Gibberellins and the photoperiodic control of stem elongation in the long-day plant Agrostemma githago L.

Agrostemma githago is a long-day rosette plant in which transfer from short days (SD) to long days (LD) results in rapid stem elongation, following a lag phase of 7–8 d. Application of gibberellin A₂₀ (GA₂₀) stimulated stem elongation in plants under SD, while 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618, an inhibitor of GA biosynthesis) inhibited stem elongation in plants exposed to LD. This inhibition of stem elongation by AMO-1618 was overcome by simultaneous application of GA₂₀, indicating that GAs play a role in the photoperiodic control of stem elongation in this species. Endogenous GA-like substances were analyzed using reverse-phase high-performance liquid chromatography and the d-5 corn (Zea mays L.) assay. Three zones with GA-like activity were detected and designated, in order of decreasing polarity, as A, B, and C. A transient, 10-fold increase in the activity of zone B occurred after 8–10 LD, coincident with the transition from lag phase to the phase of rapid stem elongation. After 16 LD the activity in this zone had returned to a level similar to that under SD, even though the plants were elongating rapidly by this time. However, when AMO-1618 was applied to plants after 11 LD, there was a rapid reduction in the rate of stem elongation, indicating that continued GA biosynthesis was necessary following the transient increase in activity of zone B, if stem elongation was to continue under LD. It was concluded that control of stem elongation in A. githago involves more than a simple qualitative or quantitative change in the levels of endogenous GAs, and that photoperiodic induction alters both the sensitivity to GAs and the rate of turnover of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - LDP long-day plant(s) - SD short day(s)     

Effects of Growth Retardants on Norway Spruce (Picea abies)

Young seedlings of Picea abies Karst, grown in nutrient solution were treated with the growth retardants Amo-1618, B-995, and CCC. These were added to the nutrient medium. B-995 and CCC retarded root and shoot growth in the concentrations 100, 10, and 1 mg/l. Growth was almost entirely inhibited by 300 mg/l, obviously due to toxicity. The effects of Amo-1618 were similar but more varying. GA counteracted the effects of all the retardants on shoot growth, but not on root growth.     

Effect of Growth Retardants on α-Amylase Production in Germinating Barley Seed

The effect of growth retarding compounds, (2-chloroethyl)trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (AMU-1618), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D) and N-dimethylamino succinamic acid (B-995) on α-amylase production in germinating barley seed was studied. Seeds were germinated in growth retardants in presence and absence of gibberellic acid (GA₃). CCC, AMO-1618 and Phosfon D inhibitedα-amylase production in germinating seed and the effect was reversed by GA₃ Phosfon D and AMO-1618 were stronger inhibitors of α-amylase production than CCC. CCC was by far the strongest inhibitor of all the other analogs tested. B-995 was comparatively only slightly inhibitory. The results reported here, when viewed in light of the results of other workers, provide good evidence that CCC, AMO-1618 and Phosfon D inhibit α-amylase production by inhibiting the synthesis of gibberellin or gibberellin-like hormone(s) during germination of barley seed. Consistent with other reports, B-995 possibly acts by other mechanism (s).     

Stem growth,flower formation,and endogenous gibberellins in a normal and a dwarf strain of Silene armeria

The physiological basis of dwarfism in a single-gene, recessive mutant of Silene armeria L. was investigated through comparison with a normal strain. Exposure of the normal strain to long days led to stem growth and flower formation while similar exposure of the dwarf strain led only to flowering, with very little stem growth. Application of gibberellin A₃ or A₄₊₇ in short days promoted stem elongation in the normal strain, but had a much lesser effect in the dwarf strain. Upon extraction and chromatographic fractionation of the endogenous gibberellins (GAs) in the normal strain of S. armeria, three zones of GA activity were found. An increase in one zone of activity was found in both strains after 1 long day. Neither the quality nor the quantity of the extractable GAs differed greatly between the dwarf and the normal strain. Vegetative dwarf scions, grafted onto fully induced, normal stocks formed flowers, but their growth habit was not changed. Thus, the lack of stem growth in response to long days in the dwarf strain appears to result from a lack of GA sensitivity in the stem tissue of these plants. However, during flower formation dwarf plants did exhibit elongation of the peduncles. This response was suppressed by the growth retardant 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618), and applied GA₃ could partially overcome this inhibition. Thus, peduncle elongation in the dwarf strain appears to be regulated by endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - SD short day(s)     

Kinetics of growth retardant and hormone interactions in affecting cucumber hypocotyl elongation

The capacities of indole-3-acetic acid (IAA) and gibberellin A₃ (GA₃) to counteract the inhibitory effects of (2-chloroethyl) trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618), and N,N-dimethylaminosuccinamic acid (B-995) on hypocotyl elongation in light-grown cucumber (Cucumis sativus L.) seedlings were investigated. One μg of GA₃ applied to the shoot tip was sufficient to completely nullify the effect of 10 μg of Amo-1618 or 25 μg of B-995 applied simultaneously to the shoot tip, and 10 μg of GA₃ completely counteracted the effect of 10⁻³ m CCC added to the root medium. One μg of IAA counteracted the effect of 10⁻³ m CCC in the root medium, but IAA did not nullify the action of either Amo-1618 or B-995. Experiments were conducted using 2 growth retardants simultaneously, which indicated that Amo-1618 and CCC inhibit a common process, namely GA biosynthesis, essential to hypocotyl elongation. However, since the effect of CCC was overcome by applications of both GA and IAA, growth retardation resulting from treatment with CCC apparently is not due solely to inhibition of GA biosynthesis. B-995 did not interact additively with either Amo-1618 or CCC, which suggests that B-995 affects a process different from those affected by the other 2 retardants. Thus, while inhibition evoked by B-995 is reversible by applied GA, the action of B-995 does not appear to be inhibition of GA biosynthesis.     

Effects of AMO-1618 on Growth, Morphology, and Gibberellin Content of Phaseolus Cocineus Seedlings

A study was made of the effects of AMO-1618 on the endogenousgibberellin (GA) levels and stem, leaf, and root growth of Phaseoluscoccineus seedlings. The data establishes that some of the effectsof AMO-1618 on the growth of Phaseolus seedlings are mediatedby factors other than an inhibition of GA biosynthesis.     

Chromatin RNA polymerase activity from soybean hypocotyls treated with gibberellic acid and AMO-1618

Chromatin isolated from control, AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine carboxylate] and gibberellic acid (GA) treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Gibberellic acid, sprayed on intact soybean hypocotyls, is shown to have enhanced the level of chromatin RNA polymerase activity while chromatin isolated from hypocotyls pretreated with AMO-1618 exhibits a lower polymerase activity relative to the control. Chromatin extracted from the treated or untreated seedlings are all sensitive to the inhibition (in varying degrees) by the presence of actinomycin D, pyrophosphate, or ribonuclease. Thus enhanced (or decreased) RNA-synthesizing capacity of chromatin in response to chemical treatments may be due to enhanced (or decreased) synthesis of RNA polymerase.     

Inhibition of stem growth and gibberellin production in Agrostemma githago L. by the growth retardant tetcyclacis

The effects of the new growth retardant tetcyclacis (TCY) on stem growth and endogenous gibberellin (GA) levels were investigated in the long-day rosette plant Agrostemma githago. Application of TCY (10 ml of a 5·10^-5M solution daily) to the soil suppressed stem elongation in Agrostemma grown under long-day conditions. A total of 10 g GA₁ (1 g applied on alternate days) per plant overcame the growth retardation caused by TCY.Control plants and plants treated with TCY were analyzed for endogenous GAs after exposure to nine long days. The acidic extracts were fractionated by high-performance liquid chromatography. Part of each fraction was tested in the d-5 maize bioassay, while the remainder was analyzed by combined gas chromatography-selected ion monitoring. The bioassay results indicated that the GA content of plants treated with TCY was much lower than that of untreated plants. The data obtained by gas chromatography-selected ion monitoring confirmed that the levels of seven GAs present in Agrostemma were much reduced in TCY-treated plants when compared with the levels in control plants: GA₅₃ (13%), GA₄₄ (0%), GA₁₉ (1%), GA₁₇ (33%), GA₂₀ (15%), GA₁ (4%), and epi-GA₁ (13%). These results provide evidence that TCY inhibits stem growth in Agrostemma by blocking GA biosynthesis and thus lowering the levels of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - HPLC high-performance liquid chromatography - TCY Tetcyclacis (5-[4-chlorophenyl]-3,4,5,9,10-pentaaza-tetracyclo-5,4,1,0^2,6,0^8,11-dodeca-3,9-diene)     

Involvement of endogenous gibberellins in potato tuber dormancy and early sprout growth: a critical assessment

The role of endogenous gibberellins (GAs) in the regulation of potato (Solanum tuberosum) tuber dormancy was examined by determining: 1. changes in endogenous GA levels during natural dormancy progression, 2. the effects of GA biosynthesis inhibitors on tuber dormancy duration and 3. the dormancy status and tuber GA levels in a dwarf mutant of potato. The tubers (cv. Russet Burbank) used in these studies were still completely dormant after 98 days of storage. Between 98 and 134 days of storage, dormancy began to end and tubers exhibited limited (< 2 mm) sprout growth. Tuber dormancy weakened with further storage and tubers exhibited greater rates of sprout growth after 187 days of storage. Tubers stored for 212 days or longer were completely non-dormant and exhibited vigorous sprout growth. Immediately after harvest, the endogenous contents of GA19, GA20, and GA1 were relatively high (0.48-0.62 ng g fresh weight(-1)). The content of these GAs declined between 33 and 93 days of storage. Internal levels of GA19, GA20, and GA, rose slightly between 93 and 135 days of storage reaching levels comparable to those found in highly dormant tubers immediately after harvest. Levels of GA19, GA20, and GA1 continued to increase as sprout growth became more vigorous. Neither GA4 nor GA8 was detected in any tuber sample regardless of dormancy status. Dormant tubers exhibited a time-dependent increase in apparent GA sensitivity. Freshly harvested tubers were completely insensitive to exogenous GAs. As postharvest storage continued, exogenous GAs promoted premature dormancy release with GA1 and GA20 eliciting the greatest response. Injection of up to 5 microg tuber(-1) of kaurene, GA12, GA19 or GA8 had no effect on dormancy release. Sprout growth from non-dormant tubers was also promoted by exogenous GA in the following sequence of activity: GA1 = GA20 > GA19. Kaurene, GA12, and GA8 were inactive. Continuous exposure of developing tubers to inhibitors of GA biosynthesis (AMO-1618, ancymidol, or tetcyclasis) did not extend tuber dormancy but rather hastened dormancy release. Comparison of tuber dormancy and GA1 content in tubers of a wild-type and dwarf mutant of S. tuberosum ssp. andigena revealed a near-identical pattern of dormancy progression in spite of the absence of detectable levels of GA1 in tubers of the dwarf sibling at any time during dormancy progression. Collectively, these results do not support a role for endogenous GA in potato tuber dormancy release but are consistent with a role for GAs in the regulation of subsequent sprout growth.     

Gibberellins and the photosensitivity of isolated embryos from non-stratified apple seeds

Summary The phytochrome system is responsible for the photosensitivity of dormant, isolated apple embryos in culture. Maximum photosensitivity occurs on the second day of culture and it is unaffected by gibberellins (GAs) in concentrations below 10^-4M. Higher concentrations of GA decrease the photosensitivity.The endogenous quantities of GA₄ and GA₇ were determined in embryos grown at white light, in darkness and in darkness following an exposure to red light. The GA₇ level remained unaffected by the light conditions, whereas the amount of GA₄ was three times higher in light or red-light-treated cultures than in the dark grown ones.Similar experiments were done using AMO-1618, an inhibitor of GA biosynthesis, which is also a strong inhibitor of apple seed germination. In this case the level of both GA₄ and GA₇ was light-independent. These experiments suggest that the phytochrome system participates in the regulation of GAs biosynthesis by mediating one of the last steps of GA₄ formation.     

QUANTITATIVE CHANGES IN GIBBERELLIN AND RNA CORRELATED WITH SENESCENCE OF THE SHOOT APEX IN THE ‘ALASKA’ PEA

Senescence of shoot apices of Pisum sativum L. ‘Alaska’ as measured by cessation of stem elongation was delayed by removal of flowers and by treatment with gibberellin A₃ and was hastened by treatment with AMO-1618 (2 isopropyl-4-dimethylamino-5-methylphenyl-1-piperi-dinecarboxylate methyl chloride). Ontogenetic changes in relative endogenous gibberellin levels and in capability of gibberellin biosynthesis in deflowered and control plants were determined indirectly by studying time-course changes in the sensitivity, as indicated by the growth response, of these plants to applied gibberellin and AMO-1618. The results of these experiments suggest that the endogenous gibberellin level varies directly with the growth rate. Analyses of total RNA and protein in shoot tips of deflowered and control plants revealed that the levels of these substances also vary directly with growth rate throughout ontogeny. It is concluded that decreases in endogenous gibberellin, RNA and protein are factors correlated with senescence of the shoot apex.     

Relationship between gibberellins, height, and stress tolerance in barley (Hordeum vulgare L.) seedlings

The relationship between gibberellin (GA) levels, height, and stress tolerance was investigated using barley (Hordeum vulgare L.) seedlings, which were exposed to heat stress (50 °C for 3 h) and a free radical generator (Paraquat). Barley cv. Perth seedlings were treated with GA-inhibitors, either a triazole or one of two acylcyclohexanediones. They were tested along with four mutants that were either responsive (MC96 and dwf1) or non-responsive (MC90 and Dwf2) to applied growth-active GAs. Analyses of seedlings, whether mutants, or treated with GA-inhibitors, gave a negative correlation between height and tolerance. We also observed that the shorter seedlings with the least amount of growth active GAs were the most tolerant to stress. The overall results of this study suggest that reduced GA levels or sensitivity to GA, with a concomitant reduction in height are important for induction of stress tolerance.     

INTERACTING ROLES OF GIBBERELLIN AND ETHYLENE IN COROLLA EXPANSION OF IPOMOEA NIL (CONVOLVULACEAE)

The relatively rapid and extensive corolla expansion in Ipomoea nil (L.) Roth cv. ‘Scarlett O'Hara’ appears to be initiated by a shift in the balance between a growth promoter (gibberellin, GA) and a growth inhibitor (ethylene) two days before anthesis. The effects of applied growth regulators in vitro, were measured as a change in area of segments from whole young corollas 16–17 mm long. Applied gibberellic acid (GA₃) strongly promoted growth, while a GA action inhibitor (ancymidol) reduced growth. Inhibitors of GA biosynthesis (AMO-1618, paclobutrazol, tetcyclasis, and chlorocholine chloride) had little effect, implying that the GAs were not being synthesized within the corolla segments, at least at this stage of corolla growth. Both the level of endogenous GAs and response of the segments to sucrose increased as the corolla size also increased from 15 to 20 mm in length. Applied 1-aminocyclopropane-l-carboxylic acid (ACC), an ethylene precursor, inhibited the response of corollas to applied GA₃. Applications of ethylene biosynthesis inhibitors (AVG and cobalt ions) promoted growth of the corolla segments. Rapid ethylene production by corollas 15–17 mm long (48 hr before flower opening) appeared to inhibit the growth that may have been induced by low levels of endogenous GAs. Older corollas (longer than 18 mm) had very low levels of ethylene production and much higher levels of GA-like substances; these changes apparently allow the corollas to begin to expand in vivo and to strongly respond to sucrose applications alone in vitro. Acid-induced growth does not seem to be an important component of corolla expansion; applied fusicoccin (a proton efflux promoter) and sodium orthovanadate (a proton efflux inhibitor) had no significant effect on growth of the 16–17 mm long corollas. Buffers (Good's and phosphate) over a wide pH range had no effect on corolla expansion. Taken together, our results indicate that the regulation of corolla expansion depends (at least in part) on compensatory shifts in the levels of two plant growth regulators [PGRs], ethylene and GA.     

Gibberellin-induced inhibition and promotion of sprouting in aerial tubers of Begonia evansiana Andr. in relation to photoperiodic treatment and tuber stage

Gibberellic-acid (GA₃) treatment, when applied within a period ranging from the start of short-day (SD) treatment until about 10 SD, GA₃ strongly inhibited formation of aerial tubers in response to SD and brought about sprouting of developing aerial tubers. In contrast, when applied after about 10 SD or more, GA₃ hastened the completion of the dormant state in the tubers and prolonged their dormancy. The dormancy-promoting effect of GA₃ on detached tubers increased with their degree of maturation. Application of growth retardants N-dimethylaminosuccinamic acid (B-9), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) and 2-chloroethyltrimethylammonium chloride (CCC) to the cuttings delayed the onset of dormancy in the aerial tuber. When the retardants were applied to detached aerial tubers, however, such a delay of dormancy was not observed, and GA₃ application did not inhibit sprouting in aerial tubers detached from CCC-treated cuttings.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short day(s) - LD long day(s) - SDP short-day plant - LDP long-day plant - CCC 2-chloroethyltrimethylammonium chloride - B-9 N-dimethylaminosuccinamic acid - AMO-1618 2-isopropyl-4-dimethyl-amino-5-methylphenyl-1-piperidine carboxylate methyl chloride     

Effects of photoperiod on growth rate and endogenous gibberellins in the long-day rosette plant spinach

The earliest visible responses of spinach plants (Spinacia oleracea L., cv. Savoy Hybrid 612) transferred from short to long days (8 hours of high intensity light supplemented with 16 hours of low intensity illumination from incandescent lamps) were upright leaf orientation and increased elongation of the petioles. The effect of long days on growth rate was direct; i.e., there was no after-effect if the plants were transferred to short days. Gibberellin A₃ applied to plants under short days had an effect similar to that of long days, whereas application of the growth retardant AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidinel-carboxylate] under long days caused a growth habit typical of short-day conditions. Gibberellin A₃ caused more stem growth in plants under long days in which the endogenous gibberellin content had been reduced by AMO-1618 than in plants under short days not treated with the growth retardant.     

The Promotion of Indole-3-acetic Acid Oxidation in Pea Buds by Gibberellic Acid and Treatment

Terminal buds of dark-grown pea (Pisum sativum) seedlings have an indole-3-acetic acid oxidase which does not require Mn(2+) and 2,4-dichlorophenol as cofactors. Oxidase activity is at least 50 times higher in buds of tall peas than in dwarf seedlings. Administration of gibberellic acid to dwarf peas stimulates both growth and indoleacetic acid oxidase activity to the same levels as in tall seedlings. By contrast, indoleacetic acid oxidation assayed in the presence of Mn(2+) and 2,4-dichlorophenol proceeds at similar rates regardless of gibberellin application. Treatment of tall peas with the growth retardant AMO-1618 reduces growth and oxidase activity. Such treated seedlings are indistinguishably dwarf. The enzyme does not appear to be polyphenol oxidase, nor do the results suggest that reduced activity in dwarf buds is due to higher levels of a dialyzable inhibitor. The peroxidative nature of the oxidase is probable.     

Narrow-band light regulation of ethylene and gibberellin levels in hydroponically-grown <Emphasis Type="Italic">Helianthus annuus</Emphasis> hypocotyls and roots

Both hypocotyl and root growth of sunflower (Helianthus annuus) were examined in response to a range of narrow-band width light treatments. Changes in two growth-regulating hormones, ethylene and gibberellins (GAs) were followed in an attempt to better understand the interaction of light and hormonal signaling in the growth of these two important plant organs. Hydroponically-grown 6-day-old sunflower seedlings had significantly elongated hypocotyls and primary roots when grown under far-red (FR) light produced by light emitting diodes (LEDs), compared to narrow-band red (R) and blue (B) light. However, hypocotyl and primary root lengths of seedlings given FR light were still shorter than was seen for dark-grown seedlings. Light treatment in general (compared to dark) increased lateral root formation and FR light induced massive lateral root formation, relative to treatment with R or B light. Levels of ethylene evolution (roots and hypocotyls) and concentrations of endogenous GAs (hypocotyls) were assessed from both 6-day-old sunflower plants either grown in the dark, or treated with FR, R or B light. Both R and B light had similar effects on hypocotyl and root growth as well as on ethylene and on hypocotyl GA levels. Dark treatment resulted in the highest ethylene levels, whereas FR treatment significantly reduced ethylene evolution for both hypocotyls and roots. R- and B-light treatments elevated ethylene evolution relative to FR light. Endogenous GA₅₃ and GA₁₉ levels in hypocotyls were significantly higher and GA₄₄, GA₂₀ and GA₁ levels significantly lower, for dark and FR light treatments compared to R and B light-treatments. The patterns seen for changes in GA concentrations indicate FR-, R- and B-light-mediated effects [differences] in the metabolism of the early C₂₀ GAs, GA₅₃ → GA₄₄ → GA_19. Surprisingly, GA₂₀, GA₁ and GA₈ levels in hypocotyls were very much reduced by treatment of the plants with FR light, relative to B and R-light treatments, e.g. the increased hypocotyl elongation induced by FR light was correlated with reduced levels of all three of the downstream C₁₉ GAs. The best explanation, albeit speculative, is that a more rapid metabolism, i.e. GA₂₀ → GA₁ → GA₈ → GA₈ conjugates occurs under FR light. Although this study provided no evidence that elevated ethylene evolution by roots or hypocotyls of sunflower is controlling growth via endogenous GA biosynthesis, there are differences between soil-grown and hydroponically-grown sunflower seedlings with regard to trends seen for hypocotyl GA concentrations and both root and hypocotyl ethylene evolution in response to narrow band width R and FR light signaling.     

Reduced de-etiolation of hypocotyl growth in a tomato mutant is associated with hypersensitivity to, and high endogenous levels of, abscisic acid.

A recessive single gene mutant, 7B-1, in tomato was originally selected for its photoperiod-dependent male sterility. The 7B-1 mutant also has some pleiotropic effects including reduced light-induced inhibition, i.e. de-etiolation, of the hypocotyl in long days (LD), increased seed size and weight, and reduced transpiration rate. These traits led us to investigate the sensitivity of 7B-1 to exogenous hormones and the interaction of these responses with daylength. In LD, but not in short days (SD), 7B-1 was more sensitive than wild-type (WT) to exogenous abscisic acid (ABA) for inhibition of seed germination, root elongation and transpiration rate. 7B-1 mutant also exhibited reduced responses to exogenous gibberellin (GA(3)) for hypocotyl elongation, and to inhibitors of GA biosynthesis for seed germination and root and hypocotyl elongation. 7B-1 hypocotyls contained a higher level of endogenous ABA than WT in both photoperiods, although ABA levels were higher in LD than in SD. In contrast, growth-active GAs, i.e. GA(1), GA(3) and GA(4), and IAA were low in the mutant hypocotyls. The 7B-1 mutant appears to be an ABA-overproducer, and the photoperiod-regulated ABA levels may be responsible for the hypersensitivity of the mutant to exogenous ABA.     

Extension growth and cold hardening of young ‘Valencia’ orange trees sprayed with AMO-1618

Rate of extension growth, as measured by height, of 2-month-old Valencia orange trees (Citrus sinensis (L.) Osbeck) on rough lemon rootstock (C. limon Burm. f.) was reduced to 0.5 mm from 5.0 mm day^–1 with 0.1% (w/v) sprays of the growth retardant AMO-1618 (4 hydroxy-5-isopropyl-2-methyl phenyl trimethyl-ammonium chloride, 1 piperdine carboxylate) every 2 weeks during 11 weeks under natural daylight in a glasshouse. Trees sprayed with AMO-1618 were 10-fold shorter, more compact in appearance, and leaves were greener and more oval shaped than those on untreated trees. There was no chemical burn. AMO-1618-sprayed trees were more cold hardy than untreated trees during controlled-temperature, cold-hardening regimes. Alone, AMO-1618 had no effect on freeze tolerance at -5.5° C. AMO-1618 also was associated with greater tree tolerance to freeze injury determined by O₂ uptake in Valencia leaves to as low as -6.7° C.This paper reports the results of research only. Mention of a trademark of a proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.