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a Panhandle Research and Extension Center, 4502 Avenue I, Scottsbluff, NE 69361
b Dept. of Agronomy, University of Nebraska-Lincoln, Lincoln, NE 68583-0915
* Corresponding author (dbaltensperger1{at}unl.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Several techniques have been developed for artificial hybridization of small grain crops (Fehr and Hadley, 1980). Some of these techniques have been successfully used in oat (Avena sativa L.) (McDaniel et al., 1967), barley (Hordeum vulgare L.), wheat (Triticum aestivum L.) (Wells and Caffey, 1956), proso millet (Panicum miliaceum L.) (Nelson, 1984), and sorghum (Sorghum bicolor L.) (Schertz and Clark, 1967), but each has limitations imposed by the wide diversity of floral structure and development among small grain crops and environmental influences.
Floral morphology and anthesis behavior make foxtail millet [Setaria italica (L.) Beauv.] one of the most difficult species to cross pollinate (Baltensperger, 1996). Foxtail millet is largely self-pollinated, with cross pollination averaging about 4% (Li et al., 1935). The inflorescence is a spike with short side branches bearing spikelets and bristles. Each spikelet consists of a pair of glumes that embrace two minute flowers (about 1 mm in length); the lower one sterile and the upper one bisexual, with three stamens and a long oval smooth ovary with two long styles, that terminate in a brush like stigma (Hector, 1936). From one to three bristles develop at the base of each spikelet (Vinall, 1924).
Anthesis in foxtail millet generally takes place near midnight and in the morning, but varies significantly with the environment (Malm and Rachie, 1971). The rate of anthesis is generally favored by low temperature and high humidity (Rangaswami Ayyangar et al., 1933; Heh et al., 1937). Similar to the time of daily anthesis, the process of anthesis in a single flower varies considerably with the environment. The time required for the opening and closing of a single flower was as long as 2.5 h in Korea (Malm and Rachie, 1971), or as short as 70 min in Hunan, China (Li et al., 1935). The difference in time has been attributed, mainly, to variations in temperature and humidity.
Because of the minuteness of flowers, delicate process of anthesis and its variation with the environment, and the time when anthesis occurs, hybridization of foxtail millet is very difficult and it has been difficult to adapt other small grain breeding techniques to the crop. Li et al. (1935) outlined a hand crossing technique for foxtail millet, but there is no evidence of its efficiency. Li et al. (1935) tested the use of hot water emasculation, as described by Stephens and Quinby (1933), but there was insufficient evidence to determine the success of the method.
The difficulties of making crosses and the lack of an efficient crossing technique have resulted in a limited number of genetic studies and, consequently, little or no genetic improvement of foxtail millet. Therefore, development of an efficient crossing technique is essential to achieve genetic and breeding objectives in foxtail millet.
The objectives of this study were to acquire knowledge of the anthesis habits of foxtail millet under Nebraska glasshouse conditions, and to develop a technique for artificial hybridization of foxtail millet.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Establishment of Plant Nursery
The seven cultivars were grown under greenhouse conditions at the University of Nebraska-Lincoln during the summer of 1994. The greenhouse was set to minimum and maximum temperatures of 18 and 29°C, respectively and 14-hour day length. Under these conditions, 10 plants per cultivar were grown in two 15-cm diameter pots containing volumetric 1:1:1:1 soil, vermiculite, perlite, and peatmoss mixture. Because the cultivars vary significantly in maturity, each was planted every three days for about 4 wk to synchronize anthesis among the cultivars. Water and nutrients (20-10-20, N-P-K) were provided as a solution twice a day, in the morning and in the evening.
Observations of Process of Anthesis
Knowledge of anthesis habits of this crop under given conditions was necessary before developing a successful crossing technique. At least five plants from each of five cultivars, PI 458628, PI 531445, Red Siberian, NESE 62, and Golden German, were observed for time required from heading to anthesis, order of anthesis on each spike and its side branches, number of flowers opened each day, daily anthesis periods, and duration between opening and closing of a single flower. The number of flowers opened each day was recorded only for the first four days. At the same time (about 0800 h) each day, the flowers were counted and then removed with a pair of fine forceps, so that there was no confusion with flowers opened the previous day.
In addition to observations made on anthesis habits, hourly temperature and relative humidity were recorded and then associated with anthesis processes, especially with daily anthesis periods and duration from opening to closing of a single flower. Two pots of five plants each from each of two cultivars, PI 458628 and Red Siberian, were moved into a growth chamber at a constant, temperature (21°C) and humidity (78%), when anthesis occurred under greenhouse conditions. After, approximately one hour, one pot from each cultivar was moved to normal greenhouse conditions and the other pot was kept in the growth chamber.
Equipment for Artificial Hand Hybridization
The equipment needed included a pair of scissors and a fine pair of forceps that were eight to 10 cm in length, paper clips, glassine bags (5 by 17 cm), fine point black or blue permanent markers, paper tags (20 by 25 mm) for identifying the cross, and magnifying glasses. The points of the forceps may be straight or curved depending on the user's preferences, but they should fit closely together and be smoothly blunted so as not to pierce the diminutive flower.
Crossing Technique
The technique described is based on some basic ideas outlined by Li et al. (1935) and Heh et al. (1937). On the second day of anthesis, the female parent was isolated from other plants and the flowers, which open on the first day, were removed with a fine pair of forceps. The preferable period to do this is in the morning between 0800 and 0900 h, because no anthesis occurs between 0800 and 1800 h. As the anthesis period approaches, the bristles of the female and the male parents are excised gently with a pair of scissors.
After the bristles were excised, emasculation was done when the first anther had just emerged and before the pollen sacs had burst. With or without the help of magnifying glasses, depending on the worker, the three anthers were carefully removed with a fine pair of forceps. Sometimes the flowers were not wholly opened, especially those of round shape (PI 458628, NESE 62, and Golden German); consequently, the anthers were not completely pushed out either. Generally, they remain adhered in the incurved edges of the palea. Under these conditions, the anthers were removed by gently inserting the forceps at each side of the palea and pushing the anther out quickly. Each emasculated flower was then immediately marked with a fine point black or blue permanent marker so that the emasculated flower was identified. After emasculation was accomplished, all unmarked flowers were removed and the head was covered with a glassine bag.
Pollination was immediately done by positioning the emasculated female spike slightly below the male spike, that was shedding pollen, and covering both spikes with a glassine bag. Pollen from the male spike showers down on the female spike affording good opportunity for fertilization. The spikes were shaken together for 2 d during the daily anthesis periods. This process of shaking provided opportunity for fertilization because the stigmas of foxtail millet remain outside the glumes and are receptive for about 48 hr. On the third day of pollination, the male spike was carefully removed and the female one was checked for any floret that may have developed later. Failure to remove such florets completely, often allowed them to develop and produce seed, which may be confused with the cross-fertilized ones. The female spike was rebagged and maintained until harvested at maturity.
Evaluation of the Crossing Technique
The crossing technique was evaluated at three levels. First, 130 flowers from two cultivars, PI 531445 and NESE 62, were emasculated and bagged without pollination, and the seed set was determined. Second, more than 6400 flowers among the seven cultivars were emasculated and pollinated in a 7 by 7 half diallel arrangement such that at least 10 spikes were pollinated from each of 21 cross-combinations. The percent seed set was then determined. Finally, random samples of 30 plants from each of 21 cross-combinations were grown under greenhouse conditions during the winter of 1994–1995 and the percentages of true hybrid plants were determined. The true hybrid plants were identified based on the six simply inherited traits described previously.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The process of opening of flowers in foxtail millet was of particular interest in connection with the crossing technique described. As the glumes began to spread, the stigmas and the anthers developed and pushed out the slit between the incurved edges of the palea. The feather like stigmas were first to emerge, but were quickly followed and overtaken by the anthers. Sometimes, some anthers remained adhered in the curved edges of the palea. This pattern was generally associated with round shaped flowers or deficiency in soil moisture. In general, however, the anthers shed pollen after they were fully extruded outside the glumes. After dehiscence, the glumes began to close up, leaving the shriveled anthers and the tip of the stigmas outside.
Anthesis in foxtail millet takes place at specific periods during the day and the periods are cultivar dependent. In Nebraska during the summer, anthesis occurred between 0100 and 0300 h and 2100 and 2300 h, for cultivars with elliptical seeds, and between 0600 and 0700 h and 1800 and 1900 h, for cultivars with round seeds. Outside these periods, very few flowers opened and practically no anthesis occurred between 0800 and 1800 h. However, as the temperature started declining by the end of August, the anthesis periods started to become longer, especially in the morning. This anthesis behavior, in addition to those reported in previous studies (Rangaswami Ayyangar et al., 1933; Li et al., 1935; Heh et al., 1937) suggests that anthesis in foxtail millet is associated not only with specific periods during the day, but also with specific locations and seasons.
The daily anthesis periods were associated with rapid changes in temperature and humidity (Fig. 1), and not with low temperature and high humidity as was concluded in previous studies (Rangaswami Ayyangar et al., 1933; Li et al., 1935). This observation was confirmed when 10 plants at anthesis stage from each of two cultivars, PI 458628 and Red Siberian, were moved into a growth chamber at a constant temperature and humidity (21°C and 78%, respectively) as anthesis occurred in the greenhouse. The flowers never opened under those conditions. However, opening occurred when half of the plants were moved into the greenhouse where the temperature was about 30°C and relative humidity about 50%.
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The rate at which flowers opened per day increased in an exponential manner during the first four days and varied with cultivars (Table 2). The largest number of flowers opened was observed in cultivar NESE 62 that had the largest spike. Similarly, the smallest number of flowers opened was recorded in cultivar PI 531445 that had the smallest spike. This suggests that in cultivars with small spikes, the number of flowers opened would be smaller than those in cultivars with large spikes, although the number of florets per spike may also influence the total number of flowers opened each day.
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The average percentage of seed set among the 21 cross-combinations ranged from 58 to 90% and averaged about 75% (Table 3). The lowest percentages were recorded in all the crosses with Golden German as male parent. The low percentages of seed set in these crosses may be associated with the amount and, probably, the viability of pollen. Although there are no records on the amount and viability of pollen of the seven cultivars used in this study, the anthers and the quantity of pollen in Golden German were smaller than those in the other cultivars. In addition, the anthers in this cultivar generally remained adhered to the curved edges of the palea. Therefore, the availability of pollen may have been limited and may have contributed to the low percentage of seed set.
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The percentages of true hybrid plants identified among 30 F1 plants from each of 21 cross-combinations (Table 4) show that self-fertilization does occur and did vary between zero and 17%. Mostly, however, less than 10% self-fertilization was observed. In fact, more than 92% of the plants among the 21 cross-combinations resulted in true hybrids. These results, in addition to low percentage of seed set on non-pollinated checks and about 75% of seed set among the 21 cross-combinations, clearly prove the effectiveness of the hybridization technique.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication August 22, 2000.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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  M. M. Siles, W. K. Russell, D. D. Baltensperger, L. A. Nelson, B. Johnson, L. D. Van Vleck, S. G. Jensen, and G. Hein Heterosis for Grain Yield and Other Agronomic Traits in Foxtail Millet Crop Sci., November 1, 2004; 44(6): 1960 - 1965. [Abstract] [Full Text] [PDF]
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