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Phytomorphology 59 (3 & 4) 2009, 116-120 PHYTOMORPHOLOGY z July-December 2009

Comparative Studies of Morphological and Anatomical Characteristics

of Siliqua in Shatter-Susceptible and Resistant Brassica Cultivars
Navjyot Kaur, Neelam Setia, R.C. Setia and S.S. Banga1
Department of Botany, Punjab Agricultural University, Ludhiana-141004, India

ABSTRACT
The mechanism of siliqua shattering was studied in shattering-susceptible (SS) cultivars of Brassica napus (GSL-1,
GSL-2 and GSC-5) by comparing their morphological and anatomical characteristics with shattering-resistant (SR)
cultivars of B. juncea (cv. PBR-210) and B. carinata (cv. PC-5). The examination of siliqua surface revealed that SS
cultivars had distinct wide valve margins at apical, middle and basal positions of the siliqua than SR cultivars. The
length and width of siliqua were greater in SS cultivars as compared to SR cultivars. Anatomical studies in SS cultivars
revealed the presence of a dehiscence zone characterized by thin walled parenchyma cells placed between the valves
and replum tissue. However, this zone was composed of thick walled and was lignified in SR cultivars. Further, SR
cultivars had greater transectional area of main vascular bundle than SS cultivars providing the structural basis of resistance
to siliqua shattering in Brassica species.

Key words: Dehiscence zone, main vascular bundle, shattering, valve margins

Abbreviations: DAA - Days after anthesis, MVB - main vascular bundle, TI - terminal inflorescence, SR - shattering-
resistant, SS - shattering-susceptible

Author for correspondence: R.C. Setia, e-mail - rc_setia@yahoo.co.in

Introduction viz. GSL-1, GSL-2 and GSC-5 were susceptible to

In oilseed brassicas, the shattering or dehiscence of shattering, and the shattering percentage recorded in
siliquae, before or during harvest is an economically these cvs. was 30, 36 and 18, respectively. The purpose
significant problem that leads to considerable loss of of the present investigation was to compare the
seed yield (MacLeod, 1981). The shattering of siliqua morphological and anatomical features of the siliquae
is reported to be closely associated with its structural of the resistant and susceptible cultivars of Brassica spp.
features (Kadkol et al., 1986; Spence et al., 1996). At so as to find out phenological markers, which could be
maturity, the drying of siliquae causes separation of their used as selection criteria for breeding programmes.
walls from a centrally located structure, the replum, Material and methods
which is situated between the walls, triggered
Seeds of Brassica napus L. (cvs. GSL-1, GSL-2 and
mechanically by wind vibration (Austin, 1986). The
GSC-5), B. juncea Czern & Coss (cv. PBR-210) and
siliqua walls, which are joined to replum, exhibit varying
B. carinata A. Braun (cv. PC-5) were procured from
amount of lignified tissue which possibly determines
Department of Plant Breeding, Genetics and
the extent of shattering in different Brassica species
Biotechnology, Punjab Agricultural University,
(Kadkol et al., 1984, 1986; Østergaard et al., 2006). In
Ludhiana. The plants were raised in small plots according
recent years, efficient breeding has resulted in the
to the recommended practices for fertilizer application,
development of shattering-resistant cultivars of some
irrigation and aphid control treatments. Observations on
Brassica species. Five cultivars belonging to three
various morphological and anatomical characters were
Brassica spp.-B. juncea, B. carinata and B. napus were
made from siliquae growing on the terminal inflorescence
selected for this study. Experiments conducted under
(TI). The siliquae were collected at 40 days after anthesis
field conditions revealed that the cultivars belonging to
(DAA) stage from basal (1st, 2nd and 3rd at base) positions
B. juncea (cv. PBR-210) and B. carinata (cv. PC-5) were
of TI. In order to ascertain the age of siliquae, the flowers
resistant to shattering, while those belonging to B. napus
growing on these positions were tagged for several days.
1 Present Address: Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana-141004, India
Navjyot Kaur et al. – Studies of Morphological and Anatomical Characteristics of Siliqua of Brassica
The surface features of siliqua were viewed under
stereozoom microscope. Angle of insertion of siliqua
on to the TI was measured using a protractor. The length
of siliquae was measured from base to beak with a
centimeter scale. The width was measured using ocular
micrometer from transverse sections cut from the middle
position of siliqua. For anatomical studies, siliquae
collected at 40 DAA stage were fixed in FAA (Berlyn
& Miksche, 1976). Hand sections of the meterial were
cut from middle portion of siliqua and stained with
safranin and fast green. Photomicrographs were taken
on Leica bright field research microscope fitted with
a Digital camera and Computer imaging systems. The
siliqua wall thickness and area of main vascular bundle
were calculated from Camera Lucida diagrams drawn
on graph paper.
Results and discussion
Morphological studies
Externally, three major morphological pattern are
distinguishable in a siliqua - the valves or siliqua walls,
the replum or the central ridge between the valves, and
the valve margins where the valves are joined to the
replum. The replum along the entire length of siliqua.
The dehiscence of siliqua occurs due to separation of
valves from replum. An examination of the siliqua
surface of different Brassica cultivars with stereozoom
microscope revealed that the siliquae of shattering-
susceptible cvs. GSL-1, GSL-2 and GSC-5 had distinct
wide valve margins (Fig. 1A-I) in all three regions
(apical, middle and basal) whereas in shatter-resistant
cvs. PBR-210 and PC-5 the valve margins were
comparatively narrow in all the three regions of siliqua
(Fig. 1J-O). Østergaard et al. (2006) also reported similar
surface characteristics of siliquae in shatter - resistant
transgenic Brassica lines, which lacked proper valve
margins compared with fruit from wild type shattering-
susceptible plants.
Further, among different Brassica cultivars,
significant variations were recorded in the siliqua angle
to TI (Fig. 2). In shattering-susceptible cultivars, the
mean siliqua angle to TI varied between 85 and 99°,
whereas, in shattering-resistant cultivars, it was about
36°. Morgan et al. (2003) found siliqua angle as an
important trait for breeding of Brassica species to produce
shatter-resistant cultivars.
Table 1 shows data on dimensional features of
siliqua and tissues. The siliquae in shatter-susceptible
cultivars were longer in length and wider in diameter
117
as compared to those of shattering-resistant cultivars.
Child et al. (2003) also reported that the siliquae of
resynthesized shatter-resistant, oilseed rape line DK142
were shorter in length than those of shatter-susceptible
commercial cultivar Apex.
Anatomical studies
The anatomical features of the siliquae of different
Brassica cultivars are almost similar as is evident from
Figure 3, and are in agreement with earlier reports
(Kadkol et al., 1986, Setia et al., 1989, Spence et al.,
1996). The main difference among different cultivars,
however, was observed in overall siliqua wall thickness
and lignification of tissues in the dehiscence zone.
Structurally the siliqua wall is essentially differentiated
into exocarp, mesocarp and endocarp. While the exocarp
represents the outer epidermis, endocarp comprises the
inner epidermis and the underlying sclerenchymatous
cell layer (Fig. 3 A-B). The mesocarp consists of several
layers of parenchymatous cells with interspersed vascular
bundles, and determines the thickness of siliqua wall.
Compared with resistant cultivars, the susceptible
cultivars had greater siliqua wall thickness and higher
transectional area of mesocarp cells (Table 1).
The single cell layered replum, which runs the
length of siliqua cavity, encloses main vascular bundle
(MVB), one at each end of the siliqua. The site of
attachment of siliqua wall to the side of MVB is referred
to as suture. The suture consists of a few cell layers
intercalated between the valves and replum tissue. In
shatter-resistant cultivars this layer of cells was thick
walled and lignified, but in shatter-susceptible cultivars
these cells were thin walled and non-lignified and formed
the separation or dehiscence zone. Further the cells in
dehiscence zone showed separation of cells due to
dissolution of middle lamella (Fig. 3C-D). However in
shatter resistant cultivars this layer of cells was thick
walled and lignified (Fig. 3E-F) Kadkol et al. (1986)
reported that shatter - susceptible and intermediate
shattering accessions of Brassica spp. had non-lignified
separation layer in the region of attachment of siliqua
wall to the replum, but this layer was completely absent
in shatter-resistant accessions of B. campestris. Pod
shatter-resistant transgenic lines 35S::FUL.2 and
35S::FUL.1 of B. juncea have also been reported to
exhibit complete absence of separation layer
differentiation in their fruits (Østergaard et al., 2006).
Further, the transectional area of the MVB also
varied considerably among different Brassica cultivars.
118 PHYTOMORPHOLOGY z July-December 2009

Fig. 1: A-O –Surface views of siliquae from three regions i.e. apical (A, D,G,J and M), middle (B, E, H, K and N) and basal (C, F, I, L
and O), as observed under stereozoom microscope. A-C. Brasica napus cv. GSL-1. Note wide valve margins (arrows). D-F. B. napus cv.
GSL-2. Note wide valve margins (arrows). G-I. B. napus cv. GSC-5. Note wide valve margins (arrows). J-L. B. juncea cv. PBR-210. Note
narrow valve margins, especially in L (arrows). M-O. B. carinata cv. PC-5. Note narrow valve margins, especially in M (arrows). (A-O.
x 7).
Table 1. Mean values of morphological and anatomical characters of siliqua at 40 DAA (days after anthesis) stage in Brassica napus
(CVS. GSL-1, GSL-2 and GSC-5), B. juncea (Cv. Pbr-210) and B. carinata (Cv. Pc-5)

Characters cv.GSL-1 cv.GSL-2 cv.GSC-5 cv.PBR-210 cv.PC-5

Angle of insertion of siliqua onto the TI (Unit?) 85±2.65 88±2.00 99±1.00 36±2.00 36±2.65
Siliqua length (cm) 5.85±0.06 5.89±0.20 6.33±0.21 4.05±0.15 5.28±0.15
Siliqua width (mm) 3.85±0.03 4.17±0.04 3.94±0.03 3.34±0.04 3.47±0.03
Siliqua wall thickness (mm) 0.77±0.05 0.85±0.01 0.78±0.03 0.64±0.02 0.45±0.02
Transectional area of a mesocarp cell (mm2) 4.84x10-3 5.33x10-3 4.20 x10-3 2.77 x10-3 2.48 x10-3
No. of cell layers in mesocarp 7 7 8 6 6
Area of main vascular bundle (mm2) 0.258±0.005 0.236±0.002 0.317±0.003 0.325±0.003 0.316±0.004
Navjyot Kaur et al. – Studies of Morphological and Anatomical Characteristics of Siliqua of Brassica 119

The shatter-resistant cultivars showed greater

transectional area of MVB than the shatter-susceptible
cultivars (Table 1). Thus, the resistance to siliqua shatter
could be associated with the presence of larger MVB
in cvs. PBR-210, PC-5 and GSC-5 (most resistant cultivar
to siliqua shatter among the susceptible cultivars) as
compared to cvs. GSL-1 and GSL-2 (which are more
susceptible to siliqua shattering). Studies of Child et al.
(2003) revealed that resynthesized, shatter-resistant,
oilseed rape line, DK142 had stronger pods because of
larger main vascular bundles than shatter-susceptible
pods of commercial cultivar Apex.

Fig. 2: A portion of terminal inflorescence (TI) of B. napus (cvs. References

GSL-1, GSL-2 and GSC-5), B. juncea (cv. PBR-210) and B. carinata Austin, R.B. 1986. Molecular Biology and Crop Improvement,
(cv. PC-5). Note the angle of insertion of siliquae onto the TI is Cambridge University Press, London.
greater in cvs. GSL-1, GSL-2 and GSC-5 as compared to cvs. Child, R.D., Summers, J.E., Babij, J., Farrent, J.W. & Bruce, D.M.
PBR-210 and PC-5 (Scale bar=20 cm). 2003. Increased resistance to pod shatter is associated with

Fig. 3: A-F – All transverse sections of siliqua at 40 DAA stage stained with safranin and fast green. (OE-outer epidermis; MC-mesocarp;
VB-vascular bundle; SCL-sclerenchymatous layer of endocarp; IE-inner epidermis; MVB-main vascular bandle). A. Brassica napus cv.
GSL-1. Note completely differentiated siliqua wall. B. B. carinata cv. PC-5. Note relatively narrow siliqua wall as compared to A. C. B.
napus cv. GSL-1. Note valve margin region (arrow) where cells in the dehiscence zone show separation due to dissolution of middle lamella
between the cells. D. Enlarged view of C. E. B. juncea cv. PBR-210. Note differentiation of thick walled valve edge cells (arrow). F. B.
carinata cv. PC-5. Arrow indicates thick walled valve edge cells. Note the cells between valve edge cells and main vascular bundle do not
show any characteristic change that could indicate initiation of formation of dehiscence zone. (A-B. x 62.5, C. x 125, D. x 250, E. x 125,
F. x 250).
120
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