Auxin synergists in the rooting of mango cuttings

Scientia Horticulturae, 9 (1978) 381--387

381

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

AUXIN SYNERGISTS IN THE ROOTING OF I~ANGO CUTTINGS

M.K. SADHU, SANGHAMITRA BOSE and LILY SAHA

College of Agriculture, Calcutta University, Calcutta-700019 (India) (First received 14 March 1978; in revised form 30 May 1978)

ABSTRACT

Sadhu, M.K., Bose, S. and Saha, L., 1978. Auxin synergists in the rooting of mango cuttings. Scientia Hortic., 9: 381--387. Preplanting application of phenolic compounds, p-hydroxybenzoic acid, p-coumaric acid and ferulic acid, in most cases promoted auxin-induced rooting on cuttings of mango, a difficult-to-root fruit plant. The ethylene-releasing chemical CEPA (ethrel, ethephon) also stimulated rooting in the presence of auxins. Synergism was more pronounced in the presence of IBA than with IAA. To a certain extent, root-promoting effects of auxins and non-auxin chemicals were related to the utilization of carbohydrate fractions from the region of root formation. Utilization of reserve polysaccharides assumed greater importance only during the root-development phase.

INTRODUCTION

A large number of physiologically and chemically unrelated compounds such as indole, a-naphthol, ~-naphthol, phenols, growth-inhibiting and growthretarding chemicals have been found to influence the regeneration of roots on cuttings (Van Raalte, 1954; Poapst and Durkee, 1967; Hess, 1968; Gorter, 1969; Basu, 1969; Bojarczuk and Jankiewicz, 1975a). Some of these compounds interact with exogenously applied auxins, giving synergistic (more than additive) effects. Unsaturated gas, such as ethylene, has also been shown to promote rooting of cuttings singly as well as in combination with auxins (Krishnamoorthy, 1970; Roy et al., 1972). The possibilities of using auxinsynergists in the rooting of difficult-to-root plants have not been investigated. So far, most studies on synergism have been done on easy-to-root herbaceous cuttings and it remains to be seen whether the conclusions drawn from such materials would also hold true for difficult-to-root materials. The mode of action of auxin-synergists in the regeneration process is not clear. Whether the root-promoting effects of auxin synergists are related to the metabolism of carbohydrates or not, has not been reported. The present investigation, therefore, was undertaken to explore the possibility of using synergistic non-auxin chemicals in the induction of rooting on cuttings of mango, a difficult-to-root fruit plant, and also to examine the involvement

382 of these chemicals in the changes of different carbohydrate fractions in the regenerating tissue. MATERIALS AND METHODS Freshly extracted mango seeds (Mangifera indica L.) of one clone were sown outdoors in a nursery bed in June 1976. Two months after germination, when the seedlings were about 25--30 cm in length and had produced 8--9 leaves, they were harvested for the preparation of cuttings. The cuttings were 20 cm tall and 2 uppermost leaves were retained on each cutting. The non-auxin compounds were ferulic acid, p-hydroxybenzoic acid, p-coumaric acid and (2-chloroethyl)phosphonic acid (CEPA, syn. ethephon, ethrel), in the concentration of 10 -3 M, except for CEPA which was used at the concentration of 50 mg/1. The concentrations of indol-3yl-acetic acid (IAA) and indol-3yl-butyric acid (IBA) were 2 X 10 -2 M and 10 -2 M, respectively. Three hundred cuttings were first treated with each of the non-auxin chemicals (including distilled water) by dipping the base of the cuttings in 500 ml solution of the chemical. After 24 h, when most of the solution was taken up, the cuttings were removed and the solution was allowed to dry up from the surface of the cutting. The cuttings were then divided into 3 equal groups, each consisting of 100 cuttings. One group, as control, was directly planted in washed sterile sand in propagation frames under polythene cover. Of the remaining 2 groups, 1 was treated with IAA and the other with IBA. The experiment was duplicated and pooled average data were taken to calculate synergism. Data on the percentage of cuttings rooted and the number, length and dry weight of roots were recorded 21 days after planting. Synergism between the auxins and the non-auxin chemicals was calculated by subtracting individual effects of auxins and synergists from their combined effects (Gorter, 1969). The level of significance of the synergistic effect was determined statistically as interaction for root numbers by analysis of variance, using 2-way interaction tables and F test (Fisher, 1948). For estimation of different carbohydrate fractions, samples of tissues (bark and wood taken together) were collected from the root-forming region 4.0 cm above the cut end of the cuttings, on day 0 of making cuttings, and 3, 7, 14 days after planting, to be indicated as S(stage), 0, $3, S~, $14, respectively. Sugars were determined by the copper reduction method of Somogyi (1945). Reserve polysaccharides were estimated by the same method after removing soluble sugars and hydrolyzing the residue with 6N HC1 on a boilingwater bath for 3 h (Asana and Saini, 1962). RESULTS

Control cuttings did not root during the course of the investigation (Table 1).

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TABLE

1

Effect of auxins, phenolic substances and CEPA on the rooting of mango cuttings Values in parentheses denote increase (+) or decrease ( - ) in root number due to synergism or antagonism between t h e a u x i n and the respective non-auxin chemical. **Significant at P = 0.01. *Significant at P = 0.05. Non-auxin chemicals

Auxins Control

Percentage of cuttings rooted Control p - H y d r o x y b e n z o i c acid p-Coumaric acid Ferulic acid CEPA Mean number of roots per cutting Control p - H y d r o x y b e n z o i c acid

0 10 10 5 0 0.0 0.1±0.01

p-Coumaric acid

0.1±0.01

Ferulic acid

0.5_+0.03

CEPA

0.0

IAA

IBA

10 50 55 10 30

30 60 80 45 50

0.3-+ 0.02 1.4-+ 0.06 (+1.0) 2.3_+ 0.10 0.7_+ 0.05 (-0.1) 0.7+- 0.04 (+0.4)

1.5± 0.04 5.3-+ 0.37 (+3.7**) 6.2-+ 0.41 (+4.6**) 3.3_+ 0.29 (+1.3) 3.0_+ 0.33 (+1.5)

2.6± 1.9-+ 2.5± 2.4_+ 1.8±

2.0± 2.1± 2.2_+ 2.6+_ 1.4-+

(+1.9")

Mean length of roots (cm) Control p-Hydroxybenzoicacid p-Coumaricacid Ferulicacid CEPA Dry weight of roots per cutting (mg) Control p - H y d r o x y b e n z o i c acid p-Coumaricacid Ferulic acid CEPA

0.0 2.1±0.23 1.0_+0.12 2.0-+0.22 0.0 0 17 ±0.57 18 -+0.71 8 ±0.63 0

25 437 237 125 141

0.26 0.12 0.19 0.18 0.13

± 1.76 -+23.50 ± 5.72 ± 4.58 ± 2.30

215 876 560 64Y/ 244

0.14 0.18 0.18 0.23 0.08

+- 5.78 ±44.46 -+35.58 -+24.46 ± 5.45

Treatments with auxins (IAA or IBA) stimulated the formation of adventitious roots, IBA being more effective than IAA. Rooting was also promoted to some extent by all the 3 phenolic substances when applied alone. The chemicals, however, showed interesting interactions with auxins, p-Hydroxybenzoic acid and p-coumaric acid greatly synergised the rooting of IBA-treated cuttings. The synergistic effect of p-hydroxybenzoic acid on IAA-induced root formation was not significant, while p-coumaric acid showed moderate synergism with IAA (significant at P = 0.05). Ferulic acid in the presence of IBA apprec-

384

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Fig. 1. Changes in carbohydrate fractions in the root-forming region during the course of IAA (.-----~); IBA (o o); p-hydroxybenzoic acid (v []); p-hydroxybenzoic acid + IAA ( A - - A ) ; p-hydroxybenzoic acid + IBA ( a - - a ) . Stages of sampling on abscissa: initial (So) at the time of making cutting, and 3 ($3), 7 ($7) and 14 (S~4) days after planting. root formation in mango cuttings after being treated w i t h : control ( × - - × ) ;

Fig. 2. Changes in carbohydrate fractions in the root-forming region during the course of ); IAA (. 8); IBA (o- --o ); p-coumaric acid ( v - - u ) ; p - c o u m a r i c acid + IAA (A -); p-coumaric acid + IBA ( a - - a ) . Stages of sampling as in Fig. 1.

r o o t formation in mango cuttings after being treated with: control (× - - ×

iably increased the number of roots per cutting but did not influence the IAA-induced rooting. When used jointly with either IAA or IBA, CEPA almost doubled the percentage of cuttings rooted as well as the number of roots per cutting, over the respective controls. Data in Table 1 also show that most of the synergistic combinations not only increased the number of rooted cuttings and the root number per cutting, but also significantly increased the dry weight of roots. Maximum increase in dry weight of roots was recorded in p-hydroxybenzoic acid plus IBA. Each o f the phenolic substances in the presence of IBA increased the

385 length of roots more than IBA alone, whereas these substances in combination with IAA showed no appreciable effect on root length. The changes in the concentration of different carbohydrate fractions in the root-forming region of cuttings as affected by various treatments are presented in Figs. 1--4. The periods from So to $3 (0--3 days) and $3 to $7 (3--7 days) represented approximately the early and late phase of root initiation, respectively, while the period $7 to $14 (7--14 days) corresponded to the phase of root emergence and root development. It is clearly evident that, regardless of treatments, there was a reduction in the concentration of soluble sugars during all these phases of regeneration. Among the auxins, reduction was

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Fig. 3. Changes in carbohydrate fractions in the root-forming region during the course of root formation in mango cuttings after being treated with: control (x x ); IAA ( • - ~ ) ; IBA ( o - - o ) ; ferulic acid (v v); ferulic acid + IAA (A A); ferulic acid + IBA ( ~ - - A ) . Stages of sampling as in Fig. 1. Fig. 4. Changes in carbohydrate fractions in the root-forming region during the course of root formation in mango cuttings after being treated with: control (× x ); IAA (o •); IBA ( o - - o ) ; CEPA (o- --~); CEPA + IAA ( A ~ A ) ; CEPA + IBA ( ~ - - ~ ) . Stages of sampling as in Fig. 1.

386

greater with IBA than with IAA. There was little difference between non-auxins and control. The utilization of total sugars (reducing and non-reducing sugars) was highest in cuttings treated with IBA up to $7. However, sugars were utilized to a greater extent in cuttings treated with auxins plus non-auxins at $14. There were only minor changes in the concentration of reserve polysacchariaes during So to $3, with little difference between them at that stage. Subsequently, the reserve polysaccharides were utilized rapidly and the reduction was greater with auxin-synergists than with other treatments. The non-auxin chemicals, when applied separately, showed little effect on the utilization of reserve polysaccharides and the variation in the level of reserve polysaccharides in the cuttings at the later stages was largely due to the action of auxins, IBA causing slightly greater reduction than IAA. DISCUSSION The phenolic substances, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid and the ethylene-releasing compound CEPA all stimulated the production of roots on mango cuttings. This agrees with earlier findings on several other species (Basu et ah, 1969; Roy et al., 1972; Bojarczuk and Jankiewicz, 1975b). There seems to be no relationship between the activity of the IAA-oxidizing system and root formation. Thus, p-hydroxybenzoic acid and p-coumaric acid, which do not inhibit the IAA-oxidizing system, have been found to act as very effective auxin-synergists. On the other hand, ferulic acid inhibits the IAA-oxidizing system, but fails to synergise IAA-induced root formation. Moreover, the effectiveness of p-hydroxybenzoic acid and p-coumaric acid as auxin synergists is significant, as these compounds are reported to occur endogenously in mango (Choudhuri and Rudra, 1971). CEPA acted synergistically with exogenously applied IAA and IBA, but the latter was more effective. The promoting effect of ethylene in root formation was noted by Zimmerman and Wilcoxon (1935) and by Crocker et al. (1935). Our results indicate that the root-promoting effects of different treatments were related to a certain extent to the utilization of carbohydrates in the root-forming region of the cuttings. The high degree of synergism from p-hydroxybenzoic acid plus IBA and p-coumaric acid plus IBA, was associated with an appreciable reduction in the concentration of different carbohydrate fractions. Utilization of soluble sugars was appreciable even in the early-phase root initiation. Sugars were obviously needed to a greater extent for production of new cells and for increased respiratory activity in the regenerating tissue, but a close quantitative relation between carbohydrate utilization and root formation could not be obtained during the stages of regeneration. Only minor changes occurred in the concentration of reserve polysaccharide during the root initiation phase ($3).. However, at the time of development of root primordia, which act as strong metabolic sinks, hydrolytic activity assumed much importance. As a result, reserve polysaccharide level fell sharply, the

387

depletion being most pronounced in cuttings treated with auxin-synergists. The extent of polysaccharide utilization was greater under non-auxin plus IBA than under non-auxin plus IAA. The results clearly indicate the possibility of utilizing auxin synergists in rooting of difficult-to-root cuttings. REFERENCES

Asana, R.D. and Saini, A.D., 1962. Studies in physiological analysis of yield. V. Grain development in wheat in relation to temperature, soil moisture and age in sugar content of stem and in photosynthetic surface. Ind. J. Plant Physiol., 5: 128--171. Basu, R.N., 1969. Effect of auxin synergists in rooting of French bean (Phaseolus vulgaris L.) cuttings. Curr. Sci., 38: 5 3 3 - 5 3 5 . Basu, R.N., Bose, T.K., Roy, B.N. and Mukhopadhyay, A., 1969. Auxin synergists in rooting of cuttings. Physiol. Plant., 22 : 649--652. Bojarczuk, K. and Jankiewicz, L.S., 1975a. Rooting of Syringa vulgaris L. soft wood cuttings using auxins, vitamins, phenolic substances, indole, SADH and abscisic acid. Acta Agrobot., 28: 229--239. Bojarczuk, T. and Jankiewicz, L.S., 1975b. Influence of phenolic substances on rooting of soft wood cuttings of Populus alba L. and P. canascens Sin. Acta Agrobot., 28: 121--129. Choudhuri, J.M. and Rudra, P., 1971. Physiological studies on chemical control of growth and flowering in mango (Mangifera indica L.). Indian Agric., 15: 127--135. Crocker, W.A., Hitchcock, A.E. and Zimmerman, P.W., 1935. Similarities in the effects of ethylene and plant auxins. Contrib. Boyce Thompson Inst., 7: 231--248. Fisher, R.A., 1948. Statistical methods for research workers. Oliver and Boyd, Edinburgh. Gorter, C.J., 1969. Auxin synergists in the rooting of cuttings. Physiol. Plant., 22: 497--502. Hess, C.E., 1968. Internal and external factors regulating r o o t initiation. In: W.J. Whittington (Editor), R o o t Growth. Proc. 15th Easter School in Agric. Sci., Nottingham, pp. 42--53. Krishnamoorthy, H.N., 1970. Promotion of rooting in mung bean hypocotyl cuttings with ethrel, an ethylene releasing compound. Plant Cell Physiol., 11: 979- 982. Poapst, P.A. and Durkee, A.B., 1967. R o o t differentiating properties of some simple aromatic substances of the apple and pear fruit. J. Hortic. Sci., 42: 429--438. Roy, B.N., Basu, R.N. and Bose, T.K., 1972. Interaction of auxins with growth retarding, -inhibiting and ethylene producing chemicals in rooting of cuttings. Plant Cell Physiol., 13: 1123--1127. Somogyi, M., 1945. A new reagent for determination of sugars. J. Biol. Chem., 160: 61--68. Van Raalte, M.H., 1954. On the synergism of indole and indoleacetic acid in root production. Ann. Bogor., 1: 167--171. Zimmerman, P.W. and Wilcoxon, F., 1935. Several growth substances which cause initiation of roots and other responses in plants. Contrib. Boyce Thompson Inst., 7: 209--229.