Application of Method Embryo Culture in Combination with Gamma Irradiation and Ultra Sonic (Part II)

Developing of mutant lines

The Bulgarian fertility restorer lines 2574 R, 147 R, 374 R, 381 R, 377 R, American line RHA-857 and Bulgarian line with normal cytoplasm 197 B, which is highly homozygotic, were used as donor materials. A main requirement to the initial plant material used according to the methods of embryo culture in combination with ultra sonic or gamma irradiation is to be genetically pure, i.e. homozygotic to the highest possible degree. Therefore, the donor lines with very good morphological uniformity were chosen as initial material for induced mutagenesis.

Plants were grown in the field and were hand-pollinated. The immature seeds (13–16 days old) were treated with ultrasound at dose 25.5 W cm⁻² for 1, 2, 3, 5, 7, 9, 10, 11, 13 and 30 min and ionizing radiation such as gamma rays (¹³⁷Cs) at dose 8 Gy and 50 Gy (the power of the dose being 0.338 krad min⁻¹ = 3.38 Gy min⁻¹). The immature seeds were sterilized under the following conditions:

1. 1 min in 95% ethanol;

2. 15 min in bleaching solution (2.7% Cl);

3. followed by several washings with sterile distilled water.

Phytopathological evaluation

The phytopathological evaluation of the Bulgarian fertility restorer lines 2574 R, 147 R, 374 R, 381 R, 377 R, American line RHA-857, Bulgarian line with normal cytoplasm 197 B, mutant R and B lines and hybrids Rada and Yana were performed with regard to Orobanche cumana and Plasmopara halstedii at the Sunflower Phytopathology Laboratory; to Phomopsis helianthi and Phoma macdonaldii at infection fields of DAI – General Toshevo; to Septoria helianthi and Macrophomina phaseolina at natural conditions in field of DAI – General Toshevo.

The phytopathological evaluation of lines and hybrids was performed with regard to Downy mildew (Plasmopara halstedii (Farl.) Berlese & de Toni) – race 300, 330, 700 and 731. The method suggesting by Gulya et al. (1991) was used with a view to characterizing the resistance to Plasmopara halstedii. The evaluation of 50 plants from line was carried out using standard methodology:

• 0% = S (sensitive);

• 100% = R (resistant).

• 0% = S (sensitive);

• 100% = R (resistant).

Type of grey spot attack:

• 0–no symptoms;

• 1–a necrotic lesion up to 5 cm in diameter;

• 2–a necrotic lesion over 5 cm in diameter,

• 3–several merged necrotic lesions on the stem,

• 4–a stem broken at the place of infection.

Type of black spot attack:

• 0–no symptoms;

• 1–a necrotic lesion near the petiole;

• 2–several necrotic lesions on the stem;

• 3–the entire stem is covered with necrotic lesion or the stem is broken.

The evaluation for resistance to attacks of septoria (Septoria helianthi Ellis and Kellerman) was made in August at natural conditions in field when seed fill was in progress, and disease development was near its peak. The evaluation of 50 plants from line was carried out using a 1–4 scale, with values of 1 and 2 corresponding to highly and moderately resistant plants, respectively, and 3 and 4 corresponding to moderately and highly susceptible plants, respectively.

Macrophomina phaseolina resistance was evaluated at natural conditions in field of Dobroudja Agricultural Institute – General Toshevo. The evaluation of 50 plants from line was carried out using standard methodology:

• 0% = S (sensitive);

• 100% = R (resistant).

Evaluation of R and B lines for resistance to some economically important diseases and parasite on sunflower

In Bulgaria where sunflower is grown commercially, the successful production is endangered by many fungal pathogens and parasites. Losses may be severe, near 100% in parts or even entire fields under extreme circumstances. Although sunflower breeding has been very successful throughout the last decades, a number of aims remain to be achieved, e.g. resistance to various diseases and the parasite Orobanche. However, these efforts are obviously limited by the narrow genetic base of commercial sunflower which has to be enlarged by the utilization of few techniques as induced mutagenesis in particular. It is an alternative method to conventional ones.

In our study, plants resistance to various diseases and parasite Orobanche cumana were observed (Table 1). The initial R and B genotypes were susceptible. These results were confirmed during three years of evaluation.

Figure 1:

Phomopsis helianthi

Phomopsis helianthi has epiphytotic outbreaks in years with hot and moist weather during sunflower vegetation. Pathogen overwinters in plant residues in the form of peritecii. Infection became in phase 6–8 pair of leaves with ascospores of the fungus. According to Škorić (1985), Phomopsis is the most destructive pathogen of sunflower. In extreme cases, it can compromise sunflower production and cause significant yield losses.

Resistance to Phomopsis helinthi was registered at line 106 R, received after treatment of susceptible American line RHA-857 (Figure 1) with ultra sonic at dose 25.5 W cm⁻² for 3 min (Encheva, 2009b). Three years of phytopathological evaluation of the new line showed stable inheritance in the progenies.

Plasmopara halstedii is a soil-borne pathogen, its oospores serving as primary inoculums for young sunflower seedlings. The oospores (resting spores) capable of surviving for as long as 8–10 years in the soil. Till 2000 there are 10 downy mildew races existing in the world, as follows: 100, 300, 310, 330, 700, 703, 710, 711, 730 and 770 (Tourvieille de Labrouhe et al., 2000). According to Khan (2007), new races appear frequently owing to the pathogenic variability in the fungus and the selection pressure resulting from the use of resistant sunflower cultivars and fungicide seed treatment. In 2007, Gulya reported 35 races in different parts of the world. Since 2005 it was established race 330 in North-East part of Bulgaria (Shindrova, 2006).

In our work, we succeeded to receive resistant to Plasmopara halstedii lines 97 R, 99 R, 100 R and 101 R, after treatment of susceptible Bulgarian line 381 R (Figure 2) with ultra sonic at dose 25.5 W cm⁻² for 1 min and line 98 R for 3 min (Encheva and Shindrova, 2011).

Figure 2:

Plasmopara halstedii

A number of major resistance genes to downy mildew have been either identified in cultivated sunflower or introduced from wild Helianthus species (Miller, 1992). In our experiment, we prove that 100% stable resistance of the sunflower mutant lines to downy mildew (race 330) can also be obtained through induced mutagenesis, by treatment of immature zygotic embryo with ultra sonic, in particular. Resistance to Plasmopara halstedii, was observed in several plants, obtained after treatment of Bulgarian genotype 381 R with ultra sonic. This allows us to assume that there are mutable locations in the cultural sunflower genome resulting from induced mutagenesis.

Septoria helianthi winter as stroma and picnidii of plant residues. In mass attack observed tearing lamina and result was leaf drop. Infected plants have low yield, and seeds contain a lower percentage of oil (Yang et al., 1988). Resistance to Septoria helianthi was observed at lines 143 R (Figure 3) and 145 R, received after treatment of susceptible Bulgarian line 377 R with ultra sonic at dose 25.5 W cm⁻² for 1 min and 2 min, respectively (Encheva, 2013). The genetic nature and inheritance of Septoria leaf blight resistance is not known (Block, 2005).

Figure 3:

Genotype 377 R (left) susceptible to Septoria and line 143 R (right) resistant to Septoria

Orobanche cumana (Figure 4) in sunflower is one of the most important problems to solve because it causes drastic decreasing of yield. The parasite Orobanche cumana grow on sunflower roots, resulting in weak and dwindled plants, with thin steam. The parasite enhances the transpiration of damaged plants, which in drought conditions are withering, even if attacked by a small number of parasitic plants (Iliescu et al., 1998). Broomrape presents serious problems to sunflower production in Bulgaria, as well (Shindrova, 1994).

Resistance of the new lines to broomrape races E and G distributed in Bulgaria was established. The Bulgarian fertility restorer lines 2574 R, 147 R, 374 R and Bulgarian line with normal cytoplasm 197 B were susceptible to this parasite. We succeeded to receive new lines resistant to broomrape after treatment of Bulgarian line 2574 R with gamma irradiation at dose 8 Gy (line 114 R) and ultra sonic at dose 25.5 W cm⁻² for 1 min (lines 115 R and 116 R) (Encheva et al., 2003); at lines 116 R, 117 R, 118 R, 119 R and 120 R developed by treating of Bulgarian line 147 R with ultra sonic at dose 25.5 W cm⁻² for 5, 7, 9, 11 and 13 min, respectively (Encheva et al., 2008); at lines 193 R and 194 R produced after treatment of Bulgarian genotype 374 R with gamma ray at dose 50 Gy and ultra sonic at dose 25.5 W cm⁻² for 10 min, respectively (Encheva, 2009a) and at line 12003 R, developed by treating of Bulgarian line 2574 R with ultra sonic at dose 25.5 W cm⁻² for 1 min (Encheva et al., 2012a).

Figure 4:

Orobanche cumana

Resistance to Orobanche cumana was established at new lines with normal cytoplasm 74 В, 78 В, 85 В and 88 В, also. They were received after treatment of susceptible Bulgarian line 197 B with ultra sonic at dose of 25.5 W cm⁻² for 1, 3 and 5 min (Encheva et al., 2010).

Resistance to parasite broomrape was received simultaneously in several sunflower plants (developed in lines) after treatment of immature zygotic embryos of susceptible Bulgarian lines 2574 R, 147 R, 374 R and 197 B using ultra sound or gamma irradiation.

Several plants (developed in lines) received after treatment of susceptible Bulgarian line 381 R with ultra sonic showed simultaneously resistance to Plasmopara halstedii.

Resistance to Septoria helinthi was observed at two plants (developed in lines), received after treatment of susceptible Bulgarian line 377 R with ultra sonic.

Resaving of more than one resistant plant from one susceptible genotype allows us to assume that there are mutable locations in the cultural sunflower genome resulting from induced mutagenesis.

Burlov and Kostyuk (1976) and Pogorleckii and Gesele (1976) discovered that broomrape resistance was controlled by a dominant gene which was designated as Or.

Our results allow us to presume that the resistance of the mutant sunflower lines to Orobanche cumana occurred as a result from a single gene dominant mutation. Similar conclusion has been made by Christov and Nikolova (1996), analyzing the type of resistance to broomrape of mutant sunflower forms obtained through irradiation of air dry seeds with gamma rays. The authors found out that it was controlled by a single dominant gene. Resistant mutants to Verticillium with dominant genes were obtained after EMS treatment of pollen of tomato (Gavazzi et al., 1987).

Many studies show a monogenic control by a single dominant gene over sunflower resistance against race E (Ish-Shalom et al., 1993; Sukno et al., 1999), although two dominant genes (Dominguez, 1996) and one recessive gene (Ramaiah, 1987). The race F resistant population BR4, derived from wild species, was found to be under the control of a single dominant gene designated Or₆ (Perez-Vich et al., 2002). Pacureanu et al. (2004) reported a single dominant gene controlling the resistance to race F in Romania, also. More recent studies on the inheritance to resistance to the latest races in several countries have concluded the presence of one or two dominant genes (Škorić et al., 2010; Velasco et al., 2012).

Our results confirmed the conclusion of Skirvin (1978), that mutagenesis, physical or chemical, is favourable for induction of mutations in tissue cultures. It was established that the possibilities of experimental mutagenesis in using embryos at an early stage of their development are greater, as compared to air dry seeds (Atanassov, 1988).

Hybrids Rada and Yana, developed with mutant lines 12003 R and 12003 R

Evaluation for resistance to some economically important diseases and parasite on sunflower:

After long inbreeding mutant lines 12002 R and 12003 R were developed and tested for their combining ability. The results from the 3-year testing of lines 12002 R and 12003 R revealed very good combining ability in hybridization. The line 2607 A (sterile analogues of the Bulgarian inbred line) was used as a tester.

Commercial hybrid Rada is simple cross hybrid, developed by crossing of 2607 A × 12002 R. Line 12002 R was developed by embryo culture method of immature zygotic embryos in combination with gamma irradiation at doze 8 Gy (Encheva et al., 2003). Hybrid possessing immunity to the parasite Orobanche cumana population of race G (resistance of hybrid to race G was established later after screening test): immunity to Plasmopara helianhi – races 300, 330, 700 and 731, immunity to Macrophomina phaseolina and tolerance to Phomopsis and Phoma. The immunity to Orobanche was inherited from the mutant restorer line 12002 R. Mother line 2607 A, component of hybrids Rada, was susceptible to Orobanche cumana.

Commercial hybrid Yana is simple cross hybrid, developed by crossing of 2607 A × 12003 R (Encheva et al., 2012b). Line 12003 R was developed by embryo culture method of immature zygotic embryos in combination with ultra sonic at dose 25.5 W cm⁻² for 1 min. Hybrid possessing immunity to the parasite Orobanche cumana population of race G, immunity to Plasmopara helianhi – races 300, 330, 700 and 731, immunity to Macrophomina phaseolina, resistance to Phomopsis and tolerance to Phoma. The immunity to Orobanche was inherited from the mutant restorer line 12003 R, because mother line in the cross-2607 A was susceptible to broomrape.

Resistance of hybrids Rada and Yana to some economically important diseases and parasite Orobanche are a guarantee that it is suitable for mass production.