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Annals of Botany 94: 469–471, 2004
doi:10.1093/aob/mch161, available online at www.aob.oupjournals.org
SHORT COMMUNICATION
Morphological Features and Inheritance of Foliaceous Stipules of Primary Leaves in
Cowpea (Vigna unguiculata)
R . N . P A N D E Y * and P . D H A N A S E K A R
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
Received: 8 March 2004 Returned for revision: 13 May 2004 Accepted: 26 May 2004 Published electronically: 30 July 2004
Background and Aims The presence of connate foliaceous stipules of primary leaves and their inheritance in
cowpea (Vigna unguiculata) genotype EC394736 is reported for the first time.
Methods The development of foliaceous stipules (FS) and their persistence were examined throughout the growth
and developmental stages of the plants of the genotype EC394736. The shape, size, colour, texture and other
parameters were examined in the field during the period 15–50 d after sowing. The area of FS was measured using
image analysis software. The inheritance of FS was studied by making a cross between the genotype EC394763 with
rudimentary stipules (RS) and the genotype EC394736, which has connate foliaceous stipules of primary leaves. The
presence or absence of FS in plants of the F1, F2 and F3 generations was recorded.
Key Results The stipules developed along with the primary leaves in the genotype EC394736. One stipule of each
primary leaf fused with the adjacent stipule of the other primary leaf forming a foliaceous structure. These stipules
persisted on the plants for >50 d, even after the primary leaves had withered off. The F1 plants showed an absence of
FS indicating the rudimentary stipules to be dominant over foliaceous stipules. The F2 segregation into 15 (RS) : 1
(FS) indicated that duplicate recessive genes controlled the presence of the FS. This was confirmed from the
segregation pattern in the F3 generation.
Conclusions The presence of FS is a unique feature in cowpea genotype EC394736 and duplicate recessive genes
govern it. The FS can be used as a morphological marker for identification of cowpea varieties.
ª 2004 Annals of Botany Company
Key words: Cowpea, duplicate recessive genes, foliaceous stipules, inheritance, morphological marker, Vigna unguiculata.
INTRODUCTION
Cowpea (Vigna unguiculata), one of the most important
food legume crops, exhibits considerable morphological
variability in its wild as well as cultivated forms. The International Institute of Tropical Agriculture (IITA), Nigeria,
the main germplasm resource centre for cowpea, maintains
over 15 000 germplasm lines (Quin, 1997) that help scientists all over the world to identify the desired traits and
combine them to suit their needs. With a view to initiating
an improvement programme for cowpea through mutational
as well as conventional breeding approaches at our Centre,
about 130 germplasm accessions were obtained from IITA,
Nigeria through the National Bureau of Plant Genetic
Resources, New Delhi and used in various field and laboratory studies. During the course of field studies, a unique
feature in the genotype EC394736 (Pedigree: IT93K-204529)—the presence of a pair of foliaceous stipules (FS)
arranged opposite and at about right angle to the primary
leaves—was noticed. The plants of several other genotypes
in the germplasm collection belonging to the Nuclear Agriculture and Biotechnology Division (NA&BTD), Bhabha
Atomic Research Centre (BARC), Mumbai, were examined
and no such a feature was found in them, though they had
scaly separate or joint rudimentary stipules (RS). A survey
of the literature, especially on cowpea morphology and
taxonomy (Padulosi and Ng, 1997) and genetics (Fery,
1985; Fery and Singh, 1997), showed no mention of this
* For correspondence. E-mail [email protected]
unique type of FS of primary leaves. This prompted us to
characterize the FS in the genotype EC394736 and study
their inheritance. The details of the connate foliaceous
stipules of primary leaves and their inheritance in cowpea
are reported here for the first time.
MATERIALS AND METHODS
The development of the FS and their persistence were examined throughout the growth and developmental stages of
plants of the genotype EC394736. To study the stage
at which the FS developed, the unexpanded cotyledonary/
primary leaves of the seedlings were forced open and examined at 5 d after sowing in Petri dishes. The shape, size,
colour, texture and other parameters were examined in the
field during the period 15–50 d after sowing. Six stipules
from randomly selected plants were excised and studied for
their shape and size. The area of the stipules was measured
using image analysis software Biovis image plus, Ver. 1.4
(Expert Vision Labs Pvt. Ltd, Mumbai, India).
The inheritance of the foliaceous stipules was studied by
making a cross between the genotype EC394763 with RS
(Fig. 1) and the genotype EC394736 with FS (Fig. 2) of
primary leaves. The presence or the absence of the FS in
plants of the F1, F2 and F3 generations was recorded. The
progenies of only those F2 plants that produced 20 seeds or
more were carried forward in the F3 generation to ensure the
minimum number of plants (16) required for the dihybrid
ratio of 15 : 1.
Annals of Botany 94/3, ª Annals of Botany Company 2004; all rights reserved
470
Pandey and Dhanasekar — Foliaceous Stipules of Primary Leaves in Cowpea
T A B L E 1. Characteristics of the connate foliaceous stipules of
primary leaves in cowpea genotype EC394736
Parameter
Features in foliaceous stipule
Naissance/organogenesis
Along with the development
of cotyledonary leaves
Opposite
Horizontal (right angle with
reference to primary leaves)
Sessile
Persistent
Dark green
Smooth
Reticulate
Ovate to obovate
Obtuse to truncate
Entire
Acute
253 mm2 (range 114–384 mm2)
Phyllotaxy
Orientation
Petiolate/sessile
Persistence
Colour
Texture
Venation
Shape
Apex
Margin
Base
Size (total laminar area)
F I G . 1. Rudimentary stipules (RS) of primary leaves in cowpea genotype
EC394763.
FS
FS
F I G . 2. Connate foliaceous stipules (FS) of primary leaves in cowpea
genotype EC394736.
F I G . 3. Variation in size and shape of the foliaceous stipules.
RESULTS
Characteristics of the foliaceous stipules
The stipules developed along with the primary leaves in the
genotype EC394736. One stipule of each primary leaf fused
with the adjacent stipule of the other primary leaf forming a
foliaceous structure. The connate foliaceous stipules were
conspicuous as soon as primary leaves expanded. These
stipules persisted on the plants for >50 d, even after the
primary leaves had withered or dropped off. The characteristics of the foliaceous stipules present in the genotype
EC394736 are shown in Table 1 and Figs 2 and 3.
Inheritance
The three F1 plants of the cross EC394763 · EC394736
showed absence of the FS indicating the dominance of RS
over FS. In the F2 population of 131 plants, only 12 plants
showed the presence of FS, while 119 had RS (Table 2). The
segregation into RS- and FS-plants indicated a modified
dihybrid ratio of 15 : 1 with a c2 value of 188 (P =
010–020), i.e. duplicate recessive genes controlled the
presence of FS.
Of the 131 F2 plants, only 93 plants (seven with FS and 86
with RS) were carried forward in the F3 generation as plant
to row progenies. This constituted 50, 31 and 12 plants of
three respective families. The number of plants in the F3
progenies ranged from 18 to 33. In the 40 segregating progenies, eight progenies had 18–20 plants, 27 progenies had
21–29 plants and five had 30–33 plants each.
All the plants of the seven FS progenies bred true for the
FS-character, while only 46 of the 86 RS-plant progenies
Pandey and Dhanasekar — Foliaceous Stipules of Primary Leaves in Cowpea
T A B L E 2. Segregation for foliaceous stipules of primary
leaves in the F2 generation of the cross EC394763 · EC394736
Observed segregation
F2 family
1
2
3
Total
Total no.
of plants
RS
FS
c2
(15 : 1)
P
76
42
13
131
69
38
12
119
7
4
1
12
1.137
0.768
0.046
1.88
0.20–0.30
0.30–0.50
0.80–0.90
0.10–0.20
T A B L E 3. Segregation for the presence and absence of the
foliaceous stipules in the F3 plant progenies of the cross
EC394763 · EC394736
No. of plants
No. of
progenies
7
46
23
17
Total
144
1110
546
408
Ratio
RS
FS
O
E
c2
P
0
1110
515
315
144
0
31
93
Tr. Br.
Tr. Br.
16.6 : 1
3.39 : 1
15 : 1
3:1
0.305
1.06
0.50–0.70
0.30–0.50
O, Observed; E, expected; Tr. Br., true breeding.
FS and RS indicate the plants with foliaceous stipules and rudimentary
stipules, respectively.
471
be recessive though inherited monogenically (Paria and
Basak, 1994).
The stipules have been recognized as an important morphological character for identification of species or varieties. Mouli and Kale (1982) reported foliaceous stipules
to be a good identifying marker for the early maturing
groundnut TGE-1. The presence or absence of foliar stipules
was used to describe various tribes of the family Genisteae
(Josefa et al., 1998). Information on stipule development
is useful to the taxonomists (Stein, 1982).
The foliaceous stipules of the primary leaves in the cowpea genotype EC394736 will be used as a morphological
marker in the breeding programme of the NA&BTD,
BARC. The phenotypic behaviour of the stipule in the F1
could be very useful in detecting successful crosses involving the genotype EC394736 as a female parent. As this
genotype is endowed with the presence of foliaceous stipules of primary leaves, a cross between this parent and
those with rudimentary stipules as male can be taken as
successful if the F1 shows the absence of foliaceous stipules,
a recessive character. The FS character can also be transferred to any variety for identification. It could be used
during seed multiplication of cowpea varieties to check
for any inadvertent, undesirable out-crossing to other cowpea genotypes that would reduce the purity of the seed.
LITERATURE CITED
bred true for the RS character (Table 3). Of the remaining
40 progenies, 23 segregated into a ratio of 15 RS : 1 FS, and
17 into 3 RS : 1 FS. The segregation of the 86 RS plant
progenies in the F3 generation fitted in the expected genetic
ratio of 7 (true breeding for RS) : 4 (segregating into 15 RS :
1 FS) : 4 (segregating into 3RS : 1 FS) with the c2 value of
2394 (P = 030–050).
DISCUSSION
The segregation pattern of the foliaceous stipules observed
in the F2 and F3 generations confirmed the involvement of
duplicate recessive genes in their development.
It is proposed that the gene symbols for foliaceous
stipules of primary leaves be fos1 and fos2. The genotype
EC394736 with foliaceous stipules can thus be represented
as fos1fos1fos2fos2.
Studies on the inheritance of stipules in general, and
foliaceous stipules of primary leaves in particular, are
very few and reports are scanty. Small stipule size in
American chestnut has been reported to be incompletely
dominant over large stipule size in Chinese chestnut and
is controlled by two genes (Hebard, 1994), implying that the
large stipule size is a recessive character. This conforms
with the present authors’ findings that recessive genes
control the large size of the foliaceous stipules. In jute
also, the foliaceous stipule character has been reported to
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Singh SR, Rachie KO, eds. Cowpea research, production and utilization. Chichester, UK: John Wiley and Sons, 25–62.
Fery RL, Singh BB. 1997. Cowpea genetics: a review of the recent literature.
In: Singh BB, Mohan Raj DR, Dashiell KE, Jackai LEN, eds. Advances
in cowpea research. Ibadan, Nigeria: IITA. Co-publication of
International Institute of Tropical Agriculture (IITA) and Japan
International Research Center for Agricultural Sciences (JIRCAS),
13–29.
Hebard FV. 1994. Inheritance of juvenile leaf and stem morphological traits
in crosses of Chinese and American chestnut. Journal of Heredity
85: 440–446.
Josefa L, Antonio DJ, Trinidad R, Ana OO. 1998. Seedling morphology
in Genisteae (Fabaceae) from south-west Spain. Botanical Journal of
Linnean Society 128: 229–250.
Mouli C, Kale DM. 1982. An early maturing groundnut (Arachis
hypogaea) with foliaceous stipule marker. Current Science 51:
132–134.
Padulosi S, Ng NQ. 1997. Origin, taxonomy, and morphology of Vigna
unguiculata (L.) Walp. In: Singh BB, Mohan Raj DR, Dashiell KE,
Jackai LEN, eds. Advances in cowpea research. Ibadan, Nigeria: IITA.
Co-publication of International Institute of Tropical Agriculture
(IITA) and Japan International Research Center for Agricultural
Sciences (JIRCAS), 1–12.
Paria P, Basak SL. 1994. Inheritance of some induced mutant characters in
jute (Chorchorus olitorius L.). Indian Journal of Genetics and Plant
Breeding 54: 347–350.
Quin FM. 1997. Introduction. In: Singh BB, Mohan Raj DR, Dashiell KE,
Jackai LEN, eds. Advances in cowpea research. IITA, Ibadan, Nigeria:
Copublication of International Institute of Tropical Agriculture (IITA)
and Japan International Research Center for Agricultural Sciences
(JIRCAS), ix–xv.
Stein OL. 1982. Stipule arrangement in the genus Caesalpinia (Leguminosae). Botanical Journal of Linnean Society 84: 289–294.