全 文 ：长白山杜鹃花属种间杂交亲和
性的研究
李西时 1，巴春影 1*，曹后男 1*，宗成文 1，曲柳 1,
张吉子 2 （1. 延边大学农学院， 吉林延边
133002;2.延边农业科学院，吉林延边 133002）
摘 要 为了探索长白山杜鹃花属种间杂交
的亲和性及种间杂交在种质创新中的可行性，
以 5 种长白山杜鹃花属植物为试材，进行种间
正反杂交，并对杂种胚进行离体培养。 结果表
明， 杜鹃花属种间正反杂交亲和力有显著差
异，以牛皮杜鹃为母本，与迎红杜鹃、白花迎红
杜鹃、亚布力杜鹃杂交有较高的亲和性，与母
本的自然授粉坐果率无显著差异 ， 分别为
92.55%、94.14%、92.42%， 杂种胚的萌发率分
别为 13.60%、11.59%、4.12%；杂种苗生长势微
弱，呈白化状态，有败育迹象，经继代培养 2～3
次后，生长势得到一定的恢复，且分化率明显
高于牛皮杜鹃种胚苗，但植株的颜色仍是黄绿
色。而反交时，坐果率明显降低，坐果率最高的
组合为亚布力杜鹃×牛皮杜鹃， 为 36.51%；与
迎红杜鹃的杂交坐果率为 6.52%；而与大字杜
鹃的杂交坐果率为 0， 但反交时亚布力×牛皮
杜鹃的种胚萌发率极显著地高于正交 ，为
39.81%，而且种胚苗生长健壮 ，无需继代 ，可
直接进行炼苗移栽。
关键词 牛皮杜鹃（Rhododendron chrysanthum
Pall.）；种间杂交亲和性；种胚培养
基金项目 延边大学 2015 年大学生创新创业
训练计划项目，暨第七届本科生科研项目资助
（ydbksky2015252）。
作者简介 李西时（1993-），男，山东日照人，在
读本科生，专业:园艺，E-mail：1028620689@qq.com。
*通讯作者，E-mail：15144589828@163.com。
收稿日期 2015-04-08
修回日期 2015-06-29
Interspecific Cross Compatibility of
Rhododendron in Changbai Mountain
Xishi LI1, Chunying BA1*, Hounan CAO1*, Chengwen ZONG1, Liu QU1, Jizi ZHANG2
1. College of Agronomy, Yanbian University, Yanbian 133002, China;
2. Academy of Agricultural Sciences of Yanbian, Yanbian 133002, China
Supported by Students’ Innovation and Entrepreneurship Training Program of Yanbian
University in 2015 (ydbksky2015252).
*Corresponding author. E-mail: 15144589828@163.com
Received: April 8, 2015 Accepted: June 29, 2015A
Agricultural Science & Technology, 2015, 16(8): 1617-1620
Copyright訫 2015, Information Institute of HAAS. All rights reserved Agronomy and Horticultrue
R hododendron chrysanthumPall. is a small green shrub,and it has funnel-shaped
corollas and light yellow or white flow-
ers. Rh. chrysanthum Pall. is very
beautiful, and can be developed as
flower, leaf and fruit ornamentals. It is
a rare broadleaf evergreen in North-
east with cold and wind resistance and
water- and soil-maintaining ecological
functions [1]. However, Rh. chrysan-
thum Pall. is narrowly distributed in
China, and it is only distributed in the
National Nature Reserve of Changbai
Mountain. Due to excessive digging,
Rh. chrysanthum Pall. has been listed
as a protected and endangered
species of China[2-3]. Rh. chrysanthum
Pall. has low seed germination rate
and low transplanting survival rate. In
addition, Rh. chrysanthum Pall. is a
cold loving plant, and it can grow and
develop in the low temperature
(above 0℃) environment. Under con-
ditions of high temperature and low
humidity, Rh. chrysanthum Pall.
grows slowly, and is also vulnerable
to infestations of pests and diseases,
greatly limiting the exploitation and u
tilization of Rh. chrysanthum Pall.
Currently, the studies on Rh. ch-
rysanthum Pall. in China only focuses
on tissue culture [4 -5], medicinal ingre-
dients [6-8], anatomical structure [9], and
so on. There have been almost no re-
ports on domestication of Rh. chr-
ysanthum Pall. In this study, some
species of Rhododendron in Changbai
Abstract In order to explore the interspecific cross compatibility of Rhododendron
and the feasibility of interspecific cross in germplasm innovation, reciprocal crosses
among five species of Rhododendron in Changbai Mountain were made. In addition,
the hybrid embryos were cultured in vitro. The results showed that the species of
Rhododendron in Changbai Mountain had significant differences in reciprocal cross
compatibility. When Rh. chrysanthum Pall. was used as female parent, it showed
high cross compatibilities with Rh. mucronulatum Turcz. (92.55%), Rh. mucronulamm
Turcz. f. album Nakai (94.14%) and Yabuli Rhododendron (92.42%), and no signifi-
cant differences were found in fruit setting rate by self-pollination. The germination
rates of hybrid embryos were 13.60%, 11.59% and 4.12%, respectively. The hybrid
seedlings had weak growth vigor and albino status and were trended to be aborted.
After 2-3 generations of subculture, the growth vigor of hybrid seedlings were re-
covered gradually, and their differentiation rate was also significantly higher than that
of Rh. chrysanthum Pall. seedlings. But the hybrid seedlings were still yellowish
green. In reciprocal crosses, the fruit setting rates were significantly reduced. The
combination with the highest fruit setting rate (36.51%) was Yabuli Rhododendron ×
Rh. chrysanthum Pall. The fruit setting rates of reciprocal crosses between Rh. chrysan-
thum Pall. and Rh. mucronulatum Turcz. and Rh. chrysanthum Pall. and Rh. schlip-
penbachii Maxim were 6.52% and 0, respectively. However, the hybrid embryo’s
germination rate (39.81%) of Yabuli Rhododendron × Rh. chrysanthum Pall. was
significantly higher than that of their original cross. Moreover, the hybrid seedlings
grew strongly and could be directly transplanted without subculture.
Key words Rh. chrysanthum Pall.; Interspecific cross compatibility; Embryo culture
DOI:10.16175/j.cnki.1009-4229.2015.08.013
Agricultural Science & Technology 2015
Table 1 Fruit setting rates of different hybrid combinations at harvesting stage
Hybrid combination Pollinated flower number Setting-fruit number Fruit setting rate∥%
N×H 255 236 92.55
N×B 222 209 94.14
N×Y 132 122 92.42
H×N 230 15 6.52
Y×N 126 46 36.51
D×N 141 0 0
N×N 202 195 96.53
N, Rh. chrysanthum Pall.; Y, Yabuli rhododendron; B, Rh. mucronulamm Turcz. f. album
Nakai; H, Rh. mucronulatum Turcz.; D, Rh. schlippenbachii Maxim.
Mountain were used as materials, and
the interspecific cross compatibilities
were discussed. A number of hybrid
seedlings were obtained though ma-
ture embryo culture, laying material
foundation for germplasm innovation
of Rhododendron plants in Changbai
Mountain.
Materials and Methods
Materials
The test materials included
Rh . chrysanthum Pall., Rh. mu-
cronulatum Turcz., Rh. mucronulamm
Turcz. f. album Nakai, Yabuli rhodo-
dendron and Rh. schlippenbachii
Maxim.
Methods
Collection of pollen At the big bud
stage, the flower buds that were about
to open were collected and brought
back to laboratory. The anthers were
isolated and dried at room tempera-
ture. Subsequently, the pollen were
collected and preserved in pollen bot-
tles at -20℃.
Reciprocal crosses among Rhodo-
dendron spp. in Changbai Mountain
The interspecific crosses of Rh. chr-
ysanthum Pall. (♀)×Rh. mucronula-
tum Turcz. (♂), Rh. chrysanthum Pall.
(♀ )×Rh. mucronulamm Turcz. f. al-
bum Nakai (♂) and Rh. chrysanthum
Pall. (♀)×Yabuli rhododendron (♀)
were made by artificial emasculation
method in Small Tianchi Lake in
Changbai Mountain in June, 2011. Af-
ter hybridization, the plants were all
bagged for isolation and marked. All
the bags were removed 10 d later. The
fruit setting and development situa-
tions were observed once a week. Be-
fore the cracking of ripen fruits (late
August), the fruit setting rates were in-
vestigated, and the fruits were harvest-
ed for seed separation at room tem-
perature. The separated seeds were
preserved at 4℃ for use.
The back crosses of Rh. mu-
cronulatum Turcz. (♀)×Rh. chrysan-
thum Pall. (♀), Yabuli rhododendron
(♀)×Rh. chrysanthum Pall. (♀) and
Rh. schlippenbachii Maxim ( ♀ ) ×
Rh. chrysanthum Pall.(♀) were made
in the Botanical Garden of Yanbian
Academy of Agricultural Sciences in
late April, 2012. The test methods
were as described above.
Fruit setting rate =Setting-fruit
number/Pollinated flower number ×
100%.
In vitro culture of mature embryos
of different hybrid combinations
Referring to previous test results[10], the
mature embryos of the control (natu-
rally pollinated Rh. chrysanthum Pall.)
and all the hybrid combinations were
all disinfected in sterile room. The
seeds were first washed with sterile
water twice. They were soaked in
75% alcohol for 30 s. Subsequently,
0.1% mercuric chloride was mixed
with the 75% alcohol, and the seeds
were further soaked for 5 min. After
rinsed with sterile water 4-5 times, the
seeds were inoculated in the medium
(pH 5.4) composed of Read, GA (2.0
mg/L), sucrose (30 g/L) and agar (7
g/L) and cultured on a culture shelf.
The culture conditions were as follows:
25 ℃ at day, 20 ℃ at night, 1 500-
2 000 lx of light intensity, 12 h/d of
photoperiod. The seed germination
and seedling growth situations were
observed 20 d later, and the germina-
tion rates were investigated 60 d later.
Germination rate =Germinated
embryo number/Inoculated embryo
number×100%.
The embryo seedlings of hybrid
combinations were very thin and weak.
To get all the hybrid individuals after
seedling transplanting, the obtained
embryo seedlings were inoculated in
the propagation culture medium com-
posed of modified MS, ZT (0.2 mg/L)
and IBA (0.5 mg/L). More than 10
generations of subculture were carried
out for each seedling plant. The culture
conditions were as described above.
Results and Analysis
Fruit setting rates of different hy-
brid combinations at harvesting
stage
As shown in Table 1, the fruit set-
ting rates of hybrid combinations with
Rh. chrysanthum Pall. as the female
parents were 92.55% , 94.14% and
92.42% , respectively, which showed
no significant differences with that of
natural pollination, indicating high in-
terspecific cross compatibilities of
Rhododendron. The fruits of all the hy-
brid combinations began to grow large
2 weeks after the pollination, which
was a litter earlier than that of fruits of
the natural hybrid combination. No
significant differences were found in
development speed and state between
artificial and natural hybrid combina-
tions in later period. Till maturity stage,
a lot of capsules were obtained (Fig.1).
The fruit setting rates of all the hybrid
combinations with Rh. chrysanthum
Pall. as the male parents were signifi-
cantly lower than those of the original
crosses. The fruit setting rates of the
three hybrid combinations were
6.52%, 36.51% and 0, respectively. At
the harvesting stage, the fruit setting
rate of Rh. schlippenbachii Maxim by
self pollination was also found to be 0.
In vitro culture of mature embryos
and subculture of embryo seedlin-
gs of different hybrid combinations
In vitro culture of mature embryos
Before the in vitro culture of hybrid
embryos, their morphology was first
Fig.1 Fruits of hybrid combination of Rh.
chrysanthum Pall. × Rh. mucronula-
tum Turcz.
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Agricultural Science & Technology2015
Table 2 Germination situations of mature embryos of different hybrid combinations after a
60-d in vitro culture
Hybrid
combination
Inoculated
embryo number
Germinated
embryo number
Germination
rate∥%
Germination
starting time∥xth d
N×H 240 32 13.60 20
N×B 117 14 11.59 25
N×Y 240 10 4.12 26
H×N 106 0 0 -
Y×N 108 43 39.81 15
N×N 60 6 11.67 26
N, Rh. chrysanthum Pall.; Y, Yabuli rhododendron; B, Rh. mucronulamm Turcz. f. album
Nakai; H, Rh. mucronulatum Turcz.
observed under an optical microscope
(Fig.2). The seeds of Rh. chrysanthum
Pall. by natural pollination were most
plump, followed by those of N×H, and
the seeds of N ×B and N ×Y were
smaller. The in vitro culture of mature
hybrid embryos showed that there
were significant differences in embryo
germination rate between interspecific
original cross and back cross. Table 2
showed that the embryos of N (♀)×H
(♂) germinated earliest, and the em-
bryos began to germinate as early as
20 d after the inoculation. After a 60-d
culture, the germination rate of em-
bryos of N (♀)×H (♂) reached 13.6%,
followed by that of N (♀ ) ×B (♂ )
(11.59% ), which was close to that of
Rh. chrysanthum Pall. by natural polli-
nation, and the germination rate of
embryos of N (♀)×Y (♂) was relative-
ly low (4.12%). The growth of embryos
of N (♀ )×Y (♂ ) was weaker, and
some of the seedlings were transpar-
ent (Fig.3). In back crosses, the germi-
nation rate of embryos of H (♀)×N (♂)
was 0. The germination rate of em-
bryos of Y (♀)×N (♂) (39.81%) was
significantly higher than that of their o-
riginal cross, and with the proceeding
of subculture, the leaves of embryo
seedlings became greener and the
embryo roots became more devel-
oped. The growth of embryo seedlings
of Y (♀)×N (♂) was better than those
of all the reciprocal hybrid combina-
tions, and it was even better than that
of embryo seedlings of Rh. chrysan-
thum Pall. by natural pollination.
Subculture of embryo seedlings
The hybrid seedlings obtained by em-
bryo culture had weak growth and light
yellow leaves. To avoid loss of materi-
al, the obtained embryo seedlings
were inoculated into the subculture
medium. Among all the hybrid combi-
nations, the growth of hybrid seedlings
of N×Y was weakest, and some of the
hybrid seedlings were almost trans-
parent. With the increased generations
of subculture, the leaf color of hybrid
seedlings was restored to some ex-
tent, but most of them were just yel-
lowish green. In the hybrid seedlings,
most of the buds were light yellow or
albino, but their differentiation capacity
was significantly higher than that of
embryo seedlings of Rh. chrysanthum
Pall. by natural pollination. The signifi-
cantly enhanced differentiation ca-
pacity might be caused by strong het-
erosis in hybrids (Fig.4). After propa-
gation culture, only leaf color and leaf
area of embryo seedlings of Rh.
chrysanthum Pall. were improved, but
none new adventitious buds were ob-
served. The differentiation capacities
of embryo seedlings of the other three
hybrid combinations were relatively
high. It was showed that the differen-
tiation capacity of the embryo
seedlings was significantly higher
than their elongation capacity, so the
overall plant height was relatively low
(Fig.5).
N, Rh. chrysanthum Pall.; Y, Yabuli rhodo-
dendron; B, Rh. mucronulamm Turcz. f. al-
bum Nakai; H, Rh. mucronulatum Turcz.
Fig.2 Morphology of seeds under optical
microscope
N, Rh. chrysanthum Pall.; Y, Yabuli rhododendron; B, Rh. mucronulamm Turcz. f. album
Nakai; H, Rh. mucronulatum Turcz.
Fig.3 Germination situations of mature embryos of different hybrid combinations after a 60-
d in vitro culture
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Agricultural Science & Technology 2015
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
N, Rh. chrysanthum Pall.; Y, Yabuli rhododendron; B, Rh. mucronulamm Turcz. f. album
Nakai; H, Rh. mucronulatum Turcz.
Fig.5 Growth status of embryo seedlings after three generations of subculture
Discussion
The test results showed that the
fruit setting rates of the three hybrid
combinations with Rh. chrysanthum
Pall. as female parents were all higher
than 92.00% . Moreover, large num-
bers of capsules were obtained from
the three reciprocal hybrid combina-
tions. It is suggested that Rh.
chrysanthum Pall. has high interspe-
cific cross compatibilities with Rh. mu-
cronulatum Turcz., Yabuli rhododen-
dron and Rh. schlippenbachii Maxim.
But in back crosses, there were great
differences in fruit setting rate among
all the hybrid combinations. In addition,
there were also great differences in
fruit setting rate between original and
back crosses. In cross breeding, em-
bryo culture is an effective way to over-
come the sterility of distant hybridiza-
tion and to grain interspecific hybrid
embryos[11]. This test proves that the
interspecific cross of Rhododendron
is feasible, and it provides a new
pathway for germplasm innovation.
Interspecific cross of Rhododendron
can not only take advantage of
Rhododendron resources in Chang-
bai Mountain but also get a lot of hy-
brid rhododendrons, which will not
only enrich, as well as protecting
Rhododendron germplasm resources
in Changbai Mountain, but also lay a
foundation for future studies on
germplasm resources and detection
of genetic diversity.
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Fig.4 Growth status of albino seedlings in
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