Induced somaclonal variation in regenerated banana plants (Musa acuminate L.) cv. Dwarf

Cavendish

Fatemeh Ghasemali 1, Farah Farahani 2, Masoud Sheidai3, Taher Nejad Satari1

1 Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad

University (IAU), Tehran, Iran.

2 Department of Microbiology , Qom Branch, Islamic Azad University, Qom, Iran.

3 Department of Biology, Faculty of Biological Sciences, Shahid Beheshti University, Evin,

Tehran, Iran.

Corresponding Author:

Farah Farahani

Address: Department of Microbiology , Qom Branch, Islamic Azad University, Qom, Iran.

E-mail: farahfarahani2000@yahoo.com

Tel: +982144122070

Cell Tel: +989122778171

1

Induced somaclonal variation in regenerated banana plants (Musa acuminate L.) cv. Dwarf

Cavendish

F. Ghasemali 1, F. Farahani 2, M. Sheidai3, T. Nejad Satari1

1 Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad

University (IAU), Tehran, Iran.

2 Department of Microbiology , Qom Branch, Islamic Azad University, Qom, Iran.

3 Department of Biology, Faculty of Biological Sciences, Shahid Beheshti University, Evin,

Tehran, Iran.

Geneconserve 14 (56): 32-55

2

Abstract

Somaclonal variation is genetic variation that occurs due to tissue culture in plants. The

occurrence of somaclonal variation can be detected by morphological, cytological and molecular

approaches. Somaclonal variation may produce regenerated plants with desirable genetic and

morphological characteristics. The aim of prresent study was to induce somaclonal variation in

“Dwarf Cavandish” cultivar of banana. Therefore, the meristematic tissues of this cultivr were

cultured on BM1, BM2, BM3 and BM4 media. The media conatined MS medium plus

phytohormones. Roots from mother plants and somatic embryos of regenerated plantlets were

used for cytological study. After 4 months, the callus were not initiated in BM1, BM3 and BM4

media and meristematic tissues produced plantlets. The longest length of shoot and root were

observed in BM1 and BM3. The somatic embryos were transferred to the medium for embryo

differentiation. They mature somatic embryos placed on BM5 (MS medium with BA (3 mg/l),

IAA (2 mg/l), 0.5 g/l charcoal) medium. Somatic embryos gave rise to plantlets. The percentage

of regenerated plantlets from somatic embryos were 85-90%. Approximately 100% of the

chromosome counts showed a count of 3x=2n=33 in the mother plants. Prometaphase and

metaphase chromosomes differed in the number of chromosome. The regenerated plants from

somatic embryos showed 50% triploidy, 30% diploidy and 20% anuploidy.

Keywords: banana, cytogenetic, Dwarf Cavendish, somatic embryo.

Abbreviation: BA: Benzyl adenin; IAA: Indol Acetic Acid; MS: Murashig & Skoog (1962);

NAA: Naphtol Acetic Acid; 2,4-D: 2,4 Dichloro Phenoxy Acetic Acid;

3

Introduction

Banana (Musa spp.) is one of the most important tropical fruits in the world trade. It is a staple

food for nearly 400 million people (Novak, 1992). In many countries, banana and plantain

represent the major fruit exports and are essential sources of income for national economies. It

has world production of about 64.6 million metric tons (Anonymous, 2001). Banana is a staple

food for nearly 400 million people, howwever, its production is limited primarily by viral and

fungal diseases as well as insect and nematode pest problems (Sasson, 1997).

The application of classical methods of breeding for both disease and pest resistance has resulted

in only limited success due to the long generation times for banana and the high sterility and

triploid nature of most cultivated bananas (Sasson, 1997). Although in vitro culture of banana has

been extensively used to quickly propagate vegetative clones of many genotypes (Vuylsteks and

De Langhe, 1985; Das et al., 1998), many obstacles remain to be overcome before an efficient

banana regeneration protocol suitable for genetic transformation is developed.

Plants regenerated through organogenesis are not appropriate for genetic transformation since

many chimeric plants are produced. The integration of genetic engineering into breeding

programs may provide powerful tools to overcome these limitations by introducing specific

genetic changes that can be utilized for banana improvement within a short period of time.

However, these applications require reliable plant regeneration protocols for banana Vuylsteks

and De Langhe, 1985; Das et al., 1998).

Somatic embryogenesis, the process whereby either a single somatic cell or clusters of cells

develop into embryos, is a useful approach for in vitro plant regeneration of many species. This

technique in the genus Musa was used to develop high-performance micropropagation

4

techniques and plant regeneration systems useful for genetic improvement (Khalil et al., 2002,

Khalil and Elbanna, 2004).

In Musa spp., somatic mutations (Samson, 1982) and somaclonal variations (Vuylsteke, 1989;

Sandoval et al., 1991) have been resulted in genome instability. Furthermore, viral particles have

been reported to interact with the Musa genome to destabilize the genome, especially under in

vitro culture environments (Sasson, 1997).

Though naturally occurring genetic changes do occur in the genomes of plants, their rates are

slow. In vitro systems can enhance the mutation rate due to additional selection pressure

enforced in these methods on the cultured plant materials. These changes are manifested as

somaclonal variations. Somaclonal variations are not altogether undesirable since some may

serve as novel raw material (genetic diversity) for further crop improvement.The problem,

however, is that generating somaclonal variants is unpredictable since the type and extent of

variation or even synergistic processes forming them are random events (Larkin and Scrowcroft,

1981).

Osuji et al. (1997) noted that the instability at the genotype level of Musa compromises the

conventional idea of using phenotypic characters for molecular marking of Musa material.

Several research efforts have looked into unraveling the genotypic constitution of Musa plants,

relying on molecular cytogenetic techniques (Kosina and Helslop-Hrrison 1996; Osuji et al.,

1997, 1998).

We describe here an efficient and reproducible protocol with high frequency regeneration for

somatic embryogenesis via solid culture that lead to mass production of plantlet to improve

Musa spp. AAA group cv. Dwarf Cavendish and also report the occurrence of somaclonal

variation as revealed by cytogenetic analysis.

5

Materials and Methods

Plant material and culture initiation

“Dwarf Cavandish” cultivar of banana (Musa spp.) of the AAA group was used. Shoots (stem

with 2-3 cm) were selected and washed with 1 % (v/v) detergent solution for 5 min and surface

sterilized by 10% NaOCl2 for 15 min. They were finally rinsed three times with sterile water. In a

sterile petri dish, the outer leaves were peeled with forceps until meristemic tissues of 0.5- 1 cm

in length were obtained (Farahani et al. 2011). The meristematic tissues were isolated for

cultures.

Callus initiation

Meristematic tissues explants were isolated and cultured on solid BM1, BM2, BM3 and BM4

media. The solid media conatined macro- and micro-nutrients and vitamins of Murashige and

Skoog (1962) plus phytohormones and and 30 gl-1 sucrose with pH adjusted to 5.8 (Table 1).

The cultures were incubated in the growth chamber at 25 ºC for four weeks. The meristematic

tissues became swollen four weeks after cultures were initiated in BM2 medium. In this time,

they could be seen as whitish tissue protruding from tissues. The swollen tissues were kept in

BM2 medium, carefully excised from the mother tissue, and transferred to fresh medium.

Primary somatic embryos were produced after tissues were transferred to fresh media for four

months. Compact white calluses and friable embryogenic tissues with globular structures

containing primary somatic embryos were formed.

6

Differentiation of embryos, embryo germination and plantlet formation

Embryo development and germination were achieved by culturing the embryonic solid culture on

BM2 medium, then transferred to the medium designated BM5 (Farahani and Majd 2012),

which contained MS salts, MS vitamin, BA (3 mg/l), IAA (2 mg/l), 0.5 g/l charcoal, sucrose (30

g/l), and agar (7 g/l). The cultures were incubated at 25 °C with a photoperiod of 16 h light and

8 h dark for four weeks.

Cytogenetic Studying

Root tips of the mother plants and somatic embryos regenrated plantlets were used for

cytological study. An aqueous solution of 0.02% (w/v) 8-hydroxyquinoline was used to pre-treat

the root tips for 45-60 min in the dark since this chemical is photosensitive. A solution of

absolute ethanol and glacial acetic acid in the ratio of 3:1 (v/v) was used to fix the root tips for at

least 24 h at room temperature. Fixed roots were then stored in 70% ethanol until used for slide

preparation (Bakry and Shepherd, 2008; Osuji et al., 1996).

Cytological preparations were covered with cover-slips, Slides were observed with an oil

immersion objective lens and a Leitz Diaplan phase contrast microscope. The mitotic cells

identified with metaphase or prometaphase stages were used for chromosome counting. Good

root cells at prometaphase were photomicrographed to reflect chromosome number and

morphology. A Leica Wild MPS 52 microscope camera was used to photograph good plates,

using appropriate filters.

7

Results and Discussion

Culture initiation and callus development

The meristematic domes from apical sprouted buds were used as explants in combination with

different culture media, Meristematic tissues were isolated and cultured on BM1, BM2, BM3 and

BM4 media culture, They were compared together for callus initiation, differentiation of

embryos and embryo germination and plantlet formation.

On the other hand, explants selection could be a key factor for determining success or failure in

an embryogenesis protocol. In case of somatic embryogenesis in edible bananas and plantains,

three main principle sources have been used. These are 1- the explants, rhizome sections, leaf

bases, 2- scalps from cauliflor like multibuds and 3- immature female and male flowers (Novak

et al., 1989). However, the use of meristematic domes from sprouted buds in combination with

the culture medium permitted to obtain somatic embryos, similar to those obtained by Lopez

Torres et al. (2005) who used this explants type for somatic embryogenesis in plantain (AAB).

Schoofs et al. (1999) stated that high quality cauliflower-like meristems may be obtained from a

few days up to more than a year, although, some cultivars (Musa spp.) show a recalcitrant

response for developing somatic embryogenesis. Callus formation with embryogenic structures

was characterized in diploid banana (‘Calcutta 4´, Musa AA) Using scalps from cauliflower-like

meristems as starting material in the embryogenic process; a very low formation frequency of

calli with embryogenic structures was obtained. 0.8% (Lopez Torres et al., 2012).

After four months of incubation, the callus were not initiated in BM1, BM3 and BM4 media

cultures. However, meristematic tissues produced plantlets after 8 weeks media culture.

8

The longest length of shoot and root were observed in BM1 and BM3 media cultures (11.5 cm

and 9.1 cm, respectively). The highest mean number of shoot, leaves and root were obtained in

BM4, BM1 and BM3 treatments (5, 1.3 and 2, respectively).

The mineral composition of the media can play vital roles in somatic embryogenesis.

Unfortunately, little effort has been extended to evaluate the effect of different basal nutrient

formulations in somatic embryo induction (Merkle et al., 1995). Full-strength MS or 2MS

medium was found to be more effective than the other media used for induction and growth of

somatic embryos. This may be due to the presence of a high level of nitrogen, particularly the

reduced form (NH4+), in MS medium (Varisai et al., 2004).

Several studies have shown the influence of some amino acids like Glutamine and L-proline on

the development of somatic embryogenesis as rate regulator in the protein synthesis during the

morphogenetic process. (Lopez Torres et al., 2012). Somatic embryogenesia induction, growth

and maturation were decreased when level concentration of glutamine was high (50 to 100 mg l-

1) (Varisaiet al., 2004).

Growth of tissues was significantly stimulated by the addition of amino acids and vitamins

specifically glutamine and biotin. This stimulation may be attributed to the role of organic

nitrogen as a growth-limiting factor in date palm cultures. The inclusion of glutamine decreased

the culture lag phase, which indicated that glutamine was much readily assimilated than

inorganic nitrogen. Purves and Brown (1978) reported that glutamine plays an important role in

nitrogen assimilation as it intermediates in the transfer of ammonia into amino acids, glutamine

and asparagines interact with cell-auxin balance. Indole acetylglutamate and indole

acetylaspartate were found to be common forms of bound auxins.

9

2,4-D concentration (BM4 medium) was not determinant for callus formation with embryogenic

structures in the range of 2 mg l-1. However, embryogenic response was observed when BM4

medium were enriched by L-proline. Bieberach (1995) used MS medium supplemented with 2,4-

D and L-proline to induce callus with embryogenic structures from male inflorescences in

banana cv. ‘Grande naine’ (Musa spp. AAA) and these embryogenic structures were not observe

in banana cv. ‘Dwarf Cavendish (Musa spp. AAA).

Likewise, cytokinins also play an important role in induction and development of somatic

embryos in peas (Pisum sativum) (Kysely and Jacobsen 1990) and banana plants (Khalil et al.,

2002). Zeatin (0.219 mg/l) were essential for the stages of embryogenesis-induction and the

proliferation of somatic embryos (Komamine, 2003).

The meristematic segments became swollen 4 weeks after cultures were initiated in BM2

medium. Initially, these cultures were very heterogeneous and contained large translucent cells as

well as small dense cells. Upon frequent subculture at 3-4 weeks intervals, and subsequently at

5-week intervals, the cultures became more uniform and only contained clusters of small tightly

packed cells with a dense cytoplasm. Somatic embryogenesis was also observed characterized

by typical globular stage embryos directly from as small, hyaline protuberances on the surface of

the clusters. The embryos developed with a green plumule (Fig. 1a-h).

When BM2 was used the highest number of clones and percentage of embryos (Globular,

torpedo and mature) were obtained under photoperiod 16/8 condition at 25 ºC. Somatic embryos

initially appeared small and rapidly enlarged into distinct globular structure, which passing

through recognizable torpedo structure. Somatic embryos initially globular and torpedo observed

was noted within 2-3 weeks after aspirated on BM5 regeneration medium.

10

The features of somatic embryos (translucent and whitish) obtained in this study were similar to

those obtained earlier in banana (Cote et al.,1996; Escalant et al., 1994; Strosse et al., 2006).

Plantlets regeneration

Mature somatic embryos, which differentiated on BM2 medium were separated from the culture

mass and placed directly on MB5 (MS supplemented with 3 mgl-1 BA, 2 mg/l IAA, 0.5 g/l

charcoal and 30 g/l sucrose). Somatic embryos gave rise to small plantlets (shoots and roots)

within 4 weeks. The percentage of germination and development of completely regenerated

banana plants from somatic embryos reported in this study was 85-90%, that is significantly

higher than those reported by Novak et al. (1989) (1.5-12%); Dhed (1991) (10-23%), Cote et al.

(1996) 3-20%, Grapin et al. (1990) 10-20 % and Navarro et al. (1997) (13-25 %). Our results are

even higher than to those of Kosky et al. (2002) who reported 89.3 % germination using cell

suspension using a bioreacter, and higher than Khalil et al. (2002) who reported 89.5 %

germination percentage using secondary somatic embryos. We have successfully regenerated

plants using the system described in this paper via solid medium culture.

Khalil et al. (2002) obtained approximately 90% germination for development of somatic

embryos into plantlets, and these were subcultured onto MS medium plus 0.1% activated

charcoal 1 mg/l BA and 1 mg/l IAA where complete plantlets developed. Morphologically

normal banana plants developed from all the regenerated plantlets, the first of which were

produced within 6 months of culture initiation. In Musa acuminata „Mas‟ (of AA genomic

group), plant regeneration from embryogenic suspension cultures was achieved (Jalil et al.,

2003).

11

Meenakshi et al. (2011) studied the induction of somatic embryogenesis from young immature

male inflorescences of the banana cultivar Lal Kela (red banana) on medium (MA 1991)

supplemented with 2,4-D.

Callus exhibited embryogenic stages and for the development of complete plantlets, globular

stage embryos were transferred to different levels of BA medium. The higher concentrations BA

(10 μM to 20 μM) showed complete conversion with shoot and root development. Somatic

embryos were vitrified on culture with high BA concentration (beyond 20 μM) and the embryos

become malformed and dried after 35 days.

The increased sensibility of the tissues to higher concentrations of BA and IAA may modify

physiological and developmental processes. The addition of these compounds to the media,

keeping a high cytokinin/auxin ratio rises the rates of cell divisions, leading to the production of

multiple meristems, as already stated by Zaffari et al. (2000), and those tissues (i.e., the apical

shoots) are embryogenically more competent (Strosse et al., 2006; Ramirez-Vililalobos and De

Garcia, 2008).

Cytogenetic Results

Approximately 100% of the chromosome counts showed a count of 3x = 2n = 33 in the mother

plants. Although there were phenotypically distinguishable descriptors for the somaclonal

variants in each case, none were associated with any structural or numerical chromosomal

abnormality. The chromosome morphology was not different in the normal regenerates and was

low different in their somaclonal variants. The chromosomes were aggregated in metaphase of

mitotic division. There were a few cases of variation in number, which were adjudged to be

aneuploid cases in regenerated plants.

12

Prometaphase and metaphase chromosomes were differences in number of chromosome within

and between the materials used in this study. Tables 2 shows the chromosome number variability

in the somaclonal variants. The regenerated plants from somatic embryos showed 50% triploidy.

The chromosomes were not contracted and not well-spread around the cell with a small vertical

dispersion. We observed 30% diploidy and 20% anuploidy. The mother plants were 100%

triploid, the chromosomes.

Variability in chromosome number and morphology is more common in somatic cell cultured in

vitro than in natural environment (Bayliss, 1980; Larkin and Scowcroft, 1981; Choy and Teoh,

2001; Gordian et al., 2007). This is one of the possible reasons for somaclonal variation

occurring in tissue culture. In vitro environment affected mitotic instability in bananas, as the

mean frequency of aberrant metaphase cells was significantly higher in somaclonal variants than

in mother plants.

Vuylsteke and Swennen (1992) reported tissue culture leads to somaclonal variation in Musa.

Our results showed numerical changes in chromosomes between the mother plants and

somaclonal regenerants of the banana cv. Dwarf Cavendish used in this study.

13

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18

Figure 1- Somatic embryogenesis derived from meristematic tissues of the banana cv.

Dwarf Cavendish

a) The meristematic tissue as explants

b)Primary somatic embryos; nodular ones were frequent in all explants

c) Maturation somatic embryos developed from meristematic tissues from T2

d) Primary differentiated somatic embryos

e) Mature of somatic embryos and differentiated to plantlets (Bar = 5 mm)

f) Regenerated plantlets from mature of somatic embryos (Bar = 10 mm)

g) Elongated of regeneration plantlets (Bar = 10 mm)

h) Proliferation of regenerated plantlets in MS medium plus 3 mg/l of BA and 2 mg/l IAA (Bar =

10 mm)

19

20

Figure 2- Metaphasic cells of Musa acuminate cv. Dwarf Cavendish A) somaclonal variants with

30 chromosomes B) 14 chromosomes, C) 9 chromosomes, D) aggregated chromosomes

21

A B

CD

Figure 3- Musa acuminate cv. Dwarf Cavendish A) Triploid cell of mother plant with 33

chromosomes, B) Triploid cell of regenerated plant with 33 chromosome, B1) Diploid cell of

regenerated plant with 22 chromosomes, B2) Anuploid cell of regenerated plant with 23

chromosomes

22

A B

B1B2

B1 B2

Table 1- Medium culture with supplemented with hormones, amino acids and vitamins for

somatic embryogenesis and plantlets of regeneration

Treatment BM1 BM2 BM3 BM4 BM5

Basal medium MS 2MS MS MS MS

IAA (mg/l) 1 _ 2 1 2

BA (mg/l) 3 22.5 0.4 _ 3

NAA(mg/l) _ _ _ 1 _

2,4-D (mg/l) _ _ _ 2 _

Biotin (mg/l) _ _ _ 1 _

Glutamine (mg/l) _ _ _ 100 _

Malt extract (mg/l) _ _ _ 100 _

References Uma et al.,

2001

Schoops et

al., 2005

Badawy et

al., 2005

Vijoen et

al.,

2006and

Meenakshi

et al., 2011

Meenakshi

et al., 2011

MS (Murashig & skoog, 1962),

Table 2- Chromosome changes of somaclonal variants

23

24

% polyploidy

(Regenerated plants

from somatic embryos)

% poliploidy (Mother plants)Number of chromosome

(metaphase period)

501002n=3x=33 (Triploid)

30_2n=2x=22 (Diploid)

20_2n=2x+1=23 (Anuploid)