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Photosynthesis and freeze tolerance comparisons of the newly released “ambersweet” hybrid with “valencia” orange
Em'irosmemal and E~perimental Bottoms, \qJl 33, No. 3, pp. 391 395, 1993
I)1)98 8472i93 $6.0()~ 0.00 Pclgamon Press Lid
Printed in (;rca( Britain.
P H O T O S Y N T H E S I S A N D FREEZE T O L E R A N C E C O M P A R I S O N S OF THE NEWLY RELEASED "AMBERSWEET" HYBRID W I T H "VALENCIA" O R A N G E JOSEPH C. V. VU* and GEORGE YELENOSKY
U.S. Department of Agriculture, Agricultural Research Service, Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803, U.S.A.
(Received 10 November 1992; accepted in revisedJbrm 30 December 1992) Vu J. C. V. and YELENOSKY G. Photosynthesis and freeze tolerance comparisons of the newly released "Ambersweet" hybrid with "Valencia" orange. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 33, 391--395, 1993. Two-year-old trees of "Ambersweet", a newly released citrus hybrid of "Clementine" tangerine (Citrus reticulata Blanco)x "Orlando" tangelo (C. paradisi Macf. x C. reticulata) x midseason sweet orange [C. sinensis (L.) Osbeck], were tested and compared for photosynthetic performance and chilling tolerance in controlled-temperature chambers, using "Valencia" sweet orange [C. sinensis (L.) Osbeck] trees of the same age and on similar rootstocks. In vitro activities of ribulose bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase and concentrations of chlorophyll and nonstructural carbohydrates of Ambersweet leaves were lower than for Valencia leaves during nonhardening and cold-hardening conditions. Both varieties, however, had similar rates of leaf photosynthesis. Freeze tests at - 6 . 7 and - 8 ° C tbr 3 hr showed that cold hardiness of Ambersweet trees was comparable to that of Valencia sweet orange. It is expected that Ambersweet would compete tavorably with Valencia as well as other existing sweet orange types for new world markets.
Key word~: Ambersweet citrus hybrid, photosynthesis, cold tolerance.
INTRODUCTION
ONE of the m a j o r goals of the U.S. D e p a r t m e n t of A g r i c u l t u r e - - A g r i c u l t u r a l Research Service citrus b r e e d i n g p r o g r a m is to develop commercially a c c e p t a b l e c o l d - h a r d y citrus to ameliorate d e v a s t a t i n g freeze losses which have occurred in F l o r i d a since the 1800s. Billions of dollars have been lost because of recurring freezes
and today, h u n d r e d s of thousands of hectares of once prime citrus-producing l a n d lie idle in north central F l o r i d a because of five m a j o r freezes in the 1980s. T h e A m b e r s w e e t h y b r i d was developed in the 1960s by C. J. H e a r n of the U S D A - A R S citrus breeding p r o g r a m at the A. H. W h i t m o r e F o u n d a t i o n F a r m near O r l a n d o , Florida, and was released in 1989. (3i " A m b e r s w e e t " resulted from
* To whom all correspondence should be addressed at: USDA-ARS, Agronomy Physiology Laboratory, Building 164, University of Florida, Gainesville, FL 32611, U.S.A. Abbreviation.s: CER, CO2 exchange rate; PEPCase, phosphoenolpyruvate carboxylase; PPFD, photosynthetic photon flux density; Rubisco, ribulose bisphosphate carboxylase-oxygenase. 391
392
J.C.V.
VU and G. YELENOSKY
crossing cold-hardy "Clementine" tangerine (Citrus reticulata Blanco) x cold-hardy "Orlando" tangelo (C. paradisi Macf. x C. reticulata) with a less cold-hardy midseason sweet orange, C. sinensis (L.) Osbeck. The fruit peels easily, resembles navel orange in size and color, and has high economic potential for fresh fruit and juice processing. c3~Ambersweet is classified by the Florida Citrus Commission as an orange based on fruit traits and chemical analyses of the juice, i4 Classification by the U.S. Food and Drug Administration is pending review to ensure compliance with common standards of identity to competing products. In central Florida, Ambersweet harvest ranges from mid-October through December. This relatively early harvest, before damaging freezes usually occur in Florida in late December, January and early February, is a major advantage over "Valencia" sweet orange, C. sinensis (L.) Osbeck. Valencia accounts for about 35% of the state's 1.3 billion dollars annual citrus crop, '2i but is highly vulnerable to devastating freeze losses because of an April through May harvest season. Plantings of Ambersweet in 1992 are expected to exceed five million field and nursery trees in Florida, which is nearly as many as all other citrus varieties developed by the U.S. Department of Agriculture combined. It is thought by some citrus growers that Ambersweet, characterized by early harvest fruit, vigorous growth and dense canopy, and equally good pertbrmance on a variety of commercial rootstocks, may some day co-exist and even replace Valencia orange as a major citrus cultivar in central Florida. Citrus is generally classified as a cold-tender evergreen with a subtropical and tropical origin, and has potential to cold acclimate during progressively cooler temperatures above freezing, i9) However, citrus cold acclimation is considerably less than that of temperate tree fruit crops, and it is unclear what physiological factor(s) are responsible for citrus freeze survival. Many physicochemical changes during acclimation are implicated with supercooling being a strong freeze avoidance factor, ~1i and photosynthetic activity !5) and carbohydrate accumulation seemingly important in water stress-osmotic relationships in the tolerance of citrus tissues to ice. 'q2~ Essentially, nothing is known about these factors in Ambersweet or its parentage, other than
hybrids with Clementine as a parent having a relatively high percentage for being cold hardy. :j:~i Preliminary observations of tree per[brmance under field conditions suggest that Ambersweet may possess better cold tolerance compared to some other existing sweet orange types in central Florida including Valencia. No freeze tests or physiological-biochemical studies, however, have been conducted on this new citrus hybrid. If the chilling-tolerance traits were preserved in Ambersweet, then some exceptional characteristics in leaf photosynthetic pertbrmance might also be expressed, since maintenance of the photosynthetic capacity has been hypothesized as one of the key factors in the ability of plants to acclimate and/or survive from adverse environmental stresses, including cold temperatures. The present study was therefore conducted to evaluate the photosynthetic performance and sugar accumulation characteristics, as well as the fi'eezing tolerance and supercooling capability of Ambersweet, compared to the well-established, mildly cold-hardy Valencia sweet orange.
MATERIALS
AND METHODS
Test trees of Ambersweet hybrid and Valencia sweet orange were propagated on three different rootstocks-- Carrizo ci trange (C. sinensis x Poncirus lriJbliata L. Raf.), Cleopatra mandarin (C. reticulata) and sour orange (C. aurantium L.). Single trees were grown in 15-1 plastic pots containing sandy soil and maintained under natural daylight greenhouse conditions. Maximum day and minimum night temperatures in the greenhouse were 32 and 20°C, respectively, and relative humidity ranged from 35% (day) to 98% (night). Maximum photosynthetic photon flux density (PPFD) at mid-day was about 1000 /~mol m 2 s-~ at the top of the trees. Trees were watered daily and fertilized monthly with a 12 N 2.6 P 5 K solution containing micronutrients. An equal number of uniform-appearing, 2year-old trees (2 years from grafting), about 100 cm in height and 1 cm in stem diameter, of each scion variety on the three rootstocks was arbitrarily assigned simultaneously to 4 weeks of cold hardening and 4 weeks of nonhardening in two separate programmed temperature chambers
PHOTOSYNTHESIS AND FREEZE TOLERANCE IN CITRUS used for citrus cold-hardening tests, i9/Cold hardening consisted of20°C, 12-hr days with approximately 400/2mol m x s l P P F D at the top of the trees, and 10°C nights for two consecutive weeks, followed by 2 weeks of 15°C days and 4°C nights. N o n h a r d e n i n g was at similar light conditions with 30°C days and 20°C nights for 4 weeks. Relative humidity fluctuated from 40 to 60% in both chambers during hardening treatments. At the end of the 4-week treatment and immediately before freeze tests, determinations of COx exchange rates (CER), activities of ribulose bisphosphate carboxylase-oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase), and contents of chlorophyll and carbohydrates were made. C E R was measured on singleattached, fhlly developed top leaves inside the temperature-controlled chambers where trees were maintained, using the L I - C O R 6200 Portable Photosynthesis SystemJ 7i Fully developed top leaves from three trees of each of the two varieties on each of the three rootstocks were sampled and analyzed according to previously published procedures fbr Rubisco and PEPCase, (5/ carbohydrates i7; and chlorophyll, il) Freeze tests were performed thereafter in a separate controlledtemperature c h a m b e r with 50 _10~}"o relative humidity and no light. Trees of each variety on three rootstocks, nine for Ambersweet and nine for Valencia for each temperature-freeze test, were equilibrated at 2°C for 3 hr immediately before temperature was lowered at a rate of 5°C/hr to - 6 . 7 and - 8 ° C for 3 hr, and thawed at 5°C/hr to original starting temperature (2°C). Freeze-tested trees were kept at room temperature (25°C) for an additional 3 hr before being returned to the greenhouse for 5 weeks of freeze-ii~jury observations. Injury was based on the n u m b e r of leaves abscised or killed and the length of stem dieback relative to total length of scion. T h e approximate temperature of the scion stem and apparent m o m e n t that ice started to form in the wood (nucleation temperature), were based on the release of latent heat of freezing (exotherm). This was indicated by copper-constantan thermocouples (36-gauge, 0.12-mm diameter) inserted 2-3 m m into the central vascular system of a leaf trace at mid-stem level. Thermocouples were connected to an automated
393
data collection system accurate to + 0 . 1 ° C at 22°C ambient, and a resolution of0.015°CJ 1°:~ RESULTS AND DISCUSSION
The influence of three different rootstocks was not discernible in statistical analyses, and data acquired for Ambersweet and Valencia for each specific measurement were combined across rootstocks (Tables 1, 2). Ambersweet and Valencia had similar rates of leaf photosynthesis, which averaged 4.5 and 10.3 /~mol m x s i for coldhardened and nonhardened leaves, respectively (Table 1). Leaf chlorophyll concentrations, however, were approximately 50% less in Ambersweet than in Valencia. This agreed with observations that Ambersweet trees generally have lighter green foliage than Valencia, regardless of growth conditions or time of the year. Aetivity of PEPCase, an important enzyme in organic acid metabolism in citrus leaves, flower buds and y o u n g fruits,/8/was also less in Ambersweet leaves and averaged 62~'o of the activity tbund in Valencia leaves during both nonhardening and coldhardening conditions (Table 1). The three-fold increase in PEPCase activity after the cold-hardening treatment seems a c o m m o n response of sweet orange and other citrus varieties to cold temperatures. ~5'6;Such a change with chilling was not observed for Rubisco, the major photosynthetic carboxylating enzyme responsible for fixing ambient COx in citrus leaves. Rubisco activity in Ambersweet leaves was about 84°,~ that of Valencia at nonhardening and cold-hardening conditions (Table 1). C a r b o h y d r a t e concentrations were also lower in Ambersweet than in Valencia leaves. Soluble sugars were about 80~/o, and starch was 35°/'0 at cold-hardening and 65~)o at nonhardening temperatures tbr Ambersweet leaves, as compared with Valencia leaves. Cold hardening for 4 weeks did not save the leaves, but stems survived with little to no injury after the 3-hr freeze tests at - 6 . 7 and - 8 ° C for both citrus varieties (Table 2). This stem tolerance, however, was largely lost at - 10°C for 1 hr, and dieback was more than 80% of scion height (data not shown). T h e ability of Ambersweet and Valencia trees to cool 4-6°C below the water freezing point before ice apparently started to form in the wood supports previous data on
394
J.C.V.
VU and G. YELENOSKY
Table 1. CO2 exchange rates ( CER), chlorophyll ( Chl), ribulose biJphosphate carboxylase-oxygenase ( Rubisco), phosphoenolpyruvate carboxylase ( PEPCase) and carbohydrates of 2-year-old Arnbersweet (Arab) and Valencia (Val) orange trees grown under controlled cold-hardened and nonhardened chamber conditions Analysis of variance Cold-hardened Variable CER (/tmol m 2 s -~) Chl (mg g ~fresh wt) Rubisco (/mlol g fresh wt hr ') PEPCase (/~mol g-~freshwthr ') Soluble sugars (rag g - ' dry wt) Starch (rag g t dry wt)
Nonhardened
Amb
Val
Amb
Val
Variety
Treatment
Treatment x variety
4.4 (0.1)
4.6 (0.2)
10.2 (0.3)
10.4 (0.3)
NS
-~
NS
1.6 (0.1)
3.1 (0.1)
1.7 (0.1)
3.2 (0.1)
~
NS
NS
260.4 (7.0)
297.6 (5.9)
256.8 (13.1) 319.2 (2.7)
t
NS
NS
94.4 (3.2)
153.7 (10.0)
33.7 (0.7)
52.6 (0.9)
1"
~
*
60.4 (6.2)
80.4 (4.9)
55.4 (2.0)
62.8 (3.8)
*
*
NS
23.4 (4.2)
67.1 (6.5)
41.4 (6.5)
64.1 (5.4)
~
NS
NS
Mean values and S.E.M. (parentheses) of three to 25 measurements are presented. * Significant at P < 0.05 level. I Significant at P < 0.01 level. NS, not significant. supercooling in citrus. I< T h e g e n e r a l t r e n d of lower n u c l e a t i o n temperatures in c o l d - h a r d e n e d t h a n in n o n h a r d e n e d trees associates l o n g e r freeze-avoidance with cold h a r d e n i n g , a n d freeze i n j u r y in this s t u d y was n o t i c e a b l y greater w h e n ice was in the wood for 2 hr or m o r e ( T a b l e 2). Benefits of l o n g e r fi'eeze-avoidance a n d less
freeze i n j u r y seemingly are m o r e likely to occur in freezes w h e r e t e m p e r a t u r e s do not go below - 6 ° C a n d t e m p e r a t u r e m i n i m a persist for 1 hr or less. F r o m this limited study with y o u n g trees using c o n t r o l l e d - t e m p e r a t u r e c h a m b e r s , we did not find a n y obvious characteristic i n d i c a t i n g that
Table 2. Freeze tolerance of 2-year-old Ambersweet (Arab) and Valencia (Val) orange trees grown under controlled coldhardened and nonhardened chamber conditions Cold-hardened Variable 1. - 6 . 7 ° C / 3 hr a. Nucleation temperature (°C) b. Ice duration in scion wood (hr) c. Leaves killed ('~/o) d. Rootstock wood dieback (°4,) O/ e. Scion wood dieback (,o) 2. - 8 ° C / 3 hr a. Nucleation temperature (°C) b. Ice duration in scion wood (hr) c. Leaves killed (o,;~) O,' d. Rootstock wood dieback (,o) O/ e. Scion wood dieback ('o)
Amb
Val
Nonhardened Amb
Val
- 6.0 (0.8) 2.8 (0.5) 100 0 2 (2)
- 6.2 (0.4) 2.8 (0.2) 89 (10) 0 0
- 5.4 (0.5) 3.1 (0.1) 100 10 (10) 100
- 5.4 (0.6) 3.0 (0.1) 100 0 100
- 5 . 2 (1.0) 5.2 (0.3) 100 0 5 (5)
5.4 (0.4) 5.2 (0.2) 100 0 5 (3)
- 4 . 5 (0.7) 5.4 (0.2) 100 30 (30) 100
- 5 . 3 (0.6) 5.2 (0.1) 100 33 (33) I0
Mean values and S.E.M. (parentheses) of three to nine determinations are presented.
PHOTOSYNTHESIS AND FREEZE TOLERANCE IN C I T R U S A m b e r s w e e t was more c o l d - h a r d y t h a n Valencia. Both varieties performed c o m p a r a b l y in photosynthetic c a p a b i l i t y a n d cold hardiness, regardless of freeze-avoidance (supercooling) or freezetolerance (ice stress) in the tissues. Controllede n v i r o n m e n t tests with y o u n g citrus trees, however, m a y not always reflect actual performance of older a n d bigger trees in the field. M a t u r e trees of A m b e r s w e e t performed equally well h o r t i c u l t u r a l l y on " C l e o p a t r a " m a n d a r i n , sour orange a n d Carrizo citrange u n d e r n a t u r a l field conditions. However, they did not perform similarly in cold hardiness. Trees on Carrizo citrange sustained stem kill in tops that killed the u p p e r 30-45 cm of growth d u r i n g the 1983 freeze, while those on C l e o p a t r a m a n d a r i n and sour orange sustained no stem kill. !3! It will, therefore, take years of evaluation in the field u n d e r a variety of conditions to d e t e r m i n e w h a t role A m b e r sweet will p l a y in the F l o r i d a citrus i n d u s t r y and world markets in light of free-trade initiatives by the U.S. M o r e d a t a on fertilization requirements, d r o u g h t a n d w a t e r l o g g i n g susceptibility, a n d resistance to insects and diseases are needed for this new citrus. Since y o u n g trees of A m b e r s w e e t do not have exceptional cold hardiness, plantings of this h y b r i d on sites in north central F l o r i d a considered too cold for V a l e n c i a or " H a m l i n " orange should be taken with more precautions and careful provisions for freeze protection. Current information, however, indicates that A m b e r sweet is expected to compete favorably with, and possibly outperfbrm, existing sweet orange types for new markets. Disclaimer Mention of a trademark, warranty, proprietary product or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
395
REFERENCES
1. ARNON D. I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant. Physiol. 24, 1 15. 2. FREIE R. L. (1992) Florida Agricultural Statistics Citrus Summary 1990 1991. FL. Agric. Stat. Serv., Orlando, FL. 3. HEARN C. J. (1989) Yield and fruit quality of 'Ambersweet' orange hybrid on different rootstocks. Proc. FL State Hort. Soc. 102, 75 105. 4. STANLEY D. (1992) When is an orange not an orange? Agric. Res. 40, 18-20. 5. Vu J. C. V. and YELENOSKY G. (1987) Photosynthetic characteristics in leaves of 'Valencia' orange (Citrus sinensis (L.) Osbeck) grown under high and low temperature regimes. Environ. exp. Bot. 27, 279-287. 6. Vu J. C. V. and YELENOSKY G. (1989) Growth and photosynthesis of citrus trees subjected to soil flooding and cold temperature. Plant. Physiol. 89, S-973. 7. Vu J. C. V. and YELENOSKY G. (1991) Photosynthetic responses of citrus trees to soil flooding. Physiol. Plant. 81, 7-14. 8. Vu J. C. V., YELENOSKYG. and McDONALD R. E. (1990) Proteins, non-structural carbohydrates and organic acids in the flowers of sweet orange (Citrus sinensis (L.) Osbeck). Environ. exp. Bot. 30, 505-513. 9. YELENOSKVG. (1978) Cold hardening 'Valencia' orange trees to tolerate -6.T)C without injury. J. Am. Soc. Hort. Sci. 103, 449 452. 10. YELENOSKY G. (1985) Cold hardiness in citrus. Hort. Rev. 7, 201-238. 11. YELENOSKY G. (1991) Apparent nucleation and freezing in various parts of young citrus trees during controlled freezes. HortScience 26, 576-579. 12. YELENOSKY G. and Guy C. L. (1989) Freezing tolerance of citrus, spinach, and petunia leaf tissue. Plant Physiol. 89, 444-451. 13. YOUNG R. and HEARN C. J. (1972) Screening citrus hybrids for cold hardiness. HortScience 7, 14-18.
I)1)98 8472i93 $6.0()~ 0.00 Pclgamon Press Lid
Printed in (;rca( Britain.
P H O T O S Y N T H E S I S A N D FREEZE T O L E R A N C E C O M P A R I S O N S OF THE NEWLY RELEASED "AMBERSWEET" HYBRID W I T H "VALENCIA" O R A N G E JOSEPH C. V. VU* and GEORGE YELENOSKY
U.S. Department of Agriculture, Agricultural Research Service, Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803, U.S.A.
(Received 10 November 1992; accepted in revisedJbrm 30 December 1992) Vu J. C. V. and YELENOSKY G. Photosynthesis and freeze tolerance comparisons of the newly released "Ambersweet" hybrid with "Valencia" orange. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 33, 391--395, 1993. Two-year-old trees of "Ambersweet", a newly released citrus hybrid of "Clementine" tangerine (Citrus reticulata Blanco)x "Orlando" tangelo (C. paradisi Macf. x C. reticulata) x midseason sweet orange [C. sinensis (L.) Osbeck], were tested and compared for photosynthetic performance and chilling tolerance in controlled-temperature chambers, using "Valencia" sweet orange [C. sinensis (L.) Osbeck] trees of the same age and on similar rootstocks. In vitro activities of ribulose bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase and concentrations of chlorophyll and nonstructural carbohydrates of Ambersweet leaves were lower than for Valencia leaves during nonhardening and cold-hardening conditions. Both varieties, however, had similar rates of leaf photosynthesis. Freeze tests at - 6 . 7 and - 8 ° C tbr 3 hr showed that cold hardiness of Ambersweet trees was comparable to that of Valencia sweet orange. It is expected that Ambersweet would compete tavorably with Valencia as well as other existing sweet orange types for new world markets.
Key word~: Ambersweet citrus hybrid, photosynthesis, cold tolerance.
INTRODUCTION
ONE of the m a j o r goals of the U.S. D e p a r t m e n t of A g r i c u l t u r e - - A g r i c u l t u r a l Research Service citrus b r e e d i n g p r o g r a m is to develop commercially a c c e p t a b l e c o l d - h a r d y citrus to ameliorate d e v a s t a t i n g freeze losses which have occurred in F l o r i d a since the 1800s. Billions of dollars have been lost because of recurring freezes
and today, h u n d r e d s of thousands of hectares of once prime citrus-producing l a n d lie idle in north central F l o r i d a because of five m a j o r freezes in the 1980s. T h e A m b e r s w e e t h y b r i d was developed in the 1960s by C. J. H e a r n of the U S D A - A R S citrus breeding p r o g r a m at the A. H. W h i t m o r e F o u n d a t i o n F a r m near O r l a n d o , Florida, and was released in 1989. (3i " A m b e r s w e e t " resulted from
* To whom all correspondence should be addressed at: USDA-ARS, Agronomy Physiology Laboratory, Building 164, University of Florida, Gainesville, FL 32611, U.S.A. Abbreviation.s: CER, CO2 exchange rate; PEPCase, phosphoenolpyruvate carboxylase; PPFD, photosynthetic photon flux density; Rubisco, ribulose bisphosphate carboxylase-oxygenase. 391
392
J.C.V.
VU and G. YELENOSKY
crossing cold-hardy "Clementine" tangerine (Citrus reticulata Blanco) x cold-hardy "Orlando" tangelo (C. paradisi Macf. x C. reticulata) with a less cold-hardy midseason sweet orange, C. sinensis (L.) Osbeck. The fruit peels easily, resembles navel orange in size and color, and has high economic potential for fresh fruit and juice processing. c3~Ambersweet is classified by the Florida Citrus Commission as an orange based on fruit traits and chemical analyses of the juice, i4 Classification by the U.S. Food and Drug Administration is pending review to ensure compliance with common standards of identity to competing products. In central Florida, Ambersweet harvest ranges from mid-October through December. This relatively early harvest, before damaging freezes usually occur in Florida in late December, January and early February, is a major advantage over "Valencia" sweet orange, C. sinensis (L.) Osbeck. Valencia accounts for about 35% of the state's 1.3 billion dollars annual citrus crop, '2i but is highly vulnerable to devastating freeze losses because of an April through May harvest season. Plantings of Ambersweet in 1992 are expected to exceed five million field and nursery trees in Florida, which is nearly as many as all other citrus varieties developed by the U.S. Department of Agriculture combined. It is thought by some citrus growers that Ambersweet, characterized by early harvest fruit, vigorous growth and dense canopy, and equally good pertbrmance on a variety of commercial rootstocks, may some day co-exist and even replace Valencia orange as a major citrus cultivar in central Florida. Citrus is generally classified as a cold-tender evergreen with a subtropical and tropical origin, and has potential to cold acclimate during progressively cooler temperatures above freezing, i9) However, citrus cold acclimation is considerably less than that of temperate tree fruit crops, and it is unclear what physiological factor(s) are responsible for citrus freeze survival. Many physicochemical changes during acclimation are implicated with supercooling being a strong freeze avoidance factor, ~1i and photosynthetic activity !5) and carbohydrate accumulation seemingly important in water stress-osmotic relationships in the tolerance of citrus tissues to ice. 'q2~ Essentially, nothing is known about these factors in Ambersweet or its parentage, other than
hybrids with Clementine as a parent having a relatively high percentage for being cold hardy. :j:~i Preliminary observations of tree per[brmance under field conditions suggest that Ambersweet may possess better cold tolerance compared to some other existing sweet orange types in central Florida including Valencia. No freeze tests or physiological-biochemical studies, however, have been conducted on this new citrus hybrid. If the chilling-tolerance traits were preserved in Ambersweet, then some exceptional characteristics in leaf photosynthetic pertbrmance might also be expressed, since maintenance of the photosynthetic capacity has been hypothesized as one of the key factors in the ability of plants to acclimate and/or survive from adverse environmental stresses, including cold temperatures. The present study was therefore conducted to evaluate the photosynthetic performance and sugar accumulation characteristics, as well as the fi'eezing tolerance and supercooling capability of Ambersweet, compared to the well-established, mildly cold-hardy Valencia sweet orange.
MATERIALS
AND METHODS
Test trees of Ambersweet hybrid and Valencia sweet orange were propagated on three different rootstocks-- Carrizo ci trange (C. sinensis x Poncirus lriJbliata L. Raf.), Cleopatra mandarin (C. reticulata) and sour orange (C. aurantium L.). Single trees were grown in 15-1 plastic pots containing sandy soil and maintained under natural daylight greenhouse conditions. Maximum day and minimum night temperatures in the greenhouse were 32 and 20°C, respectively, and relative humidity ranged from 35% (day) to 98% (night). Maximum photosynthetic photon flux density (PPFD) at mid-day was about 1000 /~mol m 2 s-~ at the top of the trees. Trees were watered daily and fertilized monthly with a 12 N 2.6 P 5 K solution containing micronutrients. An equal number of uniform-appearing, 2year-old trees (2 years from grafting), about 100 cm in height and 1 cm in stem diameter, of each scion variety on the three rootstocks was arbitrarily assigned simultaneously to 4 weeks of cold hardening and 4 weeks of nonhardening in two separate programmed temperature chambers
PHOTOSYNTHESIS AND FREEZE TOLERANCE IN CITRUS used for citrus cold-hardening tests, i9/Cold hardening consisted of20°C, 12-hr days with approximately 400/2mol m x s l P P F D at the top of the trees, and 10°C nights for two consecutive weeks, followed by 2 weeks of 15°C days and 4°C nights. N o n h a r d e n i n g was at similar light conditions with 30°C days and 20°C nights for 4 weeks. Relative humidity fluctuated from 40 to 60% in both chambers during hardening treatments. At the end of the 4-week treatment and immediately before freeze tests, determinations of COx exchange rates (CER), activities of ribulose bisphosphate carboxylase-oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase), and contents of chlorophyll and carbohydrates were made. C E R was measured on singleattached, fhlly developed top leaves inside the temperature-controlled chambers where trees were maintained, using the L I - C O R 6200 Portable Photosynthesis SystemJ 7i Fully developed top leaves from three trees of each of the two varieties on each of the three rootstocks were sampled and analyzed according to previously published procedures fbr Rubisco and PEPCase, (5/ carbohydrates i7; and chlorophyll, il) Freeze tests were performed thereafter in a separate controlledtemperature c h a m b e r with 50 _10~}"o relative humidity and no light. Trees of each variety on three rootstocks, nine for Ambersweet and nine for Valencia for each temperature-freeze test, were equilibrated at 2°C for 3 hr immediately before temperature was lowered at a rate of 5°C/hr to - 6 . 7 and - 8 ° C for 3 hr, and thawed at 5°C/hr to original starting temperature (2°C). Freeze-tested trees were kept at room temperature (25°C) for an additional 3 hr before being returned to the greenhouse for 5 weeks of freeze-ii~jury observations. Injury was based on the n u m b e r of leaves abscised or killed and the length of stem dieback relative to total length of scion. T h e approximate temperature of the scion stem and apparent m o m e n t that ice started to form in the wood (nucleation temperature), were based on the release of latent heat of freezing (exotherm). This was indicated by copper-constantan thermocouples (36-gauge, 0.12-mm diameter) inserted 2-3 m m into the central vascular system of a leaf trace at mid-stem level. Thermocouples were connected to an automated
393
data collection system accurate to + 0 . 1 ° C at 22°C ambient, and a resolution of0.015°CJ 1°:~ RESULTS AND DISCUSSION
The influence of three different rootstocks was not discernible in statistical analyses, and data acquired for Ambersweet and Valencia for each specific measurement were combined across rootstocks (Tables 1, 2). Ambersweet and Valencia had similar rates of leaf photosynthesis, which averaged 4.5 and 10.3 /~mol m x s i for coldhardened and nonhardened leaves, respectively (Table 1). Leaf chlorophyll concentrations, however, were approximately 50% less in Ambersweet than in Valencia. This agreed with observations that Ambersweet trees generally have lighter green foliage than Valencia, regardless of growth conditions or time of the year. Aetivity of PEPCase, an important enzyme in organic acid metabolism in citrus leaves, flower buds and y o u n g fruits,/8/was also less in Ambersweet leaves and averaged 62~'o of the activity tbund in Valencia leaves during both nonhardening and coldhardening conditions (Table 1). The three-fold increase in PEPCase activity after the cold-hardening treatment seems a c o m m o n response of sweet orange and other citrus varieties to cold temperatures. ~5'6;Such a change with chilling was not observed for Rubisco, the major photosynthetic carboxylating enzyme responsible for fixing ambient COx in citrus leaves. Rubisco activity in Ambersweet leaves was about 84°,~ that of Valencia at nonhardening and cold-hardening conditions (Table 1). C a r b o h y d r a t e concentrations were also lower in Ambersweet than in Valencia leaves. Soluble sugars were about 80~/o, and starch was 35°/'0 at cold-hardening and 65~)o at nonhardening temperatures tbr Ambersweet leaves, as compared with Valencia leaves. Cold hardening for 4 weeks did not save the leaves, but stems survived with little to no injury after the 3-hr freeze tests at - 6 . 7 and - 8 ° C for both citrus varieties (Table 2). This stem tolerance, however, was largely lost at - 10°C for 1 hr, and dieback was more than 80% of scion height (data not shown). T h e ability of Ambersweet and Valencia trees to cool 4-6°C below the water freezing point before ice apparently started to form in the wood supports previous data on
394
J.C.V.
VU and G. YELENOSKY
Table 1. CO2 exchange rates ( CER), chlorophyll ( Chl), ribulose biJphosphate carboxylase-oxygenase ( Rubisco), phosphoenolpyruvate carboxylase ( PEPCase) and carbohydrates of 2-year-old Arnbersweet (Arab) and Valencia (Val) orange trees grown under controlled cold-hardened and nonhardened chamber conditions Analysis of variance Cold-hardened Variable CER (/tmol m 2 s -~) Chl (mg g ~fresh wt) Rubisco (/mlol g fresh wt hr ') PEPCase (/~mol g-~freshwthr ') Soluble sugars (rag g - ' dry wt) Starch (rag g t dry wt)
Nonhardened
Amb
Val
Amb
Val
Variety
Treatment
Treatment x variety
4.4 (0.1)
4.6 (0.2)
10.2 (0.3)
10.4 (0.3)
NS
-~
NS
1.6 (0.1)
3.1 (0.1)
1.7 (0.1)
3.2 (0.1)
~
NS
NS
260.4 (7.0)
297.6 (5.9)
256.8 (13.1) 319.2 (2.7)
t
NS
NS
94.4 (3.2)
153.7 (10.0)
33.7 (0.7)
52.6 (0.9)
1"
~
*
60.4 (6.2)
80.4 (4.9)
55.4 (2.0)
62.8 (3.8)
*
*
NS
23.4 (4.2)
67.1 (6.5)
41.4 (6.5)
64.1 (5.4)
~
NS
NS
Mean values and S.E.M. (parentheses) of three to 25 measurements are presented. * Significant at P < 0.05 level. I Significant at P < 0.01 level. NS, not significant. supercooling in citrus. I< T h e g e n e r a l t r e n d of lower n u c l e a t i o n temperatures in c o l d - h a r d e n e d t h a n in n o n h a r d e n e d trees associates l o n g e r freeze-avoidance with cold h a r d e n i n g , a n d freeze i n j u r y in this s t u d y was n o t i c e a b l y greater w h e n ice was in the wood for 2 hr or m o r e ( T a b l e 2). Benefits of l o n g e r fi'eeze-avoidance a n d less
freeze i n j u r y seemingly are m o r e likely to occur in freezes w h e r e t e m p e r a t u r e s do not go below - 6 ° C a n d t e m p e r a t u r e m i n i m a persist for 1 hr or less. F r o m this limited study with y o u n g trees using c o n t r o l l e d - t e m p e r a t u r e c h a m b e r s , we did not find a n y obvious characteristic i n d i c a t i n g that
Table 2. Freeze tolerance of 2-year-old Ambersweet (Arab) and Valencia (Val) orange trees grown under controlled coldhardened and nonhardened chamber conditions Cold-hardened Variable 1. - 6 . 7 ° C / 3 hr a. Nucleation temperature (°C) b. Ice duration in scion wood (hr) c. Leaves killed ('~/o) d. Rootstock wood dieback (°4,) O/ e. Scion wood dieback (,o) 2. - 8 ° C / 3 hr a. Nucleation temperature (°C) b. Ice duration in scion wood (hr) c. Leaves killed (o,;~) O,' d. Rootstock wood dieback (,o) O/ e. Scion wood dieback ('o)
Amb
Val
Nonhardened Amb
Val
- 6.0 (0.8) 2.8 (0.5) 100 0 2 (2)
- 6.2 (0.4) 2.8 (0.2) 89 (10) 0 0
- 5.4 (0.5) 3.1 (0.1) 100 10 (10) 100
- 5.4 (0.6) 3.0 (0.1) 100 0 100
- 5 . 2 (1.0) 5.2 (0.3) 100 0 5 (5)
5.4 (0.4) 5.2 (0.2) 100 0 5 (3)
- 4 . 5 (0.7) 5.4 (0.2) 100 30 (30) 100
- 5 . 3 (0.6) 5.2 (0.1) 100 33 (33) I0
Mean values and S.E.M. (parentheses) of three to nine determinations are presented.
PHOTOSYNTHESIS AND FREEZE TOLERANCE IN C I T R U S A m b e r s w e e t was more c o l d - h a r d y t h a n Valencia. Both varieties performed c o m p a r a b l y in photosynthetic c a p a b i l i t y a n d cold hardiness, regardless of freeze-avoidance (supercooling) or freezetolerance (ice stress) in the tissues. Controllede n v i r o n m e n t tests with y o u n g citrus trees, however, m a y not always reflect actual performance of older a n d bigger trees in the field. M a t u r e trees of A m b e r s w e e t performed equally well h o r t i c u l t u r a l l y on " C l e o p a t r a " m a n d a r i n , sour orange a n d Carrizo citrange u n d e r n a t u r a l field conditions. However, they did not perform similarly in cold hardiness. Trees on Carrizo citrange sustained stem kill in tops that killed the u p p e r 30-45 cm of growth d u r i n g the 1983 freeze, while those on C l e o p a t r a m a n d a r i n and sour orange sustained no stem kill. !3! It will, therefore, take years of evaluation in the field u n d e r a variety of conditions to d e t e r m i n e w h a t role A m b e r sweet will p l a y in the F l o r i d a citrus i n d u s t r y and world markets in light of free-trade initiatives by the U.S. M o r e d a t a on fertilization requirements, d r o u g h t a n d w a t e r l o g g i n g susceptibility, a n d resistance to insects and diseases are needed for this new citrus. Since y o u n g trees of A m b e r s w e e t do not have exceptional cold hardiness, plantings of this h y b r i d on sites in north central F l o r i d a considered too cold for V a l e n c i a or " H a m l i n " orange should be taken with more precautions and careful provisions for freeze protection. Current information, however, indicates that A m b e r sweet is expected to compete favorably with, and possibly outperfbrm, existing sweet orange types for new markets. Disclaimer Mention of a trademark, warranty, proprietary product or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
395
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