production of haploids plants from anther culture of musa paradisiaca cv. ‘puttabale’

International Journal of Research and Scientific Innovation (IJRSI) | Volume IV, Issue III, March 2017 | ISSN 2321–2705

www.rsisinternational.org

Production of Haploids Plants from Anther Culture of

Musa Paradisiaca cv. ‘Puttabale’

K. Girish Kumar1, V. Krishna

2*, Venkatesh

3, R. Shashikumar

4, H. S. Arunodaya

5 1, 2, 4, 5

Department of P. G. Studies and Research in Biotechnology, Bioscience Block, Kuvempu University, Jnana Sahyadri,

Shankaraghatta‐577 451, Shivamogga (Dist.), Karnataka, India.

3Department of Biochemistry Indian Institute of Science Bangalore. Karnataka, India

* Corresponding author: V. Krishna

Abstract: Haploid plants were regenerated from the anther

callus of banana Musa paradisiaca (AB) cv. Puttabale. The

highest frequency of callus induction (90%) was observed

at the concentration of 3mg/l 2, 4-D . After 20 days of

incubation organization of embyroids were organised

from the callus mass. Interaction of 4mg/l BAP and 0.4

mg/l IAA provoked shoot growth of the embryoids and

well organised roots were developed at the concentration

of 0.6 mg/l NAA and the media was agumented with 0.2%

activated charcoal. Flow cytometry study was carried out

to analyse the DNA content of the regenerated haploid

plants. The results of the investigation reported the

efficient production of haploid plants from the anther

culture.

Key words: Musa paradisica, puttabale, anther culture,

haploid plants

I. INTRODUCTION

anana and plantain (Musa spp.) are the world’s major

food crop for millions of people. Many breeders

improved the quality by manipulating the ploidy of this crop

(Pillay et al., 2003). But the conventional breeding method is

laborious and took more generations for the development of

haploid plants. The production of haploids through anther

culture has many advantages since it does not imply the

inbreeding depression caused by selfing and does not involve

pollinator genome contamination. Many factors are involved

and interact in the optimization of the anther culture protocol

to produce haploid plants; therefore it is necessary to consider:

pre‐treatment of anthers, genotype effects, the stage of

pollen development, culture conditions and induction of

embryos and regeneration of plants. Anther or microspore

cultures have been found to be the most efficient techniques

for obtaining a large number of haploid plants (De Buyser and

Henry.,et al 1980). Haploid production is extremely important

for crop improvement and eradication of pathogens (Brown

and Thorpe et al., 1995) and dramatically improves the yield.

(Badoni and Chauhan et al., 2010). Anther or microspore

cultures has proved to be the most efficient techniques for

obtaining a large number of haploid plants (Buyser and Henry

et al., 1980) and valuable in plant breeding and genetics

studies (Custódioa et al., 2005). The establishment of

homozygous lines of new varieties is possible in a short

period of time, hence provide a useful tool to accelerate plant

breeding successions (Devaux and Li et al., 2001; Zhang et

al., 2011). Haploids can obtained through natural

parthenogenesis or androgenesis. Artificial induction of

ovaries (Muren et al., 1989), ovules (Hansen et al., 1994),

anthers (Foroughi et al., 1982), microspores (Kohler and

Wenzel et al., 1985) is also a promising tool for haploid

production.

In the genus Musa, accurate determination of the

ploidy by phenotypic traits, including stomatal size, density,

and pollen size, and by chromosome counting is difficult and

laborious, mainly due to strong genotypic influences (Hamill,

1992; Vandenhout et al., 1995; Van Duren et al., 1996;

Dolezel, 2004). Musa paradisiaca cv. Puttabale (AB group)

belongs to family Musacae and an indigenous banana cultivar

of Karnataka, India. Previously we published the high

frequency plant regeneration protocol from shoot tip, leaf and

immature male floral explants. No reports available on

haploid production from this plant. This paper deals with the

in vitro haploid plant regeneration from anther culture of

banana cultivar Puttabale.

II. MATERIALS AND METHOD

Explants Collection And Sterlization

Banana cv puttubale inflorescence of appropriate

stage was collected from farmyard of Shimoga district,

Karnataka. The inflorescence were disinfected in 2% sodium

hypochloride solution for 15 min, followed by 8-10 rinses in

sterile distilled water.The immature male flowers are detached

from bract, tepals aseptically. Male flowers contained five

stamens with fully developed anthers. Anthers from the

stamens was carefully isolated and transfered into pertriplates.

Anthers with uninucleated microspore stage was determined

using 1% aceto-carmine stain at this stage anthers were

inoculated on to the callusing media.

Plant Regeneration

B



The callusing media consisted of MS media

constituents agumented with 40 g/l sucrose, 8 g/l agar,

160 mg/l adenine sulfate, 100 mg/l tyrosine and the growth

regulator 2, 4-D tested at the range of 1 to 6 mg/l. The

pH was adjusted to 5.8 before autoclaving at 121ºC for

20 min. The cultures were incubated at 25 ± 2ºC with

12 hrs photoperiod and the callogenic frequency

was evaluated after 30 days of incubation. Regenerated

androgenic embryos like structures developed from callus

were transferred to proliferation medium containing different

conentrations of BAP (2 to 6 mg/l) and IAA( 0.2 to 0.6 mg/l)

the media was agumented with 100mg/l of adanine sulphate

and tyrosin. The well grown elongated shoots were

aspectically transferred to the medium containg 0.25 to

o.75mg/l NAA and without hormones fortified with 0.2%

activated charcoal for the proper development of root system.

The plantlets from culture bottles are moved from the

laboratory later they are shifted to green house for primary

hardening where they are first gently washed free of agar

medium. The well rooted plants were washed with 0.2% of

bovastin solution then followed by primary hardening in

cocopeats, maintained in polythene house for a period of two

weeks. During this period, 90-95 % humidity is maintained

for the initial 6-8 days under diffused light, later they were

maintained at 70 %, humidity and 26 °C Then transferred to

green house for secondary hardening in a polythene bags

containing red soil, sand and cattle manure in the ratio of

1:1:2 respectively. The regenarents grew to the height of 10

to 15 cm were transferred to field condition.

Determination of Ploidy Level

The flow cytometric technique was followed to

determine the ploidy level of the anther-derived plants. Leaf

pieces (1×1 cm) from in vitro plants derived from anther

cultures were chopped with a razor blade in 600 μl buffer

solution consisting of 45 mM MgCl2, 30 mM Tri-sodium

citrate (Na3C6H5O7, 2H2O), 20 mM MOPS, 1% triton X-

100, 10 mM sodium-metabisulfite (Na2S2O5) (pH 7) to

obtain a nuclei suspension. The whole sample was sieved

through a 40-μm mesh nylon filter and stained with 6 μg ml-1

DAPI solution. A diploid parental plant was used as an

internal standard. Nuclei analysis was carried out by using

Partec CA II flow cytometer following the method of Assani

et al., 2003.

III. RESULTS AND DISCUSSION

The anthers isolated aseptically were inoculated on to

the callusing media (1 mg/l. 2,4D to 6 mg/l. 2,4D) showed

callogenic response within a week of incubation. Prior to

inoculation anthers were selected at the diod or tetraoid stage

of meiosis (Fig A) Optimum callogenesis noticed at the

concentration of 3 mg/l. 2,4D (Table.1). On callusing media

anthers began to swell and the size increases to three folds.

After ten days of incubation, callogenic mass noticed from the

dorsal side while the anther wall cells turns black (Fig B).

After 20 days of culture organization of embyronic nodules

were appeared from the sprouted callus mass. The formation

of androgenic embryroids from the protruded callus was

similar to the findings of Assani et al., 2002.

The interaction of BAP at higher levels (2.0 – 6.0

mg/l) and IAA at lower levels (0.2 ‐ 0.6 mg/l) provoked the

growth and development of the enbryonic mass in to shoot

buds. The mean number of shoots organized at each

combination of BAP and IAA is shown in the (Table 1). The

shoot multiplication was optimized at the concentration of 4

mg/l BAP and 0.4 mg/l IAA with a mean of 7.10±0.88 shoots

per callus. shoot buds. The caulogenic mass with multiple

shoots were subjected to colchecin treatment with a range of

(0.025% and 0.05%). Then they were transferred to the

development media consisted of 0.6 mg/l NAA and 0.2%

activated charcoal. The well rooted shoots were maintained

on the media for two to three cycles, then they were

transferred to the rhizogenic media.

The induction of roots depends on the composition of

mineral nutrients and growth regulators. Shoot buds were

transferred to MS with 0.6 mg/l NAA, 0.2% activated

charcoal initiated roots from the base of microshoots ( Fig E),

after three weeks of culture. The well rooted plantlets (Fig H)

were hardened primarily in coco peats containing equal

proportion of garden soil, sand, and cattle dung manure in

polythene house for a period of two weeks (Fig I) and then

they were subjected to secondary hardening in perforated

polythene bags in green house (Fig J). The presence of diploid

plants was also observed among the regenerants. While this

could be a consequence of the regeneration of diploid anther

tissues such as anther wall or connective tissue, in monocots

the regeneration of somatic anther tissue has been very rarely

reported. The other possibility is the spontaneous

chromosome doubling in haploid cells. The possible factors

leading to spontaneous doubled-haploid plants could be

nuclear fusion in the early divisions of the microspores,

endomitosis, endoreduplication or multipolar mitosis during

the callus phase (Chen et al.1982; De Buyser and Henry1986).

The polar transport of auxin is essential for the establishment

of bilateral symmetry during embryogenesis in

dicotyledonous and monocotyledonous species (Fisher,c, et

al.1996).

Androgenesis has been traditionally divided in two

main stages, namely induction and regeneration. In the former

one, somatic cells acquire embryogenic characteristics by

means of a complete reorganization of the cellular state,

including physiology, metabolism and gene expression

(Feher, A. et al. 2002). In the present study also androgenic

embryoids developed from the primary callogenic mass and

upon subculture on the cytokinin supplemented media

embryoids grew up in to plantlets.

Many authors have reported that the developmental

stage of anthers can drastically affect plant regeneration

because microspores respond only at a specific development

stage, which ranges from the tetrad, early and mid-uninucleate

to the early binucleate stage. As such anther of the Lilium ×



‘Enchantment’, excised at the uninucleate microspores stage

could form callus and regenerate bulblets (Niimi et al., 2001).

Medium is another important factor affecting anther culture.

Custódio et al. (2005) reported that the best medium for calli

induction of carob tree was MS media supplemented with 2.3

μM 2, 4-D + 8.2 μM TDZ. Plant regeneration from anther of

banana (Assani et al., 2003) was obtained in 4.4 μM BAP +

2.3 μM.

While genotype or days in vitro are factors that favor

the frequency of appearance of DNA polyploid plants or even

DNA aneuploids (Tremblay et al. 1999; Endemann et al.

2001; Currais et al. 2013), Therefore, the changes observed in

nuclear DNA content might be caused by the effect of growth

regulators used in the culture media. We noticed that synthetic

auxin 2, 4-D might be involved in embryoid formation from

the microspore mass, because it was found to be a factor

favouring changes in the genome of in vitro regenerated plants

(May and Sink 1995; Jin et al. 2008; Clarindo et al. 2008).

Additional assays would be useful to confirm the possible role

of 2,4-D as a factor promoting the variations observed, as well

as NAA or BAP, which were also mentioned as possible

mutagenic agents (Lim and Loh 2003; Mishiba et al. 2006;

Barow and Jovtchev 2007).

The histogram developed from the result of

cytometric analysis (Fig. 2 .A and B) showed a prominent

peak of nuclei in PI fluorescent intensity in Gi l and G1 stages

of interphase of the doubled diploid cells. The intensity of the

nuclei in the interphase of doubled diploid plant and it is two

folds as compared to haploid cells. Extensive fluorescent

emissions at higher intensities are the indicative of

populations of nuclei at increased ploidy levels. Flow

cytometric analyses was performed by measuring the leaf

DNA intensity of the in vitro regenerated plants. The report

of Jin et al. (2008) suggested that detection of plants with

lower nuclear DNA intensity value is due to the existence of

aneuploid plants. This assumption is based on the fact that

many of the changes that can be detected by FCM are

associated with variations in the number of chromosomes

(Clarindo et al. 2008; Currais et al. 2013). These variations

might be enough to indicate the complete or partial loss of a

chromosomes during doubling process (Pfosser et al. 1995;

Roux et al. 2003).The flow cytometric studies enlightened

many investigators to detect plants with values of nearly

twice the nuclear DNA content, suggesting a possible in vitro

polyploidization (Barow and Jovtchev 2007).

IV. CONCLUSION

Anther culture is the classical method employed in

the regeneration of haploid plants but in conventional

breeding production of haloid plant require screening of many

generations and it is difficult in pseudo fruit crops like banana.

The results of the investigation reported here showed the

efficient production haploid plants from the anther culture of

Musa paradisica cv. Puttabale

Fig. 1 High frequency regeneration of plantlets from anther explants of Musa paradisica cv. Puttabale.

A. Uninuleate stage of anther. B. Development of anther callus. C and D.

Androgenic embryos formed from the calli. E and F. Shoot growth of the embroids. G. Growth of haploid plants. H. Root induction from the haploid

plants. I. Primary hardening plants. J. Secondary hardened haploid plants.

A

B

C D

E F

G H

I J



Figure 2

Histograms of relative DNA content obtained after flow cytometric analysis of nuclei isolated from (A) leaf tissue of the diploid banana plant, and (B) leaf tissue of the haploid banana plant

Table 1 Effects of growth regulators on frequency of anther callogenesis and plant regeneration from Musa paradisiaca cv.

Puttabale

Plant growth regulators mg/L Frequency of callus formation per

explant

%

Number of multiple shoots per propagule

Mean ± S.D

2, 4 -D mg/L BAP mg/L IAA mg/L

1

2

3

4

5 6

-

- -

-

- -

-

- -

-

- -

-

- -

F Value

-

- -

-

- -

2

2 2

3

3 3

4

4

4

5

5 5

6

6 6

-

- -

-

- -

0.2

0.4 0.6

0.2

0.4 0.6

0.2

0.4

0.6

0.2

0.4 0.6

0.2

0.4 0.6

16.66

36.66

90.00

56.66

43.33 33.33

-

- -

-

- -

-

- -

-

- -

-

- -

-

- -

-

- -

0.90±0.74

1.60±0.70 1.30±0.95

2.00±0.47

3.00±0.67 2.10±0.57

3.20±1.14

7.10±0.88

4.40±1.71

4.00±1.15

5.50±0.97 4.60±1.35

3.30±1.16

5.40±1.58 4.20±1.03

27.3

The value of combination consisted of mean ± S.D. of 10 replicates.

The F-value is significantly different when p< 0.05.



REFERENCES

[1]. Assani , F. Bakry ,F. Kerbellec, R. Haicour G. Wenzel, B.

Foroughi-Wehr (2003) Afza R, Shen M, Zapata J, Xie J, Fundi H, Lee K, Bobadilla E, Kodym A(2000)., Effect of spikelet

position on rice anther culture efficiency., Plant Sci. 153:155-159.

[2]. Assani A, Bakry F, Kerbellec F, Haicour R, Wenzel G, Foroughi B (2003)., Production ofhaploids from anther culture of banana

[Musa balbisiana (BB)]., Plant Cell Rep., 21: 511-516.

[3]. Assani A, Haicour R, Wenzel G, Cote FX, Bakry F, Foroughi- Wehr B, Ducreux G, AguillarME, Grapin A (2001) Plant

regeneration from protoplasts of dessert banana Grande Naine

(Musa spp., Cavendish sub-group AAA) via somatic embryogenesis. Plant Cell Rep 20:482–488.

[4]. Assani A, Haïcour R, Wenzel G, Foroughi-Wehr B, Bakry F, Côte

FX, Ducreux G, Ambroise A, Grapin A (2002) Influence of donor material and genotype on protoplast regeneration in banana and

plantain cultivars (Musa spp.). Plant Sci 162:355–362.

[5]. Barow M, Jovtchev G (2007) Endopolyploidy in plants and its analysis by flow cytometry.In: Dolezel J, Greilhuber J, Suda J

(eds) Flow cytometry with plant cells. Wiley,

Weinheim,Bronsema F, Van O, Prinsen E, Van L(1998)., Distribution of (14C) dichlorophenoxyacetic acid in cultured

zygotic embryos of Zea mays L., JPlant Growth Regul.,17:81–88.

[6]. Barow M, Jovtchev G (2007) Endopolyploidy in plants and its analysis by flow cytometry. In: Dolezel J, Greilhuber J, Suda J

(eds) Flow cytometry with plant cells. Wiley, Weinheim, pp

349–372. [7]. Chen Z, Qian C, Qin M, Xu X, Xiao Y (1982) Recent advances in

anther culture of Hevea brasiliensis (Muell.-Arg.). Theor Appl

Genet 62:103–108. [8]. Clarindo WR, de Carvalho CR, Arau´jo FS, de Abreu IS, Otoni

WC (2008) Recovering polyploid papaya in vitro regenerants as

screened by flow cytometry. Plant Cell Tissue Organ Cult 92:207–214.

[9]. Currais L, Loureiro J, Santos C, Canhoto JM (2013) Ploidy

stability in embryogenic cultures and regenerated plantlets of tamarillo. Plant Cell Tissue Organ Cult 114:149–159.

[10]. Custódioa L, Carneirob MF, Romanoa A (2005). Microsporogenesis and anther culture in Ceratonia siliqua L.).

Scientia Horticulturae, 104: 65-77.

[11]. De Buyser J, Henry Y (1980) Induction of haploid and diploid plants through in vitro anther culture of haploid wheat (n=3x=21).

Theor Appl Genet 57:57–58.

[12]. De Buyser, J. and Henry, Y., Wheat production of haploids, performance of doubled haploids, and yield trials. Biotechnology

in Agriculture and Forestry, Transgenic Crops I (ed. Bajaj, Y. P.

S.), Springer–Verlag, Berlin, 1986, vol. 2, pp. 73–88 [13]. Devaux H, Li P (2001). Enhancement of microspore culture

efficiency of recalcitrant barley genotypes 20: 475.

doi:10.1007/s002990100368 [14]. Dolezel and Batros,.J. Dolezel, J. Batros (2005) Plant DNA flow

cytometry and estimation of nuclear genome sizeAnnals of

Botany, 95 (2005), pp. 99–110. [15]. Endemann, M., K. Hristoforoglu, T. Stauber and E. Wilhelm.

2001. Assessment of age-related polyploidy in Quercus robur L.

somatic embryos and regenerated plants using DNA flow cytometry. Biol. Plant. 44:339–345.

[16]. Feher A, Pasternak T, Otvos K, Miskolczi P, Dudits D(2002).,

Induction of embryogenic competence in somatic plantcells: a review., Biol Bratislava.,57:5–12.

[17]. Fisher C, Neuhaus G (1996)., Influence of auxin on the

establishment of bilateral symmetry in monocots., Plant J.,9:659–669.

[18]. Foroughi-Wehr B, Friedt W, Wenzel G (1982) On the genetic improvement génétique du bananier (Musa spp.). PhD thesis,

Ecole Nationale Agronomique de Rennes, France Jin S, Mushke

R, Zhu H, Tu L, Lin Z, Zhang Y, Zhang X (2008) Detection of somaclonal variation of cotton (Gossypium hirsutum) using

cytogenetics, flow cytometry and molecular markers. Plant Cell

Rep 27(8):1303–1316. [19]. Hamill, S.D. Hamill, M.K. Smith, W.A.(1992) Dodd In vitro

induction of banana autotetraploids by colchicine treatment of

micropropagated diploids Australian Journal of Botany, 40 (1992), pp. 887–896.

[20]. Hansen AL, Plever C, Pedersen HC, Keimer B, Andersen SB (1994) Efficient in vitro chromosome doubling during Beta

vulgaris ovule culture. Plant Breed 112:89–95.

[21]. Jin S, Mushke R, Zhu H, Tu L, Lin Z, Zhang Y, Zhang X (2008) Detection of somaclonal variation of cotton (Gossypium

hirsutum) using cytogenetics, flow cytometry and molecular

markers. Plant Cell Rep 27(8):1303–1316. [22]. Kerbellec F (1996) Etablissement d’une technique d’androgenèse

pour l’amélioration génétique du bananier (Musa spp.).

[23]. Kohler F, Wenzel G (1985) Regeneration of isolated barley

microspores in conditioned media and trials to characterise the

responsible factor. J Plant Physiol 121:181–191.

[24]. Lim WL, Loh CS (2003) Endopolyploidy in Vanda Miss Joaquim (Orchidaceae). New Phytol 159:279–287.

[25]. May RA, Sink KC (1995) Genotype and auxin influence direct

somatic embryogenesis from protoplasts derived from embryogenic cell suspensions of Asparagus officinalis L. Plant

Sci108:71–84.

[26]. Mishiba K, Tawada K, Mii M (2006) Ploidy distribution in the explant tissue and the calluses induced during the initial stage of

internode segment culture of Asparagus officinalis L. In Vitro

Cell Dev Biol Plant 42:83–88. [27]. Murashige T, Skoog F (1962) A revised medium for rapid growth

and bioassays with tobacco tissue cultures. Physiol Plant 15:473–

497. [28]. Muren RC (1989) Haploid plant induction from unpollinated

ovaries in onion. Hortic Sci 24:833–834.

[29]. Niimi Y, Han D, Fujisaki M (2001)., Production of virus-free plantlets by anther culture of Lilium×‘Enchantment’., Scientia

Horticulturae.,90: 325-334.

[30]. Pfosser M, Amon A, Lelley T, Heberle-Bors E (1995) Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat–

rye addition lines. Cytometry 21:387–393.pp 349–372.

[31]. Roux N, Toloza A, Radecki Z et al (2003) Rapid detection of aneuploidy using flow cytometry. Plant Cell Rep 21:483–490.

[32]. Tenhola T, Tanhuanpä P, Immonen S (2005)., The effect of cold

and heat treatments on the anther culture response of diverse rye genotypes., Euphytica.,145: 1-9.

[33]. Tremblay L, Levasseur C, Tremblay F M (1999) Frequency of

somoclonal variations in plant of black spruce (Picea mariana) and white spruce (P.glauca, pinaceae) derived from somatic

embryogenesis and identification of some factors involved in

genetic instability.Am J Bot86:1373- 1381 [34]. Van Duren et al., M. Van Duren, R. Morpugo, J. Dolezel, R.

Afza.(1996) Induction and verification of autotetraploids in

diploid banana (Musa acuminata) by in vitro techniquesEuphytica, 88 (1996), pp. 25–34.

[35]. Vandenhout et al.,H. Vandenhout, R. Ortiz, D. Vuylsteke, R.

Swennen, K.V. Bai(1995)Effect of ploidy on stomatal and other quantitative traits in plantain and banana hybrids Euphytica, 83

(1995), pp. 117–122.

production of haploids plants from anther culture of musa paradisiaca cv. ‘puttabale’

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