A PROMISING ALTERNATIVE WAY OF UTLIZING POL CMS FOR HYBRID BREEDING IN BRASSICA NAPUS L

A PROMISING ALTERNATIVE WAY OF UTLIZING POL CMS FOR HYBRID BREEDING IN BRASSICA NAPUS L

Guang-sheng Yang¹⁾,Zhi-hong Duan²⁾,Ting-dong Fu¹⁾,Chang-sheng Wu²⁾

¹⁾The National key laboratory of crop genetics and improvement, Department of Agronomy, Huazhong Agricultural University, Wuhan 430070, P. R. China. Email: gsyang@public.wh.hb.cn, futing@public.wh.hb.cn

²⁾ Hubei high quality rapeseed development and service center, Wuhan 430070, P. R. China

ABSTRACT

A ecotypical male fertile-sterile line AB₁ was selected out from the progeny of a cross between two Brassica napus lines. AB₁ was male fertile when it was sown at Wuhan in Autumn, but it became sterile when it was sown at Kunming or Xining in Summer. Some investigations showed that the fertility was probably related to the temperature during the time of the flower’s development. Genetical analyses showed that the fertility of AB₁ could be restored by pol CMS restorers, and the fertility restoring was controlled by one pair of dominant genes. RFLP and mitochondrial DNA(mt DNA) fragment polymorphism analyses showed that the cytoplasm of AB₁ was the same as the pol CMS male sterile lines. Anatomical observations showed that there was no pollen sac differentiation in the male sterile anther of AB₁ when it was sown in the summer. At present, a few lines from AB₁ with double low quality and good agronomy performance have been developed.

KEY WORDS: Brassica napus L.; Ecotypic male sterility; Polima cytoplasmic male sterility; Genetics; Breeding.

1. INTRODUCTION

Many previous tests for heterosis in Brassica napus L showed that there was significant yield heterosis in F₁ varietal hybrids. The heterosis level in spring type was about 20%~30%, and in winter was about 30%~40%. Therefore, Hybrid breeding has been regarded as an important way for further increasing the seed yield of rapeseed in the world since 1970s.

A key point of hybrid breeding is to find an effective and economical system of producing F₁ seed in large scale. At present, a few systems have been used in rapeseed in the world. They are cytoplasmic male sterility (CMS), genic male sterility (GMS), self-incompatibility(SI) and Chemical induced male sterility(CHA). Among them, CMS is considered as the most important system.

Up to now, pol CMS (Fu,1981) and ogu CMS(Ogura 1968) are still the important CMS systems for rapeseed hybrid breeding in the world. The main disadvantage of pol CMS is its unstable male sterility. The male sterile lines could become partial fertile at relative lower or higher temperature situations (Yang and Fu, 1987). But it is possible to select out a relative stable pol CMS line through many careful selections. The main problem for ogu CMS is its fertility restoring gene genetical linked tightly to the genes with high content of glucosinonates (Renard et al.,1997).

Self-incompatibility is also an important system in hybrid seed production. A few SI hybrids have been registered in the word. The main question for SI is the reproduction of SI parent lines in large scale.

In China, Genic male sterility is also an important way for hybrid seed production. Three GMS systems have been used in hybrid breeding, and a few hybrids have been registered. The main problem is that there is about 50% fertile plants in the mother lines.

Further more, a few institutions conducted investigations on using germicides to producing F₁ seed, but the main restriction factor is lacking in germicides with high inducing male sterility effect and no environment pollution.

This paper will present a new system (ecotypic male sterility) for F₁ seed production in rapeseed.

2 MATERIALS AND METHODS

2.1 Materials

The used varieties and lines of Brassica napus L were listed in table 1. The two mitochondrial DNA (mt DNA) probes atp6 and orf222 were provided by Professor Dr. Gregory G. Brown, in the Department of Biology, McGill University, Canada.

TABLE 1. NAMES AND ORIGINS OF MATERIALS

Names

Functions

Origins

AB₁

Restorer 10

Restorer 5200

1141B

1238B

KenC1

6223B

Huaye

Xiangai B

Xin-4

Xin-4-1A

Westar S

Westar F

Ecotypical male sterile line

pol CMS restorer

pol CMS maintainer

pol CMS restorer

pol CMS maintainer

pol CMS line

Westar with nap cytoplasm

Westar with normal cytoplasm

Huazhong Agricultural University(HAU)

HAU

Popular Science Center of Agro-reclamation, Shaanxi(PSCAS)

PSCAS

Hunan Academy of Agricultural Science (HAAS)

HAAS

Sichuan Academy of Agricultural Science (SAAS)

HAU

McGill University

2.2 Methods

2.2.1 Genetic analysis of the ecotypical male sterility Reciprocal crosses between AB₁ and pol CMS maintainers and restorers were made. The fertility of all F₁s were investigated in both the summer sowing situation at Kunming and the autumn sowing situation at Wuhan. The fertility of the F₂s and BC₁s populations from the reciprocal crosses between AB₁ and pol CMS restorers were also evaluated at the same situations.

2.2.2 mt DNA polymorphism analyses buds and flowers (5~10g) greenhouse grown plants of each material were used for isolation of mt DNA as described by Kemble (1987). Four restriction endonucleases EcoR, Hind , Pst and Sal were used to digest the mt DNA. The mt DNA fragments were separated on 0.7% (W/V)agarose gel for overnight, and analyzed and photographed under UV light. Then the mt DNA fragments were transferred on to nylon membranes with 0.5N NaOH solution. Finally, the membranes were probed with atp6 which could distinguish CMS systems from pol CMS (Singh and Brown,1990) or orf222 (H'Homme et al,1997) which could distinguish CMS systems from either nap CMS or pol CMS, respectively.

2.2.3 Anatomical observation of the anther development of AB₁ Buds, from the plants of AB₁ which were planted in two growth chambers with temperature 30/20 or 15/5respectively, were collected, and anthers' developmental stages were checked with a light microscope. Anthers at different development stages were picked up and put into 3% glutaraldehyde solution (3% glutaraldehyde,0.5M H₃PO₄, pH=7.2)immediately and fixed for 12~24 hours, postfixed in 1% OsO₄ for 3 hours, dehydrated in the series of acetone solutions (30%,50%,70%, 90%,100%) three times, embeded with Epon182, and cut into pieces with 2~3m thin by LKB-V super thin cutting-machine. The pieces were dyed with toluidine blue and observed and photographed under a light microscope.

2.2.4 Light and temperature tests In the Spring of 1994, five plants of AB₁ and its hybrid (AB₁x1141B) were planted in the growth chambers with temperature of 15/5,20/10,25/15and 30/20(day and night)and light of 12 hours. In the spring of 1996, five plants of AB₁ and its hybrid (AB₁1141B) were planted in the growth chambers with the same temperature and light of 11 hours and 16 hours. The fertility of each plant was checked after flowering.

3 RESULTS

3. 1 The discovery of ecotypic male sterility and the development of AB₁

In the summer of 1986, one F₃ line from a cross between KenC₁Xiangai B was discovered to be male sterile on the experimental farm of Yunnan Academy of Agricultural Science, located at suburb of Kunming City. In the autumn of 1986, the remained seed of this F₃ line was sown at Wuhan again. In the spring of 1987, the individual plants in this F₃ lines showed fertility segregation, among which three plants were fertile (5 grade), and the others were partially sterile with variant fertility (3~4 grades). The three fertile plants were selfed, and the F₄ seed from each F₃ plant was divided into two parts. One part of each F₄ seed was sown in Kunming in May, 1987. Fertility observation showed that two F₄s were male sterile, and the other was partially sterile. In the Autumn of 1987, the left seed of the two F₄s was sown at Wuhan again. After twice selfing at wuhan and fertility investigations at Kunming from 1988 to 1989, a ecotypic male sterile line AB₁ in Brassica napus was developed.

3.2 The fertility of progenies from crosses between AB₁ and pol CMS maintainers and restorers

3.2.1 The fertility of F₁s from crosses between AB₁ and pol CMS maintainers and restorers The fertility of the F₁s from reciprocal crosses between AB₁ and pol CMS maintainers and restorers in the summer sowing situation at Kunming and in the autumn sowing situation at Wuhan was listed in table 2. It showed that all F₁s were fertile at the both situations when AB₁was used as a male parent in the hybridizations. But when AB₁ was a female parent in the hybridizations, the result was different. The F₁s from the crosses between AB₁ and pol CMS restorers were fertile at the both situations, and the F₁s from the crosses between AB₁ and pol CMS maintainers were sterile at the

summer sowing situation and were partially sterile at autumn sowing situation. This result suggested

TABLE 2. THE FERTILITY OF F₁s BETWEEN THE MALE FERTILE-STERILE LINE AB₁AND POL CMS RESTORERS AND MAINTAINERS

Variety

AB₁()

Variety

AB₁()

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

AB₁

Restorer 10

Restorer 5200

Huaye

KenC₁

F(5)

F(6)

S(1)

F(6)

F(5)

F(6)

S(1)

F(6)

1141B

1238B

6223B

Xiangai B

Xin-4

PS(4)

PS(5)

S(0)

S(1)

F(6)

that the variant fertility of AB₁ at different situations was related to its cytoplasmic genotype, and all restorers of pol CMS could be used as restorers of AB_1.

3.2.2 The fertility of F₂ and BC₁ progenies from the crosses between AB₁ and pol CMS

restorers The fertility of F₂ and BC₁ progenies from the crosses between AB₁ and pol CMS restorers Huaye and Restorer 10 at summer sowing situation at Kunming and at autumn sowing situation at Wuhan was evaluated and the result was listed in table 3. When AB₁ was used as the female parent, the fertility segregation of the F₂s and BC₁s of the two crosses agreed with the inheritance of one pair of major genes in the summer sowing situation, but did not accorded with this rule in the autumn sowing situation. When AB₁ was used as the male parent, the F₂ and BC₁ from Restorer 10AB₁ and the BC₁ from HuayeAB₁ did not show fertility segregation, and the F₂ from HuayeAB₁ showed fertility segregation in agreement with the inheritance of one major gene in the summer sowing situation but did not in the autumn sowing situation. These results suggested further that the variant fertility of AB₁ in different situations be related to its cytoplasmic genotype. Pol CMS restorers Huaye and Restorer 10 had one pair of fertility restoring genes. The reason why the fertility segregation of the F₂s and BC₁s from the crosses AB₁Huaye and AB₁Restorer 10 did not agreed with the inheritance rule of one major gene in the autumn sowing situation is probably that AB₁, Huaye and Restorer 10 had low temperature sensitive genes in their nuclei.

TABLE 3. SEGREGATION OF THE FERTILITY OF F₂ ABD BC₁ PROGENIES FROM THE CROSSES BETWEEN AB₁ AND POL CMS RESTORERS

Crosses

Progenies

Locations

F/PS+S

AB₁Huaye

HuayeAB₁

AB₁Restorer10

Restorer10AB₁

F₂

BC₁

F₂

BC₁

F₂

BC₁

F₂

BC₁

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

Wuhan

Kunming

5. 75:1

3.07:1

6. 50:1

1.15:1

3. 69:1

3.07:1

23:0

19:0

28. 0:1

3.21:1

44. 0:1

1.30:1

56:0

41:0

38:0

35:0

3:1

1:1

3:1

1:1

<0.05

>0.90

<0. 01

0. 75-0.90

0.50-0.75

>0.90

<0. 01

>0.90

<0. 01

0.50-0.75

The fertility investigations of the F₁s from the crosses between AB₁ and pol CMS maintainers and of the F₁s, F₂s and BC₁s from the crosses between AB₁ and pol CMS Restorers suggested that AB₁ be probably a pol CMS line.

3.3 Comparison of the restriction pattern and RFLP analysis of mt DNA

The restriction patterns of mt DNA digested with EcoR, Hind ,Pst or Sal were analyzed.AB₁ had the same pattern as pol CMS line Xin-4-1A, but had different pattern as Westar S and Xin-4 which have nap cytoplasm, and Westar F which has normal (or cam) cytoplasm.

It was reported that atp6/orf224 was associated with pol CMS, and orf222 was associated with nap CMS. Therefore, if atp6 and orf222 are used as probes to hybridize the mt DNA digested with some restriction endonucleases, it could be easy to tell the cytoplasmic differences between the AB₁, pol CMS, nap CMS and normal cytoplasm. Southern hybridization result showed that AB₁ had the same RFLP pattern as pol CMS line Xin-4-1A, but had different pattern as Westar S, Westar F and Xin-4.

The results of the restriction fragment pattern and RFLP analysis of mt DNA suggested that AB₁ have probably pol cytoplasm.

3.4 Anatomical characteristics of AB₁’s anther development

Anthers of AB₁ in the growth chamber with temperature 15/5 had the same development procedure as those of the normal rapeseed lines. But in the growth chamber with temperature 30/20, they had the same development procedure as those of pol CMS line Xin-4-1A. The development of the anthers stopped at the stage of archesporial cell, without pollen sac differentiation.

3.5 Fertility tests of AB₁ and its hybrid (AB₁ x 1141B) under the controlled light length and temperature situation

In the Spring of 1994, the Fertility of AB₁ and its hybrid (AB₁ x 1141B) was tested in growth chambers with light length 12 hours and temperature 15/5,20/10,25/15 or 30/20, respectively(Table 4). AB₁ was fertile and its hybrid (AB₁1141B) was partially fertile at the temperature 15/5; AB₁ was partially fertile and its hybrid (AB₁1141B) was partially sterile at the temperature 20/10; AB₁ was partially sterile and its hybrid (AB₁1141B) was completely sterile at the temperature 25/15;And AB₁ was sterile and its hybrid (AB₁1141B) was completely sterile at the temperature or 30/20.In the Spring of 1996, the Fertility of AB₁ and its hybrid (AB₁1141B) was tested again in growth chambers with light length 11 or 16 hours and temperature 15/5,20/10,25/15 or 30/20, respectively(Table 4).The same result was obtained at the light length both 11 and 16 hours. These two results suggested that light length have probably no considerable influence on the fertility of AB₁ and its hybrid (AB₁1141B), and temperature be the main environmental factor influencing the fertility of AB₁ and its hybrid (AB₁1141B),the higher the temperature, the complete the male sterility. They also suggested that the influence of temperature on the fertility be related to the genotypes or the number of the temperature sensitive genes in the nuclei of the materials. In some range, the more the number of the temperature sensitive genes, the weaker the influence of the temperature.

TABLE 4. THE FERTILITY OF AB₁ AND ITS HYBRID(AB₁X1141BAT DIFFERENT TREATMENTS OF TEMPERATURE AND LIGHT LENGTH IN GROWTH BOXES

Years

The length

of light/h

Materials

Temperature

15/5

0/10

25/15

30/20

1994

1996

AB₁

F₁

AB₁

F₁

AB₁

F₁

F(5)

PF(4)

F(5)

PF(4)

F(5)

PF(4)

PS(2)

PF(4)

PS(2)

PF(4)

PS(2)

S(0)

PS(3)

S(0)

PS(3)

S(0)

S(1)

S(0)

S(1)

S(0)

S(1)

S(0)

4. DISCUSSION

Breeding practices had proved that the pol CMS lines could be divided into three groups, i.e., high temperature male sterile lines, low temperature male sterile lines and relatively stable male sterile lines(Fu et al,1989). Therefore, it is possible to develop ecotypic male sterile lines from pol CMS lines by selection. The genetic analysis, mt DNA restriction fragment length polymorphism comparison and anatomical observation of anther’s development in this study had proved that AB₁ was a pol CMS line.

The main environmental differences between the summer sowing at Kunming or Xining and the autumn sowing at Wuhan during the development of rapeseed buds, is temperature and day length. At the summer sowing situation, the mean daily temperature is relative higher (about 20) and the day length is relative longer (more than 14 hours)during June-July; But at autumn sowing situation, the mean daily temperature is relative lower (lower than 10) and the day length is relative shorter (shorter than 12 hours)during January-February. It seems that day length or temperature might be the factor influencing the fertility of AB₁. The results under the growth chambers with the controlled light length and temperature suggested that temperature be the main factor which could influence the fertility of AB₁, and day length have no considerable effect on the fertility.

Some investigations on the fertility of F₁s, F₂s and BC₁s from the crosses between AB₁ and some pol CMS maintainers at the summer sowing and winter sowing situations showed that the number of temperature sensitive genes in the nucleus were related to the influence of temperature on the ecotypic male sterility (Yang et al,1995; 1997). Therefore, we can utilize the temperature difference between the summer sowing situation at Xining or Kunming and the autumn sowing situation at Wuhan, developing pol CMS ecotypic male sterile lines which have a certain number of temperature sensitive genes and are nearly fertile at the autumn sowing situation and completely sterile at the summer sowing situation. We started a breeding program of pol CMS ecotypic male sterile lines at 1993. At present, several ecotypic male sterile lines of Brassica napus with double low qualities have been developed.

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