Agricultural
Communication
Biosci. Biotech. Res. Comm. 11(3): 481-485 (2018)
Studies on days to calli appearance in Ethiopian
mustard,
Brassica carinata
Shitole Ajit Mohanrao* and Vedna Kumari
Department of Crop Improvement, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, India, 176062
ABSTRACT
The effects of seven genotypes and their cross combinations, two basal media i.e., B5 and MS media, two different
sucrose concentrations i.e., 3% and 4% sucrose and three combinations of hormones viz. HM
1
, HM
2
and HM
3
and
their interactions on days to calli appearance in Brassica carinata were analyzed by using CPCS software. Analysis
of variance revealed that, out of four factors, only genotypes had signi cant effect on days to calli appearance. Nine
out of eleven interactions viz., genotypes x hormones, genotypes x media, hormones x media, genotypes x hormones
x media, hormones x sucrose, genotypes x hormones x sucrose, genotypes x media x sucrose, hormones x media x
sucrose and genotypes x hormones x media x sucrose showed signi cant effect on days to calli appearance.
KEY WORDS: B
5
, DAYS TO CALLI APPEARANCE, HORMONES, MS, SUCROSE
481
ARTICLE INFORMATION:
*Corresponding Author: mr.ajitshitole@gmail.com
Received 20
th
July, 2018
Accepted after revision 25
th
Sep, 2018
BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 CODEN: USA BBRCBA
Thomson Reuters ISI ESC / Clarivate Analytics USA and
Crossref Indexed Journal
NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196
© A Society of Science and Nature Publication, Bhopal India
2018. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
DOI: 10.21786/bbrc/11.3/18
INTRODUCTION
Oilseed crops are the backbone of Indian agricultural
economy and occupy an important position in daily
diet, being a rich source of fats and vitamins. India is
the second largest rapeseed-mustard growing country
and accounts for 21.7% area in the world after China.
Among oilseeds, rapeseed-mustard is the second most
important oilseed crop of the country after groundnut
and plays a signi cant role in Indian oil economy by
contributing about 28.6% to the total oilseed production
(Shekhawat et al., 2014). Rapeseed-mustard is the third
important oilseed crop in the world after soybean (Gly-
cine max) and palm (Elaeis guineensis Jacq.). The crop
occupies an area of 33.58 million ha with a total annual
production of 67.76 million tonnes and productivity
2018 kg/ha. In production, India ranks third after China
(22.9%) and Canada (19.7%). The global production of
rapeseed-mustard oil is around 12-14 million tonnes. In
India, the crop occupies an area of 6.50 million ha with a
total production of 8.02 million tonnes and productivity
of 1262 kg/ha (Priyamedha et al., 2017).
482 STUDIES ON DAYS TO CALLI APPEARANCE IN ETHIOPIAN MUSTARD BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Shitole Ajit Mohanrao and Vedna Kumari
Over the last decades, researchers have made great
efforts in developing biotechnology methods to facili-
tate the breeding of Brassicas. Research studies indi-
cated that the modern biotechnology will have a major
impact in two areas. Firstly, it provides a new range of
techniques enabling the ef cient selection of favour-
able variants in plant breeding programmes. Secondly,
it provides the opportunity to improve germplasm by
increasing its diversity beyond conventional genetic
limitations. Due to the relative ease of genetic transfor-
mation, Brassica oilseed crops have been amongst the
rst subject to study the full range of modern biotech-
nology methods (Abraha et al., 2008).
Conventional methods for breeding crop plants require
more than six to seven years of continuous efforts to get
true breeding lines after following hybridization approach,
a time consuming process (Morrison and Evans, 1988).
Hence, biotechnological tools including anther culture,
hold a great promise in accelerating the pace of breeding
programme (Guha and Maheshwari, 1964). In vitro tech-
nique of anther culture helps to achieve homozygosity
very quickly (Snape, 1989). Anther culture of potential F
1
generation genotypes can be used to facilitate regenera-
tion of stable recombinant inbreds in one to two years
thereby saving time and resources for their further use
directly as commercial cultivars and/or in structural and
functional genomics. The object of this study was to
investigate the response of different genotypes and their
cross combinations for days to calli appearance.
MATERIALS AND METHODS
The anther culture work was carried out in the Molecular
Cytogenetics and Tissue culture Laboratory of Depart-
ment of Crop Improvement, CSK HPKV, Palampur dur-
ing Rabi 2010-11. The material used and methodology
adopted to achieve the objectives of the investigation are
given below.The material used for anther culture studies
comprised of four elite genotypes and their three cross
combinations (Table 1). Suf cient numbers of plants of
aforementioned four genotypes and their cross combi-
nations were raised in the pots. In order to have avail-
ability of anthers over a long period of time, plants were
raised in ve lots at an interval of 15 days each. For
anther culture, orets from
plants were clipped off when
the size of bud was about 2-4 mm. The bud size was
earlier established on the basis of presence of majority
of the microspores at late uninuclate to early binucleate
stage as studied by squashing of anthers in a drop of
1% acetocarmine. The orets of appropriate size were
collected in 50 ml test tubes containing distilled water.
The orets collected at aforementioned stages were
treated with 70% ethanol for 10-15 seconds under asep-
tic conditions in a laminar air ow chamber. The o-
rets were then surface sterilized with 0.1% HgCl
2
for 3-5
minutes with intermittent shaking followed by three
washings with sterile distilled water. Florets were blot
dried and opened under aseptic conditions with the
help of sterile forceps and the six anthers were clipped
off from each oret without damaging the anther wall.
About 60 anthers were cultured in each pre-sterilized
petri plate containing about 25 ml of culture medium.
Two basal media viz. B
5
(Gamborg et al. 1968) and MS
(Murashige and Skoog 1962) were used for callus induc-
tion. Each of these medium was supplemented with two
different sucrose concentrations i.e., 3% and 4% sucrose
and each of these sucrose concentrated media was also
supplemented with three combinations of hormones viz.
HM
1
, HM
2
and HM
3
(Table 2). All the media were sup-
plemented with 0.8% agar.The experiments on different
callus induction media were replicated thrice involving
different media and plant growth hormones. Anthers of
all four genotypes and their crosses were plated in a rep-
licated fashion. If there was any contamination, replat-
ing of the particular treatment was done to complete the
experiment under uniform conditions. All the cultured
plates were sealed with para lm wax and kept under
dark at 25 ± 1°C until calli were developed. The Days to
calli appearance was calculated as follows: Days to calli
appearance = Number of days taken for calli appearance
Table 1. List of genotypes and their cross combinations under anther culture study
Sr. No Genotype Parentage
1 Jayanti Developed through irradiation from the parent variety HC-1
2 P-18
Advanced generation mutant obtained through treatment of
Jayanti seeds with 0.3% EMS (Pre-Soaked)
3 P-51
Advanced generation mutant obtained through treatment of
Jayanti seeds with 0.3% EMS (Pre-Soaked)
4P
(2)2
Advanced generation mutant obtained through treatment of
Jayanti seeds with 90 kR dose of gamma radiations
5 Jayanti X P-18 -
6 Jayanti X P-51 -
7 Jayanti X P
(2)2
-
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STUDIES ON DAYS TO CALLI APPEARANCE IN ETHIOPIAN MUSTARD 483
Shitole Ajit Mohanrao and Vedna Kumari
Table 2. Different media, hormones and sucrose concentration used for
calli index
Medium
Sucrose
Conc.
Hormone
Designation Name and Concentration
B
5
3% HM
1
NAA (1.0 mg/l)
B
5
3% HM
2
BAP (2.0 mg/l) + NAA (2.0 mg/l)
B
5
3% HM
3
2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
B
5
4% HM
1
NAA (1.0 mg/l)
B
5
4% HM
2
BAP (2.0 mg/l) + NAA (2.0 mg/l)
B
5
4% HM
3
2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
MS 3% HM
1
NAA (1.0 mg/l)
MS 3% HM
2
BAP (2.0 mg/l) + NAA (2.0 mg/l)
MS 3% HM
3
2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
MS 4% HM
1
NAA (1.0 mg/l)
MS 4% HM
2
BAP (2.0 mg/l) + NAA (2.0 mg/l)
MS 4% HM
3
2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
Table 3. ANOVA for days to calli appearance in different genotypes of Brassica carinata and
their hybrids involving different media, hormones and sucrose concentration
Source of variation df
Mean
Squares
CD
(5%)
CV
(%)
Genotypes 6 28.23** 0.47
11.00
Hormones 2 1.38 NS
Genotypes x Hormones 12 5.17** 0.82
Media 1 0.25 NS
Genotypes x Media 6 4.67** 0.67
Hormones x Media 2 21.60** 0.44
Genotypes x Hormones x Media 12 5.04** 1.16
Sucrose 1 0.25 NS
Genotypes x Sucrose 6 0.93 NS
Hormones x Sucrose 2 63.65** 0.44
Genotypes x Hormones x Sucrose 12 5.21** 1.16
Media x Sucrose 1 1.02 NS
Genotypes x Media x Sucrose 6 12.06** 0.94
Hormones x Media x Sucrose 2 113.34** 0.62
Genotypes x Hormones x Media x Sucrose 12 7.19** 1.64
Error 168 1.02
**Signi cant at P ≤ 0.01
from the day of culturing of anthers. Data on days to
calli appearance were analyzed in Factorial Completely
Randomized Design (CRD) to obtain the effect of various
treatments and their interactions using statistical CPCS
software.
RESULTS AND DISCUSSION
Analysis of variance for days to calli appearance involv-
ing different parameters is presented in Table 3. Out of
four factors, only genotypes had signi cant effect on
Hadi M. Hamaza AL-Mayali and Sadiya Aziz Anah
484 STUDIES ON DAYS TO CALLI APPEARANCE IN ETHIOPIAN MUSTARD BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Table 4.1. Effects of media and genotypes on days to calli appearance
Media
Genotypes
Jayanti P
(2)2
P-51 P-18 Jayanti
x P
(2)2
Jayanti x
P-51
Jayanti
X P-18
Mean CD
(P≤0.05)
MS 10.50 8.39 9.39 8.44 8.39 9.50 8.44 9.01 NS
(Media)
B
5
11.44 11.06 8.56 8.83 8.44 8.89 8.83 9.44
Mean 10.97 9.72 8.97 8.64 8.42 9.19 8.64
CD (P≤0.05) = 0.47 (Genotypes)
CD interaction = 0.67 (Genotypes x Media)
Table 4.2. Effects of hormones and genotypes on days to calli appearance
Hormonal
combination
Genotypes
Jayanti P
(2)2
P-51 P-18 Jayanti x
P
(2)2
Jayanti x
P-51
Jayanti x
P-18
Mean CD
(P≤0.05)
HM
1
13.08 9.75 10.00 10.75 9.33 10.50 10.75 10.60 NS
(Hormones)
HM
2
9.67 9.42 7.17 6.67 7.25 7.08 6.67 7.70
HM
3
10.17 10.00 7.17 8.50 8.67 10.00 8.50 9.00
Mean 10.97 9.72 8.11 8.64 8.42 9.19 8.64
CD (P≤0.05) = 0.47 (Genotypes)
CD interaction= 0.82 (Genotypes x Hormones)
Table 4.3. Effects of sucrose and genotypes on days to calli appearance
Sucrose
Genotypes
Jayanti P
(2)2
P-51 P-18
Jayanti x
P
(2)2
Jayanti x
P-51
Jayanti x
P-18
Mean
CD
(P≤0.05)
3 % 10.17 10.39 8.72 8.56 8.78 9.00 8.56 9.17
NS
(Sucrose)
4 % 11.78 9.06 9.22 8.72 8.06 9.39 8.72 9.28
Mean 10.97 9.72 8.97 8.64 8.42 9.19 8.64
CD (P≤0.05) = 0.47 (Genotypes)
CD interaction = NS (Genotypes x Sucrose)
Table 5.1. Effects of sucrose and hormones on days to
calli appearance
Sucrose
Hormonal Combination
HM
1
HM
2
HM
3
Mean CD
(P≤0.05)
3 % 10.88 7.67 8.95 9.17 NS
(Sucrose)
4 % 10.31 7.74 9.79 9.28
Mean 10.60 7.70 9.37
CD (P≤0.05) = NS (Hormones)
CD interaction = 0.44 (Hormones x Sucrose)
days to calli appearance. Nine out of eleven interactions
viz., genotypes x hormones, genotypes x media, hor-
mones x media, genotypes x hormones x media, hor-
mones x sucrose, genotypes x hormones x sucrose, gen-
otypes x media x sucrose, hormones x media x sucrose
and genotypes x hormones x media x sucrose showed
signi cant effect on days to calli appearance. From the
Tables 4.1, 4.2 and 4.3, it is pertinent that the effects of
media, hormones and sucrose were found to be non-
signi cant on all seven genotypes which indicated that
different genotypes behaved similar in different media,
hormonal combinations and sucrose concentrations for
days to calli appearance. However, the genotype Jayanti
x P
(2)2
recorded lowest days to calli appearance on dif-
ferent media and sucrose concentrations. Likewise, the
genotype P-51 took lowest days to calli appearance on
different hormonal combinations. The effects of sucrose
and hormones, hormones and media and media and
sucrose on days to calli appearance are presented in
Tables 5.1, 5.2 and 5.3, respectively. The results revealed
that the effects of sucrose and hormones, hormones and
Hadi M. Hamaza AL-Mayali and Sadiya Aziz Anah
BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS STUDIES ON DAYS TO CALLI APPEARANCE IN ETHIOPIAN MUSTARD 485
Table 5.2. Effects of hormones and media on days to
calli appearance
Hormonal
combination
Callusing Media
MS B
5
Mean
CD
(P≤0.05)
HM
1
10.05 10.95 10.50
NS
(Hormones)
HM
2
7.40 8.00 7.70
HM
3
9.38 9.36 9.37
Mean 8.94 9.44
CD (P≤0.05) = NS (Media)
CD interaction = 0.44 (Media x Hormones)
Table 5.3. Effect of media and sucrose on
days to calli appearance
Media
Sucrose
3% 4% Mean
CD
(P≤0.05)
MS 8.98 9.03 9.01
NS
(Media)
B
5
9.35 9.52 9.44
Mean 9.17 9.28
CD (P≤0.05) = NS (Sucrose)
CD interaction = NS (Sucrose x Media)
media and media and sucrose were found to be non-
signi cant which indicated that days to calli appearance
were not affected signi cantly by different media, hor-
monal combinations and sucrose concentrations.
CONCLUSION
The factors such as media, hormones, sucrose and
their interactions viz., genotypes x sucrose and media
x sucrose had non-signi cant effects on days to calli
appearance which indicated that the genotypes behaved
similar in different media, hormonal combinations and
sucrose concentrations.
ACKNOWLEDGMENTS
Authors are very much thankful to the Department of
Crop Improvement, CSK Himachal Pradesh Krishi Vish-
vavidyalaya, Palampur for providing all the essen-
tial facilities and moral support to conduct the whole
research programme and to obtain its signi cant nd-
ings.
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