Research & Reviews: A Journal of Crop Science and Technology

Volume 1, Issue 3, December 2012, Pages 1-8

__________________________________________________________________________________________

ISSN: 23193395 STM Journals 2012. All Rights Reserved
Page 1
Immature Inflorescence Culture and Plantlet Regeneration in Some Land Mark
Indian Wheat Varieties (Triticum Aestivum L. Em Thell)

V. K. Mishra*
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi-221005
*Author for correspondenceE-mail: mishravkbhu@gmail.com

1. INTRODUCTION

The transformation of all major cereals has
now been achieved opening the way for
genetic engineering of new plants with
modified agronomic traits, such as herbicide
resistance, biotic and/or abiotic stress
resistance, and grain quality and composition
[1]. In addition, somaclonal variation and
in vitro selection offers new possibility of
isolation of genetically useful variants [24].
Wheat (Triticum aestivum L.) is one of the
major staple food crops grown worldwide [5].
Since it being having a large genome size
(approximately 17000 Mb) thus makes the
improvement method genetically challenging.
Not all wheat species respond well in tissue
culture, and within the same genotype, tissue
culture response may differ from explants to
explants [6]. Both mature and immature
embryos have been used extensively in tissue
culture protocols, but mature embryos were
found to be a better choice because of round
the year availability of explant [710].
However, immature embryos are better
explants source when regeneration is
considered but they require time and growth
facilities [11]. Nevertheless, it has been first
choice for efficient induction of somatic
embryogenesis and gene transfer [1218].
However, immature inflorescences have also
been shown to provide a practical source of
young tissues for somatic embryogenesis and
plant regeneration as they are harvested at an
ABSTRACT

The present study reports immature inflorescence culture and plantlet regeneration in some land mark
wheat varieties i.e. HUW 206, HUW 234, Sonalika, and Kalyan Sona HD 2184 and UP 2338. Immature
inflorescence was taken out from the wheat plants when they were of 1020 mm in length. Double
strength MS medium supplemented with casein hydrolysate, 200 mgL
-1
, glutamine, 500 mgL
-1
and 2, 4-
D, 2 mgL
-1
was found most suitable for callus induction and embryogenic callus formation. The
frequency of callus induction was high ranging from 6080 percent and the cultivars did not differ
significantly in their response towards callus induction. However, frequency of embryogenic callus
formation was significantly different among the six cultivars tested, ranging from 14 to 64 percent.
Plantlet regeneration was obtained on MS medium supplemented with indole-3-acetic acid (IAA),
1 mgL
-1
and zeatin 1 mgL
-1
. The study indicated that genotypic effect was more pronounced for both
embryogenic callus formation and plantlet regeneration. Embryogenic callus formation and plantlet
regeneration seems to be positively correlated. In the immature inflorescence culture of wheat time
related response was observed in plantlet regeneration which significantly varied with duration of
culture. All the variety tested retained their regeneration potential up to four month of culture. Plantlet
regeneration occurred via somatic embryogenesis but organogenesis also occurred occasionally.

Keywords: Immature Inflorescence, Tissue Culture, Somatic Embryogenesis, Wheat
Research & Reviews: A Journal of Crop Science and Technology
Volume 1, Issue 3, December 2012, Pages 1-8
__________________________________________________________________________________________

ISSN: 23193395 STM Journals 2012. All Rights Reserved
Page 2
early stage than immature embryos and can be
considered as an alternative source of explants
and also has been most frequently used as
target tissue for genetic transformation
[19 21].

Most of the reports on somatic embryogenesis
and regeneration in wheat are involving
immature embryos were shown to be genotype
dependent [2224] and influenced by
component of media [2527]. It is indicated
that tissue culture responses are influenced by
the genotypes, explants source, geographical
origin and physiological status of the donor
plants, the culture medium, and the
interactions between them [28]. Though many
protocols have been developed but tissue
culture of wheat seems to be very much
genotypic dependent response [6].

In the recent years, immature inflorescence has
been reported as alternative explants source for
gene transfer in wheat [20, 29].
Morphogenesis in inflorescence culture seems
to be complex, and generally several tissue-
culture factors determine the success of
regeneration and transformation, which are:
genotype, physiological state of the
explants/donor plants, culture conditions, and
medium composition etc. The present study
was aimed at testing immature inflorescence
culture response of land mark Indian wheat
varieties and experiments were also designed
to long- term embryogenic and regeneration
potential in order to exploit it for in vitro
selection.
2. MATERIALS AND METHODS

Six land mark Indian wheat varieties, i.e.
HUW 206, HUW 234, Sonalika and Kalyan
sona, HD 2184 and UP 2338 were used in the
experiments. Immature inflorescence up to 10
20 mm in length was taken out from young
shoots prior to the emergence of the flag leaf.
The outer leaves were removed and the surface
of inner leaves were wiped out with 70%
ethanol (v/v) and the explants was surface
sterilized by transfer to detergent, 70% ethanol
(v/v), calcium hypochlorite (0.5% w/v) and
several changes in sterile water. Following
this, inflorescence was dissected out and cut
into sections of 12 mm aseptically and were
placed on the surface of the medium, and
incubated in dark/or in light (40 W, 1500 lux
fluorescent tube).

The medium for callus induction and
maintenance of embryogenic callus was
modified MS (Murashige and Skoog, 1962)
medium following the protocol of [30].
Modified MS medium [31] with double
concentration of MS inorganic salts, single
strength MS vitamins, 200 mgL
-1
casein
hydrolysate, 500 mgL
-1
glutamine, 20 gL
-1

sucrose, 2 mgL
-1
2, 4- dichlorophenoxy acetic
acid (2, 4-D), and 8 gL
-1
agar, pH 6.0 prior to
autoclaving, and then medium was sterilized at
15 psi, 121C

for 15 min. For plantlet
regeneration, a modified MS medium
following the protocol of [32] was used. The
MS basal medium was supplemented with
indole-3-acetic acid at concentration 1 mgL
-1

Research & Reviews: A Journal of Crop Science and Technology
Volume 1, Issue 3, December 2012, Pages 1-8
__________________________________________________________________________________________

ISSN: 23193395 STM Journals 2012. All Rights Reserved
Page 3
and zeatin 1 mgL
-1
and gelled with agar 8 gL
-1
.
The pH of the medium was adjusted to 6.0
prior to autoclaving at 15 psi, 121C

for
15 min.

3. STATISTICAL ANALYSIS

After four weeks of culture, callus induction
was scored on percent basis, number of
inflorescence sections formed callus per total
numbers of sections. Fifty explants were used
for initiation of callus in each cultivar. For
observation on embryogenic callus formation,
12 mg of callus-clumps from each cultivar
was sub-cultured to fresh medium for growth
and proliferation. After 28 days of culture,
embryogenic callus was identified as opaque,
yellowish/ or off-white, compact and nodular
organized callus mass. Percent of embryogenic
callus formation was scored on the basis of
number of embryogenic callus per total
number of callus clumps, 50 clumps in each
cultivar. Embryogenic callus was transferred
to regeneration medium. After four weeks of
transfer on regeneration medium, percent
plantlet regeneration was scored on the basis
of number of plants regenerated per total
number of callus clumps. The study was
conducted as a separate experiment for callus
induction, embryogenic callus formation and
plantlet regeneration. Data were analyzed in 2
x n Chi-square (
2
) test (P=0.05), sample size,
n= 50 per cultivar.



4. RESULTS AND DISCUSSION

Efficient plant regeneration from cultured cells
and tissues is required for the successful
application of modern biotechnology in crop
improvement. Therefore, the success of cell
and tissue culture research depends upon
reliable callus culture and plant regeneration
procedures [33]. Genotype and culture media
play an important role in success of plant
tissue culture response in wheat [32, 34]. Both,
organogenesis and somatic embryogenesis
have been reported from the immature
inflorescence indicating complex
morphogenetic behaviour of the explant [35].
In the study, explants were taken at the stage
when immature inflorescence has rachis and
differentiating spikelets. After 15 days of
culture initiation, calli were seen emerging
from the cut surface of rachis and areas of
spikelets. The developmental stage of the
spike is critical for successful callus induction
in cereals [33]. In bread wheat, inflorescence
collected at the pollen mother cell (PMC)
stage or just prior to meiosis has been found
suitable for callus induction [30]. In the
experiment, high frequency callus induction
was observed in all cultivars on MS medium,
double ionic strength supplemented with
casein hydrolysate 200 mgL
-1
, glutamine
500 mgL
-1
, and 2, 4-D 2 mgL
-1
(Table I). The
double ionic strength of MS medium with
casein hydrolysate, glutamine and 2, 4-D has
been reported to suppress precocious
generation of somatic embryos and promote
normal maturation of the embryos [25, 30].
Research & Reviews: A Journal of Crop Science and Technology
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Supplementation of the media with casein
hydrolysate, glutamine and double ionic
strength of the medium was considered
suitable for long-term maintenance of
embryogenic potential [27].

Microscopic examination of callus showed a
friable and embryogenic callus which is
characterized by the presence of compact
nodular organized mass, yellowish in
appearance when cultured in light and off-
white colour in dark culture (Figure 1). In
histological examination, somatic embryos
were seen at various developmental stages,
such as early and late globular stage, and
bipolar embryos. In the present experiment,
embryogenic callus formation varied from 14
to 64 percent and was significantly different
among the cultivars (Table- I). Plantlet
regeneration was obtained on MS medium
containing IAA, 1 mgL
-1
and zeatin 1 mgL
-1
. It
is reported that IAA and zeatin
supplementation resulted in increase in
frequency of plantlet regeneration in
inflorescence culture of wheat. In the study,
frequency of plantlet regeneration ranged from
1036 percent and was significantly different
among the cultivars (Table I).

Table I : Percent Callus Induction, Embryogenic Callus Formation and Plantlet Regeneration in
Immature Inflorescence Culture of Land Mark Indian Wheat Varieties.
Sample size n=50; *= significant at 5% level; ns= not significant at 5% level;
A
+
modified Ozias-Akins and Vasil (1982) medium; B
+
Gosch-Wackerle et al. (1979);
Means followed by same letters are not significantly different in 2 x 2 Chi-square (
2
) test (P 0.05).

S.No.

Cultivars

Percent
Callus induction
A+
Embryogenic callus
formation
A+

Plantlet regeneration
B+

1 HUW206 74.00
ab
28.00
bc
14.00
bc

2 HUW234 70.00
ab
36.00
c
20.00
abc

3 Sonalika 64.00
ab
30.00
b
16.00
bc

4 Kalyansona 60.00
b
14.00
c
10.00
c

5 HD 2184 80.00
a
40.00
b
26.00
ab

6 UP 2338 74.00
ab
64.00
a
36.00
a

Chi square (
2
) 9.7143
ns
31.9121* 16.125*
Research & Reviews: A Journal of Crop Science and Technology
Volume 1, Issue 3, December 2012, Pages 1-8
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ISSN: 23193395 STM Journals 2012. All Rights Reserved
Page 5





























Fig. 1: Immature Inflorescence Culture of Bread Wheat: 1). Embryogenic Callus Cultured in Light,
Off-White, and Yellow, Nodular Callus; 2). Embryogenic Callus Cultured under Dark Condition;
3). Plantlet Regeneration at Early Stage of Development, 4. Regenerated Plantlets.

The results of the study indicated that there
exist a positive correlation between
embryogenic callus formation and plantlet
regeneration. The cultivars having high
embryogenic frequency have shown high
regeneration capacity. There are reports of low
Research & Reviews: A Journal of Crop Science and Technology
Volume 1, Issue 3, December 2012, Pages 1-8
__________________________________________________________________________________________

ISSN: 23193395 STM Journals 2012. All Rights Reserved
Page 6
frequency plantlet regeneration in
inflorescence culture of wheat [30, 35]. Barro
et al. (1999) [34] has reported 4050 percent
regeneration capacity in inflorescence culture
with exception of some varieties with no
regeneration to some with high regeneration
capacity. Maddock et al. (1983) [22] reported
significant influence of genotype on frequency
of plantlet regeneration in inflorescence
culture of wheat. Amirova et al. (2002) [36]
stated that a reproducible and genotype-
independent system for the long term
regeneration in wheat tissue culture was
developed. Typically for cereal crops, highly
totipotent cell lines are often derived from
friable embryogenic (Type II) callus tissues, in
most of these crops and wheat, Type II
cultures occur at low frequencies and/ or
difficult to establish and maintain [37]. Wheat
regeneration was found to be determined by
polygenic system [38], and genes on 5A, 1B,
4D chromosomes and also genes on 2A, 3A,
3B, 4B, 6B and 1D chromosomes influence
somatic in vitro morphogenesis in wheat [39].

In the present study, all the cultivars retained
their regeneration potential up to four months
of culture initiation, and thereafter
regeneration potential declined very sharply
(Table I). In the study, most of the regenerants
were obtained via somatic embryogenesis
showing simultaneous formation of shoots and
roots on a single medium. However, in some
instances only shoot formation was observed
on the regeneration medium, and the roots
were developed afterwards on transfer to
strength MS medium. This indicated that in
some instances organogenesis also takes place
but it was rare in occurrence.

5. CONCLUSION

Plantlet regeneration in inflorescence culture
of some land mark Indian wheat varieties
occurred via somatic embryogenesis.
Varieties used in the study showed high
frequency callus induction, and were having
moderate embryogenic and regeneration
potential. The varieties with high embryogenic
frequency have shown high regeneration
capacity. It was also observed that
regeneration potential of embryogenic calli
decreased significantly with culture duration
and declined sharply after four months of
culture initiation. The study also revealed that
varying response of high yielding varieties
towards embryogenic callus formation and
plantlet regeneration and genotype dependent
response in immature inflorescence culture of
wheat.

ACKNOWLEDGEMENTS

We would like to thank the School of
Biotechnology, and Department of Genetics
and Plant Breeding, Banaras Hindu University,
Varanasi for providing all necessary facility
for the research work. We are also thankful to
DBT for sanctioning SAP fellowship to the
Department which made it possible to carry
out this work.

Research & Reviews: A Journal of Crop Science and Technology
Volume 1, Issue 3, December 2012, Pages 1-8
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ISSN: 23193395 STM Journals 2012. All Rights Reserved
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