MICROPROPAGATION OF ELITE GENOTYPES OF CASTANEA SATIVA (MILL

200 | P a g e J u n e 2 1 , 2 0 1 4 MICROPROPAGATION OF ELITE GENOTYPES OF CASTANEA SATIVA (MILL.) Dorothy T. Tchatchoua,, Evangelos Barbas and Filippos A. (Phil) Aravanopoulos 1 Laboratory of Forest Genetics and Tree Breeding, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, Thessaloniki, PO Box 238, GR54124. ‡ Present Address: Department of Agriculture, Animal Husbandry and Derived Products, Higher institute of the Sahel (ISS), University of Maroua. Cameroon. E-mail:d.tchatchoua@yahoo.com 1 Laboratory of Forest Genetics and Tree Breeding, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, Thessaloniki, PO Box 238, GR54124. E-mail: vbarbas@for.auth.gr 1 Laboratory of Forest Genetics and Tree Breeding, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, Thessaloniki, PO Box 238, GR54124. E-mail: aravanop@for.auth.gr ABSTRACT


INTRODUCTION
European chestnut (Castanea sativa Mill.) is one of the more widespread broad-leaved tree species in southern Europe and Asia Minor where it is used for high quality timber and starch containing nut production in managed forests and fruit orchards. It appears as natural (in autochthonous pure and mixed with other broadleaves stands) and naturalized forest, coppice forest and orchards, the latter dedicated to nut production, representing an important and vital integral part of the rural economy (Aravanopoulos et al. 2001). Moreover, C. sativa presents considerable ornamental qualities and in places not favourable for nut production (central Europe, the United Kingdom, parts of North America), is used as an ornamental tree (Plants for a Future 2014). Its qualities include a majestic dome shaped canopy, yellow-white and strongly fragrant catkins produced in the summer, green spiked burs that contain the nut which ripe in the fall, deeply serrated, large and dark green foliage turning golden bronze before falling and a twisted, ribbed, retiform bark that takes a conspicuous form with age (FAO (2014).
However, C. sativa is under pressure from a number of environmental and anthropogenic threats, that include diseases such as chestnut blight caused by Cryphonectria parasitica (Murr) and ink disease caused by Phytophthora cinnamomi (Rand), as well as environmental degradation of natural ecosystems. Specially designed European-wide breeding programs aim at developing genetically improved chestnuts for a multitude of applications including timber production, nut production, ecosystem stabilization and ornamental use, calling for the selection and use of novel cultivars and varieties (Fernandez-Lopez et al. 2005, Tchatchoua and Aravanopoulos 2010a, 2010b). Quantitative genetics results from a provenance-progeny test located at Taxiarchis, Greece which is part of a European-wide provenance-progeny trials network (Fernandez-Lopez et al. 2005), indicated that plus-trees from Greek provenances could be considered as elite trees and can be used in further multipurpose chestnut breeding programs for the genetic improvement of wood and nut production, as well as aesthetic form (Tchatchoua and Aravanopoulos 2010b). Henceforth, tissue culture propagation methods were initiated on elite genotypes for clonal selection procedures. Tissue culture allows elite individuals to be directly tested as clones, (instead of indirect testing, typical of progeny tests). Besides saving time, since it does not require trees to produce seed, this strategy also allows a higher number of individuals to be tested per unit of time and space. In addition, the potential of vegetative propagation techniques to deliver high genetic gains is evident, as they offer the possibility of capturing the total genetic variation (i.e. both additive and non-additive components).
Vegetative propagation of chestnut has proved to be challenging. Rooting of greenwood and hardwood cuttings has proved largely unsuccessful (Cummins 1970). Vegetative propagation by layering was developed for some clonal rootstocks (Ridley and Beaumont 1999) however this method is laborious and time-consuming. Various grafting and budding methods have been applied in chestnut, being heavily dependant on factors such as time of grafting, age, growth vigour of rootstock, quality and quantity of scion, graft incompatibility and susceptibility to chestnut blight (Ferrini and Pisani 1994). Grafting results have been generally inconsistent (Osterc et al. 2005). Tissue culture can provide an adequate method for mass propagation of elite genotypes in C. sativa. In the past decades tissue culture application in C. sativa has recorded some successes (Rodriguez 1982

Origin of explants
Scions were collected from stump sprouts of eight mature plus-trees from two old-growth natural C. sativa populations located in Mt. Paiko and Mt. Hortiatis, Greece. Tree diameter was measured by a Nestle Best Nr 300060 calliper, tree height with a Vertex IV and Transponder T3 instrument and a large increment borer was employed to record tree age ( Table 1). The scions were dipped in small plastic jugs for sprouting in a glass house of the Laboratory of Forest Genetics and Tree Breeding, Aristotle University of Thessaloniki, Greece. After sprouting, young shoots about 2-3 cm long were excised from the axillary meristem put in labelled plastic bags and transferred to the laboratory.

Phase 1: Sterilization and inoculation of explants
Leaves were removed from the young shoots and washed thoroughly in running tap water. They were sterilized in a 0.15% solution of HgCl2 for 2 min, rinsed three times in sterile deionised water and single-node segments were placed in tubes containing 50ml of MS (Murashige and Skoog 1962) medium for shoot initiation. Each culture tube contains five single -nodes with four repetitions per genotype. All media were supplemented with 30 g l 1 of sucrose, 7.1 g l 1 agar, vitamins, micronutrients and Fe-EDTA of Murashige and Skoog (1962), 0.1 mg l 1 of BAP (6-Benzylaminopurine). The pH was adjusted to 5.6 prior to autoclaving. The propagation chamber was maintained at 25 o C and 16 hours of photoperiod, under white fluorescent lamps throughout the experiment..

Phase 2: Multiplication phase
Multiplication was developed by axillary shoot production. Stem segments at a length of at least 2 cm, bearing at least one axillary or apical bud, were excised and inserted vertically into the multiplication medium.
The culture media used were: MS (Murashige and Skoog, 1962) with half strength of nitrates (MS-½NO3) (Vieitez et al. 1983), ½MS and MS. BAP (6-benzylaminopurine) concentrations used as growth regulators were: BAP 0.1 mg l 1 , BAP 0.2 mg l 1 and BAP 0.5 mg l 1 . The shoots were sub-cultured every four weeks for multiplication and elongation under same condition of light as described above. The number of explants used per medium per BAP concentration was 20. In total, 180 combinations (20 x 3 x 3) were used for the three media and three hormone concentrations per subculture. Four subcultures were carried out and during each sub-culturing the recorded variables were: (a) number of shoots produced, (b) length of the longest shoot segments, which indicates the possibility of obtaining shoots that can be used directly for rooting and (c) percentage of responsive explants, which indicates how explants respond to the different treatments applied (Fig. 1).

Phase 3: Rooting
Shoots greater than 2 cm long were excised and placed in sterilised vermiculite where a solution of ½MS medium supplemented with five (0.0 mg l 1 , 0.10 mg l 1 , 0.25 mg l 1 , 0.50 mg l 1 , and 1.0 mg l 1 ) different concentrations of NAA was added. The number of replications employed for the rooting experiment was 16 for each treatment and a total sample size of 80 was produced during this stage with only one factor considered, i.e. the different concentrations of NAA in the vermiculite medium used. The variables measured after eight weeks were: (a) number of roots, (b) length of each root and (c) percentage of rooted shoots. Rooted shoots were removed from the agar medium, washed in running tap water and transferred in plastic pots for acclimatisation in a mixture of perlite and vermiculite.

Statistical analysis
One-way analyses of variance (ANOVA) were carried out. The data set was balanced with approximately normal distribution, hence no transformation was required. The type III model at the 5% significant level was employed and the means among factors were compared using the Duncan Multiple Range Test (DMRT) where appropriate. The SPSS (version 15) statistical software package was used (SPSS 2006).

Effect of genotype on Castanea sativa tissue culture
The selected individuals were mature or over-mature plus-trees of high growth and aesthetic value. The age of the trees ranged between 47 and 500 years old, with an average of 193.4 years; tree height averaged 16 m and diameter 61.6 cm ( Table 1). All eight mature elite plus-trees were successfully introduced in tissue culture. For shoot regeneration, the number of shoots for the P-2 and P-8 elite-trees, was observed to be very high compared to the other plus-trees that were used (Table 2, Fig. 2). A genotype effect was also observed regarding growth of the elite trees in culture. The effect J u n e 2 1 , 2 0 1 4   Usually, juvenile explants are most readily established in culture, while they grow and proliferate at a more rapid rate than adult explants tissue. Nevertheless, the genotypic effect in this experiment resulted in the proliferation of only the P-2 elite mature tree, regarding in vitro multiplication. In the genus Castanea, Chevre and Salesses, (1987) found that truly juvenile shoots tips from seedlings could be explanted and propagated by shoot culture, but adult material (for example of the hybrid C. crenata x C. sativa) require to be rejuvenated in advance. C. sativa shoots tips isolated from seedlings obtained by embryo culture, resulted in shoots which proliferated more rapidly and rooted more easily than those derived from axillary bud explants (Piagnani and Eccher 1988). Moreover, Chevre et al. (1983) noted a difference in the axillary shoots obtained from juvenile and mature buds of this species.

Effect of media and concentrations on shoot length and shoot number of Castanea sativa
The analysis of the effects of media and concentrations on the size of the longest shoots and the number of shoots produced, showed that both media and their concentrations had highly significant effects. Among the different media employed, MS was the best followed by MS-½NO3 (Fig. 3, 4, Table 3). The best results regarding BAP concentrations were obtained at 0.1 mg l 1 BAP, followed by 0.2 mg l 1 BAP, while the least effective was 0.5 mg l 1 BAP J u n e 2 1 , 2 0 1 4 (Fig. 5, Table 4). The medium and concentration that produced the longest shoots also produced the largest number of shoots. While MS was the best medium identified, it also indicated the present of vitrified shoots. This result indicates that a high concentration of nitrate (1650 mg l 1 NH4NO3) in the medium favourably influenced the number of shoots produced. However, negative effects of a high NO3 -/ NH4 + ratio on in vitro growth were mentioned by Vieitez et al., (1983) in chestnut, Gitonga et al. (2008) in Macadamia integrifolia, and more generally by Griffis et al. (1983) in fruit trees, and McCown and Sellmer (1987) in woody species. On the other hand, high proliferation rates on media containing low total nitrogen levels and low nitrate/ammonium ratios were observed (Miranda-Fontaina and Fernandez-Lopez 2001, Piagnani andEccher 1988, Riffaud andCornu 1981), in contrast to the present findings. The best medium for clones and hybrids of C. sativa studied by Miranda and Fernandez-Lopez (2001) was the GD medium (Gresshoff and Doy 1972), while the MS-½NO3 medium was the second best on the number of shoots per explant as in the present study. Mullins (1987) recorded the highest multiplication rate for C. sativa clones using a combination of MS-½NO3 and BA (0.1 mg l 1 ).  With regards to the different concentrations of BAP used, it is noted that BAP has been found to promote axillary bud proliferation in Castanea in (Vieitez and Vieitez 1980). Riffaud and Cornu (1981) also used successfully a 4 x 10 -6 M BAP concentration in Prunus avium micropropagation. The effect of cytokinins on tissue or organ cultures can vary according to the particular compound used, the type of culture, and the variety of plant from which it was derived and whether the explant is derived from juvenile or mature tissue (Sanchez et al. 1997). In Corylus avellana, 5 mg l 1 BAP gave the best rate of shoot multiplication from juvenile explants, but 10 mg l 1 zeatin was required for nodal sections of plants in the adult phase (Messeguer and Mele 1987). In many species shoots formation can be stimulated by a balance between auxin and cytokinin as demonstrated by Grinblat, (1972) in Citrus. J u n e 2 1 , 2 0 1 4

Effect of NAA concentration on in-vitro rooting of Castanea sativa
We investigated different concentration of NAA for rooting ability using the P-2 elite tree. Both the highest mean number of roots and the mean length of roots were recorded in a concentration of 1.0 mg l 1 NAA and 0.25 mg l 1 respectively (Fig. 6, Table 5). In addition, the highest percentage (56.25 %) of rooting was recorded in a concentration of 0.5 mg l 1 NAA (Fig. 7, Table 5). Among the concentrations of NAA employed, the lowest number of roots was observed in 0.25 mg l 1 NAA, the shortest root length was observed in 1.0 mg l 1 NAA and the lowest percentage of roots was observed in 0.25 mg l 1 NAA. The analysis of variance showed that the number of roots produced differed significantly among NAA concentrations (p=0.002), while for root length there was no significant difference among NAA concentrations. Similar rooting percentages were obtained in microcuttings of Castanea dentata by dipping in 5-10 mM indolebutyric acid for one minute and cultivated in half strength MS basal medium and adding 0.2 g l 1 charcoal for two weeks. In cultures established from juvenile and mature-phase debladed petioles, auxin induces cell division in explants of both mature and juvenile origin, but only the latter further develops forming adventitious roots (Murray et al. 1994). Differences observed in rooting response of easy to root and difficult to-root apple cultivars concerned the capacity to rapidly organize new cells into primordial and not just the capacity to reactivate cell division after wounding and auxin treatment (Collet et al. 1994). Auxin has been known to be involved in the process of adventitious root formation (Haissig and Davis 1994), while the interdependent physiological phases comprising the rooting process are associated with changes in endogenous auxin concentration (Heloir et al. 1996). This work, to the best of the authors' knowledge, is the first reported on elite mature trees from Greek populations.

CONCLUSION
The main objective of this work was to develop a tissue culture protocol for different Castanea sativa mature and over-mature plus-tree genotypes of high growth and aesthetic value that originated from two Greek populations. Results indicated that micropropagation of selected mature genotypes, such as the 500 years old P-2 elite mature tree from Mt. Paiko, is achievable and can provide a useful tool for the mass propagation of selected mature plus trees for wood and nut production, ornamental use, as well as for tree improvement purposes. This study also indicates that further research on sources of explants, media, hormones and their concentrations will be highly promising for the successful multiplication of elite mature trees.

ACKNOWLEDGEMENTS
Financial support from the Hellenic Scholarship Foundation (IKY) to DTT in order to pursue a doctoral program with FAA is gratefully acknowledged.