WO2014210607A1 - Bms1 compositions and methods of use - Google Patents

Abstract

The invention provides for RNAi vectors comprising a fragment of the BMS1 polynucleotide sequence and transgenic plants, e.g. transgenic sorghum plants, comprising said RNAi vectors. The invention also provides for methods of using the RNAi vectors of the invention to silence BMS1 gene expression or activity in a transgenic plant, such as a transgenic sorghum plant.

Description

BMSl COMPOSITIONS AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Folkerts, O. et al, U.S. Provisional Application No. 61/841,249 "BMSl COMPOSITIONS AND METHODS OF USE" filed June 28, 2013, incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides for RNAi vectors comprising a fragment of the BMSl polynucleotide sequence and transgenic plants, e.g. transgenic sorghum plants, comprising said RNAi vectors. The invention also provides for methods of using the RNAi vectors of the invention to silence BMSl gene expression or activity in a transgenic plant, such as a transgenic sorghum plant. The invention also provides for methods related to producing lines of sorghum plants wherein at least one sterile pedicular floret is converted to a fertile floret and seed therefrom.

GOVERNMENT SUPPORT

[0003] Not applicable.

COMPACT DISC FOR SEQUENCE LISTINGS AND TABLES

[0004] Not applicable.

BACKGROUND OF THE INVENTION

[0005] All citations are incorporated herein by reference.

Sorghum

[0006] Sorghum (such as the commercially common Sorghum bicolor) is a tropical grass that can be grouped into three basic types: (i) grain, (ii) forage, and (iii) sweet sorghum (Monk, 1980). Over 22,000 varieties of sorghum exist throughout the world (Jackson and al, 1980). Sorghum-sudangrass hybrids are intermediate in plant size between sorghum and sudangrass. Sorghum is indigenous to Africa. [0007] Sorghum has many advantageous biological characteristics, including a high photosynthetic rate and high drought tolerance. Sorghum can grow under intense light and heat. In addition, sorghum plants have a waxy surface which reduces internal moisture loss and facilitates drought resistance.

[0008] Compared to corn, sorghum suffers harsh environmental conditions successfully, including especially low water and high heat situations (Bennett et al., 1990). However, sorghum grain yields are typically lower than corn, which limits adoption of sorghum cultivation in many corn-growing regions.

Sorghum grain

[0009] Grain sorghum is historically one of the major cereal crops used for human food. This ancient crop (an Assyrian ruin from 700 B.C., for example, contains a carving depicting sorghum) (Bennett et al., 1990); however, its introduction into the US is uncertain, although as a crop it found its way to Europe around 60 A.D. While the first sorghum crop was first grown by a William Prince of New York in 1853, the US Department of Agriculture (USDA) did not release a sorghum seed until 1857, from which time many more sorghum materials have been released and introduced into the US (Bennett et al., 1990). Through the work of breeders, such as Sieglinger, Quinby, and Stephens, shorter types and hybrids were developed, dramatically increasing yields and the success of the crop (Bennett et al., 1990). In 2012, the USDA Foreign Agricultural Service's Production, Supply and Distribution (PSD) online reported 38,805 million hectares of harvested grain sorghum (database accessed May 29, 2013).

Inflorescence biology

[0010] Sorghum grain is produced on a head, the inflorescence, which is a panicle that is anywhere from 3 inches to 20 inches long to 2 to 8 inches wide. The seed branches are borne in whorls or clusters. At each node on an inflorescence branch are two spikelets: a fertile sessile spikelet and an infertile (or occasionally staminate) pedicelled spikelet. At the end of each inflorescence branch is a terminal fertile sessile spikelet and two infertile pedicelled spikelets. The fertile sessile spikelet consists of a short floral axis. Two glumes cover the seed at maturity. The glumes house two florets: an upper one that is fertile, while the lower one is usually sterile (Bennett et al., 1990). Occasionally, the sterile floret of the sessile spikelet will be functional, and even upon rare occasions, a third functional flower may be present (Karper, 1931). Ayyangar and Rao also observed sorghum varieties that had pedicelled florets that set seed: two varieties from East Africa and one from Bihar, India (Ayyangar and Rao, 1935). Karper and Stephens reported that Sieglinger had reported to them during his development of grain sorghum hybrids that he had observed instances of seed-bearing pedicelled spikelets, especially apparent in feterita (Sorghum bicolor x Sudan hybrid (Beclin et al., 2002)). After the fertile spikelets mature, the sterile spikelets fall off (Bennett et al., 1990).

[0011] The molecular basis for the development of fertile pedicelled spikelets and pedicellate seed is not understood.

[0012] Ancient barley has similar inflorescence biology to sorghum. Barley has three single-flowered spikelets at each node of the rachis, with three spikelets produced alternately on opposite sides (Forster et al., 2007). In Hordeum vulgare ssp. spontaneum, the wild progenitor of cultivated barley, the outer two lateral spikelets at each branch node are sterile and the spike produces two rows of seeds.

However, during the domestication of cultivated barley (Hordeum vulgare ssp. vulgare), six-rowed spikes evolved, where all three spikelets at a branch node are fertile and the spike thus appears to have six rows of grains (Ramsay et al., 2011).

[0013] The barley six-row phenotype is controlled by VRSl, a homeodomain-leucine zipper (HD-ZIP) Class 1 homeobox gene on chromosome 2H (Komatsuda et al., 2007). This gene is also associated with reducing the number of tillers in barley (Kirby and Riggs, 1978; Lundqvist et al., 1997). Map-based cloning of VRSl has identified various vrsl alleles as loss-of-function mutations of this H D-ZIP transcription factor. HvHox2 on the short arm of barley chromosome 2H appears to be the ancestral gene from which VRSl (also known as HvHoxl ) was derived and transposed to the long arm of chromosome 2H. Comparative mapping appears to confirm this hypothesis, with both local gene and marker colinearity and presence of HvHox2 homologs in syntenic chromosomal regions of rice, Brachypoodium, maize, and wheat. However, while there is a high degree of marker and gene colinearity between the short arm of barley chromosome 2H and syntenic regions of these species, there is no corresponding VRSl homolog present at this interval in any of the non-barley grasses. This is consistent with the hypothesis that the gene duplication and transposition of VRSl is an event that occurred after the divergence of barley from other grass species. While the wild-type Vrsl.b allele encodes a transcriptional repressor that inhibits the development of fertile lateral spikelets (the two- rowed phenotype), loss of function alleles result in the six-row phenotype (Komatsuda et al., 2007). However, the Vrsl.b allele phenotype can be modified by INTERMEDIUM (INT) genes (Lundqvist and Lundqvist, 1988). When these modifiers are homozygous, a partial or complete six-row phenotype is observed (Ramsay et al., 2011).

[0014] Recently, stable and heritable sorghum mutants are produced in which the development arrest of the pedicellate spikelets is released has been reported (US20140068798, "Multi-Seed Mutant of Sorghum for Increasing Grain Yield"). In these mutants, all spikelets, both sessile and pedicellate, develop into flowers and produce mature seeds, thereby significantly increasing seed production and yield in comparison to wild-type sorghum.

SUMMARY OF THE INVENTION

[0015] In one aspect, the invention provides for methods of converting at least one sterile pedicular floret to a fertile floret in sorghum, the methods comprising introducing a polynucleotide construct into a sorghum plant cell that upon activation or expression, converts at least one sterile pedicular floret to a fertile floret in a sorghum plant comprising the transgenic sorghum plant cell. In this aspect, the construct can decrease the level of BMS1 expression in a sorghum plant comprising the transgenic sorghum plant cell compared to the level of BMS1 expression in a control, non-transgenic sorghum plant. Reducing BMS1 expression can comprise reducing the level of an mRNA in the sorghum plant comprising the transgenic sorghum plant cell, wherein the mRNA is encoded by a polynucleotide having at least 70% sequence identity to a nucleic acid sequence of SEQ ID NO:4, and by expression of an RNAi vector comprising a fragment of at least 20 contiguous nucleotides of a sequence having at least 90% sequence identity to SEQ ID NO:4. The RNAi vector can comprise a polynucleotide having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:6-8. The methods can further comprise the step of screening the sorghum plants comprising the transgenic sorghum plant cell for a reduction of BMS1 expression by comparing the BMS1 expression in the transgenic plant to a control, non-transgenic sorghum plant.

[0016] The invention provides for an RNAi vector comprising a BMS1 polynucleotide, BMS1 sequence variant polynucleotide, a fragment of at least 20 contiguous nucleotides of a BMS1 polynucleotide or a fragment of at least 20 contiguous nucleotides of a SMSlsequence variant polynucleotide. These RNAi vectors will facilitate silencing of the BMS1 gene in transgenic plant cells and in transgenic plants which are transformed with the RNAi vectors of the invention. For example, silencing of the BMS1 gene is accomplished by reducing the level of BMS1 mRNA transcript in the transgenic plant or transgenic plant cell through expression of the RNAi vector in said plant or plant cell.

[0017] The RNAi vectors of the invention comprise a polynucleotide having at least 70%, sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs:4, 6, 7, and 8. In addition, the invention provides for RNAi vectors comprising at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs:4, 6, 7, and 8. The invention also provides for RNAi vectors comprising a polynucleotide having a nucleic acid sequence of SEQ ID NO: 4, 6, 7 or 8 or a fragment thereof which is at least 20 contiguous nucleotides. The RNAi vectors may comprise a polynucleotide having a nucleic acid sequence that is a fragment of at least 25, 30, 35, 40, 45, 50, 75, 100, 110, 120, 140, 150, 160, 170 , 180, 190, 200, 204, 220, 250, 300, 350, 359, 360, 367, 400 or 500 contiguous nucleotides of SEQ ID NO: 4, 6, 7 or 8. The RNAi vectors may comprise a polynucleotide having a nucleic acid sequence that is a fragment of at least 25, 30, 35, 40, 45, 50, 75, 100, 110, 120, 140, 150, 160, 170 , 180, 190, 200, 204, 220, 250, 300, 350, 359, 360, 367, 400 or 500 contiguous nucleotides of a BMSl polynucleotide sequence variant such as a nucleotide sequence that is at least 70%, 90% or 95% identical to SEQ ID NO: 4, 6, 7 or 8.

[0018] The invention also provides for plant cells comprising any of the RNAi vectors of the invention. The invention also provides for a plant part comprising any of the RNAi vectors of the invention, such plant parts include seeds and apedicellate florets.

[0019] The invention further provides for transgenic plants comprising any of the RNAi vectors of the invention. For example, the invention provides for Sorghum sp. plants comprising any of the RNAi vectors of the invention. The invention also provides for Sorghum bicolor plants comprising any of the RNAi vectors of the invention. In particular, the invention provides for transgenic plants, such as Sorghum sp. plants and Sorghum bicolor plants, that have the BMSl gene silenced such the level of BMSl expression is decreased compared to the level of BMSl expression in a control, non-transgenic plant, wherein expression is decreased by reducing the level of mRNA transcript in the plant and the decrease is accomplished by any of the RNAi vectors of the invention. For example, the invention provides for transgenic plants and plant cells wherein expression of a BMSl gene is decreased by at least 90% or 95% when compared to a non-transformed plant cell.

[0020] The invention also provides for seeds and other plant parts of a transgenic plant comprising any of the RNAi vectors of the invention.

[0021] The invention also provides for methods for silencing BMSl gene in a transgenic plant such as a transgenic Sorghum plant or a transgenic plant cells, such as a transgenic Sorghum plant cell, comprising decreasing the level of BMSl expression compared to the level of BMSl expression its level in a control, non-transgenic plant by reducing the level of an mRNA in the transgenic plant, wherein the mRNA is encoded by a polynucleotide having at least 70% sequence identity to a nucleic acid sequence of SEQ ID NO:4, and by expression of an RNAi construct comprising a fragment of at least 20 contiguous nucleotides of a sequence having at least 90% sequence identity to SEQ ID NO:4.

[0022] These methods may be carried out with any of the above-described RNAi vectors of the invention. For example, the methods may be carried out with an RNAi vector comprising a

polynucleotide having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:6-8, or an RNAi vector comprising a polynucleotide having at least 95% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:6-8 or an NAi vector comprising a polynucleotide having at least 98% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:6-8. The methods of the invention also may be carried out with an RNAi vector comprising a polynucleotide selected from the group consisting of SEQ ID NOs:6-8 or a fragment thereof that is at least 20 contiguous nucleotides of any one of SEQ ID NOs:6-8.

[0023] In addition any of the methods described above may further comprise the step of screening the transgenic plants for a reduction of BMS1 expression by comparing the BMS1 expression in the transgenic plant to a control plant.

[0024] The invention also provides for methods of increasing grain yield of a sorghum plant, comprising converting at least one sterile pedicular floret to a fertile floret. For example the converting step of this method may be accomplished by expression of any one of the RNAi vectors of the invention in the sorghum plant.

[0025] In another aspect, the invention further provides for methods of creating a sorghum B line wherein at least one sterile pedicular floret is converted to a fertile floret comprising the steps of creating Fl plants comprising crossing a sorghum elite B line to a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret; selfing the Fl plants to create F2 plants and selecting for those plants wherein at least one sterile pedicular floret is converted to a fertile floret; and selfing the selected F3 plants and selecting for desirable agronomic traits, thereby recovering B line plants, wherein at least one sterile pedicular floret is converted to a fertile floret. Furthermore, in this aspect, the B line plants may be selfed to create progeny and the progeny plants crossed to a sorghum cytoplasmic male Al sterile line.

[0026] In yet another aspect, the invention provides methods of making sorghum R lines wherein at least one sterile pedicular floret is converted to a fertile floret, comprising crossing a sorghum R line with a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret to create Fl plants; selfing the Fl plants to create F2 plants, and selecting for those F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret; selfing the F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret to create F3 plants. The method can further comprising selfing the F3 plants to create F4 plants.

[0027] In another aspect, the invention is directed to methods of producing a hybrid sorghum seed, the methods comprising the steps of crossing a sorghum B line, wherein at least one sterile pedicular floret is converted to a fertile floret, with a sorghum R line, and recovering the produced hybrid seed. [0028] The invention also provides for methods of producing a hybrid sorghum seed, the method comprising crossing a B line of the invention with an R line of the invention.

[0029] In another aspect, the invention provides for methods of testing a sorghum B line wherein at least one sterile pedicular floret is converted to a fertile floret, comprising crossing said sorghum B line with an R line and assessing phenotype and yield.

[0030] In another aspect, the invention provides methods of amplifying sorghum R line seed wherein at least one sterile pedicular floret is converted to a fertile floret, comprising selfing said sorghum R line and recovering the seed.

[0031] In yet another aspect, the invention is directed to methods of amplifying sorghum B line seed wherein at least one sterile pedicular floret is converted to a fertile floret, comprising crossing said sorghum B line with an A line and recovering the seed.

[0032] In the amplifications of the R line or B line, the amplification may be sufficient for pilot testing or commercial production of a sorghum hybrid wherein at least one sterile pedicular floret is converted to a fertile floret. Such amplifications can be done in a winter nursery.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0033] FIG. 1 shows the three exemplary hairpin RNAi vectors designed to silence BMS1 in sorghum: a) pHAN-BMSl- 5'UTR, b) pHAN-BMSl-ORF and c) pHAN-BMSl-3'UTR. The vectors use three different regions of the BMS1 gene as targets for mRNA transcript degradation via the post-transcriptional gene silencing pathway.

DETAILED DESCRIPTION OF THE INVENTION

I. INTRODUCTION

[0034] The inventors reasoned that a VRS1 functional homolog is likely to exist in sorghum at some non-syntenic locus, and that mutation of this functional homolog leading to loss of function would result in conversion of sterile florets to fertile ones, resulting in at least a doubling of seed and grain yield in sorghum. The sorghum functional homolog of VRS1 is called Bicolor Maintainer of Sterilityl (BMS1).

[0035] BMS1 is likely also derived from an HD-ZIP transcription factor similar in sequence to HvHox2 or VRS1. In barley, HvHox2 is expressed at a low level in all tissues. In contrast, VRS1 is only expressed in immature inflorescences, consistent with its specialized role in controlling floret morphogenesis.

Therefore, among a collection of sorghum HD-ZIP Class 1 transcription factors, the identification of BMSl can be facilitated by comparative gene expression analysis in a range of tissues that includes immature inflorescences.

[0036] In embodiments of the invention, proof of identifying BMSl is accomplished by NAi-mediated down-regulation of the candidate gene, which, depending on the degree of penetrance achieved among different transgenic events, should result in a range of morphological phenotypes consistent with conversion of sterile florets to fertile florets and increased seed set. In addition to inducing post- transcriptional gene silencing by RNAi, artificial microRNAs (amiRNAs) may be used to specifically target one or more VRS1 functional homologs, including BMSl (Eamens and Waterhouse, 2011; Ossowski et al., 2008; Schwab et al., 2006; Warthmann et al., 2008; Waterhouse and Helliwell, 2003).

[0037] Alternatively, targeted mutagenesis can be used to effect a complete loss of function of the candidate gene via deletion, substitution, or insertion of DNA in the gene or its regulatory elements, (Curtain et al., 2012; Gao et al., 2010; Lloyd et al., 2005; Voytas, 2013) which results in quantitative loss of sterile florets and their conversion to fertile florets that bear seed after pollination.

[0038] Moreover, in a combination of these approaches, targeted mutagenesis may be used to replace a portion of BMSl with DNA sequences that cause its transcript to assume a hairpin structure which acts as an RNAi or amiRNA that now causes post-transcriptional silencing of that gene and its homologs, for example in a cross intended to make a hybrid seed. Similarly, an endogenous miRNA locus could be modified by targeted mutagenesis to add, or replace a native sequence with a SMSl-homologous region resulting in an amiRNA at that locus which acts to post-transcriptionally silence BMSl and/or its homologs.

[0039] RNAi-, miRNA-, or amiRNA-based constructs act as dominant traits, which allows for accelerated trait assessment, for example in a range of test crosses designed to discover modifiers of kernel size that could then be recombined in commercial inbred lines or hybrids carrying the multi-seed trait. Moreover, as a dominant-acting trait, both hybrid seed production and inbred development are simplified by use of RNAi or amiRNA. In hybrid seed production, only one inbred parent needs to carry the trait for its expression in Fl seed, which creates flexibility in testing and production of new hybrid combinations. Similarly, development and genetic improvement of inbred parent lines is simplified because only one parental lineage requires conversion and introgression of the trait. II. MAKING AND USING THE INVENTION

A. Identification of a VRSl functional homolog in sorghum

[0040] The genomic sequence of barley VRSl (the wild-type Vrsl.b allele) is shown in Table 1 (SEQ ID NO:l), the mRNA sequence is shown as SEQ ID NO:3 in Table 2, and the polypeptide sequence is shown as SEQ ID NO:2 in Table 3. Using the polypeptide sequence of SEQ ID NO:2, the inventors queried sorghum sequence databases using standard procedures and identified candidate genes that are expressed according to expressed sequence tag (EST) data in floral tissues. Of these candidate genes, the BMSl locus was chosen to be the gene Sb02g037560. The polynucleotide sequence (SEQ ID NO:4) and the polypeptide sequence (SEQ ID NO:5) of BMSl are shown in Tables 4 and 5, respectively.

TABLE 1

Barley Vrsl genomic sequence (Vrsl.b allele) (SEQ ID NO:l) gtcataactc ggcaaacata gattagacag aattttctga gttcttatct agaggaactc 60 gatgaacttg aggcattgtc gaggttcttc ctttcaccga gtactttttt gcgtgtacta 120 ggcaaatata tgaagtttgt gagtttcgga tcaccaccga gtgcaagttt ggaccaaact 180 tgacaaatac ataagtttgg cgagctccga atgaaatgaa ctctgcaaaa gaatagaact 240 cggcgcaaaa ccagattcta atagtgtgtg aatttttggg ctgttttgta taaatatgat 300 gaaacttagt aaaatttcac tcaggtcaat gctaatgtgg agagtaaata aaaaatgaag 360 ggagtacttg gctgcatcat atgtttgccc ccgatcacct tcacatctcc ccgtccggac 420 ggcctggatc ggaaagcact cagccggagc cccgccggcg cttgccgttg ggtacctctg 480 ccacctattt atattacccc taggtctctc cctggagaca cgcactcccc tccttcaact 540 agtgctttgc ggcccgtggt cctcctctcg atccagttcc tgagcacacc aacaggcaac 600 agaacaacct accgtgtctc ccctccaatc tcctcacgat cccttctttc cctcagatcc 660 gaaccgaaag catggacaag catcagctct ttgattcatc caacgtggac acgactttct 720 tcgcggccaa tggtacacac gacgccgcgc gcgcccggtc tttgcgcatg cgatgatgca 780 gctgcagtag cttcagtttc accggccagg acacgcatgt gatgacgttt tttccattct 840 gtgtttgtat gtgcaggcac ggcgcagggg gataccagca agcagagggc gcggcgcagg 900 cggcggaggt cggcgaggtg cggcggaggg gatggtgacg gtggggagat ggacggagga 960 ggggacccca agaagcggcg gctcaccgac gagcaggccg agattctgga gctgagcttc 1020 cgggaggacc gcaagctgga gacagcccgc aaggtgtatc tggccgccga gctcgggctg 1080 gaccccaagc aggtcgccgt gtggttccag aaccgccgcg cgcgccacaa gaacaagacg 1140 ctcgaggagg agttcgcgag gctcaagcac gcccacgacg ccgccatcct ccacaaatgc 1200 cacctcgaga acgaggtatg cttgctcgca tacactcaca ctggcttaca tatggcgctg 1260 cacatctgca gttcctctcc gttcttgaac atgcttactg acaaacatat ggccagctgc 1320 tgaggctgaa ggagagactg ggagcgactg agcaggaggt gcggcgcctc aggtcggcag 1380 ctgggagcca cggggcatct gtggatggcg gacacgccgc tggcgccgtt ggcgtgtgcg 1440 gcgggagccc gagctcgtcc ttctcgacgg gaacctgcca gcagcagccg ggtttcagcg 1500 gggcagacgt gctggggcgg gacgatgacc tgatgatgtg cgtccccgag tggtttttag 1560 catgaattag agtttatgct ggctaagccg atagcagcgt ggtcgagtgt tttttagcat 1620 gaaatcagat ctccatctcc cataaaatag ccgagatagc tgctgccgcc gccaaatcct 1680 ctatagggct tcaagatcgg cagaaacctc tagaaatcat ctcccccctc cggaaaagtc 1740 gcctctattt gtctccattg cccgcgatgc agcatccggt atagctgcta agacaggccg 1800 cccctaaatc gtttctccag cgattttaat ctttggtttt tagcctgtat atatgggctg 1860 tgatttgaag ttgagacgag ctggacatca actgcacgct gatcgattac tattctagtt 1920 tggcatagtg ttaattaagt ttggatgatc tctaggcgtg cgttaagtat gtagatagtg 1980 ttgattaatg gcaaaagctt gcaagttaag tgtagtattg gcagctctct tgaagatcaa 2040 atatgatgtg tgttatcatt tgatgatata tattttactt cagccgtaaa tagtcttctt 2100 agggaagcac tgtccatgta tgtgctggta gttggcattc atctttc 2147

TABLE 2

Barley Vrsl m NA sequence (Vrsl.b allele) (SEQ ID NO:3)

acgcactccc ctccttcaac tagtgctttg cggcccgtgg tcctcctctc gatccagttc 60 ctgagcacac caacaggcaa cagaacaacc taccgtgtct cccctccaat ctcctcacga 120 tcccttcttt ccctcagatc cgaaccgaaa gcatggacaa gcatcagctc tttgattcat 180 ccaacgtgga cacgactttc ttcgcggcca atggcacggc gcagggggat accagcaagc 240 agagggcgcg gcgcaggcgg cggaggtcgg cgaggtgcgg cggaggggat ggtgacggtg 300 gggagatgga cggaggaggg gaccccaaga agcggcggct caccgacgag caggccgaga 360 ttctggagct gagcttccgg gaggaccgca agctggagac agcccgcaag gtgtatctgg 420 ccgccgagct cgggctggac cccaagcagg tcgccgtgtg gttccagaac cgccgcgcgc 480 gccacaagaa caagacgctc gaggaggagt tcgcgaggct caagcacgcc cacgacgccg 540 ccatcctcca caaatgccac ctcgagaacg agctgctgag gctgaaggag agactgggag 600 cgactgagca ggaggtgcgg cgcctcaggt cggcagctgg gagccacggg gcatctgtgg 660 atggcggaca cgccgctggc gccgttggcg tgtgcggcgg gagcccgagc tcgtccttct 720 cgacgggaac ctgccagcag cagccgggtt tcagcggggc agacgtgctg gggcgggacg 780 atgacctgat gatgtgcgtc cccgagtggt ttttagcatg aattagagtt tatgctggct 840 aagccgatag cagcgtggtc gagtgttttt tagcatgaaa tcagatctcc atctcccata 900 aaatagccga gatagctgct gccgccgcca aatcctctat agggcttcaa gatcggcaga 960 aacctctaga aatcatctcc cccctccgga aaagtcgcct ctatttgtct ccattgcccg 1020 cgatgcagca tccggtatag ctgctaagac aggccgcccc taaatcgttt ctccagcgat 1080 tttaatcttt ggtttttagc ctgtatatat gggctgtgat ttgaagttga gacgagctgg 1140 acatcaactg cacgctgatc gattactatt ctagtttggc atagtgttaa ttaagtttgg 1200 atgatctcta ggcgtgcgtt aagtatgtag atagtgttga ttaatggcaa aagcttgcaa 1260 gttaagtgta gtattggcag ctctcttgaa gatcaaatat gatgtgtgtt ate 1313

TABLE 3

Barley Vrsl polypeptide sequence (Vrsl. b allele) (SEQ ID NO:2)

MDKHQLFDSS NVDTTFFAAN GTAQGDTSKQ RARRRRRRSA RCGGGDGDGG EMDGGGDPKK 60 RRLTDEQAEI LELSFREDRK LETARKVYLA AELGLDPKQV AVWFQNRRAR HKNKTLEEEF 120 ARLKHAHDAA ILHKCHLENE LLRLKERLGA TEQEVRRLRS AAGSHGASVD GGHAAGAVGV 180 CGGSPSSSFS TGTCQQQPGF SGADVLGRDD DLMMCVPEWF LA 222

TABLE 4

Sorghum BMS1 polynucleotide sequence (SEQ ID NO:4)

atggaggaat aegaeggget ctttccttcc gcctacgtgg actcctcctc atccctcctg 60 gtgcccaacg gcacggcgca gggggagagg ccgagagcgc ggcgcaggag gcgtcgagca 120 ccgaggtgcg gcggcggcgg cgacctggac gggggagggg accccaagaa gcggcggctg 180 agegacgage aggtagagat gctggaactg agettceggg aggageggaa gctggagacc 240 ggccggaagg tgcacctggc cgccgagctc ggcctcgacc ccaagcaggt cgccgtctgg 300 ttccagaacc gccgcgcccg ccacaagagc aagctgeteg aggaggagtt cgccaagctc 360 aagcaggcac acgacgccgc catcctccac aaatgccacc ttgagaacga ggtgatgagg 420 ctgaaggaga ggctggtgct cgecgaggag gagctgaege gtttcagatc cgeggggage 480 cacgccgtct ccggtgacgg cggagacatc atgggccgtg ccgtctgcag cgggagcccg 540 agetcategt tctcgacagg cacctgccac cagccgggcg tegaegtegg eggeggegat 600 cacctggggg acgacgacca gctgctctac gttcctgact atgcctacgc tgacaacagc 660 gtggtcgagt ggtttagcct gtatggactg atgtaa 696

TABLE 5

Sorghum BMS1 polypeptide sequence (SEQ ID NO:5)

MEEYDGLFPS AYVDSSSSLL VPNGTAQGER PRARRRRRRA PRCGGGGDLD GGGDPKKRRL 60

SDEQVEMLEL SFREERKLET GRKVHLAAEL GLDPKQVAVW FQNRRARHKS KLLEEEFAKL 120

KQAHDAAILH KCHLENEVMR LKERLVLAEE ELTRFRSAGS HAVSGDGGDI MGRAVCSGSP 180

SSSFSTGTCH QPGVDVGGGD HLGDDDQLLY VPDYAYADNS VVEWFSLYGL M 231 B. Silencing BMS1 in sorghum with RNAi

[0041] The invention includes methods of silencing the BMS1 gene, wherein a sorghum plant is transformed with nucleic acids capable of silencing a BMS1 gene. Silencing BMS1 can be done conveniently by sub-cloning a BMS1 targeting sequence, such as one of the polynucleotides of SEQ ID N0s:6-8 (Table 6), into RNAi vectors or using an RNAi vector comprising a fragment of at least 20 contiguous nucleotides of a sequence having at least 90% sequence identity to SEQ ID NO:4. Exemplary fragments of SEQ ID NO: 4 are portions of the 5' UTR such as SEQ ID NO: 6, a portion of the open reading frame of SEQ ID NO: 4 that is not highly conserved such as SEQ ID NO: 7,or portions of the 3' UTR such as SEQ ID NO: 8.

TABLE 6

Exemplary BMS1 targeting sequences

SEQ ID Sequence

NO:

6 ctggtgcctt gccctgtggt cctcgctccg tccttaatcc acttctcctg ctgcctgccc 60 agcaccacac cacaccatcc cgtctcctct ccccggccag cctccctctc tctttctctg 120 tcctcagtcc tcctctcacg cttgacaagg agagagaaag gtgtagcagc agctagctga 180 tccggccggg agcgcaggca aagg 204

7 tcgaggagga gttcgccaag ctcaagcagg cacacgacgc cgccatcctc cacaaatgcc 60 accttgagaa cgaggtgatg aggctgaagg agaggctggt gctcgccgag gaggagctga 120 cgcgtttcag atccgcgggg agccacgccg tctccggtga cggcggagac atcatgggcc 180 gtgccgtctg cagcgggagc ccgagctcat cgttctcgac aggcacctgc caccagccgg 240 gcgtcgacgt cggcggcggc gatcacctgg gggacgacga ccagctgctc tacgttcctg 300 actatgccta cgctgacaac agcgtggtcg agtggtttag cctgtatgga ctgatgtaa .^' 359

8 tcgatcggca gattcattct agcagctagc tactcatcgt agtgtgtcat tgctgtgctg 60 aattgctgat ttctaagtgc gtgtgcttaa tcatatgtac ataaatcaag ttgttaatta 120 gtggcaaaat atagccagct accagcaact agtacttgaa ttcagatgtc tgttactata 180 ctgcgtgcta cccacattgg aatctttttt tttccccctt cactactata tatagtcttt 240 cgtgagctga tgagcaagta ctatgcatgt gctagctgac aatcacatcc ttgctacgtg 300 ggaaggtaac atttgaacaa aaaaatatat attcaaatag tatacataca atttcgaggt 360 tcatgtc 367 [0042] RNA interference (RNAi) in plants (i.e., post-transcriptional gene silencing (PTGS)) is an example of a broad family of phenomena collectively called RNA silencing (Hannon, 2002). The unifying features of RNA silencing phenomena are the production of small (21-26 nt) RNAs that act as specificity determinants for down-regulating gene expression (Djikeng et al., 2001; Hamilton and Baulcombe, 1999; Hammond et al., 2000; Parrish and Fire, 2001; Parrish et al., 2000; Tijsterman et al., 2002; Zamore et al., 2000) and the requirement for one or more members of the Argonaute family of proteins (or PPD proteins, named for their characteristic PAZ and Piwi domains) (Fagard and Vaucheret, 2000; Hammond et al., 2001; Hutvagner and Zamore, 2002; Kennerdell et al., 2002; Martinez et al., 2002; Pal-Bhadra et al., 2002; Tabara et al., 1999; Williams and Rubin, 2002).

[0043] Small RNAs are generated in animals by members of the Dicer family of double-stranded RNA (dsRNA)-specific endonucleases (Bernstein et al., 2001; Grishok et al., 2001; Ketting et al., 2001). Dicer family members are large, multi-domain proteins that contain putative RNA helicase, PAZ, two tandem ribonuclease III (RNase III), and one or two dsRNA-binding domains. The tandem RNase III domains are believed to mediate endonucleolytic cleavage of dsRNA into small interfering RNAs (siRNAs), the mediators of RNAi. In Drosophila and mammals, siRNAs, together with one or more Argonaute proteins, form a protein-RNA complex, the RNA-induced silencing complex (RISC), which mediates the cleavage of target RNAs at sequences with extensive complementarity to the siRNA (Zamore et al., 2000).

[0044] In addition to Dicer and Argonaute proteins, RNA-dependent RNA polymerase (RdRP) genes are required for RNA silencing in PTGS initiated by transgenes that overexpress an endogenous mRNA in plants (Zamore et al., 2000), although transgenes designed to generate dsRNA bypass this requirement (Beclin et al., 2002).

[0045] Dicer in animals and CARPEL FACTORY (CAF, a Dicer homolog) in plants also generate microRNAs (miRNAs), 20-24-nt, single-stranded non-coding RNAs thought to regulate endogenous mRNA expression (Park et al., 2002). miRNAs are produced by Dicer cleavage of stem-loop precursor RNA transcripts (pre- miRNAs); the miRNA can reside on either the 5' or 3' side of the double-stranded stem. Generally, plant miRNAs have far greater complementarity to cellular mRNAs than is the case in animals, and have been proposed to mediate target RNA cleavage via an RNAi-like mechanism (Llave et al., 2002; Rhoades et al., 2002).

[0046] In plants, RNAi can be achieved by a transgene that produces hairpin RNA (hpRNA) with a dsRNA region (Waterhouse and Helliwell, 2003). Although antisense-mediated gene silencing is an RNAi-related phenomenon (Di Serio et al., 2001), hpRNA-induced RNAi is more efficient (Chuang and Meyerowitz, 2000). In an hpRNA-producing vector, the target gene is cloned as an inverted repeat spaced with an unrelated sequence as a spacer and is driven by a strong promoter, such as the 35S CaMV promoter for dicots or the maize ubiquitin 1 promoter for monocots. When an intron is used as the spacer, essential for stability of the inverted repeat in Escherichia coli, efficiency becomes high: almost 100% of transgenic plants show gene silencing (Smith et al., 2000; Wesley et al., 2001). RNAi can be used against a vast range of targets; 3' and 5' untranslated regions (UTRs) as short as 100 nt can be efficient targets of RNAi (Kusaba, 2004).

[0047] For genome-wide analysis of gene function, a vector for high-throughput cloning of target genes as inverted repeats, which is based on an LR clonase reaction, is useful (Wesley et al., 2001). Another high-throughput RNAi vector is based on "spreading of RNA targeting" (transitive RNAi) from an inverted repeat of a heterologous 3' UTR (Brummell et al., 2003a; Brummell et al., 2003b). A chemically regulated RNAi system has also been developed (Guo et al., 2003).

[0048] Virus-induced gene silencing (VIGS) is another approach often used to analyze gene function in plants (Waterhouse and Helliwell, 2003). RNA viruses generate dsRNA during their life cycle by the action of virus-encoded RdRP. If the virus genome contains a host plant gene, inoculation of the virus can trigger RNAi against the plant gene. This approach is especially useful for silencing essential genes that would otherwise result in lethal phenotypes when introduced in the germplasm. Amplicon is a technology related to VIGS (Waterhouse and Helliwell, 2003). It uses a set of transgenes comprising virus genes that are necessary for virus replication and a target gene. Like VIGS, amplicon triggers RNAi but it can also overcome the problems of host-specificity of viruses (Kusaba, 2004).

[0049] In addition, siRNAs and hpRNAs can be synthesized and then introduced into host cells. The polynucleotides of SEQ ID NOs:6-8 can be prepared by conventional techniques, such as solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, CA; USA), DuPont, (Wilmington, DE; USA), Genescript USA (Piscataway, NJ, USA), GeneArt/ThermoFisher Scientific (Waltham, MA, USA) or Milligen (Bedford, MA; USA). Modified polynucleotides, such as phosphorothioates and alkylated derivatives, can also be readily prepared by similar methods known in the art. The polynucleotides of SEQ ID NOs:6-8 can also be generated by conventional PCR of genomic DNA from sorghum. 1. RNAi vectors

[0050] Excellent guidance can be found in Preuss and Pikaard regarding RNAi vectors (Preuss and Pikaard, 2004). In some embodiments, RNAi vectors are introduced using Agrobacterium tumefaciens- mediated delivery into plants. Several families of RNAi vectors that use Agrobacterium tumefaciens- mediated delivery into plants are widely available. All share the same overall design, but differ in terms of selectable markers, cloning strategies and other elements (Table 7). A typical design for an RNAi- inducing transgene comprises a strong promoter (as well-known to those of skill in the art, such as Cauliflower Mosaic Virus 35S promoter (CaMV 35S)) driving expression of sequences matching the targeted mRNA(s). These targeting sequences are cloned in both orientations flanking an intervening spacer, which can be an intron or a spacer sequence that will not be spliced. For stable transformation, a selectable marker gene, such as herbicide resistance or antibiotic resistance, driven by a plant promoter, is included adjacent to the RNAi-inducing transgene. The selectable marker gene plays no role in RNAi but allows transformants to be identified by treating seeds, whole plants or cultured cells with herbicide or antibiotic. For transient expression experiments, no selectable marker gene would be necessary. In constructs for use in A. tumefaciens-mediated delivery, the T-DNA is flanked by a left border (LB) and right border (RB) sequence that delimit the segment of DNA to be transferred. For stable transformation mediated by means other than A. tumefaciens, LB and RB sequences are irrelevant (Preuss and Pikaard, 2004).

TABLE 7

Exemplary vectors for stable transformation for hpRNA production

pFGC5941 pMCG161 pHannibal pHELLSGATE

Organism Dicots Monocots Dicots Dicots

Cloning Method restriction restriction restriction GATEWAY^®

digest/ligation digest/ligation digest/ligation recombination

(Invitrogen)

Bacterial Selection Kanamycin chloramphenicol ampicillin Spectinomycin and chloramphenicol

Plant Selection Basta Basta (none) geneticin dsRNA promoter CaMV 35S CaMV 35S CaMV 35S CaMV 35S

Inverted repeat ChsA intron Waxy intron Pdk intron Pdk intron spacer [0051] Two vectors are especially useful, pHANNIBAL and pHELLSGATE (Helliwell et al., 2005; Wesley et al., 2001). pHELLSGATE vectors are also described in U.S. Patent No. 6,933,146 and US Patent

Publication 2005/0164394. The pHANNIBAL vector has an E. coli origin of replication and includes a bacterial selection gene (ampicillin) and a strong promoter (CaMV 35S) upstream of a pair of multiple cloning sites flanking the PDK intron. This structure allows cloning sense and antisense copies of target sequence, separated by the intron. The pHELLSGATE vectors facilitate high-throughput cloning of target sequences directly into an Agrobacterium vector by taking advantage of Gateway recombination technology. The efficiency of pHELLSGATE vectors provides a potential advantage for large scale projects seeking to knock down entire categories of genes. In pHELLSGATE2, the target sequences are incorporated into the T-DNA region (the portion of the plasmid transferred to the plant genome via Agrobacterium-mediated transformation) via the aatB site-specific recombination sequence.

pHELLSGATE8 is identical to pHELLSGATE2 but contains the more efficient aatP recombination sites.

[0052] Another set of NAi vectors originally designed for Arabidopsis and maize are freely available through the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH) and were donated by the Functional Genomics of Plant Chromatin Consortium (Gendler et al., 2008). Vectors pFGC5941 and pMCG161 include within the T-DNA a selectable marker gene, phosphinothricin acetyl transferase, conferring resistance to the herbicide Basta, and a strong promoter (CaMV 35S) driving expression of the RNAi-inducing dsRNA. Introduction of target sequences into the vector requires two cloning steps, making use of polylinkers flanking a Petunia chalcone synthase intron, an overall design similar to pHANNIBAL. Other ChromDB RNAi vectors, such as pGSA1131, pGSA1165, pGSA1204, pGSA1276, and pGSA1252, pGSA1285, offer kanamycin or hygromycin resistance as plant selectable markers, instead of Basta resistance, and a non-intronic spacer sequence instead of the chalcone synthase intron. The ChromDB vectors are based on pCAMBIA plasmids developed by the Center for Application of Molecular Biology to International Agriculture (CAMBIA; Canberra, Australia). These plasmids have two origins of replication, one for replication in Agrobacterium tumefaciens and another for replication in E. coli. Thus, all cloning steps can be conducted in E. coli prior to transformation (Preuss and Pikaard, 2004).

2. Design of targeting sequences (Preuss and Pikaard, 2004)

[0053] RNAi vectors are typically designed such that the targeting sequence corresponding to each of the inverted repeats is 300-700 nucleotides in length; however, a stretch of perfect complementarity larger than 14 nucleotides appears absolutely required; 20 nucleotides is a convenient minimum.

Success is more easily achieved when the dsRNA targeting sequence is 300-700 nucleotides. Exemplary targeting sequences of the invention include those of SEQ ID NOs:6-8, and those having at least 90%- 99% sequence (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) identity thereto (Table 6), as well as any 20 contiguous nucleotides of SEQ ID NO:4 (Table 4) or those having at least 90%-99% sequence (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%) identity thereto.

[0054] Naturally-occurring miRNA precursors (pre-miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other.

[0055] In hpRNAs, one portion of the duplex stem is a nucleic acid sequence that is complementary to the target mRNA. Thus, engineered hpRNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The two stem portions are about 18 or 19 to about 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In plant cells, the stem can be longer than 30 nucleotides. The stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides.

[0056] hpRNAs of the invention include the sequences of the desired siRNA duplex. The desired siRNA duplex, and thus both of the two stem portions in the engineered RNA precursor, are selected by methods known in the art. These include, but are not limited to, selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from the target gene mRNA sequence from a region 100 to 200 or 300 nucleotides on the 3' side of the start of translation. In general, the sequence can be selected from any portion of the mRNA from the target gene (such as that of SEQ ID NO:4; Table 4).

3. Inactivation of BMSl via targeted mutagenesis

[0057] Suitable methods for BMSl inactivation include any method by which a target sequence-specific DNA-binding molecule can be introduced into a cell. Preferably such agents are, or are operably linked to, a nuclease which generates double-stranded cuts in the target DNA. Such agents can include meganucleases, homing endonuceases, zinc finger nucleases, or TALENs (Transcription Activator-Like Effector Nucleases) (Curtain et al., 2012; Gao et al., 2010; Lloyd et al., 2005; Voytas, 2013). Double- stranded DNA breaks initiate endogenous DNA repair mechanisms, primarily non-homologous end- joining, that can result in the deletion or insertion of one, a few, or many nucleotides at the site at which the double-stranded break occurred. These insertions or deletions can result in loss of function of the target gene through introduction of frameshift, nonsense, or missense mutations.

4. Methods for delivering polynucleotides to plants and plant cells

[0058] Suitable methods include any method by which DNA can be introduced into a cell, such as by Agrobacterium or viral infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment.

[0059] Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Fromm et al., 1985; Wong and Neumann, 1982) and the gene gun (Fynan et al., 1993; Johnston and Tang, 1994); (3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988; Eglitis et al., 1988; Lu et al., 1993); and (4) receptor-mediated mechanisms (Curiel et al., 1991; Curiel et al., 1992; Wagner et al., 1992).

[0060] Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. The introduction of DNA by electroporation is well-known to those of skill in the art. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made susceptible to transformation by mechanical wounding. To effect

transformation by electroporation one can use either friable tissues such as a suspension culture of cells or embryogenic callus, or alternatively one can transform immature embryos or other organized tissues directly. Cell walls are partially degraded of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounded in a controlled manner.

[0061] Microprojectile bombardment, shoots particles coated with the DNA of interest into to plant cells. In this process, the desired nucleic acid is deposited on or in small dense particles, e.g., tungsten, platinum, or preferably 1 micron gold particles, that are then delivered at a high velocity into the plant tissue or plant cells using a specialized biolistics device, such as are available from Bio- ad Laboratories (Hercules, CA). The advantage of this method is that no specialized sequences need to be present on the nucleic acid molecule to be delivered into plant cells.

[0062] For bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos, seedling explants, or any plant tissue or target cells can be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.

[0063] Various biolistics protocols have been described that differ in the type of particle or the manner in that DNA is coated onto the particle. Any technique for coating microprojectiles that allows for delivery of transforming DNA to the target cells can be used. For example, particles can be prepared by functionalizing the surface of a gold oxide particle by providing free amine groups. DNA, having a strong negative charge, binds to the functionalized particles.

[0064] Parameters such as the concentration of DNA used to coat microprojectiles can influence the recovery of transformants containing a single copy of the transgene. For example, a lower concentration of DNA may not necessarily change the efficiency of the transformation but can instead increase the proportion of single copy insertion events. Ranges of approximately 1 ng to approximately 10 pg, approximately 5 ng to 8 μg or approximately 20 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 pg, 2 μg, 5 μg, or 7 μg of transforming DNA can be used per each 1.0-2.0 mg of starting 1.0 micron gold particles.

[0065] Other physical and biological parameters can be varied, such as manipulation of the

DNA/microprojectile precipitate, factors that affect the flight and velocity of the projectiles,

manipulation of the cells before and immediately after bombardment (including osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells), the orientation of an immature embryo or other target tissue relative to the particle trajectory, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. Physical parameters such as DNA concentration, microprojectile particle size, gap distance, flight distance, tissue distance, and helium pressure, can be optimized.

[0066] The particles delivered via biolistics can be "dry" or "wet." In the "dry" method, the MC DNA- coated particles such as gold are applied onto a macrocarrier (such as a metal plate, or a carrier sheet made of a fragile material, such as mylar) and dried. The gas discharge then accelerates the macrocarrier into a stopping screen that halts the macrocarrier but allows the particles to pass through. The particles are accelerated at, and enter, the plant tissue arrayed below on growth media. The media supports plant tissue growth and development and are suitable for plant transformation and regeneration. These tissue culture media can either be purchased as a commercial preparation, or custom prepared and modified. Examples of such media include Murashige and Skoog (MS), N6, Linsmaier and Skoog, Uchimiya and Murashige, Gamborg's B5 media, D medium, McCown's Woody plant media, Nitsch and Nitsch, and Schenk and Hildebrandt. Those of skill in the art are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation, pH, and incubation temperatures can be optimized.

[0067] Those of skill in the art can use, devise, and modify selective regimes, media, and growth conditions depending on the plant system and the selective agent. Typical selective agents include antibiotics, such as geneticin (G418), kanamycin, paromomycin; or other chemicals, such as glyphosate or other herbicides.

[0068] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Dafny-Yelin et al. provide an overview of

Agrobacterium transformation (Dafny-Yelin and Tzfira, 2007). Agrobacterium plant integrating vectors to introduce DNA into plant cells is well known in the art, such as those described above, as well as others (Rogers et al., 1987). Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences (Jorgensen et al., 1987; Spielmann and Simpson, 1986).

[0069] A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Homozygous transgenic plants can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants for the targeted trait or insertion.

[0070] In some methods, Agrobacterium carrying the gene of interested can be applied to the target plants when the plants are in bloom. The bacteria can be applied via vacuum infiltration protocols in appropriate media, or even simply sprayed onto the blooms.

[0071] For RNA-mediated inhibition in a cell line or whole organism, gene expression can be conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, basta, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95%, 99%, or 100% as compared to a cell not treated. Lower doses of injected material and longer times after administration of NAi agent can result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%/o, 90%, or,95% of targeted cells). Quantitation of gene expression in a cell can show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition can be determined by assessing the amount of gene product in the cell; mRNA can be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide can be detected with an antibody raised against the polypeptide sequence of that region. Quantitative PCR techniques can also be used.

C. Methods related to producing lines of sorghum plants wherein at least one sterile pedicular floret is converted to a fertile floret and seed therefrom.

[0072] The sorghum plants produced by the methods of the invention can be crossed with other sorghum lines, such as an elite line, to generate fertile hybrids that exhibit the multi-seeded phenotype and increased yield. A variety of sorghum lines may be crossed, especially those that produce large- seeds. Desirable germplasm is available from GRIN - Germplasm Resources Information Network (www.ars-grin.gov). In producing a hybrid, for example, first a sorghum plant is prepared from seed of a plant produced by the methods of the invention, and crossed with a plant of a second sorghum variety to generate Fl plants. The Fl plants are selfed to generate subsequent generation (F2) sorghum plants (hybrids), some of which will exhibit the multi-seeded phenotype wherein pedicellate spikelets produce viable seed, which are selected and recovered. These hybrid subsequent generation plants which exhibit a phenotype wherein pedicellate spikelets produce viable seed may be optionally further selfed, with progeny exhibiting the multi-seeded phenotype selected and retained.

[0073] General techniques for the production of sorghum hybrids are well-known and are described, for example, by Bading et al. (U.S. Pat. No. 8,212,126) and by Bennet et al. (Bennett et al., 1990) and may involve the steps of: (1) planting in pollinating proximity seeds of a first and second parent sorghum plant; (2) cultivating or growing the seeds of the first and second parent sorghum plants into plants that bear flowers; (3) emasculating the flowers of either the first or second parent sorghum plant, i.e.

physically removing the anthers from the florets prior to blooming of the flowers so as to prevent pollen production or preventing dehiscence of pollen from anthers by introduction and maintenance of a high humidity environment by bagging a panicle or portion of a panicle with a plastic bag prior to blooming (a "wet pollination emasculation") or by using as the female parent a male sterile plant, thereby providing an emasculated parent sorghum plant; (4) allowing natural cross-pollination to occur between the first and second parent sorghum plants or mechanically moving pollen from the pollen parent to the pollen sterile seed parent; (5) harvesting seeds produced on the emasculated parent sorghum plant; and, where desired, (6) growing the harvested seed into a sorghum plant, which may be a hybrid sorghum plant.

[0074] In one embodiment, methods of creating a sorghum B line wherein at least one sterile pedicular floret is converted to a fertile floret comprise the steps of creating Fl plants comprising crossing a sorghum elite B line to a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret; selfing the Fl plants to create F2 plants and selecting for those plants wherein at least one sterile pedicular floret is converted to a fertile floret; and selfing the selected F3 plants and selecting for desirable agronomic traits (such as seed size, stay-green, etc.), thereby recovering B line plants, wherein at least one sterile pedicular floret is converted to a fertile floret. Furthermore, the B line plants may be selfed to create progeny and the progeny plants crossed to a sorghum cytoplasmic male Al sterile line to create a cytoplasmic male sterile B line.

[0075] In an embodiment, methods of making sorghum lines wherein at least one sterile pedicular floret is converted to a fertile floret, comprise crossing a sorghum R line with a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret to create Fl plants; selfing the Fl plants to create F2 plants, and selecting for those F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret; selfing the F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret to create F3 plants. The method can further comprising selfing the F3 plants to create F4 plants.

[0076] In another embodiment, methods of producing a hybrid sorghum seed, the methods comprise the steps of crossing a sorghum B line, wherein at least one sterile pedicular floret is converted to a fertile floret, with a sorghum R line, and recovering the produced hybrid seed.

[0077] In another embodiment, methods of producing a hybrid sorghum seed, the method comprise crossing a B line of the invention with an R line of the invention.

[0078] In an embodiment, methods of testing a sorghum B line wherein at least one sterile pedicular floret is converted to a fertile floret, comprise crossing said sorghum B line with an R line and assessing phenotype and yield. [0079] In an embodiment, methods of amplifying sorghum R line seed wherein at least one sterile pedicular floret is converted to a fertile floret, comprise selfing said sorghum R line and recovering the seed.

[0080] In yet another embodiment, methods of amplifying sorghum B line seed wherein at least one sterile pedicular floret is converted to a fertile floret, comprise crossing said sorghum B line with an A line and recovering the seed.

[0081] In the amplifications of the R line or B line, the amplification may be sufficient for pilot testing or commercial production of a sorghum hybrid wherein at least one sterile pedicular floret is converted to a fertile floret. Such amplifications can be done in a winter nursery.

[0082] Sorghum crops have been classically bred through techniques that take advantage of the plants method(s) of pollination. A plant is considered "self-pollinating" if pollen from one flower can be transmitted to the same or another flower, whereas plants are considered "cross- pollinated" if the pollen has to come from a flower on a different plant in order for pollination to occur.

[0083] Plants that are self-pollinated and selected over many generations become homozygous at most, if not all, of their gene loci, thereby producing a uniform population of true breeding progeny. A cross between two homozygous plants from differing backgrounds or two different homozygous lines produces a uniform population of hybrid plants that is likely to be heterozygous at a number of the gene loci. A cross of two plants that are each heterozygous at a number of gene loci will produce a generation of hybrid plants that are genetically different and are not uniform.

[0084] Sorghum plants are self-pollinating plants, but they can also be bred by cross- pollination. The development of sorghum hybrids requires the development of pollinator parents (fertility restorers) and seed parent inbreds using the cytoplasmic male sterility-fertility restorer system, the crossing of seed parents and pollinator parents, and the evaluation of the crosses. Pedigree breeding programs combine desirable traits; in the present application the desirable trait being the multi-seeded phenotype. This trait is introduced into the breeding pool from one or more lines, such that new inbred lines are created by crossing, followed by selection of plants with the desired trait, followed by more crossing, etc. New inbreds are crossed with other inbred lines (e.g., elite plant lines).

[0085] Pedigree breeding starts with the crossing of two genotypes, such as a line wherein at least one sterile pedicular floret is converted to a fertile floret, and an elite sorghum line. If the original two parents do not provide all of the desired characteristics, then other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations, the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection.

[0086] Typically, in the pedigree method, five or more generations of selfing and selection are practiced (e.g., SI, S2, S3, S4, S5, etc.).

[0087] Backcrossing is used to improve a plant line. Backcrossing transfers a specific desirable trait from one source to another that lacks the trait. This is accomplished by, for example, crossing a donor (to an inbred line. The progeny of this cross is then crossed back (i.e. backcrossing) to the elite inbred line, followed by selection in the resultant progeny for the desired trait. Typically, following five or more backcross generations with selection for the desired trait the progeny are typically heterozygous for the locus (loci) controlling the desired phenotype, but will be like the elite parent for the other genetic traits.

[0088] The last backcrossing is typically selfed in order to give a pure breeding progeny for the gene being transferred.

[0089] In current hybrid introgressive sorghum breeding programs, new parent lines are developed to be either seed-parent lines or pollen-parent lines, depending on whether or not they contain fertility- restoring genes; the seed-parent lines do not have fertility restoring genes and are male-sterile in certain cytoplasms (also known as "A-line" plants) and male fertile in other cytoplasms (also known as "B-line" plants), whereas the pollen-parent lines are not male sterile and do contain fertility restoring genes (also known as " -line" plants). The seed-parent lines are typically created to be cytoplasmically male sterile such that the anthers are minimal to non-existant in these plants thereby requiring cross- pollination. The seed-parent lines will only produce seed, and the cytoplasm is transmitted only through the egg. The pollen for cross pollination is furnished through the pollen-parent lines that contain the genes necessary for complete fertility restoration in the Fl hybrid, and the cross combines with the male sterile seed parent to produce a high-yielding single cross hybrid with good grain quality.

[0090] Typically, this cytoplasmic male sterility-fertility restorer system is performed for the production of hybrid seed by planting blocks of rows of male sterile (seed-parent) plants and blocks of rows of fertility restorer (pollen-parent) plants, such that the seed-parent plants are wind pollinated with pollen from the pollen-parent plant. This process produces a vigorous single-cross hybrid that is harvested and planted by the consumer. [0091] Male sterile, seed-parent plants can also be created by genetically breeding recessive male- sterile nuclear genes into a particular population, however the cytoplasmic male sterility- fertility restorer system is typically the system used for breeding hybrid sorghum.

DEFINITIONS

[0092] "BMS1 sequence variant polynucleotide" or "BMS1 sequence variant nucleic acid sequence" means a BMS1 sequence variant polynucleotide having at least about 60% nucleic acid sequence identity, more preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence of SEQ ID NO: 4. Variants do not encompass the native nucleotide sequence.

[0093] Ordinarily, BMS1 sequence variant polynucleotides are at least about 8 nucleotides in length, often at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 35, 40, 45, 50, 55, 60 nucleotides in length, or even about 75-200 nucleotides in length, or more.

[0094] "Consisting essentially of a polynucleotide having a % sequence identity" means that the polynucleotide does not substantially differ in length, but in sequence. Thus, a polynucleotide "A" consisting essentially of a polynucleotide having 80% sequence identity to a known sequence "B" of 100 nucleotides means that polynucleotide "A" is about 100 nts long, but up to 20 nts can vary from the "B" sequence. The polynucleotide sequence in question can be longer or shorter due to modification of the termini, such as, for example, the addition of 1-15 nucleotides to produce specific types of probes, primers and other molecular tools, etc., such as the case of when substantially non-identical sequences are added to create intended secondary structures. Such non-identical nucleotides are not considered in the calculation of sequence identity when the sequence is modified by "consisting essentially of."

[0095] The specificity of single stranded DNA to hybridize complementary fragments is determined by the stringency of the reaction conditions. Hybridization stringency increases as the propensity to form DNA duplexes decreases. In nucleic acid hybridization reactions, the stringency can be chosen to either favor specific hybridizations (high stringency). Less-specific hybridizations (low stringency) can be used to identify related, but not exact, DNA molecules (homologous, but not identical) or segments.

[0096] DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2) the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4) the temperature of the reaction, and (5) the presence of certain organic solvents, such as formamide, which decreases DNA duplex stability. A common approach is to vary the temperature: higher relative temperatures result in more stringent reaction conditions. Ausubel et al. (1987) provide an excellent explanation of stringency of hybridization reactions (Ausubel, 1987).

[0097] "Elite plant" refers to any plant that has resulted from breeding and selection for superior agronomic performance.

[0098] "F-generation" and "filial generation" refer to any of the consecutive generations of plants, cells, tissues or organisms after a biparental cross. The generation resulting from a mating of a biparental cross (i.e. two parents) is the first filial generation (designated as "Fl") in reference to a seed and its plant, while that resulting from crossing of Fl individuals is the second filial generation

(designated as "F2") in reference to a seed and its plant, and so on.

[0099] "Hybrid" refers to the offspring or progeny of genetically dissimilar plant parents or stock produced as the result of controlled cross-pollination as opposed to a non-hybrid seed produced as the result of natural pollination.

[00100] An "isolated" molecule (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to a molecule that is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[00101] "Linker" refers to a DNA molecule, generally up to 50 or 60 nucleotides long and composed of two or more complementary oligonucleotides that have been synthesized chemically, or excised or amplified from existing plasmids or vectors. In a preferred embodiment, this fragment contains one, or preferably more than one, restriction enzyme site for a blunt cutting enzyme and/or a staggered cutting enzyme, such as BamHI. One end of the linker is designed to be ligatable to one end of a linear DNA molecule and the other end is designed to be ligatable to the other end of the linear molecule, or both ends may be designed to be ligatable to both ends of the linear DNA molecule

[00102] "Non-protein expressing sequence" or "non-protein coding sequence" means a nucleic acid sequence that is not eventually translated into protein. The nucleic acid may or may not be transcribed into NA. Exemplary sequences include ribozymes or antisense RNA.

[00103] "Nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide which can be derivitized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g, 6-(2- amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8- chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7- deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs (Herdewijn, 2000).

[00104] "Operably linked" means a configuration in which a control sequence, e.g., a promoter sequence, directs transcription or translation of another sequence, for example a coding sequence. For example, a promoter sequence could be appropriately placed at a position relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence.

[00105] "Percent (%) nucleic acid sequence identity" with respect to BMS1 sequence- nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the BMS1 sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalig (DNASTA ) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[00106] When nucleotide sequences are aligned, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:

% nucleic acid sequence identity = W/Z ^' 100

where

W is the number of nucleotides cored as identical matches by the sequence alignment program's or algorithm's alignment of C and D

and

Z is the total number of nucleotides in D. [00107] When the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[00108] "Phenotype" or "phenotypic trait(s)" refers to an observable property or set of properties resulting from the expression of a gene. The set of properties may be observed visually or after biological or biochemical testing, and may be constantly present or may only manifest upon challenge with the appropriate stimulus or activation with the appropriate signal.

[00109]The term "plant part" includes a pod, root, sett root, shoot root, root primordial, shoot, primary shoot, secondary shoot, tassle, panicle, arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath, node, internode, bud furrow, leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis, vascular tissue, organ, protoplast, crown, callus culture, petiole, petal, sepal, stamen, stigma, style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or embryo. Other exemplary plant parts are a meiocyte or gamete or ovule or pollen or endosperm of any of the preceding plants. Other exemplary plant parts are a seed, seed-piece, embryo, protoplast, cell culture, any group of plant cells organized into a structural and functional unit or propagule.

[00110] A "polynucleotide" is a nucleic acid polymer of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNA mimetics (such as, PNAs), and derivatives thereof, and homologues thereof. Thus, polynucleotides include polymers composed of naturally occurring nucleobases, sugars and covalent inter-nucleoside (backbone) linkages as well as polymers having non- naturally-occurring portions that function similarly. Such modified or substituted nucleic acid polymers are well known in the art and for the purposes of the present invention, are referred to as "analogues." Oligonucleotides are generally short polynucleotides from about 10 to up to about 160 or 200 nucleotides.

[00111] "Polypeptide" is a chain of amino acids connected by peptide linkages. The term "polypeptide" does not refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term "exogenous polypeptide" is defined as a polypeptide which is not native to the plant cell, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the plant cell by recombinant DNA techniques.

[00112] "Progeny" refers to generations of a plant, wherein the ancestry of the generation can be traced back to said plant. [00113] A "promoter" is a DNA sequence that allows the binding of RNA polymerase (including RNA polymerase I, RNA polymerase II and RNA polymerase III from eukaryotes) and directs the polymerase to a downstream transcriptional start site of a nucleic acid sequence encoding a polypeptide to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region.

[00114] A "promoter operably linked to a heterologous gene" is a promoter that is operably linked to a gene that is different from the gene to which the promoter is normally operably linked in its native state. Similarly, an "exogenous nucleic acid operably linked to a heterologous regulatory sequence" is a nucleic acid that is operably linked to a regulatory control sequence to which it is not normally linked in its native state.

[00115] "Regulatory sequence" refers to any DNA sequence that influences the efficiency of transcription or translation of any gene. The term includes sequences comprising promoters, enhancers and terminators. Similarly, an "exogenous regulatory sequence" is a nucleic acid that is associated with a gene to which it is not normally associated with its native state.

[00116] "RNA analog" refers to an polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. Oligonucleotides can be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog can comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phophoroamidate, and/or phosphorothioate linkages. RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA interference.

[00117] "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

[00118] "RNAi vectors" refer to a construct designed to carry and express an RNA interference polynucleotide in a host cell, such as a sorghum cell, and which will decrease expression of the gene of interest or silence the gene of interest. NAi vectors include vectors comprising RNAi, microRNAs (miRNAa), hairpin RNA (hpRNA) or artificial microRNA (amiRNA).

[00119] A "screenable marker" is a gene whose presence results in an identifiable phenotype. This phenotype may be observable under standard conditions, altered conditions such as elevated temperature, or in the presence of certain chemicals used to detect the phenotype. The use of a screenable marker allows for the use of lower, sub-killing antibiotic concentrations and the use of a visible marker gene to identify clusters of transformed cells, and then manipulation of these cells to homogeneity. Preferred screenable markers of the present include genes that encode fluorescent proteins that are detectable by a visual microscope such as the fluorescent reporter genes DsRed, ZsGreen, ZsYellow, AmCyan, Green Fluorescent Protein (GFP) and modifications of these reporter genes to excite or emit at altered wavelengths. An additional preferred screenable marker gene is lac.

[00120] Alternative methods of screening for modified plant cells may involve use of relatively low, sub- killing concentrations of a selection agent (e.g. sub-killing antibiotic concentrations), and also involve use of a screenable marker (e.g., a visible marker gene) to identify clusters of modified cells carrying the screenable marker, after which these screenable cells are manipulated to homogeneity. As used herein, a "selectable marker" is a gene whose presence results in a clear phenotype, and most often a growth advantage for cells that contain the marker. This growth advantage may be present under standard conditions, altered conditions such as elevated temperature, specialized media compositions, or in the presence of certain chemicals such as herbicides or antibiotics. Use of selectable markers is described, for example, in (Broach et al., 1979). Examples of selectable markers include the thymidine kinase gene, the cellular adenine phosphoribosyltransferase gene and the dihydrylfolate reductase gene, hygromycin phosphotransferase genes, the bar gene, neomycin phosphotransferase genes and phosphomannose isomerase, among others. Preferred selectable markers in the present invention include genes whose expression confer antibiotic or herbicide resistance to the host cell, or proteins allowing utilization of a carbon source not normally utilized by plant cells. Expression of one of these markers should be sufficient to enable the survival of those cells that comprise a vector within the host cell, and facilitate the manipulation of the plasmid into new host cells. Of particular interest in the present invention are proteins conferring cellular resistance to kanamycin, G418, paramomycin, hygromycin, bialaphos, and glyphosate for example, or proteins allowing utilization of a carbon source, such as mannose, not normally utilized by plant cells.

[00121] "Small interfering RNA" ("siRNA") (or "short interfering RNA") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) that is capable of directing or mediating NA interference. An effective siRNA can comprise between about 15-30 nucleotides or nucleotide analogs, between about 16-25 nucleotides, between about 18-23 nucleotides, and even about 19-22 nucleotides.

[00122] "Specifically hybridize" refers to the ability of a nucleic acid to bind detectably and specifically to a second nucleic acid. Polynucleotides specifically hybridize with target nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding by nonspecific nucleic acids.

[00123]To hybridize under "stringent conditions" describes hybridization protocols in which nucleotide sequences at least 60% homologous to each other remain hybridized.

[00124] An RNAi agent having a strand which is "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

[00125] "Sorghum" means Sorghum bicolor (primary cultivated species), Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum rundinaceum,, Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum carinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum, and Sorghum vulgare (including but not limited to the variety Sorghum vulgare var. sudanens also known as sudangrassj. Hybrids of these species are also of interest in the present invention as are hybrids with other members of the Family Poaceae.

[00126] A "targeting" sequence means a nucleic acid sequence of BMS1 sequence or complements thereof can silence a BMS1 gene. Exemplary targeting sequences include SEQ ID NOs:6-8. A target sequence can be selected that is more or less specific for a particular Sorghum

[00127] "Transformed," "transgenic," "modified," and "recombinant" refer to a host organism such as a plant into which an exogenous or heterologous nucleic acid molecule has been introduced, and includes meiocytes, seeds, zygotes, embryos, endosperm, or progeny of such plant that retain the exogenous or heterologous nucleic acid molecule but which have not themselves been subjected to the

transformation process. [00128] "Transgene" refers to any nucleic acid molecule that is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell. Such a transgene can include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or can represent a gene homologous to an endogenous gene of the organism. Transgene also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNAs, that encode one or more engineered NA precursors, to be expressed in a transgenic organism, e.g., plant, that is partly or entirely heterologous, i.e., foreign, to the transgenic plant, or homologous to an endogenous gene of the transgenic plant, but which is designed to be inserted into the plant's genome at a location that differs from that of the natural gene. A transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, operably linked to the selected sequence, and can include an enhancer sequence.

[00129] Comparing a value, level, feature, characteristic, property, etc. to a suitable control means comparing that value, level, feature, characteristic, or property to any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. A suitable control can be a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNAi agent of the invention into a cell or organism. A suitable control can be a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. A control can also be a predefined value, level, feature, characteristic, property, etc.

EXAMPLES

[00130]The following examples are meant to only exemplify the invention, not to limit it in any way. One of skill in the art can envision many variations and methods to practice the invention.

Example 1 - Identification of BMS1 and generation of RNAi vectors

1.1 Identification of BMS1

[00131] In order to identify a functional homolog of Vrsl from the sorghum genome, the protein sequences of HvHox2 and Vrsl from barley were used as queries in a BLASTP search (Altschul et al., 1990) with the subject restricted to the predicted proteins of Sorghum bicolor genome. The sorghum sequences with the highest identity to HvHox2 or Vrsl were selected for further analysis. All of the genes recovered with the highest scores in the BLASTP search encoded proteins with the homeodomain and the leucine-zipper motifs characteristic of Class 1 HD-ZIP transcription factors (Hu et al., 2012). An alignment of the predicted Vrsl-like proteins from sorghum with HvHox2 and Vrsl protein sequences from barley showed that the encoded protein lengths and characterized motifs were well-conserved between these members of the Class 1 HD-ZIP family. An examination of the chromosomal location of the sorghum candidate genes revealed that Sb02g037560 (BMSl; SEQ ID NO:4, shown in Table 4) is syntenic with HvHox2 according to available physical and genetic map data.

1.2 Analysis of expression profiles of candidate BMSl genes

[00132] Because a candidate gene for floral repression would necessarily have to be expressed in florets, the tissue-specific expression profiles of the sorghum HD-ZIP candidate genes were examined using the publically available EST databases and using a microarray dataset previously produced by the inventors. Genes that were specifically and highly expressed predominantly in or only in developing inflorescences were ranked as highest priority for RNAi targets. Genes that shared high sequence identity with HvHox2 or Vrsl, but were not expressed in florets at any stage were eliminated from the pool of candidate genes. Based on a combination of different lines of evidence from protein alignments, gene expression analyses, conserved protein motifs, and synteny with HvHox2 and/or Vrsl, Sb02g037560 (BMSl) (SEQ ID NO:4) was determined to be the Vrsl-like gene from sorghum.

1.3 Production of DNA elements for RNAi vectors

[00133] Three fragments from the genomic sequence of BMSl were used in three different RNAi constructs. The three fragments were localized in the 5' untranslated region (5'UTR; SEQ ID NO:6), the open reading frame (ORF; SEQ ID NO:7), and the 3'UTR (SEQ ID NO:8), respectively and shown in Table 6. Previous experiments by the inventors have shown that the promoter of the rice actin (OsActV2) gene is a strong constitutive promoter in sorghum. This promoter was chosen to drive the expression of the RNAi cassette. The terminator sequence of the Arabidopsis pyruvate kinase 2 gene (AtT6) was chosen to terminate transcription.

[00134]The sorghum BMSl DNA elements are amplified by PCR from genomic DNA isolated according to the standard protocol described in "Isolation of High-Molecular-Weight DNA Using Organic Solvents to Purify DNA" (Green et al., 2012). The conditions for the PCR are as follows: 200ng (2μΙ) of genomic DNA is used as template in a 50μΙ reaction containing 25μΙ of Q5^® High-Fidelity 2X Master Mix (New England BioLabs Ipswich, MA, US), ΙμΙ each of forward and reverse primers (final concentration of 200ng each), and 21μΙ nuclease-free water. The PCR is run on a 2720 Thermocycler (Applied Biosystems, Life

Technologies, Grand Island, NY, US) under the following temperature conditions: initial denaturation of 95 °C for 5 min, to be followed by 35 cycles of 95 °C for 30 sec, 55-60 °C (depending on the primer combination) for 30 sec, and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The primers used for amplification of target DNA sequences (including the necessary restriction enzyme sites at the ends of the amplicon) are shown in Table 8. The rice actin promoter (OsActV2) and Arabidopsis terminator (AtT6) are synthesized and cloned into the pUC57 vector. The promoter and terminator elements with the correct restriction sites are then amplified using PCR using synthesizer-provided vector as template, following the same PCR conditions as described above, using the primers described in Table 9 (SEQ ID NOs:9-12). All PCR products and digested vector fragments are purified from a 1% TAE/agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Germantown, MD).

TABLE 8

Primers for amplifying target sequences

Amplification SEQ ID Sequence

target SEQ ID NO NO:

6 13 (F) gactagtcta gactcgagct ggtgccttgc cctgtggtcc 40

14 (R) gactagatcg atggtacccc tttgcctgcg ctcccggc 38

7 15 (F) gactagtcta gactcgagtc gaggaggagt tcgccaagc 39

16 (R) gactagatcg atggtacctt acatcagtcc atacaggcta aaccac 46

8 17 (F) gactagtcta gactcgagtc gatcggcaga ttcattctag c 41

18 (R) gactagatcg atggtaccga catgaacctc gaaattgtat gtatac 46

TABLE 9

Promoter and terminator PCR primers

SEQ ID NO: Sequence

9 (F) gactaggagc tcatcgaggt cattcatatg cttgagaag 39

10 (R) gactagctcg agcgtctacc tacaaaaaag ctccgca 37

11 (F) gactagtcta gaagcttctt ttaagatggg atgtctttaa 40

12 (R) gactaggcgg ccgcacattt agggtcagtt tttttggtcg 40

1.4 Vector Construction A modified version of the pHANNIBAL vector (Wesley et al., 2001) is created in order to incorporate the desired DNA elements for the BMS1 RNAi experiments. DNA elements are swapped out of or added to the original pHANNIBAL vector one by one via iterative digestion and ligation steps, extensively described in (Green et al., 2012), and in the New England BioLabs Catalog and Technical Reference Manual, 2012. First, the CaMV 35S promoter from pHANNIBAL is replaced with the OsActV2 promoter by digestion with Sac\ and Xho\ restriction enzymes, making the vector pHan-OsAct. Next, the OCS terminator from pHan-OsAct is replaced with the AtT6 terminator by digestion with Xba\ and Not\, making the RNAi base vector pHan-OsAct-AtT6. At this point, a plant selectable marker cassette (for example, the Yatl promoter driving expression of the Nptll gene for Geneticin resistance) can be inserted into the pHan-OsAct-AtT6 plasmid at the Not\ restriction site or, alternatively, an additional plasmid containing a plant selectable marker cassette may be co-bombarded with pHan- OsAct-AtT6. Plasmid pHan-OsAct-AtT6 thus consists of the OsActV2 promoter upstream of the PDK intron, which is itself flanked by two unique multiple cloning sites (Aval, Xho\, EcoR\, and Kpn\ upstream of the intron and Cla\, Hind\\\, BamW\, and Xba\ downstream), followed by the AtT6 terminator. The three different BMSl target fragments are generated by PCR with unique restriction sites designed into the primers, allowing for directional cloning of the fragments into pHan-OsAct-AtT6. The sense fragment (5' -> 3') of each target is inserted into Xho\ and Kpn\ sites of pHan-OsAct-AtT6, upstream of the PDK intron. Next, the antisense fragment (3' -> 5') is inserted into the Cla\ and Xba\ sites of pHan-OsAct- AtT6, downstream of the PDK intron. The three final vectors for the RNAi experiments, pHan-BMSl- 5'UTR, pHan-BMSl-ORF and pHan-BMSl-3'UTR, can be seen in Figure 1. The complete vectors are confirmed by sequencing.

[00135] In addition to the three RNAi vectors previously described, we will produce another version of the RNAi cassettes using the native promoter of BMSl to silence the gene in the tissues it would normally be expressed in. Additionally, we will also produce a version of the RNAi cassettes using a flower-specific promoter. This will be crucial if constitutively silencing the BMSl gene is particularly detrimental to plant development or even lethal.

Example 2 Production of transgenic sorghum with down-regulated BMSl expression

[00136] In order to obtain transgenic plants with down-regulated BMSl, we will transform sorghum with the three BMSl RNAi vectors described in Example 1. In addition to transforming sorghum with the BMSl RNAi vectors, we will also transform control plants with the base vector, pHan-OsAct-T6. We will use either particle bombardment (and co-bombard the pHan-BMSl vectors with a second plasmid containing the plant selection cassette Yatl:Nptll:AtT6) or Agrobacterium-mediated transformation (after subcloning the RNAi cassettes into a binary vector suitable for Agrobacterium-mediated transformation) to introduce the RNAi vector DNA into the genome of wild-type sorghum. These standard techniques are well known to those of skill in the art. Potentially transformed events will be cultured under Geneticin selection consisting of 20mg/L G418 for two weeks, then 40mg/L G418 for two weeks, and finally 60mg/L G418 for a further two weeks. Resistance to this antibiotic is conferred by the plant selectable marker that will be co-bombarded with pHan-BMSl-5'UTR/ORF/3'UTR plasmids, so any untransformed tissue should be killed on the selective agar plates. Selective pressure will be maintained through the stages of regeneration and rooting to ensure a minimum number of escapes. Regenerated callus and subsequent plants will be screened for the RNAi cassette by PCR using the primers of SEQ ID NOs found in Table 10. The same DNA extraction and PCR techniques described in Example 1.3 will be used for screening the transgenic events.

TABLE 10

Event screening primers

SEQ ID NO: Sequence

19 (F) ttttttttcc gtctcggtct eg 22

20 (R) gatcttgcgc tttgttatat tagca 25

[00137] Example 3. Characterization of transgenic plants

[00138] After potential transgenic events have been screened for the RNAi cassette using the primers of SEQ ID NOs: 19 and 20, they will be transferred from selective in vitro culture to soil and maintained until maturity in a controlled environment. Throughout development, the T₀ lines of transgenic plants, including all three of the BMS1 RNAi lines and the control lines containing the empty base vector will be constantly monitored for phenotypic differences. Since BMS1 is only expressed in developing spikes in wild-type plants, we do not expect to see any phenotypic differences between the control and experimental plants during vegetative development. Upon the transition from vegetative to reproductive growth, the control and RNAi lines will be carefully examined for any differences in inflorescence, spikelet or floret morphology. Inflorescences at early stages of development will be examined under a microscope to assess developmental differences between control and experimental lines. Specifically, the nature of the pedicellate florets in the RNAi lines will be carefully noted. Nearly mature inflorescences will be reproductively isolated (bagged) prior to anthesis to ensure self- pollination. At reproductive maturity, the number of fertile sessile florets and fertile pedicellate florets per spike will be quantified and compared between the control and RNAi lines. After self-pollination, seed set will be carefully assessed. In addition to quantifying seed set in the transgenic lines, we will also assess the quality of the seed. Grain size and grain viability will be compared between the transgenic and control lines. These observations will show whether any RNAi-induced fertile pedicellate florets are able to generate viable seed. At reproductive maturity, we expect that the RNAi lines will produce statistically more fertile pedicellate florets than the control lines. Furthermore, we expect the RNAi lines to produce statistically more grain than the control lines, which will show that at least a significant number of the pedicellate florets were fully fertile.

Example 4. Molecular characterization of transgenic plants

[00139] In order to confirm that the transfected RNAi cassettes are functional in the transgenic plants, we will assay transcript abundance of BMSl by RT-PCR in the RNAi and control lines. Various tissue types will be harvested from developing and mature plants from both transgenic and control lines. We will include the following tissue types in the RT-PCR assay: developing leaves, mature leaves, mature stem, developing entire inflorescences, developing sessile florets, developing pedicellate florets, mature sessile florets, and mature pedicellate florets. RNA will be extracted from these tissues using the RNeasy Plant Mini Kit (Qiagen). Using the RNA as template, cDNA and subsequent RT-PCR products will be generated in a single step using the OneStep RT-PCR Kit (Qiagen). The primers for the BMSl RT-PCR product (Table 11) were designed to be specific to BMSl (they will not amplify other HD-ZIP genes from sorghum) and they were designed to span the first intron of BMSl, thus preventing amplification from genomic DNA. Also, the primers were designed to amplify a region of the ORF of BMSl that was not used as the RNAi target, in order to avoid any possible amplification from transcripts derived from the trangene from the pHan-BMSl-ORF construct.

TABLE 11

BMSl RT-PCR primers

SEQ ID NO: Sequence

21 (F) ctacgtggac tcctcctcat ccct 23

22 (R) ctacctgctc gtcgctcagc c 21

[00140] We anticipate that there will be low to no RT-PCR amplification in any of the vegetative tissues in either the control lines or the RNAi lines. This is because BMSl is not normally expressed in these tissues in wild-type plants, so there will be no endogenous transcript for post-translational gene silencing to act upon. However, in the reproductive tissues, we expect to see a difference in RT-PCR amplification. In the control lines, we anticipate a strong BMSl signal in the developing entire inflorescences and developing pedicellate florets and we anticipate a strong to weaker signal in the mature pedicellate florets. This is because if BMS1 is a floral repressor, we expect it to be expressed in florets that will end up sterile at maturity (the pedicelled florets). Furthermore, in the control lines, we do not expect to see a signal in the developing sessile florets or mature sessile florets, because these florets are fully fertile. On the other hand, in the NAi lines, if we have fully blocked BMS1 transcript accumulation via PTGS, we anticipate that there will be no BMS1 signal detected in any tissue.

Combined with the phenotypic characterization described above, we will show that a lack of BMS1 transcript will correlate with an increase in number of fertile pedicellate florets and a subsequent increase in grain number in the BMS1 RNAi lines.

[00141] Alternatively, antibodies that specifically bind the BMS1 polypeptide will be used to evaluate BMS1 gene expression and to determine the overall efficiency of the RNAi vector in the plant cell.

Antibodies to BMS1 polypeptides may be obtained by immunization with purified BMS1 polypeptide or a fragment thereof, or with BMS1 peptides produced by biological or chemical synthesis. Suitable procedures for generating antibodies include those described in Hudson and Hay, Practical Immunology, 2nd Edition, Blackwell Scientific Publications (1980).

[00142] Polyclonal antibodies directed toward a BMS1 polypeptide generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of BMS1 polypeptide or BMS1 peptide and an adjuvant. After immunization, the animals are bled and the serum is assayed for anti-BMSl polypeptide antibody titer.

[00143] Monoclonal antibodies directed toward a BMS1 polypeptide are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridism methods of Kohler et al., Nature, 256:495-497 (1975) and the human B-cell hybridism method, Kobo, J. Immune., 133:3001 (1984); Brooder et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987).

TABLE OF ABBREVIATIONS

Abbreviation Term Abbreviation Term

VRS Barley six row AP Alkaline phosphatase

BMS Bicolor maintainer of sterility LacZ Beta galactosidase

USDA United States Department of GUS Beta glucuronidase

Agriculture

PSD Production, Supply and CAT Chloramphenicol

Distribution acetyltransferase

INT INTERMEDIUM GFP Green fluorescent protein

RT-PCR Real-time polymerase chain HRP Horseradish peroxidase reaction

RNAi RNA interference Luc Luciferase

amiRNA Artificial microRNAs NOS Nopaline synthase

PTGS Post-transcriptional gene OCS Octopine synthase

silencing

siRNA Small interfering RNA PTGS Post-transcriptional gene silencing

RISC RNA-induced silencing complex

RdRP RNA-dependent RNA

polymerase

hpRNA Hairpin RNA

CaMV Cauliflower Mosaic Virus

UTR Untranslated region

LB Left border

RB Right border

TALENs Transcription Activator-like

Effector Nucleases

AHAS Acetohydroxyacid synthase All references are herein incorporated in their entireties. REFERENCES

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Claims

CLAIMS We claim

1. A method for converting at least one sterile pedicular floret to a fertile floret in sorghum, the method comprising introducing a polynucleotide construct into a sorghum plant cell that upon activation or expression, converts at least one sterile pedicular floret to a fertile floret in a sorghum plant comprising the transgenic sorghum plant cell.

2. The method of claim 1, wherein the construct decreases the level of BMSl expression in a sorghum plant comprising the transgenic sorghum plant cell compared to the level of BMSl expression in a control, non-transgenic sorghum plant.

3. The method of claim 2, wherein decreasing the level of BMSl expression comprises reducing the level of an m NA in the sorghum plant comprising the transgenic sorghum plant cell, wherein the mRNA is encoded by a polynucleotide having at least 70% sequence identity to a nucleic acid sequence of SEQ ID NO:4, and by expression of an RNAi vector comprising a fragment of at least 20 contiguous nucleotides of a sequence having at least 90% sequence identity to SEQ ID NO:4.

4. The method of claim 3, wherein the RNAi vector comprises a polynucleotide having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:6-8.

5. The method of any one of claims 3-4, further comprising the step of screening the sorghum plants comprising the transgenic sorghum plant cell for a reduction of BMSl expression by comparing the BMSl expression in the transgenic plant to a control, non-transgenic sorghum plant.

6. A method of creating a sorghum B line wherein at least one sterile pedicular floret is converted to a fertile floret comprising the steps of

creating Fl plants comprising crossing a sorghum elite B line to a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret;

selfing the Fl plants to create F2 plants and selecting for those plants wherein at least one sterile pedicular floret is converted to a fertile floret; and

selfing the selected F3 plants and selecting for desirable agronomic traits, thereby recovering B line plants, wherein at least one sterile pedicular floret is converted to a fertile floret.

7. The method of claim 6, further comprising selfing the B line plants to create progeny; and crossing the progeny plants to a sorghum cytoplasmic male Al sterile line.

8. A method of making sorghum R line wherein at least one sterile pedicular floret is converted to a fertile floret, comprising

crossing a sorghum R line with a sorghum line wherein at least one sterile pedicular floret is converted to a fertile floret to create Fl plants;

selfing the Fl plants to create F2 plants, and selecting for those F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret;

selfing the F2 plants wherein at least one sterile pedicular floret is converted to a fertile floret to create F3 plants.

9. The method of claim 8, further comprising selfing F3 plants to create F4 plants.

10. A method of producing a hybrid sorghum seed, the method comprising the steps of crossing the B line of claim 6 with the R line of claim 8, and recovering the produced hybrid seed.

11. A method of testing the sorghum B line of claim 6, comprising crossing the sorghum B line with an R line and assessing phenotype and yield.

12. A method of amplifying seed of the sorghum R line of claim 8, comprising selfing the sorghum R line and recovering the seed.

13. A method of amplifying seed of the sorghum B line of claim 6, comprising crossing the sorghum B line with an A line and recovering the seed.

14. The method of claim 12 or 13, wherein the amplification is sufficient for pilot testing of a sorghum hybrid.

15. The method of claim 12 or 13, wherein the amplification is sufficient for commercial production of a sorghum hybrid.

16. The method of claim 12 or 13, wherein the amplification is performed in a winter nursery.