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

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.

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

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 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

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.

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

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.

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Table 2: Identification of inbred behavior for maintenance and restoration of diverse CMS

sources of sunflower

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

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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.

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).

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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).

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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;

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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.

B. 50% unstained pollenAll stained pollen

E. Unstained pollen

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)

C. All unstained pollen D. All unstained pollen

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

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.

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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.

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(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.

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.

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.