Note: Descriptions are shown in the official language in which they were submitted.
CA 02748306 2011-06-23
Title: Breeding Method for Brassica Napus L
Self-incompatibility Two-line Hybrid
Technical Field
The invention relates to a breeding method for Brassica Napus L.,
specifically, relates to a breeding method for Brassica napus L.
self-incompatibility two-line hybrid.
Background Art
A large number of researches in China and abroad indicate that the
inter-varietal hybrid of Brassica Napus L. exhibits a great deal of heterosis.
Currently, the ways of utilizing heterosis of Brassica Napus L. include
cytoplasmic male sterility (CMS), genic male sterility (GMS),
self-incompatibility (SI) and chemical hybridization. Genic male sterility
(GMS) of Brassica Napus L. has an advantage of stable sterility, however its
fertility is likely to segregate, when producing seeds, 50% of the fertile
plants
in the sterility line needs to be pulled out at first flowering stage of
Brassica
Napus L.; that will reduce the yield of seed production and increase the cost
of
seed production, further, since it is generally difficult to completely pull
out the
fertile plants, that will affect the purity of seeds and increase production
risk. The
way of chemical hybridization has such shortages as environment pollution
and low purity of the hybrid. At present, Pol cms is the well-known most
valuable cytoplasmic male sterility line of Brassica Napus L. in the world;
however, its fertility is apt to be affected by environmental conditions, and
the breeding processes of maintainer line and restorer line are complicated
and have long periods, and have potential risk of negative effects of
cytoplasma.
Comparing with other ways of utilizing heterosis of Brassica Napus L.,
self-incompatibility hybrid of Brassica Napus L. has advantages including
short breeding period, being easy to screen a restorer line, no negative
effects
of cytoplasma and high yield of seed production. Some possible ways of
utilizing self-incompatible lines of Brassica napus L. to prepare hybrids have
been proposed previously, such as self-incompatibility single-cross hybrid
(self-incompatible line A x self-incompatible line B), self-incompatibility
CA 02748306 2011-06-23
three-line hybrid ((self-incompatible line x maintainer line) x restorer
line),
self-incompatibility three-way cross hybrid ((self-incompatible line A x
self-incompatible line B ) x restorer line) and self-incompatibility double
cross hybrid ((self-incompatible line A x self-incompatible line B) x
(self-incompatible line C x self-incompatible line D)) (Breeding and
utilization of rapeseed hybrid, page 142-145, editor in chief, FU Tingdong;
Hubei Science and Technology Press, August 2000). Although these ways have
been proposed for many years, due to the limit of breeding method for
Brassica napus L. self-incompatibility hybrid, currently the breeders have not
bred any Brassica napus L. self-incompatibility hybrid which is suitable for
large scale popularization in agricultural production. Aiming at the
drawbacks of existing breeding methods for Brassica napus L.
self-incompatibility hybrid, the present invention provides a breeding method
for Brassica napus L. self-incompatibility two-line hybrid. The method is
expected to breed Brassica napus L. self-incompatibility hybrids which are
suitable for large scale popularization in agricultural production, thus
promoting heterosis utilization of Brassica Napus L.
Contents of the Invention
The object of the present invention is to overcome drawbacks of the
prior art, and provide a breeding method for Brassica napus L.
self-incompatibility two-line hybrid. The invention is a breakthrough and
innovation based on the existing three-line breeding method for rapeseed. The
technical solution of the invention generally includes breeding and
propagation
of Brassica napus L. self-incompatible lines, breeding and propagation of
Brassica napus L. self-incompatibility restorer lines, breeding of Brassica
napus
L. self-incompatibility two-line hybrid and method of seed production thereof.
The invention will promote application of the Brassica napus L.
self-incompatibility two-line system in current heterosis breeding.
The invention is carried out through the following technical solutions:
A breeding method for Brassica napus L. self-incompatibility two-line
hybrid, comprising the following steps:
2
CA 02748306 2011-06-23
(1) breeding of Brassica napus L. self-incompatible line: Brassica
napus L. self-incompatible line S-1300 as female parent (see: Breeding of
Brassica napus L. double low (low erucic acid, low glucosinolates)
self-incompatible lines, MA Chaozhi, et al, Journal of Huazhong Agricultural
University, 1998 , 17 (3): 211-213) was crossed with Brassica napus L.
self-compatible line Hua Shuang No. 3 (also referred to as Huayou Shuang
No. 3, they are actually the same variety; the rapeseed variety was bred by
Huazhong Agricultural University in Wuhan, Hubei province of China, which
was approved by Hubei Provincial Crop Variety Approval Committee on
March 26, 1998, certificate number: Hubei seed certificate No. 156; it was a
new variety of Brassica napus L. which has been openly popularized) to
obtain F1 seeds; the self-compatibility of F1 plants were measured by
compatible index method, and the self-incompatible F1 plants were
subjected to bud-pollination to obtain F2, the self-compatibility of F2 plants
were measured, and the self-incompatible F2 plants were selected and
subjected to selfing by artificial bud-pollination to obtain F3 seeds; F3
seeds
were sowed and the F3 family lines were selected, all individual plants of the
selected lines were selfed in bags and 3-5 individual plants selected from
every line were subjected to selfing by artificial bud-pollination to obtain
F4
seeds; F4 seeds from F3 family lines in which the compatible index of
individual plant is less than 1 were sowed and the F4 family lines were
selected, all individual plants of the selected lines were selfed in bags and
3-5 individual plants selected from every line were subjected to selfing by
artificial bud-pollination to obtain F5 seeds, the F5s from F4 family lines
with compatible index of less than 1 were selected as the desired Brassica
napus L. self-incompatible lines; the F3 family lines with unstable
self-incompatibility were repeatedly subjected to the above steps until a
Brassica napus L. self-incompatible line with stable self-incompatibility
was selected; the applicant thus obtained Brassica napus L.
self-incompatible line 07-P63-6 YU 34 through the above steps;
(2) breeding of Brassica napus L. self-incompatibility restorer lines:
3
CA 02748306 2011-06-23
=
Brassica napus L. self-incompatible line S-1300 as female parent was
crossed with self-compatible rapeseed line Zheyou 18 (bought from China
Zhejiang Academy of Agricultural Sciences), Huyou 17 (bought from China
Shanghai Academy of Agricultural Sciences) and Hui 5900 (the term "Hui"
means restorer) (see: Genetic Diversity of Parents for Hybrid Breeding in
Brassica napus L. Detected by RAPDs and RFLPs, MA Chaozhi, et al., Acta
Agronomica Sinica, 2003, 29 (5): 701-707) to obtain F1 seeds; F1 seeds were
sowed and the self-compatibility of F1 was investigated; said self-compatible
rapeseed lines were sowed at the same time, and their yield and yield-related
traits were investigated, the breeding materials with good field performance,
wherein the compatible index of F1 combination from that breeding material
and said Brassica napus L. self-incompatible line S-1300 was over 10, were
selected as target restorer lines, i.e., Hui Zheyou 18, Hui Huyou 17 and Hui
5900;
(3) breeding of Brassica napus L. self-incompatibility two-line hybrid:
Brassica napus L. self-incompatible line S-1300 and its improved
self-incompatible line 07-P63-6 YU 34 as female parents were crossed with the
self-incompatibility restorer lines Hui Zheyou 18, Hui Huyou 17 and Hui
5900 as male parents to breed two-line hybrid; F1 combinations and the
contrast varieties were sowed in field at the same time according to
randomized block design, and agronomic traits, yield, disease resistance,
growth period and quality traits of the plants were investigated to select the
hybrid combination with strong heterosis for subsequent use;
(4) propagating of Brassica napus L. self-incompatible lines: the means
of early sowing, low density cultivation and high fertilizer were used to
cultivate strong seedlings of Brassica napus L. self-incompatible lines, 3%
NaC1 solution was sprayed in blossoming of Brassica napus L. under isolated
condition with the aid of honeybee pollination, the compatibility index of
each plant was investigated and the seeds of the plant with compatibility
index of less than 1 were harvested as breeder seeds of Brassica napus L.
self-incompatible line, the seeds were kept in refrigerator at -20 C for
4
CA 02748306 2011-09-20
subsequent use; a small amount of breeder seeds were sowed in the next
sowing season, 3% NaCl solution was sprayed in blossoming of Brassica
napus L. under isolated condition with the aid of honeybee pollination to
propagate the self-incompatible lines, and the original seeds of Brassica
napus L. self-incompatible lines were obtained;
(5) propagating Brassica napus L. self-incompatibility restorer lines:
Propagation methods of restorer lines utilizing heterosis such as Brassica
napus L. cytoplasmic male sterility, or genic male sterility, or chemical
hybridization were used to propagate the Brassica napus L.
self-incompatibility restorer lines (see: Breeding and utilization of rapeseed
hybrid, pages 145-150, editor in chief, FU Tingdong; Hubei Science and
Technology Press, 1995);
(6) producing the seeds of Brassica napus L. self-incompatibility
two-line hybrid: Brassica napus L. self-incompatible line obtained from step
(1) was served as female parent, Brassica napus L. self-incompatibility
restorer line obtained from step (2) was served as male parent, wherein the
female parent and male parent plants were planted under artificial isolated
condition or in natural isolated area in a row ratio of 2:1 or 3:1 or 4:1, in
blossoming of the Brassica napus L. plants, honeybees were used to
supplement pollination, and at the end of final flowering stage of Brassica
napus L. the plants of male parent rows were removed, the female parent row
plants were harvested at mature stage of Brassica napus L. to obtain seeds of
Brassica napus L. self-incompatibility two-line hybrid.
The advantages of the invention are as follows:
1. The breeding of Brassica napus L. self-incompatibility two-line
hybrid solves the technical problem that Brassica napus L.
self-incompatibility hybrid was not able to popularize and cultivate in a
great
area in agricultural production.
2. Compared with the existing breeding method for Brassica napus
L. three-line hybrid, only female parent and male parent are needed for the
Brassica napus L. self-incompatibility two-line hybrid, maintainer line and
temporary maintainer line are not involved, thus the invention has the
5
CA 02748306 2011-06-23
advantages of simplified breeding process, short breeding period and high
breeding efficiency.
3. Compared with the existing breeding method for Brassica napus L.
three-line hybrid, the method for propagation of female parent (Brassica
napus L. self-incompatible lines) is simple.
4. Breeding method for Brassica napus L. self-incompatibility restorer
lines (male parent) is simple. Since the vast majority of conventional
varieties /lines of Brassica napus L. can restore the self-incompatibility of
Brassica napus L. self-incompatible lines, thus readily selecting an excellent
hybrid combination.
5. The yield of seed production for Brassica napus L.
self-incompatibility two-line hybrid is high and the cost of seed production
is
low.
For more detailed technical solutions, see "Specific Mode for Carrying
out the Invention".
Description of Drawings
Figure 1 is the technical road map according to the invention.
Figure 2 shows Brassica napus L. self-incompatible line S-1300 after
selfing in bags and the seeding states of its hybrid F1 in the present
examples,
figures a-d indicates that Brassica napus L. self-incompatible line S-1300
after selfing in bag hardly produces seeds; figure e indicates the seeding
state
of F1 hybrid developed by crossing Brassica napus L. self-incompatible line
S-1300 as female parent and conventional Brassica napus L. line Zheyou 18
as male parent after selfing in bags, which normally produces seeds.
Figure 3 is the propagation effect of Brassica napus L.
self-incompatible line S-1300 under isolation shelter condition.
Figure 4 is the production state of the seed of Brassica napus L.
self-incompatibility two-line hybrid (Brassica napus L. self-incompatible
line S-1300 as female parent and Zheyou 18 as male parent with female
parent and male parent in a row ratio of 2:1) under isolation shelter
condition.
6
CA 02748306 2011-06-23
Figure 5 is the production state of the seed of Brassica napus L.
self-incompatibility two-line hybrid (Brassica napus L. self-incompatible line
S-1300 as female parent and Zhongshuang No. 10 (an excellent Brassica napus
L. variety bred by Research Institute of Oil Plants, Chinese Academy of
Agricultural Sciences, Wuhan in Hubei province, China, which had been widely
popularized in agricultural production) as male parent with female parent and
male parent in a row ratio of 2:1 under condition of natural isolated area,
Figure
5 shows the seedling stage of Brassica napus L.
Figure 6 is the production state of the seeds of Brassica napus L.
self-incompatibility two-line hybrid (Brassica napus L. self-incompatible line
S-1300 as female parent and Brassica napus L. variety Zhongshuang No. 10 as
male parent with female parent and male parent in a row ratio of 2:1) under
condition of natural isolated area, Figure 6 shows the pod mature stage of
Brassica napus L.
Specific Mode for Carrying out the Invention
In terms of the biological materials involved in the following examples,
it should be understood according to the part of "Contents of the Invention"
that the applicant has made a statement to the disclosed way of obtaining the
biological materials used in examples 1-2 for breeding (also referred to as
crossing) (see: Certificate of Compliance 1-2). Owing to the limitation of
space, said contents of Certificate of Compliance are omitted in examples 1-2,
however, it should not be construed as lacking the description of the
biological material in the invention.
Example 1
1. Breeding of Brassica napus L. self-incompatible lines, which
comprises the following steps:
(1) Brassica napus L. self-incompatible line S-1300 as female parent was
crossed with Brassica napus L. self-compatible line Hua Shuang No. 3 to
obtain seeds of the first generation of hybrid (F1)=
(2) The F1 seeds obtained from step (1) were sowed. The main
7
CA 02748306 2013-04-09
inflorescences of F1 plants were subjected to selfing in bags during the
flowering period of Brassica napus L. and the self-compatibility of F1 was
estimated. If F1 exhibits self-compatibility, then F1 was subjected to selfing
in
bags; if F1 exhibits self-incompatibility, then F1 was subjected to selfing by
bud-pollination; the F2 seeds were obtained;
(3) The F2 seeds obtained from step (2) were sowed. Each F2 plant was
selfed in bags during the flowering period of Brassica napus L. The
self-incompatible plants were selected and selfed by artificially
bud-pollination. The seeds of each F2 plants selfed by bud-pollination were
harvested.
(4) The F2 seeds of selfed by bud-pollination from step (3) with
compatibility index of less than 1 were sowed to obtain F3 family line. F3
family line was selected depending on the breeding objectives such as growth
situation in seedling period and growth period. All the individual plants of
the
selected F3 family line were selfed in bags, and each plant with excellent
self-incompatibility was selected to be selfed by artificially bud-
pollination.
The seeds from F3 family line selfed by bud-pollination with compatibility
index of less than 1, were taken as the seeds of F4 family line during mature
stage.
(5) The seeds of F4 family line were sowed. F4 family line was selected
depending on breeding objectives such as growth situation at the seedling
stage
and growth period. All individual plants of the selected F4 family line were
selfed in bags and the individual plants with excellent self-incompatibility
were selected to be selfed by bud-pollination with hand. All the F4 family
lines,
whose compatibility index of individual plant is less than 1, were namely the
new Brassica napus L. self-incompatible lines.
(6) If there was no F4 family line whose compatibility index of individual
plant is less than 1, the step (4) was repeated until a new Brassica napus L.
self-incompatible line with stable self-incompatibilty (referred to herein as
07-P63-6 YU 34) was selected. Through the above steps, the applicant has
obtained Brassica napus L. self-incompatible line 07-P63-6 YU 34.
8
= CA 02748306 2011-06-23
2. Breeding of Brassica napus L. self-incompatibility restorer lines,
which comprises the following steps:
(1) Brassica napus L. self-incompatible line S-1300 as female parent
was crossed with self-compatibility Brassica napus L. varieties Zheyou 18,
Huyou 17 and Hui 5900 to obtain seeds of the first generation of hybrid (F1).
(2) The F1 seeds from step (1) were sowed. All the F1 plants were selfed
in bags during the flowering period of Brassica napus L. The compatibility
index of F1 plants was evaluated during the mature stage.
(3) Self-compatible Brassica napus L. lines were planted in the field at
the same time when the step (2) was carried out, and their yield and
yield-related traits were investigated.
(4) Combining the results of steps (2) and (3), the breeding materials
with good field performance were selected, namely the Brassica napus L.
self-incompatibility restorer lines, wherein the compatibility index of F1
combination obtained by crossing the breeding material and said Brassica
napus L. self-incompatible line S-1300 is over 10. Through the above steps
the applicant has obtained Brassica napus L. self-incompatibility restorer
lines Hui Zheyou 18, Hui Huyou 17 and Hui 5900.
3. Breeding of Brassica napus L. self-incompatibility two-line hybrid,
which comprises the following steps:
(1) Brassica napus L. self-incompatible lines S-1300 and 07-P63-6 YU
34 were respectively as female parents crossed with the self-incompatibility
restorer lines Hui Zheyou 18, Hui Huyou 17 and Hui 5900 as male parents to
obtain F1 hybrid combination.
(2) According to randomized block design, F1 combinations and the
contrast varieties were sowed in test field at the same time, with 2-3
replications, 3-5 rows in one area, row length of 1.9m, row width of 25cm
and spacing in the plants of 17cm. During the mature stage of Brassica napus
L., 10-15 plants were selected randomly from each area and the other plants
in each area were harvested. Agronomic traits including plant height,
9
CA 02748306 2011-06-23
=
branching part and yield per plant, disease resistance, growth period and
yield
in each area for each combination were investigated based on the breeding
objectives, and the quality traits of the harvested seeds including oil
content
and erucic acid content were analyzed.
(3) The F1 combinations which met the breeding objectives were namely
the strong heterosis combinations available for the next experiment. Through
the above steps, the strong heterosis combinations according to the invention
increased the production by 8-12% than that of the contrast variety
Zhongyouza No. 2 (an excellent rapeseed variety bred by Research Institute
of Oil Plants, Chinese Academy of Agricultural Sciences, approved and
popularized widely over China).
4. Propagating of Brassica napus L. self-incompatible lines, which
comprises the following steps:
(1) Brassica napus L. self-incompatible line S-1300 was sowed every
year from later August to early September under isolated conditions with
spacing in the rows of 2 m and spacing between plants of 1 m in winter
rapeseed-planting areas along the middle and lower reaches of Yangtze river,
China, and Brassica napus L. self-incompatible line with large plant shape
and branches as many as possible was bred. Each plant of Brassica napus L.
self-incompatible line S-1300 was encased with a 60-mesh nylon breeze tent
at bolting stage, and honeybee pollination was deployed in the tent. The main
inflorescences of the self-incompatible line were selfed in bags, and
self-compatibility of the plant of the self-incompatible line was identified.
(2) During flowering period, the plants of Brassica napus L.
self-incompatible line S-1300 under isolated condition were sprayed one time
every morning with 3% NaCl solution, and sprayed 20-30 times in total.
(3) At mature stage of Brassica napus L. self-incompatible line S-1300,
seeds of the self-incompatible line of individual plant were harvested. The
compatibility index was calculated by the formula: compatibility index =
seed number / flower number, and the plants whose compatibility index is
over 2 were weeded out. Contents of erucic acid and glucosinolates of the
CA 02748306 2011-08-25
seeds of each plant were analyzed, and the seeds with erucic acid content of
less than 1% and glucosinolates content of less than 30umol/g were selected
as breeder seeds of Brassica napus L. self-incompatible line, and the seeds
were kept in refrigerator at -20C for subsequent use.
(4) The breeder seeds obtained from step (3) were continuously sowed
in the next propagation season. In flowering period, plants of the
self-incompatible line were sprayed one time every morning with 3% NaCl
solution, and sprayed 20-30 times in total. Honeybee pollination was
deployed, and original seeds of Brassica napus L. self-incompatible line were
harvested for use in agricultural production.
5. Propagation of Brassica napus L. self-incompatibility restorer
lines:
Propagation methods of restorer lines utilizing heterosis such as
Brassica napus L. cytoplasmic male sterility, or genic male sterility, or
chemical hybridization were used to propagate the Brassica napus L.
self-incompatibility restorer lines (see: Breeding and utilization of rapeseed
hybrid, page 145-150, editor in chief, FU Tingdong; Hubei Science and
Technology Press, 1995) .
6. Seed production method for Brassica napus L. self-incompatibility
two-line hybrid:
(1) Brassica napus L. self-incompatible line as female parent was
crossed with Brassica napus L. self-incompatibility restorer line as male
parent, wherein the female parent and male parent plants were planted under
artificial isolated shelter or in natural isolated area in a row ratio of 2:1
or 3:1
or 4:1. The field management is the same as that of the local large field crop
production.
(2) In blossoming of the plants of Brassica napus L., honeybees were
arranged to assist pollination in artificial isolated shelter or natural
isolated
area.
(3) At the end of final flowering stage of Brassica napus L., the plants
11
CA 02748306 2011-06-23
of male parent rows were removed.
(4) At mature stage of Brassica napus L., the female parent row plants
were harvested to obtain seeds of Brassica napus L. self-incompatibility
two-line hybrid.
Example 2 (comparative test)
The detail steps are as follows:
1. Using compatibility index to evaluate the self-compatibility of
Brassica nap us L.
At primary flowering stage, the main branches of the investigated Brassica
napus L. plants were encased in bags. When there were 3-5 flowers blossoming
at a main branch, all the blossoming flowers at the main branch were removed
and some little flower buds at the top of inflorescence center were thinned
out;
then the main branch and two side branches were encased in a sulfate paper bag
and the bag was clipped tightly with paper clips. The paper bag was pulled
upward every two days, while patting it to shed the pollen to assistant
pollination.
If there were flowers or flower buds revealing out of the bag when the bags
were
pulled up, the flowers or flower buds should be removed immediately to avoid
harvesting impure seeds. When the pollination of almost all flowers in bags
was
finished, the bags were removed and the buds and un-pollinated flowers were
stricken off, a preliminary investigation of self-compatibility was carried
out
based on the development of rapeseed pods. After harvesting, the seeds were
threshed and the compatibility index of each plant was investigated. The
compatibility index of a plant was calculated by the formula: compatibility
index (Self-compatibility Index, SCI) = seed number /flower number.
2. Breeding of Brassica napus L. self-incompatible line 07-P63-6 YU 34
(1) In spring of 1998, the self-incompatible line S-1300 as female parent
was crossed with Hua Shuang No. 3, a semi-winter type Brassica napus L.
line in China, to obtain seeds of the first generation of hybrid (F1), in a
rapeseed testing field of Huazhong Agricultural University, Wuhan in Hubei
province, China.
12
CA 02748306 2011-06-23
(2) In autumn of 1999, F1 seeds were sowed (2 rows, 10 plants /row). In
spring of 2000, the F1 plants were selfed in bags in blossoming to evaluate
the self-compatibility of F1, and the F1 plants were selfed by artificially
bud-pollination. F1 plants were harvested in early May, and the selfed F1
plants hardly went to seed.
(3) In autumn of 2000, the seeds of the selfed F1 plants were sowed to
obtain F2 plants (Field number: 01-9-701, 2 rows /area, 10 plants /row, 20 F2
plants in total), in rapeseed testing field of Huazhong Agricultural
University, Wuhan; in spring of 2001, 8-10 individual plants of the
Brassica napus L. were selected to be selfed and selfed by bud-pollination
in blossoming; when the rapeseed pods were mature in early May, the seeds
of each selfed F2 plant were harvested separately, and the compatibility
index was investigated, the seeds of the plant whose compatibility index is
less than 1 were kept.
(4) In 2006, the Brassica napus L. self-incompatible line with stable
self-incompatibility and double-low quality was obtained through the
breeding method of steps (3)-(6) according to the invention.
In autumn of 2006, the seeds harvested from an individual plant with
low compatibility index, low erucic acid content and low glucosinolates
content, and more seeds obtained by bud-pollination, were sowed in testing
field in Huazhong Agricultural University, Wuhan, and the testing number is
YU 34; the plants were transplanted in an isolated shelter No. P63-6 in early
November, and the field number is 07-P63-6 YU 34 , the plants were
transplanted in 5 rows and has a number of about 50 in total; in spring of
2007, the shelter was encased with nylon breeze tent to isolate the foreign
pollen; all the individual plants grew normally, the number of seeds by
selfing was small, compatibility index was less than 1, the content of erucic
acid <1%, and the content of glucosinolates <30umol/g, they met the
double-low standard.
3. Breeding of Brassica napus L. self-incompatibility restorer lines
(1) In spring of 2005 in the test field of Huazhong Agricultural
13
CA 02748306 2011-06-23
University in Wuhan, and in summer of 2005 in the testing field of Northwest
China Testing Station of Huazhong Agricultural University in Hezheng
county in Gansu province, Brassica napus L. self-incompatible line S-1300
as female parent was respectively crossed with self-compatible Brassica
napus L. varieties Zheyou 18, Huyou 17 and Hui 5900 to obtain seeds of
three F1 combinations; in autumn of 2005 the F1 combinations was sowed (3
rows /area, 10 plants /row) in the rapeseed testing field of Huazhong
Agricultural University in Wuhan, and the seed-setting circumstances of each
combination were investigated in spring of 2006, it was found that the
compatibility index of each of the three F1 combinations was more than 10.
This indicated that all the male parents of the F1 combinations can restore
the
self-incompatibility of Brassica napus L. self-incompatible line S-1300.
(2) In autumn of 2005, the 3 portions of seeds of self-compatible
Brassica napus L. were sowed in a randomized block design (2 repeats, 3
rows /area, 10 plants / row) in the rapeseed testing field of Huazhong
Agricultural University; the field management is the same as that of the local
large field crop production.
(3) Plants of the three kinds of Brassica napus L. were observed to
exhibit good field performance, and the compatibility index of the
combination between the three kinds of Brassica napus L. and Brassica
napus L. self-incompatible line S-1300 was more than 10; This indicated that
they can be used as Brassica napus L. self-incompatibility restorer lines,
i.e.,
Hui Zheyou 18, Hui Huyou 17 and Hui 5900, for further combination and
comparative experiments.
4. Breeding of Brassica napus L. self-incompatibility two-line hybrid
(1) In spring of 2007, Brassica napus L. self-incompatible line S-1300
and 07-P63-6 YU 34 as female parent were crossed with 3 Brassica napus L.
self-incompatibility restorer lines, respectively (Zheyou 18, Huyou 17 and
Hui 5900) to obtain F1 combinations.
(2) In autumn of 2007, the above F1 combinations and one control
variety (Zhongyouza No. 2) were sowed in a randomized block design (2
14
CA 02748306 2011-06-23
=
repeats, 3 rows /area, 10 plants / row) in the rapeseed testing field of
Huazhong Agricultural University; the field management is the same as that
of the local large field crop production.
(3) In May 2008, 10 plants were randomly selected from each area, their
agronomic traits such as plant height, branching part and t yield per plan etc
were investigated, and the other plants were harvested to investigate the
yield
per area, and finally the quality traits were analyzed. Through the above
steps,
the yield per area of the F1 combinations according to the invention was
increased by 8-12% than that of the control variety Zhongyouza No. 2 (bred by
Research Institute of Oil Plants, Chinese Academy of Agricultural Sciences,
an excellent rapeseed variety popularized widely over China). This indicated
that the Brassica napus L. self-incompatibility hybrids have strong heterosis.
5. Propagation of Brassica napus L. self-incompatible lines
(1) Brassica napus L. self-incompatible line S-1300 was sowed every
year from later August to early September under isolated conditions with
spacing in the rows of 2 m and spacing between plants of 1 m in winter
rapeseed planting areas along the middle and lower reaches of Yangtze river,
China, and Brassica napus L. self-incompatible line with large plant shape
and branches as many as possible was bred. Each plant of Brassica napus L.
self-incompatible line S-1300 was encased with a 60-mesh nylon breeze tent
at bolting stage, and honeybee pollination was deployed in the tent. The main
inflorescences of the self-incompatible line were selfed in bags, and
self-compatibility of the plant of the self-incompatible line was identified.
(2) During flowering period, the plants of Brassica napus L.
self-incompatible line S-1300 under isolated condition were sprayed one time
every morning with 3% NaC1 solution, and sprayed 20-30 times in total.
(3) At mature stage of Brassica napus L. self-incompatible line S-I300,
seeds of the self-incompatible line of individual plant were harvested. The
compatibility index was calculated by the formula: compatibility index =
seed number / flower number, and the plants whose compatibility index is
over 2 were weeded out. Contents of erucic acid and glucosinolates of the
CA 02748306 2011-06-23
seeds of each plant were analyzed, and the seeds with erucic acid content of
less than 1% and glucosinolates content of less than 30umol/g were selected
as breeder seeds of Brassica napus L. self-incompatible line, and the seeds
were kept in refrigerator at -20 C for subsequent use.
(4) The breeder seeds obtained from step (3) were continuously sowed in
the next propagation season. In flowering period, plants of the
self-incompatible line were sprayed one time every morning with 3% NaCl
solution, and sprayed 20-30 times in total. Honeybee pollination was
deployed, and original seeds of Brassica napus L. self-incompatible line were
harvested for use in agricultural production.
6. Propagation method for Brassica napus L. self-incompatibility
restorer lines
The method is the same as propagation methods of restorer lines
including utilizing heterosis such as Brassica napus L. cytoplasmic male
sterility, or genic male sterility, or chemical hybridization. On September
25,
2006, Brassica napus L. self-incompatibility restorer line Zheyou 18 was
sowed in the seedling bed, and the plants were transplanted in an isolated
shelter No. P63-8 on November 5; in 2007 the blossoming flowers were
removed in primary flowering stage of Brassica napus L., and the shelter was
encased with a 60-mesh nylon breeze tent to isolate the foreign pollen, and
honeybee pollination was deployed in the tent; the seeds of Brassica napus L.
self-incompatibility restorer line Zheyou 18 in the isolated shelter were
harvested together in mature stage.
7. Seed production method for Brassica napus L. self-incompatibility
two-line hybrid
(1) In autumn of 2003, Brassica napus L. self-incompatible line S-1300
(female parent) and Brassica napus L. self-incompatibility restorer line
Zheyou 18 (male parent) were respectively sowed in the seedling beds; in
November 2003, the female parent (S-1300) and male parent (Zheyou 18)
were transplanted in a row ratio of 2:1 in the isolated shelter No. P63-2; the
field management is the same as that of the local large field crop production.
16
CA 02748306 2014-02-24
In 2004 honeybee pollination was deployed in the isolated shelter No. P63-2
in flowering period of Brassica napus L., at the end of final flowering stage,
the male parent plants were removed and the female parent plants were kept;
the female parent plants were harvested in mature stage of Brassica napus L.
to obtain seeds of Brassica napus L. self-incompatibility two-line hybrid.
(2) In autumn of 2007, Brassica napus L. self-incompatible line S-1300
(female parent) and Brassica napus L. self-incompatibility restorer line Hui
Zhongshuang No. 10 (male parent) were respectively planted in a row ratio of
2:1 in an isolated area; the field management is the same as that of the local
large field crop production. In 2008 at the end of final flowering stage of
Brassica napus L., the male parent plants were removed and the female
parent plants were kept; the female parent plants were harvested in mature
stage of Brassica napus L. to obtain seeds of Brassica napus L.
self-incompatibility two-line hybrid.
Remarks
Seeds of Brassica napus L. Hui 5900 are a material of Brassica napus L.
Self-incompatible Restorer Line, bred by Huazhong Agricultural University. For
the publication information of the material, see Genetic Diversity of Parents
for
Hybrid Breeding in Brassica napus L. Detected by RAPDs and RFLPs, MA
Chaozhi, et al., Acta Agronomica Sinica, 2003, 29 (5): 701-707).
Seeds of Brassica napus L. S-1300 are a material of Brassica napus L.
Self-incompatible Line, also bred by Huazhong Agricultural University. The
information of this material was published in pages 211-213 in Journal of
Huazhong Agricultural University in June 1998.
The seeds of Brassica napus L. Hui 5900 and Brassica napus L.
Self-incompatible Line S-1300, are available from Huazhong Agricultural
University until at least December 19, 2028.
17
