BARC Newslett" 168 Found,,', Day Spedall"ue 2002

andStrategies for in vitro PropagationSynthetic Seeds in BananaT.R. Ganapathi, P.Suprasanna, V.M. Kulkarni, V.A. Bapat and P.S. RaoN",leee Ageic"'t", cod 8iotechool09Y DivioioaShobh, Atomic R"eocch Ceetee

Introduction

The word banana is derived from the Arabicword far fingee. There are more than 300kinds af banana but only a few arecommercially important. Banana is the man'soldest and most valued fruit ccap. It isprized for its nutritive value with highcacbohydrates (22.2%), fibre (0.84%) andpcatein (1.1%) with less fat (0.2%) andwater (75.7%). The World production ofbanana is about 95 million tons and most of

the production is consumed locally.

In India, It is the most important fruit cropand is grown in 4.3 million hectare with atotal production of 13.9 million tonnes.Several cultivars of banana are cultivated inthe countey among which, Dwarf Cavendishand Robusta are peedominantly grownbecause of higher yields, resistance to strongwinds and shoot cropping dueation besides agood profit margin (Singh 1990). In additionto these, cultivaes such as Poovan, Rasthali,Lalkela, Safed velchi and Kaeibale monthanace also geown. Banana is severely affectedby viral (bunchy top virus, cucumber mosaicvirus, banana steeak virus), bacterial (mokoor bacterial wilt, bacterial soft rot), fungal(black sigatoka, Fusarium wilt) agentsbesides insects/pests and nematodes.

Abstract

thatoff,.of baoana ",fng

Banana is a long duration crop of one and ahalf years and is propagated vegetatively bysuckers. The production of suckers varies indifferent genotypes ranging from 5-10 per

plant per year. Crop productivity andmaturity is dependent on the size and age ofsuckers and uneven maturity extends theduration by 3-4 months. Suckers also carrysoil nematodes, disease causing ocganismssuch as bunchy top virus, leaf spot etc.,thereby affecting the crop productionconsiderably. In this regard, biotechnologicalapproaches such as cell and tissue culture,protoplast fusion and gene transfer offeras useful tools (Novak et a/ 1993,Ganapathi eta/.,. 2002). In vitro propagationof banana through shoot tip cultures IS usefulin the rapid multiplication of desirabledisease free plantlets. In addition carefulselection and updating of mother plantsresults in impcaved crop yield (Vuylsteke,1989).

Enset [Ensete superbum] is the Indian wildbanana, propagated by means of seeds anddoes not produce vegetative side suckersnaturally. The Ensets are the staple food ofalmost 10 million people in Ethiopia. Thesehave capacity to withstand severe draught,and have also been used to help ward offfamine in Ethiopia. The wild counterparts are

BARCN,w,I,"" 169 Found,,', Day Sp,cial I"u' 2002

known lor their broad genetic base and carryseveral desirable genes and thus the Ensetscould be utilized for improvement ofcultivated bananas by employing various invitro techniques (Vuylsteke and Swennen,1993; Novak et al., 1992). Conventionalpropagation of Enset is time consuming andhence there is a need for optimization oftissue culture techniques for the rapidpropagation of enset.We describe here our studies on in vitropropagation of banana and enset, low coststrategies for in vitro propagation andsynthetic seeds in banana.

Materials and Methods

Suckers of ten varieties viz., Basrai,Shrimanthi, Rasthali, Lalkela, Poovan,Ardhapuri, Karibale monthan, Safed Velchi,Mutheli and Hazari were collected and

established in the departmental greenhouse. Cultivars, Williams and Grande Nainewere obtained in the form of sterile cultures

from INIBAP's Germplasm Transit Centre,Belgium. Male inflorescences of en set were

collected from the wild plants growing on thesteep hill slopes of Sinhagad hill fort of theWestern Ghats, at an altitude of 800 m.

Shoot tips were. isolated from suckers byremoving the sheathing leaf bases and wereestablished in liquid MS medium (Murashigeand Skoog 1962) supplemented with 5 mg!1benzyl adenine (BA). After 3 weeks thesewere transferred to semi-solid medium

comprising of 2 mg!1 BA and 30 mg!1adenine sulphate (AS) and 3% sucrose. Eachshoot tip produced 3-S shoots within a spanof 3-4 weeks. Multiplication of shoots wascarried out by isolating individual shoots andsubculturing them on the same medium till asufficient number of shoots were obtained.The individual shoots isolated from these

multiple shoot cultures were used for various

experiments. Individual shoots weretransferred to medium with napthaleneaceticacid (NAA) for root induction.

Investigations on the effect of cyanobacterialextracts (CYE) were conducted with

cyanobacterial strain (Plectonema boryanumUT x 594) grown on BGll medium. Thedetailed procedures are given in Ganapathi

et al., (1994). Shoot tips were grown onmedium with CYE ( 10, 20, 40, 80 %) and

also CYE (100%) alone, along with growthregulators.

Experiments were conducted with

commercial grade sugar (CGS) and tapwater (TW) in place of sucrose and distilledwater, respectively. Multiplication of shootsand rooting of shoots was tested on mediumwith CGS (3% ) and TW. Shoots were also

encapsulated in sodium alginate (3%) andtheir conversion into plants was examinedin the above media combinations

(Ganapathi et a/., 1995). For each treatment24 shoots were used and all the experimentswere conducted under controlled conditions

of light (1000 lux), temperature (25:t2' C)and relative humidity of 65%. Rootedplantlets were transferred to poly bagscontaining good horticultural soil and farmyard manure (1:1) in the green house athigh humidity. About 8 week old hardenedplants were used for field planting atExperimental Field Facility, Trombay, BARCand Gujarat State Fertilizers Company

(GSFC) Ltd., Vadodara, Gujarat.

Results and Discussion

In vitro propagation of banana

Initially shoot tip cultures were established invitro for the induction of multiple shoots(Fig.lA). The multiple shoots showedelongation and produced roots in liquidmedia (Fig.lB). Individual shoots transferred

to rooting media formed complete plantletswithin 4 weeks (Fig. Ie). After another period

of 4 weeks, plantlets transferred to polybagsin the green house exhibited good growth bythe emergence of new leaves (Fig.1O).

More than 15,000 plants were regenerated invitro and around 4,500 plants were planted atmulti!ocations in the states of Maharashtra

and Gujarat for field trials. A field trial was

conducted at R&D farm Gujarat StateFertilizers Company, Baroda (Fig. IE) and thedata suggested an early maturity by about 6weeks and an increase in yield of 33% in thetissue cultured plants of cv. Basrai ascompared to control. The tissue culture raised

BARC N,w""",, 170 Found,,', Day Sp,cial!"u, 2002

Fig.l..

A8CD.E.FGH.

Vadodoca.

BARC New,'etter 171 Founder', Day Speda! "'ue 2002

plants grown at the Experimental Field Facilityat BARC, Trombay, showed more vigorousgrowth, early maturity and increase in bunchweight with good quality fruits (Fig.1F).

In vitro propagation of Enset

Male inflorescences were cultured on MS

medium supplemented with BA (2 109/I) andgibberellic acid (GA3 1 109/I). The extremeapical region of male inflorescencesnecrosed, but the surrounding floralprimordia showed growth and produced

profuse leafy structures initially. After 2subcultures, the proportion of leafystructures decreased substantially «10 %)and a mixture of corm tissue and multipleshoots became predominant (>90%).Longer shoots (2.9 em) were induced on MS+ BA (2 109/I) + AS (30 109/I) + GA3compared to shoots of 1.7 and 1.8 emlength on MS + BA (2 109/I) + AS (30 109/I)with and without caesin hydrolysate (500109/I) respectively (Kulkarni et al., 1997a).

Efficacy of various media tested for rootinduction showed that MSmedium alone wasineffective, whereas MS medium with NAA(Img/l) gave highest number of rootsfollowed by MS + indole-3-acetic acid (IAA)or indole-3- butyric acid (IBA). Addition ofcharcoal (0.1%) to these media did notimprove rooting efficiency. Rooted plantletsexhibited normal growth upon transfer topolybags in the greenhouse and in the field.

There have been very few reports concerningmicropropagation of Ensets. Afza et a/.,(1996) reported micrapropagatian of E.ventricosum using shoot-tips fromvegetative corms. Mathew and Philip (1997)reported regeneration using vegetative shootapices in Ensete superbum. In the this study,cultured floral apices were employed as analternative method for the in vitropropagation of enset. In the case of triploidbanana cultivars, floral apices havesuccessfully been used for the propagation ofChandrabale, Rasthali, Robusta (Doreswamyand Shahi)ram, 1989) and cvs. Monthan andRobusta (Balakrishnamurthy and SreeRangasamy, 1988). The protocol for theregeneration E. superbum using male

inflorescences will be useful for rapid in vitropropagation.

Low cost strategies for inpropagation

The main objective of this investigation wasto develop a low cost in vitro technique forthe micropropagation of banana bysubstituting laboratory grade sucrose anddistilled water with commercial grade sugarand tap water, respectively, for minimizingthe use of the expensive components of thenutrient media.

vitro

The shoot tips grown on media prepared indistilled water containing lab grade sucraseor commercial grade sugar showed theformation of multiple shoots (4-5 shoots/hoot tip) in four weeks. There was nosignificant change in the frequency ofmultiple shoot formation in both the mediaand almost 100 % of the shoots responded.In an another experiment when tap waterwas used instead of distilled water, moretime was required for the induction ofmultiple shoots and proliferation and thetime of the subculture passage had to beextended to 6 weeks.

As presented in the Table 1, the excisedshoots from multiple shoot cultures weretransferred on various media for plantletregeneration. The shoots cultured on MSmedium prepared in distilled water andcontaining either laboratory grade sucrose orcommercial grade sugar showed a goodshoot growth having 2-4 roots with lateralsin three weeks. The hoots showedcomparatively slow growth on similarmedium prepared in tap water. Thefrequency of plantlet formation was almost95% and there was no change in the numberof roots and their laterals.

Plantlets regeneration was also observed onKnop's salts with MS minor, iron andvitamins with lab grade sucrose orcommercial grade sugar, In mediumprepared either with distilled water or tapwater. The response was comparativelybetter in medium prepared in distilled water,as has been shawn in Table 1. The frequency

BARC New,le"« 172 Found«'s Day Speeial/"ue 2002

Table 1 : Effect of different media on plantlet formation from shoot tips of banana

of plantlet formation was around 75-80% onKnop's salt alone with lab grade sucrose orcommercial grade sugar.

The rooted plantlets (7-9 em in length)thus produced were transplanted into

ploybags for hardening in the green housefor about two months before field planting.

In the present study with banana tissuecultures, laboratory grade sucrose was

totally eliminated by using 3 % commercialgrade sugar and for the preparation ofmedia clean tap water of good quality was

employed instead of distilled water. Thoughall the components of MS medium were

necessary for shoot multiplication, rootingand regeneration of plantlets was achievedonly on Knop's salts (KNO3,

Ca(NO3)2.4H20, MgSO4.7H20, KH2PO4)with NAA and commercial grade sugar.These alterations in the medium would be

useful in lowering the total cost of tissuecultured bananas without affecting the rateof multiplication and quality of plants.

Effect of cyanobacterial extract (CYE)

The effect of cyanobacterial extracts(Plectonema boryanum UT x 594) was tested

on growth and multiplication of shoots fromshoot tips as well as encapsulated shoot tipsof banana. Shoot tips reared on MS mediumcontaining different concentrations of CYE(10, 20, 40 and 80% v/v), showed inductionof multiple shoot formation (2-4). Higherconcentrations (40 or 80%) exhibitedincreased frequency of multiple shootformation (Fig.2) and shoot growthcompared to other concentrations. In onetreatment, with complete elimination of MSbasal medium, shoot tips were cultured onCYE supplemented with 3% sucrose, 5 mg/lSA and 0.8% agar. Although multiple shootformation was noted in all the concentrationswith varying frequencies, the shootsexhibited slow growth.

Liquid cultures were also initiated withdifferent concentrations of CYE (10-100%v/v) to minimize the use of nutrientsand growth regulators. Among these, 10and 20 % CYE induced 2-4 shoots while

100% CYE gave as many as 8 shoots andthe shoots elongated with 2-3 pairs ofleaves. To regenerate complete plantlets,the elongated shoots were cultured on MSmedium with CYE (10, 20, 40 and 80% v/v)and plants were regenerated on all thefour concentrations with varying frequencies

Media + carbon % cultures No. of Plantletsource (3%) + forming roots/

etk:f:;' JNAA (1 mq/lJ lantlets lantletA. MS basal salts +I) sucrose/commercial grade 100 2-4 7-9

sugar in distilled waterII) sucrose/commercial grade 95 2-3 7-8

suoar in taD water

B. Knop's salts +MS minor, iron & vitamins+III) sucrose/commercial grade 85 2-3 6-8

sugar in distilled waterIV) sucrose/commercial grade 85 2-3 6-8

suoar in taD waterC. Knop's salts +V) sucrose/commercial grade 75 2-3 6-7

sugar in distilled waterVI) sucrose/commercial grade 75 2-3 6-7

suoar in taD water

Data obtained after 4 weeks' 24 exnlants ner treatment.

BARC New,lett" 173 Foond,,', Day Spedal "'ne 2002

with 1 mg/I NAA or 5mg/I BA and also ondifferent substrates likesterile absorbent cotton

( Fig. 1H), soil, filterpaper and soilrite (allmoistened with 1/4thMS salt solution) forgermination. Amongthese, White's mediumgave high frequency(100%) plantlet deve-

160 I lopment within a weekcompared to othersubstrates and MS

media. Encapsulatedshoot tips directly sownIn petri platescontaining autoclavedsoil showed the emer-

gence of shoots (10%) but failed to formcomplete plantlets.

120 140

F'g. 2 , Effect of Cyanobactec!al Ext.act (eYE) on muWple ,hootcultu.., and planetfo.mation !n banana

(Fig.2). Similar to the controls (shoots on MSmedium with 1 mg/I NAA), 100% of shootscultured on MS medium with 80% CYEdeveloped into plantlets within 4 weeks andall the plants were transplantable.

Cyanobacterial extracts ace known tostimulate somatic embryogenesis and theconversion of synthetic seeds in carrot(Wake et al., 1991, 1992) and sandalwood(Bapat et al., 1996). Although the exactfactors that are involved in such a responseare unknown, it has been postulated thatcyanobacterial extracts may synthesize awide variety of compounds including plantgrowth regulating substances (Metting andPyne 1986). In the present study, CYE hasshown promotive effects on shootmultiplication and plantlet developmentsuggesting that CYE can be employed in thein vitro propagation of banana.

Synthetic seeds

Shoot tips excised from the shoot cultures ofbananas cv. Basrai were encapsulated toprepare synthetic seeds, in 3% sodiumalginate solution prepared either in distilledwater or MS medium with 0.1% activatedcharcoal (Fig.1G) and an antibiotic mixture(Ganapathi et al., 1992). Encapsulated shoottips were placed on MS or White's media

The results revealed that the encapsulatedshoot tips can be handled like a seed andcould be useful in minimizing the cost ofproduction as 1 ml of medium is sufficient forencapsulation of a single shoot tip comparedto 15-20 ml for conversion of shoot tips intoplantlets. By directly sowing the encapsulatedshoot tips in soil, the two stage process suchas rooting and hardening can be eliminated.As compared to suckers, encapsulated shoottips present as inexpensive, easier and safermaterial for germplasm exchange,maintenance and transportation (Rao et al.,1993).

Prospects for the improvement ofbanana

Banana has assumed an important statusamong fruit crops owing to the significantresearch inputs in the areas of cellular andmolecular biology. Globally research is beingfocussed on aspects relevant to the needs ofthe developing countries. Mutation inductionusing physical as well as chemical mutagenscan be useful for the induction of geneticvariability. Towards this goal, we haveconducted research as part of theCoordinated Research Project (CRP) of

BARC New,l"'" 174 Founder', Day Spedall"ne 2002

Table 2: Biotechnological approaches for the improvement and increasingproductivity in banana

Productivity

Germplasm storage &Transportation

Fungal diseases

Viral diseases

Nematodes

Post harvest lile

Cloning of high yielding varieties

Cryopreservation, Synthetic seeds

Antifungal proteins, Antimicrobial compounds,Toxins

Coat protein genes, Replicase

Bt genes, Chitinases

Anti polygalacturonase ( PG),Anti-ethylene biosyntheticSynthase, ACC Oxidase)

genes (ACC

International Atomic Energy Agency (IAEA),Vienna, on the 'Mutation breeding andrelated biotechnologies for banana

improvement (Kulkarni et al., 1997b, Rao etal., 1999).

Somatic embryogenesis offers as an ideal

system for the production somatic embryoson a large scale for use in the preparation ofsynthetic seeds, propagation and genetictransformation. In this direction, somaticembryogenesis has already been established

in our laboratory from immature maleflowers and proliferating shoot tips(Ganapathl et a/., 1999, 2001).Embryogenic cell suspension culturesdeveloped from proliferating shoot tips havebeen useful as the best target tissue forgenetic transformation using Agrobacterium

tumefaciens (Ganapathi et a/., 2001).

Introduction of genes for fungal diseaseresistance may enable the development ofnew lines with disease resistance in banana.

One of the approaches includes theexpression of antimicrobial peptides.

Improving fruit quality with options fordelayed fruit ripening can have significanceto boost banana exports from developingcountries. Another dimension in the genetic

engineering is the production of recombinantproteins or peptides of pharmaceuticalimportance in transgenic plants. Productionof edible vaccines in an edible plant tissue,

for example, in fruits of banana can be ofgreat benefit to the developing countries togain access to the much-needed health care.Studies In this direction have already shown

some success towards obtaining transgenic

plants of banana Incorporated with usefulcharacters like disease resistance and fordeveloping edible vaccines (Ganapathi et al.,2002 ).

Acknowledgements

Authors thank Dr. R.K. Iyer, MolecularBiology and Agriculture Division, BARC, forproviding the cyanobacterial strain(Plectonema boryanum UTx 594).

References

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BARC Newslen.. 176 Founder's Day Spe<ial Issue 2002

This paper was presented the Best Poster award in micro propagation in theNational Symposium on "Plant Tissue Culture: Commercialization,Diversification of Agriculture twd PreserVtltion of Fragile Ecosystems", heldduring April 10-12, 1996, at G.B. Pant University of Agriculture and Technology,

Pantnagar

About the autho", ...Dr T.R.Ganapathl did his Ph.D. from Karnatak Unlve"ny,Dh.,wad, and joined BARC In 1991. He Iswo,klng In the Plant Cell Cultuee Teohnology Secllon of Nude., Ag,loultuee and Bloteohnology DMslon.His speolaUzatlon Is In the field of Plant IIssue wltuee. He has stand.,dlzed mlceopropagallon protocolsIn banana and the technique has been transfe"ed to user agencies. He has also estabUshed methods fa,somallo embryogenesis and agrobactenum mediated transfo,mallon In banana using embryogenic cell

oultuees. CureenOy, he is working on Genetio T,ansfa,mation fa, the In"'rporation of useful traits into ceop plants.

E1

~

Dr P. Suprasanna joined BARC in ,9g, after obtaining his Ph.D. degree in Genetics hom OsmaniaUnive"ity, Hyderabad. He Is working In the Plant Cell Culwee Technology Section of NA & BTD and isengaged in plant bio_nological eesearch on rice, banana and sugaroane. His ",nlrlbutions have beenin the areas of cell and IIssue wlture, somatic embryogenesis, in vnro selection and synthetic seeds.D, Suprasanna has to his credn. more than 60 ,esearch pubUcations in joumals and books pubUshed bynational and Intemational publlshe". His Inteeests are In using plant cell wltuces fa, unde"tandlngmechanISm of d,ffeeentiation as well as fa, genetic manipulation.

Dr VIshvas H. Kulkarni joined BARC In 1993 and subsequenOy obtained Pti.D. (BlotechnoI09Y) deg,eefrom "'ysoce Unive"ity. 1n the Plant Cell Cultuce Technology Section of NA & BTD, he has been workingon the use of tissue cultuee, molecular biological approaches in Banana and Pearl "'illet and onmutagenesis in vitro using gamma i"adiation for the ISOlation of banana mutants. He has also beenInvolved in ",lIaborabve research projects, and has guided graduate and postgraduate students far thecompletion of thei, res..,ch projects. He has pubUshed several eesearch pape", in National andInternational Journals/Symposia/Wo,kshops.

Dr V.A. Bapat is wo,klng In the a,ea of plant tissue cultuce fa, the last 31 yea". The main theust maof h05 work Is on ""oropropa9allon ond Genello hansformallon of Plants. He has effecllvely ",nt,lbutedfa, donal propagallon. suspension wlluees, protoplasts, synthello seeds and bloceact" production ofsomallo emb,yos In a peedous focest tcee - Sandalwood. He has also done extensive wack onmiceopropagation of Banana and "'ulbeeN' He has sevocal publications in National and 1ntemationaljoumals to his "edit. Cuccently he is heading the Plant Celf Culwce Technology Section.

II Dc P- S- Rao, fa"""c Head, Nude.. Agricultuce and Biotechnology Division, joined BARC in 1966. andsubsequentiy obtained his Doctoca'e in Bo"'ny hom Delhi Univecsity In 1967. As a Visiting Scientist,he did advanced ees..rch in plant mo",hogenesis at the CNRS labs In Gif-suc-Yvette, Fcanre and Inprotoplast culluee (19BI-82) at the "'ax-Planck Institute, Koln, Geemany. He Is a Fellow of theNational Academy of Sclenres of India, and National Academy of Agcioultucal Sdences. Dc. Rao hasguided semal students foc M.Sc, M. Teoh. and Ph.D. He has publ/shed mace than 170 ..searchpapees/artloles. He has been the membee of advlsocy bo..d of semal national ..search rentces.unlveesltles and govemment bodies such as Dep..tment of Bloteohnology (D8T), Dep..tment of

Sclenre & Teohnology (DST). He Is a member of sevocal professional societies In 1ndia and abroad, and Is seNlng on theeditorial board of scientific jouma/s. Cun-ent/y, D,.Rao Is the Vlce-Pcesident (Biotechnology) of Indo-American HybcidSeeds (lndia) Pvt. Ltd., Bangaloce.

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