Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. More than half of the production of major crops such as maize, rice, sorghum, rapeseed, and sunflower comes from hybrid varieties.

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Hybrid
Heterosis
Conventional breeding
Molecular breeding
Biotechnological
approaches (Transgenic)
Genome editing tools
Seed Production
Technology (SPT)
Male Sterility / Fertility
Molecular Control of Male Fertility
for Crop Hybrid Breeding

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Outline
 Hybrids cops to increase agricultural yield
 Types of male sterility
 Approaches to create male-sterile female lines
 Genetic resources to exploit heterosis in crop production
 Mitochondrial genes that control CMS
 Cytoplasmic–Nuclear interaction in CMS restoration
 The Seed Production Technology (SPT) platform
 Case study
 Summary

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Charles Darwin
 Best known for his contributions to the science branching
pattern of evolution.
 Resulted from a process that he called natural selection, in
which the struggle for existence has a similar effect to
the artificial selection involved in selective breeding.
 In the 1860s, first described heterosis in maize (Zea mays).
Gregor Mendel
 Genetic basis for inherited traits.
 Maize became the first commercialized hybrid seed crop.
Successively rice, barley, rapeseed etc.
Joseph Gottlieb Kölreuter
 The first documentation of male sterility in 1763.
 Observed heterosis, that hybrids surpassed their parents.

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 Tremendous contribution to increases agricultural yield.
 Hybrid plants possess superior traits such as better adaptability, greater
uniformity, and improved abiotic and biotic stress tolerance.
Wheat
(Triticum spp.)
3.5–15%
Barley
11%
Rice
55%
Common
bean
(Proteus
vulgaris)
47%
Foxtail
millet
(Setaria
italica)
68%
Brassica
oilseed
crops
200%
(Kim and Zhang., 2018)

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Producing hybrid seeds requires -
1. Cross-pollination of parental lines with genetic diversity.
2. The male-sterile plants must be maintained and propagated for
large-scale use as the male-sterile crossing partner.
3. Plant male sterility must be overcome in the F1 hybrid generation
to achieve a higher yield for crops whose seeds are harvested.

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Types of male sterility
1. Genetic Male Sterility (GMS)
1.1 Environmental Sensitive (EGMS)
a) Thermo sensitive genetic male sterility (TGMS)
b) Photoperiod sensitive genetic male sterility (PGMS)
1.2 Environmental non-sensitive
2. Cytoplasmic Male Sterility (CMS)
3. Cytoplasmic Genetic Male Sterility (CGMS)
4. Transgenic Male Sterility (TMS)

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Approaches to create male-sterile female lines
1. Chemicals :
 Auxins, anti-auxins, halogenated aliphatic acids, gibberellins, arsenicals,
ethepon applied as foliar sprays prior to flowering have been investigated
(McRae, 1985).
Drawbacks :
 Impacts on genotype and environment, as well as the logistical challenges of
accurate and timely chemical application.
 Some chemical applications reduced seed yields in excess of 20% because of
damage to the female flower (Newhouse et al., 1996).

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Approaches to create male-sterile female lines
2. Genetic manipulations :
 First biotechnology-based approaches to male sterility was proposed by
Mariani et al., (1990) and included tapetal-specific expression of a ribonuclease
gene, barnase, to cause complete male sterility.
 This technology is linked with the selectable marker phosphinothricin
acetyltransferase (PAT) conferring tolerance to the herbicidal active ingredient
glufosinate ammonium.
Drawbacks :
 Timely application of herbicide is essential to remove non transgenic plants,
which adds to costs and complexity.

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 Glyphosate-resistant corn was developed via expression of a glyphosate
insensitive 5-enolpyruvyl-shikimate 3- phosphate synthase enzyme (CP4-EPSPS).
 The resulting plant demonstrated sterile tassel following glyphosate
application with little to no injury to the rest of the plant.
 Roundup Hybridization System (RHS) replaces mechanical detasseling with
glyphosate spray and greatly simplifies the process of hybrid seed corn
production.

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Genetic Resources to Exploit Heterosis in Crop Production
 CMS and EGMS lines do not require emasculation and therefore are ideal female
lines for hybrid seed production.
Three Line Hybrid Seed Production System
(Kim and Zhang., 2018)

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Two-Line Hybrid Seed Production System
Nongken 58S (NK58S), O. sativa ssp. Japonica in 1973
completely male sterile when grown under long-day
conditions but male fertile when grown under short-day
conditions (Shi, M.S., 1985).
(Kim and Zhang., 2018)

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Mitochondrial Genes that Control CMS
 The mitochondrion is a semiautonomous organelle and power house of cell.
 The reported CMS DNA sequences encode small transmembrane proteins that
are 10–35 kDa in size and cytotoxic.
 The specific accumulation of cytotoxic CMS proteins within the tapetum,
frequently causes altered mitochondrial function such as ATP synthesis and
abnormal programmed cell death (PCD) in the sporophytic or gametophytic cells,
leading to male sterility.
 Until now 28 CMS genes have been identified from 13 crop species.
 e.g. in Rice – orf79, orfH79 etc.
(Chen and Liu., 2014)

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 CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental
interaction between mitochondrial gene and a conserved, essential nuclear gene.
Wild Abortive
CMS WA 352

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How CMS genes affect only male development . . . ?
 It may result from one of three possible types of expression patterns of CMS
genes :
1. Male organ (anther)–specific mRNA expression and protein accumulation
– NO CMS system
2. Constitutive mRNA expression and protein accumulation throughout
plant tissues – orf 129 expression in flowers, roots and leaves. But
functions only in male organ (Energy Deficiency).
3. Constitutive mRNA expression but specific or preferential protein
accumulation in the male organs (anthers).

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A model for mitochondrial-nuclear gene interactions in CMS/Rf systems.
(Chen and Liu., 2014)
CMS restoration by RF proteins can be
achieved at the G, R, P, or M level.

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Proposed a model: “Multi-recombination/protogene formation
/functionalization” mechanism .
Involves-
• Variations in the structure
• Sequence
• Copy number
• Function.
Formation and evolution of new CMS genes

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(Tang et al. 2017)
Multi-recombination/protogene formation
/functionalization mechanism .
Darwin’s
Principle of
descent with
modification
Microevolution

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Multiple rounds of recombination of the mitochondrial
genome during evolution
(Kim and Zhang., 2018)

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Cont. . .
PCMS Norin 26
Murai et al., 2016

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Cytoplasmic–Nuclear Interaction in CMS Restoration
 Rf genes from the restorer lines are nuclear genes that encode proteins
localized to mitochondria that play a retrograde regulatory role by suppressing
CMS defects.
 About half of the identified Rf genes encode pentatricopeptide repeat (PPR)-
containing proteins that are localized to mitochondria or plastids.
 In most cases each Rf functions specifically in a particular type of CMS system,
which has a unique phenotype caused by the specific type of mitochondrial DNA
and corresponding nuclear gene(s).

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 Until now 14 Rf genes have been isolated from 9 crop plants
(Chen and Liu., 2014)

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Fig - A typical structural arrangement of a PPR protein.
(Saha et al., 2007)
PPR proteins
 A typical PPR protein is targeted to mitochondria or chloroplasts, binds one
or several organellar transcripts, and influences their expression by altering RNA
sequence, turnover, processing, or translation.
Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein
families in land plants, with more than 400 members in most species.
 Functional studies on different PPR proteins have revealed their role fertility
restoration in CMS plants, embryogenesis, and plant development.

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Major functions of PPR proteins in plants
(Saha et al., 2007)

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Characterized cytoplasmic male sterility (CMS)/restorer (Rf ) gene systems in major
crops
(Chen and Liu., 2014)

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Cont. . .
Most CMS genes encodes :
• Mitochondrial ETC pathways
• Transmembrane proteins
(Chen and Liu., 2014)

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The Seed Production Technology (SPT) Platform
 The Barnase-Barstar system (Bayer) has been tried in many crops but has
encountered several problems.
 A transgenic construct-driven non- GM (genetically modified) system called
‘seed production technology’ (SPT) was recently developed by DuPont Pioneer.
 Developed a hybrid seed system that exploits a transgene but generates non-
transgenic seeds.
 The SPT platform consists of a transgenic maintainer line capable of
maintaining and propagating non transgenic nuclear male-sterile female lines for
hybrid seed production.
 This novel hybridization system allows the use of nuclear male sterility to
produce hybrid seeds in maize, rice, and other cross-pollinating crops.

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Case Study
The maize SPT maintainer line DP-32138-1 is a homozygous recessive male sterile
transformed with a SPT construct containing:
(i) A complementary wild-type male fertility gene to restore fertility
(ii) An a-amylase gene to disrupt pollination
(iii) Seed colour marker gene

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Experimental procedure
1. Maize transformation and molecular analyses of transformants
Male-sterile transformation-amenable maize lines, which contain the ms450-
9301 allele were developed by backcrossing with pollen from a
transformable maize genotype.
Immature embryos that segregated for the ms450-9301 allele were used for
transformation.
Agrobacterium-mediated transformation
2. Histochemical analysis, pollen in vivo germination and Microscopy
3. Seed sorting and sorter specifications (ScanMasterII 200 high-volume colour
sorter)
4. Real-time PCR of male-sterile progeny plants

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 Isolation of the Ms45 male fertility gene
 Gene Construct used
 pPG47::Bt1:zm-aa1 linked to DsReds under a constitutive maize ubiquitin (Ubi)
Promoter.

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Fig.- Pollen from transgenic plant at different developmental stages
Results : FDA
KI
Absence of detectable starch in pollen
with the presence of pPG47::Bt1:zm-aa1
DsReds

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Fig.- Pollen kernel characters of transgenic plant
Transgenic
Non Transgenic

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Schematic representation of the Seed Production
Technology (SPT) process

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 Extremely high efficacy of the SPT genetic process in preventing the transfer
of the SPT transgenes to the male-sterile female parents.
 Large-scale automated seed sorting can be employed to further help ensure
the purity of the nontransgenic male-sterile inbred parent seed being increased
and subsequently used for commercial scale maize hybrid seed production.
 The SPT process has the potential to dramatically impact hybrid maize seed
production as it eliminates a need for mechanical and/or hand detasseling.
Conclusion:

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Summary
 CMS proteins may interact with nucleus-encoded mitochondrial factors to
induce abnormal programmed cell death in tapeta and microspores, leading to
male sterility.
 CMS/Rf genes may be formed during evolution through gene duplication and
multi-recombination.
 The SPT process has potentially important applications for hybrid seed
production in other major crops including rice, sorghum and wheat, which have
flowers that are not amenable to manual emasculation.
 More CMS systems in staple crops need to be studied and applied in
agriculture to avoid genetic vulnerability in hybrid crop production that relies on
few CMS cytoplasms, such as CMS-WA in rice.

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https://www.pioneer.com/home/site/about/template.CONTENT/news-media/media-
kits/seed-production-technology/guid.B84F55B0-BE0B-6774-F216-69CE365F59A7
For more details on SPT -

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