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Reference
Stimulation of root formation in difficult-to-root woody cuttings by dithiothreitol
AUDERSET, Guy Leon, et al.
Abstract
Shoot microcuttings of Malus domestica cv. Jork 9, Malus domestica cv. Cepiland, Mandevilla (Dipladenia) sanderi, and Daphne odora cv. variegata were induced to root by the addition of 0.1-1 mM dithiothreitol following an auxin (indole-3-acetic acid) treatment to initiate root formation. The most difficult to root, Malus domestica cv. Cepiland, required higher dithiothreitol concentrations to achieve optimum rooting than did the less difficult to root, Malus domestica cv. Jork 9, Mandevilla sanderi, or Daphne odora variegata. The roots induced by the combination of dithiothreitol and auxin treatment were approximately twofold longer and more robust than those induced with auxin treatment alone. The combination treatment of auxin and dithiothreitol has the potential to be of practical importance in root initiation in difficult-to-root cuttings of both woody and herbaceous horticultural varieties.
AUDERSET, Guy Leon, et al . Stimulation of root formation in difficult-to-root woody cuttings by dithiothreitol. International Journal of Plant Sciences , 1997, vol. 158, no. 2, p. 132-135
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STIMULATION OF ROOT FORMATION IN DIFFICULT-TO-ROOT WOODY CUTTINGS BY DITHIOTHREITOL
GUY AUDERSET,* CHARLES MONCOUSIN,* JANE O'ROURKE,* AND D. JAMES MORRE' t *Centre de Lullier, Ecole d'Ingenieurs CH-1254 Jussy, Geneva, Switzerland; and tDepartment of Medicinal Chemistry
and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907
Shoot microcuttings of Malus domestica cv. Jork 9, Malus domestica cv. Cepiland, Mandevilla (Dipladenia) sanderi, and Daphne odora cv. variegata were induced to root by the addition of 0.1-1 mM dithiothreitol following an auxin (indole-3-acetic acid) treatment to initiate root formation. The most difficult to root, Malus domestica cv. Cepiland, required higher dithiothreitol concentrations to achieve optimum rooting than did the less difficult to root, Malus domestica cv. Jork 9, Mandevilla sanderi, or Daphne odora variegata. The roots induced by the combination of dithiothreitol and auxin treatment were approximately twofold longer and more robust than those induced with auxin treatment alone. The combination treatment of auxin and dithiothreitol has the potential to be of practical importance in root initiation in difficult-to-root cuttings of both woody and herbaceous horticultural varieties.
Introduction
Induction of rooting is an important step in plant propagation, especially of woody plants from cuttings or from adventitious shoots produced from callus (Lane 1978; Moncousin 1992). The use of an auxin treatment employing high concentrations of indole-3- acetic acid (IAA) or other auxins is the usual method to induce rooting in microcuttings of woody horticul- tural varieties (Moncousin 1992), although numerous other factors including shoot selection and manipula- tion strategies may eventually influence rooting (Marks and Myers 1994). However, microcuttings from many woody species are difficult or impossible to root with auxin treatment alone. Even in species that can be rooted using auxin treatment alone, the roots formed may be stunted or malformed as a result of the auxin treatment (Lane 1978).
The use of thiol compounds, alone or in combina- tion with an auxin treatment (Auderset et al. 1996), has been reported to increase rooting of herbaceous cuttings as well as Malus domestica shoots produced via organogenesis from callus. In this report, we de- scribe the use of dithiothreitol (DTT) in combination with an auxin treatment to induce rooting in micro- cuttings of shoots of Malus domestica Borkh. cv. Ce- piland, a difficult-to-root variety, and in Malus domes- tica (Borkh.) cv. Jork 9, Mandevilla (Hemsl.) Dipladenia sanderi (Woodson) and Daphne odora (Thun.) cv. variegata.
Material and methods
Rooting of microcuttings of Malus domestica, Mandevilla sanderi, and Daphne odora variegata were studied. Shoots (2 cm) were produced in vitro on the following multiplica- tion media:
1. Malus. For the trials, we used two apple cultivars, Jork 9 and Cepiland. The multiplication medium was a Murashige and Skoog (1962) mineral solution with 100 mg/L inositol, 1 mg/L thiamine-HCL, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, the plant growth regulators 6-furfurylamino
'Author for correspondence and reprints.
Manuscript received June 1996; revised manuscript received No- vember 1996.
purine (kinetin) (1 mg/L) and indole-butyric acid (IBA) (0.1 mg/L), and sucrose (30 g/L) (MS medium). The pH was adjusted to 5.5. The culture conditions were temperature, 20?-22?C; photoperiod, 16 h and fluorescent light (70 pLmol s-' m-2). To propagate the plants further, 0.5-cm apical shoots were excised and transferred to a fresh medium. After 6 wk, proliferating shoots between 2 and 4 cm in length were formed.
2. Daphne. The multiplication medium was a Woody Plant Medium mineral solution (Lloyd and McCown 1981) supplemented with vitamins (Mullin et al. 1974) and amino acids (Skoog 1944). The plant growth regulators were ki- netin (0.5 mg/L) and a-naphthylacetic acid (NAA) (0.01 mg/L). Sucrose and Difco Bacto agar were added at 20 g/L and 9 g/L, respectively. The pH was 5.5.
3. Mandevilla. The multiplication medium was based on Murashige and Skoog (1962) mineral, vitamin, and amino acid solutions as described for Malus supplemented with 4.0 mg/L 2-isopentyladenine and 0.2 mg/L IBA. Sucrose and Difco Bacto agar at 30 g/L and 7 g/L, respectively, were added. The pH was 5.7.
The rooting media were identical to the multiplication me- dia except they were growth regulator free.
To induce rooting, the shoots were first treated with auxin by incubating the base of the cutting (2-3 min) for 1 h (Man- devilla) or 2 h (Malus and Daphne) in an 8 mg/L solution of agar containing 1 mM of IAA (Mandevilla and Daphne) or 2 mM IAA (Malus). The auxin (IAA) stock solution was in 70% alcohol, pH 5.5. The auxin treatment was in plastic jars. After this auxin treatment, the microcuttings were trans- ferred to an auxin-free agar rooting medium, containing the thiol compound, dithiothreitol (DTT), at different concentra- tions. Rooting was in glass tubes closed with Magenta caps.
The microcuttings were first placed in the dark for 6 d at 260 ? 1?C and then transferred to the culture room 16/24 h light (fluorescent, 70 Lmol s-' m-2).
To investigate the timing of DTT action on rooting of Malus microcuttings, one application of DTT was during the 5 d following the auxin treatment, after which the cuttings were transferred to MS medium containing 0.1 mM DTT.
Another application of DTT after 5 d on MS medium, after which the cuttings were transferred to the medium with 0.1 mM DTT (day 5-day 21).
Each treatment consisted of 20 or 30 microcuttings. Ex- periments were repeated two times. Rooting was measured as the number of roots per cutting after 2 wk and again after 3 wk following the auxin treatment for Malus domestica cv.
Jork 9 and Malus domestica cv. Cepiland, 3 and 4 wk fol-
132
AUDERSET ET AL.-STIMULATION OF ROOT FORMATION 133
Table 1
ROOTING OF DAPHNE ODORA AFTER AUXIN TREATMENT IN THE PRESENCE OF VARYING CONCENTRATIONS OF DITHIOTHREITOL
AFTER 8 wk OF DITHIOTHREITOL TREATMENT
Dithiothreitol (mM) Rooting (%) Roots/cutting 0.00 (control) ... 18a 0.2a 0.1 ... 63 2.7 0.25 ... 54 1.4 0.50 ...30a... . 0.6
Note. Data are expressed as percentage of rooting and number of roots per cutting and mean comparison by the Dunnett test at P <
0.05. Means with the same letter were not different from the control.
lowing the auxin treatment for Daphne odora, and 4, 6, and 8 wk for Mandevilla sanderi.
Statistical data were analyzed using GBStat software one-, two-, or three-way ANOVA and mean comparisons by Dun- nett Test at P < 0.05. Percentages were converted using a standard conversion table (Dagnelie 1975). Each test result was compared with the control treatment (DTT = 0).
Results DAPHNE ODORA
Rooting was improved at low concentrations of DTT (0.1-0.25 mM) (table 1) during the 4 wk follow- ing auxin treatment.
Another aspect of using DTT was the absence of callus formation at the base of the microcuttings (fig.
1). Large callus were always developed in rooting without DTT but less so in its presence.
MANDEVILLA SANDERI
With cuttings obtained either from apical shoot seg- ments (apex) or from mononodal shoot segments, the rooting was improved by DTT after 8 wk (table 2).
After auxin treatment, incorporating DTT in the root-
Table 2
ROOTING OF MANDEVILLA SANDERI APICAL CUlTINGS Dithio-
Type of Auxin threitol Roots/ Rooting cutting treatment (mM) cutting (%) Apex ... - (control) 0.00 1.6a 13a 0.1 1.9a iSa 0.25 2.3a 15a + 0.00 3.3a 30a 0.1 4.3a 77 0.25 6.5 89 Node ... - (control) 000 O.Oa Oa
0.1 0.8 9 0.25 1.1 10 + 0.00 0.3a 2a
0.1 0.3a 5a 0.25 1.1 9 Note. Means with the same letter were not different from the control (no auxin treatment, no dithiothreitol) as analyzed by the Dunnett test at P < 0.05. The greatest rooting response was with 0.25 and 0.1 mM dithiothreitol after 8 wk of dithiothreitol treatment.
ing medium at concentrations of 0.1 or 0.25 mM ap- proximately doubled the rate of root formation. As in the Daphne experiments, callus formation was absent in cuttings treated with DTT (not illustrated).
MALUS DOMESTICA CV. JORK 9
With this easy-to-root rootstock cultivar, use of DTT consistently promoted adventitious rooting (fig. 2).
The greatest numbers of roots were obtained with 0.25 and 0.3 mM of DTT (table 3). After 3 wk, the cuttings without auxin treatment did not root either in the pres- ence or in the absence of DTT.
The application of DTT (0.1 mM) during the de- velopment of the root primordia over 5 d following the auxin treatment, resulted in the formation of 4.4
Fig. 1 Appearance of rooted cuttings of Daphne ordora after 8 wk of dithiothreitol treatment following the auxin treatment. A, Auxin alone. B, Auxin and 0.25 mM dithiothreitol. C, Auxin and 1 mM dithiothreitol. Scale bar = 1 cm.
Ar B .
Fig. 2 Appearance of rooted cuttings of Malus domestica cv. Jork 9 after 3 wk. A, Auxin alone. B, Auxin and 0.1 mM dithiothreitol.
C, Auxin and 0.25 mM dithiothreitol. D, Auxin plus 0.5 mM di- thiothreitol. Scale bar = 1 cm.
roots/cutting compared with 2.4 roots/cutting when the application was made after day 5.
MALUS DOMESTICA CV. CEPILAND
Cuttings from this cultivar of Malus were more dif- ficult to root than were those of Jork 9. At 3 wk after auxin treatment, the cuttings treated with DTT under the same conditions as Jork produced more roots than did cuttings without DTT only at 1.0 mM DTT (table 4). In the absence of an auxin treatment, on average less than one root of cutting was induced. Higher con- centrations of DTT were required for rooting of Ce- piland than were required for rooting of Jork 9.
Discussion
Few studies have been related to the ability of thiol compounds, such as reduced glutathione (GSH) and DTT, to influence differentiation in plants. In animal
Table 3
RESPONSE OF CUTTINGS OF MALUS DOMESTICA CV. JORK 9 TO INCREASING CONCENTRATIONS OF DITHIOTHREITOL AFTER
3 wk FOLLOWING THE AUXIN TREATMENT
Dithiothreitol (mM) Rooting (%) Roots/cutting 0.0 (control) ... 73a 3.4a 0.1 ... 77a 3.8a 0.15 ... ... 91 3.8a 0.2 ... 92 4.1 0.25 ... 91 4.9 0.3 ... 96 6.1 0.5 ... 77a 3.2a 1.0 ... 73a 2.5a 2.0 ... 23 0.2
Note. Means with the same letter were not different from the control as analyzed by the Dunnett test at P < 0.05.
cells, such reagents, which reduce disulfide bonds, have been extensively used to probe the potential role of protein thiols and disulfides in modulating the struc- ture and function of hormones and neurotransmitter receptors (Aronstam et al. 1978; Bottari et al. 1979;
Suen et al. 1980; Larsen et al. 1981; Sidhu et al. 1986;
Falcone and Aharony 1990; Pavo and Fahrenholz 1990).
Reports of the ability of thiol compounds to influ- ence the differentiation of higher plants also have been few (Auderset et al. 1996). With Pelargonium, a pro- moting action of rooting was seen for natural antiox- idants (vitamins C and E) or synthetic food antioxi- dants, especially EHA (E320) mixed with gallic acid (Lis-Balchin 1989).
In another report dealing with rooting in Malus, Standardi and Romani (1990) reported that rooting was decreased if antioxidants (including GSH) were added in the induction phase. The percentage of roots formed increased if the test substances were given lat- er. Standardi and Romani (1990), however, tested only very high concentrations of glutathione, concentrations much higher than those found to promote rooting (Au- derset et al. 1996).
In Malus Jork 9, our findings showed a specific ac- tion of DTT on rooting at relatively low concentrations (0.1-0.2 mM). In adventitious rooting of shoots, the
Table 4
EFFECT OF DITHIOTHREITOL CONCENTRATION ON ROOTING OF MALUS DOMESTICA CV. CEPILAND CUTTINGS AFTER 3 wk IN
RESPONSE TO AUXIN TREATMENT
Dithiothreitol (mM) Rooting (%) Roots/cutting 0.0 (control) ... 77a 2.6a 0.1 ... 73a 1.8a 0.25 .*-- 68a 1.7a 0.5 ... 79a 2.2a 1.0 ... 91 4.6 2.0 ... 70a 2.4a
Note. Means with the same letter are not different from the con- trol as determined by the Dunnett test at P < 0.05.
AUDERSET ET AL.-STIMULATION OF ROOT FORMATION 135
following pathway is accepted by many authors: (1) perception of auxin stimulation by target cells, (2) gene expression related to the formation of root mer- istems, (3) dedifferentiation of specific fields, (4) dif- ferentiation and formation of promeristems (meriste- moids), and (5) formation of roots and outgrowth. The timing and cytological evolution of the cell systems involved in these rooting processes have been well studied (Hicks 1986; Zhou et al. 1992; Auderset et al.
1994).
Step 1 can be reached by a 1-day auxin treatment with 25 FM IBA on stem disks (Welander and Paw- licki 1993) or with 2 h of 1 mM IAA treatment on shoots. Both treatments are sufficient for induction of root formation in Malus Jork 9. Other steps take place after transfer to medium without auxin-like substances.
Root induction (steps 2 and 3) occurs in the 4-5 d following the auxin treatment. Root elongation or ex- pression requires an additional 3-5 d to occur.
In our experiments, DTT was applied between steps
2 and 5. Under these conditions, DTT promoted root- ing. If the application of DTT was delayed until step 5, the DTT was much less effective.
The mechanism whereby sulfhydryl compounds might induce rooting is unknown. Their effectiveness parallels that of classical rooting induction obtained by auxins. In herbaceous plants that are easy to root, the response to DTT was to nearly double the number of roots produced in either the presence or absence of auxin treatment (Auderset et al. 1994). With woody plants, which are often more difficult to root than her- baceous plants, the rooting response was significantly enhanced by the presence of dithiothreitol during and after the auxin treatment.
Acknowledgments
This study was completed as part of a European Economic Community Bridge-Cost 87 activity and re- ceived partial support from the Office Federal de l'Education et de la Science in Berne, Switzerland.
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