r =﹣0.737** ~﹣0.877**）,在11月时胞内电阻率ri与抗寒性也呈显著负相关（r =﹣0.857**）,表明在非冷冻条件下可采用枝条的电阻率r和r1、胞外电阻率re、弛豫时间τ估测苹果砧木实生后代枝条的抗寒性。通过比较3个时期各参数的最终通径系数,得出re为估测枝条抗寒性最佳的参数。     

核桃属4 树种展叶期抗寒性鉴定

 以普通核桃（Juglans regia L.）、核桃楸（J. mandshurica Maxim.）、河北核桃（J. hopeiensis Hu）和黑核桃（J. nigra L.）4种核桃属植物的20份种质展叶期叶片为试验材料,采用电导法并配合Logistic方程确定其低温半致死温度（LT₅₀）;分析了LT₅₀与叶片解剖结构之间的相关性。结果表明,随着处理温度的降低,核桃叶片的相对电导率（REC）均呈上升趋势;供试核桃种质间的抗寒性差异较大,低温半致死温度在–8 ~ 2 ℃之间,在黑核桃、核桃楸及普通核桃中均有抗寒性强的种质资源。利用略高于大部分核桃种质LT₅₀的温度（0 ℃或–3 ℃）处理叶片,其相对电导率与低温半致死温度之间达到极显著正相关,而且相关系数高,此温度下处理12 h后叶片的相对电导率以及叶片的LT₅₀均可作为鉴定展叶期核桃种质抗寒性强弱的物理指标。核桃属不同种间的叶片结构类似,但各有特点。栅栏组织厚度和叶片总厚度与其LT₅₀之间均达到极显著负相关,可作为核桃叶片抗寒性鉴定的形态结构指标。     

菊花8个品种的低温半致死温度及其抗寒适应性

 通过测定秋冬季自然降温过程中菊花脚芽的叶片相对电导率（REC）,结合Logistic方程计算的半致死温度（LT₅₀）评价了8个不同花期菊花品种在不同降温时期的抗寒性,并通过生长恢复试验进行验证。结果表明,在自然降温过程中,8个菊花品种的低温半致死温度均随气温的下降而不断降低,但下降幅度因品种而异,从4.0～9.4℃不等。8个品种抗寒性由强到弱排序依次为'金陵黄鹤'>'银星'>'奥运晚霞'>'金陵之光'>'奥运锦云'>03（6）-16>03（6）-12>'奥运火炬',植株抗寒性与花期相关性不显著。11月底菊花脚芽恢复生长试验与当月半致死温度测定结果基本一致,表明半致死温度可作为菊花抗寒性评价的一个可靠指标。当温度降到-14℃时,供试品种的脚芽均不能恢复生长。     

Paclobutrazol application enhances antioxidant enzyme activities in pomegranate plants affected by cold stress

Paclobutrazol (PBZ) is a member of the triazole plant growth inhibitor group that is responsible for inducing tolerance to a number of biotic and abiotic stresses. An experiment was, therefore, conducted to test whether PBZ application at various concentrations (0, 25, 50, or 75 mg L^?1) through foliar spray or soil drench would protect pomegranate (Punica granatum) seedlings cv. Robab, subjected to freezing at ?3°C for 7 hours. PBZ improved the growth rate of pomegranate seedlings subjected to freezing stress and increased relative leaf chlorophyll content, chlorophyll fluorescence ratio, relative water content, soluble carbohydrate content, and enzyme activity of ascorbate peroxidase and guaiacol peroxidase compared with the control at the end of experiment. PBZ ameliorated the injury caused by freezing stress by inhibiting increases in proline content and leaf electrolyte leakage, which suggested that PBZ ameliorated the negative effect of freezing stress. The best protection was obtained from plants treated with PBZ at 50 or 75 mg L^?1. The most evaluated parameters were not affected by PBZ application method.     

Effect of pruning and plant spacing on the growth of cherry rootstocks and their influence on stem water potential of sweet cherry trees

Summary

The aims of this work are to describe the effects of pruning and planting density on growth and water relations of ungrafted and grafted sweet cherry trees. A trial with cherry rootstocks ‘Prunus avium’, ‘CAB 11E’, ‘Maxma 14’, ‘Gisela 5’ and ‘Edabriz’ was begun in 1997. Pruning severities were applied to the rootstocks (0, 30, 60 and 90% of the vegetative growth was removed corresponding to P1, P2, P3 and P4 treatments, respectively) after planting to two plant spacings (S1 = 0.25 × 1.0 m and S2= 0.45 × 1.5 m). Canopy, root growth and leaf water potential (ψ_leaf) were quantified throughout the growing season. Pruning significantly affected root length and root weight of the rootstocks. Uncut plants (P1) showed a heavier and expanded root biomass (231 g and 108 m) than the intensively pruned plants (P4) (187 g and 75 m). The greater root biomass was obtained with the spacing/pruning combination, S1/P1 (285 g), and the smaller with S1/P4 (180 g) and S2/P4 (176 g). ψ_leaf varied significantly between the rootstocks and plant spacing but not with pruning. ‘Maxma 14’ and ‘P. avium’ attained the lowest values of midday ψ_leaf, –2.28 and –2.04 MPa, but the highest values of predawn ψ_leaf, –0.29 and –0.25 MPa, respectively. Generally, with high density (S1), the rootstocks exhibited lower predawn and midday ψ_leaf. In 1998, cultivars ‘Burlat’, ‘Summit’ and ‘Van’ were grafted onto rootstocks and a trial was installed in 1999. Predawn and midday stem water potential (ψ_stem) on cherry trees, measured in 2002, were affected significantly by the rootstock/genotype combination. Cultivars grafted on ‘P. avium’ and ‘Maxma 14’ showed the less negative midday ψ_stem, –1.36 and –1.42 MPa respectively, so these rootstock genotypes perhaps induced a higher drought resistance to the scion. Recorded data show that the scion-rootstock interaction with regard to production performance under water deficits may be an important consideration in cherry tree planting strategies.     

Abscisic acid alleviates the deleterious effects of cold stress on ‘Sultana’ grapevine (Vitis vinifera L.) plants by improving the anti-oxidant activity and photosynthetic capacity of leaves

The effects of exogenous application of abscisic acid (ABA) on anti-oxidant enzyme activities and photosynthetic capacity in ‘Sultana’ grapevine (Vitis vinifera L.) were investigated under cold stress. When vines had an average of 15 leaves, 0 (control), 50, 100, or 200 µM ABA was sprayed to run-off on all leaves of each plant. Twenty-four hours after foliar spraying with ABA, half (n = 5) of the water-only control vines and half (n = 5) of each group of ABA-treated plants were subjected to 4°C for 12 h, followed by a recovery period of 3 d under greenhouse conditions (25°/18°C day/night). The remaining plants in each treatment group were kept at 24°C. Cold stress increased H₂O₂ and malondialdehyde (MDA) concentrations in vine leaves, whereas all foliar ABA treatments significantly reduced their levels. Chilled plants showed marked increases in their total soluble protein contents in response to each ABA treatment. ABA significantly increased the activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase in cold-stressed grapevine leaves. In contrast, cold stress markedly decreased the rates of leaf photosynthesis (A) and evaporation (E), stomatal conductance (g_s), and chlorophyll concentrations in leaves, but increased intercellular CO₂ concentrations (C_i) in leaves. Treatment with all concentrations of ABA resulted in lower leaf A, E, and g_s values, but higher C_i values at 24°C. However, following cold stress, ABA-treated vines showed higher leaf A, E, and g_s values, but lower C_i values compared to control vines without ABA treatment. The application of 50–200 µM ABA allowed chilled vines to recover more quickly when re-exposed to normal temperatures, enabling the vines to resume their photosynthetic capacity more efficiently following cold stress. These results showed that, by stimulating anti-oxidant enzyme systems and alleviating cold-induced stomatal limitations, ABA reduced the inhibitory effect of cold stress on the rate of CO₂ fixation in ‘Sultana’ grapevine plants.     

Reactions of strawberry plants to long-term freezing and alternate freezing and thawing

Summary

The study was conducted in two experiments, one examining the effects of alternate freezing and thawing at –12?C for 2 h and +6#x02DA;C for 24 h, and the other examining the effects of permanent freezing for 1 or 15 d at+12?C. In both experiments the plants were grown in the pre-fruiting period at a coastal or a continental location in Norway. The freezing injury increased the higher the stress, indicated by reduced growth, fruit size and yield.It was shown that ‘Korona’ was the most hardy cultivar at a short period of freezing stress and at alternate freezing and thawing, while ‘Senga Sengana’ was more freezing tolerant at long-term permanent freezing than ‘Bounty’ and ‘Korona.’ In general the results indicate that the plants induce freezing tolerance according to some factor of origin, since plants grown in the continental climate were better able to survive long-termpermanent freezing than plants grown in a coastal climate, and plants grown in a coastal climate were better able to survive alternate freezing and thawing than plants grown in a continental climate.     

Regulated deficit irrigation in potted Dianthus plants: Effects of severe and moderate water stress on growth and physiological responses

The purpose of this study was to analyze the physiological and morphological response of carnation plants to different levels of irrigation and to evaluate regulated deficit irrigation as a possible technique for saving water through the application of controlled drought stress. Carnations, Dianthus caryophyllus L. cultivar, were pot-grown in an unheated greenhouse and submitted to two experiments. In the first experiment, the plants were exposed to three irrigation treatments: (control); 70% of the control (moderate deficit irrigation, MDI) and 35% of the control (severe deficit irrigation, SDI). In the second experiment, the plants were submitted to a control treatment, deficit irrigation (DI, 50% of the control) and regulated deficit irrigation (RDI). After 15 weeks, MDI plants showed a slightly reduced total dry weight, plant height and leaf area, while SDI had clearly reduced all the plant size parameters. RDI plants had similar leaf area and total dry weight to the control treatment during the blooming phase. MDI did not affect the number of flowers and no great differences in the colour parameters were observed. RDI plants had higher flower dry weight, while plant quality was affected by the SDI (lower number of shoots and flowers, lower relative chlorophyll content). Leaf osmotic potential decreased with deficit irrigation, but more markedly in SDI, which induced higher values of leaf pressure. Stomatal conductance (g_s) decreased in drought conditions more than the photosynthetic rate (P_n). Osmotic adjustment of 0.3 MPa accompanied by decreases in elasticity in response to drought resulted in turgor less at lower leaf water potentials and prevented turgor loss during drought periods.     

低温胁迫下佛手半致死温度测定和抗寒性分析

 以‘青皮’和‘矮化’佛手(Citrus medica var. sarcodactylis Swingle)为试材,测定不同低温下处理24 h后,叶片的电解质外渗率(REC),计算半致死温度(LT₅₀);在半致死温度下设置不同处理时间,测定REC、超氧化歧化酶(SOD)活性、游离脯氨酸(Pro)和丙二醛(MDA)含量,进行抗寒性分析。结果表明：佛手REC随温度降低及处理时间延长呈“S”形上升, LT₅₀在-4~ -5℃之间;在LT₅₀下,随着处理时间的延长SOD活性呈先上升后下降的趋势, Pro和MDA含量逐渐上升;12~ 24 h为佛手在LT₅₀下冷害发生的临界时间。     

Comparison of Freeze Protection Methods for Strawberry Production in Florida

During strawberry (Fragaria × ananassa) freeze protection, the standard practice is applying sprinkler irrigation on plant canopies to prevent and reduce freezing damage. This method is highly inefficient because it uses large volumes of water. The overall goal of this study was to compare the effects of freeze protection methods on water savings, and growth and fruit weight of strawberry. Treatments consisted of the following: sprinkler heads delivering 17 L?min^–1, sprinkler heads delivering 13 L?min^–1, light-weight row covers (21 g?m^–2) on the crop canopy, light-weight row covers on 60-cm-high mini-tunnel hoops, heavy-weight row covers (31 g?m^–2) on the crop canopy, heavy-weight row covers on 60-cm-high mini-tunnel hoops, and foliar application of a polymer (Desikote). There were eight freezing and near freezing nights (≤1.1 °C) at the experimental site during both seasons with a minimum air temperature of –2.8 °C. Minimum temperature inside the row covers ranged between 0.6 to 4.4 °C at the canopy level. There were significant treatment effects on early and total marketable fruit weights. The highest early marketable fruit weight was found in plots protected with light-weight row covers with hoops, heavy-weight row covers on canopy, and foliar polymer, ranging between 5.0 to 5.5 t?ha^–1. For total marketable fruit weight, using non-irrigation methods resulted in the highest fruit weight with 23.0 t?ha^–1. In conclusion, data showed that using non-irrigation techniques provided satisfactory freeze protection under the evaluated conditions and increased total marketable fruit weight, possibly due to reduced injury of fruits and flowers when using alternative techniques.     

Moving lamps increase leaf photosynthetic capacity but not the growth of potted gerbera

To investigate the responses of leaf photosynthesis and plant growth to a moving lighting system, potted gerberas (Gerbera jamesonii H. Bolus ex J.D. Hook “Festival”) were grown under supplemental lighting in a greenhouse with either a stationary or a moving lighting system positioned above the benches. The stationary system consisted of a fixed high pressure sodium (HPS) lighting system, while the moving lighting system consisted of a moving HPS fixture attached to a cable system to move the light fixture back and forth over the crop. In both cases, the supplemental lighting was applied from 6:00 to 24:00 h with the same supplemental daily light integral (4.9 mol m⁻² day⁻¹). Moving lamps significantly increased leaf photosynthetic capacity as represented by light saturated net CO₂ exchange rate (NCER) (A_sat), light- and CO₂-saturated rate of NCER (A_max), maximum rate of Rubisco carboxylation (V_cmax), maximum rate of electron transport (J_max) and rate of triose phosphate utilization. However, in situ leaf NCER and stomatal conductance, leaf chlorophyll content index, leaf area, leaf thickness, fresh weight of plants were significantly lower under moving lighting than under stationary lighting. It is suggested that the reduced growth of plants under moving lighting might be due to (1) the overall lower light use efficiency of leaves under moving lighting than those under stationary lighting; (2) the slower response time of the photosynthetic system compared to the rate of change in light intensity under moving lighting.     

The influence of temperature on photosynthesis of different tomato genotypes

Net photosynthesis and dark respiration from whole plants of various tomato genotypes were measured in a closed system. At low irradiance (27 W m^?2) and low external CO₂ concentration (550 mg m^?3), net photosynthesis of 10 genotypes was found to vary between 0.122 and 0.209 mg CO₂ m^?2 s^?1. Correlation was observed between net photosynthesis, net uptake on a daily basis (8 h photoperiod at 20°C and 16 h nyctoperiod at 10°C), specific leaf weight and leaf area ratio. At high irradiance (243 W m^?2), high external CO₂ concentration (1480 mg m^?3) and ambient temperatures of 10, 18, 20 and 26°C, four genotypes were analysed. ‘F6 I.V.T.’ had the highest rate of photosynthesis at 10°C, while ‘Sonatine’ ranked high at 26°C. Dark respiration increased with temperature, except in the case of ‘Bonabel’ where the effect of temperature was slight.     

Effects of water stress and re watering on ribulose-1,5-bis-phosphate carboxylase activity,chlorophyll and protein contents in two cultivars of tomato

Summary

The effects of water deficit and rewatering on ribulose-1,5-bis-phosphate carboxylase activity, chlorophyll and protein content were evaluated in plants of two cultivars of tomato. During the water deficit period, values of water potential, osmotic potential and relative water content decreased along with associated decreases in RuBPcase activity, protein content and chlorophyll content, being less marked for chlorophyll content. There was a significant correlation of RuBPcase activity and protein content with components of leaf water status from plants under water stress. The associated decrease of RuBPcase, chlorophyll and protein contents with decreased osmotic potential during the development of water deficit was evident. At the time the plants reached a water potential of –1.40 to –1.56 MPa (RI: first level of recovery after water stress), one group of plants was rewatered. The rest were kept under stress until the water potential reached values of –2.30 to –2.51 MPa (RII: second level of recovery after water stress). It was observed that all of the varieties measured at both levels (RI and RII) showed a gradual recovery, reaching or even surpassing the values of control plants.     

Temperature and development in Iris × hollandica during preplanting storage. I. Leaf initiation

Summary

Leaf initiation was examined in Dutch iris bulbs during pre-planting storage temperature treatments in the dark. The number of leaves initiated before inflorescence evocation increased with increasing temperature. The base, optimum and maximum temperature for leaf initiation were established as –0.4, 13.1 and 26.7°C respectively. The rate of leaf initiation was shown to be linearly related to temperature. The average thermal-time required for each leaf to be initiated under constant temperatures was 79°Cd but leaves initiated during the transfer temperature treatments required an average of 92°Cd. Rates of leaf initiation predicted from thermal-time equations were similar to those observed in bulbs stored at the lower temperatures (2–13°C) but rates observed at warmer temperature (17–25°C) never reached the predicted high value.