Sunflower breeding and current prospects

Sunflower
Group 1
Brody Deaton
Hrithik Mangla

Introduction
• Genus: Helianthus
• One of the four most important oil
crops globally
• Originated in North America,
domesticated around 1000 B.C.
• Uses: edible oil, meal, industrial
application, non-oilseed, forage
P u t n a m , D . H . , O p l i n g e r , E . S . , H i c k s , D . R . , B u r g a n , B . R . ,
N o e t z e l , D . M . , M e r o n u c k , R . A . , D o l l , J . D . , & S c h u l t e , E . E .
( n . d . ) . A l t e r n a t i v e F i e l d C r o p s M a n u e l . S u n f l o w e r . R e t r i e v e d
N o v e m b e r 2 1 , 2 0 2 2 , f r o m
h t t p s : / / w w w . h o r t . p u r d u e . e d u / n e w c r o p / a f c m / s u n f l o w e r . h t m l
2
E n c y c l o p æ d i a B r i t a n n i c a , i n c . ( n . d . ) . S u n f l o w e r . E n c y c l o p æ d i a B r i t a n n i c a . R e t r i e v e d N o v e m b e r 2 1 , 2 0 2 2 , f r o m
h t t p s : / / w w w . b r i t a n n i c a . c o m / p l a n t / s u n f l o w e r - p l a n t
https://www.oregonlive.com/hg/201
4/08/sensational_sunflowers.html

Sunflower Producers
COUNTRY
• Russia
• Ukraine
• European Union
PERCENT OF WORLD
PRODUCTION
• 33
• 20
• 18
PRODUCTION
(1000MT)
• 17,000
• 10,100
• 9,475
https://ipad.fas.usda.gov/cropexplorer/cropview/commodityView.aspx?cropid=2224000
2/3/20XX 3

Sunflower Uses
• Black-shelled oilseed
varieties
• Peredovik
• Aritar-93
• Hysun-33
• White or grey stripped
• Human and animal consumption
• China 1
• BlackHungary
• Kolos 1
https://iowaagliteracy.wordpress.com/2017/07/03/whats-the-difference-sunflowers/
2/3/20XX 4
Ahmad, I., Jadoon, S. A., Said, A., Adnan, M.,
Mohammad, F., & Munsif, F. (2017). Response of
sunflower varieties to NPK fertilization. Pure and Applied
Biology (PAB), 6(1), 272-277.
Hladni, N., Terzić, S., Mutavdžić, B., & Zorić, M. (2017).
Classification of confectionary sunflower genotypes based on
morphological characters. The Journal of Agricultural Science,
155(10), 1594-1609.

Sunflower oil
https://www.ers.usda.gov/data-products/chart-
gallery/gallery/chart-detail/?chartId=104023
2/3/20XX 5

Helianthus
• Most common: Helianthus annus
• Helianthus genus comprises over 70
species
• 12 annual species = diploid
• 37 perennials = 25 diploid, 3 tetraploid,
6 hexploid, 3 mixaploid
6
https://www.oregonlive.com/hg/201
4/08/sensational_sunflowers.html
Jovanka, A. (2004). ROLES OF INTERSPECIFIC HYBRIDIZATION AND CYTOGENETIC STUDIES IN
SUNFLOWER BREEDING/EL PAPEL DE LA HIBRIDIZACIÓN DE INTERSPECIES E
INVESTIGACIONES CITOGENÉTICAS EN LA SELECCIÓN DE GIRASOL/RÔLES DE L’HYBRIDATION
INTERSPÉCIFIQUE ET DES ÉTUDES CYTOGÉNÉTIQUES DANS LA CULTURE DU TOURNESOL.
Helia, 27(41), 1-24.

Sunflower Cytogenetics
• Helianthus (n=17) genus is a polyploid complex composed of diploids
(2n=2x=34), tetraploids (2n=4x=68), and hexaploids (2n=6x=102) (Jovanka,
2004).
• Linkage group and individual chromosome number’s relationship is unknown,
due to lack of genetic stock and inconsistent cytogenetic studies (Feng et al.,
2013)
• H. annus: diploid 2n=34 (used for oil seed)
• H. hirsutus: tetraploid 2n=68 (wild species)
• H. tuberosus: hexaploidy 2n=102 (cultivated for its tuber)
Feng, J., Liu, Z., Cai, X., & Jan, C. C. (2013). Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by
landed BAC/BIBAC clones. G3: Genes| Genomes| Genetics, 3(1), 31-40.
Jovanka, A. (2004). ROLES OF INTERSPECIFIC HYBRIDIZATION AND CYTOGENETIC STUDIES IN SUNFLOWER BREEDING/EL PAPEL DE LA
HIBRIDIZACIÓN DE INTERSPECIES E INVESTIGACIONES CITOGENÉTICAS EN LA SELECCIÓN DE GIRASOL/RÔLES DE L’HYBRIDATION
INTERSPÉCIFIQUE ET DES ÉTUDES CYTOGÉNÉTIQUES DANS LA CULTURE DU TOURNESOL. Helia, 27(41), 1-24.
7
This Photo by Unknown Author is licensed under CC BY-NC

Cytogenetic Map (Feng et al., 2013)
• BAC clones LG15 and LG18 are on the same chromosome
• LG15 and LG18 are subtelocentric chromosomes
• RFLP maker LG12 and LG17 are in the same linkage group SSR-LG11
Feng, J., Liu, Z., Cai, X., & Jan, C. C. (2013). Toward a molecular cytogenetic map for cultivated sunflower (Helianthus
annuus L.) by landed BAC/BIBAC clones. G3: Genes| Genomes| Genetics, 3(1), 31-40. 2/3/20XX 8

Genomics
• Sunflower genome consists of highly repetitive DNA sequences
• Cytoplasm density around the chromosome limits signal intensity and physical
chromosome position
• In situ hybridization with BAC/BIBAC clones eliminate background noise
(Feng et al., 2013).
• 44 clones contain 18 RFLP linkage groups (Feng et al., 2013)
annuus L.) by landed BAC/BIBAC clones. G3: Genes| Genomes| Genetics, 3(1), 31-40.
https://www.google.com/imgres?imgurl=https%3A%2F%2Fcdn.wikifar
mer.com%2Fwp-content% 9
annuus L.) by landed BAC/BIBAC clones. G3: Genes| Genomes| Genetics, 3(1), 31-40.

Structural Make-up of Sunflower
• Morphological characters are affected by
environment and genotype, comparison should
be made using HA 89 due to its wide use as a
breeding line (Seiler, 1997).
MORPHOLOGY AND ANATOMY
• Stem and Branching
• Roots
• Inflorescence and Flowering
• Anther and Pollen Development
• Achenes
Seiler, G. J. (1997). Anatomy and morphology of sunflower. Sunflower technology and
production, 35, 67-111.
10
Brittanica web image

STEM
ROOTS
11

Inflorescence
https://www.auburn.edu/cosam/news/articles/2022/08/auburn_plant_biologists_seek_to_better_understand
_sunflower_development_and_evolution_with_750,000_nsf_award.htm
2/3/20XX 12

Anther and Pollen Development
https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.17627
https://www.grainsa.co.za/sunflowers-and-its-stages-
of-development
13

Achenes
https://science.jburroughs.org/resources
/flower/fruit3.html
14

Sunflower Breeding
methods-
• Hybridization and selection
• Mutation Breeding
• Population improvement and inbred lines
development
• Heterosis Breeding
• Marker assisted Selection
• Somatic Hybridization
• Transgenic Sunflower
This Photo by Unknown Author is licensed under CC BY-SA-NC
This Photo by Unknown Author is licensed under CC BY-NC-ND

HYBRIDIZATION AND SELECTION
• Prebreeding germplasms.
• Embryo culture and Chromosome doubling by Colchicine has facilitated the crosses between
WRs(outcrossing) and cultivated hybrids.
• Identification of cms sources.
• Herbicide Resistance – Imidazoline resistant lines to control Broomrape infestation. Sulfonylurea
Resistant lines used to control the broadleaf weeds.(Miller and Al-Khatib,2004)
• Disease Resistance – Wild H. Annus is a source of resistance to Alternaria leaf blight, Phomopsis
Blight and the rust disease.
Sample Footer Seiler, G.J., Qi, L.L. and Marek, L.F. (2017), Utilization of Sunflower Crop Wild Relatives for
Cultivated Sunflower Improvement. Crop Science, 57: 1083-1101. https://doi.org/10.2135/cropsci2016.10.0856
Text
Miller, J. and Al-Khatib, K. (2004), Registration of Two Oilseed Sunflower Genetic Stocks, SURES-1 and
SURES-2 Resistant to Tribenuron Herbicide. Crop Science, 44: 1037-
1038. https://doi.org/10.2135/cropsci2004.1037

MUTATION BREEDING
• Mutagenesis complements the natural genetic variations for the continual growth of crop improvement.
• Imidazoline tolerant variety CLEARFIELD PLUS was developed by selection from an EMS treated M2
population of 1200000 plants.(Sala et. al 2008)
• TILLING (Target Induced local lesion in Genomes) was implemented in EMS treated populations to study the
genes associated with fatty acid biosynthesis.(Sabetta et. Al 2011). Later Kumar et al. in 2013 identified SNPs
in genes like fat4 and SAD using optimized mutagenized TILLING techniques.
Sala, C.A., Bulos, M. and Echarte, A.M. (2008), Genetic Analysis of an Induced Mutation Conferring Imidazolinone Resistance in Sunflower. Crop
Sci., 48: 1817-1822. https://doi.org/10.2135/cropsci2007.11.0625
Dimitrijevic A and Horn R (2018) Sunflower Hybrid Breeding: From Markers to Genomic
Selection. Front. Plant Sci. 8:2238. doi: 10.3389/fpls.2017.02238
This Photo by Unknown Author is licensed under CC BY

60000 EMS treated and rinsed in sodium thiosulphate
seeds of BTK47 [M1] sown in field.
20 seeds/capitulum of M1 plants sown in greenhouse,
treated with Imazypyr at V2-V4 stage,8 M2 plants
selected, selfed and harvested individually
20 plants/capitulum of M2:3 selected plants grown in
greenhouse, treated and 1 family selected,
Resistant phenotypes were selfed and harvested
individually
6 M2:4 lines grown in field, screened,
selfed and harvested individually.
Clearfield plus (closely
resembling BTK47)
BTK47 used as a Check
Sala, C.A., Bulos, M. and Echarte, A.M. (2008), Genetic Analysis of an Induced Mutation Conferring Imidazolinone Resistance in Sunflower. Crop
Sci., 48: 1817-1822. https://doi.org/10.2135/cropsci2007.11.0625

POPULATION IMPROVEMENT AND INBRED LINES
DEVELOPMENT
• Sunflower being a Cross pollinated crop utilizes all of the breeding methods of the population improvement like
recurrent selection methods and mass selection. Theses methods provide elite germplasm for development of
synthetics and superior inbred lines for hybrid breeding.
• Pureline method is used to develop the inbred lines and the backcross methods are used for introgressing
desired resistance or major genes .
• Pedigree selection was used to develop the inbred lines with acquired resistance for Charcoal Rot in elite lines
from a wild H. agrophyllos source after several continuous selections from F2 to F4.(Shehbaaz et al. 2018)
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic Improvement. In: Al-Khayri, J., Jain, S.,
Johnson, D. (eds) Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham.
https://doi.org/10.1007/978-3-030-23265-8_16
Shehbaz, M., Rauf, S., Al-Sadi, A.M. et al. Introgression and inheritance of charcoal rot (Macrophomina
phaseolina) resistance from silver sunflower (Helianthus argophyllus Torr. & A. Gray) into cultivated sunflower
(Helianthus annuus L.). Australasian Plant Pathol. 47, 413–420 (2018). https://doi.org/10.1007/s13313-018-
0573-9

HEAD TO ROW AND REMNANT SEED METHOD
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic Improvement. In: Al-Khayri, J., Jain,
S., Johnson, D. (eds) Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham.
https://doi.org/10.1007/978-3-030-23265-8_16
Modified Recurrent selection
Cultivar “Peredovik”was
released in 1958

HETEROSIS BREEDING
• Discovery of cytoplasmic male sterility source in H. petiolaris by Leclereq in 1969 and the fertility restorer
genes by Kinman in 1970.
• Hybrids have higher yield performance than OPVs and synthetics due to hybrid vigour.
• In most of the hybrid seed production systems, only single source of cms PET-1 is used. Need to develop more
proficient cms sources to reduce the vulnerability of cms source and possibility of disease outbreaks
• Hybrid seed production system of sunflower contains a ratio of 4:2 of female to male lines and pollination is
facilitated through honeybee hives ( 2 hives /ha)
Wani, Shabir & Saini, Hitesh & Gupta, Vikas & Bhat, Muhammad. (2010). Present Status and Future Prospects for
Heterosis Breeding in Sunflower (Helianthus annuus L.).. Asian J. Sci.Tech. 2. 49-54.
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic
Improvement. In: Al-Khayri, J., Jain, S., Johnson, D. (eds) Advances in Plant Breeding
Strategies: Industrial and Food Crops. Springer, Cham. https://doi.org/10.1007/978-3-
030-23265-8_16

NEW CMS SOURCE AND FERTILITY RESTORER GENES
Feng, J., Jan, CC. Introgression and molecular tagging of Rf 4, a new male fertility restoration gene from wild sunflower Helianthus maximiliani L.. Theor Appl Genet 117, 241–249 (2008).
• CMS source designated as CMS GIG2
• Novel fertility restorer genes rf4

Chigeza, G., Mashingaidze, K. & Shanahan, P. Advanced cycle pedigree breeding in sunflower. II: combining ability for oil yield and its components. Euphytica 195, 183–195 (2014).
https://doi.org/10.1007/s10681-013-0985-0
ADVANCED CYCLE PEDIGREE BREEDING
Random mating in F2 followed
by 3 generations of selfing and
selection
So far largest line × tester analysis

MARKER ASSISTED SELECTION
• Various markers have been validated to be linked with traits like Oleic
acid content(ORS-728, F4-R1),disease resistance, oil content etc.
• Lili et al. used SSRs and SNP markers linked to resistance genes for two
diseases – Rust and Downy mildew ; for gene pyramiding purposes as
follows-
o 5 F2 populations were screened with codominant markers for
homozygousity of resistance genes.(reducing the size of F2:3 to be
grown)
o F2 derived F3 families were phenotypically tested for Disease resistance.
https://doi.org/10.1007/978-3-030-23265-8_16
Qi, Lili, and Guojia Ma. 2020. "Marker-Assisted Gene Pyramiding and the Reliability of
Using SNP Markers Located in the Recombination Suppressed Regions of Sunflower
(Helianthus annuus L.)" Genes 11, no. 1: 10. https://doi.org/10.3390/genes11010010
24

SOMATIC HYBRIDIZATION
Used for fusing genetically incompatible species to produce somatic hybrids. Steps are as follows-
1. Preparation of plant material – H.annus ( seeds) × H.maximiiani( apical shoots), sterilization and in vitro
cultures.
2. Protoplast isolation – Plasmolyzed by cell wall degrading enzymes followed by filteration (
Hypocotyl – H. annus and mesophyll in case of H. maximiliani)
3. Electrofusion – 3 Pulses of 1250V/cm on equal densities of protoplasts.
4. Protoplast culture and plant regeneration- embedded in agarose droplets  Microcalluses transfer to solid
differentiation media  KR-R_Ag medium  Shoots excised  Rooting  Growth chambers
5. Isozyme and RAPD Analysis  Screening for hybrids.
6. Disease resistance  Screening for sclerotinia tolerance in obtained hybrids
Strategies: Industrial and Food Crops. Springer, Cham. https://doi.org/10.1007/978-3-
030-23265-8_16
Taski-Ajdukovic K, Vasic D, Nagl N (2006) Regeneration of
interspecific somatic hybrids between Helianthus annuus L. and
Helianthus maximiliani (Schrader) via protoplast electrofusion.
Plant Cell Rep 25(7):698–704
This Photo by Unknown Author is licensed under CC
BY

Transgenic Sunflower
• Difficult to transform via Agrobacterium mediated techniques due to various limitations like chimeric expression,
regeneration of viable shoots( transformation frequency is very low<0.06-0.52%)
• Despite the difficulties the Agrobacterium-mediated technique is the preferred one followed by Particle
bombardment.
• Radonic et al. used this protocol to transform antifungal genes into Sunflower to induce Disease resistance .
• The gene encoding cis-prenyltransferase was used to transform sunflower for production of latex.(Hallahan and
Keiper-Hrynko 2007)
https://doi.org/10.1007/978-3-030-23265-8_16
Radonic LM, Zimmermann JM, Zavallo D et al (2008)
Introduction of antifungal genes in sunflower
via agrobacterium. Electron J Biotechnol 11(5):8–9

Breeding
objectives
Depends upon the area of production
and the breeding program

Oil content
• Quantitatively inherited and medium heritability(0.57). Ranges between 30-50 %. Improved by early breeding
methods employed by Pustovoit.
• A smaller seed with higher embryo size and thin testa gives larger oil recovery .
• Depends on oil bodies concentration rather than larger oil bodies.
• Improved oil yield was reported in relation to increase in harvest index, higher kernel to achene ratio, higher
kernel oil concentration.
• Negatively related to the protein content.
Lopez Pereira. (2000). Genetic improvement of sunflower in Argentina between 1930
and 1995. Part III. Dry matter partitioning and grain composition. Field Crops Research.,
67(3).
Improvement. In: Al-Khayri, J., Jain, S., Johnson, D. (eds) Advances in Plant
Breeding Strategies: Industrial and Food Crops. Springer, Cham.
https://doi.org/10.1007/978-3-030-23265-8_16
This Photo by Unknown Author is licensed under CC BY-SA

Broomrape
• Orobanche cumanais is an obligatory
parasitic plant having different races
originated in several areas of the world
• Causes upto 80% of yield losses.
• Resistance to orobanche races is found in
several Wild sunflower species.
• Resistance genes are annotated as
o1,o2,o3,o4,o5 for races 1 to 5 respectively.
Amri, Moez & Abbes, Zouhaier & Youssef, Salah & Bouhadida, Mariem & Salah, Hamadi & Kharrat, Mohamed. (2012). Detection of the parasitic plant,
Orobanche cumana on sunflower (Helianthus annuus L.) in Tunisia. AFRICAN JOURNAL OF BIOTECHNOLOGY. 11. 4163-4167.
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic Improvement. In: Al-Khayri, J., Jain, S., Johnson, D.
(eds) Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham. https://doi.org/10.1007/978-3-030-23265-
8_16

Disease Resistance
RUST POWDER MILDEW DOWNY MILDEW
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic Improvement. In: Al-Khayri, J., Jain, S., Johnson, D. (eds)
Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham. https://doi.org/10.1007/978-3-030-23265-8_16
Image Source - https://www.ndsu.edu/agriculture/sites/default/files/2021-
05/dusty%20cinnamon-brown.png
Image Source - https://live.staticflickr.com/7576/16116832188_0f615db7d0_b.jpg Image Source - https://www.ndsu.edu/agriculture/sites/default/files/2021-
05/dusty%20cinnamon-brown.png

ABIOTIC STRESS TOLERANCE
DROUGHT HEAT STRESS
Strategies: Industrial and Food Crops. Springer, Cham. https://doi.org/10.1007/978-3-030-
This Photo by Unknown Author is licensed under CC BY-ND
Image source-https://www.cypresscreeklandscapesupply.com/wp-content/uploads/2019/07/bigstock-Sunflower-
77693630.jpg
This Photo by Unknown Author is licensed
under CC BY-SA
• Production constraint.
• Assimilation and Mobilization
• Hairiness, cuticular wax and small leaf
area
• Canopy temperature depression
• Early maturing
• Gametophytic sterility
• >30℃
• Downward head position and erect
leaves

OIL QUALITY TRAITS
2/3/20XX
Sample Footer Text
Tocopherols
and
Phytosterols
Oleic acid
Stearic acid
• Antioxidant properties
• Polygenic mode of inheritance .
• Mutation breeding and Genetic transformation
• Sunflower oil contains two major fatty acids : linoleic acid(18:2) and oleic
acid(18:1).
• Higher Linoleic acid degrades oil quality under heat stress.
• Pervenent (produced by mutation) is used in several backcrosses.
• good cooking Qualities
• More healthier than palmitic acid.
• X –rays for mutation breeding- CAS-12(55% Stearic acid), CAS -29 (24.9%
stearic acid)
Rauf, S. (2019). Breeding Strategies for Sunflower (Helianthus annuus L.) Genetic Improvement. In: Al-Khayri, J., Jain, S., Johnson, D. (eds) Advances in Plant Breeding Strategies: Industrial and Food Crops. Springer, Cham.
https://doi.org/10.1007/978-3-030-23265-8_16

Future prospects
WGS TO UNRAVEL THE DESIRABLE TRAITS
CRISPR/CAS9 TO KNOCKOUT
UNDESIRABLE REGIONS
https://www.news-
medical.net/images/news/ImageForNews_709348_16488126728996589.jpg
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tbn0.gstatic.com/images?q=tbn:ANd9GcS
vYYuRDqlQe0rS4KlttfH1Jv68QoxfyXuFtQ
&usqp=CAU
33
This Photo by Unknown Author is licensed under CC BY-SA

BREEDING FOR CONFECTIONARY AND
BIOFUEL PURPOSES
BETTER PROTOCOL FOR
GENETIC ENGINEERING
https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcSm2gQ5YcRGcbaE6ZbcHVmCmgTnlBg8RfkkTg&usqp=CAU
This Photo by Unknown Author is licensed under CC BY-SA-NC

Thank You
Presenter Name- Brody Deaton and Hrithik Mangla
Email- Brody.Deaton@uga.edu and Hrithik.Mangla@uga.edu

Sunflower breeding and current prospects

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