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a USDA-ARS, Grain, Forage, and Bioenergy Research, 314 Biochemistry, Univ. of Nebraska-Lincoln, Lincoln, NE 68583-0737
b Dep. of Physics, Oklahoma State Univ., Stillwater, OK 74078
c W.H. Miner Agricultural Research Institute, Chazy, NY 12921. Joint contribution of the USDA-ARS and the University of Nebraska Agricultural Experiment Station
* Corresponding author (jeff.pedersen{at}ars.usda.gov).
ABSTRACT
Four sorghum [Sorghum bicolor (L.) Moench] genetic stocks, BN611 (Reg. No. GS-140, PI 652943), AN612 (Reg. No. GS-141, PI 652944), BN612 (Reg. No. GS-142, PI 652945), and RN613 (Reg. No. GS-143, PI 652946), with stacked brown midrib genes bmr-6 and bmr-12 were developed jointly by the USDA-ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and were released in July 2006. The genetic stocks BN611, AN612 and BN612, and RN613 were tested as Atlas bmr-6 bmr-12, Wheatland bmr-6 bmr-12, and RTx430 bmr-6 bmr-12, respectively. Release of these genetic stocks makes stacked brown midrib genes, reported to reduce the activity of two enzymes important in lignin synthesis, cinnamyl alcohol dehydrogenase (bmr-6) and caffeic acid O-methyl transferase (bmr-12), available in a common forage sorghum, a common grain sorghum seed parent, and a common grain sorghum pollen parent background. The genetic stocks have immediate application for basic research involving lignin synthesis.
Abbreviations: ADF, acid detergent fiber • ADL, acid detergent lignin • CAD, cinnamyl alcohol dehydrogenase • COMT, caffeic acid O-methyl transferase • NDF, neutral detergent fiber
Four sorghum [Sorghum bicolor (L.) Moench] genetic stocks, BN611 (Reg. No. GS-140, PI 652943), AN612 (Reg. No. GS-141, PI 652944), BN612 (Reg. No. GS-142, PI 652945), and RN613 (Reg. No. GS-143, PI 652946), with stacked brown midrib genes bmr-6 and bmr-12 were developed jointly by the USDA-ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and were released in July 2006. The genetic stocks BN611, AN612 and BN612, and RN613 were tested as Atlas bmr-6 bmr-12, Wheatland bmr-6 bmr-12, and RTx430 bmr-6 bmr-12, respectively.
An important source of fodder for feed, sorghum is being targeted for use as a bioenergy feedstock and is becoming increasingly important as a model species for basic research. Some qualities that make sorghum ideal for use as a model species are its C4 metabolism, ability to be grown as an annual, and its relatively small, diploid genome (Sarath et al., 2008). The most successful approach to genetic modification of sorghum cell wall chemistry for feed and bioenergy applications has been through incorporation of brown midrib genes, which are associated with reduced lignin and altered lignin chemical composition (Bucholtz et al., 1980; Porter et al., 1978).
Sorghum has at least three different brown midrib loci (Bittinger et al., 1981). Nineteen independently occurring brown midrib mutants were originally described (Porter et al., 1978). Three of those 19 mutants, bmr-6, bmr-12, and bmr-18, were selected by Porter et al. (1978) for further evaluation and have subsequently been more thoroughly characterized. The mutant bmr-6 reduced activity of cinnamyl alcohol dehydrogenase (CAD) (Bucholtz et al., 1980), and the allelic (Bittinger et al., 1981) bmr-12 and bmr-18 genes reduced caffeic acid O-methyl transferase (COMT) activity (Bout and Vermerris, 2003). Our laboratory previously developed and released sets of sorghum lines near-isogenic for bmr-6 and bmr-12 (Pedersen et al., 2006a,b,c) and used them to investigate effects of these individual genes on morphology, cell wall chemistry, and yield in multiple genetic backgrounds (Oliver et al., 2005a,b). This work demonstrated that genetic background had a significant effect on the phenotypic properties of both bmr genes.
Because the bmr-6 and bmr-12 genes affect different enzymes in lignin synthesis, it is reasonable to hypothesize that their combined effect could result in even greater reduction of lignin content and in greater changes in other cell wall–related traits. However, the genetic resources were unavailable to test this hypothesis. We therefore undertook the development of lines with stacked bmr-6 and bmr-12 genes near-isogenic to a subset of our previously released lines near-isogenic for the individual bmr-6 and bmr-12 genes.
The pedigrees of the resultant lines BN611, BN612, and RN613 are 00-2051 (a sister line of N598, PI 639702, synonym = Atlas bmr-6, Pedersen et al., 2006b)/00-2172 (a sister-line of ‘Atlas bmr-12’, PI 636763, Pedersen et al., 2006a); 00-1754 (a sister-line of N599, PI 639709, synonym = Wheatland bmr-6, Pedersen et al., 2006c)/00-1644 (a sister-line of N600, PI 639710, synonym = Wheatland bmr-12, Pedersen et al., 2006c); and 00-1677 (a male-sterile (ms3ms3) sister-line of N609, PI 639719, synonym = RTx430 bmr-6, Pedersen et al., 2006c)/00-2161 (a sister-line of N610, PI 639720, synonym = RTx430 bmr-12, Pedersen et al., 2006c), respectively.
Release of BN611, AN612, BN612, and RN613 makes stacked brown midrib genes reported to reduce the activity of two enzymes important in lignin synthesis, CAD (bmr-6) and COMT (bmr-12), available in a common forage sorghum, a common grain sorghum seed parent, and a common grain sorghum pollen parent background.
Methods
Development of BN611, AN612, BN612, and RN613
The genetic stocks BN611, AN612, BN612, and RN613 were developed using the pedigree method. They are F3 or F4 selections from crosses between sister lines of N598 and ‘Atlas bmr-12’, N599 and N600, and a male-sterile (ms3ms3) sister-line N609 and N610, respectively. Crossing between the RTx430 (Miller, 1984) derivatives was facilitated by the use of the nuclear male-sterility gene ms3. Hand emasculations were used to make the crosses between the ‘Atlas’ (NSL 3986; Brown et al., 1936) and ‘Wheatland’ (CIso 918; Brown et al., 1936) derivatives. Selection of individuals homozygous for both bmr-6 and bmr-12 was accomplished in the F3 generation for the Atlas derivative, and in the in F4 generation for the Wheatland and RTx430 derivatives by crossing individual plants to both AN599 (AWheatland bmr-6) and AN600 (AWheatland bmr-12) and selecting those that produced only the brown midrib phenotype in the resulting progeny. BN612 (Wheatland bmr-6 bmr-12) was male-sterilized in A1 cytoplasm by crossing to AN599 (AWheatland bmr-6) followed by four backcrosses to BN612 with homozygosity for bmr-12 selected for using progeny testing for the brown midrib trait as above.
Confirmation of Fertility Restoration and Presence of bmr-6 and bmr-12 Genes
The following test crosses were made to confirm fertility restoration and stability of the bmr genes when stacked: AN599/BN611, AN599/BN612, AN599/RN613, AN600/BN611, AN600/BN612, AN600/RN613, AN612/BN599, AN612/BN600, and AN612/BWheatland. Approximately 30 F1 seedlings from each cross were visually examined to determine midrib color in the greenhouse. Six of these F1 progeny were grown to maturity with inflorescences protected with pollinating bags and examined for presence or absence of seed set.
Field Trials
Field trials comparing BN611, BN612, and RN613 with their wild-type progenitors (Atlas, Wheatland, and RTx430, respectively) and counterparts near-isogenic for bmr-6 and bmr-12 (Atlas bmr-12 and N598, N599 and N600, N609 and N610, respectively) were conducted at the University of Nebraska Field Laboratories at Ithaca, NE, and at Lincoln, NE.
Atlas derivative trials were planted 20 May 2004 at Ithaca and 18 May 2005 at Lincoln. Plots consisted of three 7.6-m rows spaced 76 cm apart. The experimental design was a randomized complete block with five replications in each environment. Nitrogen fertilizer was applied before planting at both locations at 157 kg ha–1. At Lincoln, 3.36 kg ha–1 propachlor [2-chloro-N-(1-methylethyl)-N-phenylacetamide] and 1.1 kg ha–1 of atrazine [6-chloro-n-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4,diamine] were applied immediately after planting for weed control. No supplemental irrigation was applied. At Ithaca, atrazine was applied at 2.2 kg ha–1 immediately after planting, followed by an application of quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) and atrazine at 0.37 kg ha–1 and 1.1 kg ha–1, respectively, approximately 14 d postemergence. Supplemental irrigation (2.5 cm) was applied at Ithaca via overhead sprinklers on 3, 12, and 19 Aug. 2004. Emergence was determined by counting the number of seedlings in each row approximately 4 wk postplanting. Days to flowering was recorded at 50% anthesis. Height was measured to the top of the mature panicle before harvest. Total biomass yield was determined by harvesting the middle row of each plot using a commercial silage cutter modified for small plot use (Pedersen and Moore, 1995). Grain-free forage samples for laboratory analyses were collected by removing all panicles from one of the outside rows of each plot and harvesting in a similar manner. Subsamples were collected and oven dried (60°C) to determine dry matter for calculation of plot dry matter yields. Ithaca plots were harvested 24 Sept. 2004, and the Lincoln plots were harvested 29 Sept. 2005. All plots were at hard dough or were fully mature.
The Wheatland and RTx430 derivative trials were planted 21 May 2003 at Lincoln and 20 May 2004 at Ithaca. Plots consisted of two 7.6-m rows spaced 76 cm apart. Wheatland and RTx430 derivatives were planted in blocks to minimize border effects. The experimental design was a split plot with lines being whole plots and genotype being subplots, and four replications in each environment. Cultural practices were the same as described above. Panicles were hand-harvested at maturity; residue was then harvested and dried as described above. Panicles were dried to a uniform 14% grain moisture content and subsequently threshed to determine grain yield. Grain and residue were harvested 1 Oct. 2003 at Lincoln and 6 Oct. 2004 at Ithaca.
Laboratory Analyses
Forage and residue samples were prepared for chemical analyses by grinding in a Wiley mill (2-mm screen; Arthur H. Thomas Co., Philadelphia, PA) and were analyzed sequentially for neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) content using an ANKOM 200 fiber analyzer (ANKOM Tech. Corp., Fairport, NY) (Vogel et al., 1999).
Statistical Analyses
The data were analyzed using SAS software (SAS Institute, 2000–2004). Environments and replications were considered random. Least square means were calculated using PROC MIXED, and differences of least square means tested for significance using the PDIFF option of PROC MIXED.
Characteristics
BN611 resembles Atlas with white caryopses color, no tannin-containing testa in the grain, normal and white endosperm, awnless florets, purple necrotic lesion color, and juicy culms, and it does not restore fertility in A1 cytoplasm. Averaged over environments, BN611 was 4 d later in maturity (83 d vs. 79 d) and 64 cm shorter in height (214 cm vs. 278 cm) than Atlas (Table 1 ). Seedling emergence of BN611 was slightly higher than for Atlas. Biomass yield of BN611 was significantly lower (10.5 t ha–1 vs. 17.7 t ha–1) compared with Atlas. Neutral detergent fiber and ADF of BN611 and Atlas were not significantly different. Acid detergent lignin was significantly lower in BN611 (35 g kg–1) compared with N598 (Atlas bmr-6, 45 g kg–1) and Atlas bmr-12 (41 g kg–1), which were both significantly lower in ADL content than wild-type Atlas (54 g kg–1). Given that both single mutants show decreases in ADF compared with wild-type Atlas, the lack of further reduction in ADF content in the double mutant BN611 might at first glance appear surprising. However, this is readily explained by equivalent NDF content of BN611 and Atlas and the larger decrease in ADL in BN611 compared with the decrease observed between N598 and Atlas bmr-12, and wild-type Atlas. Complete comparisons of BN611 with N598 (Atlas bmr-6), Atlas bmr-12, and wild-type Atlas are shown in Table 1.
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When crossed to various bmr-6 or bmr-12 testers, BN611, AN612, BN612, and RN613 had brown midrib progeny indicating that both bmr genes are present and stable in these four genetic stocks (Table 4 ). Fertility restoration in selfed F1 progeny of these same crosses confirm A-, B-, and R- reactions as indicated by the genetic stock prefixes (Table 4).
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Seed of these genetic stocks is maintained and distributed by the USDA-ARS, Grain, Forage, and Bioenergy Research Unit, Department of Agronomy, University of Nebraska, Lincoln, NE 68583-0737, and will be provided without cost to each applicant on written request. Genetic material of this release will be deposited in the National Plant Germplasm System where it will be available for research purposes, including development and commercialization of new cultivars. It is requested that appropriate recognition be made if these genetic stocks contribute to 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 January 29, 2008.
References
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