Michael G. Bausher

Control of in vitro contamination of explants from greenhouse- and field-grown trees

SummaryControlling fungal and bacterial contamination of woody plant material can be extremely difficult. Isothiazolone biocides and sodium dichloroisocyanurate have been used singly and in combination to reduce microbial contamination in bud explants derived from greenhouse- and field-grown citrus trees. Explants from greenhouse-grown trees were effectively disinfested (<5% vs. 85% contamination) using a ‘standard’ disinfestation (SD) procedure followed by culture on medium containing 5 ml l−1 Plant Preservative Mixture, or by the SD procedure but substituting 300 ppm sodium dichloroisocyanurate for 48 h for the treatment with 1.05% NaOCl in the SD procedure. Disinfestation of explants from field-grown trees was less effective than explants from the greenhouse, but was improved (10% vs. 47% contamination) by using a combination of the SD procedure with 1.05% NaOCl, or 100 or 300 ppm sodium dichloroisocyanurate followed by culture onto medium containing 5 ml l−1 Plant Preservative Mixture.

Influence of growth regulator treatments on dry matter production, fruit abscission, and14C-assimilate partitioning in citrus

Experiments were performed to monitor (1) uptake and translocation of foliar-applied microdroplets of14C hormones and (2) effects of multiple growth regulator sprays on foliar and fruit growth variables and photosynthate partitioning in Valencia orange trees (Citrus sinensis (L.) Osbeck). The uptake of14C-sucrose,14C-paclobutrazol (PP333), and14C-napthaleneacetic acid (NAA) in 6-month-old greenhouse-grown trees exceeded that of14C-abscisic acid (ABA) and14C-benzyladenine (BA) 48 h after microdroplet application.14C-sucrose transport from the application site was much greater than any other source, especially14C-BA. In a second study, 2-year-old Valencia orange trees were maintained under field conditions and were sprayed to foliar runoff (3 × /week for 3 weeks) with BA, NAA, ABA, PP333, and gibberellic acid (GA3) at 100 μM during flowering and early fruit set. Select branches were then briefly exposed to14CO2 and harvested 24 h later. Both GA3 and BA sprays promoted foliar growth. BA also stimulated fruit growth, whereas GA3 sharply increased fruit dry weight while fruit number decreased. BA and GA3 enhanced14C assimilate export by the foliage to the developing fruit, and GA3 was especially active in promoting fruit sink intensity (14C/dry wt). The other compounds (NAA, ABA, PP333) restricted foliar and fruit growth. They also inhibited transport of14C assimilate from the leaves to the fruit. Results indicate that foliar-applied growth regulators can influence source-sink relations in citrus early in reproductive development by manipulating photoassimilate production and partitioning.

Morphological changes incitrus associated with relatively high concentrations of paclobutrazol

Progressively higher concentrations of Paclobutrazol-markedly reduced germination of Valencia sweet orange (Citrus sinensis (L.) Osbeck) seed and induced significant changes in the morphology, growth, and development of roots of Valencia seedlings and rough lemon (C. limon (L.) Burm. f.) leaves. Threshold concentrations for significant visible effect to radical change ranged from 103 to 105 ppm (2.84×10−4 to 2.84×10−2 M) (ai). Initial change was readily evident in reduced lateral and fibrous root development at the lower concentrations (103 ppm). Higher concentrations (105 ppm) resulted in no secondary root formation and progressive basal enlargement terminating in a bulbouslike apex of the primary root. Lack of secondary roots suggests disruption in the pericycle or severe inhibition of meristematic initial cells. Root system changes were not visibly indicated in shoot growth other than strong inhibition of extensions.

Physiological effects of temperature and growth regulators on foliar chlorophyll, soluble protein, and cold hardiness in citrus

Leaf chlorophyll (Chl, A, B) and total soluble protein were assayed in greenhouse-grown 1.5-year-old trees of 2 citrus types, trifoliate orange (Poncirus trifoliata (L.) Raf.) and sour orange (Citrus aurantium L.) exposed to 12 h (day/night) photoperiods in growth chambers under high (30°/21°C, day/night; noncold-hardening) and low (16°/5°C; cold-hardening) temperature regimes. Trees were sprayed 2 × per week for 5 weeks with one of the following solutions at 100 μM: napthaleneacetic acid (NAA), paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol) (PPP333), benzyl-adenine (BA), abscisic acid (ABA), gibberellic acid (GA3), minerals only (N, P, K, S, Ca, Mg) and BA (+) minerals. NAA, PP333, ABA and GA3 decreased Chl A, B and soluble protein in both citrus types under cold-hardening conditions in contrast to increases with the use of BA and BA (+) minerals especially in trifoliate orange. Both BA and GA3 increased Chl A, B and protein synthesis under high temperature in both citrus types. Under noncold-hardening temperatures, GA3 enhanced Chl A, B but sharply reduced foliar protein concentration. Dieback of both cultivars following exposure to temperatures down to −6.7°C was decreased 7% by NAA sprays during noncold-hardening temperatures. Cold tolerance of noncoldhardened trifoliate orange trees was also improved with ABA and PP333. Foliar sprays of NAA (sour orange) and PP333 and BA (+) minerals (trifoliate) increased cold tolerance of cold-hardened trees by 8%. Results indicate that spray applications of growth regulators influence physiological factors associated with foliar functioning and cold tolerance in citrus during different temperature regimes.SummaryGrowth promoters (BA) and inhibitors (NAA) have the potential to promote cold hardines through either a strong stimulatory effect on foliar physiology or a marked inhibition of growth in general. This suggests that each growth regulator may possess an independent role in the cold-hardiness phenomenon and may also interact with physiological processes other than soluble protein and chlorophyll metabolism. The relationship between soluble protein levels in citrus foliage and the degree of cold hardiness remains uncertain and is essentially unresolved pending more specific qualitative research.

Purification and N-terminal sequence of the major leaf protein (trifoliatin) from trifoliate orange

Abstract The major protein found in the leaves, bark and hardened roots of trifoliate orange ( Poncirus trifoliata L. Raf.) is a glycoprotein ( M r 24 000). Characteristics of trifoliate orange protein, now called trifoliatin, are consistent with those reported for other plant storage proteins, the low sequence similarity with other leaf proteins indicates that trifoliatin may represent a new class of vegetative proteins. Levels of glycoprotein vary inversely with leaf age and temperature [6] and can reach levels up to 65% of total leaf protein. The purification of this protein was accomplished by a combination of free zone isoelectric focusing and chromatography techniques. The N-terminal sequence determined by Edman (P. Edman and G. Begg, Eur. J. Biochem., 1 (1967) 80) degradation shares little to no sequence similarity with other known leaf proteins. The partial sequence of trifoliatin has the highest sequence similarity with sporamin, the major tuberous root storage protein in sweet potato. Trifoliatin has no protease activity to casein hydrolysate or inhibitory activity to trypsin or α-chymotrypsin.

Allergenic Cross-Reactivity between Callistemon citrinis and Melaleuca quinquenervia Pollens

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH4B) and melaleuca (NH4M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH4B and NH4M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH4B1-NH4B4 and NH4M1-NH4M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH4B1 and NH4M1, respectively, contained allergenic components. These allergenic components, NH4B1 and NH4M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.

Activity of ribulose-1,5-bisphosphate carboxylase in the leaf tissues of blight-affected ‘Valencia’ orange trees (Citrus sinensis (L.) Osbeck)

Abstract The activities and kinetic properties of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were measured in leaf extracts of ‘Valencia’ orange trees. Enzyme preparations were made from healthy (control) trees and from trees affected by citrus blight, a disease of unknown cause. Activity of RuBPCase decreased in blighted trees as compared with the control, and this decrease was most obvious in the afternoon. The enzyme from leaves of control and blighted trees showed no differences in affinities for CO 2 and RuBP, having a K m (CO 2 ) of 25.8 μM and a K m (RuBP) of 210 μM. The V max values of the enzyme in blighted trees, however, were only 71 and 74% of those of the control with respect to HCO 3 − and RuBP concentration, respectively. Zinc concentration was about sixfold higher in trunk wood and twofold higher in leaf tissues of blight-affected trees than control trees. Activity of carbonic anhydrase in leaves of blighted trees was about 69% of the control. Reduced RuBPCase activity is considered one of multiple factors contributing to reduced photosynthetic rates and decreased growth and productivity of blight-affected citrus trees.