1. Fertility restoration and maintenance of male sterility in different CMS sources of
sunflower (Helianthus annuus L.)
OR
Confirmation of fertility restoration through cytological (pollen) study in different CMS
sources of sunflower (Helianthus annuus L.)
H.P. Meena*and A. J. Prabakaran1
*Corresponding author: Scientist (Plant Breeding), Directorate of Oilseeds Research,
Rajendranagar, Hyderabad, India-30 (Email ID: hari9323@gmail.com)
1
Principal Scientist, Directorate of Oilseeds Research, Rajendranagar, Hyderabad, India-30
ABSTRACT
Seven cytoplasmic male sterile (CMS) lines of sunflower (Helianthus annuus L.) with
PET-1 (H. petiolaris) cytoplasm and one with IMS (H. lenticularis) cytoplasm sources were
crossed with twenty seven testers to assess their maintainer/restorer behavior. In this study we
compared two methods (visual observation vs pollen study) for classifying the
maintainer/restorer into different category. All the inbreds were categorized into maintainer and
restorer based on visual observation (pollen presence or absence) as well as through cytological
observation (pollen study). We observed that cytological study is better for testing fertility
restoration compare to visual observation. While 22 inbreds maintained sterility of CMS PET 1,
28 inbreds restored their fertility. The second CMS line and IMS 852A, was maintained by all
the inbreds indicating involvement of different gene(s). Only one inbred restored their fertility.
Most of the commercial sunflower hybrids are been produced using CMS PET 1. Now with the
identification of restorers for CMS IMS 852A, new more productive commercial hybrids can be
produced. Efforts should be made to locate restorers for CMS GIG 1 for its utilization in
production of sunflower hybrids.
INTRODUCTION
Hybrid breeding has developed successfully in sunflower over the last 40 years since the
identification of cytoplasmic male sterility among progenies of the interspecific cross Helianthus

2. petiolaris x Helianthus annuus by Leclercq (1969) and the subsequent discovery of pollen
fertility restoration genes (Kinman, 1970; Leclercq, 1971; Vranceanu and Stoenescu, 1971). This
source (PET-1 cytoplasm), of cytoplasmic male sterility has proved to be very stable and is used
almost exclusively in breeding programmes throughout the world since late 1970s, when it
replaced the NMS system for producing hybrid seeds that was being used in several European
countries till the early 1970s.
Nevertheless, frequent use of the same sterile cytoplasm increases the genetic
vulnerability of the present sunflower hybrids to diseases and pests. In order to minimize such a
risk, new sources of cytoplasmic male sterility and corresponding fertility restorers are essential
to increase the genetic diversity of the commercial hybrids. Inspite of the fact that new CMS
sources continue to be discovered (Serieys, 2002), there are hardly any reports of their utilization
for commercial hybrid production. The reluctance is presumably due to a lack of superior CMS-
restorer combinations, as well as the time consuming conversion programs of CMS and
restoration genes into inbred lines (Jan et al., 2006).
Success in heterosis breeding is largely dependent on the development of inbreds having
broader genetic base. In general, inbreds with high combining ability and per se performance are
either converted into CMS lines or fertility restorer lines for their future use in hybrid breeding
programmes. Keeping this in view superior inbreds were evaluated for their maintainer and
restorer behaviour, with the objective of identifying diverse sources of CMS maintainers and
restorers. We herein make use of the easy method proposed by Chaudhary et al. (1981), for
ascertaining the pollen fertility of crosses; leading to the identification of selected superior
inbreds as maintainers and restorers of two diverse CMS sources, for the practical use of these
inbreds in future sunflower breeding programme to augment the genetic diversity of sunflower
hybrids. The present study was under taken to find out the fertility restoration ability and
maintainer reaction of the twenty seven testers on the eight CMS lines.

3. MATERIALS AND METHODS
Plant material
The breeding material comprised of two diverse cytoplasmic male sterile sources of
sunflower viz., H. petiolaris and H. lenticularis. In this study we used seven CMS lines from
PET-1 background viz., CMS-852A, CMS-7-1A, CMS-234A, CMS-2A, COSF-1A, COSF-7A,
CMS-10A and one CMS from IMS background viz., IMS-852A maintained by DOR, Hyderabad,
and twenty seven advanced inbred lines (testers) i.e., seven inbreds viz., AKSF-51-6-21, AKSFI-
49-3, AKSFI-49-4, AKSFI-46-2, AKSFI-78, AKSFI-42-1 and AKSFI-52-2 from Akola,
(Maharashtra) center, other three advanced breeding lines i.e., HOHAL-17, HOHAL-25 and
HOHAL-37 from Ludhiana, (Punjab) centre; eight inbred lines i.e., CSFI-5134, CSFI-5055,
CSFI-5261, CSFI-5133, CSFI-5185, CSFI-5033 and CSFI-5075 from Coimbatore, (Tamil Nadu)
centre; other two inbreds i.e., RHAGKVK-1 and RHAGKVK-2 from Bangalore, (Karnataka)
center and five other advanced lines i.e., IB-50, IB-60, IB-61, IB-67 and IB-101 from DOR,
Hyderabad and Selection-I and NDLR-06 from Latur, (Maharashtra) and Nandyal,(Andhra
Pradesh) centres respectively.
Field experiment
Three rows each of the eight cytoplasmic male sterile lines from PET-1 and lenticularis
cytoplasmic backgrounds, and two rows each of the twenty seven testers (inbreds) were planted
during the rabi season of the year 2012-13, with a row to row and plant to plant spacing of 60 cm
x 30 cm. A row length of 4.5 m was maintained. Staggered sowings of male parents, twice at
weekly interval, was done to synchronize the flowering. Recommended agronomic practices
were followed. The heads of male sterile lines and the inbreds were covered with cloth bags at
the ray floret stage i.e., just before the commencement of flower opening. The eight CMS lines
from two different CMS sources were crossed to all the twenty seven inbreds in a line x tester
fashion. Crossing was done by collecting pollen from the inbreds in a petridish with the aid of a
small brush which was applied on five florets each of the corresponding CMS lines between 8 to
11 am and the procedure repeated till the opening of all disc florets. Precautions were taken to
avoid possible contamination. F1 seeds from each of the 216 crosses were collected separately at

4. maturity for assessing the fertility restoration of the 27 inbreds on the 8 CMS lines. The
identification of inbred behaviour, with respect to maintenance and restoration of the
cytoplasmic male sterile sources of sunflower involved in the present study, was conducted
during the kharif season of the year 2013-14 in the Narkhoda Research Farm, DOR, Hyderabad.
F1 seeds from the 216 crosses were planted in a randomized block design (RBD) in replicated
experiment with 2 replications. Two rows of 3 m for each F1 entry were planted maintaining a
row to row distance of 60 cm and a plant to plant distance of 30 cm.
Observation for pollen fertility and sterility
We classified hybrids as maintainer/restorer based on visual observation (pollen present
or absent) as well as through cytological study (pollen study). Based on visual observation, the
pollen parents leading to sterile crosses were classified as maintainers, while those that gave
fertile crosses were classified as restorers of the corresponding CMS lines. Pollen fertility
percentage was calculated by classifying pollen grains as sterile or fertile following
(Chaudharyet al., 1981). For pollen study anthers were collected from all the fertile F1 hybrids.
Pollen grains were treezed out of the anther on glass slide. The fertile and sterile pollen grains
were counted under a light microscope. The pollen fertility was calculated as the ratio between
the number of fertile (round and darkly stained) and sterile (yellow, sheveal, partial stained or
unstained) pollen grain in the microscopic field (Figure 1). Based on fertility, plants were
classified effective as restorers (> 90% pollen fertility), Partial restorers (20-80% pollen fertility),
partial maintainer (1-20% pollen fertility) and effective maintainers (< 1% pollen fertility or no
pollen).
RESULTS AND DISCUSSION
It can be shown from Table 1 that twenty two inbreds namely; RHA 348, 7-1 B, 234 B,
302 B, 378 B, 851 B, 852 B, HA 341, HA 380, GP 290, GP 2008, GP 2111, GP 761, GP 898, M
307-2, M 1008, M 1015, M 1026, DRM 34-2, DRM 70-1, NDOL 87 and LTRR 1 produced
sterile F1s on the CMS PET 1 and CMS ARG 1 sources. Further, all fifty inbreds produced
sterile F1s on CMS GIG 1 as well. Though a minute fraction of aborted pollens (sterile pollens)
was also observed, it can be seen from the Table 1 that twenty eight (RHA 271, RHA 273, RHA

5. 274, RHA 297, RHA 298, RHA 341, RHA 344, RHA 345, RHA 346, RHA 356R, RHA 587,
RHA 859, RHA 6D-1, HAM 161, HAM 174, HAM 175, HAM 180, SF 206, SF 207, SF 208, SF
211, SF 216, BLC P6, PARRUN 1329, RES 834-1, RCR 8297, R 83 R6 and NDLR-1) out of
fifty inbreds produced sufficient fertile F1s with CMS PET 1 and CMS ARG 1. The inbreds
which produced sterile F1s were classified as maintainers, while the ones that produced fertile
F1s were classed as restorers of the respective CMS sources (Table 2).
The present findings agree with the conclusion of Spirova (1990), regarding the
infrequency observed for fertility restoration. This is more obvious in case of CMS GIG 1;
whose fertility was not at all restored by any of the 50 inbreds evaluated in the present study.
Moreover it is known that the restorer of one CMS source may act as a maintainer of other CMS
types. Furthermore, the observation that while all 50 inbreds acted as maintainers of the
cytoplasmic male sterility of GIG 1, 28 common inbreds acted as restorers of both CMS lines
(PET 1 and ARG 1), suggests that while ARG 1 is similar to the French CMS source; PET 1, the
cytoplasm of GIG 1 is different. Petrov and Nenov (1992), drew similar conclusions regarding
the differences of three new CMS sources with the French CMS source PET 1. That the two
CMS sources PET 1 and ARG 1 had different reactions than that of GIG 1; the third CMS source
in the present study, to the inbreds, further indicates a distinct mechanism of cytoplasmic male
sterility operating in CMS GIG 1 (Jan, 2000).
The same pollen parent exhibited different type of fertility restoration behavior in
different CMS line combinations have been found in the material under study. Such type of
results obtained may be due to the minor gene(s) with additive gene action with the cytoplasmic
gene of different CMS line.
The results also revealed that RHA 274 is able to fully restore the fertility of CMS PET 1
and CMS ARG 1, whereas it failed to restore the fertility of CMS GIG 1. These results are in
agreement with those of Havekes et al. (1991). RHA 274, (H. petiolaris restorer line) restorer
with higher oil percentage, has also been found to completely restore the fertility of mutant CMS
HA 89 lines produced by treating maintainer line HA 89 with mitomycin C and streptomycin
(Jan and Rutger, 1988). Likewise RHA 274 has also been observed to restore the fertility of
CMS PI 432513 (Jan and Vick, 1997, 1998, 2007). The restoration of CMS PET 1 and ARG 1 by

6. RHA 274 observed in the present investigations, along with the above reports by various
workers, suggests that RHA 274 is a useful restorer and, this inbred which likely carries Rf1; the
dominant H. petiolaris restorer gene (Jan and Vick, 2007), should be used in hybrid sunflower
breeding programme, especially for higher oil content.
In the present investigations, sufficient restorers were observed for PET 1 and ARG 1.
Since no restorers for CMS GIG 1 could be identified, the cytoplasm of CMS GIG 1 is indicated
to be distinct from those of commercially used PET 1 cytoplasm as well as that of ARG 1. Hence
it is safe to conclude that, while PET 1 can continue to be utilized for the production of
commercial sunflower hybrids, promising hybrids with a different cytoplasm of ARG 1 can also
be obtained. Similarly chances do exist for the development of hybrids with CMS GIG 1
cytoplasmic background. This surmise is based on the fact that Havekes et al. (1991) have
already recovered 50, 81 and 100% male fertile F1 plants derived out of crosses between CMS
GIG 1 and the male fertility restorer lines namely; RGIG 1, RPET 2 and RHA 294, respectively.

7. Table 1: Frequency of F1 sterile pollens (SP) and fertile pollens (FP) from anthers of H. annuus
plants after crossing CMS PET 1 and IMS with 30 inbred testers

8. To be useful for hybrid seed production, a CMS line needs complete male sterility and
female fertility. That male sterility of CMS GIG 1 is stable and completely maintained by 50
different inbred testers, is confirmed in the present experiment. Thus further selection; at least for
fertility restoration of the CMS source GIG 1 is necessary before it can be used for commercial
hybrid production. However since this CMS source (GIG 1), is from H. giganteus, a species
different from the cultivated sunflower (H. annuus), it remains to be seen whether, it will
contribute towards reduction of the genetic vulnerability of worldwide sunflower hybrids, by
providing an alternative to the CMS PET 1 cytoplasm.
Nevertheless, efforts toward identification of different restorers for CMS GIG-1 are
desirable for greater genetic diversity to be used in the development of new restorer inbred lines
(Gimenez and Fick, 1975) and the hybrids. The different CMS lines and their concerned
maintainer and restorer inbreds of sunflower can be utilized directly in maintenance breeding and
hybrid development programme.
ACKNOWLEDGMENT
The authors are thankful to the All India Coordinated Research Project (Sunflower)
centres Bangalore, Akola and Coimbatore for providing the valuable inbred lines. I am extremely
thankful to Dr. A. J. Prabakaran for guiding me and providing cytological facility for pollen
study.
REFERENCES
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Christov, M., (1992). New sources of male sterility and opportunities for their utilization in
sunflower hybrid breeding. Helia, 15: 41-48.
Gimenez, D.J. and Fick, G. (1975). Fertility restoration of male sterile cytoplasm in wild
sunflower. Crop Sci., 15: 724-726.

9. Havekes, F.W., J.F. Miller and C.C. Jan, (1991). Diversity among sources of cytoplasmic male
sterility in sunflower. Euphytica, 55: 125-129.
Heiser, C.B. (1982). Registration of Indiana-1 CMS sunflower germplasm. Crop Sci., 22: 651-
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a Helianthus annuus landrace PI-432513. Proceedings of the Sunflower Research
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Jan, C.C. and B.A. Vick, (1998). Cytoplasmic male sterility in sunflower landrace PI 432513 and
the inheritance of fertility restoration. Proceedings of the Sunflower Research Workshop,
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Jan, C.C. and Vick, B.A. (2007). Inheritance and allelic relationships of fertility restoration genes
for seven new sources of male-sterile cytoplasm in sunflower. Plant Breed., 126: 213-217.
Jan, C.C. and Rutger, J.N. (1988). Mitomycin C- and streptomycin-induced male sterility in
cultivated sunflower. Crop Sci., 28: 792-795.
Jan, C.C., (2000). Cytoplasmic male sterility in two wild Helianthus annuus L. accessions and
their fertility restoration. Crop Sci., 40: 1535-1538.
Jan, C.C., J.F. Miller, B.A. Vick and G.J. Seiler, (2006). Performance of seven new cytoplasmic
male-sterile sunflower lines from induced mutation and a native American variety. Helia,
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breeding programmes. Proceedings of the 4th International Sunflower Conference, June
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Leclercq, P., (1969). Une sterilite male cytoplasmique chez le tournesol. Ann. Amelior. Plant.,
19: 99-106.

10. Leclercq, P., (1971). La sterilite male cytoplasmique de tournesol. I. Premieres etudes sur la
restoration de la fertilite. Ann. Amelior. Plant., 21: 45-54.
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genus helianthus. Helia, 15: 49-52.
Serieys, H., (2002). Identification, study and utilization in breeding programs of new CMS
sources, in FAO Subnetwork. Proceedings of Sunflower Subnetwork Progress Report FAO
Rome Italy 7-9 October.
Spirova, M., (1990). Genetic nature of male sterility in sunflower from a source of cultivated
sunflower. Comptes Rendus de I’ Academie Bulgare des Sci., 43: 113-116.
Vranceanu, A.V. and F.M. Stoenescu, (1971). Pollen fertility restorer gene from cultivated
sunflower (Helianthus annuus L.). Euphytica, 20: 536-541.
Whelan, E.D.P. and W. Dedio, (1980). Registration of sunflower germplasm composite crosses
CMG-1, CMG-2 and CMG-3. Crop Sci., 20: 832-832.

11. Table 2: Identification of inbred behavior for maintenance and restoration of diverse CMS
sources of sunflower

12. PET 1 (Helianthus petiolaris); R: Restorer; ARG (Helianthus argophyllus)., M: Maintainer; GIG
1 (Helianthus giganteus), Results are pooled observations of F1s (CMS x inbreds), from a
replicated experiment with three replications, for fertility reactions of inbreds
Modern sunflower breeding began with development of F1 hybrids after the discovery of
cytoplasmic male sterility (Leclercq, 1970) and fertility restorer genes (Kinmann, 1970). The
first reliable cytoplasmic male sterile source was isolated by Leclercq (1969) from the
interspecific cross Helianthus petiolaris Nutt. × Helianthus annuus and designated as PET-1
cytoplasm (Serieys, 1987).
Serieys, H.A., 1987. Genetic evaluation and use of Helianthus wild species and their use in
breeding programme. FAO Subnetwork Report, 1984-1986, pp. 1-23.
The pollen grains were uniformly plumpy and did not clump indicating the normal development
of pollen. The percentage of fertile pollen ranged from ……….in the F1 hybrids.
The percentage of stained pollen and or the percentage of typical aborted pollen should be used
as an essential index for determining plant fertility. Most research confirms that pollen fertility
could be a main criterion for assessing fertility. Percentage of fertile pollen was the most reliable
criterion for fertility (Hu, 1983).
Hu, Jinguo (1983). Exploratory research on the criterion used for studying the inheritance of
CMS in rice. J. Huazhong, Agric. Coll., 2(3):
Main factors affecting fertility restoration:
Genetic diversity: Genetic diversity includes differences in sterile cytoplasm and backgrounds of
maintainers and restorers. Isogenic male sterile (MS) lines with different sterile cytoplasms may
belongs to different sterile types and have different restorers.

13. The genetic background of maintainers influences the fertility of F1 hybrids having the same
sterile cytoplasm. For example, fertility restoration of WA type MS line Zhe-Shan-97A was
easier than of WA type Er-Jiu-Nan-1A (Li and Yiao, 1982).
The genetic background of restorers apparently influences fertility restoration. When restorer IR-
24, IR-26, IR-28 and Gu-154 were crossed with the same male sterile (MS) line, the fertility of
F1 hybrids revealed that the restorers differed in fertility restoring ability. Especially under
unfavorable climate, the seed setting rate of hybrids derived from IR-28 and Gu-154 was lower
than the hybrid derived from IR-24.
Environmental variation: Environmental factors, particularly temperature, greatly influence
fertility restoration. Seed setting rate may be drop when unfavorably high or low temperature
occurs during the pollen mother cell meiosis stage or heading stage. Chinese research showed
that temperature and moisture affect fertility restoration. Hybrids derived from different MS lines
and restorers differ in their reactions to environmental variations (Li and Yiao, 1982).
Li, Zebing and Yiao, Yihua (1982). Hybrid rice research and practice. Shanghai Technological
Press, China.
This variation among male-fertility-restored plants may be due to genetic background or
environmental factors such as high temperatures at critical times of flower development (Barham
and Munger, 1950; Meer and Bennekom, 1969).
Barham, W.S. and H.M. Munger. (1950). The stability of male sterility in onion. Proc. Amer.
Soc. Hort. Sci. 56 : 401–409.
Meer, Q.P. and J.L. Bennekom. (1969). Effect of temperature on the occurrence of male sterility
in onion (Allium cepa L.). Euphytica, 18 : 389–394.
Similar variation was found in the Tournefortii male sterility of rapeseed (Brassica
napus), in which some genotypes showed different degrees of male-fertility-restoration and were
explained as interactions with genetic background (Pahwa et al., 2004).

14. Pahwa, R.S., S.K. Banga, K.P.S. Gogna, and S.S. Banga. 2004.Tournefortii male sterility system
in Brassica napus. Identification, expression and genetic characterization of male fertility
restorers. Plant Breed. 123 : 444–448.
Environmental factors (such as nutritional and water deficiencies or high temperatures), pests
(insects or diseases), and/or other genetic factors could affect pollen production (Barham and
Munger, 1950; Delph et al., 2007; Monosmith, 1928). Barham and Munger (1950) studied
temperature effects on pollen production in S-cytoplasmic male-sterile lines and found that high
temperatures after emergence of scapes increased the amount of viable-appearing pollen;
however, no selfed seeds were produced on these S-cytoplasmic plants. In chive (Allium
schoenoprasum), a restorer locus has been reported that produces viable pollen at high
temperatures (Engelke et al., 2004).
Delph, L.F., P. Touzet, and M.F. Bailey. (2007). Merging theory andmechanism in studies of
gynodioecy. Trends Ecol. Evol. 22 : 17–24.
Monosmith, H.R. (1928). An investigation of the histological development and inheritance of
male sterility in a clone of Allium cepa L. PhD Diss., Univ. California, Berkeley.
Engelke, T., D. Gera, and T. Tatlioglu. (2004). Determination of the frequencies of restorer- and
maintainer-alleles involved in CMS1 and CMS2 in German chive varieties. Plant Breed.
123 : 51–59.
There is an improved understanding of the importance of a balance between genes for pollen
fertility restoration in male parent and fertility enhancing genes that affect the case of
restoration in the female. It may be possible to identify CMS female that exhibit partial
fertility in a favourable (Shallow sterile) environment. Such lines in hybrid combination
may interact with restorer genes to provide a highly fertile hybrid that is stable over a range
of climatic conditions.
Restoration is also influenced by environmental conditions, with cool conditions around
flowering favoring sterility and high temperatures favouring fertility (Downs and Marshall 1971;
Brooking 1976, 1979).

15. Brooking IR (1976) Male sterility in Sorghum bicolor (L.) Moench induced by low night
temperature. I. Timing of the stage of sensitivity. Aust. J. Plant Physiol., 3 : 589–596.
Brooking IR (1979) Male sterility in Sorghum bicolor (L.) Moench induced by low night
temperature. II. Genotypic differences in sensitivity. Aust. J. Plant Physiol., 6 : 143–147.
Downs RW, Marshall DR (1971) Low temperature induced male sterility in grain sorghum. Aust.
J. Agric. Res. Animal Husb., 11: 352–356.
Do you consider that different cytoplasm require entirely different sets of restoring genes (yes).
Restorers of one CMS line were found to be maintainers of other CMS lines and vice versa, it
can be concluded that specific restorer genes exist for a specific cyto-sterility system.
However, it is possible that certain restorer lines would restore the fertility of two different
cytosterile lines because they posses restorer genes for the two cytosterility systems.
Have you observed any environmental conditions (or plant growth regulators) that will restore
pollen fertility in CMS lines
Ans. For stable CMS lines, we have found none. For unstable A lines, there may be.

16. Figure 1. A). All stained pollen (Complete restorer). B). 50% unstained pollen (Partial restorer)
C&D). All unstained pollen (Maintainer), and E). 50% stained pollend (Partial restorer)
A. CMS-234A x RHAGKVK-2 B. CMS-10A x AKSFI-46-2
B. IMS-852A x IB-61 D. IMS-852A x AKSFI-49-3
E. CMS-10A x AKSFI-46-2 (Partial restorer)
B. 50% unstained pollenA. All stained pollen
C. All unstained pollen D. All unstained pollen
E. Unstained pollen

17. Table 3: Classification of F1 population based on pollen fertility in sunflower
S. No. Class Pollen fertility per cent
1 Fertile > 80
2 Partially fertile 50.1-80
3 Partially sterile 1.1-50
4 Sterile 0

18. Step 1. (10,000 to 15,000)
5000 - 8000
Individual plant
selections
Based on uniform maturity, plant
height, head shape, size, seed filling
ability
Oil and hull analysis.
1,000-1,200 heads
Selected for first year nursery
(2RRT)
Step 2. First year nursery (Progeny row trial) Aportion of seed Kept as reserve
C C C C C C
Agronomic, seed quality, disease
and seed yield, oil content
evaluation, 150-200 heads selected
for second year nursery.
Second year nursery (Remant seeds used)
C C C C C C
Second year evaluation of
characters. 20-50 heads selected for
cross pollination nursery
Cross-pollination nursery Seeds from Cross-pollination
nursery are used for preliminary
and competitive strain testing
Best families IP harvested separetely, Lab Analysis
Best plants retained Remaining seeds
for next breeding cycle bulked family wise
(Isolation)
Preliminary strain testing Preliminary increase of best
cultivars conducted on the basis of
competitive testing
Competitive cultivars
testing
State cultivar testing
Diagram of pustovoit “ Method of Reserves” for improvement of sunflower cultivars.
Superior
cultivars
populations
or hybrids

19. Table 1. Mapping of fertility restorer genes in sunflower
Trait Gene/locus Linkage group
(LG)
Population/s, line/s References
Pollen fertility
restoring genes
Rf1 LG 13 HA89 x RHA266,
CX x RHA266,
PAC2 x RHA266
Gentzbittel et al. 1995, 1999
msc1 LG 7 CP73 x PAC1,
GH x PAC2
Gentzbittel et al. 1995, 1999
msc1 LG P GH x PAC2 Mestries et al. 1998
Rf1 LG 13 RHA325 x HA342 Horn et al. 2003;
Kusterer et al. 2005
Rf3 LG7 RHA340 x ZENB8 Abratti et al. 2008
Rf1 LG13 RHA439 x
cmsHA441
1. Yue et al. 2010
Rf6 LG3 - 2. Liu et al. 2013
3.
Liu, Z., Wang, D., Feng, J., Seiler, G.J., Cai, X.and Jan, C.C. (2013). Diversifying sunflower
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gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers. Plant Breeding,
129(1): 24-28.
Gentzbittel, L., Vear, F., Zhang, Y.X., Bervillé, A. nad Nicolas, P. (1995). Development of a
consensus linkage RFLP map of cultivated sunflower (Helianthus annuus L.). Theor Appl
Genet 90: 1079–1086.
Gentzbittel, L., Mestries, E., Mouzeyar, S., Mazeyrat, F., Badaoui, S., Vear, F., Tourvieille de
Labrouhe, D., Nicolas, P. (1999). A composite map of expressed sequences and phenotypic
traits of the sunflower (Helianthus annuus L.) genome. Theor Appl Genet 99: 218–234.
Mestries, E., Gentzbittel, L., Tourvieille de Labrouhe, D., Nicolas, P. and Vear, F. (1998).
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sunflower (Helianthus annuus L.) using molecular markers. Mol Breed 4: 215–226.

20. Horn, R., Kusterer, B., Lazarescu, E., Prüfe, M. and Friedt, W. (2003). Molecular mapping of the
Rf1 gene restoring pollen fertility in PET1-based F1 hybrids in sunflower (Helianthus
annuus L.). Theor Appl Genet 106: 599–606.
Kusterer, B., Horn, R. and Friedt, W. (2005). Molecular mapping of the fertility restoration locus
Rf1 in sunflower and development of diagnostic markers for the restorer gene. Euphytica
143: 35–42.
Abratti, G., Bazzalo, M.E. and León, A. (2008). Mapping a novel fertility restoration gene in
sunflower. Proc 17th Int Sunflower Conf, vol 2, Córdoba, Spain, pp 617–621.
Fertility restoration of dominant nuclear genes is essential for hybrid breeding based on CMS to
obtain high yields of seeds. One to four dominant restorer genes have been described depending
on the material (Serieys 1996). However, in most of the elite sunflower lines, the two dominant
nuclear genes Rf1 and Rf2 are responsible for fertility restoration (Leclercq 1984). As Rf2 is
present in nearly all inbred lines, including maintainers of CMS, the Rf1 gene is most important
for sunflower hybrid breeding. Molecular markers linked to different fertility restoration genes
have been identified, and some of these genes have been mapped to LG 13 (Rf1, for PET1
cytoplasm from H. petiolaris) and to LG 7 (Rf3) (Gentzbittel et al. 1995; Horn et al. 2003; Yu et
al. 2003, Hamrit et al. 2006b; Abratti et al. 2008). Identifying molecular markers tightly linked to
this gene will be useful in marker-assisted selection to develop maintainer and restorer lines.
Hamrit S, Engelmann U, Schnabel U, Warber D, Kurutz S, Kusterer B, Lazarescu E, Özdemir N,
Friedt W, Abratti G, Leon A, Horn R (2006b) Comparative mapping of restorer genes
restoring pollen fertility in the presence of different CMS cytoplasms in the genus
Helianthus. In: Proc 7th Eur Conf Sunflower Biotechnol, 3–6 Sept 2006, Gengenbach,
Germany, p 10.
Yu JK, Tang S, Slabaugh MB, Heesacker A, Cole G, Herring M, Soper J, Han F, Chu WC,
Webb DM, Thompson L, Edwards KJ, Berry ST, Leon A, Olungu C, Maes N, Knapp SJ
(2003) Towards a saturated molecular genetic linkage map for cultivated sunflower. Crop
Sci 43: 367–387.
Leclercq, P. (1984). Identification de gènes de restauration de fertilité sur cytoplasms stérilisants
chez le tournesol. Agronomie 4: 573–576.

21. Gundaev, A.I. (1971). Basic principles of sunflower selection. In: Genetic Principles of Plant
Selection. (In Russian). Nauka, Moscow, USSR, pp 417–465.
Nuclear male sterility (NMS) in sunflower was first discovered by Kuptsov in 1934
(Gundaev 1971).
Cytoplasmic male sterility (CMS) and its fertility restoration (Rf) genes are critical tools
for hybrid seed production to utilize heterosis. To produce the hybrid seeds, the CMS plants are
crossed with restorer lines that have the Rf1 (restorer of fertility) gene to obtain fertile plants.
The ability of molecular markers linked to this locus facilitates the introgression of this gene in
different lines of breeding programs for developing new restorer lines.
The two most important developments were the dramatic increase in seed oil percentage
achieved by breeders in the former Soviet Union (FSU), and the development of a cytoplasmic
male sterility system, combined with fertility restoration by nuclear genes, which enabled the
commercial production of hybrid seed (see for review, Fick and Miller 1997).
Alternative CMS/Rf gene systems could expand the diversity of the sunflower crop, and reduce
the risks inherent with using a single CMS/Rf system. Also, identification and characterization of
additional CMS/Rf gene systems will enrich knowledge of the interactions between cytoplasm
and nuclear genes.
Screening elite breeding lines for effective and genetically diverse maintainers and
restorers for different CMS lines is important in developing new CMS lines and productive
hybrids. The success of hybrids based on new cytoplasm depends not only on restoration ability
but also on the nuclear genes for exploitation of heterosis.
A high percentage of pollen fertility restoration, stable restoring ability over
locations/season, and good combining ability are the important key attributes needed to ensure
commercially viable hybrid technology. For developing new cytoplasmic male sterile (CMS)
lines, it is important to screen the locally adapted elite breeding lines for genetically diverse
maintainers and promising restorers with wide genetic base for developing commercial hybrids.

22. The diversification of CMS sources may also be useful to optimize the utilization of
genetic resources in breeding programs by “changing’ the restorer status of an inbred line (i.e., a
restorer genotype of one cytoplasm may be a male sterility maintainer of a second one).
All the CMS sources utilized in the experiment showed diversity among themselves, thus
broadening the genetic base of the CMS lines, which could be safely included in breeding
programme thereby mitigating the vulnerability of the lines to various insect pests and diseases.
Actually, research efforts for developing heterotic groups with high yields and reduced
genetic vulnerability to ever changing environmental stress and diseases are often jeopardized
due to lack of alternative CMS and the associated restorers.
The PET-1 cytoplasm controlling sterility has no apparent adverse effects on agronomic
or seed oil characteristics (Velkov and Stoyanova, 1974), and has proven widely successful in
production of hybrid seed. However, suitable fertility restorers are not available for all of these
sources and several sources have negative effects on seed yield, and other plant and seed
characteristics (Petrov, 1992a; Serieys, 1992; Havekes et al., 1991).
Velkov, V. and Stoyanova, Y. (1974). Biological peculiarities of cytoplasmic male sterility and
schemes of its use. P. 361-365. In Proc. 6th
Int. Sunflower Conf., Bucharest, Romania, 22-
24 July. Int. Sunflower Assoc., Paris, France.
Serieys, H. (1992). Cytoplasmic effects on some agronomical characters in sunflower. P. 1245-
1250. In Proc. 13th
Int. Sunflower Conf., Pisa, Italy, 7-11 September. Int. Sunflower
Assoc., Paris, France.
Petrov, P. (1992a). Effect of various cytoplasmic male sterility sources (CMS) on some
sunflower qualities. P. 1211-1215. In Proc. 13th
Int. Sunflower Conf., Pisa, Italy, 7-11
September. Int. Sunflower Assoc., Paris, France.
Havekes, F.W.J., Miller, J.F. and Jan, C.C. (1991). Diversity among sources of cytoplasmic male
sterility in sunflower (Helianthus annuus L.). Euphytica, 55: 125-129.