SCREENING FOR IRON CHLOROSIS TOLERANCE OF IN VITRO PYRUS ROOTSTOCK GERMPLASM

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1 SCREENING FOR IRON CHLOROSIS TOLERANCE OF IN VITRO PYRUS ROOTSTOCK GERMPLASM Sugae Wada, Department of Hortiulture, Oregon State University, 4017 Agriulture & Life Siene Bldg. Corvallis Oregon Barbara M. Reed, United States Department of Agriulture, Agriultural Researh Servie, Peoria Road Corvallis Oregon ABSTRACT Fruit trees grown in alareous soils in arid and semiarid regions are iron (Fe) defiient, produing symptoms known as Fe-hlorosis. In vitro sreening for pear rootstoks to determine whih are tolerant to the Fe-hlorosis from diverse Pyrus germplasm ould be major importane to fruit growers for preventing hlorosis, reduing eonomi losses and the need for expensive and environmentally unfavorable soil amendments. This study was to develop a standardized in vitro testing and sreening method for resistane to Fe hlorosis. In vitro Fe-stress onditions were indued for the plantlets of eah rootstok by modifying the Fe 3+ helate ethylendiamine di (o-hydroxyphenylaeti) aid (EDDHA) and potassium biarbonate (KHCO 3 ) onentrations in pear rootstok (PRS) medium. Data was evaluated for six ategories; quality, shoot length, perent dead leaves, olor, and SPAD index. Photos were taken for the visual assessment. The most effetive treatment was PRS medium with 1 mm eah of Fe-EDDHA and KCO 3 at ph 7.0. The plant responses indiated that this treatment an be used as a definitive protool with evaluation using the SPAD readings to determine tolerant or suseptible genotypes. Medium ph redution for eah genotype was not effetive for determining tolerant ultivars, but the SPAD index and the visual assessments were definitive. The most tolerant was P. ommunis OHxF87 followed by P. ommunis Horner 4, P. betulifolia OPR 260 and P. spinosa. Suseptible ultivars were Winter Nelis, OPR 113 and Pyriam. This in vitro sreening tool an be used to determine whih rootstoks should be onsidered as possible andidates for field trials, reduing time and resoures needed to test all available rootstoks INTRODUCTION Fruit trees grown in alareous soils in arid and semiarid regions are iron (Fe) defiient, produing symptoms known as Fe hlorosis. The nutritional status of field plants an be deteted by leaf mineral analysis; however, leaf analysis has serious limitations for evaluating atual iron defiieny. There is often no orrelation between iron ontent in

2 leaves and the degree of Fe-hlorosis, making it one of the most omplex nutritional defiienies. Massive appliation of syntheti Fe helates to soils are the most ommon treatment for orreting Fe-hlorosis in fields, but this reates the risk of negative environmental impats(pestana et al., 2003), and is expensive (Álvarez-Fernández et al., 2005). In Europe, an inrease in Fe-hlorosis symptoms diretly orrelated with sharp redutions in fruit yield per tree, fruit size and quality and there is a serious need for early diagnosis of this ondition (Abadía et al., 2000). Fortunately, in vitro-grown pear and quine shoots with no root system are able to respond to iron defiieny and biarbonate enrihed onditions and provide biohemial and physiologial responses that an be measured (Donnini et al., 2008). In vitro responses of miropropagated shoots to low Fe onditions are similar to those of mature trees in the field. Obvious differenes exist among speies in the ability to redue Fe and to aidify the growth medium (Dolet-Sanjuan et al., 1990). Pyrus amygdaliformis adapted more easily to Fe-limiting onditions found with alkaline soils, and had a higher ability to redue Fe and to aidify the medium than the more sensitive quine rootstoks, Cydonia oblonga (Lombard and Westwood 1987). Roots and shoots showed similar adaptive responses to Fe stress, indiating that Fe defiieny an be readily expressed in shoots as well as in the roots. A speies that adapts well to Felimiting onditions suh as alkaline soils exhibits enhaned ability to redue Fe and to aidify the medium ompared to sensitive speies (Dolet-Sanjuan et al., 1992). A SPAD hlorophyll meter provides an unbiased, quantitative measure of leaf hlorosis (Álvarez-Fernández et al., 2005) and hlorosis-tolerant Prunus rootstoks in the field showed high or very high SPAD values (Jiménez et al., 2008). In vitro sreening for pear rootstoks to determine whih are tolerant to the Fe hlorosis from available Pyrus germplasm ould be of major importane in regions with alareous soils for preventing hlorosis, reduing eonomi losses and reduing the need for soil amendments. OBJECTIVES Develop a standardized in vitro testing and sreening method for Fe hlorosis for Pyrus rootstoks. Test a wide range of pear rootstok germplasm. Disseminate findings and protools to growers and ommerial labs.

3 PROCEDURES Plant Materials: Pear rootstok ultures were established on Pyrus rootstok (PRS) medium developed in earlier studies (Wada et al., unpublished). Fully grown shoot ultures (4 week subulture) were planted with five shoots per ontainer without removing the base. Three repliations and three separate experiments were run (Otober / November / Deember) for Winter Nelis, OHxF87, Horner 4 and OPR 260 (n=45). Due to slower multipliation, two experiments were run for P. spinosa set in Ot. and De.(n=30), and one for Pyriam in Ot. and one for OPR113 in De.(n=15) (Table 1). Table 1. Genotypes tested Otober through Deember, Genotype Loal number Speies (subfamily) Otober November Deember Winter Nelis P. ommunis X X X OHxF P. ommunis X X X Horner P. ommunis X X X OH-11 Pyriam P. ommunis X OPR P. betulifolia X X X OPR P. betulifolia X P. spinosa P. spinosa (Amygdaloideae) X X Indution of Fe hlorosis: In vitro Fe-stress onditions were indued for the plantlets of eah rootstok by modifying the Fe 3+ helate ethylendiamine di (ohydroxyphenylaeti) aid (EDDHA) and potassium biarbonate (KHCO 3 ) onentrations in PRS medium. Two initial tests with six P. ommunis: OHxF69, 87,97, 513, Pyro 2-33, and Horner 10 for the first, the seond for OHxF 87, 97, 513, Pyro 2-33 Horner 10 and Fox 11, were used to identify a definitive testing range for the hemials and ph. Fe-defiient to Fe-suffiient onditions: To ompare responses to Fe-defiient or Fe suffiient onditions by adding 1 mm eah of Fe-EDDHA and KHCO 3, shoot ultures of eah genotype were grown on three ontainers of eah treatment for 4 weeks (n=15). Data were taken at 4 weeks. Eah genotype have three repliates and the experiment were done three times (n=45). Testing ontinued using the six modified treatments (all at 1 mm) with more genotypes inluding other speies. Trt 1: MS-Fe onentration with no KHCO 3 at ph 5.7 (Control 1) Trt 2: MS-Fe onentration with 1 mm KHCO 3 at ph at 6.3 Trt 3: 1 mm Fe-EDDHA with no KHCO 3 at ph at 5.7 (Control 2) Trt 4: 1 mm Fe-EDDHA and 1 mm KHCO 3 at ph at 5.7

4 Trt 5: 1 mm Fe-EDDHA and 1 mm KHCO 3 at ph at 6.3 Trt 6: 1 mm Fe-EDDHA and 1 mm KHCO 3 at ph at 7.0 The ph range: The medium ph was adjusted to 5.7 for T1, T3, and T5; 6.3 for T2 and T5; and 7.0 for T6. High or low target ph was ahieved using 4N hydrohlori aid (HCL) or 1N sodium hydroxide (NaOH). The ph of the medium was measured before and after autolaving and at the end of the growth period. Data taken: Categories evaluated were quality ratings (1 low, 3 high), shoot length, perent dead leaves, olor ratings (1 low, 3 high), and SPAD readings (< 25 yellow or light green, > 25 green, >30 dark green) (data not shown). Mean responses of 15 plants from three repliates (n=45) were taken 4 weeks after planting. Data were pooled and analyzed using SAS (ver. 9.3 Cary, NC: SAS Institute). Models were determined for six responses (quality, shoot length, perent dead leaves, olor rating, and SPAD index). RESULTS The initial tests showed that the ph range should be adjusted to 5.7 or 6.3 with the initial ph no higher than 7.0 as the most extreme ph ondition for in vitro system. An adjusted ph at 8.0 was too high for shoot survival. Potassium biarbonate at 1 mm should be the highest onentration. It was not meaningful to test a high onentration suh as 10 mm KHCO 3 for this in vitro system as all the ultures died. The standard MS iron onentration or 1 mm Fe EDDHA were both within range for ontinued testing. Removing the base of shoots when planting was too harsh on the high KHCO 3 media and growth was poor for all. The final sreening tests resulted in data from six ategories and all had signifiant P- values between 0.02 to < Medium ph substantially hanged after autolaving when KHCO 3 was added. Control media remained lose to the original ph. Spent medium ph largely varied by genotype (Table 2). The highest ph was originally at 7.0 but reahed 9.58 after autolaving the medium. Redution of ph in the spent medium ranged from a derease of 0.34 in T6 by OHxF87 to a derease of 1.95 in T5 by Winter Nelis (Figure 1). These dereases were not useful for evaluating the pear rootstok tolerane. Table 2. Medium ph values at three points in the experiment. Treatment Adjusted medium ph Fresh medium ph after autolaving Spent medium ph After 4 wks (average) 1 MS iron (EDTA) MS iron+khco Fe-EDDHA

5 4 Fe-EDDHA+KHCO Fe-EDDHA+KHCO Fe-EDDHA+KHCO ph redution by genotype Winter Nelis OHxH 87 Horner 4 OPR 260 P. spinosa Pyriam OPR 113 ph 5.5(EDTA) ph 9.3(EDTA) ph 5.5(EDDHA) ph 8.4(EDDHA) ph 9.3(EDDHA) ph 9.6(EDDHA) Figure 1. The medium ph redution by eah genotype 4 week after planting. The ph values represent the fresh medium ph after autolaving. Final medium ph for eah treatment is shown Table 2. Chlorophyll ontent Chlorophyll ontent in leaves was determined by the SPAD readings (Figure 2). The higher the hlorophyll ontent, the healthier the leaf, even under stress on the high ph medium (T2 and T6). Treatment 6, the EDDHA medium with the highest ph (9.6), ould be used to determine tolerant or suseptible genotypes by the SPAD readings. The most tolerant was P. ommunis OHxF87 followed by P. ommunis Horner 4, P. betulifolia OPR 260 and P. spinosa. Suseptible ultivars were Winter Nelis, OPR 113 and Pyriam. The visual assessment evaluated the overall ondition of the shoot ultures (Figure 3). Shoots for all genotypes on the MS ontrol treatment T1 (MS iron) were green and atively growing and all exept OPR 113 were good on T3 (EDDHA iron). Higher ph treatments T2, T4 and T5 produed aeptable growth exept for OPR 113. The main differenes in plant growth were seen on T6 with the highest ph. This treatment

6 resulted in distint visual hanges in the shoots on some genotypes and ould be used to differentiate the tolerane or suseptibility of the genotypes. Chlorophyll ontent (SPAD reading) a b b d b b a WinterNelis OHxF87 Horner4 OPR260 P. spinosa Pyriam OPR113 d T1 T2 T3 T4 T5 T6 Figure 2. Chlorophyll ontent of the six pear rootstoks after growth on six treatments. The means separation values are by T-test (SAS 9.3). SPAD data (Figure 2) ombined with visual assessment (Figure 3) indiated that OH F 87, Horner 4 and OPR 260 were tolerant; OPR 113 was moderately tolerant and Pyriam and Winter Nelis were suseptible. DISCUSSION This study identified Fe-hlorosis tolerant and suseptible rootstoks using an in vitro system. The ombination of 1 mm eah of Fe-EDDHA and KHCO 3 reated the suffiient medium ph range from 5.49 to 9.58 after autolaving for this sreening test. The most effetive treatment was 1 mm Fe-EDDHA with 1 mm KHCO 3 at ph 7.0 and it an be used as a definitive protool for pear rootstok Fe-hlorosis sreening. Medium ph was redued at different rates by eah genotype (Figure 1) but it did not definitively indiate tolerane. The SPAD readings and the visual assessments used together were definitive. This study defining an in vitro sreening tool for pear rootstoks an be used to determine whih rootstoks should be onsidered as andidates for field trials, reduing the time and ost needed to test all available andidates.

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