Recipient Organization
FLORIDA A&M UNIVERSITY
(N/A)
TALLAHASSEE,FL 32307
Performing Department
Agricultural Research
Non Technical Summary
Currently research on chestnut focuses on the development of vegetative propagation systems and this satisfies the demand for elite genotypes that provide high-quality timber and/or nuts and resistance to ink disease and chestnut blight, caused by the fungi Phytophthora cinnamomi and Cryphonectria parasitica, respectively. Chestnut species are difficult-to-root, grafting is the most frequent conventional propagation technique used. However, other methods for layering and cutting have recently been improved and are also used in nurseries to propagate ink-disease-resistant Euro-Japanese hybrids. Reliable in vitro regeneration systems that allow clonal propagation can also be used as an alternative to conventional vegetative propagation methods.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
(1) To optimize micro propagation protocols based on axillary shoot developments for F1 hybrids chestnut trees2) To induce multiple shoot formation from cotyledonary nodes and define protocols for induction, maturation and germination of somatic embryos
Project Methods
Methods and ProcedureThe micropropagation of chestnut via axillary shoots will involve four stages: 1) initiation (in vitro shoot growth on primary explants); 2) shoot proliferation; 3) shoot rooting; and 4) plantlet acclimatization (hardening)..Culture MediaGresshoff and Doy mineral medium (GD; 1972) will be used for material of both juvenile and mature origin. Apical necrosis and chlorosis will be avoided, and general appearance improved, with either GD or Murashige and Skoog medium (MS; 1962) with half strength nitrates (Vieitez et al. 1986; Sánchez et al. 1997a; Gonçalves et al. 1998; Ballester et al. 2001). However, in protocols developed for C. dentata, Woody Plant Medium (Lloyd and McCown, 1981) will also be used both for culture initiation (supplemented with 1mg/l 6-benzyladenine, BA) and for shoot proliferation (supplemented with 0.2 mg/l BA)Explant Preparation and Culture InitiationThe primary explants from which chestnut shoot cultures will be initiated are shoot tips and nodes bearing 1 or 2 axillary buds. After excision from the source plant (juvenile or mature tree), they will be sterilized and established in vitro on the initiation medium.Juvenile Plant MaterialInitial explants will be obtained from juvenile plants. Seedlings obtained conventionally will be grown in the greenhouse or, preferably, in a climate chamber so as to reduce explant contamination rates (Vieitez et al. 1986). Active growing shoots will be collected from plants between a few weeks and a few months old and will be used for culture initiation Alternatively, initial explants may be taken from seedlings obtained by in vitro germination of zygotic embryonic axes (Vieitez and Vieitez, 1980). In either case, the frequencies of responsive explants, shoot multiplication rates and rooting rates will be high.Juvenile Parts of Mature TreesCuttings 15-20 cm long will be taken in winter from shoots emerging from the base of the trunk, and will be stored at 4ºC until forced to flush in a climate chamber, primary explants will then be taken from the flushed shoots. The use of a climate chamber is both logistically advantageous, allowing primary explants to be obtained throughout the year, and also helps keep contamination rates low (5-15%).Steps1. Basal shoots or stump sprouts will be harvested from selected trees growing in the field in late autumn or winter, and cuttings 15-20 cm long will be taken.2. The cuttings will be immersed for 1 h in a 3.4 g/l solution of Cupravit (50% copper oxychloride), left to dry for 24 h, packed tightly in plastic bags, and stored at 4ºC for 2-6 months, until use.3. The cuttings will be taken out of cold storage, arranged upright in water or in moistened perlite in trays, and forced to flush in a growth cabinet at 24ºC and 90% relative humidity (RH) under a 16 h photoperiod (95-100 μmol m-2s-1 provided by cool-white fluorescent lamps).4. After 15-20 days of flushing, shoots 1-4 cm long will be collected as the source of explants.5. The collected shoots will be stripped of leaves and surface-sterilized by successive immersion for 30 s in 70% ethanol and for 8-10min in sodium hypochlorite solution (Millipore Chlorine Tablets, 0.6% active chlorine) containing 2 or 3 drops of Tween 80.6. The shoots will be rinsed three times with sterile distilled water.7. The disinfected shoots will be sectioned at 5 mm long shoot tips and nodes (primary explants).8. The explants will be placed upright in 20 ×150 mm culture tubes containing 15 ml of initiation medium.9. After 24 h, the primary explants will be moved to a different site within the same tube to reduce the deleterious effects of exudation and blackening of the medium. Thereafter, the explants will be transferred to fresh medium every 2 weeks until 6 weeks after the initiation of culture.10. Shoots produced on primary explants will be excised and subcultured to achieve clonal shoot multiplication cultures.Shoot Proliferation and MaintenanceNew shoots obtained in vitro by 6-8 weeks of culture of primary explants will be excised, divided into 0.8-1.0 cm segments (including shoot tips), and subcultured into 500 ml glass jars (9 explants/jar) containing 70ml of shoot multiplication medium. In vitro stabilization will be achieved within 4-8 subculture cycles, and cultures will be maintained for years if healthy, vigorous shoots are always used for subculture.RootingChestnut is a difficult-to-root species, and the rooting and acclimatization of shoots obtained in vitro are crucial for mass production of viable plants by micropropagation. We will use 3 options for inducing roots on micropropagated chestnut shoots (Sánchez et al. 1997a;Gonçalves et al. 1998):culture for 5-7 days in rooting medium containing 3 mg/l indole-3-butyric acid (IBA)dipping the basal end of the shoots for 1-2 min in 1 g/l IBA solution;culture for 24 h in rooting medium containing 25 mg/l IBA.After one of the three procedures has been carried out, the auxin-treated shoots will be transferred either to a substrate mixture, or to auxin-free root expression medium consisting of basal rooting medium containing1% activated charcoal.HardeningThe adaptation of micropropagated chestnut plants to ex vitro conditions is generally long and difficult. Therefore several techniques will be used to harden the plants before they are taken to the green house.Molecular Marker AnalysisThe genetic stability of in vitro regenerated plants is an essential requisite to maintain clonal identity. Although shoot tips are in principle genetically stable, micropropagation protocols should include confirmation that true-to-type plants are being produced. Methods for early detection of genetic variation include morphological observations, cytological methods and DNA analysis (Rani and Raina, 2003). In the case of chestnut, screening for DNA polymorphism by RAPD analysis has provided evidence supporting the genetic stability of Castanea sativa ×C. crenata hybrids propagated in vitro; after in vitro multiplication of axillary shoots for more than 4 years, comparison of RAPD patterns of in vitro cultures with those of the plants of origin showed no polymorphism between the former and the latter (Carvalho et al. 2003). Assessment of genetic stability is also required following cryopreservation (Harding, 2004). For chestnut, the analysis of genetic integrity of cryopreserved shoots has been performed by RAPD analysis (San-José et al. 2005): in experiments on three genotypes using DNA obtained from fresh young leaves, RAPD profiles generated with forty 10-mer primers showed no differences between non-cryopreserved cultures and cultures that had been recovered following cryopreservation. Therefore, we will use these genetic techniques to test for genetic stability of the produced cultures.