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Acta Botanica Sinica
植 物 学 报
2004, 46 (4): 451－456
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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)
Abst r act : 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 Blike genes in Arabidopsis and Antirrhinum.
Ke y wo rds:
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 ternin g of o rgan t ype, o rgan number and o rgan form
(Yan ofsky, 1995). Based on some gen es 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
mut ation s which affect organ number, organ s hape and
regio nal differentiation within floral organ s. For example,
mu tation s in cla va ta 1 (clv; Clark et al., 1993; 1997),
clavata3 (clv3; Clark et al., 1995), ettin (ett; Sessions et al.,
1997), and periana nthia (pan; Running and Meyerowitz,
1996) increase organ numb er within the flower, whereas
mutations in tousled (tsl; Ro e 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 u nclear wh ether the molecular mechanism fo r dicot
floral organ development is also suitable to monocot species.
Rice (Oryza sativa) is not o nly one of the mos t importan t
cro ps in t h e wo rld , b ut als o an ideal mo del p lan t fo r
research es on the development al molecu lar biolo gy 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 demon strated floral organ morph ogenesis, anatomical 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 progeny 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 su bsp. japon ica Kat o), Maylielle (O. sa tiva sub sp.
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 mu tant were
observed under a microscope before flowering. Meanwhile,
200 florets were investigated and some images of themwere
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:<[email protected]>.
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Ａｃｔａ　Ｂｏｔａｎｉｃａ　Ｓｉｎｉｃａ　植物学报　Ｖｏｌ．４６　Ｎｏ．４　２００４
recorded.
1.3 Observation by scanning electron microscopy (SEM)
Procedures for the scanning electron micrographs were
do ne ess en tially according t o Feng et al. (1995) an d
Mizukami and Ma (1992) with some modification s. Young
panicles at various stages were fixed in a fixative solution
of buffered glutaraldehyde (3%) for 24 h, dehyd rated 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 photo graphed 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 forceps t o make p ollen g rain s s pilled ou t. Un der o pt ical
microscope, the pollen fertility was examined according to
its morpho logy and its stainin g 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, th e different ap pearan ce 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 abnormal 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 between the leafy palea and lemma (Figs.4－6) and most floret s cont ained three, four or five stamens. Statis tical results from 200 floret s showed that th e floret s containing
three, four an d five st amens accounted fo r 37.5%, 34.5%
and 18%, res pectively. Occas ionally, one sin gle filament
was tipped with two anthers (Fig.10). One to four ovaries in
the s ame floret often develop ed at a differen t stage (Fig.
11). According to the analysis, the florets possessing one,
two an d three ov aries 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 qu antit y and size of b ulged tiss ues
(Figs.5, 7, 8) or stamen/pistil-like organ s (Fig.9) and the
florets wit h bulged tissu es 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 stamen and pistil primordium differentiation (Figs.16－21). A
wild floret consists of six st amens an d one pistil, an d six
stamen primordia seem undulant and evenly distribu ted
(Fig.16), while the distributions of stamen and pistil primordia 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 med ial
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 t he lo ng hull mut ant. Following co rollaries
can be established for the low seed setting rate: (1) relation
with pollen sterility, the pollen fertility of long hull mutant
was lo wer (62.46%, Fig.12) th an that of the control (90%,
Fig.13); (2) results from ovary malformat ion; (3) spikelets
incompletely closed , which cau se female organs to lose
water (Bai et al., 2000).
2.4 Inheritance of the floret mutant
F1 h ybrids were made fro m t he cross es b etween t he
long hull mutant and three rice varieties, namely Jodan (O.
sativa subsp. japonica Kato), Maylielle (O. sativa sub sp.
tropic japonica Kato ), and s huhui-527 (O. sat iva sub sp.
indica Kato). Observed with pollen fertility an d seed setting 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
(χc2 =0.09－0.37, P > 0.05). Thus, the mutant trait was controlled by sing le recess ive gene. The gene is temporarily
designated as lh (long hull).
Table 1
Frequency of mutant in F2 population
No. of
normal
plants
Jodan/lh
194
Lh/Maylielle
109
Shuhui 527/lh 278
Crosses
3
No. of
mutant
plants
54
43
88
Expected
ratio
χc2
P
3:1
3:1
3:1
0.37
0.21
0.098
0.1－0.95
0.1－0.5
0.5－0.95
Discussion
In the past decade, there have been great advances in
our und erstanding of flower develo pment. This p rogress
ZHANG Xu-Mei et al.: Morphogenesis, Anatomical Observation and Genetic Analysis of a Long Hull Floral Organ Mutant
in Rice
(wig) in Arabidopsis. wig mutant plants show an increase
in organ number similar to those seen in pan mutants, with
extra sepals and pet als, and some effects o n stamen and
carp el number as well. Sessio ns et al. (1997) isolated ett
gene which is related to floral organ number. Ettin mu tation s have p leio tro pic effect s on Arab ido psis flo wer
development, causing increases in perianth organ formation,
decreases in stamen number and anther formation, and apical-basal patterning defects in the gynoecium. In rice, floral
org an mu tants are comparatively fewer, s o only several
loci that affect floral organ number have b een described,
including dl, fon1, fon2, mp 1, 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 rou gh investig ations 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 structures were seen. These phenotypes are very similar to the
phenotype of B loss-of-function mutant. Fu rthermore, 58.
86% of the florets had 3－5 stamens, lemma/palea-like structure but one pistil. The phenotypes that are caused by l h
gene mutation is very similar to those of ett, pan and w ig
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;
Kh ush an d Librojo , 1985) and lhs1（lo ng hu ll s terile;
Kinoshita et al., 1977; Niikura et al., 1992; Jeon et al., 2000）
mut ants, have b een found, and ph enoty pes of them are
controlled by a single ressesive gene. Khu sh 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,
lon g palea-2) genes. In o ur exp eriment, th e effects o f l h
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 mo re pistils, and the development of ovaries is o ften at a different stag e, whereas lh s
florets with two pistils are occasional. Furthermore, one or
more bulged tissues appear in 34.5% florets. Sometimes
th ere are stamen/ pis til-like organs , t wo anthers on o ne
sin gle filament in lh mut ant. Lh gene is pro bably a new
g en e t h at affect s flo ral o rg an n umb er an d regio nal
differentiation. The allelic relationships of this lh gene with
t h e o t h er g en es rep o rt ed p rev io u s ly n eed t o b e
investigated.
Acknowledgements: We thank YA N Zhi-Bin for helps
wit h anatomical o bserv ation and XIE Lin for helps with
scanning microscopic observation. We also thank Professor XU Zheng-Jun and Professor NIU Ying-Ze for helpful
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discussions of the manuscript.
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