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Indole acetic acid and abscisic acid levels in new shoots and fibrous roots of citrus scion-rootstock combinations
Scientia Horticulturae 84 (2000) 245±254
Indole acetic acid and abscisic acid levels in new shoots and fibrous roots of citrus scion-rootstock combinations Katsuji Nodaa,d, Hitoshi Okudab, Isao Iwagakic,* a
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1112, Japan b Department of Citriculture, National Institute of Fruit tree Science, Nakachyo, Okitsu, Shimizu, Shizuoka-ken 424-0292, Japan c Faculty of Agriculture, Shizuoka University, Kariyado, Fujieda, Shizuoka-ken 426-0001, Japan d Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan Accepted 17 May 1999
Abstract Citrus rootstocks are important for the growth of scion varieties, but it is not clear how they regulate scion vigor. We studied three citrus rootstock cultivars; `Flying Dragon' (Poncirus trifoliata var. monstrosa) a dwarfing rootstock, `Swingle' citrumelo (P. trifoliata Citrus paradisi) a vigorous rootstock, and trifoliate orange (P. trifoliata) as the control. Tender buds from new shoots of `Eureka' lemon were cleft grafted on etiolated rootstock seedlings. Eighteen months after grafting, the dry matter of each part of the young grafts was measured. Top weight was the greatest on `Swingle' citrumelo and smallest on `Flying Dragon', but there was no distinct difference in root growth among the rootstocks. Endogenous indole acetic acid (IAA) and abscisic acid (ABA) were measured in the new shoots and fibrous roots. The IAA level in the new shoots was highest in `Swingle' citrumelo and lowest in `Flying Dragon'. The ABA level in the new shoots was highest in `Flying Dragon' and lowest in `Swingle' citrumelo. Both the IAA and ABA levels in the fibrous roots were highest in the strains of Poncirus trifoliata and lowest in `Swingle' citrumelo. The vigorous rootstock `Swingle' citrumelo had the highest T±R and IAA±ABA ratios in the new shoots. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Rootstock; IAA; ABA
* Corresponding author. Tel.: +81-54-641-9500; fax: +81-54-644-4641. E-mail address: [email protected] (I. Iwagaki). 0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 0 - 1
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1. Introduction Trifoliate orange is the most common citrus rootstock, accounting for about 95% of all the citrus rootstocks used in Japan. Citrus trees grafted on trifoliate orange have comparatively small tree canopies and produce consistent high quality fruit. In spite of the merits of trifoliate orange, new types of rootstocks, vigorous and/or dwarf, for new scion varieties and innovative culture systems are needed. Plant growth regulators have important functions in plant behavior. Lockard and Schneider (1981), who summarized the existing information on the role of the rootstock in the dwarfing of grafted apple trees, suggested that a dwarfing mechanism is triggered by plant growth regulators. It is important to clarify the relationship that exists between the top and root in scion-rootstock combinations. The findings should contribute to the future development of new rootstocks. Auxin generally is considered as a plant growth promoter. Exogenously applied Indole 3-acetic acid (IAA) strongly promoted stem elongation over a long period in intact light-grown seedlings of both dwarf and tall peas (Yang et al., 1993), but auxin generally inhibits root elongation (Pilet and Saugy, 1987; Tanimoto and Watanabe, 1986). Abscisic acid (ABA) is regarded not only to be an inhibitor of elongation (Bensen et al., 1988; Sakurai et al., 1985; Yadava and Dayton, 1972) but a growth promoter (Bradford, 1983; Weston, 1976). A negative correlation between root growth and endogenous ABA was reported (Pilet and Saugy, 1987), and exogenous ABA inhibited the tip growth of excised roots (Gaither et al., 1975). The roles of IAA and ABA in scion-rootstock interaction are not fully known. We examined the levels of endogenous IAA and ABA in citrus scionrootstock combinations and here discuss the effect of the rootstock on scion growth. 2. Material and methods 2.1. Plant materials Three citrus rootstock varieties were used: `Swingle' citrumelo (Poncirus trifoliata Citrus paradisi) a vigorous rootstock, `Flying Dragon' (Poncirus trifoliata var. monstrosa) a dwarfing rootstock, and trifoliate orange (Poncirus trifoliata) as the standard rootstock. In May 1996, tender buds from new shoots of `Eureka' Lemon (Citrus limon var `Eureka') were cleft grafted on etiolated rootstock seedlings which had been seeded in 6 cm plastic pots and grown in a dark incubator at 298C for 21 days. After grafting, three replications of 10 plants of each scion-rootstock combination were grown in a glass house. In June 1996,
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the plants were transplanted to 9 cm diameter plastic pots, and in May 1997 retransplanted to 15 cm diameter plastic pots. Eighteen months after grafting (October 1997), plants no. 1, 3 and 5, in order of growth performance, underwent destructive harvest and dried in an oven at 708C to obtain the dry matter weight. Twenty-four months after grafting (April 1998) 30 mm tips of new shoots and twenty-five months after grafting (May 1998) fibrous roots were collected from the remaining plants. The collected samples were immediately lyophilized then stored in a freezer at 308C until the analysis of IAA and ABA. 2.2. IAA and ABA analysis The lyophilized materials were homogenized in 90% acetone (with 100 mg/l buthylated hydroxyl toluene) with polytron, after which 200 ng of 13 C6 -IAA and D6-ABA was added as the internal standard. IAA and ABA were extracted three times for 1 h at 48C, and the extracts filtered and dried. The dried samples were treated three times with pH 8.5 K-phosphate buffer in an ultrasonic bath and then filtered. Five-tenths of a gram of PVPP (polyvinylpolypyrrolidone) was added, after which the samples were stirred and then filtered. The extracts were concentrated to about 5 ml, adjusted to pH 2.5 with 1N HCl, then filtered through a nylon disk filter (25 mm diameter, 45 mm pore size). The extracts were partitioned three times with half volume of ethyl acetate. The ethyl acetate phase was collected and dried, after which it was treated three times with 400 ml of 1 : 9 ethyl acetate, hexane in an ultra sonic bath and then dried. The dried sample was dissolved in methanol and then methylated with diazomethane and dried. The methylated sample was dissolved in 50 ml ethyl acetate, then injected into GC/MS by the EI ionization method. This procedure was repeated three times for each sample; once to obtain the total ion current (TIC) chromatogram and twice using selected ion monitoring (SIM). The selected ion for IAA was m/z 130 and for ABA m/z 190, their respective main peak ions. 3. Results 3.1. Plant growth Grafts were grown in a glass house, and dry matter was measured 18 months after grafting. The dry weight of the total above-ground part was greatest for `Swingle' citrumelo and smallest for `Flying Dragon' rootstock. Dry weights of the leaves and stems showed the same pattern as that of the total top weight (Table 1). There were no significant differences among the rootstocks for root dry matter in total plant dry matter. The top±root ratio (T±R ratio) order, however, was highest for `Swingle' citrumelo and lowest for `Flying Dragon'.
248
Rootstock
Dry matter (g) Above ground
Flying Dragon Trifoliate orange `Swingle' citrumelo
Leaf
Stem
Total
6.38 0.16a 6.90 0.31ab 7.07 0.20b
2.60 0.12a 2.90 0.17ab 3.26 0.19b
8.98 0.25a 9.80 0.46ab 10.33 0.38b
Root
Total
T±R ratio
8.20 0.33a 7.85 0.29a 7.52 0.30a
17.18 0.53a 17.65 0.70a 17.85 0.57a
1.10 0.04a 1.25 0.05b 1.39 0.06c
Data are means standard error (n9); different letters indicate significant difference (P < 0.05).
K. Noda et al. / Scientia Horticulturae 84 (2000) 245±254
Table 1 Dry leaf, stem and root matter of `Eureka' lemon, 18 months after grafting on three rootstocks
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Fig. 1. IAA (A) and ABA (B) levels in new shoots of `Eureka' lemon, 24 months after grafting on three citrus rootstocks. Each column shows the means (s.e., n6) of the measurements.
3.2. IAA and ABA levels in above-ground plant parts Twenty-four months after grafting, IAA and ABA levels were measured in the new shoots of the grafts (Fig. 1). Endogenous IAA levels were highest for `Swingle' citrumelo and lowest for `Flying Dragon'. These IAA levels in the new shoots were correlated with scion growth. The ABA level in the new shoots was lower on the vigorous rootstock than on the dwarf rootstock. Assuming that IAA is a growth promoter and ABA an inhibitor in the above-ground parts, we calculated the IAA±ABA ratio (Fig. 2). It was highest in `Swingle' citrumelo and lowest in `Flying Dragon'. 3.3. IAA and ABA levels in under-ground plant parts Fibrous roots were collected 25 months after grafting, and the IAA and ABA levels measured (Fig. 3). IAA levels in the fibrous roots were highest in `Flying Dragon', followed by trifoliate orange then `Swingle' citrumelo. ABA levels in `Flying Dragon' and trifoliate orange also were higher than in `Swingle' citrumelo. Endogenous IAA and ABA levels in the fibrous roots of the rootstocks were negatively correlated with shoot growth and the endogenous IAA levels in shoots.
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Fig. 2. IAA±ABA ratios in new shoots of `Eureka' lemon. Each column shows the means (s.e., n6) of the measurements.
4. Discussion 4.1. Plant growth Wheaton et al. (1991) reported that seven-year-old `Hamlin' and `Valencia' oranges, `Murcott' tangor and `Redblush' grapefruit trees grafted on `Swingle'
Fig. 3. IAA (A) and ABA (B) levels in fibrous roots of `Eureka' lemon, 24 months after grafting on three citrus rootstocks. Each column shows the means (s.e., n6) of the measurements.
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citrumelo made large tree canopies and those grafted on `Flying Dragon' made the smallest canopies in their experiment on the effects of three rootstocks on tree growth. We obtained similar results. The weight of the dry matter of `Eureka' lemon was greatest on `Swingle' citrumelo and lowest on `Flying Dragon', which agrees with other reports and practical experience. There were no distinctive differences in the dry root matter and dry total plant matter of the three rootstocks. Takahara et al. (1994) reported that in Iyo rootstocks which showed poor top growth there were low T±R ratios. Our findings support this, the T±R ratio of `Swingle' citrumelo being highest and that of `Flying Dragon' lowest. In apple, the rootstock is reported not to affect the T±R ratio (Beakbane, 1956; Rogers and Booth, 1959), but in citrus the T±R ratio may be affected by rootstock traits. 4.2. IAA and ABA levels in above-ground plant parts Auxin is important for controlling growth, the size of shoots, and possibly, the size of roots (Lockard and Schneider, 1981). Yang et al. (1993) found that exogenously applied IAA strongly promoted stem elongation over the long-term in intact, light-grown seedlings of both dwarf and tall peas. Endogenous auxin levels were reported as higher in the shoots and apices of seedlings of tall than that of dwarf peas (Law and Davies, 1990). Inoue et al. (1982) showed that the endogenous IAA contents in seedlings of 12 barley strains were correlated with the growth rate of coleoptile length. Endogenous IAA typically is greatest in the apex and actively growing regions (Heremans et al., 1986; Ortuno et al., 1990; Law and Davies, 1990; Iino and Carr, 1982). In our experiment, the apical portion of new shoots (30 mm) was used for the IAA and ABA measurements. Results for `Eureka' lemon showed a positive correlation between the endogenous IAA levels and the dry matter of the above-ground parts on the three different rootstocks, indicative that endogenous IAA may have promoted the shoot growth of `Eureka' lemon. Another plant growth regulator, ABA, generally is regarded as an inhibitor of elongation. In our experiment the endogenous ABA content was negatively correlated with shoot growth. It has been reported to be associated with stunted growth of squash (Sakurai et al., 1985) and soybean (Bensen et al., 1988) hypocotyls. Levels of ABA-like substances were found to be higher in the roots, leaves and stems of dwarf apple seedlings than in vigorous seedlings (Yadava and Dayton, 1972). Although there is some evidence that ABA is an inhibitor, in contrast it has been reported to promote tomato shoot elongation (Weston, 1976). Bradford (1983) also reported that ABA promoted stem elongation when applied to ABA-deficient, mutant tomato plants. Moreover, its level was found to be higher in young regions of asparagus from which buds would sprout (Kojima et al., 1993). Although the role of ABA in scion growth is still controversial, our
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findings for `Eureka' lemon support the hypothesis that a high ABA level may depress shoot growth. 4.3. IAA and ABA levels in under-ground plant parts High concentrations of exogenous IAA or ABA inhibited the growth of excised pea root tips and the elongation of lettuce roots (Gaither et al., 1975; Tanimoto and Watanabe, 1986). Pilet and Saugy (1987) reported a negative correlation between root growth and the endogenous content of IAA or ABA in maize roots. We also found a negative correlation between citrus scion growth and endogenous IAA and ABA in the fibrous roots. According to Ohwaki and Tsurumi (1976), basipetal transport of IAA occurs mainly in the outer part of the root, whereas acropetal transport occurs mainly in the inner part. They suggested that basipetally transported IAA, passes through the outer part of the intact root, inhibiting its elongation. High concentrations of endogenous IAA in roots would inhibit root elongation, and there is more ester-linked than free IAA in maize roots (Saugy and Pilet, 1987). To counter inhibition of root elongation, IAA in vigorous rootstock roots, such as `Swingle' citrumelo, may be converted to esters or other conjugates and transported basipetally. Moreover, IAA metabolism may be more active in roots of vigorous than dwarf rootstocks. In spite of the differences among the three citrus rootstocks in the IAA and ABA levels in their fibrous roots, there was no significant difference in root dry matter. The comparatively short period of cultivation (about two years after grafting) may not have been long enough to show a difference, and there may be limitations in using a potted plant model to determine the actual relationship between plant growth regulators and root growth. 4.4. Interrelationship between scion and rootstock The `Eureka' lemon grafted on `Swingle' citrumelo had a large amount of IAA in its new shoots and a small amount of IAA in the fibrous roots. The latter suggests that there is active IAA metabolism or conversion to conjugates in the roots. Esters and other conjugates of IAA may be transported to the scion and reconverted to IAA. IAA esterification in the roots could have a protective role in preventing peroxidative attacks (Cohen and Bandurski, 1987). Dwarf rootstock, on the other hand, had a large amount of IAA in its fibrous roots, and its IAA metabolite transport to the scion may be less than in vigorous rootstocks. As for ABA, our results suggest that a high concentration of it may cause growth inhibition in the above and under-ground parts of citrus plants. The IAA±ABA ratio in new shoots of `Eureka' on vigorous `Swingle' citrumelo rootstock was higher than that for the dwarf rootstock `Flying Dragon'. Moreover, the vigorous rootstock `Swingle' citrumelo has a larger T±R ratio than
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`Flying Dragon'. The large amount of photosynthetic assimilates produced by citrus trees on vigorous rootstock with high IAA±ABA and T±R ratios supports the vigor of the plant as a whole. 5. Conclusion Scion growth of `Eureka' lemon was best on `Swingle' citrumelo, then on trifoliate orange, followed by `Flying Dragon'. There were no distinctive differences in root growth among the three rootstocks. The dwarf rootstock `Flying Dragon' induced a low level of IAA in the new shoots and high levels of IAA and ABA in the fibrous roots. The concentrations of IAA and ABA in the fibrous roots were negatively correlated with scion growth. As compared with dwarf rootstock, vigorous rootstock induces a higher IAA±ABA ratio in new shoots and a higher T±R ratio. References Beakbane, A.B., 1956. Possible mechanism of rootstock effect. Ann. Appl. Biol. 44, 517±521. Bensen, R.J., Boyer, J.S., Mullet, J.E., 1988. Water deficit-induced changes in abscisic acid, growth, polysomes, and translatable RNA in soybean hypocotyls. Plant Physiol. 88, 289±294. Bradford, K.J., 1983. Water relation and growth of the flascca tomato mutant in relation to abscisic acid. Plant Physiol. 72, 251±255. Cohen, J.D., Bandurski, R.S., 1987. The bound auxins: protection of indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta 139, 203±208. Gaither, D.H., Lutz, D.H., Forrence, L.E., 1975. Abscisic acid stimulates elongation of excised pea root tips. Plant Physiol. 55, 948±949. Heremans, S., Van Onckelen, H.A., De Greff, J.A., 1986. Longitudinal gradients of indole-3-acetic acid and abscisic acid in the hypocotyl of etiolated bean seedlings. J. Exp. Botany 37, 1525± 1532. Iino, M., Carr, D.J., 1982. Estimation of free, conjugated, and diffusible indole-3-acetic acid in etiolated maize shoots by the indolo-a-pyrone fluorescence method. Plant Physiol. 69, 950±956. Inoue, M., Sakurai, N., Kuraishi, S., 1982. Growth regulation of dark-grown dwarf barley coleoptile by the endogenous IAA content. Plant Cell Physiol. 23, 689±698. Kojima, K., Kuraishi, S., Sakurai, N., Itou, T., Tsurusaki, K., 1993. Spatial distribution of abscisic acid and 2-trans-abscisic acid in spears, buds, rhizomes and roots of asparagus (Asparagus officinalis L). Scientia Horticulturae 54, 177±189. Law, D.M., Davies, P.J., 1990. Comparative indole-3-acetic acid levels in the slender pea and other pea phenotypes. Plant Physiol. 93, 1539±1543. Lockard, R.G., Schneider, G.W., 1981. Stock and scion growth relationships and the dwarfing mechanism in apple. Hortic. Rev. 3, 315±375. Ohwaki, Y., Tsurumi, S., 1976. Auxin transport and growth in intact roots of Vicia faba. Plant Cell Physiol. 17, 1329±1342. Ortuno, A., Sanchez-Bravo, J., Moral, R., Acosta, M., Sabater, F., 1990. Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls. Physiol. Plant. 78, 211±217.
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Pilet, P.E., Saugy, M., 1987. Effect on root growth of endogenous and applied IAA and ABA. Plant Physisol. 83, 33±38. Rogers, W.S., Booth, G.A., 1959. The roots of fruit trees. Scientia Horticulturae 14, 27±34. Sakurai, N., Akiyama, M., Kuraishi, S., 1985. Roles of abscisic acid and indoleacetic acid in the stunted growth of water-stressed, etiolated squash hypocotyls. Plant Cell Physiol. 26, 15±24. Saugy, M., Pilet, P.E., 1987. Changes in the level of free and ester indole-3yl-acetic acid in growing maize roots. Plant Physiol. 85, 42±45. Takahara, T., Ogata, T., Kawase, K., Iwagaki, I., Muramatsu, N., Ono, S., Yoshinaga, K., Hirose, K., Yamada, Y., Takatsuji, T., Uchida, M., 1994. Effect of rootstock on growth and fruit quality of `Otani Iyokan' (Citrus iyo hort ex Tanaka). Japan Bull. Fruit Tree Res. Stn. 26, 39±60 (In Japanese with English summary). Tanimoto, E., Watanabe, J., 1986. Automated recording of lettuce root elongation as affected by auxin and acid pH in a new rhizometer with minimum mechanical contact to roots. Plant Cell Physiol. 27, 1475±1487. Weston, G.D., 1976. Effect of abscisic acid on root and shoot growth of tomato. HortScience 11, 22±23. Wheaton, T.A., Castle, W.S., Whitney, J.D., Tuckar, D.P.H., 1991. Performance of citrus scion cultivars and rootstocks in a high-density planting. HortScience 26, 837±840. Yadava, U.L., Dayton, D.F., 1972. The relation of endogenous abscisic acid to dwarfing capability of East Malling apple rootstocks. J. Am. Soc. Hort. Sci. 97, 701±705. Yang, T., Law, D.M., Davies, P.J., 1993. Magnitude and kinetics of stem elongation induced by exogenous indole-3-acetic acid in intact light-grown pea seedlings. Plant Physiol. 102, 717±724.
Indole acetic acid and abscisic acid levels in new shoots and fibrous roots of citrus scion-rootstock combinations Katsuji Nodaa,d, Hitoshi Okudab, Isao Iwagakic,* a
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1112, Japan b Department of Citriculture, National Institute of Fruit tree Science, Nakachyo, Okitsu, Shimizu, Shizuoka-ken 424-0292, Japan c Faculty of Agriculture, Shizuoka University, Kariyado, Fujieda, Shizuoka-ken 426-0001, Japan d Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan Accepted 17 May 1999
Abstract Citrus rootstocks are important for the growth of scion varieties, but it is not clear how they regulate scion vigor. We studied three citrus rootstock cultivars; `Flying Dragon' (Poncirus trifoliata var. monstrosa) a dwarfing rootstock, `Swingle' citrumelo (P. trifoliata Citrus paradisi) a vigorous rootstock, and trifoliate orange (P. trifoliata) as the control. Tender buds from new shoots of `Eureka' lemon were cleft grafted on etiolated rootstock seedlings. Eighteen months after grafting, the dry matter of each part of the young grafts was measured. Top weight was the greatest on `Swingle' citrumelo and smallest on `Flying Dragon', but there was no distinct difference in root growth among the rootstocks. Endogenous indole acetic acid (IAA) and abscisic acid (ABA) were measured in the new shoots and fibrous roots. The IAA level in the new shoots was highest in `Swingle' citrumelo and lowest in `Flying Dragon'. The ABA level in the new shoots was highest in `Flying Dragon' and lowest in `Swingle' citrumelo. Both the IAA and ABA levels in the fibrous roots were highest in the strains of Poncirus trifoliata and lowest in `Swingle' citrumelo. The vigorous rootstock `Swingle' citrumelo had the highest T±R and IAA±ABA ratios in the new shoots. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Rootstock; IAA; ABA
* Corresponding author. Tel.: +81-54-641-9500; fax: +81-54-644-4641. E-mail address: [email protected] (I. Iwagaki). 0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 8 0 - 1
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1. Introduction Trifoliate orange is the most common citrus rootstock, accounting for about 95% of all the citrus rootstocks used in Japan. Citrus trees grafted on trifoliate orange have comparatively small tree canopies and produce consistent high quality fruit. In spite of the merits of trifoliate orange, new types of rootstocks, vigorous and/or dwarf, for new scion varieties and innovative culture systems are needed. Plant growth regulators have important functions in plant behavior. Lockard and Schneider (1981), who summarized the existing information on the role of the rootstock in the dwarfing of grafted apple trees, suggested that a dwarfing mechanism is triggered by plant growth regulators. It is important to clarify the relationship that exists between the top and root in scion-rootstock combinations. The findings should contribute to the future development of new rootstocks. Auxin generally is considered as a plant growth promoter. Exogenously applied Indole 3-acetic acid (IAA) strongly promoted stem elongation over a long period in intact light-grown seedlings of both dwarf and tall peas (Yang et al., 1993), but auxin generally inhibits root elongation (Pilet and Saugy, 1987; Tanimoto and Watanabe, 1986). Abscisic acid (ABA) is regarded not only to be an inhibitor of elongation (Bensen et al., 1988; Sakurai et al., 1985; Yadava and Dayton, 1972) but a growth promoter (Bradford, 1983; Weston, 1976). A negative correlation between root growth and endogenous ABA was reported (Pilet and Saugy, 1987), and exogenous ABA inhibited the tip growth of excised roots (Gaither et al., 1975). The roles of IAA and ABA in scion-rootstock interaction are not fully known. We examined the levels of endogenous IAA and ABA in citrus scionrootstock combinations and here discuss the effect of the rootstock on scion growth. 2. Material and methods 2.1. Plant materials Three citrus rootstock varieties were used: `Swingle' citrumelo (Poncirus trifoliata Citrus paradisi) a vigorous rootstock, `Flying Dragon' (Poncirus trifoliata var. monstrosa) a dwarfing rootstock, and trifoliate orange (Poncirus trifoliata) as the standard rootstock. In May 1996, tender buds from new shoots of `Eureka' Lemon (Citrus limon var `Eureka') were cleft grafted on etiolated rootstock seedlings which had been seeded in 6 cm plastic pots and grown in a dark incubator at 298C for 21 days. After grafting, three replications of 10 plants of each scion-rootstock combination were grown in a glass house. In June 1996,
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the plants were transplanted to 9 cm diameter plastic pots, and in May 1997 retransplanted to 15 cm diameter plastic pots. Eighteen months after grafting (October 1997), plants no. 1, 3 and 5, in order of growth performance, underwent destructive harvest and dried in an oven at 708C to obtain the dry matter weight. Twenty-four months after grafting (April 1998) 30 mm tips of new shoots and twenty-five months after grafting (May 1998) fibrous roots were collected from the remaining plants. The collected samples were immediately lyophilized then stored in a freezer at 308C until the analysis of IAA and ABA. 2.2. IAA and ABA analysis The lyophilized materials were homogenized in 90% acetone (with 100 mg/l buthylated hydroxyl toluene) with polytron, after which 200 ng of 13 C6 -IAA and D6-ABA was added as the internal standard. IAA and ABA were extracted three times for 1 h at 48C, and the extracts filtered and dried. The dried samples were treated three times with pH 8.5 K-phosphate buffer in an ultrasonic bath and then filtered. Five-tenths of a gram of PVPP (polyvinylpolypyrrolidone) was added, after which the samples were stirred and then filtered. The extracts were concentrated to about 5 ml, adjusted to pH 2.5 with 1N HCl, then filtered through a nylon disk filter (25 mm diameter, 45 mm pore size). The extracts were partitioned three times with half volume of ethyl acetate. The ethyl acetate phase was collected and dried, after which it was treated three times with 400 ml of 1 : 9 ethyl acetate, hexane in an ultra sonic bath and then dried. The dried sample was dissolved in methanol and then methylated with diazomethane and dried. The methylated sample was dissolved in 50 ml ethyl acetate, then injected into GC/MS by the EI ionization method. This procedure was repeated three times for each sample; once to obtain the total ion current (TIC) chromatogram and twice using selected ion monitoring (SIM). The selected ion for IAA was m/z 130 and for ABA m/z 190, their respective main peak ions. 3. Results 3.1. Plant growth Grafts were grown in a glass house, and dry matter was measured 18 months after grafting. The dry weight of the total above-ground part was greatest for `Swingle' citrumelo and smallest for `Flying Dragon' rootstock. Dry weights of the leaves and stems showed the same pattern as that of the total top weight (Table 1). There were no significant differences among the rootstocks for root dry matter in total plant dry matter. The top±root ratio (T±R ratio) order, however, was highest for `Swingle' citrumelo and lowest for `Flying Dragon'.
248
Rootstock
Dry matter (g) Above ground
Flying Dragon Trifoliate orange `Swingle' citrumelo
Leaf
Stem
Total
6.38 0.16a 6.90 0.31ab 7.07 0.20b
2.60 0.12a 2.90 0.17ab 3.26 0.19b
8.98 0.25a 9.80 0.46ab 10.33 0.38b
Root
Total
T±R ratio
8.20 0.33a 7.85 0.29a 7.52 0.30a
17.18 0.53a 17.65 0.70a 17.85 0.57a
1.10 0.04a 1.25 0.05b 1.39 0.06c
Data are means standard error (n9); different letters indicate significant difference (P < 0.05).
K. Noda et al. / Scientia Horticulturae 84 (2000) 245±254
Table 1 Dry leaf, stem and root matter of `Eureka' lemon, 18 months after grafting on three rootstocks
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Fig. 1. IAA (A) and ABA (B) levels in new shoots of `Eureka' lemon, 24 months after grafting on three citrus rootstocks. Each column shows the means (s.e., n6) of the measurements.
3.2. IAA and ABA levels in above-ground plant parts Twenty-four months after grafting, IAA and ABA levels were measured in the new shoots of the grafts (Fig. 1). Endogenous IAA levels were highest for `Swingle' citrumelo and lowest for `Flying Dragon'. These IAA levels in the new shoots were correlated with scion growth. The ABA level in the new shoots was lower on the vigorous rootstock than on the dwarf rootstock. Assuming that IAA is a growth promoter and ABA an inhibitor in the above-ground parts, we calculated the IAA±ABA ratio (Fig. 2). It was highest in `Swingle' citrumelo and lowest in `Flying Dragon'. 3.3. IAA and ABA levels in under-ground plant parts Fibrous roots were collected 25 months after grafting, and the IAA and ABA levels measured (Fig. 3). IAA levels in the fibrous roots were highest in `Flying Dragon', followed by trifoliate orange then `Swingle' citrumelo. ABA levels in `Flying Dragon' and trifoliate orange also were higher than in `Swingle' citrumelo. Endogenous IAA and ABA levels in the fibrous roots of the rootstocks were negatively correlated with shoot growth and the endogenous IAA levels in shoots.
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Fig. 2. IAA±ABA ratios in new shoots of `Eureka' lemon. Each column shows the means (s.e., n6) of the measurements.
4. Discussion 4.1. Plant growth Wheaton et al. (1991) reported that seven-year-old `Hamlin' and `Valencia' oranges, `Murcott' tangor and `Redblush' grapefruit trees grafted on `Swingle'
Fig. 3. IAA (A) and ABA (B) levels in fibrous roots of `Eureka' lemon, 24 months after grafting on three citrus rootstocks. Each column shows the means (s.e., n6) of the measurements.
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citrumelo made large tree canopies and those grafted on `Flying Dragon' made the smallest canopies in their experiment on the effects of three rootstocks on tree growth. We obtained similar results. The weight of the dry matter of `Eureka' lemon was greatest on `Swingle' citrumelo and lowest on `Flying Dragon', which agrees with other reports and practical experience. There were no distinctive differences in the dry root matter and dry total plant matter of the three rootstocks. Takahara et al. (1994) reported that in Iyo rootstocks which showed poor top growth there were low T±R ratios. Our findings support this, the T±R ratio of `Swingle' citrumelo being highest and that of `Flying Dragon' lowest. In apple, the rootstock is reported not to affect the T±R ratio (Beakbane, 1956; Rogers and Booth, 1959), but in citrus the T±R ratio may be affected by rootstock traits. 4.2. IAA and ABA levels in above-ground plant parts Auxin is important for controlling growth, the size of shoots, and possibly, the size of roots (Lockard and Schneider, 1981). Yang et al. (1993) found that exogenously applied IAA strongly promoted stem elongation over the long-term in intact, light-grown seedlings of both dwarf and tall peas. Endogenous auxin levels were reported as higher in the shoots and apices of seedlings of tall than that of dwarf peas (Law and Davies, 1990). Inoue et al. (1982) showed that the endogenous IAA contents in seedlings of 12 barley strains were correlated with the growth rate of coleoptile length. Endogenous IAA typically is greatest in the apex and actively growing regions (Heremans et al., 1986; Ortuno et al., 1990; Law and Davies, 1990; Iino and Carr, 1982). In our experiment, the apical portion of new shoots (30 mm) was used for the IAA and ABA measurements. Results for `Eureka' lemon showed a positive correlation between the endogenous IAA levels and the dry matter of the above-ground parts on the three different rootstocks, indicative that endogenous IAA may have promoted the shoot growth of `Eureka' lemon. Another plant growth regulator, ABA, generally is regarded as an inhibitor of elongation. In our experiment the endogenous ABA content was negatively correlated with shoot growth. It has been reported to be associated with stunted growth of squash (Sakurai et al., 1985) and soybean (Bensen et al., 1988) hypocotyls. Levels of ABA-like substances were found to be higher in the roots, leaves and stems of dwarf apple seedlings than in vigorous seedlings (Yadava and Dayton, 1972). Although there is some evidence that ABA is an inhibitor, in contrast it has been reported to promote tomato shoot elongation (Weston, 1976). Bradford (1983) also reported that ABA promoted stem elongation when applied to ABA-deficient, mutant tomato plants. Moreover, its level was found to be higher in young regions of asparagus from which buds would sprout (Kojima et al., 1993). Although the role of ABA in scion growth is still controversial, our
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findings for `Eureka' lemon support the hypothesis that a high ABA level may depress shoot growth. 4.3. IAA and ABA levels in under-ground plant parts High concentrations of exogenous IAA or ABA inhibited the growth of excised pea root tips and the elongation of lettuce roots (Gaither et al., 1975; Tanimoto and Watanabe, 1986). Pilet and Saugy (1987) reported a negative correlation between root growth and the endogenous content of IAA or ABA in maize roots. We also found a negative correlation between citrus scion growth and endogenous IAA and ABA in the fibrous roots. According to Ohwaki and Tsurumi (1976), basipetal transport of IAA occurs mainly in the outer part of the root, whereas acropetal transport occurs mainly in the inner part. They suggested that basipetally transported IAA, passes through the outer part of the intact root, inhibiting its elongation. High concentrations of endogenous IAA in roots would inhibit root elongation, and there is more ester-linked than free IAA in maize roots (Saugy and Pilet, 1987). To counter inhibition of root elongation, IAA in vigorous rootstock roots, such as `Swingle' citrumelo, may be converted to esters or other conjugates and transported basipetally. Moreover, IAA metabolism may be more active in roots of vigorous than dwarf rootstocks. In spite of the differences among the three citrus rootstocks in the IAA and ABA levels in their fibrous roots, there was no significant difference in root dry matter. The comparatively short period of cultivation (about two years after grafting) may not have been long enough to show a difference, and there may be limitations in using a potted plant model to determine the actual relationship between plant growth regulators and root growth. 4.4. Interrelationship between scion and rootstock The `Eureka' lemon grafted on `Swingle' citrumelo had a large amount of IAA in its new shoots and a small amount of IAA in the fibrous roots. The latter suggests that there is active IAA metabolism or conversion to conjugates in the roots. Esters and other conjugates of IAA may be transported to the scion and reconverted to IAA. IAA esterification in the roots could have a protective role in preventing peroxidative attacks (Cohen and Bandurski, 1987). Dwarf rootstock, on the other hand, had a large amount of IAA in its fibrous roots, and its IAA metabolite transport to the scion may be less than in vigorous rootstocks. As for ABA, our results suggest that a high concentration of it may cause growth inhibition in the above and under-ground parts of citrus plants. The IAA±ABA ratio in new shoots of `Eureka' on vigorous `Swingle' citrumelo rootstock was higher than that for the dwarf rootstock `Flying Dragon'. Moreover, the vigorous rootstock `Swingle' citrumelo has a larger T±R ratio than
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`Flying Dragon'. The large amount of photosynthetic assimilates produced by citrus trees on vigorous rootstock with high IAA±ABA and T±R ratios supports the vigor of the plant as a whole. 5. Conclusion Scion growth of `Eureka' lemon was best on `Swingle' citrumelo, then on trifoliate orange, followed by `Flying Dragon'. There were no distinctive differences in root growth among the three rootstocks. The dwarf rootstock `Flying Dragon' induced a low level of IAA in the new shoots and high levels of IAA and ABA in the fibrous roots. The concentrations of IAA and ABA in the fibrous roots were negatively correlated with scion growth. As compared with dwarf rootstock, vigorous rootstock induces a higher IAA±ABA ratio in new shoots and a higher T±R ratio. References Beakbane, A.B., 1956. Possible mechanism of rootstock effect. Ann. Appl. Biol. 44, 517±521. Bensen, R.J., Boyer, J.S., Mullet, J.E., 1988. Water deficit-induced changes in abscisic acid, growth, polysomes, and translatable RNA in soybean hypocotyls. Plant Physiol. 88, 289±294. Bradford, K.J., 1983. Water relation and growth of the flascca tomato mutant in relation to abscisic acid. Plant Physiol. 72, 251±255. Cohen, J.D., Bandurski, R.S., 1987. The bound auxins: protection of indole-3-acetic acid from peroxidase-catalyzed oxidation. Planta 139, 203±208. Gaither, D.H., Lutz, D.H., Forrence, L.E., 1975. Abscisic acid stimulates elongation of excised pea root tips. Plant Physiol. 55, 948±949. Heremans, S., Van Onckelen, H.A., De Greff, J.A., 1986. Longitudinal gradients of indole-3-acetic acid and abscisic acid in the hypocotyl of etiolated bean seedlings. J. Exp. Botany 37, 1525± 1532. Iino, M., Carr, D.J., 1982. Estimation of free, conjugated, and diffusible indole-3-acetic acid in etiolated maize shoots by the indolo-a-pyrone fluorescence method. Plant Physiol. 69, 950±956. Inoue, M., Sakurai, N., Kuraishi, S., 1982. Growth regulation of dark-grown dwarf barley coleoptile by the endogenous IAA content. Plant Cell Physiol. 23, 689±698. Kojima, K., Kuraishi, S., Sakurai, N., Itou, T., Tsurusaki, K., 1993. Spatial distribution of abscisic acid and 2-trans-abscisic acid in spears, buds, rhizomes and roots of asparagus (Asparagus officinalis L). Scientia Horticulturae 54, 177±189. Law, D.M., Davies, P.J., 1990. Comparative indole-3-acetic acid levels in the slender pea and other pea phenotypes. Plant Physiol. 93, 1539±1543. Lockard, R.G., Schneider, G.W., 1981. Stock and scion growth relationships and the dwarfing mechanism in apple. Hortic. Rev. 3, 315±375. Ohwaki, Y., Tsurumi, S., 1976. Auxin transport and growth in intact roots of Vicia faba. Plant Cell Physiol. 17, 1329±1342. Ortuno, A., Sanchez-Bravo, J., Moral, R., Acosta, M., Sabater, F., 1990. Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls. Physiol. Plant. 78, 211±217.
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