Induced somaclonal variation in regenerated banana plants ... · Induced somaclonal variation in...
Transcript of Induced somaclonal variation in regenerated banana plants ... · Induced somaclonal variation in...
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: [email protected]
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
References
Anonymous A (2001). FAOSTAT Database. Food and Agriculture Organization of the United
Nations, Rome.
Bakry F and Shepherd K (2008). Chromosome count on banana root tip squashes. Fruits. 63(3):
179-181.
Bayliss MW (1980). Chromosomal variation in plant tissues in culture. Int. Rev. Cytol., 11A
(Suppl.): 113-114.
Bieberach C (1995). Embriogénesis somática y regeneración de plantas en cultivares de Musa
spp. Tesis presentada en opción al grado científico de Magister Scientiae. CATIE, Turrialba,
Costa Rica; 6p
Choy MK and Teoh SB (2001). Mitotic instability in two wild species of bananas (Musa
acuminata and M. balbisiana) and their common cultivars in Malaysia. Caryologia. 54(3): 261-
266.
Cote FX, Domergue R, Monmarson S, Grapin A, Schwendiman J and Teisson C (1996). Somatic
embryogenesis in plantain banana. In Vitro Cell Dev Biol. Plant. 32: 66-71.
Das A., Paul A.K. & Chaudhuri S. 1998. Banana tissue culture variation in response of
four genotypes. Horticulture, 11: 13-20.
Dhed’A D, Dumortier F, Panis B, Vuylstteke D and Delanghe E (1991). Plant regeneration in cell
suspension cultures of the cooking banana cv. Bluggoe (Musa spp. AAB Group). Fruits. 46:125–
135.
Escalant JV, Teisson C and Cote FX (1994). Amplified somatic embryo-genesis from male
flowers of triploid banana and plantain cultivar (Musa spp.). In Vitro Cell Dev. Biol. 30: 181-
186.
14
Farahani F and Majd A (2012). Comparison of liquid culture methods and effect of temporary
immersion bioreactor on growth and multiplication of banana (Musa, cv. Dwarf Cavendish).
African Journal of Biotechnology. 11(33): 8302-8308.
Gordian C, Obute P and Aziagba C (2007). Evaluation of Karyotype Status of Musa L.
Somaclonal Variants (Musaceae: Zingiberales). Turk. J. Bot. 31: 143-147.
Grapin A, Ortiz J, Lescot T, Ferriere N and Cote F (1990). Recovery and regeneration of
embryogenic cultures from female flowers of False Horn Plantain. Plant Cell Tissue and Organ
Culture. 61(3): 237-244.
Khalil SM, Cheah KT, Perez EM, Perez DA and Hu JS (2002). Regeneration of banana (Musa
spp. AAB cv. Dwarf Brazilian) via secondary somatic embryogenesis. Plant Cell Report. 20:
1128- 1134.
Khalil SM and Elbanna AAM (2004). Highly efficient somatic embryogenesis and plant
regeneration via suspension cultures of banana (Musa spp.). Arab J. Biotech. 7(1): 99-110.
Komamine A (2003). My way with plant cell cultures: significance of experimental systems in
plant biology. In vitro Cellular and Developmental Biology-Plant. 39(2): 63-74.
Kosina R and Heslop-Harrison JS (1996). Molecular cytogenetics of an amphiploid trigeneric
hybrid between Triticum durum, Thinopyrum distichum and Lophopyrum elongatum. Ann. Bot.
78: 583-589.
Kosky RG, Silva M, De F, Perez LP, Gilliard T, Martnez FB, Vega MR, Milian RC and Mendoza
EQ (2002). Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid
medium and scaledup in a bioreactor. Plant Cell Tissue Organ Cult. 68: 21-26.
Kysely W and Jacobson HJ (1990). Somatic embryogenesis from pea embryo and shoot apices.
Plant Cell Tiss. Org. Culture. 20, 7-14.
15
Jalil M, Khalid N and Othman R (2003). Plant regeneration from embryogenic suspension
cultures of Musa acuminata cv Mas (AA). Plant Cell Tissue & Organ Culture. 75(3): 209-214.
Larking TJ, Scrowcroft WR (1981). Somaclonal variation – a novel source of variability from
cell cultures for plant improvement. Theor. Appl. Genet. 60: 197.
Lopez Torres J, Montano N, Gkosky R, Rayas A, Reinaldo D and Cabrera M (2005). Nueva
alternativa para el desarrollo de la embriogénesis somática en plátanos viandas (AAB).
Memorias Congreso Internacional Biotecnología y Agricultura (Bioveg 2005). Cabrales G (ed).
Centro de Bioplantas. Ciego de Avila, Cuba.
Lopez Torres J, Kosky RG, Perezl NM, Damicela RA, Cabreral AR, Joval MC, Medero Vegal
SPV, Perezl MB and Roux N (2012). New explant for somatic embryogenesis induction and
plant regeneration from diploid banana (‘Calcutta 4´, Musa AA). Biotecnología Vegetal. 12(1):
25 - 31,
Meenakshi S, Shinda BN and Suprasanna P (2011). Somatic embryogenesis from immature male
flowers and molecular analysis of regenerated plants in banana, LAL KELA (AAA). (Versión
electronic Journal of Fruit and Ornamental Plant Research. 19(2): 15-30.
Merkle SA, Parrott WA and Flinn BS (1995). Morphogenic aspects of somatic embryogenesis.
In: Thorpe, T. A., ed. In vitro embryogenesis in plants. Dordrecht: Kluwer Academic Publishers,
155–203.
Murashige T and Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco
tissue culture. Physiol Plant. 15: 473-497.
Navarro C, Escobedo RM and Mayo A (1997). In vitro plant regeneration from embryogenic
cultures of a diploid and a triploid, Cavendish banana. Plant Cell Tissue Organ Cult. 51: 17-25.
16
Novak FJ (1992). Musa (bananas and plantains). - In Biotechnology of Perennial Fruit Crops
(F.A. tfammerschlag and R.E. Litz, eds), pp. 449-4,88. CAB International, Walling-ford.
Novak FJ, Aftza R, Van Dure M, Dallos MP, Conger B and Xiaolang T (1989). Somatic
embryogenesis and plant regeneration in suspension cultures of dessert (AA and AAA) and
cooking (ABB) bananas (Musa spp.). Biotechnology. 7: 154-159.
Osuji JO, Okoli BE and Ortiz R (1996). An improved procedure for mitotic studies of the
Eumusa section of the genus Musa L. (Musaceae). Infomusa. 5: 12–14.
Osuji JO, Crouch J, Harrison G and Heslop-Harrison JS (1997). Identification of the genomic
constitution of Musa L. lines (bananas, plantains and hybrids) using molecular cytogenetics.
Annals of Botany. 80: 787–793.
Osuji JO, Crouch J, Harrison G and Heslop-Harrison JS (1998). Molecular cytogenetics of Musa
species, cultivars and hybrids: Location of 18S-5.8S – 25S and 5S rDNA and Telomere-like
sequences, Ann. Bot. 82: 243-248.
Purves WK and Brown HM (1978). Indoleacetaldehyde in cucumber seedlings. Plant Physiol. 6:
104–106.
Ramirez-Villalobos M and De Garcia E (2008). Obtainment of embryogenic cell suspensions
from scalps of the banana CIEN-BTA-03 (Musa sp., AAAA) and regeneration of the plants.
Electronic Journal of Biotechnology. 11(5): Special Issue.
Samson JA (1982). Tropical Fruits. New York: Longman.
Sandoval J, Tapia A, Muller L and Villabobos V (1991). Obervaciones sobre la variabilidad
encontrada en plantas micropropagades de Musa cv. Falso Cuerno AAB. Fruits. 46: 533-539.
17
Sasson A (1997). Importance of tropical and subtropical horticulture, future prospects of
biotechnology in tropical and subtropical horticulture species. International Society for
Horticultural Sciences (ISHS) Leiden Acta Hortic. 460:12-26.
Schoofs H, Panis B, Strosse H, MA M., Lopez Torres J, Roux N, Dolezel J and Swennen R
(1999). Bottlenecks in the generation and maintenance of morphogenic banana cell sus-pensions
and plant regeneration via somatic embryogenesis therefrom. Infomusa. 8(2):3-7.
Strosse H, Schoofs H, Panis B, Anre E, Reyiniers K and Swennen R (2006). Development of
embryogenic cell suspensions from shoot meristematic tissue in bananas and plantains (Musa
spp.). Plant Science. 170(1): 104-112.
Varisai M, Wang CS, Thiruvengadam M and Jayabalan N (2004). In vitro plant regeneration via
somatic embryogenesis through cell suspension cultures of HORSEGRAM [MACROTYLOMA
UNIFLORUM (LAM.) VERDC.]. In Vitro Cell. Dev. Biol. Plant. 40:284–289.
Vuylsteke D and De Langhe E (1985). Feasibility of in vitro propagation of bananas and
plantains. Trop Agric. 62: 323-328.
Vuylsteke DR (1989). Shoot-tip culture for the propagation, conservation and exchange of Musa
germplasm. International Board for Plant Genetic Resources, Rome.
Vuylsteke D and Swennen R (1992). Biotechnology approaches to plantain and banana
improvement at IITA. In: Biotechnology: Enhancing Research on Tropical Crops in Africa.
International.
Zaffari G, Kerbauy G, Kraus J and Romano E (2000). Hormonal annelid histological studies
related to in vitro banana bud formation. Plant Cell, Tissue & Organ Culture. 63(3): 187-192.
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)