Plant Cell Rep (2006) 25: 879–886 DOI 10.1007/s00299-005-0108-z
CELL BIOLOGY AND MORPHOGENESIS
S. Valladares · C. S´anchez · M. T. Mart´ınez · A. Ballester · A. M. Vieitez
Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis Received: 15 September 2005 / Revised: 30 November 2005 / Accepted: 3 December 2005 / Published online: 18 March 2006 C Springer-Verlag 2005
Abstract Somatic embryogenesis was induced in expanding leaf explants excised from epicormic shoots forced from branch segments taken at four different times of year from a mature oak (Quercus robur L.). Branch segments 2– 4 cm in diameter produced most shoots when collected in March. Somatic embryos were induced on explants derived from branches of all collection dates, although collection in November seemed to afford the best results. Germination and conversion ability of embryos of embryogenic lines derived from six oak trees depended heavily on genotype, conversion rates ranging from 0 to 70%. RAPD analyses found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryo derived plantlets and the trees of origin. The embryogenic system used in this study appears to be suitable for true-to-type clonal propagation of mature oak genotypes. Keywords Genetic stability . Pedunculate oak . Quercus robur . RAPD analysis . Somaclonal variation . Somatic embryogenesis Abbreviations BA: 6-benzylaminopurine . MS: Murashige and Skoog . NAA: 1-naphthaleneacetic acid . RAPD: random amplified polymorphic DNA Introduction Quercus robur L. is one of the most important broadleaf forest trees of central and western Europe, both ecologiCommunicated by: H. L¨orz S. Valladares · C. S´anchez · M. T. Mart´ınez · A. Ballester · A. M. Vieitez () Instituto de Investigaciones Agrobiol´ogicas de Galicia, CSIC, Avda. de Vigo s/n, Apartado 122, 15080 Santiago de Compostela, Spain e-mail:
[email protected] Tel.: +34-981590958 Fax: +34-981592504
cally and because of its valuable timber. However, its improvement, and the conservation of high-value genotypes, is limited by long rotations, inadequate seed production relative to demand, the impossibility of storing seeds for long periods, and difficulties in vegetative propagation (Savill and Kanowski 1993). In such circumstances, in vitro propagation provides an essential means of multiplication of selected genotypes. Somatic embryogenesis is a potentially powerful tool for improvement of forest trees, with applications for largescale clonal propagation, genetic transformation, or for provision of cryopreservable tissue, and can also be of use for studies of embryogenesis and embryo development. The integration of this technology into oak improvement programmes could be very beneficial. The feasibility of initiating embryogenic lines from immature zygotic embryos has been shown in various oak species (Wilhelm 2000; Kim 2000; Mauri and Manzanera 2003), but such material is of unproven genetic value, as are somatic embryos initiated from leaf explants excised from young seedlings (Fern´andez-Guijarro et al. 1995; Rancillac et al. 1996; Cuenca et al. 1999). Induction of somatic embryogenesis from leaves of mature oak trees has been investigated for Q. ilex (F´eraud-Keller and Espagnac 1989) and Q. suber (Hern´andez et al. 2003a). In a previous study (Toribio et al. 2004), we obtained somatic embryos from the leaves of epicormic shoots forced from branch segments collected from several hundred-year-old Q. robur trees, establishing embryogenic lines which were proliferated by secondary embryogenesis. The ability to use such tissues rather than those of genetically unproven seedlings offer an important advantage for rapid propagation of elite genotypes with valuable traits. However, the embryogenic system reported by Toribio et al. (2004) was still of limited usefulness, because of embryogenesis induction was strongly affected by genotype (source tree). The induction of somatic embryogenesis can be influenced by the physiological or developmental stage of the donor tree from which the initial explants are directly or indirectly obtained, and in particular by whether tissue collection takes place in the dormant season or not (Bonga
880
2004). Since in our earlier study (Toribio et al. 2004), most branches were collected in February, before new flush growth began, our first aim in the study described here was to determine whether this timing was critical for the establishment of embryogenic cultures. The genetic stability of in vitro regenerated plants is an essential requisite for large-scale clonal forestry. Methods for early detection of genetic variation include morphological observations, cytological methods, and DNA analysis (Rani and Raina 2003). In particular, the examination of randomly amplified DNA sequences for polymorphism (RAPD analysis) has been used to ascertain the genetic stability of embryogenic systems for tree species such as the Gymnosperms Picea mariana (Isabel et al. 1993), Picea glauca (De Verno et al. 1999), Picea abies (Fourr´e et al. 1997) and Pinus taeda (Tang 2001) and the Angiosperms peach (Hashmi et al. 1997), mango (Jayasankar et al. 1998) and date palm (Javouhey et al. 2000). In studies of oak species, RAPD analysis has not detected genetic variation within embryogenic lines of Q. suber (Gallego et al. 1997), Q. serrata (Ishii et al. 1999) or Q. robur (S´anchez et al. 2003), or between these lines and the somatic embryo derived plantlets. Since in all the above-mentioned studies of in vitro genetic variation in Quercus the embryogenic lines were initiated from either zygotic embryos or very young seedlings, variation with respect to the tissues of origin was not investigated. In the case of lines initiated from mature tissue, genetic variation with respect to the tissue of origin is of greater interest, and since initiation from such tissue probably involves the proliferation and de-differentiation of already well-differentiated cells, the risk of such variation may not be negligible. Hornero et al. (2001), using AFLP markers, observed polymorphisms between leaves of mature Q. suber trees and somatic embryos derived therefrom in two of the three cases they examined. The purpose of this study was to compare the abilities of branches of a mature Q. robur tree collected at different times of year to yield leaf explants suitable for induction of somatic embryogenesis. We also evaluated somatic embryos from embryogenic lines established from six different mature Q. robur trees with respect to their ability to germinate and be converted into plantlets. In addition, we performed RAPD analyses to assess both the genetic fidelity within embryogenic lines established from three of
these trees, and the molecular conformity to the trees of origin of these lines and of plantlets generated therefrom. Material and methods Plant material Crown branches from a hundred-year-old specimen of Q. robur growing in Pontevedra, Spain (CR-0) were collected on February 7, March 13, May 16 and November 11, 2003. The branches were cut in 25–30 cm segments ranging from 0.5 to 4.0 cm in diameter that were placed upright in moistened perlite and were forced to flush in a growth chamber at 25◦ C and 90% relative humidity under 16 h photoperiod (95–100 µmol m−2 s−1 provided by cool-white fluorescent lamps). Between 30 and 80 branch segments were used per collection date, grouped in four diameter classes (Table 1). The proportion of branch segments producing newly flushed shoots, the numbers of shoots per segment and their lengths were recorded after 22 days in the growth chamber. Shoot number data were subjected to two-way analysis of variance with branch segment diameter and collection date as the two main factors. The significance of differences between means was estimated using the Dunnett test. Induction of somatic embryogenesis Expanding leaves 0.5–1.5 cm long were taken from the forced shoots as initial explants and were disinfected by immersion for 30 s in 70% ethanol followed by 3 min in sodium hypochlorite solution (Millipore Tabletsr , 0.25% active chlorine) containing two drops of Tween 20, after which they were rinsed three times in sterile distilled water. Two leaves were placed, abaxial side down, in Petri dishes 6 cm in diameter containing 15 ml of MS medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose, 0.6% (w/v) Vitroagar (Pronadisa), 21.48 µM naphthaleneacetic acid (NAA), 2.22 µM benzylaminopurine (BA) and 500 mgl−1 casein hydrolysate (induction medium 1). The dishes were then sealed with Parafilmr and left in darkness at 25◦ C for 6 weeks. After this period, the explants were transferred to 9 cm Petri dishes containing 25 ml of
Table 1 Effects of branch segment diameter and collection date on flushing capacity of oak branch segments (%) and mean number of shoots per segment (N) Collection date
February March May November Overall means
Branch diameter (cm) 0.5–1.0 1–2 % N %
N
2–3 %
N
3–4 %
N
100 66.7 40.0 73.3 70.0
8.5±8.1 6.1±3.3 4.3±0.4 7.6±1.4 6.6±1.6 b
100 100 100 100 100
13.2±9.3 23.8±6.9 12.4±1.8 11.3±2.6 15.2±5.0 c
100 100 100 100 100
24.0±13.5 43.0±0.0 25.9±2.6 7.0±0.0 24.9±12.7 d
3.5±2.1 2.5±2.4 2.0±0.6 2.6±0.6 2.7±0.5 a
80.0 100 81.6 89.5 87.8
Overall means % N 93.3 92.6 79.0 85.7
12.3±8.8 18.9±16.1 11.2±9.3 7.1±1.8
Data were recorded 3 weeks after branch segments were placed in the growth chamber. Value of mean number of shoots followed by different letters are significantly different (p
