Immature Inflorescence Culture-format.pdf

Research & Reviews: A Journal of Crop Science and Technology

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

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ISSN: 2319–3395© STM Journals 2012. All Rights Reserved

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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 correspondence E-mail: [email protected]

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 [2–4].

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 [7–10].

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 [12–18].

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 10–20 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 60–80 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

mailto:[email protected]



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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 [22–24] and influenced by

component of media [25–27]. 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 1–2 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, 121°C

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



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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, 121°C

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,

1–2 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 (χ 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].



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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

10–36 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 (χ2) test (P≤ 0.05).

S.No.

Cultivars

Percent

Callus induction A+

Embryogenic callus

formation A+

Plantlet regeneration

B+

1 HUW206 74.00ab

28.00 bc

14.00bc

2 HUW234 70.00ab

36.00 c 20.00

abc

3 Sonalika 64.00ab

30.00 b 16.00

bc

4 Kalyansona 60.00b 14.00

c 10.00

c

5 HD 2184 80.00a 40.00

b 26.00

ab

6 UP 2338 74.00ab

64.00a 36.00

a

Chi square (χ2 ) 9.7143

ns 31.9121* 16.125*



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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



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frequency plantlet regeneration in

inflorescence culture of wheat [30, 35]. Barro

et al. (1999) [34] has reported 40–50 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 ¼

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.



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