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a USDA-ARS, Crop Germplasm Research Unit, 430 Heep Center, Texas A&M Univ., College Station, TX 77843-2474
b USDA-ARS, Grassland, Soil, and Water Res. Lab., 808 E. Blackland Rd., Temple, TX 76502
c Texas Agric. Exp. Stn., 3507 Hwy. 59E, Beeville, TX 78102
* Corresponding author (byron.burson{at}ars.usda.gov).
ABSTRACT
The switchgrass (Panicum virgatum L.) germplasm line TEM-LoDorm (Reg. No. GP-98, PI 636468) was developed by the USDA-ARS in cooperation with the Texas Agricultural Experiment Station and was released in May 2007. TEM-LoDorm has reduced post-harvest seed dormancy and was developed to provide breeders with a germplasm source to improve germination and stand establishment. The cultivar Alamo was used as a base population, and TEM-LoDorm was developed using four cycles of recurrent selection. During the first three cycles, recently harvested seed from about 200 entries were germinated to evaluate immediacy of germination. Seedlings from seed that germinated within 3 to 14 d were used to establish a polycross nursery to produce seed for the next cycle of selection. Twenty-four plants in the cycle 3 crossing block that produced seed with the most rapid germination rate were dug and used to establish cycle 4 crossing blocks. Over a 2-yr period, germination of recently harvested seed from most of these 24 plants was significantly (P < 0.05) higher than seed from unselected Alamo; some plants were 10 or more times higher than Alamo. Equal quantities of seed from these 24 plants were bulked to constitute TEM-LoDorm germplasm.
Abbreviations: PPFD, photosynthetic photon flux density
Switchgrass (Panicum virgatum L.) is a warm-season, perennial bunchgrass that is native to North America. Historically, it has been used as a forage and conservation grass, but more recently, the grass has received considerable attention as a bioenergy crop for the production of cellulosic ethanol (Vogel, 2004). Because of its potential as a renewable biofuels crop, interest in the grass is increasing and the land area being planted to switchgrass is expanding. However, establishment of desirable stands can be a problem because post-harvest seed dormancy causes low germination and slow seedling development (Beckman et al., 1993; Zarnstorff et al., 1994). Post-harvest dormancy in recently harvested seed of some switchgrass cultivars can be as high as 95%, and the seed can require up to 2 yr of after-ripening to become germinable (Shen et al., 2001). This lengthy after-ripening period can be reduced by cold stratifying the seed (Zarnstorff et al., 1994; Shen et al., 2001) or treating the seed with different chemicals or growth regulators (Haynes et al., 1997; Tischler et al., 1994; Zarnstorff et al., 1994). Stratification is the most practical approach to break dormancy because it can be accomplished simply by planting the seed, provided the soil environmental conditions meet the stratification requirements. However, this also can be problematic. If switchgrass seed are planted into moist, cool soil in the spring, several weeks of stratification are required before germination is initiated, but uncontrollable environmental events can negatively impact stratification and subsequent germination. If seed are planted into a dry soil, for example, stratification is initiated only after a rainfall event, and the soil temperature must be low enough to chill the seed before germination. If it does not rain until late spring or early summer, the soil temperatures can be too high for stratification to occur, and germination is delayed until the temperature and moisture conditions are favorable, which may not occur until the following spring.
Even if the conditions for stratification are met and the seed germinate, seedlings emerging in late spring or early summer face the prospect of unreliable rainfall, which also can negatively impact stand establishment. An approach that can circumvent these uncontrollable environmental events is to artificially stratify the seed before planting, but this also can be problematic. If the seed are artificially stratified but the weather or soil conditions are not conducive to planting, the seed must either be placed in cold storage or quickly dried to prevent sprouting. Unfortunately, drying stratified seed can cause some of them to revert back to a dormant state (Shen et al., 2001).
An approach that would improve germination and stand establishment and circumvent the potential problems associated with cold stratification is to plant seed with reduced post-harvest dormancy. If switchgrass seed with low post-harvest dormancy existed, it would be possible to establish desirable stands when the environmental conditions are conducive for germination and seedling growth. This even could be accomplished using recently harvested seed. The objective of this research was to develop switchgrass germplasm with reduced post-harvest seed dormancy.
Materials and Methods
TEM-LoDorm (Reg. No. GP-98, PI 636468) was developed by the USDA-ARS in cooperation with the Texas Agricultural Experiment Station and was released in May 2007. It was developed from ‘Alamo’ switchgrass, a cultivar that has the greatest potential as a biofuel crop of all available switchgrass cultivars adapted to the southern United States (Sanderson et al., 1996). Alamo is a polymorphic, tetraploid (2n = 4x = 36), lowland ecotype that was collected by the USDA Soil Conservation Service (currently the USDA-NRCS) in 1964 from a native population growing near George West, TX, and was released in 1978 by the USDA Soil Conservation Service and Texas Agricultural Experiment Station (currently Texas AgriLife Research) (Alderson and Sharp, 1994).
Because switchgrass is highly self-sterile and is wind pollinated, TEM-LoDorm was developed using recurrent selection techniques. Four cycles of selection were utilized to develop this germplasm. During the first three cycles, fresh seed were harvested, bulked, threshed, and placed in a germinator within 1 or 2 wk after collection. These seed were evaluated for immediacy of germination. For each cycle, the seed were placed on moist germination blotters in petri dishes, and these were put into a germinator that was maintained at 12 h of light (photosynthetic photon flux density [PPFD] 5 µmol m–2 s–1) at 35°C and 12 h of darkness at 20°C.
During the first cycle of selection, 150 seedlings from the seed that germinated within 14 d were selected and saved. The early germinating seedlings were transplanted into a commercial soil mix in 3-cm by 4-cm by 6-cm compartments in plastic flats and were eventually transplanted into the field to establish a crossing block to produce seed for the next cycle of selection. A more stringent selection pressure was imposed on the seedlings used to establish the crossing blocks for cycles 2 and 3. Only those seedlings that germinated within 7 d were selected for the second cycle, and by the third cycle, only seed exhibiting outward signs of germination within 3 d after being placed in the germinator were selected. One hundred and fifty and 163 seedlings were selected and used to establish the cycle 2 and cycle 3 polycross nurseries, respectively. For the fourth cycle of selection, seed were harvested from 131 of the 163 plants in the cycle 3 crossing block and seed from each individual plant were kept separate. One hundred seed from each of the 131 seed lots were germinated immediately after harvest, and the 24 plants that produced seed with the highest and most rapid germination were identified. Vegetative material of these 24 plants was dug from the cycle 3 polycross nursery and each plant was cloned into eight ramets. In April 2001, four ramets of each plant were transplanted into a crossing block near Temple, TX, and the other four ramets were used to establish a crossing block near College Station, TX. At both locations, each block was planted in a randomized complete block design with four replications, and these were used as the cycle 4 crossing blocks. Open-pollinated seed were harvested from all flowering plants in both nurseries in autumn 2001 and 2002. A small block, consisting of 50 unselected Alamo plants, also was established at Temple and College Station, TX, in spring 2001 to provide seed to be used as a control. At each location, both crossing blocks (cycle 4 and unselected Alamo) were planted on the same soil type (Temple: Houston Black clay [fine, smectitic, thermic Udic Haplusterts]; College Station: Ships clay [very-fine, mixed, active, thermic Chromic Hapluderts]) but at a sufficient distance from one another to prevent cross-pollination. Seed were harvested from each of the 96 (intermated) plants in the cycle 4 crossing block and were bulked by entry number. All seed harvested from the unselected Alamo plants were bulked together at each location.
To measure progress in reducing post-harvest dormancy, we compared the rate of germination of recently harvested bulked seed from each of the 24 selected clones to recently harvested seed from unselected Alamo plants produced at both locations over two seed production years. Because of variation in flowering date of the plants in the cycle 4 crossing block, seed from all 24 entries were not always available at the same time. Since we were interested in determining the germination potential of recently harvested seed, percentage germination was determined within 1 or 2 wk following seed harvest, rather than waiting until seed were available from all entries. Consequently, adequate seed for testing were collected from only 13 of the entries at Temple and College Station in 2001 (Table 1 ), but sufficient seed were collected from five additional entries in 2002 (Table 2 ). Seed also were collected from the remaining six entries but not in sufficient quantity to meet the replication criteria of this experiment.
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The seed collected at Temple in 2001 were divided into three lots and used to determine the effect of different storage conditions on dormancy and germination. One lot was germinated immediately after collection, and the other two lots were stored under different conditions. One lot was placed in a cold-storage vault (15°C) for 6 mo, and the other lot was stored in a freezer (–20°C) for 6 mo. Seed collected at College Station in 2002 were divided into two lots. One lot was germinated immediately after collection. The other seed lot was stored in a freezer at –20°C for 6 mo before it was germinated. Data from these experiments provide insight as to how different storage conditions affect seed dormancy and also provide additional information on the progress that was made in reducing dormancy.
All data were subjected to analysis of variance using PROC GLM procedures (SAS Institute, 1999). Mean separations were made on the basis of Duncan's multiple range test at the 0.05 probability level. All data were transformed for analysis using the arcsin square-root transformation to achieve homogeneity of variances; however, the actual percentage germination means are shown in the data tables. Pearson's correlation coefficient was calculated to determine the relationship between germination at alternating and constant temperatures.
Results and Discussion
Seed Germination
The germplasm line TEM-LoDorm is unique because it has reduced post-harvest seed dormancy in recently harvested seed to improve germination and stand establishment of switchgrass.
Fresh seed from 13 of the 24 genotypes in TEM-LoDorm and unselected Alamo that were harvested in autumn 2001 at Temple and College Station, TX, were divided into two lots and germinated at alternating (35°C/20°C) and constant (30°C) temperatures (Table 1). The percentage germination for all 13 genotypes was significantly higher than unselected Alamo regardless of which germination regime was used or where the seed were produced (Table 1). These data clearly show the progress made in reducing post-harvest dormancy in these 13 genotypes. Germination of the Temple grown seed at alternating temperatures was 90% or higher for 7 of the 13 entries and was 80% or higher for all 13 entries, whereas germination averaged only 28% for the unselected Alamo seed (Table 1). These are very high germination percentages for switchgrass, and these data show that significant progress was made in reducing post-harvest dormancy in these entries. When the seed produced at College Station were germinated at alternating temperatures, the percentage germination for all 13 entries and unselected Alamo was lower than the seed produced at Temple, but the germination of all entries was significantly higher than Alamo (Table 1). When seed produced at both locations were germinated at a constant temperature (30°C), the percentage germination was lower than when germinated at alternating temperatures, except for entry 130 seed produced at College Station (Table 1). However, germination of all the entries at a constant temperature, regardless of location, also was significantly higher than Alamo (Table 1). The relationship between germination at constant versus alternating temperatures differed between locations but was higher for alternating temperatures, which is characteristic of panicoid grasses (Hyder et al., 1971). The Pearson correlation coefficient for germination at the two temperature regimes was 0.2314 (P = 0.4468) for the Temple seed and 0.9129 (P < 0.0001) for the College Station seed. Although these experiments were not designed to specifically address this issue, the data suggest a strong genotype x location x temperature interaction influencing germination.
The germination pattern of the recently harvested seed produced at Temple during autumn 2002 (Table 2) was similar to that observed for the 2001 seed (Table 1) in that the percentage germination for all entries was significantly higher than unselected Alamo. Data in Table 2 include the percentage germination of five entries—3, 18, 128, 142, and 161—that were not included in Table 1 and demonstrate that these additional genotypes have less post-harvest seed dormancy than Alamo. However, the percentage germination for essentially all entries of the 2002 seed (Table 2) was lower than that observed for the 2001 seed (Table 1). The 2002 summer was much drier than the 2001 summer, and although the crossing blocks received supplemental irrigation on two occasions, the lower germination could be the result of adverse environmental conditions during pollination and seed development.
Combined data in Tables 1 and 2 demonstrate that 18 of the 24 genotypes in TEM-LoDorm switchgrass have less post-harvest dormancy than Alamo. Seed were collected from the remaining six entries but not in sufficient quantity to meet the replication criteria of this experiment. However, seed from these six entries were germinated, and all had less post-harvest dormancy than Alamo (data not shown).
Effect of Cold Storage on Germination
The 2001 Temple seed (11 of the original 13 entries) stored for 6 mo at 15°C and –20°C were germinated at a constant temperature (30°C); the results are presented in Table 3
. Not enough seed of entries 35 and 145 were available to include them in this study. Analysis of variance indicated significant (P < 0.001) entry, storage condition, and entry x storage condition effects. Germination generally was similar for cold room–stored (15°C) and fresh seed, except it was significantly higher for stored seed of entries 30, 40, and 60 than fresh seed, while for entry 93, germination of cold room–stored seed was significantly lower than that of fresh seed. Mean values over all entries for fresh and cold room–stored seed were not significantly different (51.1 vs. 54.6%, respectively). A different pattern was observed for the frozen seed (–20°C), with means over all entries being 17.6%, significantly lower than the other two categories. Germination of frozen seed was never higher than either fresh seed or seed stored at 15°C and in most cases was lower (Table 3). More important, these data demonstrate that whether stored at 15°C or –20°C, germination of these 11 entries was significantly higher than Alamo. The only exception was entry 93 seed stored at –20°C.
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Phenotype and Winter Hardiness
TEM-LoDorm plants are polymorphic and appear similar to Alamo switchgrass. They essentially are indistinguishable from one another. TEM-LoDorm germplasm was grown at Temple, TX, for six winters, and no winter damage was observed. Because Alamo is adapted to plant hardiness zone 6, TEM-LoDorm should have sufficient cold tolerance to grow as far north as this hardiness zone. Seed production for this release is similar to that of Alamo.
Availability
Seed of TEM-LoDorm has been deposited in the National Plant Germplasm System, where small quantities are available for research purposes, including the development and commercialization of new cultivars. Vegetative material of the 24 genotypes that make up TEM-LoDorm will be maintained by the USDA-ARS, Southern Plains Agricultural Research Center, Crop Germplasm Research Unit, at College Station, TX, and will be used to produce additional seed of this germplasm line. Small quantities of seed from this seed increase block also can be obtained from the corresponding author. Appropriate recognition is requested if this germplasm is used in the development of a new breeding line or cultivar.
Footnotes
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication July 25, 2008.
References
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