Turkish Journal of Field Crops, 2012, 17(2):124-128 
 
 
THE SHORT BREEDING CYCLE PROTOCOL EFFECTIVE ON DIVERSE  
GENOTYPES OF SUNFLOWER (Helianthus annuus L.) 
 
 
*  
Nazan DAGUSTU , Gamze BAYRAM , Mehmet SINCIK , Melek BAYRAKTAROGLU 
 
 
Uludag University Agricultural Faculty, Department of Field Crops, Bursa, TURKEY
*Corresponding author: ndagustu@uludag.edu.tr 
 
Received: 14.06.2012 
 
ABSTRACT 
 
Immature embryo culture of sunflower (Helianthus annuus L.) was studied for shortening the generation time 
in breeding programs. The seed development from pollination to maturity in sunflower takes 50-60% (60 
days) of the life cycle duration (120-150 days). This technique allows the production of fertile plants from 
immature embryos of 11 sunflower genotypes. Immature embryos of 10-12 days after pollination were 
dissected from seed grown plants (SGP), were transferred into MS medium allowing shoot and root 
development for 5-10 days. Young plantlets were transferred to soil, developed to maturity and were then self 
pollinated and set seed. The first cycle of immature embryo raised plants (IERP) was obtained. The four cycles 
of IERP were obtained from immature embryo culture technique in contrast to one generation per year with 
conventional breeding. The majority of cultured embryos developed into vigorous plantlets with 3-6 leaves. 
Out of 1320 immature embryos, the average response of the explants were 92.1% (1216). The 75% of the 
developed plantlets had vigorous roots and were transplanted into viol containing 1:1:2 peat: perlite: soil 
mixture (v/v) at 24 ± 2oC in 16h/8 h (light/dark) in the growth chamber. The only 70.3% of them was grown to 
maturity, self-pollinated and set seed. The overall result was average 40-45 regenerated and matured plants 
per 100 immature zygotic embryos. The regenerated plants also showed no morphological changes. The 
analysis of variance for all agronomic characters (plant height, head diameter, number of leaves, stem 
diameter, number of branches and seed number per head) taken from the mean of four generations in vitro 
grown plants resulted in significant differences among genotypes at 5% level. All the agronomic characters 
examined at in vitro regenerated plants decreased compared to field grown plants. 
 
Key words: fertile plant regeneration, immature embryo, shortening the breeding cycle, sunflower. 
 
INTRODUCTION A field grown sunflower crop requires at least 3-4 
months from sowing to maturity for reseeding the next 
 Sunflower (Helianthus annuus L.) is one of the 
generation. Of this time, seed development from 
most important oil seed crops in both Turkey and the 
pollination to maturity takes 2 months of the life cycle 
world. Development of hybrids which have valuable 
duration (Hahne, 2002). In vitro immature embryo culture 
agronomic performances is an important target in 
reduces the flowering and seed maturation, shortening the 
sunflower breeding program. Sunflower is a highly cross-
entire lifecycle, thus increasing the number of generation 
pollinated crop and a breeding program of these crops 
that can be produced annually. The length of one cycle is 
involves several breeding cycles. Obtaining homozygous 
quite often limiting factor for performing a breeding aim 
parental lines for hybrid seed production in short period of 
in a reasonable period of time. This system can also 
time are of great importance for commercial hybrid 
reduces the cycle of backcross and selfing of lines, thus 
production. In general sunflower plants were grown from 
breeding process speed up and overcome seed dormancy 
seeds once a growing season in the field conditions in 
problem. Consequently, reducing the duration of one cycle 
many countries. Therefore, only one generation a year is 
should have a great importance for the breeder to obtain 
taken during classical breeding program. The homozygous 
seed propagated cultivars, lines and new source of 
lines and conversion of an inbred to a cytoplasmic male 
maintainers (B), restorers (R) and cytoplasmic male sterile 
sterile (CMS) line can be obtained within minimum 6 
(CMS) lines.  
generations by conventional methods (Jambhulkar, 1995; 
Hu et al., 2008). On the other hand, it is also known that  Application of new technologies such as in vitro 
sunflower seeds keep germination capacity about 6 days tissue culture methods in sunflower (anther culture, 
after pollination, becomes dormant 16 days after microspore culture, ovule culture, immature embryo 
pollination (Maiti et al., 2006), remain dormant for 45-60 culture etc.) accelerates the breeding process. Embryo 
days (Jambhulkar, 1995) and seed maturation in sunflower culture has been used successfully by plant breeders to 
takes 50-60% of the life cycle duration (Serieys, 1992).  solve the problems of seed set, seed dormancy, slow seed 
germination, inducing embryo growth in the absence of 
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symbiotic partner, shortening the breeding cycle, rapid were then placed in 31 by 51 cm plastic multipot trays of 
seed viability test, obtaining rare hybrids and homozygous 48 pots per tray containing sterile 1:1:2 peat:perlite:soil 
lines, haploid production (Yeung et al., 1981; Chandler mixture (v:v). The trays were covered with a plastic bag 
and Beard, 1983; Gürel et al., 1991; Zhong et al., 1995; and incubated for a period of 7-10 days in the growth 
o
Bhojwani and Razdan, 1996; Torresàn et al., 1996; Saji chamber at 25 ± 2 C in the light (16 h/ 8 h day/night), 
and Sujatha, 1998; Raghavan, 2003). By using embryo after which the plantlets were transferred to 320 X 270 
culture, breeding cycle of sunflower (Jambhulkar, 1995;  mm (16 lt) pots with regular soil, keeping 5 plants per pot 
Torresàn et al., 1996), artichoke (Cravero and Cointry, at the same growth chamber. Three pots were set up for 
2007), wheat (Sharma and Gill, 1982), soybean (Roumet each genotype.  The plantlets were irrigated with sterile 
and Morin, 1997), orchids, roses and banana (Yeung et al., water 3 times weekly to maintain a high humidity 
1981),  iris (Randolph, 1945) azalea (Michishita et al., environment and fertilized every 10 days. Plantlets had 
2001) were shortened from 1 to 3 years to a few months flowers approximately 7-8 weeks after sowing and set 
depended on the plants. The sunflower embryo culture seed.  
system was first developed for interspecific hybridization 
The regeneration response of immature embryos was 
by Chandler and Beard (1983). Jambhulkar (1995) 
scored after 2 weeks, evaluating the growth of the shoot, 
developed a rapid embryo-raised plant system for 
root and their intensity. The number of plantlets 
sunflower production from immature embryos, which 
regenerated and mature plants obtained were recorded. 
allow five cycles in 316 days. The purpose of the present 
Each genotype consisted of 10-15 petri dishes (9 cm) with 
study is to increase the generation time via immature 
10 embryos in each. Observations on efficiency of the 
embryo culture. This technique was developed for rapid 
immature embryos (%), embryos developing plantlets 
propagation of parental lines (B, R and CMS) and hybrids 
(number) were evaluated.  
by speeding up the breeding process in sunflower 
industry. A number of growth parameters (head production, 
50% of flowering dates, pollination dates, embryo 
MATERIALS AND METHODS 
emasculation dates, transfer to soil dates) and agronomic 
The 11 sunflower genotypes (Record 109/Sanay 3-5 B, traits (plant height, head diameter, stem diameter, number 
Record 109/Sera B, BGC0565, IMIN3, RHA 04, RHA 07, of leaves per plant, number of branches, number of seeds 
RHA 14, RHA 16, IMICMS6 X IMIN6, T0910131-2, per head) were determined for each of the  individual 
T091241-3) were obtained from 3 different sources plants in the field and plants grown in pots. Five plants 
(sunflower collections of Field Crops Department, Uludağ were randomly selected from each genotype grown from 
University, Bursa; Trakya Agricultural Research Institute, original seeds in the field conditions. Mean data were 
Edirne; TMT Ltd. Şti., Tekirdağ, Turkey). These presented with standard deviations. For in vitro grown 
genotypes were chosen for their early flowering and plants, 3 pots with 5 plants each were subjected to 
maturing capacity and the shortest plant height within variance analysis in the completely randomized design 
population. Sunflower collections were planted into 5 m with JUMP statistical program. The agronomic 
lines with 0.65 m row spacing and 0.3 m plant-plant performances of in vitro grown plants were determined by 
spacing at Uludağ University, Agricultural Research and the mean of four generations. The significance of 
Experimental Station in 2010. genotypes was calculated at the 0.05 probability level by 
the Student’s t-test. The experiments were carried out for 
The plants were hand-pollinated, and maintained until 
2 years to obtain reliable results. 
the seeds were set. The first regeneration cycle has started 
from seed grown plants (SGP). Ten to 12 days after RESULTS AND DISCUSSION 
pollination the heads were brought into the laboratory. 
The first cycle of immature embryo raised plants 
The achenes placed at the outer 3-4 rows of the nd rd
(IERP) had taken 85-90 days after sowing. The 2 , 3  and 
inflorescence were separated. For homogenize the th
then 4  cycle of IERP were obtained from in vitro grown 
embryos, achenes with dark color had been selected. The 
plants. Immature seeds removed as early as 10-12 days 
embryos with pericarps were surface sterilized in 20% 
after pollination were successfully cultured in vitro. All of 
commercial bleach with a few drops of detergent for 15 
the cultured genotypes developed and produced plantlets 
minutes. They were then rinsed three times in sterile 
at the first stage in MS medium. Within two weeks, 
distilled water. The immature embryos were excised from 
developing embryos mainly grew plantlets with strong 
the pericarps, endosperm and embryo sac for producing 
roots (2-6 cm and branching) and stem with 6-8 leaves (~1 
the first cycle of plants and placed into Murashige and 
cm) as described by Freyssinet and Freyssinet (1988).  
Skoog (MS, 1962) medium with 2% sucrose and 0.8% 
agar at pH 5.6-5.7 as described by Jambhulkar (1995). All The well developed plantlets were selected to transfer 
studies were carried out under sterile conditions. The petri into viol containing peat:perlite:soil mixture for 
dishes were sealed with parafilm to prevent moisture loss acclimatization. The plants were kept for two weeks in 
o
and placed in the light (16h/8h day/night) at 25 ± 2 C and growth chamber. Almost all of the plantlets grew healthy 
cultured until plantlets production. Within 1-2 weeks of in the viols in green house conditions and were 
culture in MS, developing embryos grew plantlets with phenotypically similar to original seeds. They were 
strong roots (2-6 cm and branching) and stem (3-5 cm) generally short, often branched with a flower on each 
with 6-8 leaves (~1 cm). Plantlets washed agar debris branch. The only 15 of them placed into pots (5 plantlets / 
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pot). They survived to flower and set seed after 8 weeks. medium, 85% was in peat:perlite:soil mixture and the only 
The premature flowering did not occur in vitro. Mature 77.5% of them was grown to maturity, self-pollinated and 
plants exhibited normal phenotype and were fertile. When set seeds. This study reports the results of improvement of 
the unshaped and small plantlets were transplanted to soil, the growing sunflower plants from in vitro immature 
they did not survive. Days from culturing to harvesting of embryo culture in the growth chamber. 
mature seeds were almost 80-95 days depended on the 
The mean values for agronomic performances of SGP 
genotype. 
and IERP sunflower genotypes are given in Table 1 and 
Out of 1320 immature embryos, the average response Table 2.  Although there were no big morphological 
of the explants was 92.1% (1216) showing morphogenesis changes in in vitro regenerated plants, they showed 
with a range of 90-100%. The majority of cultured variations for plant height, head diameter, stem diameter, 
embryos developed into plantlets like Jeannin and Hahne number of branches, number of leaves per plant and 
(1991).  Of the embryos that placed into embryo growth number of seed per head.  
 
Table 1. The agronomic performances (plant height, head diameter and number of leaves) of seed grown and in vitro grown plants in 
sunflower genotypes. 
Plant height 
Genotypes Head diameter (cm) Number of leaves  (no) 
(cm) 
 SGP IERP* SGP IERP* SGP IERP* 
RHA04 93.0  ± 2.6 40.8ef 7.5 ± 0.2 2.06de 21.0 ± 1.1 16.4bc 
RHA 07 97.4 ± 3.8 41.2ef 9.0 ± 0.3 2.14de 22.4 ± 0.5 17.0bc 
RHA 14 84.8  ± 3.4 60.6b 6.1 ± 0.3 4.50a 21.2 ± 0.4 17.3bc 
RHA 16 58.8 ± 6.0 51.04c 5.2 ± 1.0 2.06de 18.6 ± 1.9 18.3ab 
T0910131-2 70.4 ± 1.9 41.8ef 11.0 ± 0.6 2.32cd 24.6 ± 2.4 20.2a 
T091241-3 120.0 ± 8.4 61.2b 10.6 ± 1.4 2.82bc 18.4 ± 1.6 18.3ab 
IMIN3 75.8  ± 5.8 41.9ef 7.5 ± 1.1 1.54f 22.4 ± 1.4 16.2c 
IMICMS6 X IMIN6 78.8 ± 4.1 37.9f 7.5 ± 0.8 1.77ef 22.8 ± 0.9 15.6c 
Record 109/Isera 70.2 ± 3.1 45.2de 5.3 ± 0.1 2.37cd 19.0 ± 0.5 17.0bc 
Record 109/Sanay 3-5(B) 81.4 ± 7.9 69.1a 7.8  ±0.9 2.67bc 20.8 ± 0.8 16.8bc 
BGC0505 68.0 ± 2.4 48.4cd 4.5 ± 0.2 3.08b 18.6  ± 0.9 16.6bc 
*the mean of 4 generations were taken from in vitro grown plants 
 
Table 2. The agronomic performances (stem diameter, number of branches and number of seed per head) of seed grown and in vitro 
grown plants in sunflower genotypes. 
Genotypes Stem diameter (cm) Number of branches (no) Number of seed per head (no) 
 SGP IERP* SGP IERP* SGP IERP* 
RHA04 0.46 ± 0.0 0.38c-e 14.6 ± 0.7 3.12bc 558.3 ±33.5 16.3e 
RHA 07 0.54 ± 0.0 0.47bc 8.6 ± 0.5 3.31b 579.3 ±148.9 41.5ab 
RHA 14 1.86± 0.0 1.28a 7.4 ± 0.4 4.30a 646.3 ±167.8 72.7a 
RHA 16 0.86± 0.0 0.36de 8.0 ± 0.7 1.79ef 172.7 ±58.0 22.1c-e 
T0910131-2 2.04 ± 0.2 0.43b-d 2.8  ± 1.4 1.04g 841.7 ±160.9 17.7de 
T091241-3 1.64 ± 0.2 0.39b-e 3.6 ± 0.4 1.50fg 787.0 ±133.4 30.1b-e 
IMIN3 0.56± 0.2 0.47bc 10.4 ± 0.5 2.56cd 382.0 ±58.9 32.2a-c 
IMICMS6 X IMIN6 0.88± 0.0 0.41b-e 10.6 ± 0.9 2.44c-e 535.3±21.5 18.8de 
Record 109/Isera 0.60± 0.0 0.49b 7.6 ± 0.7 3.11bc 415.7±84.0 39.3a-c 
Record 109/Sanay  
          0.40± 0.0 0.31e 10.4 ± 1.2 2.13d-f 494.7±27.1 35.8a-c 
3-5(B) 
BGC0505 0.72± 0.0 0.43b-d 7.0  ± 0.3 3.80ab 508.7±81.1 38.9a-c 
*the mean of 4 generations were taken from in vitro grown plants 
 
The seed grown plants had generally higher agronomic plant height (37.9 cm). The plant height of seed grown 
performances compared to in vitro grown plants as plants varied between 58.8 and 120.0 cm. It was lower 
reported by Jeannin and Hahne (1991). In general, the than 100 cm except T091241-3 (120.0 cm). The 
plant height, stem diameter, number of seed per head, regenerated plants had minimum 1.54 and maximum 4.5 
head diameter decreased when in vitro technique was used cm head diameter at harvest stage. The similar results with 
as indicated by Encheva et al., (2003) and Gopalkrishnan reduced head diameter of sunflower after in vitro grown 
(1993). The possible reason for decreasing of the conditions were published by Encheva et al., (2003). The 
agronomic performances is growing conditions of the size of the individual flowers chanced from a few ray 
plants. The plant height of the individual sunflower plants flowers surrounding a small number of disk flowers to 
obtained from in vitro culture varied between 37.9 and sunflower heads of reasonable size with more than 70 
69.1 cm as it was revealed earlier by Hahne (2002). seeds in this study. The seed production of SGP varied 
Record 109/Sanay A 3-5 had the highest plant height from 173 to 842 while in vitro grown plants had 16-73 
(69.1 cm) while IMICMS6 X IMIN6 had the shortest seeds (average of 24 seeds) under our growth conditions.  
126 
All the plants transferred to soil produced flower, Based on the results from the present study, the time 
contained both fertile and sterile seeds. When the plants required to recover immature embryos averaged 3 months. 
were harvested, all the heads produced seeds changing The length of time includes the following steps: 1) 2 
from 16.3 to 72.7 in total depended on the genotype. weeks in in vitro conditions for production of plantlets 2) 
Similarly, Gopalkrishnan et al., (1993) observed that the 2 weeks acclimatization period from in vitro to growth 
vegetative phase of embryo derived plants was very short room chamber conditions, 3) 4-5 weeks of plant growth in 
resulting in small plants (12-25 cm), flower buds and early the green house to flowering and pollen shed, 4) 2 weeks 
flowering compared to field-grown plants. The possible to allow the seeds to fill and discarded immature embryo. 
reason for decreasing of the agronomic performances is 
This study reports the results of improvement growing 
growing conditions of the plants.  
sunflower plants from in vitro immature embryo culture in 
One of our study conducted at field conditions in the growth chamber and green house conditions. This 
Marmara region showed that the agronomic features of system is available for use by sunflower breeders and 
sunflower in the field conditions was very high (Göksoy et researches to create hybrids, parental lines in a very short 
al., 2003). Our results indicated that it is possible to time at breeding studies. 
produce viable seeds from in vitro grown plants and to 
ACKNOWLEDGMENT 
obtain four generations a year.  
 
 The overall result was average 54 regenerated This research was supported by the Scientific 
and matured plants per 563 excised immature embryos. Research Projects Unit of Uludag University (Project No: 
Although agronomic characters of in vitro regenerated 2009/28). The author is grateful to the Scientific Research 
plants decreased, more than 16 seeds were harvested for Projects Unit of Uludag University for financial support. 
each genotype and majority of them was viable.  
Therefore, it was sufficient for continuing to the next LITERATURE CITED 
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