Advances in

HorticultureBiotechnologyR E G E N E R A T I O N S Y S T E M S

Volume I: FRUIT CROPS, PLANTATION CROPS AND SPICES

H.P. SinghV.A. ParthasarathyK. Nirmal Babu

Westville Publishing HouseNew Delhi

© Publishers

First Edition 2011

ISBN 978-81-85873-65-7

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted, by anymeans, electronic, mechanical, photocopying, recording, or otherwise, without written permission from thepublisher.

Published by Mrinal Goel, Westville Publishing House47, B-5, Paschim Vihar, New Delhi – 110 063, IndiaTel: 011-25284742, Fax: 011-25267469Email: westville_2002@yahoo.co.in

Printed at Salasar Imaging Systems, New Delhi

H. P. Singh, V. A. Parthasarathy and K. Nirmal Babu (2011) Advances in Horticulture Biotechnology — Regeneration Systems — Fruit Crops, Plantation Crops and Spices (Volume I), pp 484, Westville PublishingHouse, New Delhi

Foreword

Indian agriculture made a rapid stride, converting the country from food scarce tosufficiency. But challenges for the Indian agriculture in 21st century are much greater thanbefore. The growing population has to be fed and surplus has to be produced with decliningland, water and threat of climate change. The horticulture, which includes fruits, vegetables,spices, flowers, and medicinal and aromatic plants, has proved beyond doubt its potentialityfor gainful diversification. Initiatives taken by Government and other stakeholders have impactedthe development in terms of increased production, productivity and availability of horticulturalcrops. The emerging trend worldwide and also in the country is indicative of a paradigm shiftin dietary needs of the people with rise in the income, which demand for more horticulturalproduce. Since the growing of horticultural crops is rewarding to the farmers in terms of returnsper unit area, the sector is expected to contribute significantly for food and nutritional security,employment opportunity and poverty alleviation.

The Indian Council of Agricultural Research is the premier agency which pioneeredsystematic research on agricultural crops in the country. Horticulture research in India receivedvery little attention till the 3rd five year plan. To-day, the horticultural research in the country isbeing carried out at ten ICAR institutes (with 24 regional stations) and 5 Directorates and 8National Research centres (on major crops). Area specific, multi-disciplinary research is alsobeing conducted fewer than 13 -All India Co-ordinated Research Projects at 215 centres locatedat various research Institutes, and State Agricultural Universities. In addition, several net workprojects are now in operation. Research on horticulture is also being undertaken at severalmulti-crop, multi-disciplinary Institutes. Departments of Horticulture in 34 AgriculturalUniversities, three deemed to be Universities and one full fledged University of Horticultureand Forestry are also engaged in horticultural research. As a result, the country now has asound research infrastructure in horticulture to meet the growing needs and expectations of thefast developing horticulture industry. Among the various areas of research being carried outworld wide, Biotechnology stands out as the frontier area of research. India is endowed with astrong manpower in the area of Biotechnology. With the first horticultural transgenic developedin tomato in USA in 1980 (Flavr Savr), the research has led to the development of a large numberof transgenics in potato, squash, corn and recently in brinjal in India. Resent advances in thefields of molecular genetics and recombinant DNA technology has opened up new opportunitiesin agriculture, medicine, and industry and environment protection. The ability to move genesacross sexual barriers has led to heightened interest in the conservation and sustainable and

Foreword

Biotechnology stands out as the frontier area of research for crop improvement. The firsthorticultural transgenic developed in tomato in USA in 1980 (Flavr Savr) has been followedby a large number of transgenics developed in potato, squash, corn and recently in brinjal inIndia. Recent advances in the fields of molecular genetics and recombinant DNA technologyhas opened up new opportunities in agriculture, medicine, industry and environment protection.The ability to move genes across sexual barriers has led to heightened interest in the conservationand sustainable and equitable use of biodiversity. Since biodiversity is the feedstock for plant,animal and microbial breeding enterprises, its conservation becomes more important for effectiveuse. The publication of genome maps of human and Arabidopsis has opened up new vistas inthe area of genomic research and its application. Today we have genome maps available for alarge number of plants and animals including perennial crops like date palm, oil palm andpeach. Of course, we have to go miles before we develop maps for many of the tropical fruitsand vegetables.

There is little doubt that the biotechnology has opened up uncommon opportunities forenhancing the productivity, profitability, sustainability and stability of major cropping systemsand has also created scope for developing crop varieties tolerant/resistant to biotic and abioticstresses through an appropriate blend of GM Mendelian and molecular breeding techniques.These advances have also led to the possibility of undertaking anticipatory breeding to meetpotential changes in temperature, precipitation and sea level as a result of global warming.Marker Assisted Selection has now become an integral part of breeding programmes. Manynovel and powerful markers such as SSRs and SNPs are available to the breeders for preciseand rapid transfer of desired traits. The development of QTLs in perennial crops has becomeeasier with the development of association mapping procedures. It is time that our scientiststake advantage of these tools and apply them for improvement of the crops native to India.

While the benefits are clear, there are concerns about the short and long term impact ofGMOs on the environment, biodiversity and human and animal health. There are also equityand ownership issues in relation to biotechnological processes and products. Thus, there isneed for transparent and truthful risk-benefit analysis in relation to GMOs, on a case-by-casebasis. Biotechnology offers opportunities for converting our biological wealth into economicwealth and new employment opportunities on an environmentally and socially sustainablebasis.

(iv)

equitable use of biodiversity, since biodiversity is the feedstock for plant, animal and microbialbreeding enterprises. Transgenic research should not be undertaken in crops/commodities whereour international trade may be affected Cultivation of GM crops should not be permitted indesignated “Agro-Biodiversity Sanctuaries”. Besides, we should ensure strict safety proceduresso that the food becomes safe for consumption with no public outcry.

In the areas of genomics and bioinformatics, the publication of genome map of human andArabidopsis has opened up vistas in the area of genomic research and today we have genomemaps available for a large number of plants and animals. Genome maps of perennial crops likeDate palm, oil palm and peach are already available. Of course, we have to go miles before wedevelop maps for many of the tropical fruits and vegetables. Very good facilities for genomicsare available in India.

Marker Assisted Selection has become a part of many of the breeding programmes. Manynovel and powerful markers such as SSRs and SNPs are available to the breeders. The developmentof QTLs in perennial crops has become easier with the development of association mappingprocedures. It is time that many of the institutes should develop mapping population for thispurpose. It would not be long before we have the linkage and even genome map for most of thecrops native to India. In addition it also helps in generating employment opportunities andenhancing standards for global competitiveness. There is little doubt that the biotechnology hasopened up uncommon opportunities for enhancing the productivity, profitability, sustainabilityand stability of major cropping systems. It has also created scope for developing crop varietiestolerant/resistant to biotic and abiotic stresses through an appropriate blend of Mendelian andmolecular breeding techniques. It has led to the possibility of undertaking anticipatory breedingto meet potential changes in temperature, precipitation and sea level as a result of global warming.While the benefits are clear, there are concerns about the short and long term impact of GMOs onthe environment, biodiversity and human and animal health. There are also equity and ownershipissues in relation to biotechnological processes and products. Thus, there is need for transparentand truthful risk-benefit analysis in relation to GMOs, on a case-by-case basis. Biotechnologyoffers opportunities for converting our biological wealth into economic wealth and newemployment opportunities on an environmentally and socially sustainable basis.

With the vast available literature scattered over various sources, it is difficult for researchersand students to know the status of science of biotechnology of various horticultural crops. Theattempt by Dr.H.P.Singh, Deputy Director General (Horticulture), ICAR, New Delhi is highlya visionary approach. I am impressed by the volume of talent we have in India on biotechnology.He has tried to collate all the work done on these crops in five thematic areas, namely,regeneration systems, molecular markers, gene cloning, transgenics and diagnostics. The seriesof volumes on these thematic areas will be highly useful to all plant biotechnologists in generaland horticulturists in particular.

Dr R.S. Paroda

With the vast available literature scattered over various sources, it is difficult for researchersand students to remain abreast of all the biotechnology related developments in varioushorticultural crops. I compliment the efforts of Dr. H.P. Singh, Deputy Director General(Horticulture), ICAR, New Delhi for compiling and editing the series of the books entitled“Advances in Horticulture Biotechnology”. Commendable efforts have been made in thistreatise to collate all the work done on horticultural crops in five thematic areas, namely,regeneration systems, molecular markers, gene cloning, transgenics and diagnostics. The serieswill be highly useful to all plant biotechnologists in general and horticulturists in particular.

R.S. ParodaChairman

Trust for Advancement of Agricultural ScienceNew Delhi, India

Preface

Systematic research on fruits, vegetables and ornamental crops in India began in 1954with the initiation of independent institutions and programmes. The research agenda is designedrelevant to national plans and priorities for the horticulture development. Today, 10 ICARinstitutes with 27 regional stations, 5 Directorates, 7 national research centres, 15 all Indiacoordinated research projects (AICRPS) with 223 research stations, 1 full fledged universityof horticulture, 25 state agricultural universities and 7 multi-disciplinary institutes of the ICARare engaged in horticulture research. In addition, a few R&D establishments of crop/commodityboards and private sectors are providing research support to Indian horticulture. Besides, ICARoperates over 330 ad-hoc research projects. Presently there are 29 revolving fund schemes andmany network projects both within ICAR and outside ICAR. It operates a massive seed projectencompassing all ICAR institutes and SAUs to meet the growing demand of quality seedmaterials. Research system in horticulture is now geared to provide necessary technologicalsupport to the expanding horticulture industry. The strength of horticulture biotechnology interms of man power and infrastructure is great in India. We have to channelize this for effectiveimprovement of horticulture crops and have to keep in mind, the tools of biotechnology can beused across various disciplines and on various crops.

Production of quality planting materials is fast becoming an important input in diseasemanagement programmes especially in production of disease/virus free plants. Many accreditedlaboratories are being established to test the genetic fidelity and virus indexing ofmicropropagated plants. Tissue culture raised plants also become an important source forcolonization and delivery of bio-control agents. In addition, efficient plant regeneration systemsare fast becoming important for in vitro manipulations and transgenic pathways for cropimprovement.

In the present series, volume 1 deals with available information on the micropropagationand plant regeneration technologies developed for fruit crops, plantation crops and spiceswhile volume 2 deals with vegetables, ornamentals and tuber crops. These are comprehensivelycovered with respect to individual crops by experienced scientists. Information on variousexplants sources and media combinations are also presented in a concise form.

We are thankful to the contributors for their sincere effort to prepare comprehensive reviewon the plant regeneration systems of different crops published in this volume. We gratefully

(vi)

acknowledge their contributions. We are also grateful to many others for their participationand help in this publication. We hope this volume would be of use to students and researchersin the field of biotechnology of fruit crops, vegetable crops, ornamentals, tuber crops, plantationcrops and spices.

New Delhi H. P. SinghV. A. Parthasarathy

K. Nirmal Babu

Contents

Foreword ........................................................................................................................................ iiiPreface ............................................................................................................................................ vContributors ................................................................................................................................... xvAcronyms ..................................................................................................................................... xvii

1. Biotechnology in Horticulture – Regeneration Systems — Status, Progress and Future ................................................................................. 1Introduction ................................................................................................................................ 1Micropropagation and Commercialisation ................................................................................ 1Production of Virus Free Plants ................................................................................................. 5Plant Regeneration and Somatic Embryogenesis ....................................................................... 5Somaclonal Variation and In vitro Selection ............................................................................. 6In vitro Flowering, In vitro Pollination and Embryo Rescue ..................................................... 7Development of Haploids .......................................................................................................... 7Synthetic Seeds .......................................................................................................................... 7Protoplast Culture and Development of Somatic Hybrids ........................................................ 7Conservation of Germplasm through In vitro Conservation and Cryopreservation .................. 8Long term Storage of Pollen .................................................................................................... 11Production of Secondary Metabolites ...................................................................................... 12Future Thrusts .......................................................................................................................... 12

FRUIT CROPS

2. Banana ........................................................................................................................... 15Introduction .............................................................................................................................. 15Micropropagation ..................................................................................................................... 16Hardening ................................................................................................................................. 20Production of Virus Free Planting Materials ........................................................................... 21Safe Germplasm Movement — Technical Guidlines ............................................................... 26Somaclonal Variation ............................................................................................................... 28Macropropagation .................................................................................................................... 33Somatic Embryogenesis ........................................................................................................... 34Organogenesis .......................................................................................................................... 43Embryo Culture and Embryo Rescue ....................................................................................... 44

(viii)

Anther Culture .......................................................................................................................... 46Protoplast Culture .................................................................................................................... 50Conservation of Musa Germplasm .......................................................................................... 52Germplasm Exchange and Linkages ........................................................................................ 56Future Perspectives .................................................................................................................. 56

3. Mango ............................................................................................................................. 73Introduction .............................................................................................................................. 73In vitro Propagation ................................................................................................................. 74Somatic Embryogenesis ........................................................................................................... 75Organogenesis .......................................................................................................................... 83Embryo Rescue ........................................................................................................................ 83Shoot Bud Culture .................................................................................................................... 84Hardening ................................................................................................................................. 85In vitro Germplasm Conservation ............................................................................................ 86Future Perspectives .................................................................................................................. 86

4. Papaya ............................................................................................................................ 91Introduction .............................................................................................................................. 91Micropropagation ..................................................................................................................... 91Plant Regeneration ................................................................................................................... 94Protoplast Culture .................................................................................................................... 96Anther Culture .......................................................................................................................... 97Embryo Rescue ........................................................................................................................ 98In vitro Conservation ............................................................................................................... 99Future Perspectives ................................................................................................................ 100

5. Guava ...........................................................................................................................103Introduction ............................................................................................................................ 103Micropropagation ................................................................................................................... 104Callus Culture ........................................................................................................................ 108Somatic Embryogenesis ......................................................................................................... 111Embryo Culture ...................................................................................................................... 113Anther Culture ........................................................................................................................ 113In vitro Conservation ............................................................................................................. 114Future Perspectives ................................................................................................................ 115

6. Citrus ............................................................................................................................121Introduction ............................................................................................................................ 121Micropropagation ................................................................................................................... 121Plant Regeneration ................................................................................................................. 125Rooting and Acclimatization ................................................................................................. 130Somatic Embryogenesis ......................................................................................................... 133Protoplast Culture .................................................................................................................. 138Production of Somatic Hybrids .............................................................................................. 140Rootstock Improvement ......................................................................................................... 142Scion Improvement ................................................................................................................ 145Seedlessness ........................................................................................................................... 148

(ix)

In vitro Auto tetraploid production ........................................................................................ 148Embryo Rescue ...................................................................................................................... 149Haploid Production ................................................................................................................ 150Rootstock Breeding ................................................................................................................ 152Mutation Breeding and Mutant Selection .............................................................................. 156Rootstock Breeding via Embryo Rescue ............................................................................... 157In vitro Conservation ............................................................................................................. 157Cryo Preservation ................................................................................................................... 159Future Perspectives ................................................................................................................ 161

7. Grape ............................................................................................................................173Introduction ............................................................................................................................ 173Micropropagation ................................................................................................................... 174Pathogen Elimination and Production of Virus Free Planting Material ................................ 175Plant Regeneration ................................................................................................................. 175Hybrid Embryo Rescue .......................................................................................................... 177Haploid and Dihaploid Production ........................................................................................ 178In vitro Conservation ............................................................................................................. 180Future Perspectives ................................................................................................................ 181

8. Aonla ............................................................................................................................185Introduction ............................................................................................................................ 185Micropropagation ................................................................................................................... 185Plant Regeneration ................................................................................................................. 188Future Perspectives ................................................................................................................ 189

9. Apple ............................................................................................................................191Introduction ............................................................................................................................ 191Micropropagation ................................................................................................................... 191Organogenesis and Somatic Embryogenesis ......................................................................... 194Embryo Rescue ...................................................................................................................... 197In vitro Conservation ............................................................................................................. 198Future Perspectives ................................................................................................................ 199

10. Pear ...............................................................................................................................203Introduction ............................................................................................................................ 203Micropropagation ................................................................................................................... 203Organogenesis ........................................................................................................................ 206Synthetic Seeds ...................................................................................................................... 207Haploid and Dihaploid Production ........................................................................................ 208In vitro Conservation ............................................................................................................. 208Future Perspectives ................................................................................................................ 208

11. Peach ............................................................................................................................213Introduction ............................................................................................................................ 213Shoot Culture ......................................................................................................................... 214Micropropagation ................................................................................................................... 215Embryo Rescue ...................................................................................................................... 218

(x)

Somatic Embryogenesis and Organogenesis ......................................................................... 218Future Perspectives ................................................................................................................ 220

12. Plum .............................................................................................................................223Introduction ............................................................................................................................ 223Organogenesis and Regeneration ........................................................................................... 224Micropropagation ................................................................................................................... 226Embryo Rescue ...................................................................................................................... 227Cryo Preservation ................................................................................................................... 228Future Perspectives ................................................................................................................ 229

13. Pomegranate ................................................................................................................233Introduction ............................................................................................................................ 233Micropropagation ................................................................................................................... 233Organogenisis ......................................................................................................................... 234Embryogenesis ....................................................................................................................... 237Problems in Micropropagation .............................................................................................. 239Anther Culture ........................................................................................................................ 240In vitro Polyploidy ................................................................................................................. 240Conservation .......................................................................................................................... 240

14. Annona .........................................................................................................................243Introduction ............................................................................................................................ 243Micropropagation ................................................................................................................... 243Problems in Micropropagation .............................................................................................. 247Organogenesis ........................................................................................................................ 248Protoplast Culture .................................................................................................................. 248Embryo Rescue ...................................................................................................................... 248Haploids ................................................................................................................................. 248Triploids ................................................................................................................................. 249Mycorrhiza ............................................................................................................................. 249

15. Bael, Sapota and Wood Apple ...................................................................................253Bael ...............................................................................................................................253

Micropropagation ................................................................................................................... 253Organogenesis .......................................................................................................................... 25Embryogenesis ....................................................................................................................... 257

Sapota ...........................................................................................................................257Micropropagation ................................................................................................................... 257Organogenesis ........................................................................................................................ 258Embryogenesis ....................................................................................................................... 259

Wood Apple .................................................................................................................260Micropropagation ................................................................................................................... 260Organogenesis ........................................................................................................................ 260Embryogenesis ....................................................................................................................... 262

16. Ber ................................................................................................................................265Introduction ............................................................................................................................ 265

(xi)

Micropropagation ................................................................................................................... 265Organogenesis ........................................................................................................................ 270Embryogenesis ....................................................................................................................... 271Embryo Culture and Embryo Rescue ..................................................................................... 272Elimination of Viruses and other Microbes ........................................................................... 274Polyploidy .............................................................................................................................. 275Conservation .......................................................................................................................... 276

17. Datepalm ...................................................................................................................... 279Introduction ............................................................................................................................ 279Micropropagation ................................................................................................................... 279Organogenesis ........................................................................................................................ 280Embryogenesis ....................................................................................................................... 282Problems Micropropagation .................................................................................................. 287Production of Disease and Pest free Plantlets ....................................................................... 289Germplasm ............................................................................................................................. 290Embryo Culture ...................................................................................................................... 290Haploids ................................................................................................................................. 290In vitro Conservation ............................................................................................................. 290Future Perspectives ................................................................................................................ 293

PLANTATION CROPS

18. Coconut ........................................................................................................................299Introduction ............................................................................................................................ 299Production of Homozygous lines ........................................................................................... 302In vitro Screening for Stress Tolerance .................................................................................. 302Embryo Culture for Safe Movement of Germplasm .............................................................. 302Cryopreservation .................................................................................................................... 306

19. Arecanut....................................................................................................................... 313Introduction ............................................................................................................................ 313Somatic Embryogenesis ......................................................................................................... 314Embryo Rescue ...................................................................................................................... 316

20. Oil Palm ....................................................................................................................... 319Introduction ............................................................................................................................ 319Oil Palm Tissue Culture ......................................................................................................... 320Constraints in Micropopagation............................................................................................. 323Protoplast and Anther Culture ................................................................................................ 328In vitro Conservation and Cryopreservation .......................................................................... 328Genetic Transformation .......................................................................................................... 329Somaclonal Variation ............................................................................................................. 331Future Perspectives ................................................................................................................ 334

21. Cashew ......................................................................................................................... 343Introduction ............................................................................................................................ 343Micropropagation ................................................................................................................... 343

(xii)

Micrografting ......................................................................................................................... 351Somatic Embryogenesis and Organogenesis ......................................................................... 351Embryo Rescue ...................................................................................................................... 352Future Perspectives ................................................................................................................ 353

22. Cocoa ............................................................................................................................357Introduction ............................................................................................................................ 357In vitro Multiplication ............................................................................................................ 358Embryo Rescue ...................................................................................................................... 363In vitro Conservation ............................................................................................................. 365Future Perspectives ................................................................................................................ 366

SPICES

23. Black Pepper ................................................................................................................369Introduction ............................................................................................................................ 369Micropropagation ................................................................................................................... 369Plant Regeneration through Shoot Organogenesis ................................................................ 376Plant Regeneration through Somatic Embryogenesis ............................................................ 382Cell Suspension Cultures ....................................................................................................... 387Protoplast Culture .................................................................................................................. 389Anther Culture ........................................................................................................................ 389In vitro Conservation ............................................................................................................. 390Future Perspectives ................................................................................................................ 391

24. Cardamom ...................................................................................................................395Introduction ............................................................................................................................ 395Micropropagation ................................................................................................................... 395Plant Regeneration from Callus Cultures .............................................................................. 397Characterization of Somaclones ............................................................................................ 398Anther Culture ........................................................................................................................ 399Protoplast Culture .................................................................................................................. 399Synthetic Seeds ...................................................................................................................... 400Genetic Transformation .......................................................................................................... 400Conservation of Genetic Resources ....................................................................................... 400Future Perspectives ................................................................................................................ 402

25. Turmeric ......................................................................................................................405Introduction ............................................................................................................................ 405Micropropagation ................................................................................................................... 406Micro Rhizomes ..................................................................................................................... 409Plant Regeneration- Organogenesis and Somatic Embryogenesis ........................................ 411Cell Suspension Culture ......................................................................................................... 414Field Evaluation ..................................................................................................................... 415In vitro Conservation ............................................................................................................. 415Germplasm Exchange ............................................................................................................ 416Cryo Preservation ................................................................................................................... 416

(xiii)

Future Perspectives ................................................................................................................ 416

26. Ginger ...........................................................................................................................421Introduction ............................................................................................................................ 421Micropropagation ................................................................................................................... 421Field evaluation ...................................................................................................................... 424Microrhizomes ....................................................................................................................... 425Plant Regeneration from Callus Cultures .............................................................................. 427Somaclonal Variation ............................................................................................................. 429Induction of Systemic Resistance .......................................................................................... 430In vitro Polyploidy ................................................................................................................. 431Anther Culture ........................................................................................................................ 431Inflorescence Culture and In vitro Development of Fruit ...................................................... 432Protoplast Culture .................................................................................................................. 433Genetic Transformation .......................................................................................................... 434Synthetic Seeds ...................................................................................................................... 435Germplasm Conservation ....................................................................................................... 435Production of Secondary Metabolites .................................................................................... 437Future Perspectives ................................................................................................................ 438

27. Tree Spices ...................................................................................................................443Introduction ............................................................................................................................ 443Micropropagation ................................................................................................................... 444Plant Regeneration in Tree Spices ......................................................................................... 446Synthetic Seeds ...................................................................................................................... 447In vitro Conservation ............................................................................................................. 447Production of Secondary Metabolites .................................................................................... 447Future Perspectives ................................................................................................................ 447

28. Seed Spices ...................................................................................................................451Introduction ............................................................................................................................ 451Application of Tissue Culture ................................................................................................ 451Germplasm Enrichment through Tissue Culture .................................................................... 452Somatic Embryogenesis and Synthetic Seed Technology: .................................................... 452In vitro Conservation of Germplasm ..................................................................................... 453In vitro Flowering ................................................................................................................... 453In vitro Androgenesis and Double Haploids .......................................................................... 453In vitro Mutagenesis .............................................................................................................. 457In vitro Screening for Biotic and Abiotic Stresses ................................................................. 457Production of Secondary Metabolites and Biotransformation .............................................. 458

Index .............................................................................................................................463

Contributors

Anitha KarunCentral Plantation Crops Research InstituteKasaragod – 671 124Kerala

Anju BajpaiCentral Institute for Sub-Tropical HorticultureRehmankhera P.O. Lucknow – 227 107Uttar Pradesh

Hare KrishnaCentral Institute of Temperate HorticultureMukteshwar – 263 138Uttarakhand

Jayanthi M.Director of Oil Palm ResearchPedavegi – 534 450Andhra Pradesh

Leela SahijramIndian Institute of Horticultural ResearchHessarghatta Lake, Bangalore – 560 089Karnataka

Madhu KamleCentral Institute for Sub-Tropical HorticultureRehmankhera, P.O., Lucknow – 227107Uttar Pradesh

Mandal P.K.Director of Oil Palm ResearchPedavegi – 534450Andhra Pradesh

Maneesh MishraCentral Institute for Sub-Tropical HorticultureRehmankhera P.O.Lucknow – 227107Uttar Pradesh

Malhotra S. K.Indian Council of Agricultural ResearchKrishi Anusandhan Bhawan – II, Pusa CampusNew Delhi – 110 012

Minoo D.Indian Institute of Spices ResearchCalicut – 673 012Kerala

Mir J. I.Central Institute of Temperate HorticultureRengreth – 190 007SrinagarJammu & Kashmir

More T. A.Central Institute for Arid HorticultureBeechwalBikaner – 334006Rajasthan

Nazeer AhmedCentral Institute of Temperate HorticultureRengreth – 190 007SrinagarJammu & Kashmir

(xvi)

Neelam ShuklaCentral Institute for Sub-Tropical HorticultureRehmankhera P.O.Lucknow – 227 107Uttar Pradesh

Nirmal Babu K.Indian Institute of Spices ResearchCalicut – 673 012Kerala

Parthasarathy V. A.Indian Institute of Spices ResearchCalicut – 673 012Kerala

Rajesh PatiCentral Institute for Sub-Tropical HorticultureRehmankhera P.O., Lucknow – 227107Uttar Pradesh

Ramakrishnan Nair R.Indian Institute of Spices ResearchCalicut – 673 012Kerala

Ramesh ChandraCentral Institute for Sub-Tropical HorticultureRehmankhera, P.O., Lucknow – 227 107Uttar Pradesh

Reshi T. A.Central Institute of Temperate HorticultureRengreth, Srinagar – 190 007Jammu & Kashmir

Saraswathi M. S.National Research Centre for BananaTiruchirapalli – 620102Tamil Nadu

Selvarajan R.National Research Centre for BananaTiruchirapalli – 620 102Tamil Nadu

Senthil Kumar R.Indian Institute of Spices ResearchCRC AppangalaKarnatakaKerala

Sheeja T. E.Indian Institute of Spices ResearchCalicut – 673 012Kerala

Singh DhurendraCentral Institute for Arid HorticultureBeechwal, Bikaner – 334 006Rajasthan

Singh, H.P.Indian Council of Agricultural ResearchKrishi Anusandhan Bhawan – II, Pusa CampusNew Delhi – 110012

Sivalingam P.N.Central Institute for Arid HorticultureBeechwal, Bikaner – 334 006Rajasthan

ThimmappaiahDirectorate of Cashew ResearchPuttur – 574 202, Dakshin KannadaKarnataka

Uma S.National Research Centre for BananaTiruchirapalli – 620 102Tamil Nadu

Verma M. K.Central Institute of Temperate HorticultureRengreth – 190 007, SrinagarJammu & Kashmir

VijayaKumari N.National Research Center for CitrusShankar Nagar P.O, Nagpur – 440010Maharashtra

Acronyms

2, 4-D : 2,4-dichlorophenoxyacetic acid2iP : N-Isopentenyl amino purineABA : Abscissic AcidAC : Acivated charcoalACC : Acetyl-coA-carboxylasaAFLP : Amplification Fragment Length PolymorphismAIPUB : Association for Improvement in Production and Utilisation of Bananaals : Acetolactate synthase (ALS)AMF : Arbuscular mycorrhizal fungiANOVA : Analysis of varienceAP-PCR : Arbitarily primed PCRBA : 6-benzyladenineBAP : 6-benzyl aminopurinebt : Bacillus thuringiensiscat : Chloramphenicol acetyltransferasechs : Chalone synthaseCF : Culture filtrateCIRAD : Centre d cooperation Internationale en Resherche Agronomique pour le

diveloppementCMS : Cytoplasmic male sterilitycp : Coat Proteincry : Insecticidel Crystal Proteins (ICP)CW : Coconut waterDAF : DNA amplification fingerprintingDAS–ELISA : Double antibody sandwich– ELISADFMA : α- difluoromethyl argentineDFMO : α- difluoromethyl ornithinedhfr : Dihydropholate reductase (DHFR)DMSO : DimethylsulphoxideECS : Embryogenic cell suspensionEDTA : Ethelyenediamine tertra acetic acidELISA : Enzyme linked Immunosorbent assayepsp : EPSP synthase (EPSPS)FACS : Fluorescence activated cell sorterFDA : Fluorescein diacetateGA : Gibberlic acidgent : Gentamycin acetyl transferase (GENT)gus : α-glucuronidase (GUS)

(xviii)

Hepes : N-2-hydroxy ethane piperazine-N’-2-ethanesulphonic acidhyg : Hygromycin phosphotransferase (HYG)I50 : Inhibitor concentration resulting in 50% inhibitionIAA : Indole acetic acidIBA : Indole 3- butyric acidICAR : Indian Council of Agricultural ResearchIITA : International Institute of Tropical AgricultureINIBAP : International Network for Improvement of Banana and PlantainIPGRI : International Board for Plant Genetic Resources (Presently Bioversity International)ISSR : Inernal Simple Sequence RepeatsK- Medium : Knudson orchid mediumKin : Kinetin (6- furfuryl amine)lac : α -galactosidase (LAC)LS medium : Linsmair and Skoog mediumMES : 2-(N-morpholino) ethanesulphonic acidMIC : Minimum concentration resulting in 100% inhibitionMS medium : Murashige and Skoog mediumNAA : Naphthalene acetic acidNASH : Nucleic acid spot hybridisationNBPGR : National Bureau of Plant Genetic Resourcesnos : Nopaline synthase (NOS)nptII : Neomycin phosphotransferase (NPT II)NRCB : National Research Center for Bananaocs : Octopine synthase (OCS)PAGE : Polyacrylamide gel electro phoresis

PCPA :

P-chlorophenoxy acetic

acid

PCR : Polymerase chain reactionPCV : Packed cell volumePDA : Potato dextrose agarPEG : Polyethylene glycolPGR : Plant genetic resourcesPLB : Protocorm like bodiesppm : Parts per millionPPV : Plum pox virusPVP : PolyvinylpyrolidoneQTL : Quantitative trait lociRAPD : Random amplified polymorphic DNARH : Relative humidityRT- PCR : Reverse transcriptase -PCRSA : Salicylic acidSCV : Settled cell volumeSDS : Sodium dodecyl sulphateSH medium : Schenk and Hildebrandt mediumSSR : Simple (short) sequence repeatTDZ : ThidiazuronTERI : Tata Energy Rresearch InstituteTRIA : TriacontanolTTC : 2,3,5-triphenyl tetrazolium chlorideV/V : volume/volumeV/W : volume/weightWPM : Woody Plants Medium (Lloyd & Mc Cowan)

3

MangoRamesh Chandra, Rajesh Pati and Maneesh Mishra

IntroductionThe mango (Mangifera indica. L.) belongs to family Anacardiaceae. It is one of the most

important fruit crops of the world, especially of Asia. The centre of diversity for the genusMangifera is in South East Asia. It is the most widely cultivated species within the genus andhas a natural distribution in southeast Asia. It is grown in almost all states of India and comprisesabout 42% of area under fruits. It is grown up to an altitude of 1500 MASL but the fruiting ispoor above 7000 MASL. The annual world production of mango is more then 14.6 milliontons. Mango fruits are considered as one of the most delicious of all the fruits. The ripe fruit isconsumed as a table fruit as dessert or often between meals and is also processed for preparingpulp, juices, squashes and jams. Premature fruit is used for extraction of tannins and in theproduction of other astringent products, viz., chutneys, curries, cold drinks, pickles, etc. Mangostones are used for raising seedlings, oil, fiber and as animal feed. The kernel is a rich sourceof carbohydrate; calcium, fat and starch can be produced for industrial purposes. Branches andwood are utilized for fuel and timber. The centre of origin and diversity of the genus Mangiferais now firmly established in South East Asia. Malay Peninsula, the Indonesian archipelago,Thailand, Indo-China and the Philipines are the centers of diversity of Mangifera species(Mukherjee 1985; Bompard 1989; Kostermans and Bompard 1993).

Mango is highly heterozygous crop and seed propagation does not ensure true to typeprogeny. Among the different vegetative propagation methods, the most common are grafting,budding and enarching. These methods are time consuming, labor intensive, and dependent onthe nature. Therefore, the quality of planting material is always in short supply to the orchardists.The standardization of micro-propagation techniques in mango will facilitate in making availablelarge quantities of elite planting material to the orchardists. Mango seems to be difficult plantmaterial with respect to tissue culture response. However, the world over owing to the definite

74 Advances in Horticulture Biotechnology (Vol-1)

advantage of micropropagation, efforts have been made to micropropagate mango with littlesuccess. The main problem in tissue culture of mango is the development of axenic cultures,phenol leaching, browning of tissue explant, poor conversion rate of somatic embryoids andpoor shoot and root development. However, efforts are being continued to overcome theseproblems.

Efficient gene delivery system requires an efficient in vitro regeneration system.Furthermore, in vitro propagation also facilitates rapid multiplication of superior clones withina short span. Polyembryonic mango genotypes particularly those which are exploited asrootstocks for their desirable attributes are exclusively propagated by seed that give rise tolimited number of clonal seedlings; though identical to mother plant. This is another area,where potentialities of micropropagation can be explored to resolve the problem of clonalrootstocks.

In vitro PropagationIn vitro nucellar embryogenesis is the efficient tool for clonal micropropagation. It involves

in vitro somatic embryo formation from nucellar tissue cultured on nutrient mediumsupplemented with various growth hormone levels. The embryo developed from single cellsand gradually converted to different embryo stages viz. globular, heart shaped, torpedo shapedand cotyledonary shaped and finally give rise to complete plant formation. However, all thesestages are not so difficult in case of zygotic embryogenesis. In vitro nucellar embryogenesishas a unique feature in producing not only true to type plants but also disease free plants. It hashigh multiplication rate rather than other micro-propagation techniques. In vitro nucellarembryogenesis has been extensively employed in several fruit trees, considering horticulturalapplication in clonal propagation. This technique has been employed in both mono and polyembryonic trees which includes, Ribes rubrum (Zatyko et al., 1975); Vitis vinifera (Mullinsand Srinivasan, 1976); Malus domesticum (Eichholtz et al., 1979); Eriobotrya japonica (Litz,1985); Syzigium cuminii (Yadava et al., 1990) and Theobroma cacao (Sondahal, 1991) andMusa spp. In mango, the performance of scion is affected by the quality of root stock. Theproblem can be alleviated if genetically uniform root stocks could be made available to suitdifferent agro climates and also compatible scion variety. This will help in raising uniformorchards of trees.

Tissue culture research has been actively pursued in mango for the last 20 years in respectto developing regeneration protocols for clonal multiplication and also for genetic transformationstudies. Of the various in vitro approaches pursued for cloning the mango trees, success hasbeen achieved with only nucellus explants. All attempts to culture its vegetative explants hasbeen unsuccessful. Rao et al. (1981) has been able to induce only callus tissue and rootingfrom cotyledon explants from mango. However, no regenerants differenciated, while explantsturned black and died. Later, shoot regeneration from leaf explant has been reported,(Raghuvanshi and Srivastava, 1995). However, they explored the morphogenic potential ofmature leaf explants. Callus was initiated from mature leaf explant and later subcultured ontoMS medium for multiple shoot formation. Liquid shake culture was used to reduce phenolicexudation and subsequent necrosis of explants. Multiple shoots were transferred to rooting

Mango 75

medium fortified with 9. 8 µM IBA, only 20% of the cultured explants developed roots on thismedium. However, results could not be repeated.

One of the major problem in nucellar embryogenesis is excessive necrosis or browning ofintervening proembryonic or embryonic tissue and proembryos, which progressively increasesafter every subculture, resulting in reduction of number of potential embryos that can be convertedinto plantlets and adversely affecting on clonal propagation of mango. Despite several changesin media composition (lowering the concentration of major salts, increasing the concentration ofvitamins and adding organic supplements, like coconut water and yeast extracts) and in the physicalenvironment of incubation, such a situation continue to prevail in nucellar embryogenesis inmango (Litz, 1986 and Pantena, et al., 2002). Mango tissues darken very quickly in vitro as aresult of action of enzyme polyphenol oxidase activity (Litz and Vijayakumar, 1988).

A serious problem encountered in the in vitro nucellar embryogenesis in certain mangovarieties of the fasciation of embryos, that is embryogenic structures appeared as aggregationof embryos, their non separation and pluricotyly. Ethylene has been reported to promotevitrification, flaccidity, several abnormalities and embryonal fasciation in vitro. To checkfasciation of embryos and promote individual embryo formation and proliferation, variousethylene inhibitors, like Salicylic acid, ancymidol, AgNO

3, AVG has been found useful to

suppress ethylene accumulation in cultures (Litz and Yurgalevitch, 1997 and Chi et al., 1991).

Somatic EmbryogenesisIn comparison to other horticultural crops, mango has been a relatively “hard to deal crop”

for tissue culture. Nucellus has been utilized as the primary explants. Nucellus derived plantsare generally free from viruses and other endophytic disease causing organisms, due to theabsence of vascular connection between the surrounding maternal tissue and the nucellus. Thework on mango micropropagation is summarized in Table 3.1. Litz et al. (1982) were first toreport induction of embryogenic mango cultures from nucellar tissues of some polyembryoniccultivars after one to two months from ovule cultures excised form 40 to 60 days old fruitlets.Subsequently, the suitable conditions for induction of embryogenic cultures from nucellus ofmonoembryonic mango cultivars were defined (Litz., 1984). In general it was found that 2-3cm long fruits were gave higher somatic embryogenesis frequency rather than small andbigger one. Litz., 1984 reported that somatic embryos particularly in monoembryonic mangocultivars would eliminate systemic pathogens and avoid catastrophic loss frequently occurringin clonally propagated genotypes owing to diseases and environmental stresses under tropicalenvironment. This approach has been described in detail both in polyembryonic (Litz., et al.,1982, 1998; Dewald et al., 1989a; Patena et al., 2002) and monoembryonic genotypes (Litz,1984; Jana et al., 1994; Pliego-Alfaro et al., 1996a; Laxmi et al., 1999; Thomas, 1999; Ara et al.,2000b; Deore et al., 2000; Chaturvedi et al., 2004a, b; Rivera-Dominguez et al., 2004). Though,complaisance has been observed more in the former group. Somatic embryogenesis is a rapidregeneration procedure and is essential for genetic transformation (Mathews et al., 1993).

The induction of embryogenic cultures in mango is primarily dependent upon themorphogenetic potential of the nucellus. In polyembryonic genotypes, adventitious embryosdifferentiate from competent cells, which are present within the nucellus (Litz., 2003).

76 Advances in Horticulture Biotechnology (Vol-1)

Numerous developmental abnormalities, such as polycotyledony, fasciation, the absenceof bipolarity and formation of secondary embryos from hypocotyls were described, whichcontribute to the failure of embryos to develop to maturity normally (Dewald et al., 1989b). Inaddition, the problems of precocious germination and progressive necrosis have also preventedthe efficient recovery of mango plantlets (Dewald et al., 1989a). In an attempt to mitigate theseproblems, Dewald et al. (1989a) investigated the suitable conditions for optimizing the inductionand maintenance of embryogenic cultures and for maturation of somatic embryos in detail.Currently, the protocols employed broadly have four stages, viz., induction of somatic embryos,maintenance (proliferation), maturation and germination of somatic embryos into plantlets.Precocious germination of somatic embryos could be minimized by the addition of 3.0 ìMABA and 6.0% sucrose. Dewald et al. (1989b) thereafter proposed the suitability of liquidshake culture for the maintenance of embryogenic culture in mango. They observed that modifiedB5 medium to be optimum for induction with 6% sucrose and 20% coconut water. Also theirimportance for developing somatic embryos was quantified. Despite the higher rate ofembryogenesis from nucellar tissues, it has not given success in cvs. Chausa and Anwar Rataul.This was attributed to excessive phenolic exudation from nucellus explant in the medium (Usmanet al., 2005). Pati et al. (2005) reported that B5 Major salts + MS minor salts + vitamin andiron sources + 2, 4-D 1.0 mg l-1 + glutamine 400 mg l-1, 100 mg l-1 ascorbic acid + spermidine1.0 mg l-1 and malt extract 500 mg/l was found to be best for induction and proliferation ofsomatic embryos in Kurakkan.

Induction of Embryogenic Cultures

Two to three cm long (30-60 days old) fruits were harvested after pollination are suitablefor induction of somatic embryogenic culture. Fruits were washed under running tap water forhalf an hour and then dipped in solution containing 0.1% carbenzamine (bavestin) + 2-3 dropsof tween-20 along with 0.2% PVP (Polyvinylpyrrolidone) for one hour. These fruits were againwashed under running tap water followed by sterilization with 0.1% mercuric chloride (HgCl

2)

for 15 minutes followed by washing with double distilled water for 5-6 times. The fruits weredipped in 70% alcohol for a few seconds and then flame sterilized at the time of inoculationunder laminar air flow. Fruits were cut longitudinally into 2 halves, after removing the zygoticembryo from the immature seed, ovular halves containing nucellus tissue was scooped outfrom pericarp. Aseptic explants derived from surface sterilized fruits or seeds were inoculatedin the media with the cut face away from the semi-solid nutrient medium. The cultures weretransferred under dark condition in the culture room.

Induction of somatic embryos from the nucellus was done by many workers (Litz et al.,1982; Dewald et al., 1989a; Pliego-Alfaro et al., 1996a; Ara et al., 1999; Singh et al., 2002;Sulekha and Rajmohan, 2004). Ara et al. (2004) transferred monoembryonic ovules to an MSmedium consisting half-strength major salts and chelated iron + full strength microsalts andvitamins, 6% sucrose, 400 mg/l L-glutamine, 100 mg/l ascorbic acid, 0.8% agar and with1.0 mg l-1 2, 4-D. Dewald et al. (1989a); Litz et al. (1993 and 1995) optimized the somaticembryo production from nucellar tissue, the nucellus was transferred onto a sterile embryoinduction medium consisting of B5 major salts (without ammonium sulphate), MS minor salts

Mango 77

Fig. 3.1. Various stages of Somatic embryogenesis in mango – induction, maturation and hardening

B

D

F

H

A

C

E

G

78 Advances in Horticulture Biotechnology (Vol-1)

and organic components, L-glutamine, sucrose, ascorbic acid, 4.52 to 9.04 µM 2, 4-D and 2.0g l-1 gellan gum.

Ara et al. (2000b) employed the same medium except the use of half strength Fe-EDTAand the absence of ascorbic acid in medium. Chaturvedi et al. (2004a) observed that nucellartissue under different developmental stages responded differently to the same gelled nutrientmedium used for induction of embryogenesis. The youngest fruits (2.5 cm) required 0.25mg/l BAP, 1.0 mg l-1 NAA, while for the oldest fruits (5.0 cm), 0.5 mg l-1 2iP was sufficient forembryogenesis.Nucellar cultures are incubated in darkness at 25 °C and regularly transferredonto a fresh medium until darkening due to phenol exudation from explant has ceased completely(Litz, 2003). On the other hand, Patena et al. (2002) shifted the basal medium from B5 mediumto BP medium (Barba and Patena’s formulation) in eight strains of Carabao and two unidentifiedvarieties to effectively control the browning. Lad et al. (1997) demonstrated that embryogeniccompetence in ‘Carabao’ was acquired exposure to 2, 4-D for at least 28 days. Embryogenicnucellar tissue was apparent following culture for approximately 30 days onto induction medium.The embryogenic response is strongly cultivar dependent. On the basis of their embryogenicresponse, Litz et al. (1998) classified some varieties as highly embryogenic (polyembryonicHindi and Parris), moderately embryogenic (monoembryonic Lippens and Tommy Atkins) anddifficult-to-regenerate (polyembryonic Nam Doc Mai). They also demonstrated that nurse culturederived from Parris can improve the embryogenic response of non-embryogenic cultures.Manzanilla Ramiriez et al. (2000) compared the induction responses of three cultivars andobserved that ‘Ataulfo’ (polyembryonic) more embryogenic than either ‘Tommy Atkins’(monoembryonic) or ‘Haden’ (Monoembryonic) in that order. Litz and Yurgalevitch (1997)suggested that differential regulation of ethylene biosynthesis and the enzyme spermidinesynthase in mango may be major contributing factors in controlling induction of somatic embryosin vitro and confirmed the effects of spermidine in induction of embryogenic competence inmango cultures.

Proliferation and Maintaince of Somatic Embryos

Sustained cultures in most cultivars is possible on induction medium (Litz, 2003).Embryogenic cultures of many cultivars exhibit significantly higher proliferation in suspension.It was observed that culture darkening was slower in liquid than on solid medium (Dewald etal., 1989a). Both cytokinin and auxin are important for proliferation and maintenance of mangoglobular embryogenic masses (Litz, 2003). Deore et al. (2000) reported that the cytokinin isnot essential for induction, but it is important for stimulating organization of the apical meristemduring maturation. Good callus induction in medium supplemented with 0.1 mg l-1 kinetin +2.0 mg l-1 2, 4-D. Embryogenic cultures of “Parris” grown in liquid maintenance mediumcontaining 4.0 to 8.0 µm 2, 4-D and 4.6 µM kinetin produced significantly early and highernumber of heart shaped somatic embryos (Dewald et al., 1989a). Ara et al. (2004) suggestedthe use of 1.0 mg l-1 2, 4-D and 1.0 mg l-1 NAA, either alone or with 1.0 mg l-1 kinetin forenhancement of proliferation of the pro-embryogenic cultures (PEC) in liquid as well as solidmedia. Gross developmental abnormalities in mango somatic embryos can be prevented up tosome extent by careful manipulation of the culture conditions during PEM maintenance and

Mango 79

somatic embryo development. Maintenance as suspension culture as well is cultivar dependent(Litz et al., 2000). Proliferation of embryogenic cultures has been achieved by production ofsecondary embryos from PEMs (Dewald et al., 1989a; Litz et al., 1993). Over the time, gradualloss of morphogenetic potential of embryogenic culture occurs.

Maturation of Somatic Embryos

Main problem in normal development of somatic embryos include precociousgermination, secondary embryogenesis, etc. from hypocotyl of germinating embryos and theabsence of biopolarity. For subsequent germination of somatic embryos into normal seedlings,the control of developmental changes is indispensable. In early stage of somatic embryoinduction 2, 4-D is necessary but continuous presence of 2, 4-D in maintenance mediumexerts a considerable negative influence on the somatic embryos production by interferingits development beyond globular stage. Zimmerman (1993) was suggested that the progressionfrom the globular to heart shape stage are triggered only in the absence of auxin. Initiation ofmaturation including the development of bipolarity in globular embryos followed by thedifferentiation of cotyledons can be stimulated by transfer of embryogenic cultures frommaintenance to maturation medium. Laxmi et al. (1999) as well observed maximum embryoproduction on ½ strength MS medium supplemented with 20. 0 µM BAP devoid of 2, 4-D.Maturation of somatic embryo often has been accompanied by gradual necrosis of cotyledonand hypocotyl. Addition of malt extract, casein hydrolysate and reducing agent is not effective(Litz, 1984). Medium supplemented with coconut water (20%) delayed the necrosis andenhanced the production of somatic embryos (Litz, 1984; Dewald et al., 1989a). Accordingto Dewald et al. (1989a), the maturation medium consisted of modified B5 major salts, MSminor salts and organics, L-glutamine, casein hydrolysate and 20% coconut water. ABA incombination with coconut water stimulated a higher somatic embryos production in 3.0%sucrose contaning medium (Dewald et al., 1989b). Singh et al. (2001) suggested addition of100 µM ABA to hormone free regeneration medium to improve frequency of embryogenesisas well as percentage of normal dicotyledonary embryo induction. The apical meristem ofglobular embryo organizes faster when the maturation medium is supplemented with eitherkinetin or 6-benzylamino purine at 1.0 to 3.0 mg l-1 (Litz et al., 1993; Lad et al., 1997).Addition of kinetin at 4.65 µM or benzyl adenine (BA) at 4.44 µM to the maturation mediumstimulated the development of cotyledons (Litz and Gomez-Lim, 2005). Somatic embryosthat shows necrotic disorder are unable to reach maturity (Mathews et al., 1992 and Monsaludet al., 1995). This has also been a limiting factor for the development of highly embryogenicsuspension cultures. Pliego-Alfaro et al. (1996a) overcome this problem with pulsing of 750to 1750 µM ABA, 7.5 to 12.5% mannitol and their combination. Monsalud et al. (1995)achieved reversion of hyperhydricity of mango somatic embryos in two ways; partialdehydration of heart shaped somatic embryos under controlled conditions at high relativehumidity for 24–48 h and by increasing the concentration of gelling gum from 2–6%.Precocious germination, another problem in somatic embryogenesis can be inhibited byaddition of 100 µM ABA by Pliego-Alfaro et al. (1996b). The control of precocious rootformation by the use of ABA was also described by Monsalud et al. (1995). Transfer ofsomatic embryos from ABA containing medium onto without ABA containing medium, it

80 Advances in Horticulture Biotechnology (Vol-1)

stimulates highly synchronized germination. Thomas (1999) also obtained embryo maturationin the presence of ABA at 1.0 mg l-1. Ara et al. (1999) observed that ABA at 0. 004 and 0.02µM had no significant effect on germination percentage and plantlet development but delayedthe germination by three weeks. Thomas (1999) suggested reduced application of sugar with1.0 mg l-1 ABA for the maturation of early or late heart stage and early cotyledonary embryo.Litz and Gomez-Lim (2005) also advocated reduction in concentration of sucrose in maturationmedium from 6–4%.

Germination and Conversion into Plantlets

It is observed that the 1.0–1.5 cm long cotyledionary embryos showed higher germinationefficiency rather than small ones. Litz, (2003) and Thomas (1999) reported that the lowsugar concentration i.e. 2.0% was gave higher germination frequency in matured somaticembryos. Laxmi et al. (1999) also advocated the lowering of sucrose concentration andaddition of GA3 and N6-benzylamino purine for improved somatic embryo germination.Germination was achieved on a medium with B5 major salts, MS minor salts and organics.The conversion efficiency could be enhanced by high light intensity, i.e. 160 µmol m2s1 in aCO

2 enriched atmosphere (20,000 ppm) (Litz, 2003). Dewald et al. (1989a, b) and Laxmi et

al. (1999) reported that the best germination of somatic embryos was found in gelrite andphytagel respectively rather then other gelling agents. However, Chaturvedi et al. (2004b)advocated the need to use liquid state of nutrient medium for development, maturation,germination and conversion (plantlet formation) of cotyledonary embryos. BM2 basal mediumalong with 0. 1 mg l-1 abscisic acid + 100 mg l-1 polyethylene glycol and 0.1 mg l-1 IAA wasfound to be good for development, maturation and germination of embryos. 1.5 and 2.0 cmlong cotyledionary embryos were gave maximum germination percentage (94%). But only80% of the germinated embryos converted into plantlets, with well-developed green leaves.The in vitro raised plantlets showed approximately 50% success in hardening. Xiao et al.(2004) reported direct somatic embryogenesis and plantlet regeneration from cotyledon ofimmature zygotic embryos. Conversion of somatic embryos was found on a mediumcontaining 23 µM kinetin. Ara et al. (2000a) described a protocol for regeneration of mangoplantlets isolated form PEMs (Pro Embryonic Masses) in a suspension culture derived fromthe nucellar callus of mango cv. Amrapali. The dividing protoplasts were transferred to amedium with growth regulators for development of microcalli, which later produced somaticembryos. The mature somatic embryos were germinated into well-developed seedlings andsubsequently transferred to soil.

Table 3.1. In vitro responses of Mangifera indica

S.No Explant Media Response References

1. Cotyledon MS + Kinetin or NAA & CW Callus, organogenesis Rao et al., 19812. Ovules and Modified MS + BA & CW Somatic embryogenesis Litz et al., 1982

nucellus3. Nucellus Modified MS + 2, 4-D or NAA Callus, PEM, somatic Litz 1984

or BA or 2iP embryogenesis

(Contd.)

Mango 81

Modified MS + CW S. E. maturation, germination4. Nucellus Modified MS + 2, 4-D, Spermidine, Callus, PEM, somatic Litz and Schaffer 1987

Putrescine or Spermine Embryogenesis5. Nucellus Modified mod. MS + 2, 4-D Callus, PEM De-Wald et al., 1989 a,b

Modified B5 + 2, PEM4-D & KinetinModified B5 + CW Somatic embryogenesisModified B5 + CW S.E. maturation, germination& ABA

6. Nucellus Modified MS + 2, 4-D Callus, PEM Mathews and Litz 1990Modified B5 + 2, 4-D PEMModified B5 + CW Somatic embryogenesis,

S.E. maturation7. Nucellus. Modified B5 + 2, 4-D Callus, PEM Litz et al., 1991

Modified B5 + CW or Kinetin & BA Somatic embryogenesis,S. E. maturation

8. Nucellus. Modified B5 + 2, 4-D Callus, PEM Mathews et al., 1992Modified B5 + BA Somatic embryogenesis

9. Shoot tips & B5, r WPM Combinations of BA, Kinetin, Shoot formation (organogenesis) Yang and Ludders 1993axillary buds. Zeatin or 2iP with IAA, IBA, NAA or GA3

10. Nucellus MS + 2, 4-D & GA3 PEM, direct somatic Jana et al., 1994embryogenesis

Modified MS + ABA, CW S.E. maturationMS + BA Germination

11. Nucellus Modified B5 + 2, 4-D Callus, PEM Monsalud et al., 1995Modified B5 + BA Somatic embryogenesisModified B5 + BA & ABA or CW S. E. maturation

12. Young fully MSexpanded leaves MS + BA, or Kinetin with IAA or NAA Shoot formation (organogenesos) Reghuvanshi and

and elongation Srivastava 1995MS + IBA Root formation (organogenesis)

13. Nucellus Modified MS + 2, 4-D Callus, PEM Pliego-Alfaro et al.,1996a, b

Modified B5 + CW & ABA Somatic embryogenesis,S.E. maturation

14. Nucellus Modified B5 + 2, 4-D Callus, PEM Cruz-Hernandezet al., 2000

15. Nucellus Modified B5 + 2, 4-D Callus, PEM, SomaticModified B5 + None or 2, 4-D Somatic embryogenesis Lad et al., 1997Modified B5 + BA or Kinetin S. E. maturation

16. Nucellus Modified B5 + 2, 4-D + DCGA or Callus, PEM, Somatic Litz and YurgalevitchAVG or MGBG or ACC embryogenesis 1997

17. Immature/mature Modified MS WPM, B5 or RO Limited explant survival Thomas and Ravindrashoot tips, axillary 1997buds

18. Nucellus Modified MS + 2, 4-D PEM, Somatic embryogenesis Ara et al., 1998Modified B5 + GA3 S.E. maturation, germinationModified B5 + IAA, NAA Or IBA Root formation (organogenesis)

19. Nucellus Modified B5 + 2, 4-D PEM Litz et al., 1998

(Contd.)

S.No Explant Media Response References

82 Advances in Horticulture Biotechnology (Vol-1)

20. Nucellus Modified MS + 2, 4-D Callus, PEM Ara et al., 1999Modified B5 Somatic embryogenesisModified B5, with GA3 & ABA S. E. maturation, germination

21. Nucellus MS, B5 or RO + 2, 4-D and GA3 Callus, PEM Thomas 1999Modified B5 or RO + 2, 4-D & GA3 or CW S o m a t i c

embryogenesis.Modified B5 + ABA & CW S. E. maturation, germinationModified B5 + ABA, CW & TDZ or BA GerminationModified B5 + NAA or IBA and No secondary rooting observedPaclobutrazol or GA3

22. Nucellus Modified MS + 2, 4-D Callus Ara et al., 2000a,bModified B5 PEMModified B5 + 2, 4-D Protoplast formation and

micro-calli formation.Modified B5 + 2, 4-D, NAA, IBA Callus, Somatic embryogenesisor KinetinModified B5 + None or GA3 Germination

23. Ovule halves Modified B5 + 2, 4-D Callus, PEM Cruz-Hernandezet al., 2000

24. Nucellus Modified MS + 2, 4-D, BA, NAA, Callus, PEM, Somatic Deore et al., 2000embryogenesis

25. Nucellus Modified B5 or BA + 2, 4-D Callus, PEM, Somatic Patena et al., 2002Embryogenesis.

26. Nucellus Modified MS or Modified B5 + 2, Callus, PEM, Ara et al., 20044-D, NAA or Kinetin

27. Nucellus Modified B5 + 2, 4-D Callus, PEM, Rivera-Dominguezet al., 2004

Modified B5 + BA Somatic embryosModified B5 + CW S. E. maturation, germination

28. Nucellus, MS + 2, 4-D, GA3 & CW Callus and direct somatic embry- Sulekha andembryosmass, ogenesis from nucells and embryo Rajmohan 2004floral parts & leaf mass onlysegments

29. Immature Modified B5 or Modified MS + IBA Direct somatic embryogenesis Xiao et al., 2004cotyledonsfrom or 2, 4-D, & GA3zygotic embryos Modified B5 + Kinetin & CW S.E. maturation, germination

30. Zygotic embryos, MS or B5 + 2, 4-D Callusing only from Usman et al., 2005nucellus orshoot tips zygoticembryos

31. Shoot tips MS or B5 Limited explant survival/plant Hare Krishna et al.,regeneration 2008

32. In vitro shoot G medium + BA + Zeatin + 2iP 2.0 + shoot tip establishment and Yang and Ludderstip culture IAA + IBA 0. 5 mg/l Casein elongation 1993

Hydrolysate33. Shoot bud MS + IAA, Kinetin Bud break and shoot growth Chandra et al., 2004

Est. = establishment medium; Main. = maintenance medium; Emb. = embryogenesis medium; Mat. = maturation medium; Germ. =germination medium; B5 = (Gamborg et al., 1968); MS = (Murashige and Skoog); WPM = Woody Plant Medium (Lloyd and McCown,1980); G = (Yang et al., 1984); RO = Rugini Olive medium (Rugini, 1984); BP = Barba & Patena formulation (Patenaet al., 2002); S. E. = Somatic embryos (s).

S.No Explant Media Response References

Mango 83

OrganogenesisIt is very hard to regenerate rooting in field-grown shoots, explants, which is attributed to

high phenolics exudation (Krishan et al., 2008) and systemic contamination (Rocha Monteirode Andrade et al., 2009) during culture establishment. Rao et al. (1981) first time inducedrooting from callus which was initiated from mango cotyledons on MS medium supplementedwith kinetin and NAA. However, development of shoots was not observed. Later on Singhet al. (1991) reported callus induction from different explants such as epicotyl segment, leafpetiole and shoot tip excised from aseptically germinated embryo. Higher callus was recordedon epicotyl segment, while direct root organogenesis was noted in epicotyl and shoot tip culturewith low level of 2, 4-D. Thomas and Ravindra (1997) tried to establish shoot tip culture insome mango genotypes. Their study indicated that several problems such as phenolic exudation,medium discoloration and explant browning are interrelated and are influenced by differentfactors like medium, genotype, explant, season and decontamination treatment. The problemsassociated with field-grown shoots can be avoid by utilizing in vitro grown shoots, which aremore responsive to culture conditions to in vitro culture. Ara et al. (1998) devised a two stepprotocol for in vitro rooting of microshoots obtained from nucellar somatic embryos. 24 hpulse treatment with 5.0 mg l-1 IBA , was found most effective with respect to rooting and rootgrowth. The balance between the use of explant derived from field-grown trees and in vitrogrown shoots can be struck by employing greenhouse grown shoots. Reuveni and Golubowicz(1997) tried the small internodes of greenhouse grown mango for culture initiation but werenot able to promote shoot formation in established cultures. Krishna, et al. (2006) also triedglasshouse raised shoot segments of cv. Amrapali but was able to achieve only callusing on cutend and sprouting of axillary bud. Further shoot proliferation could not be achieved and culturessurvived for more than six months. They also used in vitro germinated seedlings of Kurukkanfor initiation of shoots along with callusing on cut end was observed. These cultures alsosurvived for more than six months. Shahin et al. (2003) reported that stem node explant wasbetter than shoot tips with respect to shoot proliferation. The highest proliferation was notedon modified WPM supplemented with 20 g l-1 sucrose, 30 mg l-1 adenine + 2 mg l-1 iso-pentyladenine + 0.5 mg l-1 IBA + casein hydrolysate or 30 mg l-1 adenine + 0.2 or 0.5 mg l-1

IBA. Modified WPM medium supplemented with 30 mg l-1 adenine + 1.0 mg l-1 BA + 4.0 mg l-1

IAA registered highest rooting percentage and root number.

Embryo RescueEmbryo culture can be employed to rescue hybrid embryo of mango. In mango heavy fruit

drop is a major problem and hence embryo culture can improve breeding efforts. Sawke et al.(1990) reported that several factors associated with fruit drop include nutritional deficiency,moisture stress, hormone imbalance, climatic factors, lack of fertilization and attack of diseasesand pests. Research done in this aspect shows that the region of abscission layer formation ispre-determined. Cells are detached in abscission zone by dissolution of middle lamella (Chadha,1959). For in vitro regeneration, 35–45 day-old immature fruits was shown to significantlyimprovement in mango breeding (Nathhang, 1999).

84 Advances in Horticulture Biotechnology (Vol-1)

Chandra et al. (2003a, b) were successful in regenerating immature mango embryo intocomplete plantlet on MS basal medium supplemented with 9. 0 mg l-1 BA + 3. 0 mg l-1 kinetin+ 400 mg l-1 glutamine + 500 mg l-1 activated charcoal + 60 g l-1 sucrose. Complete plantletformation was observed in 72% of the cultures (Fig. 3.2). Sahijram et al. (2005) used 6–8weeks old post-pollination fruits for embryo culture. Hybrid embryos were aseptically excisedfrom immature ovules and thereafter, inoculated in vitro onto semi-solid half strength modifiedMS medium containing casein hydrolysate (1.25 g l-1) and sucrose (4.5%). After 12–14 weeks,well-developed seedlings were transferred to nonsterile conditions (tap water in parent culturevessels) under identical environmental conditions for initial hardening-off.

Shoot Bud CultureShoot bud culture has not yielded encouraging result so far. Shoot bud culture in mango

from two years old glass house plants (Yang and Ludders, 1993) as well as 8–16 years old trees(Thomas and Ravindra, 1997) has not yielded encouraging results. The work done at CISH,

Fig. 3.2. Various stages in embryo rescue in mango

A. Different Stage of fruits showing embryo used in hybrid embryorescue, B. Initiationof shoot after 2 weeks of inoculationC. Initiationof shoot after 2 weeks of inoculation

A B

C

Mango 85

Lucknow (Chandra, et al., 2004) on mango shoot bud culture revealed that explants takendirectly from field-grown mature tree could not be regenerated in to platlets. After pre-treatmentthese shoot buds were inoculated on MS basal medium supplemented with 3. 0 mg l-1 IAA +1.0 mg l-1 kinetin + 200 mg l-1 adenine sulfate + 1.0 mg l-1 thiamine + 1 0 mg l-1 riboflavin +100 mg l-1 casein hydrolysate + 400 mg l-1 glutamine + 100 mg l-1 PVP + 100 mg l-1 ascorbicacid + 60 mg l-1 sucrose. These cultures remained green up to 8–10 weeks. Shoot buds of cv.Amrapalli showed the best response with 48. 92% cultures remaining green and with the openingof leaf primordial even after 4 weeks of inoculation. Differentiation of shoot buds into plantletswas achieved but plantlets did not grow beyond the 3-4 leaf primordial stage. Shoot establishmentand subsequent proliferation could not be achieved. In vitro shoot tip grafting or micrografting(Sahijram et al., 2009) need to be attempted to multiply some of the important or newly releasedvarieties of mango.

HardeningHardening of in vitro raised plantlets before transplantation to soil is a very crucial step

determining successful transplantation to soil and their ex vitro survival. It is well documentedin literature that in vitro raised plantlets have divergent leaf anatomy from the normal grownplants, i.e., malfunctioning of stomata, poor development of cuticular wax and of palisadetissue and with low photosynthetic efficiency, mainly due to high humidity (90%) and sugarcontaining medium obtained in vitro. Thus, when they have to switch over to autotrophic modeafter transfer to soil, involving normal photosynthetic activity and without transpiration anddesiccation for which they need to be acclimatized. The acclimatization period during transitionfrom culture to greenhouse may vary from 15 days to 1 month, depending on the nature andhardness of plant species (Ziv et al., 1987; Donnelly and Tisdall, 1993 and Hazarika, 2003).

Successful acclimatization of in vitro raised mango plantlets is very intractable propositioneluding success in the true sense of the term. Various strategies have been attempted by manyworkers, but none of them worked. Litz (1993) reported nurturing of in vitro raised plantletson a sugar free medium and exposure of cultures and plantlets to high level of CO

2, so as to

increase the photosynthetic efficiency before their plantation to soil. Although such plantletsdeveloped thick leaves but failed to survived under greenhouse conditions. Dewald et al. (1989b)suggested periodic application of macro and micro elements as foliar spray for suspecting theirroots being only partially or largely malfunctional. Infect the in vitro formed roots of mangoplantlets through appeared normal have been very brittle and fragile and mostly lacked lateralroots not to mention root hair.

In vitro raised plantlets of mango, the ex vitro survival of in vitro raised plantlets is theprime unsolved aspect of in vitro cloning, of mango. Consistent failure of in vitro raised mangoplantlets to survive under even controlled conditions of greenhouse or hardening chambers hasbeen faced by virtually at the investigator working on this problem (Chaturvedi et al., 2004).Despite use of known procedure of hardening including the one which has been successful incase of most difficult plant like jojoba for its transplant success (Chaturvedi and Sharma,1989) and a large array of potting mixtures were used, maximum ex vitro survival duration ofin vitro raised plantlets of mango varieties has been ca 4 months (Chaturvedi et al., 2003).

86 Advances in Horticulture Biotechnology (Vol-1)

During this period the surviving plants showed symptom of blackening of stem and its softeningat the level of potting mixture, blackening and necrosis of shoot tip and leaf tip and its curlingwhich within a short time lead to shrinking of stem and finally death of plants.

In vitro Germplasm ConservationMango seeds are highly recalcitrant and cannot be stored for long time. Therefore, tissue

culture methods can be an ideal approach, (Engelmanns, 1991). Pliego-Alfaro et al. (1996a, b)cultured somatic embryos on mannitol and ABA supplemented medium, which suppressed thegrowth and the cultures could be extended up to three months. Further, they suggested thatmedium term germplasm storage can be made in mango through in vitro means. Convertingsomatic embryos to ‘Synthetic seed’ or ‘Synseeds’ by encapsulation could possibly be utilizedas a means for germplasm storage and transportation of elite germplasm. Desiccated somaticembryos can be stored for several months without any significant decline in their germinabilitye.g., in alfalfa, somatic embryos desiccated to 10–15% were stored at room temperature for1 year (McKersie and Bowley, 1993). Wu et al. (2003) compared three techniques forcryopreserving the embryonic mango cultures. The best result was obtained with vitrification,while the encapsulation/dehydration yielded no recovery. The synthetic seed technology offerstremendous potential in micro-propagation and germplasm conservation. In Mangifera indica(Ara et al., 1999 and Ara, 1998), the somatic embryos has been encapsulated to produce syntheticseeds. However further research is needed to optimize protocol for production of viable syntheticseeds that could be stored for longer periods and could be commercially viable.

Future PerspectivesMango seems to be difficult plant material with respect to tissue culture response. The

main problem in tissue culture of mango is the development of axenic cultures, phenol leaching,browning of tissue explant, poor conversion rate of somatic embryoids and poor shoot and rootdevelopment. Various groups have demonstrated in vitro regeneration of mango through nucellarembryogenesis. However, field establishment of micropropagated plants of mango has notbeen successful. In order to overcome problem of functional root, somatic embryos can betransformed with Agrobacterium rhizogene for development of adventitious root system. Thereis need to develop efficient acclimatization protocol for micropropagated plants of mango.

References

Ara, H., Jaiswal, U. and Jaiswal, V.S., 2004. An improved method of proliferation of proembryogenic calli of Mangiferaindica L. var. Amrapali for scale-up of somatic embryo production. Indian J. Biotechnol, 229-234.

Ara, H., Jaiswal, U., and Jaiswal, V.S., 1999. Germination and plantlet regeneration from encapsulated somatic embryosof mango (Mangifera indica L.). Plant Cell Rep., 19(2):166-170.

Ara, H., Jaiswal, U., and Jaiswal, V.S., 2000a. Plant regeneration from protoplasts of mango (Mangifera indica L.) throughsomatic embryogenesis. Plant Cell Rep., 19(6):622-7.

Ara, H., Jaiswal, U., and Jaiswal, V.S., 2000b. Somatic embryogenesis and plantlet regeneration in Amrapali and Chausacultivars of mango. Curr Sci., 78(2):164-169.

Ara, H., Jaiswal, U., and Jaiswal, V.S., 1998. Rooting of microshoots of Mangifera indica L. cv. Amrapali. Curr. Sci.,74: 259-64.

Mango 87

Ara, H., 1998. Ph. D. Thesis, Banaras Hindu University, Varanasi.Bompard, J.M., 1989. Wild Mangifera species in Kalimantan (Indonesia) and in Malaysia. Final Report. International

Board for Plant Genetic Resources, Rome.Chadha, K.L., 1959. Studies on the periodicity of fruit fall in some mango varieties of Punjab. M. Sc. (Agric.) thesis,

Punjab University. Punjab.Chandra, R., Padaria, J.C., and Srivastava, S., 2003a. Factors influencing in vitro establishment of mango shoot buds.

Indian J Plant Physiol., 9(2):136-44.Chandra, R., Padaria, J.C., Singh, R., and Pal, J.K., 2003b. In vitro establishment of in vitro raised mango plantlets.

Indian J Plant Physiol, 705-710 [special issue].Chandra, R., Padaria, J.C., and Srivastava, S., 2004. Factors influencing in vitro establishment of mango shoot buds. Ind.

J. Plant Physiol., 9(2):136-144.Chaturvedi, H.C., and Sharma, M., 1989. In vitro production of cloned plants of jojoba (Simmondsi chinensis (Link)

Scheneider) through shoot proliferation in long term culture. Plant science, 63:199-207.Chaturvedi, H.C., Agnihotri, S., Sharma, M., Sharma, A.K., Jain, M., Gupta, P., Chourasia, A., and Kidwai, N.R., 2004.

Induced nucellar embryogenesis in vitro for clonal multiplication of Mangifera indica L. var. Ambalavi: a dwarfingrootstock. Indian J Biotechnol 3:222-228.

Chaturvedi, H.C., Sharma, M., Agnihotri, S., Sharma, A.K., Jain, M., and Chourasia, A., 2004b. In vitro development andconvertibility of nucellar embryos of Mangifera indica L. Bappakai, a dwarfing rootstock. Plant Cell BiotechnolMol Biol., 5(1/2):7-14.

Chaturvedi, H.C., Agnihotri, S., Sharma, M., Sharma, A.K., Jain, M., and Gupta, P., 2004a. Induced nucellar embryogenesisin vitro for clonal multiplication of Mangifera indica L. var. Ambalavi: a dwarfing rootstock. Indian J. Biotechnol,3:22–228.

Chaturvedi, H.C., Agnihotri, S., Sharma, M., Sharma, A.K., Jain, M., Gupta, P., and Chourasia, A., 2003. In vitro controlof fascination in proliferating nucellar embryos of Mangifera indica L. var. totapuri red small for cloning. Indian JExp Biol 41:1311-1316.

Chi, G. L., Pua, E.C., and Goh, C.J., 1991. Role of ethylene on de novo shoot regeneration from cotyledionary explantsof Brassica compestris spp. Pekinesis (Lour) Olsson in virtro. Plant physiol. 96:178-183.

Cruz-Hernandez, A., Town, L, Cavallaro, A., and Botella, I.R., 2000. Transient and stable transformation in mango byparticle bombardment. Acta Hortic., 509:237-42.

Deore, A.C., Chavan, S.S., Deshpande, R.S., and Dhonushke, B.L., 2000. Factors affecting in vitro somatic embryogenesisfrom nucellar tissue of Alphonso mango. Ann Plant Physiol., 14(2):182-186.

Dewald, S.G., Litz, R.E., and Moore, G.A., 1989b. Maturation and germination of mango somatic embryos. J Am SocHortic Sci., 114:837-841.

Dewald, S.G., Litz, R.E., and Moore, G.A., 1989a. Optimizing somatic embryo production in mango. J Amm Soc HorticSci., 114:712-716.

Donnelly, D.J., and Tisdall, L., 1993. Acclimatization strategies for micropropagated plants. In: Micro-propagation ofwoody plants. (Ed. M. R. Ahuja), Forestry Sci., 41:153-166.

Eichholtz, D., Robitaille, H.A., and Hasegawa, P.M., 1979. Adventive embryony in apple. Hort Sci 14:699-700.Engelmanns, F., 1991. In vitro conservation of tropical plant germplasm. Euphytica 57:227-243.Hazarika, B.N., 2003. Acclimatization of tissue culture plants. Curr Sci. 85:1704-1712.Jana, M.M., Nadgauda, R.S., Rajmohan, K., and Mascarennhas, A.F., 1994. Rapid somatic embryogenesis from the

nucelli of monoembryonic mango varieties. In Vitro Cell Dev Biol Plant, 30:55–57.Krishna, H. Sairam, R.K., Singh, S.K., Patel, V.B., Sharma, R.R., Grover, M., nain, L. and Sachdev, A., 2008. Mango

explant browning: effect of ontogenic age, mycorrhization and pretreatment. Scientia Hort., 118:132-138.Kostermans, A.J.G.H., and bompard, J.M., 1993. The mangoes, their botany, nomenclature, horticulture and utilization.

Pub. For the International Board for Plant Genetic Resources (IBPGR) and the Linnean society of London.Lad, B.L., Jayasankar, S., Pliego-Alfaro, F., Moon, P.A., and Litz, R.E., 1997. Temporal effect of 2, 4-D on induction of

embryogenic nucellar cultures and somatic embryo development of ‘Carabao’ mango. In Vitro Cell Dev BiolPlant, 33:253-257.

Laxmi, D.V., Sharma, H.C., Kirti, P.B., and Mohan, M.L., 1999. Somatic embryogenesis in mango (Mangifera indica L.).Curr Sci., 77(10):1355-8.

88 Advances in Horticulture Biotechnology (Vol-1)

Litz, R.E., Gomez-Lim, M.A., Jayashankar, S., and De Moura Barros L., 2000. Biotechnology for mango improvement andpropagation. In: Recent Advances in Mango Research (Eds. Srivastava, R.P.). Prem Printing Press, Lucknow.

Litz, R.E., and Gomez-Lim, M.A., 2005. Mangifera indica mango. In: Biotechnology of Fruit and Nut Crops (Eds. Litz,R.E.) pp. 40-61. CABI Publishers.

Litz, R.E., Hendrix, R.C., Moon, P.A., and Chavez, V.M., 1998. Induction of embryogenic cultures as affected by genotype,expanting, 2, 4-D and embryogenic nurse culture. Plant Cell Tissue Organ Cult., 53:13-18.

Litz, R.E., Knight, R.K., and Gazit, S. 1984. In vitro somatic embryogenesis from Mangifera indica L. Sci Hortic.,22:233-40.

Litz, R.E. and Schaffer, B., 1987. J. Plant Physiology, 128:251-258.Litz, R.E. and Jaiswal, V.S., 1991. Micro-propagation of tropical and subtropical fruits. In Micro-propagation Technology

and Application. (Eds R.H. Zimmerman and Debergh, P.). Kluwer academic publishers, Dordecht. pp. 247-264.Litz, R.E., Knight, R.K., and Gazit, S., 1982. Somatic embryos from cultured ovules of polyembryonic Mangifera indica L.

Plant Cell Rep 1:264-266.Litz, R.E., Mathews, V.H., Moon, P.A., Pliego-Alfaro, F., Yurgalevitch, C., and Dewald, S.G., 1993., Somatic embryos of

mango (Mangifera indica L.). In: Synseeds Applications of Synthetic Seeds to Crop Improvement. (Eds.Redenbaugh, K.) pp. 409-25 Boca Raton: CRC Press.

Litz, R.E., Moon, P.A., Monsalud, M.J., Jayasankar, S., and Mathew, H., 1995. Somatic embryogenesis in Mangiferaindica L. (mango). In: Somatic Embryogenesis in Woody Plants, vol. 2 (Eds. Jain, S.M., Gupta, P.K. and Newton,R. J.) pp. 341-356. Dordrechet, Academic Publishers.

Litz, R.E., and Vijayakumar, N., 1988. In vitro somatic embryogenesis from the nucellus of mango. Acta Hortic231:473-275.

Litz, R.E., and Yurgalevitch, C., 1997. Effects of 1 aminocylopropane-1-carboxylic acid, aminoethoxyvinylglycine,methylglyoxal bis-(guanylhydrazone) and dicyclohexyl ammonium sulfate on induction of embryogenic competenceof mango nucellar explants. Plant Cell Tissue Organ Cult., 51:171-176.

Litz, R.E., 2003. In vitro regeneration and transformation of mango. In: Plant Genetic Engineering. (Eds. Jaiwal, K. andSingh, R.P.) p. 23-40. LLC: Sci. Tech. Publishers.

Liz, R.E., 1985. Somatic embryogenesis in tropical fruit trees. In: Tissue culture in forestry and Agriculture. Eds. R. R.Henke, K.W. Hughes, M.J. Constantin and A. Hollaender, Plenum Publicating Co., New York. pp. 179-193.

Liz, R.E., Jarret, R.L., and Asokan, M.P., 1986., Tropical and subtropical fruits and vegetables. In: Tissue culture as aPlant Production System for Horticultural Crops. (Eds. R.H., Zimmerman, R.J., Griesbach, F.A., Hammerschlagand R.H.) pp. 237-251. Lawson, Martinus-Nijhoff Publishers, Dordrecht.

Litz., R.E., Mathews, H., Bharathan, N., and Narayan, K.R., 1990. Transformation of somatic embryos of mango.Proceedings of the VII International Congress on Plant Tissue and Cell Culture. Amsterdam. p. 67 (Abstract).

Mathews, H., Litz, R.E., Wilde, D.H., Merkela, S., and Wetzstein, H.Y., 1992. Stable integration and expression of bita-glucuronidase and NPT-II genes in mango somatic embryo. In Vitro Cell Dev Biol Plant., 28:172-178.

Maethews, H. and Litz, R. E., 1990. Mango. In : Biotechnology of Perennial Fruit Crops, Biotechnology in Agriculture, No.8. Eds. F. A. Hammesschlag and R. E. Litz, CAB International, University Press, Combridge, U.K. pp. 433-448.

Mathews, H., Litz, R.E., Wilde, D.H., and Wetzstein, H.Y., 1993. Genetic transformation of mango. Acta Hortic 341:93-97.Manzanilla Ramiriez, M.A., Robles Gonzales, M.M., and Medina Urrutia, V.M., 2000. Advances sobre embriogenesis

somatica en mango Ataulfo Haden. In: Control de Floracion Y Mejoramiento Genetico (Eds Medina Urrutia, V.M.,Perez Barraz, H. and Chavez Contreras X). pp. 47–56 Tecoman, Mexico: INIFAP.

McKersie, B.D., and Bowley, S.R., 1993. Synthetic seeds of alfalfa. In: Synseeds: Applications of Synthetic Seeds toCrop Improvement. (Ed. Redenbaugh K.). pp. 231–255 Boca Raton, FL: CRC Press.

Monsalud, M.J., Mathews, H., Litz, R.E., Gray, D.J., 1995. Control of hyperhydricity of mango somatic embryos. PlantCell Tissue Organ. Cult., 42:195-206.

Mullins, M.G., and Srinivasan, C., 1976. Somatic embryos and oplantlets from an ancient clone of grapewine (cv. CabernetSauvignon) by apomyxix in vitro. J. Exp. Bot., 27:1022-1030.

Mukherjee, S.K., 1985. Systematic and ecogeographical studies on crop gene pool: I Mangifera L., International Boardfor Plant Genetic Resources, Rome, Italy, pp 86.

Mango 89

Nathhang, N.T., 1999. Studies on embryo rescue in mango (Mangifera indica L.) M. Sc. thesis, IARI, New Delhi. pp. 156.Patena, L.F., Carlos-Refuerzo, L.R., and Barba, R.C., 2002. Somatic embryogenesis and plantlet regeneration in mango

(Mangifera indica L.). In Vitro Cell Dev Biol Plant, 38(2):173-177.Pati, R., Mishra, M., Bajpai, A. and Chandra, R., 2005. In vitro regeneration of mango (Mangifera indica L.) cv. Kurrukan.

In: National Symposium on “Plant Biotechnology: New Frontiers” to be held at CIMAP, Lucknow, India, during18-20th November. pp. 55.

Pliego-Alfaro, F., Litz, R.E., Moon, P.A. and Gray, D.J., 1996a. Effect of abscisic acid, osmolarity and temperature on invitro development of recalcitrant mango nucellar embryos. Plant Cell Tissue Organ. Cult., 44:53-61.

Pliego-Alfaro, F., Monsalud, M.J., Litz, R.E., Gray, D.J., and Moon, P.A., 1996b. Effect of abscisic acid, osmolarity andpartial desiccation on the development of recalcitrant mango (Mangifera indica L.). Plant Cell Tissue Organ Cult.,44:63-70.

Raghuvanshi, S.S., and Srivastava, A., 1995. Plant regeneration of Mangifera indica using liquid shaker culture to reducephenoloc exudation. Plant Cell Tisss Org. Cult., 41:83-85.

Rivera-Dominguez, M., Manazanilla-Ramerez, M.A., Robles-Gonzalez, M. and Gomez-Lim, M.A., 2004. Induction ofsomatic embryogenesis and plant regeneration of ‘Ataulfo’ mango (Mangifera indica L.). Plant Cell Tiss. Org.Cult., 4586:1-4.

Rao, A.N., Sin, Y.M., Kathagoda, N., and Hutchinson, J.F., 1981. Cotyledon tissue Culture of some tropical trees. In:Proceeding of. COSTED Symposium on Tissue Culture of Economically Important Plants. (Ed. Rao, A.N.).pp. 124-137 Costed Singapore.

Reuveni, O., and Golubowicz, S., 1997. Trials of using in vitro techniques for vegetative propagation of mangoes. ActaHortic., 455:496-504.

Rivera-Dominguez, M., Manzanilla-Ramirez, M.A., Robles-Gonzalez, M., and Gomez-Lim, M.A., 2004. Induction of somaticembryogenesis and plant regeneration of ‘Ataulfo’ mango (Mangifera indica L.). Plant Cell Tissue Organ Cult79(1):101-104.

Rocha Monteiro de Andrade, S., Faleiro, F.G., Batista Teixeira, J. de Queiroz Pinto, A.C., Radel, G. , Rocha Cordeiro,M.C., Vargas Ramo, V.H., 2009. Uses of alternative antibiotics to control bacterium growth in mango explants820(1):319-324.

Sahijram, L., Bollamma, K.T., Naren, A., Soneji, J.R., Dinesh, M.R., and Halesh, G.K., 2005. In vitro embryo rescue inmango (Mangifera indica L.) breeding. Indian J. Hortic., 62(3):235-237.

Sawke, D.P., Ramtake, J.R., and Deshmukh, M.T., 1990. Irrigation and mulching studies in mango. InternationalSymposium. Natural Resources Management for Sustainable Agriculture, p. 101, 6-10 February, New Delhi.

Shahin, M.F.M., Wafaa, H.W., El-Hammady, A.M., and Haggag, L.F., 2003. In vitro propagation of mono and poly-embryonicmango cultivars. Arab Univ. J. Agric. Sci., 11(1):343-357.

Singh, S.K., Sharma, H.C., Goswami, A.M., and Singh, S.P., 2001. Abscisic acid aided normal somatic embryogenesisfrom nucellar tissue in mango cv. Amrapali. In: Recent Trends in Horticultural Research. (Eds. Rai, M.; Kumar,S.; Singh, R. and Prasad, V.S.R.K.). pp. 161-164.

Singh, S.K., Sharma, H.C., Singh, S.P. and Kishore, P.B.K., 1991. Callus induction and root initiation from explants ofzygotic embryos in mango hybrids. Plant Tissue Culture and Biotechnology: emerging trends. Proceeding ofSym., pp. 129-133.

Singh, S.K., Sharma, H.C., and Singh, S.P., 2002. In vitro pol-yembryony in mono-embryonic mango cultivars (Mangiferaindica L.). In: Sustainability of Hill Agriculture: Emerging Trends and Possible Solutions. (Ed. Kapoor, A.C.)pp. 295-299.

Sulekha, G.R., Rajmohan, K., 2004. Relative response of varieties and explants in the induction of somatic embryogenesisin mango (Mangifera indica L.). South Indian Hortic., 52(1-6):5-12.

Thomas, P., and Ravindra, M.B., 1997. Shoot tip culture of mango, influence of media, genotype, explant factors, season,and decontamination treatments on phenolic exudation, explant survival and axenic culture establishment.J. Hortic. Sci., 72:713-722.

Thomas, P., 1999. Somatic embryogenesis and plantlet regeneration from nucellar tissue of monoembryonic mango.J. Hortic. Sci. Biotechnol., 74(1):135-139.

Usman, M., Jaskani, M.J., Rehman, M.Y., Fatima, B. and Khan, I.A., 2005. In vitro regeneration of mono-embryonicmango cultivars. Int. J. Biol. Biotechnol., 2(1):23-27.

90 Advances in Horticulture Biotechnology (Vol-1)

Wu, Y.J., Hunag, X.L., Xiao, J.N., Li, X.J., Zhou, M.D., and Engelmann, F., 2003. Cryopreservation of mango (Mangiferaindica L.) embryogenic cultures. Cryo-lett., 24(5):303-314.

Xiao, J.N., Huang, X.L., Wu, Y.J., Li, X.J., Zhou, M.D., and Engelmann, F., 2004. Direct somatic embryogenesis inducedfrom cotyledons of mango immature zygotic embryos. In Vitro Cell Dev. Biol. Plant., 40(2):196-199.

Yadava, U., Lal, M., and jaiswal, V.S., 1990. In vitro micro-propagation of the tropical fruit tree Syzigium cumanii L. PlantCell. Tiss. Org. Cult., 21:87-92.

Yang, Z., and Ludders, P., 1993. Effect of growth regulator and media on in vitro shoot tip culture of different cultivars ofmango (Mangifera indica L.) rootstocks. Acta Hortic., 341:240-247.

Zatyko, J.M., simon, L., and Szabo, C.S., 1975. Induction of polyembryoni in cultivated ovulte of red currant. Plant SciLett., 4:822-825.

Zimmerman, J.L., 1993. Somatic embryogenesis: a model of early development in higher plants. Plant Cell.,5:1411-1423.

Ziv, M., Schwartz, A., and Fleminger, D., 1987. Malfunctioning stomata in vitreous leaves of carnation (Dianthuscaryphyllous) plant propagated in vitro, implications for hardening. Plant Sci., 52:127-134.