Morphology. H1roSh1 INOUE and University, Miki-Cho,

J. Japan. Soc. Hort. Sci. 58(2) : 267--274. 1989. Effects of Temperature on the of the Roots of Trifoliate Orange Morphology Budded with and Physiology Satsuma Mandarin ROedhy POERWANTO*, Faculty of Agriculture, Kagawa H1roSh1 INOUE and University, Miki-Cho, Ikuo KATAOKA Kita-Gun, Kagawa 761-07 Summary One-year-old satsuma mandarin (Citrus unshiu Marc. cv. Okitsu Wase) trees on trifoliate orange rootstocks were grown in growth chambers with constant temperatures of 15, 20, 25 and 30 C and under field conditions for 6 or 8 months. The effects of temperature on root elongation, root hair development, physiological activity and free proline accumulation in the fibrous roots were investigated. Root growth was greatly restricted at 15 C. At 30 C roots grew most vigorously, and were the longest and the heaviest. They also developed finer fibrous roots and more root hairs than at all other treatments. Length of the root hairs increased with rising temperature. The most common type of root hairs was papillate at 15 C and cylindrical in the other treatments. TTC reducing activity of the fibrous roots of trees at warm temperature (above 20 C and in the field) rapidly increased from April to June, and then decreased. However, the activity at 15 C was constant from April to June, and slightly increased from June to December. Free proline accumulated more in fibrous roots at 15 C than in other treatments. Fibrous roots of trees grown in the field accumulated much proline in December. Introduction The growth patterns of shoots and roots of citrus trees are influenced by thermal environment during development. Controlling temperature is the most important point on the `House Mikan' (satsuma mandarin growing in heated plastic houses) management. Many researches have been conducted to observe the influence of temperature on the growth of top parts of citrus trees(3, 10, 13, 18). However, there is little information on the temperature effects on citrus root growth and morphological characteristics. Minimum temperature for development of citrus roots is 12 C, optimum is 25 to 26 C and maximum is 37 C, with slight variation among different species (8). High temperatures (30/25 C, day/night) particularly stimulated root growth of rooted cuttings of navel or- Received for publication May 13, 1988. Part of this paper was presented at the 1987 Chugoku-Shikoku district meeting of Japan. Soc. Hort. Sci. * Present address : Department of Agronomy, Faculty of Agriculture, Bogor Agricultural University, Jl. Pajajaran, Bogor, Indonesia. ange (13). Bevington and Castle (3)also reported that the rate of root elongation of rough lemon and Carrizo citrange budded with Valencia orange increased with rising soil temperature, and the most intense root growth occurred when soil temperatures were above 27 C. The presence of root hairs has been observed in several citrus species(5, 9,11,19). However, the favorable conditions for their generation have not been clearly determined(20). Girton (8) found that root hairs of sweet and sour orange seedlings were most abundant at approximately 33 C. Root hairs of citrus species are relatively shorter than those of other tree species (7). Among citrus rootstocks used in Japan, i. e. trifoliate orange (Poncirus trifoliata (L.) Raf.), Yuzu (Citrus junos Seib, ex Tanaka) and Natsumikan (C. natsudaidai Hayata), root hairs of trifoliate orange seedlings were the shortest (19). In order to get basic information on the roles of temperature in satsuma mandarin culture under heated plastic houses, we studied the effects of temperature on root growth and roothair development of trifoliate orange budded 267

268 R. POERWANTO, H. INOUE AND I. KATAOKA with satsurna mandarin. In addition, physiological activity of the roots and free proline accumulation in the fibrous roots were investigated. Materials and Methods Uniform one-year-old `Okitsu Wase' satsuma mandarin trees on trifoliate orange were used. The trees were subjected to different ambient temperatures of 15, 20, 25, 30 C and field conditions. Trees in each treatment grew in constant temperature growth chambers (750 relative humidity and natural day length), except for those under field conditions. Experiment I was conducted from April 1 to November 17, 1986. The trees were grown in 60 cm (high) X 40 cm (wide) X 20 cm (front to rear) glass-walled root observation chambers filled with a mixture of granite soil and bark "compost (2: 1). They were fertilized with ammonium sulfate, superphosphate and potassium sulfate equivalent to 9.8 gn, 9.8 gp205 and 9.8 gk20 per tree. The length of the roots which appeared on the glass windows of the chamber was recorded at 5-day intervals. Experiment II was conducted from April 1 to December 10, 1985 with four trees per treatment. The trees were grown in unglazed pots (30 cm in diameter), filled with granite soil and bark compost (2: 1), and fertilized with Foil cakes, ammonium sulfate, superphosphate :and potassium sulfate equivalent to 4.5 g N, _2.1 g P205 and 1.75 g K20 per tree. At the end of the experiment, all trees were harvested. The roots were separated into fibrous roots (diameter<2 mm), small roots (diameter 2'5 :mm), medium roots (diameter 5-'10 mm), large roots (diameter> 10 mm) and taproots. Fresh and dry weight and length of the roots were measured. The length of small, medium, large roots and taproots was measured with a rule. Total length of fibrous roots was estimated from their weight. Length of subsamples of fibrous roots was estimated by modified form (22) of Newman intercept line method (16) with slight modification (18). The apical parts of actively growing roots were :sampled on December 10, 1985, fixed in 4% glutaraldehyde and post-fixed in 2% osmium, and dehydrated in ethanol as described previously(18,19). Then the ethanol was ex- changed with iso-amyl acetate and criticalpoint-dried. These samples were coated with gold and viewed using Akashi Mini SEM. Experiment III was conducted from April 1 to December 1, 1987 with 12 trees per treatment. Trees were grown in 175 mm (diameter) X 199 mm (depth) Wagner's pots, filled with the same soil as described above and fertilized with 10 gram of compound fertilizer 8-8-8 (N- P-K) on June 26 and August 17, 1987. Three trees per treatment were harvested just before temperature treatments were started (April 1) and on each sampling date (June 11, August 11, October 11 and December 1, 1987). One gram of the fibrous roots was sampled for TTC test, which was done according to Yoshida(23). Free proline accumulation in the fibrous roots was determined using the method of Bates et al. (2) with slight modification (14, 18). Results Root and Shoot Growth Roots grew rapidly at 30 C. In contrast, root growth was greatly restricted at 15 C. Through the observation windows, it was noted that root growth alternated with shoot growth (Fig. 1). That is, the rate of root growth increased just after shoot growth ceased. This pattern was observed in all treatments, except at 15 C, where root growth was not related to that of the shoot (only spring flush occurred). Total lengths of the new roots which had appeared on the observation windows by early November, 1986 were 1, 852 cm in the field and 213, 1, 089, 1, 459 and 2, 437 cm at 15, 20, 25 and 30 C, respectively. Morphological Characteristic of the Roots The length of medium size roots and taproots was not significantly different (Table 1). Fibrous roots were the longest at 30 C ; their length was about seven times more at 30 C than at 15 C. Total length of the roots showed the same tendency as the fibrous roots, because fibrous roots contributed more than 98% of total length of the root system (Table 1). Dry weight of fibrous roots increased with rising temperature (Table 1). On a dry weight basis, fibrous roots contributed about 3457% of the root system. Dry weight of fibrous roots increased with season (Fig. 2), but slowly at 15 C. At 20 C and above, the growth rate of

TEMPERATURE ON MORPHOLOGY AND PHYSIOLOGY OF TRIFOLIATE ORANGE ROOTS 269 Fig. 1. Effect of temperature on the growth pattern of shoots and roots (Experiment J). Table 1. Effect of temperature on length and dry weight of roots (Experiment II).

270 R. POERWANTO, H. INOUE AND I. KATAOKA Fig. 2. Effect of temperature on the dry weight of fibrous roots during April to October (Experiment III). A- -A 15 C A- -A 20 C O -----O 25 C 30 C E -E Field Different letters (at same sampling date) indicate significant differences at 5 0 level. fibrous roots was slow at first, but increased in June and than decreased in August. Under field conditions the growth rate of fibrous roots was slight at first, then gradually increased, and reached a maximum between August and October (Fig. 2). The trees grown at higher temperature also produced more fibrous roots, which were finer Fig. 3. Effect of the fibrous temperature on the appearance of roots (Experiment II). than those at lower temperatures (Fig. 3). In Experiment II, the length/dry weight ratios of fibrous roots were 1.45±0.05 cm/mg in the field and 0.78±0. 005; 1.46 ±0. 18 ; 1.63±0. 06 and 1.81 f 0.08 cm/mg at 15, 20, 25 and 30 C respectively. Root hairs were found on fibrous roots of trees in all treatments. They were the most abundant on fibrous roots of trees at 30 C and the least on those at 15 C. There were various shapes of root hairs. The most common type Fig. 4. Effect of temperature on root hair development (Experiment II).

TEMPERATURE ON MORPHOLOGY AND PHYSIOLOGY OF TRIFOLIATE ORANGE ROOTS 271 Table 2. Effect of temperature on some 'anatomical characteristics of the fibrous roots (Experiment II). Fig. 5. Effect of temperature on TTC reducing activity of roots during April to October (Experiment III). 0-0 15 C A --A 20 C 0- ----0 25 C... 30 C L 1-L] Field Different letters (at same sampling date) indicate significant differences at 5 0 level. Fig. 6. Effect of temperature on the accumulation of free proline in the fibrous roots during June to October (Experiment III). Different letters (at same sampling date) indicate significant differences at 5 0 level. of root hair was papillate at 15 C and cylindrical at 20 C and above. Conical root hairs were partly observed in all treatments (Fig. 4). Length of root hairs incresed with rising temperature. However, diameter of root hairs was not influenced by temperature. The length ranged from 6 to 36 pm at 15 C, 7 to 98 pm at 20 C, 10 to 93 pm at 25 C, 10 to 197 pm at 30 C, and 10 to 72 pm in the field (Table 2). Physiological Activity of Root Physiological activity of roots was indicated by measuring triphenyltetrazolium chloride (TTC) reducing activity. TTC reducing activity in the roots of trees grown in warm temperature (in growth chamber above 20 C and in the field) increased rapidly from April to June, and then decreased. On the other hand, the activity at 15 C was low at first, and then gradually increased slightly (Fig. 5). Free Proline Accumulation in Fibrous Roots Figure 6 shows that free proline accumulated by the fibrous roots was much higher at 15 C than at other treatments. At 15 C it was about 6 to 44 times higher than at 30 C. At 15 and 20 C it increased steadily from June to Dece mber. In other treatments, its accumulation was relatively constant, except in the trees grown under field conditions, where the accumulation greatly increased in December. Discussion It seems evident from these results that the roots of trifoliate orange budded with satsuma mandarin may be markedly influenced by temperature treatments. Growth and act ivity of the roots and development of root hairs were

272 R. POERWANTO, H. INOUE AND I. KATAOKA greatly restricted at 15 C. The root observation chambers showed that the rate of root growth at 20 C was much greater than at 15 C. Probably the roots at 15 C differ qualitatively from those above 20 C. Previous studies on the growth of citrus roots(3, 8,10) indicated that no root growth was evident at soil temperatures below 12 or 14 C, and growth was limited at temperatures below 20 or 22 C. The limited root growth at 15 C may be due to inhibition of cell division by low temperature(15). Above 20 C, the growth and activity of the roots increased as temperature rose from 20 to 30 C. Temperature of 30 C was apparently optimum for growth of trifoliate orange (budded with satsuma mandain) roots. The optimum temperature conformed to those reported on the other citrus species. Girton(8) reported that the optimum temperature for root elongation of grapefruit seedlings was 26 C, sour orange seedlings was 27 C and sweet orange seedlings was 24 C. Bevington and Castle(3) reported that most growth of rough lemon and Carrizo citrange (budded with Valencia orange) roots took place when the soil temperature was above 27 C. Root branching and length also decreased at low temperature(15). Thus, the surface area of feeder root-soil contact also decreased at 15 C. The surface area of feeder root-soil contact is responsible for absorption of water and minerals. The absorbing surface of the root depends on number of root branchings, length and size of the feeder roots, and the availability of root hairs. Results showed that the roots at 15 C had the narrowest absorbing surface area. As the length of the fibrous roots and root hairs increased when temperature rose from 20 to 30 C, the absorbing surface of the root also increased. Temperatures between 20 and 30 C were apparently favorable for root hair development. Girton(8) found the optimum temperature for root hair production was 34 C for sour orange, 31 C for sweet orange, and 26 C for grapefruit. The common type of root hairs was papillate at 15 C and cylindrical at other treatments. Generally, cylindrical root hairs were longer and in higher density than papillate hairs. Papillate root hairs are considered to be dormant, or to have recently resumed growth (5). There is a close relationship between TTC reducing activity and respiration, because the TTC test measures succinate dehydrogenase activity on the respiration system(23). Kubota et al. (12) found a correlation between TTC reducing activity and growth of new roots of grape vine. Aimi and Fujimaki(1) found that high TTC reducing activity usually appeared in the younger parts of roots. TTC reducing activity of the roots at 15 C was low in June and slightly increased in August and October. Its activity in other treatments increased in theperiod of April-June, and then decreased. When the trees were harvested on June 11, many new roots had already formed in the field and in above 20 C growth chambers. In contrast, no new roots were found at 15 C. The roots of trees at 15 C accumulated much more free proline than at other treatments. Accumulation of free proline is frequently observed in trees subjected to environmentall stress. Higher accumulation of free proline in the roots grown at 15 C, could be due to temperature stress. Great accumulation of free proline in the fibrous roots apparently occurred when the growth of fibrous roots was restricted. Accumulation in the fibrous roots of trees grown in the field also greatly increased, as field temperature dropped toward December. The work of Ozturk et al. (17) showed that plants tend to accumulate proline when they are exposed to low soil temperature. Results from all temperature treatments showed that the roots of citrus trees, different from deciduous trees, grew after spring shoot growth and root growth alternated with shoot. growth. This result conformed to the report of Inoue and Harada (10), although 'we used larger root observation chambers with one tree per chamber. Shoot growth was probably one of the factors hampering intensity of root growth. Bevington and Castle (3) also reported that the number of growing roots and the rate of root elongation declined during periods of shoot elongation. Our results also showed the balance between root and shoot growth at all temperature treatments. Root and the shoot growth were great at 30 C. In contrast, the growth of roots at 15 C was restricted, and summer shoots did not flush. Borchert (4) defined a balance between

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