Pecan is a wind-pollinated monoecious crop with staminate flowers organized into an ament or catkin and female flowers borne on a spike (Wetzstein and Sparks, 1986). Self-pollination in pecan is usually limited by dichogamy, whereby female and male flowers mature at different times. Pecan cultivars differ with respect to maturity of the staminate and pistillate flowers, leading to both protandrous and protogynous patterns of flowering, termed heterodichogamy. Because of the heterodichogamy of pecan, breeding programs often must make use of stored pollen to achieve particular crosses. Efficient and reproducible viability testing is essential to facilitating the use of stored pollen because nut set cannot be determined until after the end of the flowering season (Sparks and Madden, 1985); thus, the use of nonviable pollen will result in the loss of the cross for the year.
Inadequate numbers of pollinators in pecan orchards can result in reduced crop set and lower yields (Marquard, 1988; Wood, 1997, 2000). In addition, lack of pollinators can result in an increase in self-pollination, which reduces nut set and nut quality (Marquard, 1988; Romberg and Smith, 1946; Wolstenholme, 1969). The development of artificial orchard pollination protocols, especially in western pecan regions where there is a lack of native pecan trees to provide alternative pollen sources, have been investigated (Bennett et al., 1986). Replicable viability tests are crucial to the continued development of artificial pollination and pollen storage protocols.
Germination tests have generally been considered to be the best in vitro indicator of pollen usefulness (Galleta, 1983). The in vitro germination test assesses the viability of a pollen sample by germinating a sample of pollen grains in an artificial media. Through this method it is possible to observe the percentage of pollen grains that develop pollen tubes after a certain period. Methods of in vitro testing vary with the species tested, degree of accuracy desired, and the purposes of the test. Typically, germination media is contained within a hanging drop or well, the media is solidified with agar or gel, or the pollen is germinated on a membrane support.
Pollen germination media must be similar to key components of the stigmatic fluid to produce maximal germination and give a true indication of the viability of the sample. Germination percentage is affected by many factors, including the concentration of Ca+2, H+, and borate in the germination media (Holdaway-Clarke et al., 2003). The effect of these three ions is not completely understood, but they may interact through changes in the extensibility of pectin in cell walls and appear to be important regulators of pollen tube growth (Holdaway-Clarke et al., 1997). The addition of boron and calcium to the germination media increases germination percentage and length of pollen tube growth in many fruit species (Galleta, 1983; Kwack, 1965). Optimal concentrations of Ca+2 and borate are high enough to impart rigidity to the pollen tube cell wall to prevent bursting in the face of cell turgor but low enough to allow the wall to stretch quickly during periods of accelerating growth (Holdaway-Clarke et al., 2003). Boron gradients may also be important in directing pollen tube growth toward the ovaries (Blevins and Lukaszewski, 1998). H+ promotes more elastic cell walls, perhaps by keeping pectin methyl esterase in a less active state and competing with Ca+2 to reduce pectin crosslinking, leading to a higher degree of pectin esterification and a weaker cell wall (Vervaeke et al., 2005). Sucrose primarily serves to control the osmotic potential of the germination media but may also provide a base for polysaccharide synthesis and metabolic energy (Kwack, 1965). Alternatively, polyethylene glycol (PEG) has been used in some systems to lower the water potential of the media and allow sucrose concentrations to be lowered, producing higher germination rates and/or more stable pollen tubes (Read et al., 1993; Subbaiah, 1984; Vasil, 1987). PEG is relatively inert chemically and cannot enter cells, whereas sucrose enters the pollen and increases already high internal concentrations (Taylor and Hepler, 1997).
Pollen storage and artificial germination protocols for pecan pollen have been investigated. Wetzstein and Sparks (1985) found that fresh pecan pollen could be germinated in a solution of 20% sucrose and 0.03% H3BO3, but pollen germination decreased rapidly during storage with less than 1% germinating after 5 d of storage. Yates et al. (1986) optimized the germination solution to 15% sucrose, 0.05% Ca(NO3)2, and 0.01% H3BO3, but also found that pecan pollen loses viability in a matter of days. Yates and Sparks (1989) found that controlled rehydration of dry pollen before placing in the germination media was vital to determining accurate germination rates. Controlled rehydration allows the phospholipids in the plasma membrane bilayer to go through phase transition before being exposed to bulk water (Crowe et al., 1989). Without this transition, the water uptake is rapid and highly damaging to plasma membranes (Dumont-Be'Boux, 1999). The previous lack of hydration of pecan pollen samples before germination tests likely led to the mistaken observation of pollen quickly losing viability when it was simply becoming more desiccated. This is supported by Yates and Sparks (1990) who found that pollen stored for up to 3 years at –80 or –196 °C produced fruit set equal to fresh pollen.
Despite these studies, germination tests of pecan pollen carried out in our breeding program and in other pollen physiology studies (L. Lombardini, personal communication) have often produced non-replicable results. We undertook these experiments to develop a pollen germination test that would give more consistent results and could be easily fit into a pecan breeding program.
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