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In vitro propagation of chestnut (Castanea sativa×C. crenata): Effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation
Scientia Horticulturae 72 Ž1998. 265–275
In vitro propagation of chestnut ž Castanea satiÕa= C. crenata /: Effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation Jose´ Carlos Gonc¸alves b
a,)
, Grac¸a Diogo a , Sara Amancio ˆ
b
a Laboratorio ´ de Biologia Vegetal, Escola Superior Agraria, ´ Castelo Branco 6000, Portugal Departamento de Botanica e Engenharia Biologica, Instituto Superior de Agronomia, Lisboa Codex 1399, ˆ ´ Portugal
Accepted 21 October 1997
Abstract In order to improve plant survival and to achieve a better understanding of the rooting process of chestnut Ž Castanea satiÕa= C. crenata. shoot cultures of mature origin, different rooting treatments were compared. For root induction, the basal ends of the shoots were dipped into 1 g ly1 IBA solution for 1 min or planted for 5 days in 3 mg ly1 IBA agar medium. For root development, the induced shoots were transferred either to auxin-free agar medium Žin vitro rooting. or to a peat:perlite substrate Žex vitro rooting.. Rooted shoots were subsequently acclimatized. After dipping induction, the rooting percentage was higher when root development was performed in vitro Ž97%. than ex vitro Ž77%.. After induction with IBA in agar medium, the root development conditions did not affect the rooting percentage Ž93% and 87%, respectively, for in vitro and ex vitro rooting.. In the acclimatization stage, 100% survival was obtained with microplants with ex vitro-developed roots, compared to only 50% for microplants with in vitro-developed roots. After the root inductive treatments, peroxidase activity in the shoots was characterized by an initial reduction during the first 12 h, followed by a transient peak at day 1. From day 4 to day 6, peroxidase activity increased. This increase was faster in the dipped shoots, but on day 8, no difference in activity could be observed between the treatments. The sequential
Abbreviations: BA: 6-Benzyladenine; IBA: Indole 3-butyric acid; MS: Murashige and Skoog mineral medium; PPFD: Photosynthetic photon flux density; PVPP: Polyvinylpolipyrrolidone ) Corresponding author. Tel.: q351 72 327535; fax: q351 72 328881; e-mail: [email protected] 0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 3 6 - 2
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anatomical changes during the rooting process were similar in both root induction treatments. The first cellular divisions were observed in some of the cambial derivative cells 24 h after auxin induction and a meristemoid became individualised by days 3–4. Identifiable root primordia with a conical shape were present after 6–8 days. Roots with organized tissue systems emerged from the stem 10–12 days after the root induction treatment. q 1998 Elsevier Science B.V. Keywords: Acclimatization; Anatomy; Chestnut; In vitro and ex vitro rooting; Peroxidase activity; Rooting
1. Introduction Chestnut is a difficult-to-root genera ŽGreenwood, 1986., and propagation by cuttings has proven to be very difficult ŽVieitez, 1974.. Successful methods for multiplication by axillary shooting and in vitro establishment have been described ŽVieitez et al., 1983, 1986.. However, both rooting and acclimatization procedures need to be improved for mass propagation for commercial purposes. Hybrid chestnut clones are used as rootstocks for grafting with Portuguese chestnut varieties, due to their ink disease resistance. In previous works, we achieved 93% rooting on some of these clones, adding IBA to the culture medium, or dipping the base of the shoot in concentrated IBA solution ŽGonc¸alves et al., 1993, 1994.. However, the survival rate of these plants during acclimatization was low ŽGonc¸alves et al., 1993, 1994., probably due to non-functionality of the in vitro-developed rooting systems. The type of root system formed depends on the physical characteristics of the rooting environment ŽNemeth, 1986., and survival in the acclimatization stage requires a functional root system. Knowledge of biochemical and anatomical events associated with root induction and expression is useful, as it will permit the improvement of rooting procedures. Peroxidase activity and its isoenzymatic pattern has been studied in relation to IAA–oxidase catabolism, and also as a marker of the successive rooting phases ŽGaspar et al., 1994.. To date, there are no data available on the relationship between peroxidase patterns and adventitious root formation on chestnut shoots. Vieitez and Vieitez Ž1983. described the anatomical rooting sequence using in vitro juvenile shoots of Castanea satiÕa Mill. but did not correlate it with biochemical changes. The aim of this study was to compare two inductive rooting treatments and two rooting environmental conditions for root development, bearing in mind that root system quality could be the key factor for acclimatization success. At the same time, the peroxidase activity and anatomical changes during the rooting process were also studied.
2. Materials and methods 2.1. Plant material and culture conditions An adult tree of C. satiÕa= C. crenata, hybrid clone M3, resistant to ink disease, was used as the explant source. The tree used had been cut back to ground level. Sprouts
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from the stumps were collected in December and chopped into 20-cm lengths. The cuttings were stored in a cold chamber at 48C until March, and were then transferred to a growth cabinet at 258C with 16-h photoperiod Ž45 " 5 m mol my2 sy1 . for the lateral buds to open. When the shoots from these buds reached 2–4 cm in length, the apices and nodal segments were removed and placed in a culture medium as described by Vieitez et al., 1983, 1986. The new shoots obtained in vitro were excised and subcultured every 4 weeks in order to produce clonal shoot multiplication cultures. The multiplication medium consisted of macronutrients of Greshoff and Doy Ž1972. with micronutrients of Murashige and Skoog Ž1962., supplemented with thiamine, pyridoxine, pantothenic acid, ascorbic acid and nicotinic acid, at 1 mg ly1 each, 100 mg ly1 inositol, 0.2 mg ly1 BA, 30 g ly1 sucrose and 7 g ly1 Difco agar. The pH was adjusted to 5.5–5.6 before autoclaving. In the last multiplication cycle, prior to rooting, MS Ž1r2 NO 3 . was used. Cultures were kept in a growth chamber at 258C day and 208C night with a 16-h photoperiod and a PPFD of 45 " 5 m mol my2 sy1 provided by cool-white fluorescent lamps. 2.2. Rooting experiments and acclimatization For rooting experiments, shoots 3–5 cm long were isolated, and the shoot-tip removed before rooting. The basal medium used for chestnut rooting contained half strength MS macronutrients, with the exception of nitrate, which was reduced to quarter strength, and all other additives as above. Roots were induced in chestnut shoots by either leaving the shoots for 5 days in basal medium supplemented with 3 mg ly1 IBA, or by dipping the base of the shoot in 1 g ly1 IBA in 16% hydroalcoholic solution for 1 min. After the induction treatments, the shoots were transferred either to glass jars with auxin-free basal medium containing 6 g ly1 activated charcoal ŽMerck, 2184. Žin vitro root development. or to a peat:perlite substrate Žex vitro root development. for roots to develop. Ex vitro root development was performed in 60 = 40 = 20 cm polystyrene boxes, containing 10 l of a 70% hydrated peat:perlite Ž1:2, vrv. sterilised mixture; the boxes were covered with transparent acrylic plastic, and plants sprayed daily with water. All cultures were incubated in a growth chamber under the same conditions as described for the multiplication stage. After 4 weeks, rooted shoots were transplanted to plastic pots filled with 200 cm3 of peat:perlite mixture Ž1:2, vrv.. Plantlets were acclimatized during the following 4-week period in controlled cabinets at 25 " 28C with a 16-h photoperiod, and a ppfd of 150 " 10 m mol my2 sy1 provided by cool-white fluorescent lamps. The relative humidity, produced by an ultrasonic fog system, was gradually reduced from 95% to 50% during the acclimatization process. 2.3. Peroxidase actiÕity The lower halves of the induced shoots with in vitro root development were collected Žan auxin-free control treatment was also performed. and were immediately frozen in liquid nitrogen and stored at y808C until extraction. Samples were ground in potas-
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sium–phosphate buffer, pH 6.1 Ž1 ml 100 mgy1 fresh weight. with PVPP Ž0.5 g gy1 of fresh weight. at 48C and centrifuged for 20 min at 20 000 rpm. Supernatant was used as the crude enzyme extract. Peroxidase activity was assayed spectrophotometrically at 258C following the oxidation of guaiacol. The reaction mixture consisted in 100 m l 20 mM guaiacol and 100 m l 10 mM H 2 O 2 in 700 m l potassium–phosphate buffer pH 6.1. One hundred microlitres of each crude enzyme extract was added, and the increase in absorbance at 470 nm was followed. Enzyme activity was expressed as absorbance per minute per mg protein Ž D Abs miny1 mgy1 protein.. Protein concentration was determined by the Bradford method ŽBradford, 1976.. 2.4. Histology The bottom 5–8 mm sections of the stem were collected daily for 14 days. These were fixed in 35% formalinrglacial acetic acidr50% ethanol Ž5:5:90, vrvrv., dehydrated in ethanol series between 60 and 95%, and embedded in LKB Historesin Kit w . Serial 7 m m thick transverse sections were cut with a rotatory microtome and stained with periodic acid Schiff ŽPAS. reaction and toluidine blue counterstain. 2.5. Experimental design and statistics For rooting experiments, 30 shoots for in vitro root development Žfive jars with six shoots each. and 60 shoots for ex vitro root development Žone box contains 60 shoots. were used, and the experiments were repeated twice. The rooting parameters, recorded 4 weeks after the start of auxin treatment, were rooting percentage, number of roots and length of the longest root per rooted shoot. Survival percentage was determined 4 weeks after transplant. A plant was considered a survivor when new leaves were formed after acclimatization. A two-way ANOVA was performed, and means were compared using the least significant difference ŽLSD. values Ž P F 0.05.. For peroxidase activity, each sample consisted of 10 basal stem pieces, and the experiment was repeated twice. For each extract, three replicates were measured. Each point is the mean of six values " standard errors. For histology, four basal pieces of the stems were processed each day.
3. Results 3.1. Rooting Microcuttings not treated with auxin failed to root. The rooting percentage was only affected by root development conditions ŽTable 1.. An average of 97% rooting was achieved using 3 mg ly1 IBA over a 5-day period as inductive medium, and in vitro root development as expressive medium. All the other combinations permitted a minimum of 77% rooting. The highest number of roots per rooted shoot was achieved with 1 g ly1 IBA during 1 min with ex vitro or in vitro root development Ž4.3 and 4.2 roots, respectively., in the same way as for 3 mg ly1 IBA with ex vitro root development
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Table 1 Results obtained on rooting experiments for chestnut clone M3 after 4 weeks, under two inductive rooting methods, 3 mg ly1 IBA during 5 days in rooting media, or by dipping in 1 g ly1 IBA during 1 min Root induction wIBAx
Time
3 Žmg ly1 .
5 days
1 Žg ly1 .
1 min
Analysis of variance Source of variation Induction Development Interaction Error
df
Root development
Rooting Ž%.
In vitro Ex vitro In vitro Ex vitro
93 a 87 ab 97 a 77 b
Number of roots 2.9 b 3.9 a 4.2 a 4.3 a
Length of longest root Žcm. 3.3 c 6.6 a 3.1 c 5.9 b
Acclimatization survival Ž%. 50 b 100 a 50 b 100 a
Sum of squares
1 1 1 176
0.04 ns 0.71) 0.18 ns 20.50
27.0 ) ) ) 11.9 ) ) 7.3 ) 251.6
873.0 ) 32 627.0 ) ) ) 287.4 ns 20 411.8
0.003 ns 9.522 ) ) ) 0.003 ns 14.741
F-test: ns, ) , ) ) , ) ) ) , nonsignificant or significant at P F 0.05, 0.01 or 0.001, respectively. Values in each column followed by different letters are significantly different according to the least significant difference ŽLSD. at P F 0.05. For in vitro root development assay, 30 shoots per treatment were used; for ex vitro root development assay, 60 shoots per treatment were used and the experiment was repeated twice. For each induction treatment, root development was performed, in vitro, i.e. in agar medium, or ex vitro, i.e. in peat:perlite mixture. The acclimatization survival was recorded 4 weeks after the transfer of rooted shoots to acclimatization conditions.
ŽTable 1.. The length of the longest root was clearly promoted by the ex vitro root development environment Ž P F 0.001.. Acclimatization of microplants with ex vitro-developed roots attained 100% survival compared to only 50% for in vitro-developed roots ŽTable 1.. Acclimatization survival was only affected by the root development conditions Ž P F 0.001.. The morphological and anatomical aspects of the in vitro and ex vitro-developed roots are quite different. In vitro roots, which develop from callus-like areas, lacked branch roots, and had a reduced number of root hairs. Ex vitro-developed roots were branched and had numerous root hairs ŽFig. 1A,C.. In transverse sections of ex vitro roots, cortical cells were more uniform than those of in vitro ones, which had intercellular spaces and were generally hypertrophic, giving the roots an abnormally large cross-sectional area ŽFig. 1B,D.. Ex vitro roots had a greater proportion of vascular tissue relative to the total area of the root cross-section ŽFig. 1B,D.. 3.2. Peroxidase actiÕity During the first two days, no real difference on peroxidase activity could be observed between the two inductive treatments, as well as in the untreated control shoots ŽFig. 2..
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Fig. 1. Comparative morphological and anatomical root systems developed under two different conditions. ŽA. root system developed in vitro; ŽB. transverse section of an in vitro-produced root; ŽC. root system developed ex vitro; ŽD. transverse section of an ex vitro-produced root. Bar indicates 1 cm in A and C, and 200 m m in B and D.
In all treatments, there was an initial decrease of specific peroxidase activity during the first 12 h, which was followed by a transient peak at day 1 ŽFig. 2.. From day 2, the peroxidase activity in the basal portions of induced shoots started to increase. Nevertheless, dipping treatment gave rise to significantly higher enzymatic activity up until day 6. During this period, peroxidase activity of the auxin-free control shoots was intermediate between both IBA treatments. On day 8, the peroxidase activity of the two inductive
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Fig. 2. Specific activity Ž D Abs miny1 mgy1 protein. of soluble peroxidases during in vitro rooting of hybrid chestnut clone M3. Rooting was induced with 3 mg ly1 IBA during 5 days in rooting medium ŽIBA 3. and by dipping in 1 g ly1 IBA solution during 1 min Ždipping..
treatments were quite similar, and was approximately 40% higher than control shoots ŽFig. 2.. 3.3. Anatomical changes On day 0, shoot sections showed a normal and typical tissue organisation of the stem anatomy ŽFig. 3A.. The vascular cambium was visible, the cambial derivatives differentiating gradually to assume the characteristics of the cellular elements of the secondary xylem and phloem. Also visible was a discontinuous ring of sclerenchyma surrounding the primary phloem. No significant differences between the two inductive treatments were observed during the anatomical study. The processes of initiation and development of adventitious roots were not synchronous; however, a similar sequence of events was apparent. The first cellular divisions were detected 24 h after auxin exposure, in both treatments, but the most visible change was the presence of certain cambial derivatives with densely stained cytoplasm with prominent nuclei and nucleoli ŽFig. 3B.. From days 3–4, the number of dividing cells increased markedly, and meristemoids were evident between the primary phloem region and the vascular cambium ŽFig. 3C.. The meristemoids developing outwards increased in volume and the number of cells increased as a result of their division and the involvement of dedifferentiating cells of the surrounding phloem and parenchyma. Meristemoids progressively proceeded to become individualised, and polarisation of the divisions gave rise to the typical pointed shape of the root primordium which was defined on days 6–8 ŽFig. 3D.. Cells of the root primordium underwent further internal differentiation, remaining as parenchyma tissue or differentiating into vascular bundles with tracheary elements ŽFig. 3E.. Periclinal divisions of cells in the outer layers of the primordium formed a tissue layer that subsequently gave rise to the root cap. Ten to twelve days after the initiation of the experiment, two or more adventitious roots emerged on the surface of the shoot base. In the control shoots, no specific anatomical changes were observed during the first
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Fig. 3. ŽA–E. Transverse sections of IBA-treated microcuttings of chestnut during in vitro root formation. ŽA. Anatomy of the shoot at day 0. ŽB. Activation of cells in the cambium and adjacent, that become transformed into meristematic cells. The arrow shows a mitotic division after 24 h of auxin exposure. ŽC. Meristemoids are formed outside the vascular cambium between days 3–4 Žarrows.. ŽD. Typical pointed shape of the root primordia defined on days 6–8. ŽE. Differentiating tracheary elements in the new root primordia on days 8–10. ŽF. Transverse section of control microcutting of chestnut, without IBA induction. Note the cell proliferation in the cortex and phloematic region given rise to an accentuated swelling of the shoot, without any other anatomical change. Abbreviations: cortex Žco., meristemoids Žm., phloem Žp., pith Žpi., root primordia Žrp., sclerenchyma Žs., tracheary elements Žte., vascular cambium Žvc., xylem Ž=.. Bar indicates 200 m m in A, 100 m m in B, C, D, F and 50 m m in E.
3–5 days. After this period, it could be observed that part of the cambium produced undifferentiated parenchyma cells in an outward direction, which progressively gave rise to a marked swelling on the shoot base, but with no other change ŽFig. 3F..
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4. Discussion The development of rootingracclimatization protocols enabling the commercial use of chestnut micropropagation is a pursued aim of the researchers ŽSanchez et al., 1997.. The results reported in this work show a clear improvement in acclimatization survival compared to previous references on chestnut acclimatization ŽKeys and Cech, 1982; Vieitez et al., 1986; Mullins, 1987; Gonc¸alves et al., 1994.. Rooting percentage was only affected by root development environment, in vitro or ex vitro, whereas the number of roots and length of the longest root were both dependent on the two studied factors ŽTable 1.. However, the rooting percentage is not always a good reference for the quantitative assessment of plant regeneration, as success is determined by the acclimatization stage. The present results make this statement very clear. The survival rate for the plants that had their root system developed under in vitro conditions was only 50%, against the 100% success rate for plants with their root system developed ex vitro. This significant difference could be due to the fact that ex vitro roots are more functional as a consequence of their anatomical characteristics, such as their greater proportion of vascular tissue relative to the total area of the root cross-section, a more regular endodermis, as well as a more differentiated vascular system. These characteristics may be advantageous both for water absorption and translocation. The occurrence of incomplete vascular connections between the shoot and the in vitro-developed roots, which restricted water movement and consequent growth, has been reported ŽGrout and Aston, 1977; McClelland et al., 1990.. According to these authors, this fact could be an important cause of the higher mortality percentages during the acclimatization stage, this physiological disorder being more pronounced if roots develop from callus-like areas of the shoot, where vascular connections are known to be weak. Our study has also shown a pronounced callus at the base of the shoots with roots developed in agar medium ŽFig. 1A.. This callus is not present on chestnut microplants with root systems developed in natural substrate ŽFig. 1C.. On the other hand, the presence of lateral roots and root hairs developed in natural substrate may have a positive influence. As a result of all of these factors, the microplants with the root system developed in a peat:perlite substrate respond fairly satisfactorily when they are placed in acclimatization conditions. It was found that for the initial 3 days, changes in specific peroxidase activity during in vitro rooting of chestnut shoots showed a similar profile to that presented by Gaspar et al. Ž1992, 1994. in vine cuttings. These authors pointed out that peroxidase activity showed a decrease reaching a minimum after 12 h, followed by a progressive increase that culminated at 72 h. Our results on chestnut showed a significant decrease on peroxidase activity after 12 h followed by a slight transient peak at 24 h, and then a slow progressive increase after day 2 until day 8. The drop in peroxidase activity between days 3–4 as reported by Gaspar et al. Ž1992, 1994. was not registered. Changes in peroxidase activity patterns similar to those described herein have been reported by others ŽDe Klerk et al., 1990; Gebhart, 1985; Pythoud and Bouchala, 1989.. In Quercus robur, a species of the same family as the chestnut, San-Jose´ et al. Ž1992. observed a gradual increase in peroxidase activity during all the rooting treatment, without any significant peak.
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The reported differences between the two auxin rooting treatments in peroxidase activity between day 4 and day 6, did not give rise to differences in the sequence of anatomical changes between the two inductive rooting methods used. The developmental histology sequence was very similar to that reported for the in vitro rooting of other woody species, such as camellia ŽSamartin et al., 1986., oak ŽSan-Jose´ et al., 1992., apple ŽZhou et al., 1992. and also referred to by Vieitez and Vieitez Ž1983. in juvenile C. satiÕa Mill. The time-course of anatomical events during root development in natural substrate was not performed, but ex vitro roots became visible at days 12–14 Ždata not reported., as with in vitro root development. In conclusion, it has been shown that to ensure high rates of micropropagated chestnut plantlet survival, the most important factor is the environmental conditions for root development, rather than the root induction methodology; although peroxidase activity varies during rooting period, it is not possible to establish a clear relationship between rooting and peroxidase activity. Acknowledgements Partial financial support of this work by JNICT under PBICrAGRr1461r92 and PRAXISr2r2.1rBIOr1064r95 projects, is gratefully acknowledged. References Bradford, M.M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein–dye binding. Anal. Biochem. 72, 248–254. De Klerk, G., ter Brugge, J., Smulders, R., Benschop, M., 1990. Basic peroxidases and rooting microcuttings of Malus. Acta Horti. 280, 29–36. Gaspar, T., Kevers, C., Hausman, J.F., Berthon, J.Y., Ripetti, V., 1992. Practical uses of peroxidase activity as a predicative marker of rooting performance of micropropagated shoots. Agronomie 12, 757–765. Gaspar, T., Kevers, C., Hausman, J.F., Ripetti, V., 1994. Peroxidase activity and endogenous free auxin during adventitious root formation. In: Lumdsen, P.J., Nicholas, J.R., Davies, W.J. ŽEds.., Physiology, Growth and Development of Plants in Culture. Kluwer Acad. Pub., Dordrecht, pp. 289–298. Gebhart, K., 1985. Self-rooted sour cherries in vitro: auxin effects on rooting and isoperoxidases. Acta Horti. 169, 341–349. Gonc¸alves, J.C., Amancio, S., Pereira, J.G., 1993. In vitro propagation comparative study of 7 chestnut hybrid ˆ clones C. satiÕa=C. crenata. In: Proc. Int. Congr. on Chestnut. Spoleto, Italy, pp. 211–214. Gonc¸alves, J.C., Amancio, S., Pereira, J.S., 1994. Rooting and acclimatization of chestnut by in vitro ˆ propagation. In: Lumdsen, P.J., Nicholas, J.R., Davies, W.J. ŽEds.., Physiology, Growth and Development of Plants in Culture. Kluwer Acad. Pub., Dordrecht, pp. 303–308. Greenwood, M.S., 1986. Rejuvenation of forest trees. Plant Growth Regulation 6, 1–12. Greshoff, P.M., Doy, C.H., 1972. Developmental and differentiation of haploid Lycopersicon esculentum Žtomato.. Planta 107, 161–170. Grout, B.W.W., Aston, M.J., 1977. Transplanting of cauliflower plants regenerated from meristem culture: I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration. Horti. Res. 17, 1–7. Keys, R.N., Cech, F.C., 1982. Propagation of American chestnut in vitro. In: Proc. USDA For Service Chestnut Research Cooperators Meeting. Morgantown, pp. 106–110. McClelland, M.T., Smith, M.A.L., Carothers, Z.B., 1990. The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants. Plant Cell Tiss. Org. Cult. 23, 115–123.
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Mullins, K.V., 1987. Micropropagation of chestnut Ž Castanea satiÕa Mill... Acta Horti. 212, 473–478. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol. 15, 473–497. Nemeth, G., 1986. Induction of rooting. In: Bajaj, Y.P.S. ŽEd.., Biotechnology in Agriculture and Forestry, Vol. 1: Trees. Springer, Heidelberg, pp. 49–64. Pythoud, F., Bouchala, A.J., 1989. Peroxidase activity and adventitious rooting in cuttings of Populus tremula L. Plant Physiol. Biochem. 27, 503–510. Samartin, A., Vieitez, A.M., Vieitez, E., 1986. Rooting of tissue cultured camellias. J. Horti. Sci. 61, 113–120. Sanchez, M.C., San-Jose, ´ M.C., Ferro, E., Ballester, A., Vieitez, A.M., 1997. Improving micropropagation for adult-phase shoots of chestnut. J. Horti. Sci. Žin press.. San-Jose, ´ M.C., Vidal, N., Ballester, A., 1992. Anatomical and biochemical changes during root formation in oak and apple shoots cultured in vitro. Agronomie 12, 767–774. Vieitez, E., 1974. Vegetative propagation of chestnut. N.Z. J. For. Sci. 4, 242–252. Vieitez, A.M., Vieitez, M.L., 1983. Secuencia de cambios anatomicos durante la rizogenesis in vitro del ´ ´ castano. ˜ Phyton 43 Ž2., 185–191. Vieitez, A.M., Vieitez, M.L., Vieitez, E., 1983. In vitro plant regeneration of mature chestnut. J. Horti. Sci. 584, 457–463. Vieitez, A.M., Vieitez, M.L., Vieitez, E., 1986. Chestnut Ž Castanea spp... In: Bajaj, Y.P.S. ŽEd.., Biotechnology in Agriculture and Forestry, Vol. 1: Trees. Springer, Berlin, pp. 393–414. Zhou, J., Wu, H., Collet, G.F., 1992. Histological study of initiation and development in vitro of adventitious roots in minicuttings of apple rootstocks of M26 and EMLA 9. Plant Physiol. 84, 433–440.
In vitro propagation of chestnut ž Castanea satiÕa= C. crenata /: Effects of rooting treatments on plant survival, peroxidase activity and anatomical changes during adventitious root formation Jose´ Carlos Gonc¸alves b
a,)
, Grac¸a Diogo a , Sara Amancio ˆ
b
a Laboratorio ´ de Biologia Vegetal, Escola Superior Agraria, ´ Castelo Branco 6000, Portugal Departamento de Botanica e Engenharia Biologica, Instituto Superior de Agronomia, Lisboa Codex 1399, ˆ ´ Portugal
Accepted 21 October 1997
Abstract In order to improve plant survival and to achieve a better understanding of the rooting process of chestnut Ž Castanea satiÕa= C. crenata. shoot cultures of mature origin, different rooting treatments were compared. For root induction, the basal ends of the shoots were dipped into 1 g ly1 IBA solution for 1 min or planted for 5 days in 3 mg ly1 IBA agar medium. For root development, the induced shoots were transferred either to auxin-free agar medium Žin vitro rooting. or to a peat:perlite substrate Žex vitro rooting.. Rooted shoots were subsequently acclimatized. After dipping induction, the rooting percentage was higher when root development was performed in vitro Ž97%. than ex vitro Ž77%.. After induction with IBA in agar medium, the root development conditions did not affect the rooting percentage Ž93% and 87%, respectively, for in vitro and ex vitro rooting.. In the acclimatization stage, 100% survival was obtained with microplants with ex vitro-developed roots, compared to only 50% for microplants with in vitro-developed roots. After the root inductive treatments, peroxidase activity in the shoots was characterized by an initial reduction during the first 12 h, followed by a transient peak at day 1. From day 4 to day 6, peroxidase activity increased. This increase was faster in the dipped shoots, but on day 8, no difference in activity could be observed between the treatments. The sequential
Abbreviations: BA: 6-Benzyladenine; IBA: Indole 3-butyric acid; MS: Murashige and Skoog mineral medium; PPFD: Photosynthetic photon flux density; PVPP: Polyvinylpolipyrrolidone ) Corresponding author. Tel.: q351 72 327535; fax: q351 72 328881; e-mail: [email protected] 0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 3 6 - 2
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anatomical changes during the rooting process were similar in both root induction treatments. The first cellular divisions were observed in some of the cambial derivative cells 24 h after auxin induction and a meristemoid became individualised by days 3–4. Identifiable root primordia with a conical shape were present after 6–8 days. Roots with organized tissue systems emerged from the stem 10–12 days after the root induction treatment. q 1998 Elsevier Science B.V. Keywords: Acclimatization; Anatomy; Chestnut; In vitro and ex vitro rooting; Peroxidase activity; Rooting
1. Introduction Chestnut is a difficult-to-root genera ŽGreenwood, 1986., and propagation by cuttings has proven to be very difficult ŽVieitez, 1974.. Successful methods for multiplication by axillary shooting and in vitro establishment have been described ŽVieitez et al., 1983, 1986.. However, both rooting and acclimatization procedures need to be improved for mass propagation for commercial purposes. Hybrid chestnut clones are used as rootstocks for grafting with Portuguese chestnut varieties, due to their ink disease resistance. In previous works, we achieved 93% rooting on some of these clones, adding IBA to the culture medium, or dipping the base of the shoot in concentrated IBA solution ŽGonc¸alves et al., 1993, 1994.. However, the survival rate of these plants during acclimatization was low ŽGonc¸alves et al., 1993, 1994., probably due to non-functionality of the in vitro-developed rooting systems. The type of root system formed depends on the physical characteristics of the rooting environment ŽNemeth, 1986., and survival in the acclimatization stage requires a functional root system. Knowledge of biochemical and anatomical events associated with root induction and expression is useful, as it will permit the improvement of rooting procedures. Peroxidase activity and its isoenzymatic pattern has been studied in relation to IAA–oxidase catabolism, and also as a marker of the successive rooting phases ŽGaspar et al., 1994.. To date, there are no data available on the relationship between peroxidase patterns and adventitious root formation on chestnut shoots. Vieitez and Vieitez Ž1983. described the anatomical rooting sequence using in vitro juvenile shoots of Castanea satiÕa Mill. but did not correlate it with biochemical changes. The aim of this study was to compare two inductive rooting treatments and two rooting environmental conditions for root development, bearing in mind that root system quality could be the key factor for acclimatization success. At the same time, the peroxidase activity and anatomical changes during the rooting process were also studied.
2. Materials and methods 2.1. Plant material and culture conditions An adult tree of C. satiÕa= C. crenata, hybrid clone M3, resistant to ink disease, was used as the explant source. The tree used had been cut back to ground level. Sprouts
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from the stumps were collected in December and chopped into 20-cm lengths. The cuttings were stored in a cold chamber at 48C until March, and were then transferred to a growth cabinet at 258C with 16-h photoperiod Ž45 " 5 m mol my2 sy1 . for the lateral buds to open. When the shoots from these buds reached 2–4 cm in length, the apices and nodal segments were removed and placed in a culture medium as described by Vieitez et al., 1983, 1986. The new shoots obtained in vitro were excised and subcultured every 4 weeks in order to produce clonal shoot multiplication cultures. The multiplication medium consisted of macronutrients of Greshoff and Doy Ž1972. with micronutrients of Murashige and Skoog Ž1962., supplemented with thiamine, pyridoxine, pantothenic acid, ascorbic acid and nicotinic acid, at 1 mg ly1 each, 100 mg ly1 inositol, 0.2 mg ly1 BA, 30 g ly1 sucrose and 7 g ly1 Difco agar. The pH was adjusted to 5.5–5.6 before autoclaving. In the last multiplication cycle, prior to rooting, MS Ž1r2 NO 3 . was used. Cultures were kept in a growth chamber at 258C day and 208C night with a 16-h photoperiod and a PPFD of 45 " 5 m mol my2 sy1 provided by cool-white fluorescent lamps. 2.2. Rooting experiments and acclimatization For rooting experiments, shoots 3–5 cm long were isolated, and the shoot-tip removed before rooting. The basal medium used for chestnut rooting contained half strength MS macronutrients, with the exception of nitrate, which was reduced to quarter strength, and all other additives as above. Roots were induced in chestnut shoots by either leaving the shoots for 5 days in basal medium supplemented with 3 mg ly1 IBA, or by dipping the base of the shoot in 1 g ly1 IBA in 16% hydroalcoholic solution for 1 min. After the induction treatments, the shoots were transferred either to glass jars with auxin-free basal medium containing 6 g ly1 activated charcoal ŽMerck, 2184. Žin vitro root development. or to a peat:perlite substrate Žex vitro root development. for roots to develop. Ex vitro root development was performed in 60 = 40 = 20 cm polystyrene boxes, containing 10 l of a 70% hydrated peat:perlite Ž1:2, vrv. sterilised mixture; the boxes were covered with transparent acrylic plastic, and plants sprayed daily with water. All cultures were incubated in a growth chamber under the same conditions as described for the multiplication stage. After 4 weeks, rooted shoots were transplanted to plastic pots filled with 200 cm3 of peat:perlite mixture Ž1:2, vrv.. Plantlets were acclimatized during the following 4-week period in controlled cabinets at 25 " 28C with a 16-h photoperiod, and a ppfd of 150 " 10 m mol my2 sy1 provided by cool-white fluorescent lamps. The relative humidity, produced by an ultrasonic fog system, was gradually reduced from 95% to 50% during the acclimatization process. 2.3. Peroxidase actiÕity The lower halves of the induced shoots with in vitro root development were collected Žan auxin-free control treatment was also performed. and were immediately frozen in liquid nitrogen and stored at y808C until extraction. Samples were ground in potas-
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sium–phosphate buffer, pH 6.1 Ž1 ml 100 mgy1 fresh weight. with PVPP Ž0.5 g gy1 of fresh weight. at 48C and centrifuged for 20 min at 20 000 rpm. Supernatant was used as the crude enzyme extract. Peroxidase activity was assayed spectrophotometrically at 258C following the oxidation of guaiacol. The reaction mixture consisted in 100 m l 20 mM guaiacol and 100 m l 10 mM H 2 O 2 in 700 m l potassium–phosphate buffer pH 6.1. One hundred microlitres of each crude enzyme extract was added, and the increase in absorbance at 470 nm was followed. Enzyme activity was expressed as absorbance per minute per mg protein Ž D Abs miny1 mgy1 protein.. Protein concentration was determined by the Bradford method ŽBradford, 1976.. 2.4. Histology The bottom 5–8 mm sections of the stem were collected daily for 14 days. These were fixed in 35% formalinrglacial acetic acidr50% ethanol Ž5:5:90, vrvrv., dehydrated in ethanol series between 60 and 95%, and embedded in LKB Historesin Kit w . Serial 7 m m thick transverse sections were cut with a rotatory microtome and stained with periodic acid Schiff ŽPAS. reaction and toluidine blue counterstain. 2.5. Experimental design and statistics For rooting experiments, 30 shoots for in vitro root development Žfive jars with six shoots each. and 60 shoots for ex vitro root development Žone box contains 60 shoots. were used, and the experiments were repeated twice. The rooting parameters, recorded 4 weeks after the start of auxin treatment, were rooting percentage, number of roots and length of the longest root per rooted shoot. Survival percentage was determined 4 weeks after transplant. A plant was considered a survivor when new leaves were formed after acclimatization. A two-way ANOVA was performed, and means were compared using the least significant difference ŽLSD. values Ž P F 0.05.. For peroxidase activity, each sample consisted of 10 basal stem pieces, and the experiment was repeated twice. For each extract, three replicates were measured. Each point is the mean of six values " standard errors. For histology, four basal pieces of the stems were processed each day.
3. Results 3.1. Rooting Microcuttings not treated with auxin failed to root. The rooting percentage was only affected by root development conditions ŽTable 1.. An average of 97% rooting was achieved using 3 mg ly1 IBA over a 5-day period as inductive medium, and in vitro root development as expressive medium. All the other combinations permitted a minimum of 77% rooting. The highest number of roots per rooted shoot was achieved with 1 g ly1 IBA during 1 min with ex vitro or in vitro root development Ž4.3 and 4.2 roots, respectively., in the same way as for 3 mg ly1 IBA with ex vitro root development
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Table 1 Results obtained on rooting experiments for chestnut clone M3 after 4 weeks, under two inductive rooting methods, 3 mg ly1 IBA during 5 days in rooting media, or by dipping in 1 g ly1 IBA during 1 min Root induction wIBAx
Time
3 Žmg ly1 .
5 days
1 Žg ly1 .
1 min
Analysis of variance Source of variation Induction Development Interaction Error
df
Root development
Rooting Ž%.
In vitro Ex vitro In vitro Ex vitro
93 a 87 ab 97 a 77 b
Number of roots 2.9 b 3.9 a 4.2 a 4.3 a
Length of longest root Žcm. 3.3 c 6.6 a 3.1 c 5.9 b
Acclimatization survival Ž%. 50 b 100 a 50 b 100 a
Sum of squares
1 1 1 176
0.04 ns 0.71) 0.18 ns 20.50
27.0 ) ) ) 11.9 ) ) 7.3 ) 251.6
873.0 ) 32 627.0 ) ) ) 287.4 ns 20 411.8
0.003 ns 9.522 ) ) ) 0.003 ns 14.741
F-test: ns, ) , ) ) , ) ) ) , nonsignificant or significant at P F 0.05, 0.01 or 0.001, respectively. Values in each column followed by different letters are significantly different according to the least significant difference ŽLSD. at P F 0.05. For in vitro root development assay, 30 shoots per treatment were used; for ex vitro root development assay, 60 shoots per treatment were used and the experiment was repeated twice. For each induction treatment, root development was performed, in vitro, i.e. in agar medium, or ex vitro, i.e. in peat:perlite mixture. The acclimatization survival was recorded 4 weeks after the transfer of rooted shoots to acclimatization conditions.
ŽTable 1.. The length of the longest root was clearly promoted by the ex vitro root development environment Ž P F 0.001.. Acclimatization of microplants with ex vitro-developed roots attained 100% survival compared to only 50% for in vitro-developed roots ŽTable 1.. Acclimatization survival was only affected by the root development conditions Ž P F 0.001.. The morphological and anatomical aspects of the in vitro and ex vitro-developed roots are quite different. In vitro roots, which develop from callus-like areas, lacked branch roots, and had a reduced number of root hairs. Ex vitro-developed roots were branched and had numerous root hairs ŽFig. 1A,C.. In transverse sections of ex vitro roots, cortical cells were more uniform than those of in vitro ones, which had intercellular spaces and were generally hypertrophic, giving the roots an abnormally large cross-sectional area ŽFig. 1B,D.. Ex vitro roots had a greater proportion of vascular tissue relative to the total area of the root cross-section ŽFig. 1B,D.. 3.2. Peroxidase actiÕity During the first two days, no real difference on peroxidase activity could be observed between the two inductive treatments, as well as in the untreated control shoots ŽFig. 2..
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Fig. 1. Comparative morphological and anatomical root systems developed under two different conditions. ŽA. root system developed in vitro; ŽB. transverse section of an in vitro-produced root; ŽC. root system developed ex vitro; ŽD. transverse section of an ex vitro-produced root. Bar indicates 1 cm in A and C, and 200 m m in B and D.
In all treatments, there was an initial decrease of specific peroxidase activity during the first 12 h, which was followed by a transient peak at day 1 ŽFig. 2.. From day 2, the peroxidase activity in the basal portions of induced shoots started to increase. Nevertheless, dipping treatment gave rise to significantly higher enzymatic activity up until day 6. During this period, peroxidase activity of the auxin-free control shoots was intermediate between both IBA treatments. On day 8, the peroxidase activity of the two inductive
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Fig. 2. Specific activity Ž D Abs miny1 mgy1 protein. of soluble peroxidases during in vitro rooting of hybrid chestnut clone M3. Rooting was induced with 3 mg ly1 IBA during 5 days in rooting medium ŽIBA 3. and by dipping in 1 g ly1 IBA solution during 1 min Ždipping..
treatments were quite similar, and was approximately 40% higher than control shoots ŽFig. 2.. 3.3. Anatomical changes On day 0, shoot sections showed a normal and typical tissue organisation of the stem anatomy ŽFig. 3A.. The vascular cambium was visible, the cambial derivatives differentiating gradually to assume the characteristics of the cellular elements of the secondary xylem and phloem. Also visible was a discontinuous ring of sclerenchyma surrounding the primary phloem. No significant differences between the two inductive treatments were observed during the anatomical study. The processes of initiation and development of adventitious roots were not synchronous; however, a similar sequence of events was apparent. The first cellular divisions were detected 24 h after auxin exposure, in both treatments, but the most visible change was the presence of certain cambial derivatives with densely stained cytoplasm with prominent nuclei and nucleoli ŽFig. 3B.. From days 3–4, the number of dividing cells increased markedly, and meristemoids were evident between the primary phloem region and the vascular cambium ŽFig. 3C.. The meristemoids developing outwards increased in volume and the number of cells increased as a result of their division and the involvement of dedifferentiating cells of the surrounding phloem and parenchyma. Meristemoids progressively proceeded to become individualised, and polarisation of the divisions gave rise to the typical pointed shape of the root primordium which was defined on days 6–8 ŽFig. 3D.. Cells of the root primordium underwent further internal differentiation, remaining as parenchyma tissue or differentiating into vascular bundles with tracheary elements ŽFig. 3E.. Periclinal divisions of cells in the outer layers of the primordium formed a tissue layer that subsequently gave rise to the root cap. Ten to twelve days after the initiation of the experiment, two or more adventitious roots emerged on the surface of the shoot base. In the control shoots, no specific anatomical changes were observed during the first
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Fig. 3. ŽA–E. Transverse sections of IBA-treated microcuttings of chestnut during in vitro root formation. ŽA. Anatomy of the shoot at day 0. ŽB. Activation of cells in the cambium and adjacent, that become transformed into meristematic cells. The arrow shows a mitotic division after 24 h of auxin exposure. ŽC. Meristemoids are formed outside the vascular cambium between days 3–4 Žarrows.. ŽD. Typical pointed shape of the root primordia defined on days 6–8. ŽE. Differentiating tracheary elements in the new root primordia on days 8–10. ŽF. Transverse section of control microcutting of chestnut, without IBA induction. Note the cell proliferation in the cortex and phloematic region given rise to an accentuated swelling of the shoot, without any other anatomical change. Abbreviations: cortex Žco., meristemoids Žm., phloem Žp., pith Žpi., root primordia Žrp., sclerenchyma Žs., tracheary elements Žte., vascular cambium Žvc., xylem Ž=.. Bar indicates 200 m m in A, 100 m m in B, C, D, F and 50 m m in E.
3–5 days. After this period, it could be observed that part of the cambium produced undifferentiated parenchyma cells in an outward direction, which progressively gave rise to a marked swelling on the shoot base, but with no other change ŽFig. 3F..
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4. Discussion The development of rootingracclimatization protocols enabling the commercial use of chestnut micropropagation is a pursued aim of the researchers ŽSanchez et al., 1997.. The results reported in this work show a clear improvement in acclimatization survival compared to previous references on chestnut acclimatization ŽKeys and Cech, 1982; Vieitez et al., 1986; Mullins, 1987; Gonc¸alves et al., 1994.. Rooting percentage was only affected by root development environment, in vitro or ex vitro, whereas the number of roots and length of the longest root were both dependent on the two studied factors ŽTable 1.. However, the rooting percentage is not always a good reference for the quantitative assessment of plant regeneration, as success is determined by the acclimatization stage. The present results make this statement very clear. The survival rate for the plants that had their root system developed under in vitro conditions was only 50%, against the 100% success rate for plants with their root system developed ex vitro. This significant difference could be due to the fact that ex vitro roots are more functional as a consequence of their anatomical characteristics, such as their greater proportion of vascular tissue relative to the total area of the root cross-section, a more regular endodermis, as well as a more differentiated vascular system. These characteristics may be advantageous both for water absorption and translocation. The occurrence of incomplete vascular connections between the shoot and the in vitro-developed roots, which restricted water movement and consequent growth, has been reported ŽGrout and Aston, 1977; McClelland et al., 1990.. According to these authors, this fact could be an important cause of the higher mortality percentages during the acclimatization stage, this physiological disorder being more pronounced if roots develop from callus-like areas of the shoot, where vascular connections are known to be weak. Our study has also shown a pronounced callus at the base of the shoots with roots developed in agar medium ŽFig. 1A.. This callus is not present on chestnut microplants with root systems developed in natural substrate ŽFig. 1C.. On the other hand, the presence of lateral roots and root hairs developed in natural substrate may have a positive influence. As a result of all of these factors, the microplants with the root system developed in a peat:perlite substrate respond fairly satisfactorily when they are placed in acclimatization conditions. It was found that for the initial 3 days, changes in specific peroxidase activity during in vitro rooting of chestnut shoots showed a similar profile to that presented by Gaspar et al. Ž1992, 1994. in vine cuttings. These authors pointed out that peroxidase activity showed a decrease reaching a minimum after 12 h, followed by a progressive increase that culminated at 72 h. Our results on chestnut showed a significant decrease on peroxidase activity after 12 h followed by a slight transient peak at 24 h, and then a slow progressive increase after day 2 until day 8. The drop in peroxidase activity between days 3–4 as reported by Gaspar et al. Ž1992, 1994. was not registered. Changes in peroxidase activity patterns similar to those described herein have been reported by others ŽDe Klerk et al., 1990; Gebhart, 1985; Pythoud and Bouchala, 1989.. In Quercus robur, a species of the same family as the chestnut, San-Jose´ et al. Ž1992. observed a gradual increase in peroxidase activity during all the rooting treatment, without any significant peak.
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The reported differences between the two auxin rooting treatments in peroxidase activity between day 4 and day 6, did not give rise to differences in the sequence of anatomical changes between the two inductive rooting methods used. The developmental histology sequence was very similar to that reported for the in vitro rooting of other woody species, such as camellia ŽSamartin et al., 1986., oak ŽSan-Jose´ et al., 1992., apple ŽZhou et al., 1992. and also referred to by Vieitez and Vieitez Ž1983. in juvenile C. satiÕa Mill. The time-course of anatomical events during root development in natural substrate was not performed, but ex vitro roots became visible at days 12–14 Ždata not reported., as with in vitro root development. In conclusion, it has been shown that to ensure high rates of micropropagated chestnut plantlet survival, the most important factor is the environmental conditions for root development, rather than the root induction methodology; although peroxidase activity varies during rooting period, it is not possible to establish a clear relationship between rooting and peroxidase activity. Acknowledgements Partial financial support of this work by JNICT under PBICrAGRr1461r92 and PRAXISr2r2.1rBIOr1064r95 projects, is gratefully acknowledged. References Bradford, M.M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein–dye binding. Anal. Biochem. 72, 248–254. De Klerk, G., ter Brugge, J., Smulders, R., Benschop, M., 1990. Basic peroxidases and rooting microcuttings of Malus. Acta Horti. 280, 29–36. Gaspar, T., Kevers, C., Hausman, J.F., Berthon, J.Y., Ripetti, V., 1992. Practical uses of peroxidase activity as a predicative marker of rooting performance of micropropagated shoots. Agronomie 12, 757–765. Gaspar, T., Kevers, C., Hausman, J.F., Ripetti, V., 1994. Peroxidase activity and endogenous free auxin during adventitious root formation. In: Lumdsen, P.J., Nicholas, J.R., Davies, W.J. ŽEds.., Physiology, Growth and Development of Plants in Culture. Kluwer Acad. Pub., Dordrecht, pp. 289–298. Gebhart, K., 1985. Self-rooted sour cherries in vitro: auxin effects on rooting and isoperoxidases. Acta Horti. 169, 341–349. Gonc¸alves, J.C., Amancio, S., Pereira, J.G., 1993. In vitro propagation comparative study of 7 chestnut hybrid ˆ clones C. satiÕa=C. crenata. In: Proc. Int. Congr. on Chestnut. Spoleto, Italy, pp. 211–214. Gonc¸alves, J.C., Amancio, S., Pereira, J.S., 1994. Rooting and acclimatization of chestnut by in vitro ˆ propagation. In: Lumdsen, P.J., Nicholas, J.R., Davies, W.J. ŽEds.., Physiology, Growth and Development of Plants in Culture. Kluwer Acad. Pub., Dordrecht, pp. 303–308. Greenwood, M.S., 1986. Rejuvenation of forest trees. Plant Growth Regulation 6, 1–12. Greshoff, P.M., Doy, C.H., 1972. Developmental and differentiation of haploid Lycopersicon esculentum Žtomato.. Planta 107, 161–170. Grout, B.W.W., Aston, M.J., 1977. Transplanting of cauliflower plants regenerated from meristem culture: I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration. Horti. Res. 17, 1–7. Keys, R.N., Cech, F.C., 1982. Propagation of American chestnut in vitro. In: Proc. USDA For Service Chestnut Research Cooperators Meeting. Morgantown, pp. 106–110. McClelland, M.T., Smith, M.A.L., Carothers, Z.B., 1990. The effects of in vitro and ex vitro root initiation on subsequent microcutting root quality in three woody plants. Plant Cell Tiss. Org. Cult. 23, 115–123.
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