COTTON In Vitro Analysis of Cotton Pollen Germination

average and record yields. Those crops having marketable vegetative structures exhibited approximately threeCrops with economically valuable reproductive structures show fold reductions in yield (Boyer, 1982). These data sugthe greatest negative discrepancy between average and record yields. gest that plants have high productivity potential but are To alleviate environmental stress–related yield reductions, a better understanding of the relative sensitivities of pollen development, deoperating well below their genetic potential. hiscence, pollen germination, pollen tube growth, fertilization, and Yield loss might be lessened by identifying and optisubsequent boll development is needed. Progress in identifying the mizing those plant protective mechanisms that could be sensitivities of these developmental stress responses has been hamused to improve stress-resistant germplasm stocks. One pered in part by the lack of a rapid and reliable method of germinating such protective mechanism is acquired thermotolerance, cotton (Gossypium spp.) pollen in vitro. The present study describes a process postulated to be closely linked to the heat the development of an in vitro cotton pollen germination system that shock response. Plants are frequently exposed to eleprovides improved pollen germination levels and pollen tube growth vated soil and air temperatures resulting in a reduction over existing methods. This procedure was used to evaluate the temin their growth, development, and ultimately productivperature sensitivity of pollen from greenhouse and field-grown cotton. ity. Subjecting them to a period of sublethal elevated The medium comprises 10% (w/v) agarose (pH 7.6), 25% (w/v) sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM temperatures induces a transient state of thermotolerMgS04·7H20, and 1.0 M A3 gibberellic acid. The medium is overlayed ance, which raises the injury threshold and protects the with a layer of 1.5% agarose before use. Optimum pollen germination plants from subsequent, otherwise lethal, high temperaand rapid tube elongation occurred between 28 and 31 C under 80% tures. This acquisition of thermotolerance is a complex relative humidity. Decreased pollen germination occurred at temperaphysiological phenomenon that has been shown to intures above 37 C, and decreased tube elongation occurred at temperavolve at least some heat shock proteins (HSPs) (Viertures above 32 C. Evaluation of pollen, taken at 1400 h from fieldling, 1991). Although varying in magnitude among plant grown cotton plants, showed that pollen from flowers exposed to cultivars, most vegetative tissues exhibit an inducible direct sunlight had reduced viability compared with pollen from flowheat shock response. Germinating pollen, however, has ers inside the canopy. This could be attributed to the fact that flowers from field-grown irrigated cotton plants exposed to full sunlight expenot been found to exhibit the heat shock protein inducrienced internal temperatures as high as 39 C, well above the 28 to tion pattern upon exposure to elevated sublethal tem31 C optimum for pollen germination. peratures and concomitantly exhibits rapid losses in viability upon heat exposure (Hopf et al., 1992). This may explain Boyer’s observation that crops with economiC species grown throughout the world experically valuable reproductive structures show the greatest ence environmental stresses that limit their growth, discrepancy between average and record yields (Boyer, development, and full expression of their genetic poten1982). tial for agronomic yield. Comparison of average crop Modest progress has been achieved in selecting cotton yields with reported record yields has shown that the varieties with improved heat tolerance by heat treatmajor crops grown in the USA exhibit annual average ment of pollen before pollination, allowing only the yields threeto sevenfold lower than record yields due more heat-tolerant pollen to be effective in subsequent to unfavorable environmental conditions (Boyer, 1982). crosses (Rodriguez-Garay and Barrow, 1988). The proAnalysis of yields from corn (Zea mays L.), wheat (Triticess of selecting pollen with improved heat tolerance cum aestivum L.), soybean [Glycine max (L.) Merr.], could be accelerated with a rapid and reliable method sorghum (Sorghum vulgare L.), oat (Avena sativa L.), of germinating cotton pollen to measure viability across barley (Hordeum vulgare L.), potato (Solanum tubersoa range of environmental stresses. Current pollen germisum L.), and sugar beet (Beta vulgaris L.) revealed that nation techniques include hanging drop culture, sitting the average yield represented only 22% of the mean redrop suspension culture, suspension culture, and surface cord yield. Crops with economically valuable reproducculture (Alexander and Ganeshan, 1989; Brewbaker and tive structures showed the greatest discrepancy between Kwack, 1963; Cheng and Freeling, 1976; Dawkins and Owens, 1993; Egea et al., 1992; Feijo et al., 1995; Frascaroli and Tuberosa, 1993; Mulcahy and Mulcahy, 1988; USDA-ARS, SPA, Plant Stress and Water Conserv. Lab., Lubbock, Pareddy and Petolino, 1992; Pundir, 1972; Shivanna and TX 79415. Received 3 June 2002. *Corresponding author ( jburke@ lbk.ars.usda.gov). Rangaswamy, 1992; Shivanna et al., 1991; Singh et al., 1992; Tanaka et al., 1995; Tupy et al., 1983; Vogt et al., Published in Agron. J. 96:359–368 (2004). 1994; Yistra et al., 1995; Zonia and Tupy, 1995). The  American Society of Agronomy 677 S. Segoe Rd., Madison, WI 53711 USA hanging drop and sitting drop cultures use only small