Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation

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1 Tree Physiology 20, Heron Publishing Victoria, Canada Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation ALESSIO GIOVANNELLI and RAFFAELLO GIANNINI Istituto Miglioramento Genetico Piante Forestali CNR, Via Atto Vannucci 13, Firenze, Italy Received October 26, 1999 Summary Gradual reinvigoration of adult chestnut (Castanea sativa M. cv. Montemarano) shoots was obtained by serial grafting onto juvenile rootstocks. The phenomenon was evaluated on the basis of percentage of primary nodes regenerating axillary shoots and length and number of shoots (> 10 mm) per primary node. In vitro growth of explants from serially grafted shoots was significantly lower than that of explants from seedlings at the end of the establishment phase. Only microshoots from seedlings and plants that had been serially grafted four times could be subcultured on proliferation medium. Repeated subculture on medium containing a low cytokinin concentration induced progressive reinvigoration of microshoots derived from plants that had been serially grafted four times. The number of axillary shoots per explant increased significantly after six subcultures. After 12 subcultures, microshoots from serially grafted plants showed an increase in stem elongation, rooting and plantlet survival. After in vitro stabilization, there was no difference in in vitro performance between microshoots derived from seedlings and serially grafted plants. Microshoots multiplied from serially grafted plants displayed only a transitory appearance of juvenile traits. Keywords: in vitro growth, juvenile traits, rooting, serial grafting. Introduction Maturation often adversely influences the capacity of woody plants for vegetative propagation. Restoration of some juvenile traits in the adult ortet, such as the capacity for rapid growth and for vegetative propagation, may represent a crucial step in the development of efficient in vitro cloning protocols. In some species, tissues with juvenile traits are directly available within the ortet (epicormic shoots, roots suckers, stump sprouts, etc.); in other species, suitable material for vegetative propagation may be obtained by reinvigoration of a donor tree. Reinvigoration (reversal of aging) consists of a transient increase in vigor and rooting ability (Pierik 1990). Grafting is one of the most commonly used techniques to stimulate reinvigoration in mature trees (e.g., Hackett and Murray 1993, Sánchez et al. 1997). Grafting on juvenile rootstock has been successfully used to reinvigorate adult ortets for subsequent use in in vitro clonal propagation programs (Fouret et al. 1985, Monteuuis 1991). The degree of reinvigoration can be improved by combining grafting with other techniques such as treatment with cytokinins (Donkers et al. 1985, Dumas 1987), severe hedging (Amerson et al. 1988), etiolation (Ballester et al. 1989) or cascade grafting (Pierik 1990). In response to repeated grafting, a gradual recovery of juvenile growth vigor, leaf morphology and rooting capacity has been observed in Pseudotsuga menziesii (Mirb.) Franco (Huang et al. 1992), Thuja plicata J. Donn ex D. Don (Misson 1987), Quercus acutissima Carruth. (Moon and Yi 1993) and Larix decidua Mill. (Ewald 1998). Unfortunately no long-term observations on this reinvigorated material have been carried out, so the stability of the recovered juvenile traits in these species is unknown. In this study, we evaluated the effects of repeated grafting on in vitro morphogenetic competence of mature chestnut (cv. Montemarano). In vitro performance and the retention of juvenile traits in microshoots derived from seedlings and reinvigorated adult shoots were observed and recorded over a 5-year period. Materials and methods Serial grafting Crown shoots, cm long, were collected in early winter from a mature tree of Castanea sativa Mill. cv. Montemarano growing in an experimental plot (Tiglio, central Italy) and selected as an ortet in a clonal propagation program. Shoots were stored for six months in the dark at 4 ± 1 C.Thefollowing spring, 50 scions each with two dormant buds were cut from the stored shoots and veneer grafted onto 10-month-old container-grown seedlings that had been forced to flush in a greenhouse. The following spring, 50 shoots were collected from the previously grafted plants and regrafted onto new 10-month-old seedlings. The procedure was repeated four times over a 4-year period. Culture initiation The source of the plant material used for culture initiation is depicted in Figure 1. Shoots, cm in length, were collected in early winter 1989 from: (a) the crown of the ortet (the same tree used for serial grafting); (b) 10-month-old seedlings

2 1244 GIOVANNELLI AND GIANNINI Shoot proliferation and rooting After eight weeks on initial medium, new axillary shoots (longer than 15 mm) were excised from the nodes and placed in 500-ml glass jars (with glass lids fixed with plastic film) filled with 125 ml of basal medium supplemented with 0.44 µmba (proliferation medium). During the proliferation phase, microshoots were subcultured every 5 6 weeks. For root induction, apical shoots mm long were dissected from the proliferating microshoots and cultured for 5 days in the dark on basal medium modified as follows: macronutrients were reduced to half strength and 14.7 µm IBA was added (rooting medium). The shoots were subsequently transferred to a solid rooting medium comprising a 1:1 (v/v) mix of sterile perlite and peat. All cultures were incubated at 25 ± 1 C in a 16-h photoperiod at an irradiance of 80 µmol m 2 s 1. After 3 weeks, the rooted plantlets were potted in a soil matrix made of peat:perlite:sand (2:1:2; v/v) and hardened-off in a misting bed for 1 2 months before final transfer outdoors. Figure 1. Schematic representation of serial grafting procedure. Shoots collected from parent chestnut tree were successively grafted four times onto juvenile rootstock (10-month-old seedlings). of open-pollinated origin derived from seeds collected from the ortet and grown in containers; and (c) 1-year-old plants serially grafted since The shoots were stored in a dark chamber at 4±1 Cfor6months and then forced to flush in a growth cabinet at 23±1 Cina16-h photoperiod at an irradiance of 50 µmol m 2 s 1. After 2 3 weeks, flushing shoots (30 50 mm long) were collected and sterilized by soaking in a 3.7 mm HgCl 2 solution for 4 min followed by two washes. The first wash was with a sterile solution of 44 mm CaCl 2 to eliminate mercuric chloride residues, and the second wash was with sterile distilled water. Nodal explants obtained from seedlings were processed in a similar way. Nodes with 2 3 buds were removed and each node was placed in a 75-ml test tube containing 25 ml of a basal medium composed of Woody Plant Medium (WPM) macronutrients, micronutrients, vitamins (Lloyd and McCown 1980), 87.6 mm sucrose and 0.6 % (w/v) Difco Bacto Agar. To induce shoot growth from axillary buds, basal medium was supplemented with 4.44 µm 6-benzyladenine (BA) and µm indole-3-butyric acid (IBA) (initial medium). The ph was adjusted to 5.8 with NaOH before autoclaving at 121 C for 20 min. Data collection and statistical analysis At the end of the in vitro establishment phase, the percentage of nodes carrying axillary shoots longer than 10 mm, the number of axillary shoots and the length of the tallest shoot produced by each responsive node were recorded. In the proliferation phase, the number of axillary shoots longer than 10 mm and the length of the tallest shoot per microshoot were recorded for four replicates of 12 microshoots. In the rooting phase, percentages of rooted shoots and shoot tip necrosis were recorded for four replicates of 18 microshoots. Acclimatization was determined as plantlet survival in 30 to 60 rooted shoots. For the proliferation, rooting and acclimatization phases, data were collected at the time of Subcultures 6, 12, 24, 36 and 48. In the establishment and acclimatization phases, significant differences (P 0.01) were estimated by an R C test of independence based on the G-test (Sokal and Rohlf 1981). In the proliferation and rooting phases, a two-way ANOVA was conducted and significant difference between means were tested by Tukey s test at P The percentage data were analyzed after arcsin transformation. The experiments on multiplication and rooting were repeated three times. Results In each successive grafting cycle, over 70% of grafts showed successful union after 2 months (Table 1). After 8 weeks on initial culture medium, axillary shoots longer than 10 mm developed only infrequently (5%) from nodes taken from ungrafted adult material, whereas nodes removed from seedlings exhibited a high percentage (82%) of shoot initiation. For explants taken from grafted plants, the highest number of responsive nodes was recorded after four successive grafts (57%). Nodes collected from adult material that had been serially grafted one to three times produced low quality, vitrified, short axillary shoots (< 10 mm) that were not subcultured because they perished rapidly. After up to three successive grafts, one to three vigorous new shoots longer than 10 mm developed from each responsive node; the elongated shoots were easily separated and subcultured on proliferation medium. Although four successive grafts improved the in vitro performance of mature primary nodes, the number of axillary shoots per responsive node was only about half that for seedling material. In the early proliferation stage, microshoots from seedlings showed higher in vitro growth and proliferation capacity than microshoots from plants serially grafted four times (Figure 2). After in vitro stabilization, microshoots from serially grafted plants showed a significant recovery of proliferation activity. In particular, the mean number of axillary shoots (> 10 mm) per microshoot doubled between TREE PHYSIOLOGY VOLUME 20, 2000

3 REINVIGORATION OF MATURE CHESTNUT BY GRAFTING AND MICROPROPAGATION 1245 Table 1. Effect of explant source on in vitro culture of primary nodes of chestnut after 8 weeks. Explants were collected from an adult ortet, serially grafted plants and 10-month-old seedlings. Percentage values followed by different letters are significantly different at the 1% level (R C test of independence based on the G-test). Values are means ± SE. The number of primary nodes contaminated with microrganism is reported in parenthesis. Explants source Grafting Nodes cultured Primary node with Mean number of Length of longest Axillary shoots viability (%) shoot development shoots (> 10 mm) shoot (mm) per vitrified (%) (%) per responsive node responsive node Adult ortet 60 (21) 5.1 a ± One grafted adult (8) 20 ab 1.2 ± ± Two grafted adult (6) 23 b 1.1 ± ± Three grafted adult (4) 38.2 bc 1.2 ± ± Four grafted adult (4) 57.1 c 1.9 ± ± Seedlings 30 (8) 81.8 d 3.8 ± ± n = 2. Subcultures 6 and 12, reaching values similar to those obtained by microshoots of seedlings. However, reinvigorated microshoots remained significantly shorter than microshoots of seedling origin (> 40 mm) during this period. Several shoots differentiated from the basal part of reinvigorated microshoots but they remained short (< 20 mm), collectively forming a cluster (Figure 3). A significant increase in length of shoots from grafted plants was recorded after Subculture 12. By Subculture 36, in vitro growth and proliferation capacity of shoots from serially grafted plants were similar to those of shoots derived from seedlings. After Subculture 36, however, there was a significant decrease in both the number and length of axillary shoots longer than 10 mm from grafted plants, whereas no decrease was recorded for axillary shoots derived from seedlings. Compared with microshoots of seedlings, microshoots of grafted plants showed reduced rooting capacity and increased apical necrosis (Figure 4). Adventitious roots differentiated from approximately 90% of juvenile microshoots, whereas microshoots from grafted plants rooted only infrequently (< 10%) during the early subcultures. A significant increase in Figure 2. Effects of 6, 12, 24, 36 and 48 subcultures on proliferation capacity and elongation of microshoots derived from grafted plants and seedlings. Different letters indicate significant differences at P 0.05 (Tukey s test); bars indicate ± SE. Figure 3. Morphology of microshoots originally collected from grafted plants (a) and seedlings (b) after 12 subcultures on proliferation medium; bar = 10 mm. TREE PHYSIOLOGY ON-LINE at

4 1246 GIOVANNELLI AND GIANNINI Figure 5. Rooted explants originally collected from seedlings (upper) and plants that had been serially grafted four times (lower) after 24 subcultures. Bar = 10 mm. Figure 4. Effects of 6, 12, 24, 36 and 48 subcultures on percentage of rooting and apical necrosis of microshoots derived from seedlings and plants that had been serially grafted four times. Different letters indicate significant differences at P 0.05 (Tukey s test). Reinvigoration was evident as a progressive increase in the reactivity of primary nodes and axillary shoots in response to successive grafting cycles. In our system, repeated grafting had only a reinvigorating effect, because the best in vitro growth of primary nodes from grafted plants remained significantly lower than that of nodal explants from seedlings. A similar reinvigoration phenomenon has been observed in Se- percentage of rooted microshoots from grafted plants was observed between Subcultures 12 and 24 (from 15 to 77%). Between Subcultures 24 and 36, rooting percentage of microshoots from serially grafted plants were not significantly different from that of microshoots of seedlings (Figure 5). After 36 subcultures, there was a partial loss of rooting capacity (< 50%) and an increase in shoot tip necrosis (> 45%) in microshoots from serially grafted plants. After a 3-week acclimatization period, plantlets derived from seedling tissue showed a greater survival rate compared with plantlets obtained from serially grafted plants (Figure 6). Subculture had a significant effect on survival of plantlets from grafted plants. A notable increase in survival of plantlets from grafted plants was recorded at Subculture 24, whereas a significant decrease was observed after Subculture 36. Discussion Repeated grafting improved the in vitro culture capacity of a recalcitrant mature chestnut tree, indicating that reinvigoration can be gradually acquired in mature chestnut trees. Thus, although the grafting method is time consuming, it provides a means of obtaining reinvigorated material that is easy to establish in vitro. Figure 6. Effects of 6, 12, 24, 36 and 48 subcultures on the survival rate of ex-vitro plantlets derived from seedlings and plants that had been serially grafted four times. Different letters indicate significant differences at the 1% level (R C test of independence based on the G-test). * n = 8; ** n = 21. TREE PHYSIOLOGY VOLUME 20, 2000

5 REINVIGORATION OF MATURE CHESTNUT BY GRAFTING AND MICROPROPAGATION 1247 quoia sempervirens (D. Don) Endl. (Huang et al. 1992) and Thuja plicata Don cv. Atrovirens (Misson and Giot-Wirgot 1985) where adult shoot tips regained juvenile-like traits, including high in vitro performance, following four successive graftings onto juvenile microshoots. Microshoots from serially grafted adult shoots needed a long period for in vitro stabilization (about 2 years), a tendency noted by McCown and McCown (1987) for some species of the Quercus and Fagus genera. Reinvigoration also occurred during the stabilization phase in microshoots from plants that had been serially grafted four times. A progressive increase in elongation and branching of microshoots, incidence of rooting, plantlet survival as well as a decrease in apical necrosis were observed with increasing number of subcultures. Similarly, Kretzschhmar and Ewald (1994) reported that repeated subculture of microshoots from 140-year-old Larix decidua trees resulted in a more juvenile appearance in shoots that rooted easily. We conclude that, in the absence of reliable biochemical and molecular markers, shoot vigor expressed as an increase in stem elongation and the restoration of high rooting capacity can be used to assess reinvigoration in woody plants (cf. Pierik 1990). In microshoots from serially grafted plants, increased rooting capacity and decreased apical necrosis were achieved after vigorous and elongated shoots with expanded leaves were induced on medium containing auxin. This recovery of vigor and associated elongation capacity are important parameters for identifying material capable of root formation (Sánchez et al. 1996). Our rooting data corroborate those of Fejo and Pais (1990) who reported that a progressive increase in rooting capacity of mature chestnut microshoots occurred only after eight subcultures. Similar results were obtained with apple cultivars (Welander 1985, Sriskandarajah et al. 1982). In contrast, Sánchez and Vieitez (1991) found that shoot proliferation increased in mature chestnut microshoots during successive subcultures, whereas no increase in rooting was recorded after two years. Similar results were reported with Camellia reticulata Lindl. (Ballester et al. 1990), a difficult-to-root species. The discrepancy in rooting capacity of microshoots among the studies may be associated with the use of explants from serially grafted plants, because microshoots from serially grafted plants showed increased rooting capacity after successive subcultures on a cytokinin-containing medium, whereas microshoots from ungrafted plants did not. Once stabilized, microshoots from serially grafted plants showed uniform and continuous growth similar to that of microshoots from seedlings; however, this in vitro performance was of a transient character, as has been reported for other species (e.g., Pierik 1990, Swartz 1991). In many species, in vitro proliferation and rooting cannot be maintained indefinitely and it is necessary to reestablish the culture after some time (Norton and Norton 1986). References Amerson, H.V., L. Frampton, R.L. Mott and P.C. Spaine Tissue culture of conifers using lobolly pine as a model. In Genetic Manipulation of Woody Plants. Eds. J.W. Hanover and D.E. Keathley, Plenum Press, New York, pp Ballester, A., M.C. Sánchez and A.M. Vieitez Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut. Physiol. Plant. 77: Ballester, A., M.C. Sánchez and A.M. Vieitez Development of rejuvenation methods for in vitro establishment, multiplication and rooting of mature trees. 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