Scientia Horticulturae 301 (2022) 111107

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Agro-morphological and molecular diversity analysis of new cytoplasmic

male sterile lines in Indian cauliflower for their use in hybrid breeding
Shrawan Singh *, Pritam Kalia, Rahul Kumar Meena, Brij Bihari Sharma, Bana Ram Parihar
Division of Vegetable Science, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

A R T I C L E I N F O A B S T R A C T

Keywords: Cauliflower is a typical thermo-sensitive crop, highly responsive to heterosis for yield and stress tolerance. A
Cytoplasmic male sterility diverse set comprising of 24 cytoplasmic male sterile (CMS) lines, developed across different maturity groups of
Hybrid breeding Indian cauliflower, using refined Ogura, Can(napus) and Tour sterile cytoplasms was studied to understand the
SSRs
morphometric and molecular changes. The established Ogura-based CMS line of ‘84 series’ (in the nuclear
Mitochondrial genome
Tropical cauliflower
background of snowball cauliflower) was used as an immediate source for conversion of fertile early- and mid-
maturity groups lines. Observations on 18 agro-morphological traits revealed significant differences among the
CMS lines, however they could not distinguish different CMS systems. The CMS system significantly reduced the
floral traits (19) in almost all the transformed lines irrespective of the maturity group, however, the reduction
was maximum in CMS line Tour(CMS 394–41–5) having Tour sterile cytoplasm. This CMS line had a peculiar
feature of ‘gripping phenomena’ for the style which drastically reduced flower size and eventually seed yield.
These CMS lines showed variation with 11 mtDNA markers and 36 genomic simple sequence repeats (gSSR), and
of these, BnTR1 perfectly-identified Can(napus) and Tour CMS systems. This is the first detailed report on the
extent of diversity observed in 24 new CMS lines developed using three diverse sterile cytoplasms in the nuclear
background of different maturity groups of Indian cauliflower. The information will be of immense significance
for immediate use in hybrid breeding and/or use as an immediate source of CMS trait for tropical cauliflowers in
other regions.

1. Introduction morphology arose in southern Italy from a heading Calabrese broccoli

Cauliflower (Brassica oleracea var. botrytis L.) is an important leading
Cole vegetable being cultivated on 1.44 million hectares with an annual
production of 26.92 million tonnes in nearly 94 countries of the world.
China (39.77%) and India (33.74%) are major producers while the
United States of America (4.63%), Mexico (2.67%), Spain (2.65%), Italy
(1.37%), Turkey (1.17%) also contribute significantly (FAOSTAT,
2019). Cauliflower is a member of ‘Cole crops’ which is known for their
diverse edible parts, taste, flavor, and nutritive values and their common
source of origin (i.e. wild diploid species Brassica oleracea var. sylvetris
L.) in the Mediterranean region. Schluz (1919) proposed Brassica cretica
as the probable ancestor of the cauliflower and Hyams (1971) consid­
ered that Syria was a place of origin. It has a close genetic affinity with
broccoli gene pool which clearly reflects its morphological resemblance
with broccoli gene pool. Smith and King (2000) studied BoCAL-a and
BOAP1 genes in Calabrese-Sicilian Purple-cauliflower lineage and pro­
posed a genetic model highlighting that the cauliflower curd
via an intermediate Sicilian type. However, leaf reminiscent of cabbage
and cauliflower could be the result of abnormalities in crossing between
early forms of branching shrub kale and other cultivars followed by
human selection (Snogerup, 1980). Thus, cauliflower is comparatively
of a later origin Cole crop and introduced in Italy by Genoese from
Levant or Cyprus around 1490, which became the center of genetic di­
versity for both cauliflower and broccoli. From here, it reached Ger­
many, France, and England in the beginning of the 17th century. During
this northward migration, diverse forms were evolved such as Italian or
originals, Cornish, Northern, Roscoff, Angers, and Snowball or Erfurt
(Crisp, 1982). Since then, the spread of germplasm of these groups
contributed to the evolution of new local types such as Indian, Austra­
lian, and Indonesian (Swarup and Chatterjee, 1972; Astarini et al.,
2006). The Indian cauliflower is characterized by short plant, long stalk,
broadly wavy and loosely arranged leaves, and flat somewhat loose,
yellowish cream curds which are devoid of self-blanch habit and highly
flavoured (Swarup and Chatterjee, 1972). It has tolerance to heat and

* Corresponding author.
E-mail address: singhshrawan@rediffmail.com (S. Singh).

https://doi.org/10.1016/j.scienta.2022.111107
Received 26 May 2021; Received in revised form 22 March 2022; Accepted 4 April 2022
Available online 26 April 2022
0304-4238/© 2022 Elsevier B.V. All rights reserved.
S. Singh et al.
high rainfall and categorized into three sub-groups based on tempera­
ture requirement for curd formation i.e. extra-early (25–30 ◦ C), early
(20–27 ◦ C) and mid-early (16–20 ◦ C). Mid-late group (12–16 ◦ C) rep­
resents an intermediate form of Indian and Snowball types and produce
seeds in plains of North India during winter season. While Snowball
types form curd at 10–16 ◦ C and does not form seeds in plains of India
(Singh and Sharma, 2001). These three groups of Indian cauliflower
share nearly three fourth of cauliflower production in India. It has been
established that the Indian type evolved as an ecotype in evolutionary
process with distinct morphotypes (Swarup and Chatterjee, 1972), and
any attempt of genomic introgression of CMS systems through European
or temperate types could result in potential changes in
morpho-biochemical traits as reported by Dey et al. (2017) in Snowball
group while introgressing Ogura sterile cytoplasm.
Cauliflower displays up to 50% heterosis (Verma and Kalia, 2016;
Dey et al., 2017), however, hybrid breeding get attention due to earli­
ness, uniformity in maturity, curd appearance, and plant traits, better
response to adaptive stresses, and nutritional traits. Of the two genetic
mechanisms i.e. self-incompatibility (SI) and cytoplasmic male sterility
(CMS) available in Cole crops, the SI system is stable in the early group
of Indian cauliflower whereas mid-group of Indian cauliflower and
snowball types lack strong SI alleles (Sharma et al., 2005). Alternatively,
the CMS system is more stable, easy to maintain, and convenient to use
in hybrid breeding and economic hybrid seed production. Unlike the SI
system, the CMS system completely eliminates possibilities of ‘natural
sibs’ in hybrids and ensures a stable and affordable genetic mechanism
for hybrid seed production in vegetable Brassicas (Kucera et al., 2006;
Wang et al., 2012; Dey et al., 2017). Currently, CMS is widely used for
hybrid breeding and seed production of cauliflower (Kaminski et al.,
2012) and other important vegetable crops such as cabbage (Fang et al.,
2004), radish (Lee et al., 2008), and broccoli (Shu et al., 2014). It is
convenient to use the CMS system in vegetable Brassicas because of the
economic utility that lies with vegetative tissues not with the seed as in
oilseed Brassicas, where the role of fertility restorer is important.
Brassica cytodeme has different sterile cytoplasm such as Ogura
(Ogura, 1968), pol (Fu et al., 1995), nap (Thompson, 1972), nig (Pear­
son, 1981), hau (Wan et al., 2008), and inap (Kang et al., 2017). Of them,
Ogura is the most commonly used sterile cytoplasm in Brassica crops
because of its stability and performance for hybrid seed yield. Original
Ogura cytoplasm caused low-temperature chlorosis in B. oleracea which
was refined by replacing chloroplast with broccoli and cabbage and this
‘refined Ogura’ served as a source for European cauliflower and later on
introgresed in Snowball cauliflower through backcross breeding (Dey
et al., 2011). The others like nap and pol were found to be sensitive at
higher temperatures (30/24 ◦ C) and resulted in partial to fully male
fertile phenotypes in the CMS line (Fan and Stefansson 1986). Hence,
relying only on one sterile cytoplasm may be disastrous, and exploring
the potential of new cytoplasm-nuclear combination from alternative
sterile cytoplasms such as Can(napus) (Chamola et al., 2013) and
‘Anand’ Tour (Yamagishi and Bhat, 2014) offers new options for the
pre-emptive strategy to counter the impact of mutation/ recombination
or lethal event due to incongruity between nuclear-cytoplasmic in­
teractions. Investigating therefore, the effect of the introgression of
diverse CMS systems into elite inbred lines of Indian cauliflower was
essential so as to utilize it in hybrid seed production.
The transfer of Ogura CMS from snowball into tropical cauliflower
was challenging because of differences in their genetic constitutions
mainly in relation to temperature response to curding traits and flow­
ering behavior (Sharma et al. 2005). Significant changes have been re­
ported earlier in curding and floral traits such as petaloid anthers,
carpeloids, crooked style, and reduced nectaries in Brassica oleracea
(McCollum, 1981), snowball cauliflower (Dey et al., 2011, 2017), and
broccoli (Sharma and Vinod, 2002) mainly due to incongruity between
nuclear genome and cytoplasmic factors of recipient and donor,
respectively. The genetic backgrounds and CMS types exhibit complex
variations for floral organs during the CMS transfer process which in
2
Scientia Horticulturae 301 (2022) 111107
turn considerably affect the efficiency of pollination and seed produc­
tion. However, it was required to investigate changes due to the intro­
gression of sterile cytoplasm in Indian cauliflower which, in fact,
represent a typical tropical type and share a maximum fraction of India’s
total cauliflower production (89.99 million tonnes) (FAOSTAT, 2019).
The CMS is governed by mitochondrial genes which depend on the
source species of the CMS system (Schnable and Wise, 1998; Hanson and
Bentolila, 2004). The mitochondrial genes such as orf138, orf224-atp6,
orf263, orf222, and orf288 are key genes of ogu (Bonhomme et al.,
1992), pol (Singh and Brown, 1993), tour (Landgren et al., 1996), nap
(L’Homme et al., 1997), and hau (Heng et al., 2015) CMS systems,
respectively. Molecular markers specific to mitochondrial DNA
(mtDNA) are useful in detecting CMS type in CMS lines/hybrids as well
as in hybrids carrying restorer genes. It has been successfully used in
B. juncea (Ashutosh et al., 2005), B. oleracea (Zhang et al., 2011; Wang
et al., 2012), B. oleracea var. italica (Shu et al., 2016), and B. rapa (Heng
et al., 2015). The markers have also been used to analyze cytoplasmic
diversity in varieties of broccoli (Shun et al. 2016), however, no such
attempt was made in Indian cauliflower, despite its great economic
significance in the tropical and sub-tropical region.
Therefore, the present investigation was aimed (i) to study the
impact of introgression of different CMS systems i.e. Ogura, Can(napus),
and Tour on morphological changes in curding, floral, and seed traits in
Indian cauliflower, and (ii) assess the molecular variations that occurred
both at mitochondrial and nuclear genomic levels in the CMS lines of
Indian cauliflower.
2. Materials and methods
2.1. Plant materials and field evaluation
Twenty-four CMS lines used in this study belong to four different
maturity groups of tropical (or Indian) cauliflower (Table 1). These are
developed by repeated backcrossing of elite inbred lines of tropical
cauliflower to source CMS line in broccoli nuclear background for 5 to
14 generations (Suppl. Table 1). These lines were developed by using
already converted cauliflower lines with refined Ogura (IARI, New
Delhi), Tour (NIPB via IIHR, Bengaluru), and Can(napus) (NIPB, New
Delhi) sterile cytoplasms (Fig. 1). Respective maintainers were also
included in the study. The CMS lines and maintainers were developed
and are being maintained by the Division of Vegetable Science, ICAR-
Indian Agricultural Research Institute (IARI), New Delhi.
The CMS lines were evaluated during 2017–18 and 2018–19 by
growing at the research farm of Division of Vegetable Science, ICAR-
IARI, New Delhi which is located at 28◦ 38′ 1’’ N and 77◦ 9′ 50’’ E with a
sub-tropical climate. The field evaluation of the CMS and maintainer
lines was done in a randomized block design (RBD) with three replica­
tions. Cauliflower genotypes are very specific to their temperature
requirement for curd initiation and development; hence the CMS lines
and their maintainers were grown according to their respective maturity
groups so that they could attain full growth and yield potential for
horticultural traits. The sowing of early, mid-early, and mid-late matu­
rity groups was done during mid-June, July end, and August end,
respectively. For early group, 42–45 days old seedlings were trans­
planted in the evening hours at 45 cm row to row and within row plant
distance to minimize transplanting shock (due to high temperature in
July month) and for mid-early and mid-late groups, 35- and 30-days old
seedlings were transplanted at a distance of 60 cm between rows and 45
cm between plants. Transplanting was done on 15–20 cm raised beds to
avoid the effect of waterlogging and facilitate furrow irrigation. The
crop was provided with a recommended dose of nitrogen (120 kg/ha),
phosphorus (60 kg/ha), and potash (50 kg/ha). Half dose of N and full
dose of P and K were applied as basal dose. Remaining half dose N was
given 35 days after transplanting by top dressing (Singh and Sharma,
2001).
S. Singh et al. Scientia Horticulturae 301 (2022) 111107

Table 1
Grouping of new CMS lines of Indian cauliflower based on their climatic requirement (based on North Indian plains conditions) (modified from Singh and Sharma,
2001).
Maturity Sowing time Month of Month of Temperature Month of Temperature CMS lines
groups* transplanting curd required for bolting and required for
maturity curding ( ◦ C) flowering flowering ( ◦ C) and
duration

Early (a) or Mid May July beginning mid Sept - 25–30 November – 19–23 ◦ C or lower for Ogura (CMS 175–8), Ogura (CMS
Extra- mid Oct December 3–4 weeks 999–98–4), Ogura (CMS MD-98–4)
early
Early (b) June Mid July Oct – mid 20–25 December- 16–22 ◦ C for 4–5 Ogura (CMS 131–98–4), Ogura (CMS
Nov January weeks 131–41–5), Ogura (CMS 131–23), Ogura
(CMS 999–41–5), Ogura (CMS 999–23),
Ogura (CMS 1817–7), Ogura (CMS AK 98–4),
Ogura (CMS MD-PD-12), Ogura (CMS
8498–10), Ogura (CMS 8441–5), Ogura
(CMS 48–98–4), Ogura (CMS 48–41–5), Tour
(CMS 394–41–5), Tour (CMS 394–98–4),
Can (CMS 98–4)
Mid-early July end August end to mid Nov- 15–20 January- 10–16 ◦ C for 5–6 Ogura (CMS 8409), Ogura (CMS 1944–309),
–August Mid-September mid Dec February weeks Ogura (CMS 8410–22), Ogura (CMS 8410),
Mid-late August end- September end mid Dec – 12–16 February – 11–16 ◦ C for 6 − 7 Ogura (CMS 8401), Ogura (CMS MD 202)
September Jan March weeks
mid

Fig. 1. Timeline of introgression and different cytoplasmic male sterility (CMS) systems (i.e. Refined Ogura, Tour and Eru) for development of novel CMS lines in
different maturity groups of Indian tropical cauliflower (Maturity group-wise CMS lines are given in Table 1).

2.2. Agro-morphological observations
Days to curd initiation (i.e. earliest visible size curd) and days to curd
maturity were observed by counting plants from each plot. Plant height
(cm), plant spread (cm), plant weight (g), curd weight (g), curd length
(or polar distance) and curd diameter (or equatorial distance) (cm) were
recorded from five random plants in each of three replications. Besides,
five random plants were tagged and 10 flowers from each plant were
observed for floral traits. Flower and petal colours were observed visu­
ally. The traits, such as bud length and bud width, flower length, floral
width, petal length and petal width (mm) were measured using Vernier
caliper (Mitutoyo Europe GmbH, Germany). Anther size (length ×
width) (mm) and style length (mm) were recorded using a ruler scale,
however, they were checked with Stereo Zoom Microscope. Nectary size
(length and width) (mm) were observed using a Zeiss stereo microscope
with Axio Cam 1Cc-1 camera (Carl Zeiss Microimaging Gmbh,
Germany). Profuseness of flowering and seed setting was observed
visually. Numbers of branches per plant, seeds per pod, and seed yield
per plant were recorded from five random plants.
2.3. DNA extraction and polymerase chain reaction
Healthy and uninfected leaves from six weeks old five random plants
of each line were collected and brought to the laboratory in liquid ni­
trogen (− 196 ◦ C) where they were kept in deep freezers at − 80 ◦ C for
further use. Genomic DNA was extracted from young leaf tissue
following the Cetyl trimethyl ammonium bromide (CTAB) procedure
(Murray and Thompson 1980). DNA quality and quantity were assessed
on a 1% (w/v) agarose gel stained with ethidium bromide (Sigma
Aldrich Chemical Pvt. Ltd, Bangalore, India) and also by using a Nano­
Drop® ND-1000 spectrophotometer. The ratio of absorbance was
recorded at 280 and 260 nm and a ratio of ~1.8 is generally accepted as

3
 Table 2

S. Singh et al.
Mean performance of CMS lines of tropical cauliflower for agro-morphological and yield traits in respective maturity groups.
CMS lines Maturity (days after Plant traits Leaf traits Root traits Curd traits Yield traits
transplanting)

Days to Days to Curd Plant Plant Gross plant No. of Leaves Leaf Root Root width Curd Curd Core Marketable Net curd Marketable Harvest
curd curd height height spread weight (g) leaves/ length width length (cm) length width length curd weight weight (g) curd yield index (%)
initiation maturity (cm) (cm) (cm) plant (cm) (cm) (cm) (cm) (cm) (cm) (g) (t/ha)

Early maturity group

Tour (CMS 64.0 79.4 24.3 42.8 45.7 796.0 20.6 14.9 12.4 11.0 109.3 9.0 11.3 5.7 490.0 397.0 17.6 49.9
394–41–5)
Ogura (CMS 54.8 70.8 21.1 45.2 50.8 902.0 20.2 16.4 14.4 9.6 102.6 8.5 10.8 5.2 429.0 359.0 15.4 39.8
8441–5)
Ogura (CMS 58.8 71.4 23.7 42.8 53.1 888.0 19.1 20.5 15.6 10.9 111.8 8.7 11.0 6.3 468.0 396.0 16.9 44.6
131–41–5)
Ogura (CMS 57.4 72.6 23.1 46.0 47.8 778.0 20.1 19.8 13.6 10.4 104.5 8.4 10.0 4.3 454.0 352.0 16.3 45.2
999–41–5)
Ogura(CMS 54.2 68.2 22.2 38.2 44.8 1078.0 18.3 16.6 12.1 11.0 96.4 9.1 11.7 4.2 464.0 362.0 16.7 33.6
48–41–5)
Ogura (CMS 57.6 71.8 26.3 58.8 68.0 1198.0 23.0 20.9 16.2 12.3 118.4 9.5 11.6 5.2 450.0 370.0 16.2 30.9
8498–10)
Tour(CMS 56.4 71.2 24.9 55.0 62.8 1198.0 20.0 18.2 13.1 10.1 94.2 10.5 11.8 6.5 536.0 446.0 19.3 37.2
394–98–4)
Ogura (CMS 49.8 66.4 22.3 46.6 58.2 834.0 20.2 21.1 15.6 10.0 91.9 9.6 9.7 5.4 428.0 330.0 15.4 39.6
131–98–4)
Ogura (CMS 53.4 70.0 25.8 50.8 55.6 842.0 20.4 20.5 14.3 9.2 92.9 9.4 10.6 6.1 480.0 370.0 17.3 43.9
999–98–4)
Ogura (CMS MD 48.6 62.8 23.2 46.0 46.5 1020.8 18.7 18.5 13.5 9.6 83.6 8.2 9.3 4.7 404.0 288.0 14.5 28.2
98–4)
Ogura (CMS AK- 52.6 65.2 23.6 46.8 52.2 992.0 19.9 17.8 14.7 8.6 82.6 9.6 11.3 6.6 488.0 392.0 17.6 39.5
4

98–4)
Ogura (CMS 53.8 65.0 23.1 50.6 48.7 1054.0 18.8 19.1 13.5 10.0 84.1 8.2 9.5 4.8 419.0 324.0 15.1 30.7
48–98–4)
Can(CMS 98–4) 59.6 72.8 24.4 56.0 57.8 994.0 21.0 21.1 15.6 8.8 82.1 8.0 12.2 4.9 422.0 322.0 15.2 32.4
Ogura (CMS 63.2 80.2 26.1 42.8 58.0 1186.0 24.3 26.0 16.4 12.8 128.3 11.4 13.1 6.6 594.0 454.0 21.4 38.3
131–23)
Ogura (CMS 64.0 80.4 26.6 55.6 61.2 1036.0 23.6 23.6 16.1 12.9 121.2 11.9 12.5 7.3 570.0 431.0 20.5 41.6
999–23)
Ogura (CMS MD- 66.8 84.0 27.3 53.1 59.6 1492.0 23.2 29.5 17.4 10.4 98.5 11.8 13.4 6.9 600.0 496.0 21.6 33.2
PD-12)
Ogura (CMS 45.8 60.2 20.3 52.0 54.6 883.0 15.2 21.0 16.0 12.8 87.5 7.4 9.0 4.0 388.0 282.0 14.0 31.9
175–8)
Ogura (CMS 56.0 70.0 25.9 47.8 55.6 1154.0 15.8 18.1 17.0 11.9 89.1 8.5 9.4 4.9 422.0 332.0 15.2 28.8
1817–7)
Group mean 56.49 71.24 24.12 48.72 54.50 1018.10 20.13 20.20 14.86 10.68 98.83 9.32 11.01 5.53 472.56 372.39 17.01 37.18
Mid-early maturity group
Ogura (CMS 64.2 83.6 17.1 53.2 67.6 1430.0 22.0 30.6 16.8 11.9 91.0 11.7 14.2 7.1 766.0 605.0 27.6 41.1

Scientia Horticulturae 301 (2022) 111107

8409)
Ogura (CMS 61.2 78.8 18.4 45.7 60.4 1564.0 19.0 29.1 15.9 10.5 87.6 12.9 13.9 6.9 819.0 664.0 29.5 44.6
1944–309)
Ogura (CMS 69.0 84.6 20.4 56.4 64.2 1404.0 21.0 24.7 21.0 12.8 82.3 13.6 15.1 7.4 870.0 678.0 31.3 44.3
8410–22)
Ogura (CMS 66.8 84.0 19.4 53.4 64.4 1525.0 20.0 28.1 19.3 11.3 70.9 13.2 14.8 7.3 850.0 660.0 30.6 43.1
8410)
Group mean 65.3 82.8 18.8 52.2 64.2 1480.8 20.5 28.1 18.3 11.6 83.0 12.9 14.5 7.2 826.3 651.8 29.8 43.3
Mid-late maturity group
Ogura (CMS 69.4 88.8 20.0 70.8 70.6 1728.0 21.0 33.1 24.2 12.8 92.0 13.8 15.5 7.5 1057.0 782.0 38.1 39.5
8401)
(continued on next page)
S. Singh et al. Scientia Horticulturae 301 (2022) 111107

pure DNA of the sample.

28.2–49.9
The polymerase chain reaction (PCR) was carried out in 15 µl reac­

index (%)
Harvest
tion volumes with 50 ng genomic DNA, 1.0 U Taq DNA polymerase, 1.0

40.0

39.8
38.6
µM of each primer, 0.6 µl of 10 mM dNTP mix, and 1.5 µl of 10X PCR
buffer having 17.5 mM MgCl2 (Hi media Laboratories, Mumbai, India).

Marketable
curd yield

14.0–39.2
The mitochondrial DNA primers used in the study were taken from Shu

(t/ha)
et al. (2014) (Suppl. Table 2). Besides, 36 polymorphic simple sequence

39.2

38.7
21.3

1.50
Yield traits

repeats (SSR) markers from genomic regions of Brassica oleracea were

also used. All primers were synthesized by and from G Biosciences, Geno

282.0–820.0
curd weight weight (g)
Technology, Inc. St. Louis USA. Amplification conditions used for tar­
Marketable Net curd

820.0

801.0
462.7

36.58
geted genomic regions were: one cycle of 95 ◦ C for 5 min; 35 cycles of 95
◦
C for 30 s, a suitable annealing temperature (60 − 65 ◦ C) for 1 min, and
72 ◦ C for 1 min; and a final cycle of 72 ◦ C for 10 min. Amplified products
388–1090.0 were resolved on 3.0% (w/v) agarose gels with 0.5% TAE buffer, stained
1090.0

1073.5
591.1

45.05 with ethidium bromide, at a constant voltage of 60 V for 3 h in hori­

(g)

zontal gel electrophoresis. A 50 bp ladder was used as a reference for

4.0–7.8

amplicon size. The gel was visualized and photographed under UV light
length
(cm)
Core

0.34
7.8

7.7
6.0

in a gel documentation unit (BioRad, USA).

Curd traits

9.0–15.9

2.4. Statistical analysis

width
Curd

(cm)

15.9

15.7
12.1

0.90

Basic statistics from the mean values of two years’ data were calcu­
7.4–14.9
length

lated using the online OPTSAT software (http://14.139.232.166/

Root width Curd

(cm)

14.9

14.4
10.4

0.94

opstat/). Pearson correlation analysis between the observed traits was

performed by the OPTSAT software. The cluster analyses for floral traits,
70.9–128.3

agro-morphological traits, and data from molecular analysis of CMS and

122.5

107.3
Root traits

(cm)

95.9

8.81

their respective fertile maintainer lines were done using DARWIN 6.0
software. The data from genomic SSR markers and all mtDNA markers
8.6–13.4

were used for cluster analysis. Each DNA band generated was visually
length
Root

(cm)

13.4

13.1
11.1

1.12

scored as an independent locus and qualitative differences in band in­

tensities were not considered. The data matrix was analysed by un­
12.1–24.2

weighted pair group method with arithmetic (UPGMA) mean cluster

width

analyses using DARWIN 6.0 software.

(cm)

23.3

23.8
16.2

0.81
Leaf
Leaf traits

14.9–33.1

3. Results
Leaves
length
(cm)

29.2

31.2
22.8

1.12

3.1. Extent of variation in CMS lines for agro-morphological traits

15.2–24.3
leaves/

3.1.1. Vegetative traits

No. of

plant

23.0

22.0
20.3

Observations on vegetative traits showed wide variation in 24 CMS

1.8

lines of Indian cauliflower as presented in Table 2. Plant height was

778.0–1956.0

highest in Ogura (CMS 8498–10), Ogura(CMS 999–23) and Tour(CMS

Gross plant
weight (g)

394–98–4) compared to that of Ogura(CMS 131–41–5) and Ogura(CMS

1956.0

1842.0
1182.6

93.96

8441–5). The CMS lines of the early group were dwarf (48.7 ± 5.5 cm)
as compared to mid-late (64.7 ± 8.6 cm) groups, while CMS lines of the
44.8–72.6

mid-early group were intermediate of both (52.2 ± 4.5 cm). A similar

spread
Plant traits

pattern was observed for plant spread, number of leaves, leaf length, and
Plant

(cm)

72.6

71.6
57.9

4.15

leaf width. Among all the CMS lines, Ogura(CMS 48–41–5), Tour(CMS
38.2–70.8

394–41–5) and Ogura(CMS PD-12) had compact plant type with narrow
height

frame. Curd height was highest in CMS lines of the early maturity group,
Plant

(cm)

58.6

64.7
50.4

3.13

maximum in Ogura (CMS MD PD-12) (27.3 cm). Maximum variation for

number of leaves per plant was observed in early group CMS lines
17.1–27.3

(15.2–24.3), minimum in Ogura (CMS 175–8), and maximum in Ogura

height
Curd

(cm)

23.2

21.6
22.7

1.48

(CMS 131–23). Root length ranged from 8.6 to 12.9 cm in early,

10.5–12.8 cm in mid-early, and 12.8–13.4 cm in mid-late groups. Ogura
60.2–88.8
Maturity (days after

maturity

(CMS 131–23) CMS line of the early group had highest root width
Days to
transplanting)

(128.3 cm) followed by Ogura(CMS MD-202) line of the mid-late group.

curd

85.0

86.9
74.8

2.49
45.8–69.4

3.1.2. Curding and yield-related traits

initiation
Days to

The performance of the tested 24 CMS lines for curd and yield traits
curd

66.8

68.1
59.2

2.59
Table 2 (continued )

is given in Table 2. These CMS lines were developed in different maturity

Ogura (CMS MD

groups viz., extra-early (3), early (15), mid-early (4) and mid-late (2).
Overall mean

Thus, they have specific temperature requirements for curd initiation

Group mean

CD (at 5%)
CMS lines

and development as 25–30 ◦ C, 20–25 ◦ C, 16–20 ◦ C and 12–16 ◦ C,

202)

Range

respectively. Three CMS lines, namely Ogura (CMS 175–8), Ogura (CMS
MD-98–4) and Ogura(CMS 999–98–4) were earliest in curd initiation

5
S. Singh et al.
Fig. 2. a-x. Floral traits of different CMS systems and changes after introgression in Indian cauliflower lines.
and maturity, hence formed curds at relatively higher temperature
range. Cream white was the predominant curd color in early group CMS
lines, while white was dominant in mid-early and mid-late groups
(Suppl. Table 1). The CMS lines CMS 131–23, CMS 999–23 and CMS
MDR-PD-12 had white compact curds. The CMS lines showed a wide
range for days to curd initiation (45.8–66.8 days after transplanting) and
curd maturity (60.2–84.0 days after transplanting).
Early group CMS lines had significantly small size curds compared to
that of mid-early and mid-late groups. Curd length was more than 12 cm
in 6 CMS lines and less than 10 cm in 14 CMS lines. Similarly, curd width
6
Scientia Horticulturae 301 (2022) 111107
was more than 14 cm in 5 CMS lines and was less than 10 cm in 7 CMS
lines. Seven CMS lines had a very small core length of less than 5 cm. The
core length to curd length ratio was in the range of 0.43 to 0.72 with
minimum in the mid-late group. Gross plant weight (GPW) ranged from
778.0 g to 1956.0 g with minimum in Ogura (CMS 999–41–5) and
maximum in Ogura(CMS-202). Among groups, early group CMS lines
had minimum average GPW (1018.1 ± 182.0 g), while it was the highest
in mid-late group CMS lines (1842.0 ± 161.2 g). Similar observations
were also recorded for marketable curd weight (MCW) and net curd
weight (NCW). Marketable curd yield (MCY) ranged from 13.97 to 39.24
S. Singh et al.
Table 3
Floral traits of CMS lines of Indian cauliflower in respective maturity groups.
t/ha. The CMS lines of the mid-late group (38.65±0.84 t/ha) had
significantly higher MCY than mid-early (29.75±1.63 t/ha) and early
group (17.01±2.31 t/ha). The high yielding CMS lines viz., Ogura (CMS
MD PD-12), Ogura(CMS 999–23) and Ogura(CMS 131–23) were in the
early-group; Ogura(CMS-8410–22) and Ogura(CMS 8410) in the mid-
early group and Ogura(MD-202) and Ogura(CMS 8401) in the mid-late
group.
3.1.3. Floral traits
Floral traits of different CMS systems are shown in Fig. 2a-x. The
extent of variation was quite high in 24 CMS lines for 18 important traits
of floral, petal, sepal, bud, stamen & anther, stigma, stalk and nectary
(Table 3). Flower length ranged from 0.54 to 1.72 cm with lowest in
Ogura (CMS 394–41–5) and highest in Ogura(CMS MD-PD-12). Flower
diameter was maximum (1.35 cm) in Ogura (CMS 131–23) and Ogura
(CMS 8498–10), while it was minimum in Ogura(CMS 394–41–5) (0.34
cm). Petal length ranged from 0.22 to 1.61 cm, however it was more
than 1.2 cm in 7 CMS lines and less than 1.0 cm in 5 CMS lines.
Maximum bud length (0.67 cm) was observed in CMS 48–98–4 and CMS
MD-PD-12. Bud diameter, however, was highest in Ogura (CMS-
999–98–4) and Ogura(CMS-MD-98–4). CMS lines were also variable for
sepal width and length. Thirteen CMS lines had petal width greater than
0.50 cm, while there was only 0.01 cm width observed in line Tour (CMS
394–41–5). Flower stalk length ranged from 0.58 to 1.29 cm. The long
and short stamen length was maximum in Ogura (CMS 8498–10). The
longest filament of long and short stamens were observed in Ogura (CMS
8498–10) and Tour (CMS 394–98–4), respectively. Style length of more
than 0.80 mm was recorded in 16 CMS lines. Size of nectaries in CMS
lines and their maintainers are shown in Suppl. Fig. 1. Nectar volume
produced by ten CMS lines was more than 3.0 µl/5 flowers, which was
highest in Ogura (CMS 131–23) also comprised the largest nectaries.
Seed yield showed wide variation in the CMS lines which ranged from
1.71 to 19.15 g.
3.1.4. Clustering of CMS lines based on agro-morphological traits
Grouping of genotypes for 19 agronomic traits (vegetative and
commercial) through the UPGMA method revealed two major clusters
7
Scientia Horticulturae 301 (2022) 111107
and four sub-clusters (Fig. 3a). Of these, one major cluster represented
CMS lines of the early maturity group, while the other mainly comprised
mid-early and mid-late group CMS lines. This grouping, although, could
differentiate CMS lines belonging to different maturity groups, but it
could not categorize them on the basis of the source of CMS cytoplasm.
This is clearly evident from the clustering of Ogura-based CMS line Ogura
(CMS 999–98–4) and Tour sterile cytoplasm-based CMS line Tour(CMS
394–41–5) in the same cluster.
Like the agro-morphological traits, the neighbor joining clustering
was performed based on 19 floral and seed traits (Fig. 3b). The extent of
variability among the CMS lines was also high based on floral traits.
Based on neighbor joining cluster analysis, 24 CMS lines were grouped
into 2 major clusters with only line Tour(CMS 394–41–5) in cluster I, and
the remaining 23 CMS lines in cluster II in two sub-clusters, in which one
sub-cluster had 13 CMS lines including Ogura, one each of the Tour and
Can(napus) CMS systems and the other sub-cluster had 10 CMS lines all
of which belonged to Ogura CMS system.
3.1.5. Correlation in floral traits
The correlation analysis in 17 floral traits and seed yield (g/plant)
observed from 24 CMS lines is given in Table 4. The development of CMS
systems from diverse sources in fertile genotypes of Indian cauliflower
affected most of the floral traits which exhibited a strong correlation
between them. Anther length and nectary size were found to be posi­
tively correlated with flower length, however, flower diameter had a
negative but non-significant correlation with anther length and nectary
length. Nectar volume is significantly correlated with nectary length and
nectar width; however, nectar volume had no-significant correlation
with seed yield. Short stamen length (cm) is significantly correlated with
nectary width and nectar volume.
3.2. Molecular analysis
Out of 11 mtDNA markers used, only one BnTR1 could distinguish all
the three CMS systems as Can (≈267 bp), Tour (≈200 bp) and Ogura
(≈237 bp) (Suppl. Fig. 2). All the fertile maintainers had only one size
amplicon Ogura ≈237 bp, thus indicating their immediate use for
S. Singh et al.
distinguishing fertile from Can and Tour-based CMS lines and among
them also. Clustering of CMS lines and fertile maintainer lines on the
basis of data from genomic and mitochondrial DNA markers clearly
distinguished CMS and fertile lines (Fig. 4). The fertile maintainer lines
were grouped in a separate cluster. Interestingly, the CMS lines of Mid-
early and Mid-late groups were grouped in a single sub-cluster, but it
had CMS lines of the early group also.
4. Discussion
Cytoplasmic male sterility (CMS) has emerged as a better option over
self-incompatibility (SI) system in hybrid breeding in Brassica vegetable
crops. It is a robust and stable mechanism across genetic backgrounds
and geographical regions. However, the impact of allo-cytoplasm on
agro-morphological traits is almost unnoticeable after attaining BC5 to
BC8 depending upon intermediate nuclear backgrounds. Although,
Ogura sterile cytoplasm was identified by Ogura in 1968 in unknown
wild Japanese radish, but it was introgressed in vegetable Brassicas and
refined for low-temperature chlorosis in the years 1970 and 1980,
respectively (Bannerot et al., 1974; Pelletier et al., 1983; Walters et al.,
1992; Sigareva and Earle, 1997). In the Indian cauliflower, however, its
introgression was initiated for the first time during later part of 1990s by
the use of two introduced CMS lines in kale (B. oleracea var. acephala)
and broccoli (B. oleracea var. italica). Among these, kale background
CMS produced flower deformities continuously up to BC6–7 while CMS
via broccoli had proper flower and seed setting (Sharma and Vinod,
2001), hence, the later one was continued which resulted in five stable
CMS lines, namely MS-01 (mid-late), MS-04, MS-05 (early), MS-09 and
MS-10 (mid-early group) (Sharma et al., 2005). Interestingly, the
earliest crossing/backcrossing were attempted at IARI Regional Station,
Katrain, because kale and snowball cauliflower did not flower in Delhi
condition due to lack of vernalization conditions which is essential for
this group for transition from vegetative to reproductive or flowering
phase (Wurr et al., 1993). After BC2–3 generations, the backcrosses were
attempted in Delhi conditions since annual habit or no chill character in
Brassica vegetables is dominant in nature. However, the recovery of the
genomic region for curding trait and removal of curding complexities (i.
e. riciness, bracting, fuzziness etc.) was quite tricky and time taking due
to their unknown genetics and environmental influence. The physio­
logical defects were prominent in earlier backcross generations which
later got overcome by targeted selection in advanced progenies of CMS
conversion. Handling such traits was essential because the background
of immediate CMS source broccoli had ‘head’ as edible portion, which
comprised green unopened flower buds while recipient cauliflower had
‘curd’ a pre-floral apical meristem as edible portion, which differ both at
physiological and molecular levels. Both are closely related for their
phylogeny and curding trait in cauliflower occurred as a result of
recessive mutations in two major genes namely BoAP1 and BoCAL1 in
Calabrese (broccoli). However, the role of another minor gene(s) can’t
be ruled out (King and Smith, 2000).
Since cauliflower is a thermo-sensitive crop, therefore temperature
along with genetic factors directly regulates curd induction and devel­
opment as well as reproductive phases (bolting and flowering). Curd
formation and development in cauliflower takes place at a temperature
range of 5 to 27 ◦ C and from investigations quantitative trait loci (QTLs)
were identified for curding related traits in response to temperature
(Thorwarth et al., 2018). The Indian cauliflower was evolved from Eu­
ropean types during the past 200 years initially by selections for curding
and flowering in north Indian plains predominantly in the ‘Cornish’
group and later in the cross progenies with genotypes of other groups
(Swarup and Chatterjee, 1972). Indian cauliflower is a novel group as it
forms curd at higher temperature (≥ 20 ◦ C) compared to that of snow­
ball group (10–16 ◦ C). The Indian cauliflower is further grouped into
early (20–30 ◦ C), mid-early (16–20 ◦ C), and mid-late (12–16 ◦ C)
maturity groups (Singh and Sharma, 2001). New genotypes in the early
maturity group have been found forming curds even at a mean
8
Scientia Horticulturae 301 (2022) 111107
temperature of 30 ◦ C. However, higher temperature adversely affects
curd proliferation and weight gain due to transpirational loss of pho­
tosynthates. Hence, early group genotypes produce small curds followed
by those from mid-early and mid-late groups (Swarup and Chattarjee,
1972; Singh and Sharma, 2001), which were also apparent in CMS lines
of different maturity groups in the present study. Similar observed were
also reported by Singh et al. (2022) in seven CMS lines from different
maturity groups of Indian cauliflower.
The CMS system showed relative delay in curd initiation and curd
maturity. However, a relative increase was noticed in leaf number, gross
plant weight and curd weight and core length. The CMS lines, however,
showed variable pattern (decrease/increase) for curd height, plant size
(high, spread), leaf length and weight, curd length, core length/curd
length ratio and harvest index. The variable pattern was primarily
appeared to be due to four factors, such as (i) genotypic variation/
relatedness in maintainer lines, (ii) diverse CMS sources and immediate
donors, (iii) difference in maturity groups of the CMS lines, and (iv)
interaction among first two factors. Interaction response of unknown
genetic factors might also contribute to the variable pattern of change.
In the early-group, the highest MCY in CMS lines, namely Ogura(CMS
MD PD-12), Ogura(CMS 99–23), and Ogura(CMS 131–23) was attributed
to lower temperature requirement for curd induction, which gave an
extended period for vegetative growth and increased number and size of
leaves due to less transpiration leading to accumulation of more
photosynthates.
The CMS lines showed wide variation for all the observed traits
suggesting diverse nature of maintainers and CMS sources. The main­
tainer lines were from three prominent groups of Indian cauliflower such
as early (18), mid-early (4), and mid-late (2). In the early-group, Tour
(CMS 394–41–5) had Tour cytoplasm which showed agronomic traits
parity with the maintainer DC 41–5. The line DC 41–5 was also trans­
formed with the Ogura system showing complete recovery of the nuclear
genome. Although, our observations were novel in terms of different
CMS systems in typical Indian cauliflower groups especially, but still the
findings are in line with those of earlier report (Dey et al.,2017), which
mentioned significant variation in Ogura CMS lines of snowball cauli­
flower. Further, most of the observed traits were under major gene(s)
control; hence, their recovery in CMS lines through backcrossing was
attained to maximum acceptable levels. However, the influence of cy­
toplasms [i.e. refined Ogura, Can, and Tour) and remnants of the nuclear
genomic region of sterile cytoplasm sources i.e. radish-kale-broccoli
system for refined Ogura, B. napus for Can, and B. tournefortii for Tour
were evident mainly for Can and Tour in BC4–8 generations.
Many proteins that accumulate in the chloroplast are encoded by the
nuclear genome, and the development transition from proplastid to
chloroplast is regulated by nuclear genes. Wu et al. (2019) reported that
the chloroplast-derived sequences are conserved among
Brassicaceae-family plants. Hence, the phylogenetic positions of ge­
nomes of different CMS sources were found close to those of wild rela­
tives. Sterile cytoplasms carry chloroplast from the wild CMS sources to
new alloplasmic combinations in B. oleracea nuclear background which
create new phenotype sometimes different from the fertile maintainer
lines. Here, in the present study, we observed such phenotype with Tour
sterile cytoplasm and cauliflower nuclear genome having complete
deformity in petals and style characters.
During CMS transformation, the alien cytoplasmic organelles
(mitochondria and chloroplasts) from CMS source gets into a new nu­
clear background. Fundamentally, these organelles provide energy and
carbon sources to cells and play role in metabolic functions such as
amino acid metabolism, hormone biosynthesis, and cellular signaling
(Berkowitz et al., 2016; van Dingenen et al. 2016). Since their small
genome largely depends on nuclear background for maintenance and
expression, therefore intensive interaction between the nucleus and
these organelles is obvious for plant growth and development. The dif­
ference in agro-morphological traits between CMS lines and their fertile
maintainers could be due to alien sterile cytoplasms (Ogura, Can, and
S. Singh et al.
Fig. 3. a-b. Grouping of the 24 CMS lines of Indian cauliflower based on 19 agro-morphological traits (a) and on 19 floral and seed traits (b) using neighbor joining
hierarchical cluster UPGMA method.
Tour) in cauliflower nuclear background. Further, during introgression,
the segments of the nuclear genome from alien sources and also im­
mediate CMS source i.e. broccoli, and then snowball cauliflower remain
in CMS lines used in the present study.
The sterile cytoplasm significantly reduced all the major floral traits
such as flower size, petal size, bud size, sepal size, stamen length, and
anther and stigma length. But, there was a variable pattern for flower
stalk length, nectary size (length, width). All the CMS lines had less
nectar volume than their fertile maintainers. Cauliflower is a honey bee-
pollinated crop and reduced flower size and nectar volume directly
affect honey bee visits To CMS lines than fertile counterparts which
manifests in reduced seed yield. Since it is a robust and stable genetic
mechanism, the entire seeds from CMS plants represent hybrid seeds
only and there is no chance of self or sib seeds as observed in self-
incompatible (SI) system.
The degree of bud abortion, floral organs shape, degree of flower
opening, nectar production, bee visits, and hybrid seed yield differed in
CMS lines with the same recurrent parent. When the CMS source was the
same and the recurrent parents were different, the degree of bud abor­
tion was different among the CMS lines, while the flowering charac­
teristics were similar (Shu et al., 2019). However, the flower organs of
the CMS lines obtained, often, show serious morphological abnormal­
ities because of incompatibility between nucleus and cytoplasm. This
resorted to degeneration or deformation of flower organs, resulting in
poor seed-setting reducing chances of obtaining good CMS lines thereby.
In the present study, we reported the extent of reduction in floral traits
viz., flower length, petal width, petal length, sepal size, stamen length,
filament length etc. These morphological variations of flower organs are
consistent with the findings in B. oleracea (Zhang et al. 2010; Wang
et al., 2012).
The CMS lines of the early maturity group had bigger size flowers
than mid-groups due to maintainer genotypes and climatic factors.
Flowering in the mid-group occurs during January end to February
months when the ambient temperature remains relatively higher than
months of November - December, the main period of flowering in the
early group. Cytoplasmic effect on flower size was clearly evident for
most of the floral traits. Of the CMS lines, Tour (CMS 394–41–5) with
Tour sterile cytoplasm produced full-size sepals witht 2–3 rudimentary
petals only even at BC9 generation, indicating its strong impact on petal
development. This CMS also had a peculiar gripping phenomenon or
9
Scientia Horticulturae 301 (2022) 111107
‘sepal gripped style’ which forces stigma to curve drastically and reduces
chances of pollinations (Fig. 2x). Although, honey bee visits were
noticed to be adequate which could be attributed to the pale green color
of sepals, the proper opening of buds at a later stage, and well-developed
nectaries. The ‘gripping’ of stigmatic surface, however, is a major
constraint for insect-pollination which adversely affect seed yield. This
phenomenon was not specific to a particular interaction i.e. Tour(CMS-
394–41–5) (CMS line) and DC 41–5 (fertile maintainer), but was also
noticed in flowering individuals of the F1 crosses between Tour(CMS
394–41–5) and 41 different fertile genotypes (13 in 2018–19; 28 in
2019–20) of cauliflower. The ‘gripping phenomenon’ also persisted in
BC2–3 generations with three different genotypes, namely DC 351aa, DC
67, Pusa Deepali (data not presented). The Tour sterile cytoplasm was
reported to be associated with abnormalities in floral parts in B. juncea
(Rawat and Anand 1979), B. napus (Stiewe and Robbelen 1994). Cardi
and Earle (1997) reported that CMS phenotype with abnormal flower
morphologies having somewhat bent pistils in somatic hybrids of
B. napus (+) B. tournefortii could be due to high content of DNA, nuclear
incompatibilities, or mitochondrial-nuclear interactions (Liu et al.,
1995). Although, Cardi and Earle (1997) observed some cybrids with
good flower morphology, but we did not find a single plant or flower
having proper floral structure despite 6–8 backcrosses in cauliflower
background and even in a large number of different nuclear back­
grounds at F1 hybrid stage, suggesting occurrence of certain unknown
mechanism or interactions.
This indicates that the Tour cytoplasm had a strong penalty on flower
traits, however, it was also observed in two other CMS systems inves­
tigated i.e. Ogura and Can(napus). The impact largely depends upon the
type of sterile cytoplasm and recipients’ nuclear genetic background.
Nectaries are essential features in flowers to attract bees and their
degree of development determines the ability of plants to secrete nectar,
affecting thereby the number of bees visiting flowers and hence the seed
yield. Therefore, the potential of nectaries to produce nectar and their
ability to attract bees are important indicators for assessing the useful­
ness of CMS lines. Shu et al. (2019) conducted a comprehensive evalu­
ation of the nectar-secreting capacity and the bee-attracting ability of 13
CMS lines of broccoli in the nuclear genetic backgrounds of 8554, 93,
213 and 93,219 and reported the positive correlation between seeds
yield per plant and the petal length, the corolla diameter, the short
stamens length, the ratio of the short stamen length: style length, the
 S. Singh et al.
Table 4
Correlation in floral traits in CMS lines of Indian cauliflower.
Characters Flower Flower Stalk Petal Petal Sepal Sepal Bud Bud Long Short Long Short Anther Stigma Nectary Nectary Nectar
length diameter length length width length width length diameter stamen stamen filament filament length length length width vol. (µl/5
(cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) length (cm) length length (cm) length (cm) (cm) (mm) (mm) flowers)
(cm) (cm)

Flower 0.621**
diameter
(cm)
Stalk length 0.232NS − 0.001NS
(cm)
Petal length 0.965** 0.640** 0.118NS
(cm)
Petal width 0.802** 0.660** 0.113NS 0.868**
(cm)
Sepal length 0.526** 0.439* 0.577** 0.475* 0.523**
(cm)
Sepal width 0.300NS 0.042NS 0.173NS 0.250NS 0.140NS 0.024NS
(cm)
Bud length 0.439* 0.288NS 0.704** 0.354NS 0.407* 0.897** 0.029NS
(cm)
Bud 0.315NS 0.107NS 0.409* 0.215NS 0.201NS 0.509* 0.334NS 0.559**
diameter
(cm)
Long stamen 0.426* 0.735** 0.081NS 0.418* 0.498* 0.439* 0.201NS 0.292NS − 0.016NS
length
(cm)
Short 0.420* 0.626** 0.202NS 0.430* 0.483* 0.518** 0.161NS 0.398NS 0.101NS 0.878**
10

stamen
length
(cm)
Long 0.285NS 0.552** − 0.012NS 0.376NS 0.397NS 0.345NS 0.070NS 0.153NS − 0.127NS 0.783** 0.868**
filament
length
(cm)
Short 0.562** 0.815** 0.129NS 0.631** 0.683** 0.461* 0.024NS 0.307NS 0.133NS 0.750** 0.799** 0.760**
filament
length
(cm)
Anther 0.164NS 0.133NS 0.180NS 0.078NS 0.206NS 0.294NS 0.176NS 0.346NS 0.179NS 0.586** 0.523** 0.237NS 0.223NS
length
(cm)
Stigma 0.503* 0.603** 0.295NS 0.533** 0.636** 0.620** 0.110NS 0.409* 0.083NS 0.572** 0.531** 0.450* 0.677** 0.244NS
length
(cm)
Nectary 0.364NS − 0.048NS 0.374NS 0.348NS 0.369NS 0.466* 0.161NS 0.589** 0.561** − 0.017NS 0.122NS − 0.059NS 0.082NS 0.302NS 0.223NS

Scientia Horticulturae 301 (2022) 111107

length
(mm)
Nectary − 0.011NS 0.012NS 0.539** − 0.129NS − 0.025NS 0.389NS 0.077NS 0.534** 0.305NS 0.145NS 0.116NS − 0.238NS 0.015NS 0.420* 0.290NS 0.397NS
width
(mm)
Nectar vol. 0.435* 0.474* 0.337NS 0.358NS 0.321NS 0.505* 0.234NS 0.360NS 0.262NS 0.541** 0.455* 0.275NS 0.344NS 0.394NS 0.378NS 0.143NS 0.297NS
(µl/5
flowers)
Seed yield 0.584** 0.681** − 0.336NS 0.648** 0.641** 0.047NS 0.148NS − 0.194NS − 0.285NS 0.526** 0.344NS 0.400NS 0.514* 0.038NS 0.442* − 0.187NS − 0.360NS 0.400NS
(g/plant)

*, ** significant at 1% and 5%, respectively. NS = non-significant.

S. Singh et al.
Fig. 4. neighbor joining similarity tree construction in CMS and their fertile maintainer lines of Indian cauliflower using mtDNA markers and genomic SSRs. Red line
indicates for maintainers.
style length, the long stamen length, the ratio of long stamen length:
style length, and the petal color and size.
Morphometric traits are rarely effective in distinguishing different
CMS systems, however, molecular markers were found to be potent for
this purpose. For this, mitochondrial genome-specific markers are useful
because the CMS system evolved due to rearrangements, novel chimeric
open reading frame (ORF) created by shuffling of endogenous sequences
and mutations in this genome. Since the mitochondrial genome size of
Ogura type was reported to be 258.42 Kb and normal mitochondrial
genome of 244.03 Kb with 80% synteny (Tanaka et al., 2012). The
differences in mitochondrial genome regions due to mutations serve as a
useful option to detect CMS systems since agro-morphological traits do
not serve the entire purpose. To explore the molecular variation in CMS
detection, the molecular markers designed from mtDNA sequences were
found to be promising. We also identified BnTR1 as a prominent marker
for the detection of Tour and Can(napus) CMS systems.
5. Conclusion
The cytoplasmic male sterility (CMS) has imparted great opportunity
for quality and affordable hybrid seed production in cauliflower, cab­
bage, broccoli, carrot, etc. In Indian cauliflower, this is the first detailed
report on development of 24 new CMS lines using three diverse sterile
cytoplasms namely Ogura, Can and Tour. These lines showed wide
variation in agro-morphological and floral traits. Introgression of alien
cytoplasm had significant influence on floral traits which was maximum
in case of Tour cytoplasm. However, the cytoplasms could not affect
agro-morphological traits of the introgressed CMS lines of Indian
cauliflower. Except Tour(CMS 394–41–5), all the CMS lines showed
proper flowering and seed setting, thus have full potential in hybrid seed
production. However, Can system is a newly introgressed cytoplasm in
cauliflower and its commercial suitability and stability in different nu­
clear background need to be investigated further. Three CMS lines Ogura
11
Scientia Horticulturae 301 (2022) 111107
(CMS 175–8), Ogura(CMS MD-98–4)) and Ogura(CMS 999–98–4) were
promising for breeding hybrids for high temperature condition (25–30
◦
C). Ogura(CMS MD PD-12) Ogura(CMS 999 − 23) and Ogura(CMS 131
− 23) of early–group; Ogura(CMS 8410–22) and Ogura(CMS8410) of mid
early group and Ogura(MD-202) and Ogura(CMS 8401) of mid- late
group were promising for yield attributes. Thus, the newly developed
CMS lines will offer great opportunity for breeding heat tolerant and
high yielding hybrids in tropical cauliflower.
Credit author statement
Shrawan Singh: Conceptualization, Methodology, Data curation,
Original draft preparation, Software.
Pritam Kalia: Conceptualization, Methodology, reviewing and
editing.
Rahul Kumar Meena: Field data and laboratory analysis.
Brij Bihari Sharma: Helped in data analysis and editing manuscript.
B R Parihar: Helped in field trials.
Availability of data and materials
Authors have data and materials which can be shared as per existing
guidelines in the country.
Code availability
Codes given for the materials are available with authors.
Authors’ contribution
SS and PK conceptualized and planned the research. RKM and BRP
assisted in field and laboratory analyses. SS monitored experiment,
analysed agro-morphological results and prepared draft manuscript.
S. Singh et al.
BBS helped in preparation of draft manuscript. PK edited the manuscript
and PK and SS finalized it. All authors read and approve the manuscript.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgements
We are thankful to Director, ICAR-IARI, New Delhi for facilities and
ICAR for funding the CRP Hybrid Technology Project (Cauliflower,
Code: CRPHT-12-142-F). We thank the Head, Division of Vegetable
Science for overall facilities and Head, Division of Floriculture &
Landscaping for providing the Zeiss stereo microscope. We thank Dr. S.
R. Sharma, Principal Scientist (Rtd.), IARI for initiating CMS develop­
ment in Indian cauliflower. We acknowledge Dr. Varalaxmi, ICAR-
Indian Institute of Horticultural Research (IIHR), Bengaloru and Dr. S.
R. Bhatt, National Institute of Plant Biotechnology (NIPB), New Delhi for
sharing the Tour and Can(napus) introgressed advance BC lines,
respectively.
Funding
All relevant funding for the research has been acknowledged.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.scienta.2022.111107.
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