WO2001006845A1

WO2001006845A1 - A method for obtaining 100 % male sterile plants to serve as the female parent in hybrid seeds production

Info

Publication number: WO2001006845A1
Application number: PCT/IL2000/000436
Authority: WO
Inventors: Shamay Izhar; Nurit Firon
Original assignee: State Of Israel/Ministry Of Agriculture
Priority date: 1999-07-25
Filing date: 2000-07-23
Publication date: 2001-02-01
Also published as: IL131095A0; AU6178500A

Abstract

The invention relates to a method for obtaining seeds that yield 100 % male sterile plants to be used as the A-line for hybrid seeds production, comprising; a) selecting male sterile plants; b) inducing transient male fertility restoration (T-MFR) in said plants; c) self-pollination or crossing among plants that undergo T-MFR in step b and produced pollen, or cross-pollination between pollen produced in step b and any suitable male sterile plants; d) collecting the seeds of the fruits developed by the pollination of step c, said seeds will yield 100 % male sterile plants.

Description

A METHOD FOR OBTAINING 100% MALE STERILE PLANTS TO SERVE AS THE FEMALE PARENT IN HYBRID SEEDS PRODUCTION

FIELD OF THE INVENTION

The present invention relates to a method for obtaining all female 100% male

sterile plants for use as the female line in hybrid seeds production. More

specifically the present invention relates to a method for obtaining seeds that

yield 100% male sterile plants by manipulation of nuclear genes. Said seeds

are obtained by self-pollination or cross pollination among male sterile plants

that undergo transient male fertility restoration treatment (T-MFR) or by cross

pollinating between the pollen produced by said plants and any suitable male

sterile plants. The T-MFR according to the present invention is induced by

subjecting the male sterile plants to controlled conditions of daytime and

nighttime temperatures for a predetermined period of time. The present

invention further relates to the pollen produced by the male sterile plants that

undergo T-MFR, to the seeds which produce 100% male sterile plants, to the

100% male sterile plants developed thereof and to the male fertile FI hybrid

seeds produced by the crossing between said 100% male sterile plants and any

other suitable male fertile C-line. The hybrid seeds produced according to the

present invention yield fertile plants. The present invention specifically relates

to said methods and seeds for plants of tomatoes and other species of the

solanaceae family. BACKGROUND OF THE INVENTION

Production of hybrid seed for commercial sale is a large developing industry

and an extremely important one for the future feeding of the world population

(Science, vol 283 pp. 310, 1999). Hybridization of plants is recognized as an

important process for producing progeny having a unique combination of the

desirable traits of the parental plant lines. Hybrid plants grown from hybrid

seed benefit from the "hybrid vigour" of crossing two genetically distinct and

carefully selected breeding lines. The agronomic performance of this progeny

is superior to both parents, in vigour, yield, and uniformity. Thus, better

performance of the hybrid seed varieties compared to open-pollinated varieties

makes the hybrid seed more attractive to farmers. The Fi hybrid plants cannot

be practically duplicated since selfing or crossing of the F 1 hybrid plants will

cause segregation of the traits according to the Mandelian inheritance.

A plant is self-pollinated when pollen of a plant fertilizes the same flower or

other flowers of the same plant. A plant is cross-pollinated if the pollen comes

from a flower of a different plant, usually by a vector (wind, insects or human.

Hybrid seeds are usually produced by crossing a female parental line with a

male parental line. In order to ensure 100% Fi hybrid seeds, the line being the

female must be 100% male sterile, otherwise self-pollination may occur,

producing seeds that will contaminate the Fi hybrid seeds. Hand or mechanical

emasculation of the female line is often done, however, this is an expensive

procedure. Only few commercial hybrid seed systems are currently known for field crops

and all of them are based on cytoplasmic male sterile parental lines being the

female parent (A-line) of the Fi hybrid seeds. For example see Frankel R. and

Galun, Pollination mechanisms, reproduction and plant breeding, Springer,

Berlin Heidelberg New York (1977), p. 281 and Kaul M.L. Male sterility in

plants Springer Verlag, Berlin Heidelberg New York (1978), p. 1005.

There is no conventional way to produce 100% male sterile (female) parents by

genetic means that are based on a single recessive gene. In conventional

systems which are based on genie male sterility, the male sterility is not 100%

and laborious efforts are needed in order to sort out (roguing) the fertile plants

in order to produce a population of 100% female plants (male sterile).

Therefore, the suggested system according to the present invention is based on

genie male sterility that could be regulated to allow transient male fertility

restoration (T-MFR) by manipulation of gene expression.

The present invention relates to a method for restoring transient male fertility

(T-MFR) in male sterile plants in order to produce 100% male sterile plants

suitable for the production of Fi hybrid seeds which are of commercial value.

Said 100% male sterile plant population is achieved by self or cross pollination

among the male sterile plants that undergo T-MFR according to the method of

the present invention and produce pollen, or by crossing the T-MFR pollen

producing plants with male sterile plants.

Such a system allows the propagation of the female line by the T-MFR

technique and enables production of seeds that produce 100% male sterile plants. In other words, the present invention assures genotypes which are 100%

male sterile at the seed stage. The plants that develop from these seeds serve as

the female line (A-line) for the production of hybrid seeds with the proper male

pollinator that is assigned by the breeder (a C line).

It is the aim of the present invention to provide a method for a transient male

fertility restoration in plants in order to produce 100% male sterile plants that

can be used as the female parents in the hybrid seed production, thus

eliminating the self-pollination in the hybrid seed production (as potential

contaminants) or the need for roguing and providing a method for low cost

production of hybrid seeds.

The present invention also enables 100% permanent male fertility restoration to

the Fj hybrid (commercial) plants. This is a prerequisite for Fi plants for most

of the major crops, in which the economical yield consists of the plant

reproductive part. In many crop species (rice, wheat etc.) such technique may

be the only way to produce Fi hybrid seeds using nuclear genes for male

sterility and not by resorting to transgenic plants.

SUMMARY OF THE INVENTION

The present invention relates to a method for obtaining seeds that yield 100%

male sterile plants to be used as the A-line for hybrid seeds production,

comprising; a) selecting male sterile plants; b) inducing transient male

fertility restoration (T-MFR) in said plants; c) self-pollination or crossing

among plants that undergo T-MFR in step b and produced pollen, or cross-pollination between pollen produced in step b and any suitable male

sterile plants; d) collecting the seeds of the fruits developed by the

pollination of step c, said seeds will yield 100% male sterile plants (all female

parent line).

According to the present invention T-MFR is produced by subjecting the male

sterile plants to controlled conditions of daytime and nighttime temperatures

for a predetermined period of time;

According to the present invention the pollination of step (c) can be done

between plants in which T-MFR was induced in step (b), being the male

parent, and plants in which T-MFR was not induced under the same

conditions, being the female parent.

The present invention further relates to the pollen produced in step (b), to the

seeds obtained by the method of the present invention that yield 100% male

sterile plants, to the 100%) male sterile plants developed thereof, to the hybrid

seeds that yield FI male fertile plants produced by the crossing between said

100% male sterile plants as A-line and any other suitable male fertile C-line of

homozygous MS-MS allele and to the F 1 male fertile plants produced thereof.

The present invention specifically relates to said methods, pollen, seeds and

plants of the solanaceae family such as tomato or petunia of any male sterility

genes which are of horticultural value and more specifically to tomato plants

having the male sterility inducing alleles in homozygous state such as ms26

ms33 or ms35 genome. However similar method can be applied to any species

having male sterility inducing gene that can be manipulated. In a preferred embodiment of the present invention the male sterile plants are

tomato plants of the female parent of varieties, such as Orit and Naama, having

a male sterility gene of tomato ms26 and the controlled temperature conditions

to induce T-MFR are: a) 1 to 5 weeks of daytime temperature of about

15-21°C and nighttime temperature lower than of 10°C, followed by b)

approximately 2 weeks of daytime temperature of approximately 26°C and

nighttime temperature of approximately 16°C.

Yet, in another preferred embodiment of the present invention the male sterile

plants are tomato plants of the female parent of varieties, such as Daniella,

having a male sterility gene of tomato ms35 and the controlled temperature

conditions to induce T-MFR are: a) 1 to 5 weeks of daytime temperature of

about 30°C and nighttime temperature of about 21°C followed by b)

approximately 2 weeks of daytime temperature of about 26°C and nighttime

temperature of about 17°C.

DETAILED DESCRIPTION OF THE INVENTION

Scheme A on page 8 describes the overall general protocol for obtaining Fj

hybrid seeds according to the method of the present invention. The method is

based on pollinating stable (msms) male sterile plants by pollen of male sterile

plants (msms) that undergo transient male fertility restoration treatment to

obtain seeds which will yield 100% male sterile plants. The so obtained 100%

male sterile plants are the female line (A-line) of the hybrid seeds. The hybrid

seeds obtained by crossing between said A-line and any suitable C-line yield

normal and fertile Fi plants which show no signs of the T-MFR treatment.

Experiments were carried out with several non-commercial and with

commercially available female lines which are based on one recessive male

sterility gene.

SCHEME A A METHOD FOR OBTAINING HYBRID SEEDS ACCORDING TO THE PRESENT INVENTION

SELECTING MALE STERILE PLANTS of msms GENOME

T-MFR TREATMENT

COLLECTING POLLEN OF ms GENOME OF TRANSIENT

FERTILE PLANTS

CROSSING BETWEEN POLLEN (ms) and STABLE MALE

STERILE PLANTS (msms)

FRUITS WITH SEEDS. THE SEEDS DEVELOP INTO:

PRODUCTION OF HYBRID SEEDS BY CROSSING WITH

C-LINE POLLEN

NORMAL HYBRID SEEDS SCHEME B

A METHOD FOR OBTAINING SEEDS THAT WILL

YIELD 100% MALE STERILE PLANTS BY TRANSIENT

MALE FERTILITY RESTORATION (T-MFR) IN

TOMATO PLANTS HAVING ms26 GENE

SELECTING MALE STERILE PLANTS of ms26ms26

T-MFR TREATMENT

1 - 5 WEEKS OF:

DAYTIME TEMPERATURE 15-21°C

NIGHTTIME TEMERATURE < 10°C

RETURNING PLANTS BACK TO STANDARD GROWING TEMPERATURES

ABOUT 3 WEEKS AFTER THE TERMINATION OF THE T-MFR TREATMENT, FLOWERING AND POLLEN APPEARANCE ARE IN

THEIR PEAK

COLLECTING POLLEN WITH ms26 GENE

POLLINATING STABLE MALE STERILE PLANTS (ms26ms26) WITH COLLECTED (ms26) POLLEN

FRUITS WITH

The following examples demonstrate the method of the present invention in

tomato plants.

EXAMPLE 1: T-MFR, production of male sterile seeds and production of

hybrid seeds in tomatoes of ms26 gemome

Scheme B on page 9 describes the protocol for the manipulation of the ms26

male sterility gene in tomatoes plants in order to obtain a 100%) male sterile

seeds.

Experiments were carried out with several commercialy available female lines

which are based on the male sterility gene ms26. The flowers of ms26ms26

genotype have certain morphology (distorted anthers with no fertile pollen)

that distinguishes them clearly from the male fertile flowers.

The procedure was carried out by the following steps:

1. Seeds of the female commercial line 1613 were sown in trays.

2. 50% of the population of the plants grown in step 1 were MS26ms26 fertile

plants and were eliminated (roguing process) in order to obtain population

of 100% male sterile plants with the ms26ms26 genotype only.

3. T-MFR induction process: The selected plants of step 2 were transferred

into a phytotron. The specific conditions to which the plants were

subjected, were normally the following temperature conditions:

a. 1 to 5 weeks of subjecting the plants to daytime temperature of about 17 ±

3°C and nighttime temperature of about 10 ± 3°C. b. About 3 weeks of the standard growing conditions for tomatoes

20-29°C/14-18°C.

4. Following the induction of T-MFR, different routes are taken for the

production of the pollen and for the growing of the female parent, both for

the production of the 100% male sterile plants:

A: Production of pollen

About 3 weeks after the T-MFR treatment (step 3a) terminates, pollen

grains develop in the anthers of about 90-100% of the plants which

underwent T-MFR. The pollen grains from these plants are collected by

any known method including the industrial method using liquid and is used

immediately or is stored for a later use. The pollen grains collection lasts

as long as they are produced on the T-MFR plants, for about 1 to 2 weeks,

depending on the T-MFR length of the induction period.

B: Growing the selected population for the female parent

Those plants that do not show signs of T-MFR during step 3 b (after

exposure for one week only) can be selected as mother plants at this stage

and are planted in a greenhouse or in the field to be the female parents of

the female A-line consisting of 100% male sterile plants. Another way of

selecting the female plants for A-line is to keep plants under T-MFR

induction conditions only for one week and then at the normal growing

conditions for tomatoes. Those plants which do not show signs of T-MFR

at all are good female plants for A-line. 5. The plants of step 4B (female parent) are pollinated by the pollen collected

at step 4A to obtain seeds of 100% male sterility (to develop the A-line

plants of the hybrid seeds).

RESULTS

1. Fertility restoration

The effect of the T-MFR is measured by 2 parameters: 1) the number of the

plants that respond to the treatment; 2) the number of fertile flowers per plant.

The production of these two parameters determines the yield of pollen that can

be obtained.

Sorting of plants according to their restored fertility was done according to the

following definitions:

S - male sterile without traces of pollen;

N.F - no flowers;

F5 - traces of pollen;

F4 - very little pollen;

F3 - medium amount of pollen as compared to fertile plants. This is usually the

stage at which the pollen is collected from the T-MFR plants;

F2 and F 1 are definition for a large amount of pollen or full fertile plants,

respectively. Such fertility degrees are usually not achieved in the T-MFR

plants.

Table 1 shows typical results of different T-MFR induction periods. Table 1: The degrees of fertility distribution of tomato plants (ms26ms26)

after T-MFR treatment:

Table 2 shows typical results of the average percentage of fertile flowers per

plant (F3 - fertility level - flowers that are suitable for collecting pollen) after 2

and 3 weeks of T-MFR induction periods, at different times after the T-MFR

treatment terminates. The test was carried out by picking an arbitrary sample

of plants. Table 2:Average percentage of fertile flowers per plant after T-MFR treatment.

2. Seeds that yield A-line plants and hybrid seeds production

Cross and self pollination was carried out among different plants that

underwent T-MFR treatment. The seeds so obtained were sown and the plants

developed from these seeds were planted in fields or greenhouses at different

locations to serve as the A-line of the hybrid seeds and were pollinated by

C-line pollen under different conditions.

The load of the seed bearing fruits on a plant is an important factor that affects

the hybrid seeds yield. Experiments were done in order to find what is the

earliest ripening level at which the hybrid seeds can be collected. The results

are summarized in Table 3. Table 3: The average sprouting rate of the seeds collected at different ripening levels

The above results show that the seeds can be collected at the orange stage, 4-6

days before full ripening, thus reducing the load of fruits per plant. Early

harvest of the fruits allow ample flowering on the plant and thus increasing the

number of fruits per plant during the season.

3. Hybrid plants

Many thousands Fj hybrid plants of the variety Orit were produced by the

hybrid seeds obtained as described above and were compared to plants that

were produced by the conventional procedure, by all the morphological

parameters. There was no difference between the two groups of plants. EXAMPLE B: T-MFR. production of male sterile seeds and production of

hybrid seeds in tomatoes of ms35 gemome

Similar experiments have been carried out with tomatoes of ms35 genome.

Induction period of 10 days with conditions of about 30°C daytime temperature

and 21 °C nighttime temperature, transient male fertility has been restored in the

plants.

Claims

1) A method for obtaining seeds that yield all female 100% male sterile plants

for use as the A-line for hybrid seeds production, comprising;

a) selecting male sterile plants;

b) inducing transient male fertility restoration (T-MFR) in said plants ;

c) self-pollination or cross-pollination among plants that undergo

T-MFR in step b and produced pollen, or cross-pollination between

pollen produced in step b and any suitable male sterile plants;

d) collecting the seeds of the fruits developed by the pollination of step

c, said seeds will yield 100% male sterile plants.

2) A method according to claim 1 wherein the transient male fertility

restoration is induced by subjecting the male sterile plants to controlled

conditions of daytime and nighttime temperatures for a predetermined

period of time.

3) A method according to claim 1 wherein the pollination of step c is done

between plants in which T-MFR was induced by step b, being the male

parent, and plants in which T-MFR was not induced under the same

conditions, being the female parent.

4) A method according to claim 1 and 2 wherein the plants are of the

solanaceae family. 5) A method according to claim 3 wherein the plants are selected from tomato

or petunia.

6) A method according to claim 4 wherein the male sterile plants are tomato

plants and wherein the male sterility is of the type containing the male

sterility genes ms26ms26 or ms33ms33 or ms35ms35 or any other tomato

male sterility genes.

7) A method according to claim 5 wherein the plants are tomato plants having

a male sterility gene ms26 and wherein the controlled temperature

conditions are as follows:

a) 1 to 5 weeks of daytime temperatures of about 15-21°C and nighttime

temperatures lower than 10°C;

followed by

b) approximately 2 to 4 weeks of daytime temperatures of approximately

26°C and nighttime temperatures of approximately 16°C.

8) A method according to claim 5 wherein the plants are tomato plants having

a male sterility gene ms35 and wherein the controlled temperature

conditions are as follows:

a) 1 to 5 weeks of daytime temperatures of about 30°C and nighttime

temperatures lower than 21°C;

followed by

b) approximately 2 to 4 weeks of daytime temperatures of approximately

26°C and nighttime temperatures of approximately 17°C. 9) The pollen produced in step b of claim 1.

10) The seeds that yield 100% male sterile plants, obtained according to

the method of claims 1 to 8.

11) The 100% male sterile plants developed from the seeds of claim 10.

12) The hybrid seeds that yield FI male fertile plants and are produced by

crossing between the plants of claim 11, being the female A-line and any

other suitable male fertile C-line of homozygous MS-MS allele.

13) The FI male fertile plants as defined in claim 12.