香蕉组织培养过程中内生菌污染的控制_英文_

23卷6期2007年11月

生　物　工　程　学　报

Chinese Journal o f Biotechnology V ol. 23　N o. 6

N ovember 　2007

Control of E ndogenous B acterial Contamination and

Micropropagation of a Traditional T able B anana (Musa spp. cv. K anthali) of B angladesh

香蕉组织培养过程中内生菌污染的控制

S oubir T itov 13, Salil K umar Bhowmik 2, Md. Sadrul Alam 1and Sarder Nasir Uddin 1

1Biotechnology and G enetic Engineering Discipline , Khulna Univer sity , Khulna 29208, Bangladesh 2Biotechnology Department , Bangladesh Agricultural Univer sity , Mymensingh 22202, Bangladesh

摘　要　研究了体外培养一种孟加拉传统香蕉(Musa spp. Cv. K anthali ) 的茎尖组织。(011%

HgCl 2处理12min ) , 接种6～15d 后外植体地下茎部分仍有微生物污染() , 的外植体。为确定无污染

培养基, 将等量外植体分别浸泡在含400mg ΠL 氨苄青霉素和200mg ΠL () 1h 。结果表明, 经抗生素处理的外植体完全没有污染, 但培养3周后不能再生, 大, 颜色由苍白转变成浅绿或深绿。, , 所有经抗生素处理过的外植体都开始死亡。:MS+410mg ΠL BA +015mg ΠL K T +15%CW , 平均生长时间为18～21d , :MS+410mg ΠL BA +210mg ΠL I AA +15%CW , 每个茎平均只萌发3～4个芽。最后, 015mg ΠL I BA 的一半浓度的MS 培养基中, 体外培养茎最大生根率达到90%。关键词　香蕉, K anthali , 体外, 茎尖, 抗生素

中图分类号　Q81313　　文献标识码　A 　　文章编号100023061(2007) 0621042207

Abstract 　Shoot tips of a traditional table banana (Musa spp. cv. K anthali ) of Bangladesh were evaluated for in vitro propagation. Initial surface sterilization (with 011%HgC l 2for 12m inutes ) of shoot tips was success ful but m icrobial contam ination (m ostly bacteria ) at the rhizomatous base of the explants was observed within 6～15days after inoculation which eventually killed 85%of inoculated explants. S o , for contam ination free culture establishment explants were soaked in tw o broad spectrum antibiotics namely am picillin and gentam icin. Cent percent contam ination free cultures were established by soaking the explants in 400mg ΠL am picillin or 200mg ΠL gentam icin for 1h. Antibiotic treated explants were found to be full contam ination free but failed to regenerate after 3weeks of culture. But some of them absorbed media for up to 2nd subculture and showed swelling of explants and some color changes from pale white to light Πdeep green. Finally , a few days after 3rd subculture , no growth of explants was observed and all treated explants eventually started to die. Am ong the untreated alive explants the best medium for single shoot development was MS +410mg ΠL BA +015mg ΠL K T +15%CW and average time required for shoot development was 18～21days. But the regeneration percentage was very low (30%) . The best medium for shoot multiplication was MS +410mg ΠL BA +210mg ΠL IAA +15%CW and only average 3～4shoots were formed per shoot. Finally , in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 015mg ΠL I BA. K ey w ords 　banana , K anthali , in vitro , shoot tip ,

antibiotic

Received :M arch 7, 2007; Accepted :April 28, 2007.

3C orresponding author. T el :+[1**********]3; E 2mail :titovbge @yahoo. com

S oubir T itov 等:C ontrol of Endogenous Bacterial C ontamination and Micropropagation of a T raditional T able Banana ……

1043

　　Banana (Musa spp. ) is the 4th largest food crop in

[1]

the w orld and affects lives of 400million people . Being parthenocarpic in nature , bananas and plantains are propagated vegetatively by means of small shoots or “suckers ”from the parent plant. But the rate of multiplication through conventional method is slow and a number of viral diseases (bunchy top virus , banana streak virus ) and other diseases are als o transmitted to new generation. Thus , the productivity of fruits decreases and finally the yield become very poor which affects national economy of the country. In Bangladesh during the last few years the production of banana is decreasing gradually from 703314tons in 1986～1987to 628425tons in 1997

[2]

～1998. It may be due to unavailability of healthy and virus free suckers. Normally four to five suckers are obtained per plants which are insu fficient to replace the affected farms with healthy germ plasm. 　　The development of micropropagation techniques has been a major focus of Musa research during the past tw o decades and such techniques have now been well

[1,3,4]

established for large scale plant production . Micropropagation of banana has been achieved [5][6]

tips and from male floral . clonal propagation by

[7]

G upta in 1986. in vitro are considered to be under s ome degree of stress and may be predisposed to direct in fection , even by bacteria not

[8]

normally pathogenic to them . The medium may contain many different bacterial nutrients , both organic constituents of the medium and exudates from the plant cells. Thus pathogens , endophytes , epiphytes and incidental contaminants may als o occur and may interface

[9]

with growth of the plant tissue . Alm ost all plant pathogenic bacteria develop m ostly in plants as parasites and partly in plant debris or in the s oil as saprophytes. There are great differences am ong bacterial species , in the degree of their development in one or other environment. These bacteria enter plants through w ounds made in roots and over winter in diseased plants or debris , vegetative propagative organs such as potato tubers and banana

[10]

rhizome . F or in vitro propagation of banana , bacterial contamination is a great problem. Although initially surface sterilization w orks , later on microbial contamination at the base of the explant is observed within 7to 15days after inoculation. Bacterial growth is als o observed around the explants in the culture media. Huge numbers of explants are destroyed in the culture due to

[11]

endogenous bacteria .

K anthali (genome AAB ) is a

[12]

traditional table banana cultivar of Bangladesh . The plant is only found in the s outhern part of the country and its population is continuously decreasing due to lack of commercial cultivation. Its production rate is relatively lower but the plant is m ore salt tolerant and disease resistant than other commercial cultivars. Therefore , the present study deals with the control of endogenous bacterial contamination and micropropagation of table banana cv. K anthali from shoot tip explants. The aims of the experimental design addresses the following aspects of in vitro development :

That sterilization procedure can be developed

which will optimize tissue survival in vitro .

That antibiotic treatment minimizes microbial

contamination.

That cytokinin and auxin ratios will facilitate tissue regeneration.

Musa spp. cv.

●●●

1　of explants

K anthali (G enome AAB ) , a leading cultivar of Bangladesh was used as the s ource material for culture establishment. The shoot tips along with a portion of rhizomatous tissue were used. In early of 2005, the experimental plants were collected from a village named Amtola in P olice Station Batiaghata , K hulna and was very near from K hulna University cam pus. The explants were prepared by rem oving the outer layer of tissues from suckers with a clean knife. The pale white tissue blocks containing the shoot tip and rhizomatous bases were surface sterilized with 011%HgCl 2for 12minutes.

112　Antibiotic treatment

Microbial contamination (m ostly bacteria ) at the rhizomatous base of the explants was observed within 6～15days after inoculation which eventually killed 85%inoculated explants. S o , for contamination free culture establishment explants were s oaked in tw o broad spectrum antibiotics namely am picillin and gentamicin. Cent percent contamination free cultures were established by s oaking the explants in 400mg ΠL am picillin or 200mg ΠL gentamicin for 1hour. 113　G row th responses

A fter the

treatment with antibiotics the shoot tips of

[13]

banana were inoculated in MS medium with varying concentrations of horm onal supplements for single shoot formation. But all of them com pletely failed to regenerate

1044

Chinese Journal o f Biotechnology 　生物工程学报　2007,V ol 123,N o 1

and s ome of them abs orbed media for up to 2

subculture. They showed swelling of explants and s ome color changes from pale white to light Πdeep green. Finally

a few days after 3subculture , no growth of explants was observed and all treated explants eventually stared to die.

From the untreated alive explants , in all concentrations of BA tissue swelling and ball like structures were formed. But the control didn ’t respond at all. Highest percentage (40%) of single shoot was formed when explants were cultured on MS +410mg ΠL BA +015mg ΠL K T +15%CW and the average length of shoot was 4150137cm. These single shoots were further subcultured on different concentrations of cytokinin (BA ) , auxin (I AA ) and 15%CW for multiplication of regenerated shoots. The growth rate was very slow and 40～45days after inoculation only highest 3～4shoots were produced in MS medium containing 410mg ΠL BA +210mg ΠL I AA +15%CW. Finally , in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 015mg ΠL I BA. 114　Computation and presentation of data

Data on different parameters from different of shoot proliferation were culture and those were recorded after 1～of The data presented in tables in the forms of percentage of explants showed proliferation Πrooting for each experiment were com puted by as follows :

Percentage of explants showed proliferation =×100%

No. of explants cultured Percentage of explants formed roots =×100%

No. of explants cultured

(C ontaminated and dead explants were excluded ) In the table , the mean data of different replications of each treatment was accom panied by Standard error of mean as an only statistical measurement which was calculated as follows :

Standard Error (S. E. ) =S n Here ,

S =Standard deviation

n =Number (viz. no. of explants , no. of culture )

Standard deviation (S. ) =Where , n =Sam ple size

2　R esults and discussion

The experiment was conducted at Plant Biotechnology Laboratory of K hulna University , Bangladesh during January to November 20051One indigenous banana plant (Musa spp. cv. K anthali ) of Bangladesh was studied in order to establish suitable protocols for in vitro plant regeneration. F or culture initiation , all the experimental explants were cultured on MS medium supplemented with different concentrations of cytokinins and auxins for prom oting m orphogenic responses. The results of the experiment are described as follows. 211　Sterilization procedure for explants

T o overcome contamination problem surface sterilization of the explants was done with 011%(W ΠV ) HgCl 2for different durations to assess the contamination percentages and viability of the explants used for in vitro culture. M ost of the explants showed bacterial 3of inoculation if they of cultured explants in fection singly and a few number of showed bacterial and fungal in fection together. None of the shoot tip explants were found contamination free after 15days when treated with 011%HgCl 2for 1, 2, 4and 6minutes. Only 20%explants were found alive and contamination free without any tissue damage when treated for 12minutes with the same concentration of HgCl 21It was found that explants were killed with the increasing duration of treatment with 011%HgCl 2s olution. 5%～25%explants were killed due to tissue injury when treated for 15minutes but highest number of explants (40%) found contamination free (Fig. 1) .

212　E ffect of antibiotic for contamination free culture establishment

Surface sterilization result showed that only 20%shoot tip explants were found alived and contamination free when treated for 12minutes. M ost of the explants died within 6～15days due to broad range of bacterial and fungal contamination. Hence , it was necessary to further sterilize the explants for 100%contamination free culture establishment. F or that purpose explants were

μimmersed in screened (with 0122m disposable filter )

antibiotics (am picillin and gentamicin ) for different durations to ensure contamination free cultures. The results show that cent percent contamination free cultures could be obtained by s oaking the explants in 400mg ΠL am picillin or 200mg ΠL gentamicin for 1hour (T able 1) .

n (n -1)

x )

S oubir T itov 等:C ontrol of Endogenous Bacterial C ontamination and Micropropagation of a T raditional T able Banana ……

1045

Fig. 1　Different m ode of fungal and bacterial contamination on surface sterilized banana shoot tip explants after 3weeks of culture

A &B:only fungal contamination ; C :both fungal and bacterial contamination ; D :only bacterial contamination.

T able 1　E ffect of different concentrations of antibiotics immersion at different duration of time to ensure contamination free cultures

N o. of

explants treated 10101010

G entamicin

10101010

Duration of treatment Πmin

[***********]20

(mg ΠC oncentration of antibiotics ΠL )

50N o 11121222

%[**************]0

N o 3556656100

%[**************]0

N o 4567610200

%4050607060N o [**************]

400

%[***********]00100

C ontamination free explants found

Antibiotics

Am picillin

213　G row th responses of Antibiotic were cultured on MS with different kinds of growth regulators (auxin and cytokinin ) . But after 3weeks of culture they were com pletely failed to regenerate. But s ome of them abs orbed media for up to

subculture and showed swelling of explants and s ome color changes from pale white to light Πdeep green. Finally

a few days after 3subculture , no growth of explants was observed and all treated explants eventually stared to die (T able 2) .

T able 2　G row th responses on antibiotics treated experimental shoot tip explants

Duration of treatment Πmin

G rowth responses

after 3weeks of culture

(mg Π(mg ΠG entamicin ΠL ) Am picillin ΠL )

200

3060

N o shoot formation N o shoot formation

400N o shoot formation N o shoot formation

C olor change from primary explant

after 3week of culture (mg Π(mg ΠG entamicin ΠL ) Am picillin ΠL )

200Pale white

to deep green Pale white to deep green

400Pale white to deep green Pale white to light green

Enlargement of explants Enlargement of explants 1st subculture (3weeks )

2nd subculture (3weeks ) Enlargement of explants Enlargement of explants

3rd subculture (3weeks ) Explants died Explants died

214　Shoot initiation , multiplication and rooting of regenerated shoots from non 2treated alived explants

F ormerly , only 20%explants were found alive and contamination free after 2weeks of culture on MS medium supplemented with different growth regulators. Those non 2antibiotics treated alive explants showed quite promising growth responses on their growth medium (Fig. 2) . The best medium for single shoot development was MS +410mg ΠL BA +015mg ΠL K T and average time required was 18～21days. F or the production of multiple shoots regenerated single shoots were cultured in MS media with

different concentrations of auxin and cytokinin. The best medium for shoot multiplication was MS +410mg ΠL BA +210mg ΠL I AA +15%CW. And with MS +410BA +210K T +210I AA +15%CW s ome promising am ount of rooting was als o observed. But overall multiplication rate was als o too low (40%) and only average 3～4shoots were formed. Finally , in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 015mg Π

L I BA (T able 3～5, Fig. 3) .

1046

Chinese Journal o f Biotechnology 　生物工程学报　2007,V ol 123,N o 16

T able 3　E ffect of different concentrations of BA in combination with KT and 15%CW for single shoot

form ation from shoot tip explants

(mg ΠG rowth regulators concentration ΠL )

(BA +K T +15%CW )

310+015310+110310+210410+015410+110410+210510+015510+110510+210

N o. of single shoots

regenerated

423865342

%of explant responded

[***********]

Days required for shoot formation

20～2520～2520～2318～2115～2117～2116～2117～2116～21

Average length of shoots

( x +S. E. )

210±0112214±0112217±0139415±0137412±0121410±0117310±0102219±0102311±0129

(mg ΠH orm onal supplements ΠL )

(BA +K T +IAA +15%CW )

310+015+210310+110+210310+210+210410+015+210410+110+210410+210+210510+015+210510+110+210510+210+210

N o. of single shoots inoculated

[***********]

N o. of single shoot showed multiplication

112123121

%of explant responded

[1**********]010

Average no. of multiple shoots Πexplant

2223222

Average time

required Πd 45～5045～5045～5040～4540～4540～4540～4540～4540～45

Intensity of

rooting

--+--++--+

　　“+”indicates very few am ount ++ount of rooting ; “-”no root formation

T able 5　E of IBA in h alf strength MS medium on adventitious root form ation

in vitro (mg ΠH orm onal supplement ΠL )

I BA

010(C ontrol ) [1**********]0

%of shootlets rooted

4060809080

N o. of roots Πshootlet

( x ±S. E. )

211±0135219±0116410±0112413±0123312±0114

Average length of

roots Πcm ( x ±S. E. )

219±0114314±0122412±0110417±0126410±0137

Days

to emergence

of roots

15～2012～1512～1510～1515～20

Fig. 2　Establishment of 100%contamination free cultures on antibiotic treated shoot tip explants (A ) and no shoot induction on MS medium supplemented with 410mg ΠL BA +015mg ΠL K T +15%CW (B ) after 3weeks of culture.

　　The experimental results indicated that initial surface

sterilization of shoot tips was success ful but microbial contamination at the rhizomatous base of the explants was observed within 6～15days after inoculation which eventually killed 85%inoculated explants. The situation

[11]

was very similar with Hadiuzzaman et al (2001) . Identification of endophytic bacteria and their elimination from banana tissue culture was als o done by s ome other

[14-17]

researchers . From the untreated alived explants different types of cytokinin , auxin and their concentrations significantly in fluenced shoot multiplication and elongation. Furtherm ore , the joint effect of BA and I AA increased shoot elongation com pared to BA alone. The optimum BA concentration does not vary significant

μam ong researchers. F or exam ple , BA at 2212m ol ΠL was

[5]

optimal in studies by Cronauer and K rikorian (1984)

[18]

μand Jarret et al (1985) and at 20m ol ΠL in a study by

[1][19]

Vuyslteke (1989) . W ong (1986) stated that

μ4414m ol ΠL BA reduced shoot multiplication. Arinaitwe

[20]

et al (2000) mentioned that shoot proliferation was cultivar dependent. R ooting in the in vitro proliferated shoots can be induced in a number of ways. F or rooting of

[21]

banana and plantain , Novak et al (1990) , Banerjee

[3]

and De 2Langhe (1985) used half strength MS +

[22]

μ110used half strength m ol ΠL I BA and H ossain (1997) MS +210mg ΠL I BA. On the other hand , Cronauer and

[5]

K rikorian (1984) used horm one 2free MS medium for rooting of banana shoots. They als o used I AA , I BA or NAA in different concentrations for root induction and reported that addition of activated charcoal (0125～215mg ΠL ) increased the average root length but not root

[23]

number. Banerjee et al (1986) found that regenerated shoots formed roots in MS s olid medium with half strength of macro 2salts with 012mg ΠL I BA in banana and plantain. In the present investigation , auxin free media and media containing one type of auxin (I BA ) at concentrations was used to in

condition.

propagation of banana , bacterial contamination is a great problem. S o , the aim was to establish a protocol for contamination free culture development using shoot tip explants. K ey factors investigated in this study include different sterilization techniques to ensure contamination free culture and the selection of appropriate growth regulator levels to achieve success ful in vitro regeneration.

Shoot tips of banana (Musa spp. cv. K anthali ) were evaluated for in vitro propagation. The initial surface sterilization (with 011%HgCl 2for 12minutes ) was success ful but microbial contamination at the base of the explant was observed within 6～15days after inoculation which eventually killed 85%inoculated explants. S o , for contamination free culture establishment explants were s oaked in 200mg ΠL gentamicin or 400mg ΠL am picillin for 1hour. Antibiotic treated explants found to be full contamination free to regenerate after 3of s abs orbed media for up

swelling of explants and from pale white to light Πdeep green.

a few days after 3subculture , no growth of explants was observed and all treated explants eventually stared to die. Am ong the untreated alived explants the best medium for single shoot development was MS +410mg ΠL BA +015mg ΠL K T +15%CW and average time required for shoot development was 18～21days. But the regeneration percentage was very low. The best medium for shoot multiplication was MS +410mg ΠL BA +210mg ΠL I AA +15%CW and average time required for production of multiple shoots from single shoot was 40～45days. Multiplication rate was als o too low and only average 3～4shoots were formed in that particular concentration. The in vitro proliferated shoots produced roots with maximum frequency in half strength of MS medium fortified with 015mg ΠL I BA.

Fig. 3　S ingle shoot induction on MS medium supplemented

w ith 410mg ΠL BA +015mg ΠL K T +15%C W (A ) and multiplication of shoots on MS medium supplemented with 410mg ΠL BA +

2mg ΠL I AA +15%CW (B )

3　Conclusion

This study examined the application of

micropropagation protocols to assist germ plasm conservation of a traditional cultivar table banana [Musa spp. cv. K anthali (G enome , AAB ) ]of Bangladesh. This has im plications for commercial explant production in large scale as it generally produces 5～6suckers from a mature m other plant per year. And for in vitro

　　The interest in Musa spp. cv. K anthali both for domestic and export sales w ould be increased in future. It is essential that an appropriate method to expedite introduction to cultivation be determined. The success of in vitro propagation methods reported that the experimental plant can be tissue cultured with further plant growth regulators experimentation. Success ful micropropagation will be of major benefit to the Agricultural Biotechnology making conventional breeding method unnecessary thus greatly assisting in the

[11]

Hadiuzzaman S , Habiba U , Reza S , Saha M L , K han MR. Development of a sustainable protocol for contamination free culture of table bananas and identification of ass ociated endogenous bacteria. In :4th International Plant T issue Culture C on ference (1st

conservation of this unique Bangladeshi plant.

Acknow ledgements 　The research w ork was carried out

on Plant Biotechnology Laboratory under Biotechnology and G enetic Engineering Discipline , K hulna University , K hulna 29208, Bangladesh. Therefore , the authors are thank ful to the pers onnel of that Laboratory for their invaluable help and support. K hulna University Research Cell sanctioned funds for conducting the research. LIST OF ABBREVIATIONS BA 　　62Benzyl adenine CW C oconut water I AA Indole 232acetic acid I BA Indole 232butyric acid K T K inetin (62furfuryl amino purine ) MS Murashige &Skoog (1962) medium NAA α2Napthaleneacetic acid W ΠV Weight per v olume REFERENCES

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Vuylsteke D. Shoot 2tip culture for the exchange of Musa germ germ plasm in [2]

for Plant

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pp. 241

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Murashige T , Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. 1962, 15:473-4971

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Cronauer SS , K rikorian AD. Rapid multiplication of banana and plantains by in vitro . Horticultural Science , 1984, 19:234-2351D oreswamy R , Sahijram L. M icropropagation of banana from male floral apices culture in vitro . Scientia Horticulturae , 1989, 40:181-1881

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23卷6期2007年11月

生　物　工　程　学　报

Chinese Journal o f Biotechnology V ol. 23　N o. 6

N ovember 　2007

Control of E ndogenous B acterial Contamination and

Micropropagation of a Traditional T able B anana (Musa spp. cv. K anthali) of B angladesh

香蕉组织培养过程中内生菌污染的控制

S oubir T itov 13, Salil K umar Bhowmik 2, Md. Sadrul Alam 1and Sarder Nasir Uddin 1

1Biotechnology and G enetic Engineering Discipline , Khulna Univer sity , Khulna 29208, Bangladesh 2Biotechnology Department , Bangladesh Agricultural Univer sity , Mymensingh 22202, Bangladesh

摘　要　研究了体外培养一种孟加拉传统香蕉(Musa spp. Cv. K anthali ) 的茎尖组织。(011%

HgCl 2处理12min ) , 接种6～15d 后外植体地下茎部分仍有微生物污染() , 的外植体。为确定无污染

中图分类号　Q81313　　文献标识码　A 　　文章编号100023061(2007) 0621042207

antibiotic

Received :M arch 7, 2007; Accepted :April 28, 2007.

3C orresponding author. T el :+[1**********]3; E 2mail :titovbge @yahoo. com

S oubir T itov 等:C ontrol of Endogenous Bacterial C ontamination and Micropropagation of a T raditional T able Banana ……

1043

　　Banana (Musa spp. ) is the 4th largest food crop in

[1]

[2]

[1,3,4]

established for large scale plant production . Micropropagation of banana has been achieved [5][6]

tips and from male floral . clonal propagation by

[7]

G upta in 1986. in vitro are considered to be under s ome degree of stress and may be predisposed to direct in fection , even by bacteria not

[8]

[9]

[10]

[11]

endogenous bacteria .

K anthali (genome AAB ) is a

[12]

That sterilization procedure can be developed

which will optimize tissue survival in vitro .

That antibiotic treatment minimizes microbial

contamination.

That cytokinin and auxin ratios will facilitate tissue regeneration.

Musa spp. cv.

●●●

1　of explants

112　Antibiotic treatment

A fter the

treatment with antibiotics the shoot tips of

[13]

banana were inoculated in MS medium with varying concentrations of horm onal supplements for single shoot formation. But all of them com pletely failed to regenerate

1044

Chinese Journal o f Biotechnology 　生物工程学报　2007,V ol 123,N o 1

and s ome of them abs orbed media for up to 2

subculture. They showed swelling of explants and s ome color changes from pale white to light Πdeep green. Finally

a few days after 3subculture , no growth of explants was observed and all treated explants eventually stared to die.

Percentage of explants showed proliferation =×100%

No. of explants cultured Percentage of explants formed roots =×100%

No. of explants cultured

Standard Error (S. E. ) =S n Here ,

S =Standard deviation

n =Number (viz. no. of explants , no. of culture )

Standard deviation (S. ) =Where , n =Sam ple size

2　R esults and discussion

212　E ffect of antibiotic for contamination free culture establishment

μimmersed in screened (with 0122m disposable filter )

n (n -1)

x )

S oubir T itov 等:C ontrol of Endogenous Bacterial C ontamination and Micropropagation of a T raditional T able Banana ……

1045

Fig. 1　Different m ode of fungal and bacterial contamination on surface sterilized banana shoot tip explants after 3weeks of culture

A &B:only fungal contamination ; C :both fungal and bacterial contamination ; D :only bacterial contamination.

T able 1　E ffect of different concentrations of antibiotics immersion at different duration of time to ensure contamination free cultures

N o. of

explants treated 10101010

G entamicin

10101010

Duration of treatment Πmin

[***********]20

(mg ΠC oncentration of antibiotics ΠL )

50N o 11121222

%[**************]0

N o 3556656100

%[**************]0

N o 4567610200

%4050607060N o [**************]

400

%[***********]00100

C ontamination free explants found

Antibiotics

Am picillin

subculture and showed swelling of explants and s ome color changes from pale white to light Πdeep green. Finally

a few days after 3subculture , no growth of explants was observed and all treated explants eventually stared to die (T able 2) .

T able 2　G row th responses on antibiotics treated experimental shoot tip explants

Duration of treatment Πmin

G rowth responses

after 3weeks of culture

(mg Π(mg ΠG entamicin ΠL ) Am picillin ΠL )

200

3060

N o shoot formation N o shoot formation

400N o shoot formation N o shoot formation

C olor change from primary explant

after 3week of culture (mg Π(mg ΠG entamicin ΠL ) Am picillin ΠL )

200Pale white

to deep green Pale white to deep green

400Pale white to deep green Pale white to light green

Enlargement of explants Enlargement of explants 1st subculture (3weeks )

2nd subculture (3weeks ) Enlargement of explants Enlargement of explants

3rd subculture (3weeks ) Explants died Explants died

214　Shoot initiation , multiplication and rooting of regenerated shoots from non 2treated alived explants

L I BA (T able 3～5, Fig. 3) .

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Chinese Journal o f Biotechnology 　生物工程学报　2007,V ol 123,N o 16

T able 3　E ffect of different concentrations of BA in combination with KT and 15%CW for single shoot

form ation from shoot tip explants

(mg ΠG rowth regulators concentration ΠL )

(BA +K T +15%CW )

310+015310+110310+210410+015410+110410+210510+015510+110510+210

N o. of single shoots

regenerated

423865342

%of explant responded

[***********]

Days required for shoot formation

20～2520～2520～2318～2115～2117～2116～2117～2116～21

Average length of shoots

( x +S. E. )

210±0112214±0112217±0139415±0137412±0121410±0117310±0102219±0102311±0129

(mg ΠH orm onal supplements ΠL )

(BA +K T +IAA +15%CW )

310+015+210310+110+210310+210+210410+015+210410+110+210410+210+210510+015+210510+110+210510+210+210

N o. of single shoots inoculated

[***********]

N o. of single shoot showed multiplication

112123121

%of explant responded

[1**********]010

Average no. of multiple shoots Πexplant

2223222

Average time

required Πd 45～5045～5045～5040～4540～4540～4540～4540～4540～45

Intensity of

rooting

--+--++--+

　　“+”indicates very few am ount ++ount of rooting ; “-”no root formation

T able 5　E of IBA in h alf strength MS medium on adventitious root form ation

in vitro (mg ΠH orm onal supplement ΠL )

I BA

010(C ontrol ) [1**********]0

%of shootlets rooted

4060809080

N o. of roots Πshootlet

( x ±S. E. )

211±0135219±0116410±0112413±0123312±0114

Average length of

roots Πcm ( x ±S. E. )

219±0114314±0122412±0110417±0126410±0137

Days

to emergence

of roots

15～2012～1512～1510～1515～20

　　The experimental results indicated that initial surface

[11]

was very similar with Hadiuzzaman et al (2001) . Identification of endophytic bacteria and their elimination from banana tissue culture was als o done by s ome other

[14-17]

μam ong researchers. F or exam ple , BA at 2212m ol ΠL was

[5]

optimal in studies by Cronauer and K rikorian (1984)

[18]

μand Jarret et al (1985) and at 20m ol ΠL in a study by

[1][19]

Vuyslteke (1989) . W ong (1986) stated that

μ4414m ol ΠL BA reduced shoot multiplication. Arinaitwe

[20]

et al (2000) mentioned that shoot proliferation was cultivar dependent. R ooting in the in vitro proliferated shoots can be induced in a number of ways. F or rooting of

[21]

banana and plantain , Novak et al (1990) , Banerjee

[3]

and De 2Langhe (1985) used half strength MS +

[22]

μ110used half strength m ol ΠL I BA and H ossain (1997) MS +210mg ΠL I BA. On the other hand , Cronauer and

[5]

[23]

condition.

swelling of explants and from pale white to light Πdeep green.

Fig. 3　S ingle shoot induction on MS medium supplemented

w ith 410mg ΠL BA +015mg ΠL K T +15%C W (A ) and multiplication of shoots on MS medium supplemented with 410mg ΠL BA +

2mg ΠL I AA +15%CW (B )

3　Conclusion

This study examined the application of

[11]

conservation of this unique Bangladeshi plant.

Acknow ledgements 　The research w ork was carried out

[1]

Vuylsteke D. Shoot 2tip culture for the exchange of Musa germ germ plasm in [2]

for Plant

G enetic

～3rd N ovember , 2001, Dhaka , Bangladesh ) . Abstracts , 2001,

pp. 241

[12]

[13]

Murashige T , Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. 1962, 15:473-4971

[14]

Van Den H ouwe I. E limination of endophytic bacteria from banana tissue culture , in :

[15]

INI BAP annual report.

1997, INI BAP

Publisher , M ontpellier , France , 1998, pp. 121

Van Den H ouwe I , G uns J , S wennen R. Bacterial contamination in

Musa shoot tip cultures. In :G al n Sa Ξco V EEd. Proceedings of

Physiologia Planatrum ,

the first international sym posium on banana in the subtropics , International S ociety for H orticultural Science , Leuven , Belgium , 1998, pp. 485-4921

[16]

[17]

[18]

Jarret R L , R odriguez W , Fernandez R. Evaluation , tissue culture propagation and dissemination of :Saba

[19]

W ong WC.

In vitro propagation of banana (Musa spp. ) :

Res ources , R ome , BBS. S tatistical Y ear Book of Bangladesh. S tatistics Division , M inistry of Planning , G ovt. of the People ’s Republic of Bangladesh , 2002, pp. 13511

[3]

Banerjee N , De 2Langhe E. A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of

Musa (banana and plantain ) . Plant Cell Report , 1985, 4:351-

3541

[4]

Israeli Y, Lahav E , Reuveni O. In vitro culture of bananas. In :G owen S (ed. ) , Bananas and Plantains , Chapman &Hall Publishers , London , 1995, pp. 147-1781

[5][6]

[7]

G upta PP.

Eradication of m osaic disease and rapid clonal

multiplication of bananas and plantains through meristem 2tip culture. Plant Cell , Tissue and Organ Culture , 1986, 6:33-391

[8][9]

Horticulturae , 1988, 225:27-371

Initiation , proliferation and development of shoot 2tip cultures on defined media. Plant Cell , Tissue and Organ Culture , 1986, 6:159-1661

[20]

Arinaitwe G, Rubaihay o PR , M agambo M JS. Proliferation rate effects of cytokinins on banana (Musa spp. ) cultivars. Scientia

Horticulturae , 2000, 86:13-211

[21]N ovak F J , A fza R , Van Duren M , Perea 2Dallos M , C onger BV , X iolang T.

S omatic embry ogenesis and plant regeneration in

suspension cultures of dessert (AA , AAA ) and cooking (AAB ) bananas. Bio ΠTechnology , 1989, 7:154-1591

[22]H ossain MS. Exploitation of in vitro technique for commercialization of banana (cv. Sagar ) plantlets. M. S. thesis , Department of Botany , University of Dhaka ,1997.

[23]Banerjee N , Vuylsteke D , De 2Langhe E. M eristem tip culture of

Musa :H istom orphological studies of shoot bud proliferation. In :

[10]Argios G N. Plant pathology , 3rd ed. Academic Press Inc. , California , US A , 1988, pp :510-5501

W ithers LA , Alders on PC , eds. Plant T issue Culture and its Agricultural Applications , London , 1986, pp. 139-1471

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