Propagation of New Chestnut Rootstocks with Improved Resistance Phytophthora cinnamomi – New Cast Rootstocks

Nowadays there is a great demand for chestnut rootstocks with improved resistance to Phytophthora cinnamomi, the causing agent of ink disease/root rot. The majority of genotypes that are being commercialized and used in the new plantations, were selected from the breeding programs of the XX century. The climate scenarios have been changing throughout the years, therefore the selection of new genotypes, better adapted to the current conditions of soil and climate, together with an effective and expedite production system, are essential to fulfill the needs of the market on better propagation materials. The INIAV's chestnut breeding program, established in 2006, aims the improvement of resistance of chestnut to biotic stresses. By taking advantage of micropropagation, this program developed a pipeline to produce new hybrid varieties with improved resistance to P. cinnamomi. Micropropagation helps to overcome the recalcitrance that chestnut tissues present to traditional clonal propagation techniques. This work describes the pipeline of production of these hybrids and characterizes the plants from in vitro to ex vitro conditions. The pipeline allows the development of healthy plants capable of robust and continuous growth in ex vitro conditions. Nowadays these hybrids are being multiplied in a large scale production system, to be released to the market in the near future.


Introduction
The European chestnut (Castanea sativa Mill.) is a multipurpose tree, having a significant economic interest for nut and wood production. Chestnut cultivars have been maintained by clonal propagation using agronomic techniques like rooting of cuttings, however, root induction in mature chestnut tissue has proven to be a very difficult process (VIEITEZ et al., 1987;VIEITEZ, 1992). In the past century, chestnut cultivars were also propagated by grafting on seedlings of C. sativa (PEREIRA-LORENZO and FERNANDEZ-LOPEZ, 1997). Nevertheless, due to the high susceptibility of this species to the oomycete Phytophthora cinnamomi (causing agent of the root rot/ink disease) (HARDHAM, 2005), the use of this technique is now compromised.
The Japanese (Castanea crenata Siebold and Zucc.) and Chinese (Castanea mollissima Blume) chestnut species are resistant to the disease, but their nuts do not have significant commercial value and they cannot be used as rootstocks due to grafting incompatibility with C. sativa (HONGWEN et al., 1994). Taking this into consideration, hybrid clones of C. sativa with the Asian species have been used as rootstocks grafting for plantations in affected areas (PEREIRA--LORENZO and FERNANDEZ-LOPEZ, 1997;GONÇALVES et al., 1998).
The available genotypes in the European market are from breeding programs from the XX century (BREISCH et al., 1995;PEREIRA-LORENZO and FERNANDEZ-LOPEZ, 1997). However, a change was projected in the distribution of the pathogen in the next years due to climate change (BURGESS et al., 2017) which might bring up different disease expressions (due to the emergence of new strains and hosts). This creates a demand for new chestnut material, more adapted to the current climatic conditions. Therefore, new genotypes with improved resistance to P. cinnamomi were selected from the INIAV's Chestnut Breeding Program initiated in 2006(COSTA et al., 2011SANTOS et al., 2015SANTOS et al., , 2016. Micropropagation has great potential as it produces healthy plant materials with robust root systems that contribute to a better adaptation in field conditions. This technology has also the potential of accelerating the delivery of plant material to the nurseries, has it is more efficient than traditional propagation methods because it can produce plants throughout the year. Several micropropagation protocols have been developed for chestnut (SEABRA and PAIS, 1993;SÁNCHEZ et al., 1997;GIOVANNELLI and GIANNINI, 2000;BALLESTER et al., 2001;TETSUMURA and YAMASHITA, 2004;OAKES et al., 2016). Taking this into consideration, the in vitro propagation system was implemented to propagate the new improved genotypes, selected from the INIAV's Chestnut Breeding Program, having a large scale production perspective to fulfill the needs of the market on better propagation materials.
The goal of this work was to describe the propagation method of the new Euro-Asian chestnut hybrids from the laboratory to the field. The in vitro development of three selected genotypes was characterized by multiplication rates and morphology in the steps prior to rooting. The root induction, development in greenhouse and development on the field were also evaluated.

Plant material
The three chestnut hybrid genotypes used for these experiments were obtained from controlled crosses and selected for their resistance to P. cinnamomi, after inoculation with the pathogen (COSTA et al., 2011, SANTOS et al., 2015, namely: variety SM904, hybrid between C. sativa x C. mollissima and varieties SC1202 and SC55, C. sativa x C. crenata hybrids.

Culture conditions and shoot propagation
In vitro multiplication was carried out as described in SANTOS et al., (2017). Shoots were placed in Murashige and Skoog (MS) medium (MURASHIGE and SKOOG, 1962) (half concentration of NH4NO3 and KNO3), supplemented with 0.1 mg/l benzyladenine (BA), 30 g/l sucrose and 8 g/l agar. Fifteen shoots were placed per vessel (Eco2Box with green filter; oval model 80 mm H; Duchefa biochemie) and cultures were maintained at 23°C under a 16 hour light photoperiod.
The multiplication rate of each genotype was obtained by counting the number of new shoots rising from the basis of the initial shoot, by the end of the subculture period (corresponding to 6 weeks). Seventy-five biological replicates were analyzed per subculture during 3 subcultures.

Elongation procedure and morphological characterization
Six-week-old shoots were transferred to full-strength Woody Plant Medium (WPM) (LLOYD and MCCOWN, 1980), 30 g/l sucrose and supplemented with 0,1 mg/l of zeatin and 8 g/L agar. The pH was adjusted to 5,6-5,8 before autoclaving at 121°C and 15 psi for 20 minutes. The containers and culture conditions were the same as described in the previous section.
The development of six-week-old shoots micropropagated in elongation media was morphologically characterized by measuring the length of the biggest shoot length (mm), the number of leaves per shoot, biggest leaf length and width (mm), dry mass (mg) (after drying at 60°C over night), moisture (%) (calculated by using the formula × 100) and stem diameter (mm).
Forty-five biological replicates were analyzed.

Rooting process
Prior to rooting, elongated shoots were subcultured on a pre-rooting medium composed by MS medium described above (without plant growth regulators) plus 3 g/L charcoal for 7-10 days (SANTOS et al., 2017) for cytokinin detoxification. Shoots were removed from the elongation medium, excised from the callus mass and placed into the new vessels for 7 days. The surface of the cut ends was then renewed by cutting 1-2mm under tap water. After this procedure shoots were dipped in a 2 g/l indole-3-butyric acid (IBA) aqueous solution, at a depth of 0.5 cm for 1 minute and placed in paperpots (previously soaked with tap water) for root development. Nine paper pots were set in closed clear plastic boxes (SteriVent Containers; High model; Duchefa biochemie) in greenhouse tunnels at 25°C with a relative humidity of 95% (controlled by humidity probes) under natural light. The number of shoots used for rooting assays was 405, 87 and 89 for SM904, SC1202, and SC55, respectively. After 3 weeks the number of rooted shoots was registered and presented as mean values.

Acclimatization and ex vitro development
Rooted plants were transferred to peat: perlite (7:3) mixture (300ml pots) and placed in an acclimatization clear tunnel at 25°C under a natural photoperiod. Relative humidity levels started at 90% and gradually decreased to 60% for 7 days. After this period, plants were transferred to the greenhouse and maintained at proximally 23ºC. Sprinkle fertigation was done once a week for 10 min. The fertilizer consisted of a 1:1 solution of GREEN LB (16 g/l) and YELLOW (16 g/l) from YaraTera KRISTALON, with an Electric Conductivity (EC) of 1.00. The height of 10 plants of each genotype was registered for 2 months and presented as mean values and Standard Error (SE) of the mean.
Plants were transferred to field conditions (GPS coordinates: 39.347494, -7.395609; field with sandy soil; pH=4.9) with a spacing of 7 x 7 m. The height of 6 trees of each genotype was registered every 6 months during 2 years to estimate the annual growth rate.

Statistical analysis
The multiplication rates were analyzed and presented as means and SE for each subculture. An analysis of variance (Kruskal-Wallis test) was performed to check for differences throughout subcultures in each genotype. Differences between genotypes multiplication were also analyzed with the same test by considering all data collected from the 3 subcultures (all 225 biological replicates per genotype). Significantly different means were identified by using the Dunn's Multiple Comparison Test (P<0.05). Data were listed as mean values and SE.
Elongation data were analyzed and presented as means and SE. The genotypes were compared for each growth parameter by performing an analysis of variance for the different parameters of growth (Kruskal-Wallis test). Significantly different means were identified by using the Dunn's Multiple Comparison Test (P<0.05).
All tests were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com.

Results and discussion
In this work, we describe in vitro and ex vitro development of 3 new chestnut hybrid varieties resistant to P. cinnamomi. This production pipeline enables chestnuts development from the laboratory to the orchard. The mass production of these genotypes aims to disclose to the market new rootstocks with improved resistance to root rot/ ink disease.
The multiplication rates for the 3 genotypes are presented in Figure 1. These data revealed significant differences among genotypes (p=0.0003) with the highest multiplication rate presented by SM904 with 2.573 new shoots. Followed by SC55 and SC1202 with 2.253 and 2.053 new shoots, respectively. These rates are in accordance with the ones described for Euro-Asian hybrids, namely from 2,5 to 3 new shoots/explant (BALLESTER et al., 2001;MIRANDA and FERNANDEZ, 2001). However, the previous works used MS medium supplemented with 0.2 mg/l BA, double the concentration used in the present work.
SM904 revealed to be the genotype with the best consistency throughout the repeated subcultures with no significant differences between them (Figure 2). Unlike SC55 (p=0.0176) and SC1202 (p=0.0169) which present a significant decrease in the number of shoots in the last subculture (Figure 2).  The elongation phase was carried out using zeatin, a plant growth regulator that has been previously described as the best for in vitro culture in Castanea species by giving the best results in terms of shoot length, number of leaves and also multiplication rate when compared to BA and Thidiazuron (TETSUMURA and YAMASHITA, 2004).
The characterization of several morphological parameters, of the chestnut hybrids, after 6 weeks of elongation is presented in Figure 3. All the genotypes presented distinct in vitro phenotypes. SM904 is characterized by having long shoots, with the smallest leaves, less dry mass and higher moisture content. SC55 shoots were the shortest and with less number of leaves. SC1202 stood out by having higher dry mass content. Three weeks after induction with 2 g/L IBA, it was possible to observe root development in all genotypes (Figure 4). SM904 rooted 315 out of 405 shoots (77.7%), SC55 69 out of 89 (77.5%) and SC1202 48 out of 87 (55.2%). Similar rooting percentages have been described for other Castanea species (XING et al., 1997;TETSUMURA and YAMASHITA, 2004;OAKES et al., 2015). The development of plant height for the 2 months in the greenhouse after acclimatization is shown in Table 1 and Figure 5. The plants' initial height was similar for the 3 genotypes (2.4 cm average). After 2 months of development, SM904 presented the highest values with 9.4 cm, in comparison with SC55 and SC1202 with 6.15 cm and 7.05 cm, respectively.  In the field, the plantation success obtained for the three genotypes was 90%. After 2 years of development on the field (Figure 6), the estimated annual growth rates for each genotype were 9.5%, 11.9% and 2.8% for SC55, SC1202, and SM904 respectively. In this work, it was possible to identify different characteristics and behavior in vitro and ex vitro culture. Globally SM904 is the best genotype in vitro, in terms of rooting rates and greenhouse growth. However, in the field, it is the one that shows the lowest annual field growth. Also, SC1202 seems to have disadvantages in vitro when comparing to the other two genotypes. Nevertheless, it has a high field growth.
Nowadays, the Portuguese Breeding Program has several ongoing experiments to better characterize the development of these rootstocks on the field. The growth in height is still being monitored for more accurate data. Irrigation tests have been also established. Initial data suggest that plant growth is improved in plants irrigated with 4 and 8 liters of water per hour. Eight months after plantation some plants were able to be grafted with C. sativa fruit varieties (Martaínha, Longal, Bária and Judia). Thirty rootstocks were grafted with a success rate of 92%. These preliminary data suggest that all genotypes, used as rootstocks, present high compatibility with the main varieties used in Portugal for nut production. The scion growth was on average of 1 m after 1 year of grafting. However, graft unions can break after years of growing (PINA et al., 2009), hence complete graft compatibility is not yet proved.
The key factors of the new hybrid varieties propagation pipeline include 1) the production of whole plants with both vigorous aerial and radicular systems; 2) a practical strategy that reduces the propagation time when compared to traditional methods of chestnut propagation, and 3) an easily applied method for the production of Castanea hybrids for the agro biotechnological industry.
In conclusion, despite all the difficulties inherent to the production process, the breeding program benefits from a reliable tissue culture protocol that enables to manage a large scale vegetative propagation of elite genotypes. Nowadays, all the selected genotypes with improved resistance to P. cinnamomi, are being produced by this method and market release is expected in the near future.