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Morphogenesis, Anatomical Observation and Genetic Analysis of a Long Hull Floral Organ Mutant in Rice


The long hull floral organ mutant in rice (Oryza sativa L.) was firstly discovered in the hybrid progeny of a wild rice (O. nivara Sharma et Shastry) and a cultivar rice (O. sativa subsp. indica Kato). The florets of the mutant plant show long, leafy paleas/lemmas that result in open hulls. A single floret consists of one to ten stamens, one to three pistils and one to five stigmas on the same ovary. Stamens/pistils-like structures and bulged tissues near ovaries were observed. Low seed setting rate was another obvious character of this mutant. According to statistical analysis, seed setting rate was 18.2%. The percentage of pollen fertility was 62.46%. The process of floral organ morphogenesis was also investigated using scanning electron microscopy (SEM), and genetic analysis indicated that mutant traits were controlled by single recessive gene (temporarily designated as lh). The possible relationships between this lh gene and other floral organ mutants reported earlier in rice are discussed. Furthermore, we deduced that this gene might represent an example of a gene required for generating proper floral organ number and also be similar to B-like genes in Arabidopsis and Antirrhinum.


全 文 :Received 7 Aug. 2003 Accepted 11 Dec. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (2001AA211081) and the Foundation of Excellent Doctorial
Dissertation, Education Administration of China (200054).
* Author for correspondence. Tel: +86 (0)28 2710888; E-mail:.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 451-456
Morphogenesis, Anatomical Observation and Genetic Analysis
of a Long Hull Floral Organ Mutant in Rice
ZHANG Xu-Mei, LI Shi-Gui*, WANG Yu-Ping, WU Xian-Jun
(Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China)
Abstract : The long hull floral organ mutant in rice (Oryza sativa L.) was firstly discovered in the hybrid
progeny of a wild rice (O. nivara Sharma et Shastry) and a cultivar rice (O. sativa subsp. indica Kato). The
florets of the mutant plant show long, leafy paleas/lemmas that result in open hulls. A single floret consists
of one to ten stamens, one to three pistils and one to five stigmas on the same ovary. Stamens/pistils-like
structures and bulged tissues near ovaries were observed. Low seed setting rate was another obvious
character of this mutant. According to statistical analysis, seed setting rate was 18.2%. The percentage of
pollen fertility was 62.46%. The process of floral organ morphogenesis was also investigated using scanning
electron microscopy (SEM), and genetic analysis indicated that mutant traits were controlled by single
recessive gene (temporarily designated as lh). The possible relationships between this lh gene and other
floral organ mutants reported earlier in rice are discussed. Furthermore, we deduced that this gene might
represent an example of a gene required for generating proper floral organ number and also be similar to B-
like genes in Arabidopsis and Antirrhinum.
Key words: Oryza sativa ; long hull mutant; anatomical structure; floral organ morphogenesis; single
recessive gene
In higher plan ts, proper floral development requires a
coordinated activity of a number of genes that control the
pat terning of o rgan type, o rgan number and o rgan form
(Yanofsky, 1995). Based on some genes which affect the
identity of an individual floral organ in Arabidopsis, Coen
and Meyerovitz (1991) pres ented ABC model which has
been widely accepted. But there are also a number of other
mutations which affect organ number, organ s hape and
regional differentiation within floral organs. For example,
mutations in clava ta1 (clv ; Clark et al., 1993; 1997),
clavata3 (clv3; Clark et al., 1995), ettin (ett; Sessions et al.,
1997), and periananthia (pan; Running and Meyerowitz,
1996) increase organ number within the flower, whereas
mutations in tousled (tsl; Roe et al., 1993; 1997), revo luta
(rev; Talbert et al., 1995), shoot meristemless (stm; Endrizzi
et al., 1996), aintegumenta (ant; Elliott et al., 1996; Klucher
et al., 1996) and wuschel (wus; Laux et al., 1996) decrease
organ number and organ size.
However, because of the lack of corresponding mutants,
it is unclear whether the molecular mechanism for dicot
floral organ development is also suitable to monocot species.
Rice (Oryza sativa) is not only one of the mos t importan t
crops in the world , but als o an ideal model p lan t fo r
researches on the developmental molecu lar biology in
monocots. Floral organs directly influence grain quality and
yield. So, it is important to study the genes for floral organ
development in rice, especially stamens and pistils. In this
study we demonstrated floral organ morphogenesis, ana-
tomical structure and genetic analysis of a long hull mutant
in rice. Obviously, new rice floral organ mutants give us an
advantage for further gene research in this field.
1 Materials and Methods
1.1 Plant materials
The long hull floral mutant was derived from the prog-
eny of an interspecific cross between Keye (Oryza nivara
Sharma et Shastry) and Zhenshan 97 (O. sativa L. s ubsp.
indica Kato). The mutant was crossed with Jodan (O. sa-
tiva subsp. japon ica Kato), Maylielle (O. sa tiva subsp.
tropic japonica Kato), Shuhui 527 (O. sativa subsp. indica
Kato). The parents, F1 and F2, were planted in the fields of
Rice Research Institute of Sichuan Agricultural University.
1.2 Anatomical observation
In summer 2002, floral structures of the mutant were
observed under a microscope before flowering. Meanwhile,
200 florets were investigated and some images of them were
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004452
recorded.
1.3 Observation by scanning electron microscopy (SEM)
Procedures for the scanning electron micrographs were
done ess en tially according to Feng et al. (1995) and
Mizukami and Ma (1992) with some modifications. Young
panicles at various stages were fixed in a fixative solution
of buffered glutaraldehyde (3%) for 24 h, dehydrated in a
series of ethanol-water (30%, 50%, 70%, 80%, 90%, 100%)
and incubated in an ethanol-isoamyl acetate mixture for 1 h.
Samples were then dried, and coated with gold. The mounted
specimens were examined, and photographed with SEM
(KYKY-1000B) at an acceleration voltage of 25 kV.
1.4 Pollen fertility
Estimation of pollen fertility was based on I2-KI s tain
method (Zhu and Yang, 1992). Pollen grains were placed on
slides with I2-KI solution, and nipped into pieces with for-
ceps to make pollen g rains s pilled ou t. Under opt ical
microscope, the pollen fertility was examined according to
its morpho logy and its staining degree. The pistil fertility
was shown with the seed setting rate.
2 Results
2.1 The morphology and anatomical structure of the
mutant florets
No difference in phenotype was observed between the
mutant and the wild type plant before heading time. After
head ing, the different appearance in florets between the
mutant and wild type was intuitive. In contrast to the wild
type, the paleas and lemmas of the mutant were leafy and
elongated (Fig.1). The spikelet was open because of abnor-
mal growth of the palea and lemma. Furthermore, palea- and
lemma-like organs appeared in some florets (Fig.2).
A wild type rice floret consists of six s tamens and one
pistil with two feather-like s tigmas (Fig.3), whereas, in an
individual mutant floret, 1-10 stamens were observed be-
tween the leafy palea and lemma (Figs.4-6) and most flo-
rets contained three, four or five stamens. Statis tical re-
sults from 200 florets showed that the florets containing
three, four and five stamens accounted fo r 37.5%, 34.5%
and 18%, res pectively. Occas ionally, one single filament
was tipped with two anthers (Fig.10). One to four ovaries in
the s ame floret often developed at a differen t stage (Fig.
11). According to the analysis, the florets possessing one,
two and three ovaries represented 56.5%, 33.5% and 8%,
respectively. Every ovary was abnormally tipped with 1-5
feather-like stigmas (Figs. 4, 7, 8). Meanwhile, in some florets,
there were different quantity and size of bulged tiss ues
(Figs.5, 7, 8) or stamen/pistil-like organs (Fig.9) and the
florets with bulged tissues and stamen/pistil-like organs
reached 34.5% and 11.5%, respectively.
2.2 Morphogenesis of the long hull mutant
No obvious difference in phenotype was found between
wild type and mutant floret primordia (Figs.14, 15). However,
different appearance was intuitive at the beginning of sta-
men and pistil primordium differentiation (Figs.16-21). A
wild floret consists of six stamens and one pistil, and six
stamen primordia seem undulant and evenly distribu ted
(Fig.16), while the distributions of stamen and pistil primor-
dia for mutant florets are irregular, and the development of
stamens is often not synchronous (Figs.18-20). Therefore,
it can be concluded that the decrease in stamen number is
correlated with the aberran t or failed initiation of medial
stamen primordia (Ses sions, 1997). Sometimes palea- and
lemma-like structu res (Fig.17) o r two florets on the same
rachilla (Fig.21) were observed.
2.3 Pollen fertility
A low s eed s etting rate of 18.2% is ano ther obvious
character for the long hull mutant. Following corollaries
can be established for the low seed setting rate: (1) relation
with pollen sterility, the pollen fertility of long hull mutant
was lower (62.46%, Fig.12) than that of the control (90%,
Fig.13); (2) results from ovary malformat ion; (3) spikelets
incompletely closed , which cause female organs to lose
water (Bai et al., 2000).
2.4 Inheritance of the floret mutant
F1 hybrids were made from the cross es between the
long hull mutant and three rice varieties, namely Jodan (O.
sativa subsp. japonica Kato), Maylielle (O. sativa subsp.
tropic japonica Kato ), and s huhui-527 (O. sat iva subsp.
indica Kato). Observed with pollen fertility and seed set-
ting rate, the F1 hybrids were completely restored to normal
morphology. Segregation of the wild type and mutant type
plants (Table 1) fits a 3:1 ratio in the th ree F2 populations
(cc2=0.09-0.37, P > 0.05). Thus, the mutant trait was con-
trolled by sing le recess ive gene. The gene is temporarily
designated as lh (long hull).
Table 1 Frequency of mutant in F2 population
Crosses
No. of No. of
Expected
cc2 Pnormal mutant
ratio
plants plants
Jodan/lh 194 54 3:1 0.37 0.1-0.95
Lh/Maylielle 109 43 3:1 0.21 0.1-0.5
Shuhui 527/lh 278 88 3:1 0.098 0.5-0.95
3 Discussion
In the past decade, there have been great advances in
our understanding of flower development. This p rogress


ZHANG Xu-Mei et al.: Morphogenesis, Anatomical Observation and Genetic Analysis of a Long Hull Floral Organ Mutant
in Rice 455
(wig) in Arabidopsis. wig mutant plants show an increase
in organ number similar to those seen in pan mutants, with
extra sepals and petals, and some effects on stamen and
carpel number as well. Sessions et al. (1997) isolated e tt
gene which is related to floral organ number. Ettin muta-
tions have p leio tropic effects on Arabidopsis flower
development, causing increases in perianth organ formation,
decreases in stamen number and anther formation, and api-
cal-basal patterning defects in the gynoecium. In rice, floral
organ mutants are comparatively fewer, s o only several
loci that affect floral organ number have been described,
including dl, fon1, fon2, mp1, mp2, ops, nsr, lhs1, and srs
(Kinoshita et al., 1977; Khush and Librojo, 1985; Niikura et
al., 1992; Nagas awa et al., 1996a; 1996b; Bai et al., 2000;
Jeon et al., 2000; Ge et al., 2001).
In this s tudy , th rough investigations of 200 mutant
florets, 41.14% florets contained 3-5 stamens and two or
more pistils. In 11.5% of the florets, stamen/pistil-like struc-
tures were seen. These phenotypes are very similar to the
phenotype of B loss-of-function mutant. Furthermore, 58.
86% of the florets had 3-5 stamens, lemma/palea-like struc-
ture but one pistil. The phenotypes that are caused by lh
gene mutation is very similar to those of ett, pan and wig
gene mutation, so we may presume that lh gene might also
have ett-like (pan- or wig-like) functions.
In rice, op (over developed palea), nsr (naked seed rice;
Khush and Librojo , 1985) and lhs1(long hu ll s terile;
Kinoshita et al., 1977; Niikura et al., 1992; Jeon et al., 2000)
mutants, have been found, and phenotypes of them are
controlled by a single ressesive gene. Khush and Librojo
(1985) reported op, nsr and lhs1 gene are allelic. Furthermore,
Thakur and Roy (1975) isolated lp-1 and lp-2 (long palea-1,
long palea-2) genes. In our experiment, the effects o f lh
mutation are partly similar to these genes, but lh gene has
some other functions. For example, 41.14% of the long hull
mutant florets have two or more pistils, and the develop-
ment of ovaries is o ften at a different stage, whereas lhs
florets with two pistils are occasional. Furthermore, one or
more bulged tissues appear in 34.5% florets. Sometimes
there are stamen/pis til-like organs , two anthers on one
single filament in lh mutant. Lh gene is probably a new
gene that affects flo ral o rgan number and regional
differentiation. The allelic relationships of this lh gene with
the o ther genes reported p rev ious ly need to be
investigated.
Acknowledgements: We thank YAN Zhi-Bin for helps
with anatomical observation and XIE Lin for helps with
scanning microscopic observation. We also thank Profes-
sor XU Zheng-Jun and Professor NIU Ying-Ze for helpful
discussions of the manuscript.
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