by Y. Chu, B.S. Sandifer, P. Ozias-Akins, Institute of Plant Breeding, Genetics & Genomics, University of Georgia, Coastal Plain Experiment Station, Tifton, GA and C.C. Holbrook USDA ARS, Tifton GA
Cultivated peanut (Arachis hypogaea L.) is an allotetraploid (2n=40) species, yet most wild peanut relatives in the same genus are diploids (2n=20). Crosses among elite cultivars have been performed routinely by peanut breeders, to stack desirable traits such as high yield, disease resistances, improved nutrition profiles, etc. (Isleib et al., 2001). Narrow genetic diversity in cultivated peanut and ample allelic diversity among wild relatives has spurred interest in using interspecific hybrids to broaden the genetic base of cultivated peanut (Bertioli et al., 2011). Multiple disease resistances have been identified in wild species such as resistance against white mold, nematodes, and leaf spots. Thus wild peanut relatives provide valuable genetic resources for peanut cultivar improvement. Cross incompatibility and ploidy barriers have limited the use of wild relatives in peanut breeding, although there are examples of introgressed traits, e.g., nematode resistance. Success can be achieved by employing unconventional methods including colchicine treatment and complex breeding pathways to introgress alien chromosome segments from wild species (Simpson, 2001). The techniques presented below are used for cultivated x cultivated crosses, but can be adapted for crossing wild x wild or cultivated x wild.
Greenhouse Hybridization Plant Growth Conditions
Greenhouse temperature should be controlled between 20◦C to 33◦C to promote peanut growth. During the growing season, infestations from thrip-vectored tomato spotted wilt virus (TSWV), spider mites, and white flies are common. Monitoring for insect/viral damage and spraying with insecticides are recommended to maintain plant health.
Before planting, peanut pods should be shelled and seeds coated with Vitavax PC (Bayer Cropscience, NC) or comparable fungicidal seed treatment. For a small amount of seeds (<30 seeds), place ¼ teaspoon of the Vitavax powder into a coin envelope (e.g. 8.8 x 15.2 cm) together with the seeds. Coat seeds well with the Vitavax powder by shaking the envelope several times. Usually three seeds per genotype are planted in each pot, and upon germination, the strongest seedling is saved for crossing. Fill a 25-30 cm pot with moist Promix (Premier Horticulture Inc., Quaketown, PA) and poke three 3-4 cm deep holes with the bottom of a 15 ml test tube (~ 1 cm in diameter) close to the center of the pot. A pinch of Bradyrhizobium powder (EMD Crop Bioscience, Milwaukee, WI) should be applied in each hole, which ensures nodulation and nitrogen fixation after germination. Place one peanut seed in each hole with the embryo axis facing downward. Cover the seeds with soil and water lightly after sowing. Germination usually is evident within 5-10 days. Ironite (12-10-10 N-P-K with 2% iron; Gro Tec, Inc, Madison, GA) can be applied 20 days after planting.
Flowers and Fruits
As a legume, the peanut flower is shaped like a typical pea flower, and each flower has one large standard (also called banner), two lateral wings, and a keel (Figure 1). The keel encloses the staminal tube at the distal end of which extends eight anthers surrounding a stigma. Four of the anther sacs are oblong-shaped, and the other four are globular. Emasculation is needed before making a cross. The peanut ovary is located inside the bracts where the hypanthium ends and joins to the reproductive branch. Within the hypanthium tube, a style elongates from the apex of the ovary and rises above the anthers to form the stigma. Peanut flowers open in the morning shortly after sunrise and wither within 5-6 hours after expansion. Upon fertilization, a peg is formed and elongates geotropically. Peanut embryos are located at the tip of the peg which enlarges and forms a pod after the peg penetrates into the soil.
Tools for Hybridization
A pair of tweezers with curved, pointed ends (Figure 2a; Cat. #18-782; Miltex, Inc. York, PA) can be used for emasculation. This design aids in opening the peanut flower buds and removing anthers without damaging other structures of the bud. Another pair of tweezers with flat ends (Figure 2b) can be used for pollination. The flat end serves as a platform to hold pollen grains and to deliver the pollen grains to the stigma on an emasculated female flower.
Method for Crossing
Peanut is a naturally self-pollinating species. However, for breeding purposes, cross-pollination must be performed by hand. Emasculation can be done any time before anther dehiscence, which is usually close to the time of petal expansion around sunrise, although this can vary with genotype. Practically, emasculation is performed between 4:00 in the afternoon and midnight. Peanut flower buds enlarge and become easier to work with as the evening progresses. If emasculation is to be performed after sunset, a headlamp (LED headlight, www.energizer.com) can be used to provide sufficient light for the operation. Use of a magnifier with an optional headlamp (OptiVisor) is preferred by some. To emasculate a female flower (Figure 3; Video 1), hold the flower bud from the banner side and remove the lower lip of the calyx with the curved tweezers. Gently move the standard and wings aside and flip the keel forward. Remove all eight anthers surrounding the stigma. Place the keel back on the stigma and close up the wings and standard. Tie a cotton thread on the hypanthium of the bud to mark the emasculated flower. At this stage, the hypanthium is usually short; therefore, care should be taken that only the hypanthium is wrapped by the thread. If neighboring stipules are tied together, new flowers can grow into the same thread making the identification of a hybrid peg difficult. Pollination is usually performed the next morning between 7:30 and 9:30. Cloudy and cool weather can delay anther dehiscence. Pollination should not be delayed later than 10:00 in the morning because a female bloom has a short life time of 5-6 hrs. To perform pollination, pick a male flower and remove the wings (Figure 4; Video 2). Squeeze the pollen grains onto the flat end of the pollination tweezers and apply them onto the tip of the stigma on an emasculated female flower.
After pollination, the greenhouse ground should be sprinkled with water to increase humidity because high humidity will improve the success of crossing. To avoid disturbance of pollen grains, the watering schedule should be moved to the evenings. Any blooms on a female plant that are not emasculated should be removed in the morning to minimize the chance of misidentifying hybrid pegs.
A peg tip emerges 5-10 days after pollination. Since the senescent flower part is still attached to the apex of the peg, the thread attached to the flower is used as a mark to identify the cross-pollinated peg (Figure 5). A wire made from phone cable components can be used to initially mark the position of the peg in a pot. As the peg extends, it can be wired permanently by bending the wire and letting the peg go through the circle on the top of the wire. Care should be taken not to damage the peg when performing the wiring step. For breeding purposes, wiring five hybrid pegs per plant usually is considered sufficient. After crossing has completed, gypsum should be applied to each pot to provide adequate calcium for pod development and improve seed quality.
A crossing number shall be assigned to each cross, e.g. C123, and the names of cultivars involved in the cross shall be expressed as female x male. In a female pot, two tags shall be placed side by side, a tag marking the genotype of the female itself and a tag with the crossing number and female x male combination. In a male pot, one tag marking the male genotype is sufficient. Ideally, each cross should involve one female-male pair rather than using multiple male plants to pollinate one female plant.
Depending on the variety, the duration between wiring a peg to pod maturation can take 60-80 days. Since the hole on the wire is much smaller than the peanut pod, the wire stays attached to the peg during the growing season and harvest. At harvest, care should be taken not to detach the pod from the peg. Harvesting the pods with wires improves the chance of hybrid recovery.
Storage of Seeds
Once the pods are harvested, they need to be dried to lower the kernel moisture content to less than 10.5% of fresh weight. This can be achieved by drying peanut at 30◦C in a ventilated oven for approximately one week. After drying, pods can be shelled and seeds placed in a plastic bag with proper labeling for storage at 4◦C for up to a year. For longer term storage, seeds should be sealed in foil bags or glass containers and stored at -18◦C. Evaluation Methods
Most cultivated peanut varieties do not have obvious visual markers that can be used to evaluate whether a cross has been achieved. More reliable determination of hybridity among putative F1 plants can be achieved with molecular markers, provided that polymorphic markers have been identified between the parents. For example, Tifguard has a normal oleic to linoleic acid ratio (O/L) in its seeds, while Florida-07 is a high O/L cultivar. A single nucleotide insertion in the coding region of the ahFAD2B gene (delta-12-desaturase or oleoyl-PC desaturase) eliminates the reductive function of this enzyme and causes the expression of high oleic acid content in the seeds of Florida-07. A gel-free single nucleotide polymorphism (SNP) assay using the HybProbe design was developed to distinguish progenies from the Tifguard x Florida-07 cross (Figure 6). This type of marker-assisted selection is PCR-based, which requires DNA extraction from leaf tissues and amplification of genomic DNA with appropriate primers for detection. Methods of DNA extraction and PCR reaction conditions have been detailed by Chu et al. (2011).
Bertioli D.J., Seijo G., Freitas F.O., Valls J.F.M., Leal-Bertioli S.C.M., Moretzsohn M.C. (2011) An overview of peanut and its wild relatives. Plant Genetic Resources-Characterization and Utilization 9:134-149.
Chu Y., Wu C.L., Holbrook C.C., Tillman B.L., Person G., Ozias-Akins P. (2011) Marker-assisted selection to pyramid nematode resistance and the high oleic trait in peanut. The Plant Genome 4:8.
Isleib T.G., Holbrook C.C., Gorbet D.W. (2001) Use of plant introductions in peanut cultivar development. Peanut Sci 28:96-113.
Simpson C.E. (2001) Use of wild Arachis species/introgression of genes into A. hypogaea L. Peanut Sci 28:114-116.