BIOTECHNOLOGY AND THE IMPROVEMENT OF CASSAVA, YAMS AND PLANTAIN IN AFRICA

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BIOTECHNOLOGY AND THE IMPROVEMENT OF CASSAVA, YAMS AND PLANTAIN IN AFRICA

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BIOTECHNOLOGY AND THE IMPROVEMENT OF CASSAVA. YAMS AND PLANTAIN IN AFRICA

Contributions from a Meeting of African Research Institutions Ibadan. Nigeria 8-9 Aug\dt 1988

INTERNATIONAL INSTITlJIE OF TROPICAL AGRICULTIJRE

Ibadan

e , 988

International Institute 01 Tropical Agriculture

Oyo Road, PMB 5320 lbadan, Nigeria TELEPHONE (022) 400300-400314 TELEX TDS ISA NG 20311 (Bol< 015)

CABLE rnoPFOUND IKEJA FACSIMILE 234-' -669185

ISBN

978-131-033-2

FOREWORD The International Institute of Tropical Agriculture (IITA) undertook a strategic study and planning exercise over the past few years. In order to sharpen the focus of Its research actMties and develop new modes of collaboration tn Its region of greatest concern. West and Central Africa. The strategic pIanntng study mobilized the best talents in African agriculture and Identified priority areas of research which require urgent attention.

In the area of commodity lmprovement. some of the new plant breeding objectives cannot be easily realized by means of conventional breeding. IITA felt the need to explore the possibilities offered in biotechnology tn order to apply non-conventional technologies in Its breeding work. particularly tn rool. tuber and plantatn crops. African agricultural research tnsUtuUons tnterested In those applications were invited to discuss the prospects at !ITA. The Meeting on the Use of Biotechnology for the Improvement of Cassava. Yams and Plantain tn Africa was held on 8 and 9 August 1988 at IlTA in Ibadan. Nigeria. with the participation of 25 experts who represented 14 tnstit utes tn 7 countrtes.

The specifiC objectives of the meettng were to: 1. Review the status of biotechnology research on cassava. yams and plantain in Africa:

2. Identify common constraints in the research and production of those crops.

3. Discuss the prospects for using advanced. non-conventional technologies to address the problems; 4. Exchange ideas and experiences among the research scientists in Africa.

The meeting agreed on a set of recommendations which would guide the respective InstltuUoIls In developing research applications of biotechnology. The present volume of contributions by the partiCipants Is Intended to promote awareness of African Initiatives among other regions of the world. and to broaden the base for fruItful collaboration.

CONTENTS KEYNUffi ADDRESS New opportunltles for crop Improvement through biotechnology

1

Gabriel B . Oguruno/a

Constraints on cassa"ll and yam research and production in Afrtca S.K. Hahn

9

Constraints on plantain and starchy banana research and production In Africa R. Swennen and S.K. Hahn

14

Germplasm preservation of tuber crops by tissue culture methods

15

Elizabeth Acheampong

Use of biotechnology In plant -quarantine processlr1g of plant Import and export materials

17

G.O. Adejare

Research strategies for Improved plantain and banana production In Africa S.A.O. Adeyemt and N. Udenst

19

Aspects of biotechnology for the Improvement of cassava (MwH/wl esculental and yams (Dtoscorea spp.) at the National Root Crops Research Institute. Umudike E .N .A. Mbanaso and L .S.O. Ene

22

Research activities of the tissue culture group at the University of Nigeria. Nsukka

26

C.E.A. Okezie

The use of biotechnology for Improvement of cassava. yams and plantain

27

D.M.Okioga

The prospects of Improving yams genetically through recombinant DNA technology G.O.Os>di

v

30

Field and laboratory research work on cassava In Zimbabwe A. I. Robertson

36

Cassava research and production constraints In Liberia

38

D.D. Wounuah

Root crop tissue culture work at the Institute of Agronomic Research, Cameroon

40

J. WuIDh

Biotechnology at IITA

41

K.S. FIscher

TIssue culture of cassava and yams at IITA

44

S.Y.C. Ng

Application of new virus detection techniques at UTA C. ThDttapptUy and H. W. Rossel

46

Biotechnology of plantain and banana D. Vuylsteke and S.K. Hahn

50

OurcOMES

Discussions on the priorities and constraints

55

Recommendations

60

ANNEX

List of participants

65

vi

NEW OPPORn1NlTIES l'OR CROP IMPROVEMENT THROUGH

mOTECHNOLOGY From the keynote

add~

GIIbrlel B. o,munola

Biotechnology Is the application of biology to the genetic Improvement of IMng systems, be It In plants for crop Improvement OT In animals fOT Improved animal health. A technique that Is basic to much of the progress In applied biology Is gene spl1ctng. In the last few years since scientists succeeded In spUclng the gene of one organism Into another, the science of genetic engineering and the evolving biotechnology have made great strtdes In genetic Improvement of great many biological systems.

Genetics Is one of man's most powerful tools. helping to revolutionize the treatment of dJseases. the feeding of the hungry. the manufacture of chemicals. the conservation of resources and the control of pollution. The fusion of microbiology and genetic engineering with traditional techniques of plant biochemistry. physiology and breeding promises benefits to society encompassing several of these areas. But none is of greater importance than imProving the world's food supply.

Genetically superior plants derived from modern crop improvement programs typically require a high level of crop management. Included In a regime of management may be the input of increasingly expensive nitrogen fert!llzer as well as the extensive use of pestiCides and herbiCides. all of whIch can result in toxic residue accumulation In the environment. In addItion . the high degree of inbreeding and the narrowtng of the genetic base of widely cultivated crops cause increasing concern about the susceptlblJlty of crops to major disease outbreaks and Imply that Important genetic tralts may be lost as world germplasm Is reduced.

With problems such as these. It Is not surprtsing that the advent of recombinant DNA technology Is generating excitement. A whole range of very speclflc plant genetic modlflca tions can now be conSidered with the use of methods that will no doubt generate a genetic diversity not naturaUy present In cultivated plants.

The same natural laws that govern the expression of DNA placed In new genetic environments through classical plant breeding apply to the expression, or lack of expression. of DNA placed in plants by recomblnanf DNA 路technology. To be successful In plant genetic engineering. we must begin to develop an understanding of the elements that control gene expression. The Significance to gene expression of precise DNA constructs Is beginning to be understood In bacterial. yeast. and even marnmaUan systems. in part because of the development of methods for IndUCing cell transfonnatlon. WIth

transformation methods evolvtng and useful genes being discussed. genetic transformation of plants can be considered realistically.

New techniques Involving DNA recombtnant can also be applJed for a more oriented genetic Improvement. Plants seem to be particularly adapted to the use of these new techniques. because there are many posslbilll1es of stngle cell/protoplast regeneration. The steps tn carrying out these transformatwns are: (a) Identification of active genes; (b) Identification of introduction vectors; (c) Construction of DNA recombinant molecules; (d) ClOning: (e) DNA Introducl1on Into the plants through the following methods: - Agrobacterium.

- DNA - eleclroporatlon.

- MlcrolnJeclion Into the zygote or the pollen tube.

- Leaf disc cuUures. Bombardment with tungsten/DNA. Large quantities of DNA can also be Introduced through: - somatic fusion.

- asymmetIical somatic fuSion.

- electrofuslon - PEG - Hlgh Ca+ and pH. - electroporation.

Screening test and vertfical10n of the expression In transformed plants are the last steps.

Plant transformation vectors

Although the transfer of cloned DNA between microorganisms Is routinely conducted In many laboratOries. the absence of convenient vector systems has to date Inhibited similar experiments with higher plants. However. rapid progress In this area has been made and a vaIiety of vectors are running Into practical use. One limitation to current vector deSign Is the lack of an Ideal transformation marker- a gene present on the vector which enables convenient Identification of transformed cells. The ability to provide a dominant selection for plant cells defiCient In alcohol dehydrogenase activity and to subsequently Identify alcohol dehydrogenase-positive revertants makes the alcohol dehydrogenase gene an attractlve marker for plant host -vector systems.

The antibiotic-reSistance genes. can be constructed and had been used as a marker. As many more convenient markers become available the development of mechanical gene Introduction methods (for example. micro-Injection or polyethylene glycol-mediated uptake of DNA by protoplasts) will be greatly facilitated. In the absence of conveniently scored markers. many plant transformation experiments have relied on natural routes of entry Into plant cells--the routes of plant pathogens. Although a variety of pathogeniC organisms may be 2

modified to serve vectorial functions as more becomes known about their mechanisms of infection and replication. efforts to date have centered on the double-strand DNA plant viruses (Caullmoviruses) and Agrobacterlum.

Rapid progress In vector construction has outdistanced two other areas of research which are critical to plant biotechnology successes. Initial transformation experlments are carried out at the level of a single cell In cult1lre. but relatively few agronomically significant crops can yet be regenerated routinely from cell culture.

This technology has been developed further. and many crops can be modified by recombinant DNA methods. The second problem Is equally significant-what genes can we transfer Into plants that w!lllmprove a crop species? Much of the development of present cultlvars has rel!ed on selection In claSSical breeding programs for polygenic characteristics such as Increased yield or protein content. wtthout an understanding of the molecular basis for such traits. In contrast. success In plant biotechnology wtIl rely. to a great degree. on a thorough knowledge of the genetics and regulation of the traits to be transferred.

A number of systems exist In plants which are being considered for manipulation through biotechnology. although a few examples demonstrate the magnitude of problems to be encountered.

Seed plOtelDs

The seeds of legumes and cereal graInS provide humans dlrectty with approximately 70 per cent of their dietary protein requirement.

Throughout seed development , storage proteins are syntheslzed and accumulated within the seed. apparently to provide a source of amino acid reserves durtng early seed germination. High levels of such protein In seeds provides an enriched amino acid source for both human and animal consumption. However, various deficiencies In certain essential amino acids do not allow either cereal grains or legumes to provide a balanced diet without supplementation of the limltlng amino acids from other sources. One widely-discussed approach for overcoming the nutritional deficiencies would be genetic englneertng of genes with encoding of the vartous proteins to Include new codons for the deficient amino acids, either by Inserting additional amino acids Into the protein. or substituting existing amino acids with those more nutrttlonally deSirable. However, there are a vartety of technical problems to resolve before such an engineering project can be successful. Structural conservation of the zein storage proteins of matze and apparent structural conservation of messenger RNA in legumes provide good examples of such problems.

To complicate matters further. there Is evidence that a conservation of nucleotide sequence In the vtcUIn genes of the seeds of various legumes may be significant to aspects of messenger RNA structural stabUlty and metabolism. If this proves correct. the genetic engineer must consider the effect of codon substitution not only on protein stabUlty but also on the folding characteristics of the messenger RNA 3

Because the storage protein systems under investigation are encoded In multlgene families. engineering of a single gene for higher levels of an amino acid would have a relattvely small effect on total seed protein composition unless the engineered gene was transcribed very actively or was amplified In the genome. A1ternattve approaches toward Improvement of seed protein composition. such as introduction of entirely novel proteins that are highly enriched In specific amino acids. can be considered. However. the problems of RNA and protein stability remain. and there IS the additional complication of obtaining accurate and high-level developmental expression of the new gene_ The transfer of genes encoding known storage proteinS to systems low In protein. or the provision of additional copies of genes to systems already prodUCing storage proteins. are further possibilities for improving seed protein quality and quantity; however. challl\elllng normal amino acid pools Into large amounts of a protein not normally present may well create serious metabolic Imbalances. not only within the protein-produCing cell but within the plant as a whole. Decreases In seed yield or alterations of other Important seed characteristics could easUy negate Improvements tn protein content.

Nitrogen fhation

Symbiotic nitrogen fIXation occurs within highly differentiated rOOI nodules formed by tnteractlons of Rhizobium with plants of the family Legumtnosae (soybean. peanut. bean. pea, for exam pIe). enabling these plants to grow without addition of nitrogenous fertUlzer. The pOSSibilities that Increased nitrogen fixation will Increase current legume yields, or that additional plant varieties could be made capable of carrying out nitrogen fIXation are being investigated from several approaches.

The formation of nodules that are effective in nitrogen fIXation depends on genetic information present In both the bacterial and host plant cells. Because Rhizobium can be easily manipulated In the laboratory. the potential for generating Improvement In the bacterial contribution to symbiotic nitrogen fixation seems high.

The potential for Improving nitrogen fIXation through genetic engtneerlng of host plants IS not as well defined. Host proteins are Involved In the plant bacterial symbiOSIS, but except for leghemoglobln (the protein responsible for proteCtion of the oxygen-sensItive nitrogenase). the fu'nctlons of host proteins specifiC for the symbiOSiS have not yet been resolved. However. there are examples of variable nltrogen-1lXtng efficiencies among legume cultlvars. ThIS suggests that exchange or alteration of the "symbiosIS genes" In plants might result tn enhanced nitrogen fIXation effiCiency.

Extension of symbiotic or asymblotlc nitrogen fixation to plants which do not now benefit from the process. would be extremely valuable. Evidence now suggests that free-living nltrogen-flxlng bacteria can be encouraged to associate with roots of cereals. enabltng 4

the plant host to receive some nitrogen through bacterial nitrogen fixation. It may be possible to genetically alter the nitrogen-fixing bacterta to bind more tightly to the roots of the cereal and thus create a more beneficial assoclatlon. 'However. creation of a new cereal symbioSIs which results In nOdulation will only be possible when more IS known about the host genes that contribute to the nodulation process.

The possibility that genes from the bactertal nIf complex can be moved Into cereals by biotechnology IS being explored.

Pest and pathogen reststance Several approaches may be used. to engineer crop plants genetically with greater Insect resIStance. A vartety of plant secondary metabolltes naturally discourages predators through various mechanisms. such as accumulatlng metabolites that mlmlc Insect hormones thereby upsetting maturation of Insects. The transfer to crop plants of genetic pathways required to synthesize such metabolites may provide resistances not present In cultivated crops. Alternatively. various polypeptlde insectiCidal toxins are In use as biological control agents. InCluding a range of toxins produced In strains of the bacteria Bacillus' thurtngtensts. While such toxins have the practical ecological advantage of being specifiC for certain insect species. they have to be applied to crops In costly spraytng programs. The prOduction of such proteins within the cells of genetically engineered plants mtght prOVIde pest resistance at both reduced. cost and with Improved enVIronmental safety over present control measures.

Stress tolerance ExtenSive research efforts are being directed toward developing a greater understanding of the phySiological. biochemical. and genetic bases for responses of plants to the environment.

Enormous genetic variability Is accumulated by plant cells proliferating In culture. Additional variability can be Induced In cultured cell populations by exposure to mutagens. This pool of genetic diversity can be examined for agronomically desirable traits at two levels of dlfferentlatlon. Populations of plants regenerated from callus cultures can be screened by conventional methods. Alternatively. selective culture conditions favoring growlh of specifiC mutant types can be applied at the cellular cell. The several charactertstlcs that have been Introduced by these methods to date are a harbinger of future contributions to be made by cell culture toward the genetic Improvement of crops.

Genetic transfer In plants through interspecific protoplast fusion Protoplasts of sexually incompatible species have been fused and In some combinations have given rise to somatic hybrid plants.

Partial eltmlnaUon of parental chromosomes from either species ts common In such hybrids. but total chromosome loss has generally 5

occurred only with phylogenetlcally unrelated palrlngs. Genetic function of one parent may be retained despite a complete loss of Its chromosomes, suggesUng tllat genetic Introgresston Is possible In the absence of complete donor chromosomes.

AppUcaUoDS

The few systems mentioned above have been w1~ely dlscussed In recent years because of the tremendous potentlal economlc Impact of Improvement. For example, the complex functions of most of these systems, Involvlng many genes of unknown Identity, make them difficult to exploit. However, the Improbablfity that we wlll see rapid successes In such complex: areas as improving plant pmteln levels or In the construction of plants that Ox their own nitrogen does not mean that plant geneUc engineering Is far from reality. Transfer of single gene traits Is technlcally feasible.

It Is likely that herbicide-resistant plants wlll soon be developed through transformatlon technology. A single new gene may be all that Is required for this trait and direct selection for transformed cells In tissue culture Is provtded by the herbICide resistance. Some pathogen resistances may be only slightly more dilllcuJt to transfer, since direct selection may again be possible for the desired resistance, both In tissue culture and In the Intact plant. Whether or not the lnJtlal plant var1eties resulting from such experlments can be easily Integrated Into practical breeding programs Is, for now, an unanswerable question-we cannot predict how such genetIC alterations w1lJ affect the metabolism of an organlsm as complex as a higher plant.

A recurring problem In considering any specifiC appUcation of &enetlc engineering In plants Is the lack of understanding of the molecular genetics Involved . Before practical appUcations can be routinely expected, basic research Is required In almost all areas of plant molecular biology. In partICular, novel approaches are needed to aid In the Identification of the genetIC components of plant characteristics. Model systems such as yeast, algae, or bacteria, whiCh are more conveniently manipulated under laboratory conditions than are higher plants, wtll be useful for some appUcations and will perhaps aid In isolation of some single or closely linked genes. More complex traits, those whICh are _lot expressed In model systems or which are polygenic In character, will need to be explored In other ways. One promlslng mechanlsm may Involve the use of transposable elements.

Plant transposons, analogous to those In prokaryotes. are genetic elements that are able to move to new locations In the plant genome.

Upon moving Into a specific genetIC locus, a transposon may alter an Identifiable gene function. With the use of recombinant DNA technology, It Is possible to Isolate and characterize DNA surrounding the site of the transposon Inserilon, thus ldentlfytng genes responsible for a specific trait. It may be possible In this way to characterize the major components of some of the more complex plant traits, where genes cannot be Identified In other ways.

6

The potenttal for Improvement of crop plants through genetic englneertng seems vast. Although only a few broad areas have been considered here. It should be apparent that the present Ilm1t on application of the many Ideas for crop Improvement Is basic understanding of the genetic components responsible for plant characteristics. Once genes necessary for valuable plant traits have been Identified. a variety of practical appl1catlons will rapidly come Into being. Initially It should be possible to develop convenient germplasm screening methods for the plant breeder. reducing the time required to organize and analyze genetlc crosses. Certainly the transfer of genes Into new plant species beyond the range of c1assJcal breeding is being attempted. and as we delve more Into plant biochemIStry. molecular biology and physiology. new applications and new approaches will naturally evolve. Plant molecular biologists can be expected to follow the leadership of scJenUsts working on the better developed animal and bacterial system; however. recent excitement In plant researcb IS certain to stimulate faster progress In plant biotechnology.

Although conventional plant breeding has had and will continue to have a tremendous Impact of geneUc Improvement. there are obstacles to rapid Improvement. particularly In the case of interspecific hybrldlzatlon. which prevents the use of disease resislance genes from wild species. In some cases. those obstacles can be overcome by some relatjve1y simple techniques such as: (al ferllltzatlon on flower buds; Ibl In uttro fertlltzatlon directly on ovules; (cl fertilization on gytloeclum. ovatIes; (d) embryo rescue.

When Interspectflc hybrtdJzatlon Is pOSSible. the technique of haploid plant induCtion through anther or mtcrospore cultures could be applJed to Fl generations In order to set directly the most favorable gene combinations.

Also for the numerous species which 'a re vegetatively propagaled. geneUc variability Is rather p()()r generally. In addition to the advantages they present as far as multiplication IS concerned. m1cropropagatlon techniqUes are very interesting for the development and use of a large quantity ol vartants due to a phenomenon called somac1onal vartatlon. ThIS provides a new source of Variability for the genetlc Improvement of those species. wblch could further be Increased considerably by attempting to get the dlfferentlatlon-prol1feratlonregeneration cycle. resulting In new plants from adventltlous buds. or by comblnlng the regeneration of one cell With mutagenic treatments such as gamma and X-rays.

7

Experts have reviewed the following major crops In UTA commodity research and have evaluated them for application of biotechnology .

Plantain. IITA research Into plantainS is among the most advanced of Its kind In the world. Mlcropropagatlon. mainly through new plant formation from what some scientists call ·protocorm". leads to the regeneration of several somaclonal variations. the spectrum of which Is not quite varted. Variability can be Increased through the combination of regeneration and mutagenic agents (X - rays and gamma rays) . Research on Isolation and production of protoplasts from cultivated and wild speCieS should result In attempts at introducing new genetic data through asymmetrical somatic fusion. This method could be used for black Sigatoka and bunchy top control.

Ca •• ava. Disease reSistance. low HeN content and drought resistance are the main constraints to cassava production. In vitro multiplication methods, with or without mutagens, should facilitate the sorting of somacJonal vartations.

Somatic embryogenesis techniques have been developed recently. Protoplasts can be Isolated and multiplied Into callus formations, although regeneration seems to be very difficult. This Is an area that requires more Intense research effort and collaboration with other laboratOries.

Yam. New seed-propagated Dtoscorea varieties will be developed soon. Cytogenetic research should be' carried out In order to identify the best parental lines and establish the origin of seeds that produce untfonn offspring, although genltors often have quite different chromosomic structures. Mlcropropagatlon, or In better words. dtfferentlatlon cycle, actively proltferated cell, seedling regeneratfon, could be a source of variations which would be probably much dtfferent from those obtained after crossing.

In conclUSion, there will continue to be need for basic research In genetic manlpulallon, biological nitrogen fixation, photosynthesIs, resistance to environmental stress and plant growth regulation .

Biotechnology In the areas of gene cloning and IdentificatioQ as well as characterJzation of dtfferent gene products will continue to have tmportant application to agriculture. [believe that while the research activities of UTA and the national fanning system pursue plant breeding objectives with biotechnological applications, a strong backup from the baSIC research labora-tories Is relevant.

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CONSTRAINTS ON CASSAVA AND YAM RESEARCH AND PRODUCTION IN AFRICA S.K. Hahn ill<

The m~or tropical root crops-cassava (ManihotescuI;.ta Crantz) and yams (Dioscorea spp.l- are wldely grown and are mostly used as subsistence staples In many parts of the tropics and subtroplcs In Africa.

They are the major source of en"cgy for well over 200 mtlllon people on the continent. The leaves of cassava are often used as a vegetable providing protein. vitamins and minerats.

A. CASSAVA RESEARCH AND PRODUcnON CONSTRAINTS

). Diseases and :lests (a) Cassava mosaic virus:

Cassava mosaic virus (CMV) . which Is widespread and IS an economically Important disease. can cause yield losses of up to 95 percent. Losses of 80 percent from CMV are common whUe losses of 20 percent are nonnal In many parts of West Africa . The causal agent of CMV Is a gemenl virus. The CMV Incidence depends on the density and activity of Bemtsla tabacl which transmitted the disease . IITA has Identlfied sources of resistance to CMV and this resistance has been successfully incorporated Into high yielding cultlvars of acceptable quality .

The mechanism of resistance to CMV Is not yet understood.

Whether resistance to CMV Is due to phYSical barriers or to antiviral factors needs to be investigated. BeSides. the pathogenlc variation of CMV needs to be studied . As for the International distribution of viruSfree clonal material. virus Indexing method should be Improved. (b) Cassava bacterial blight

Cassava bacterial blight disease (CBB) Is one of the most devastating plant bacterial diseases. CBB occurs In many countries In South America and In Africa. The causal agent of this disease is attributed to Xanthomonas campestrts pv. manihotts. Characteristic symptoms of CBB are angular, watersoaked. as well as blight , wilting. die-back. gum exudation and vascular discoloration. Local cultlvars are susceptible to CBB which often causes complete crop failure under heavy infection. Sources of resistance to eBB were Identified and resistant clones have been developed.

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(cl Other diseases Other diseases such as cassava anthracnose and cassava root rot disease are also Important. (dl Cassa[)(1 green spider mite Cassava green mites (CGM) can reduce tuber yield by up to 40 percent, espec1ally In the late planted crop. Since It was first reported In Uganda In 1972, CGM hq become a serious pest throughout the major cassava growtng areas In Africa. CGM Is a dry season pest and It IS spread by Infested planting mater1al and by wind, but It Is not clear how the mites are earned on the planting material. However, the most Important method by which CGM Is dispersed IS by wind. In the morning, adult mites lower themselves from the leaves on s1lken thread so that even low wind currents can carry them over long dIStances. ThIS may be why the mite spreads so fast at 300 Ian a year. (el Cassava mealybug Cassava mealybug (CM) can reduce tuber yield by up to 54 percent In early planled cassava and up 10 84 pen:ent In the late planted crop whJJe It causes 100 percent loss for the leaf. CM was first reporled In ZaIre In 1973 and In 1977 It was described as Phenacoccus man!hot! from specimens collected In Congo and Zaire. Since It was first dIScovered, CM Spread rapidly so that by 1980 It had covered almost all the major cassava growing countrtes on the west coast of Africa.

UTA has taken two approaches to tackle these two pests, CGM and CM, by host plant resIStant breeding and biologICal control. Resistance to CGM and CM Is associated with pubescence. The average CGM scores of pubescent types were lower than those of non-pubescent types.

Varieties that have some level of resis tance to CGM and CM have been Identified. Natural enemies of CM were found to be effiCient in controlling CM .

2. Nematodes Nematodes are becoming more and more Imporlant as human population Increases. This Is because crop rotations are becoming shorter and food crops are being planted more frequently. Whlle nemiltodes can be controlled through cultural, physical, chemical and biological methods, developing resistant cultlvars increases and stabUises yields. At the same time, these Improved cultlvars are avallable to the farmer at no extra cost to him.

The most Important nematodes of cassava are the root knot nematodes, the root-lesion nematodes, the reniform nematodes, the Spiral nematodes and the false spiral nematodes. Studies at IITA show that cassava Is an excellent host for a number of nematode species.

10

3. Quality characteristics (a) Low in protein

The protein content of cassava appears to be law. It Is often said to be the cause ofmaJnutrition In high consumption areas. (b) Cyanide in the leaves and tubers

Cassava contains hydrogen cyanide In tubers and leaves In the form of cyanogeniC glucostdes which release HCN on hydrolysis when tIssues are destroyed. No acyanogenlc cassava variety has been reported. but the level of HCN varies with variety. It Is necessary to eIlmInate this cyanide to !mprove palatability through processing with different products. .

Ic) Poor viscoelastic propertIes

Cassava flour has been tested to substitute the wheat flour In bread making In order to reduce the cost of the bread. Because of the poor vtsco e1asUc properties of cassava flour. the most suitable vartety can only substitute 20 percent of the wheat flour.

4 . l'r'oresstng

The cyanide In cassava makes It necessary to process the tubers.

This Is labor-intensive and at the same Ume peel1ng and grating cause high losses. The poor storability of cassava tubers after harvesting Is also a problem.

5. Poor genetIC studles

Very llmited genetic studies have so far being carried out on this crop.

6_ Dry matter us. .fresh yield It has been shown that high fresh tuber ylelti Is negatively correlated with dry matter yield .

7. Symbtottc organisms

Cassava can be grown under marginal soil conditions. Mycorrhlzat has been reported to be assorJated with cassava and contrtbuted to the performance of cassava under poor soli. The role of mycorrhlzaQ. association Is not clear. Other symbiotic or free l1vIng organIsms might have these contributions.

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8 . Shy jIowertng

Shy flowering In some cassava vartetles, particularly those that have desirable characterIStics. has prevented the utU!zatlon of such genetic materlals for breeding.

B. YAM RESEARCH AND PRODUCTION CONSTRAINI'S

The most economically bDportant yam species grown In Afrtca are white yam (Dloscorea rotundata), yellow yam (D. cayenensls). trlfollate yam (D. dumetorum) and water yam (D. alatol. In Afrlca, whlte yam Is grown on the largest scale and at the same tbDe is the most preferred followed by water yam and yellow yam. Africa produces 96 percent of world yam production.

Yam production requires hlgh Inputs and Is labor- intensive. The crop Is dUIicult to grow because seed yams are expensive and often unavailable, seedbed preparation Is labortous and tedious, staking Is necessary and yet staking matertaJ Is expensive and often not available, weeding Is required for 4 to 5 months and harvesting needs speclal care.

Browning of the tuber, low multlpUcation rate and sprouting after dormancy period are among the constraints. Other research and production constraints are:

1. DIseases and pests (a) Yam mosaic uirus

The causal agent for this disease Is a pot! vtrus and Is transmitted by aphids. The disease symptoms vary because of

host/pathogen interaction. Typically, mosaiC patterns and chlorosis occur. Under severe attack the plants appear stunted. Other symptoms such as leaf distortion, shoestring, vein cleartng, green vein banding, mottUng and stunting are also observed.

(bl Water yam chlorosis The causal agent of this disease Is not clear; possibly a poti viruS Is Involved. A wide range of symptoms can be observed during all stages of plant development. Typical symptoms Include chlorosis, flecking, vein clearlng, puckering, necrosis, and scorching. ThIs disease needs to be studied. (c) Yam storage

rot

This problem arose mainly from the wounding and bruising during harvesting or transporting of the tubers. Pathogens Involved are a wide range of fungi and bacteria.

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(d) Yam tuber beetles

Yam tuber beetles make feeding holes of varying shapes on yam tuber. resulting In low tuber marketabll1ty. Yam setts are attacked after planting and the vegetative development of the plants can be Umlted.

2.

NerMtodes

The nematodes attacking yams are mainly the yam lesion and rootknot nematodes. The yam nematode Is found both In roots and tubers and In the soli. Yam tubers attacked by lesion nematodes show tissue dIScoloration. dry rot and deep cracks In the surface tissue. The rootknot nematode symptoms appear as light to heavy galling of the tuber and knobby or bumpy路tuber surface. They reduce the market value of the tubers. D. dumewrum showed high resIStance to the nematode 3. Flowering

The non-flowering and the synchronization of male and female flowers are the bottleneck In yam Improvement. So far only male flowertng D. cayenesls are observed. Can we Introduce the deSirable characters from D. cayenesls to D. rotundato:? The genetlcs of this crap needs to be well studied.

4. Non'pathogenic bacteria

Non-pathogeniC bacteria were found In the leaf of yam. There are vartetal dliTerences In the amount of bacteria accommodated In the leaf.

The role of such bacteria on the host plant is not clear and further investigations are required.

5. Gennplasm preservation

Preservation of yam germplasm under field conditions Is very dliTlcult. The labor Involvement is tremendous. The most problematic Is the loss of germ plasm due to nematode allack. rot in the field and during storage and other disease attacks.

13

CONSTRAINTS ON PLANTAIN AND STARCHY BANANA RESEARCH AND PRODUC'I10N IN AFRICA

R. SwameD aDd S.K. HahD

Summary The constraints on plantain and banana research and production in Afrtca are disease and pest problems, lotIgIng of the plant, low multiplication rate, difficulty In hybridization, poor genetic study and somatic vartation In tissue culture. The disease and pest problems Include black Slgatoka disease, Panama dlsease, bunchy top viruS (both on the yield reduction and virus detection problem). nematodes and banana weevil. lodging of the plants Is mainly due to the plant type, nematodes and weevil attack. Somaclonal variation has created problems In in vitro germpJasm conservation and mlcropropagatlon.

14

GERMPIASM PRESERVATION OF TUllER CROPS

BY TISSUE C1J1,TVRE METHODS

Work Is being carried out on in vitro methods Cor the preselVation and rapid multiplication oC yam (Dio$corea spp.) and cocoyam (Xanthosoma sagfttlfolluro) germplasm in Ghana.

The yams D. rotundata and D. cayenensls are believed to have Originated In West Africa and show their greatest diversity in this region.

Ghanaian yams show a great deal oC genetic variability.

Cultivated yams In Ghana include D. rotundata, D. cayenensfS. D. alata, D. dumetorum. D. bulblfera and D. esculenta. There are many wtld Corms oC yam. some of which are gathered. This genetic diversity Is essential to Improve crops and should be preserved.

Wild habitats and local land races which are the traditional sources of genetic variability are being lost through drought. bush Ctres. land Clearance. and the abandonment of Carms by aged and retiring Carmers.

Yam germplasm collections In Ghana are under field maintenance wtth Its associated hazards of drought. bush fires. diseases and pests.

Most yam germplasm collections in Ghana were lost durtng the 1983 drought.

In vitro methods are being devised Cor germplasm storage of various yams. Cultures are being stored uSing reduced physical and reduced nutrient conditions. Shoot.路.lip culture techniques are used to maximize genetic stability. Reduced growth Is achieved by Induclng osmotlc stress. the use of reduced media. and supplying sucrose at reduced or above optimal levels.

Current activity

The current project Is to assemble samples of all exiSting yam and cassava gennplasm coUectlonB in Ghana and to preselVe them as in I.!ltro. cultures and also as greenhouse plants. At present there are eight acceSSions in culture comprising three dUTerent species, D. rotundata.

D. alata and D. dumetorum.

Fourteen other acceSSions of yam comprising six species are In the process of being cultured. These Include three wtld Corms.

Observations so far Indicate that the various yam species reqUire dtfferent media for growth. Growth of D. alata shoot tips on a simple MS basal medium supplemented with 5xlO路 7 M NAA and 5xl0路 7 M BA with 20mg/1 casein hydrolysate Is 90路100 percent. Growth DC D. dumetorum on the same medium Is only 5 percent. It is Intended to 15

work out the growth requirements for the various yam species. It Is also intended to make' addltlonal yam collections. especially wild yams, as these are not represented in the collections.

The next phase of the project is to assemble cassava and plantain gennplasm and to store them as In vitro cultures. Such cultures can be rapidly multiplied for distribution when the matertal Is required.

16

USE OF BIOTECHNOLOGY IN PLANT QUARANTINE PROCESSING OF PLANT IMPORT AND EXPORT MATERIALS

G.O.Ade)ue

Different biotechnological approaches such as tissue culture. callus culture, protoplast culture, clonal hybridization and pollen/ovule and embryo culture have been used to produce pathogenfree materials, to preserve amI dl'O&libute germp\asm mateqai'i> and. espeCially within the shortest tll= to produce hundre.d .. and even thousands of Identical plants commercially from a sIngl6 mother t~ue.. cultured iIJ lIiJro (1, 3, 4, 5, 6, 7).

The Nigerian Plant Quarantine Service (NPQSl IS fairly well equipped to carry out different biotechnological techniques In screening Imported plant materials fOT viruses, bacterta and fUngi. The use of meTlstem-tip culture In screening cassava, potatoes (Irish OT sweet). and banana/plantain against cassava mosaic virus, potato viruses and vlrolds, and bunchy-top virus respectively have been achieved and standardized. Such virus-free materials have always been Inspected, Indexed and certllled clean or healthy befoTe Telease. The Indexing methods being employed InClude infectivity assay, growing-on test, ELISA. and serological techniques especially agar gel diffUSion and micropreclpltln. The service has standardIZed the merlstem tip culture for rapid multiplication of cassava and banana (1, 2).

At present NPQS Is engaged In formulation of different media suitable for meIiSlem tip-culture for groundnut (ArachiS hypogeal. yam (DiOscorea spp.) and tomato (Lycopersicum esculentuml. Thts Is In or.der to screen and produce virus-free propagules from those crops which are In demand fTom sct.enUst$, agrtcultural ministries and local farmers.

The NPgS Is also equipped with highly trained personnel In seed pathology. Different seed health testing methods for viruses by ELISA, ISEM, DlB and electron microscopy In collaboration with !ITA and !AR&T, Ibadan are frequently used. For bacterial detection, the agar plate melhod for Isolation and characterIZation on selective media. use of near ultraviolet light (NlN) for the detection of fluorescent pathogens (bactertal and fungal). and serological tests fOT viruses and bacteria, are all in routine testing procedures. However the use of an Immunofluorescence microscope for bacteria detection Is being planned for the station. Fungi detection on seeds uSing blotter and agar plate methods and stem/compound microscopes for Isolation, Identification and characterIZation are routinely used. Finally, growing-on tests fOT pathogen Identification In the laboratory, growth chambers. glasshouses and phytotron (for extremely high risk plants) are also being used.

Biotechnology will continue to be employed in Plant Quarantine processing so as to safeguard the agrtcultural economy of the country, to continue to be a fllter organIZation and not a bottleneck to researchers, 17

scientists and agriculturists In their attempts to Improve germpIasm materials and distribute them from country to country. and finally to ensure the prompt release of disease-free. certified plant/seed materials.

REFERENCES

1.

2.

3.

4.

5.

6.

7.

AdeJare and Coutts. 1981. Eradlcatton of cassava mosaic disease from Nigerian caSli3va clones by mertstem-tIp culture. Plant Cell TIssue Organ Culture 1:25..:3211961).

Bolorunduro. Adejare. and Aluko. 1984. An investigation Into the use of tissue culture technique for mass production of banana and plantains (Musu spp.) In Nigeria. A project report for the award of HND In General Agriculture - lAR&T. Obafeml Awolowo University. lle-Ife. Dunwell 1985. Anther and OVaI)' culture. In Cereals TIssue and Cell Culture. S.W.J . Bright & M.G.K. Jones IEds) . pp.1-44. The Hague.

Martln us Nijhoff.

En,gsbro. 1983. A method to obtain pathogen free potato plants based on n,ertstem tip cult ure. Abstract of paper at the meeting of the pathology section - EAPR/EUCARPlA. Joint meeting at Aarhus.

Denmark 6-10 June.

Interna tlonal Board for Plant Genetic Resources. 1988.

Conservation and movement of vegetatively propagated gennplasm: in vitro culture and disease aspects. In lBPGR Advisory Committee Report on in vitro storage.

Kartha. 1986. ProdUCing and indexing disease-free plants. In Plant Tissue Culture and Its Agrtcultural Applications by Lyndsey A. WJth~rs and P.G. Alderson. Butterworths. London /80ston .

Ng. Rossel and Tbottapp!lly. 1987. The role of tissue culture In the '~st ablJshment of disease-free germplasm for International dis.1I:lbutJon. Paper presented at a workshop on the status and prospects of Integrated pest management for root and tuber crops In the tropics held at lITA. 25-30 October.

18

RESEARCH STRATEGIES FOR IMPROVED PlANTAIN AND BANANA PRODUCTION IN NIGERIA SAO. AdeJeml and N. UcIeJUd

The National Horticultural Research Institute (NIHORT) was established In 1976 with the mandate of conducting research Into genetic Improvement. agronomy. control of pests ,and dlseases and other problems associated with fruit. vegetable. and ornamental plant production in Nigeria.

The mandate crops of the Institute and their level of Importance are shown In the table below: Table 1. Mandate crops of the National Horticultural Research institute showing the level of Importance Order of Importance

Extert c(g tltMdJoo Cultivated Sem1-cuIUvated

National

Citrus Plantain Banana Mango Pineapple Pawpaw

lrv/ngUl gabonensts

ParkIa btglobosa

Guava

Avocado Pear Treculfa qfrIcana Dacryodes edults Teiracarptdtum cbnophorus Chrysophyllum albldum Bu/Tyospemum park"

Regional

International

Apple Pear Strawberry Gmpes Passion fruit Olive Sapota

19

The contribution ofNIHORT

Over the years. NIHORT has worked singly and in collaboration with the West African Regional Cooperative for Research on Plantain (WARCORPJ In studying the vaI10us aspects of plantain producUon In NlgeI1a. including spacing. cultural pracUces. pests and disease control. adoptable propagation techniques. nutrttlonal studies and utJllzatton.

Some of the advances made so far Include the development of a rapid mulUplication technique for plantains and bananas through the use of the spilt conn and spl1t bud techniques (SCT and SST) which give a sucker multiplicaUon ratio of 1:500 within a period of Six months and ensure as much as possible that the propagules are disease-free. SpaCIng trials have been carried out In the different agroecologlcal zones of the country and standard fertilizer recommendations for optimum productivity have been made. Utlllzation studies have shown that plantain can be used in composite flour production for confectionery.

Identified problem are.. for further resean:h

The present demand for plantain propagules Is enormous and by far exceeds supply. Conventional techniques now In use do not appear to be able to cope with thIS ever-increasing demand. NIHORT has embarked on the establ!shment of a tlssue culture laboratory to facilitate rapid multiplication of plantain/banana propagules.

There has recently been a seI10us outbreak of black Sigatoka disease of plantain/banana caused by the fungus Mycosphaerella fiJtensts. Chemical control of thIS dISease appears to be expensive or unavailable. The only hope for effective control is through extensive screening. seleCtion. breeding. and rapid multlpl1catlon of mateI1als found to be reSiStant/tolerant to the dISease.

The problem of the borer weevil (Cosmopolites sordldusl Is very serious In plantain production especially In large plantations.

Available chemicals for control are very expenSive and In many cases are not readily available.

Role of biotechnology In solving ldeIltl8ed. pmNeml

In vitro techniques exiSt for the rapid multipl!caUon. storage. conservation and exchange of valuable germ plasm mateI1als within and outside the country. Breeding of parthenocarplc plants like plantain and banana is not feaSible through conventional breeding techniques. A lasting solution lies In the use of In vitro breeding techniques to breed resiStant cultlvars.

Propoaal for further research 1.

NIHORT Is stili continuing with the search for effective chemicals for the control of the economic pests and diseases of plantain/banana In Nigeria.

20

2.

We are continuing local exploration for sources of resistance to these devastating pests and diseases.

3.

A tissue culture laboratory is being established for breeding. rapid multiplication and distribution to growers of any plantain cultlvar resistant or tolerant to black Sigatoka disease.

21

ASPECTS OF BIOTECHNOLOGY FOR THE IIIIPROVEMENT OF CASSAVA (AlANlHOT ESCULENTA) AND YAMS /DIOSCOREA SPP.) AT THE NATIONAL ROOT CROPS RESEARCH INSTnvrE. UIIllDIKE E.N.A. Mbpna so and L.S.O. EDe

The National Root Crops Res!!arch Institute (NRCRI) has a mandate to conduct research leading to Improved production, storage , processing and utilization of root and tuber crops, primarily cassava, yams, cocoyams, sweet potato, Irtsh potato and ginger. Cass ava and yams rank first and second respectively as root crops of economic Imporlance In Nigeria. Major advances have been made towards the Improvement of the quality of bolh these crops at NRCRI and elsewhere , as well as integrating them Inlo Improved and viable fanning systems.

CUIIava Nigeria produces about 12- 14 million tons of cassava. valued at approximately N2.8 billion, from between 1.9 and 2 .5 mUlion hectares of land . In experimental fields. yield up t o 20-40 tlha are easily obtainable against a poor 8-9 t/ha In farmers' fields. The defiCit In yield values highlights the unexplotted potential for routine cassava production .

NRCRl has a germplasm holding of up to 1002 accessions.

Screening. hybridization and selections have resulted In the development of many Improved cultlvars based on high tuber and dry matter yield, gaillication (quantity and quality), dtsease tolerance and adaptability to ecological wnes.

For agricultural production, cassava is propagated t hrough stem cuttings although seed propagation Is poSSible, Flowering Is frequent and regular in some cultlvars but rare or non-existent in others (8. lD), Because of the out -crossing within the species esculenta and with other Manthot species, large genetic variability exists In the natural population. Undoubtedly, many local cultlvars today are dertvatlve Qf natural hybrids. However controlled conventional breedtng work has led to the introduction of . high-yielding. disease-tolerant and lowcyanide cultlvars (5). Nonetheless. the combination of many destrable characters in one cultlvar has been rare, and yet other traits of extreme value In cassava agriculture, e.g. early maturtty. are not common.

The traditional vegetative propagation ensures the pr-eservatlon of genetic combinations over time even though multiplication Is slow and distribution of trnprovedl established cultlvars Is retarded. A rapid muitlpl1catlon technique In uitTo. developed at NRCRI. has Increased the multiplication ratio of cassava from 1: 10 to 1:40 and has proved very useful for the multiplication of breeders' stock. ,

22

However, In recent years, many workers have shown that cassava can be even more rapidly multiplied using in vItro propagation techniques (4,9,10,12). Cassava propagatJon will be completely revolutionized when faci1ltJeS for in ultro culture become more readily available. Virus-cleaning and maintenance of germplasm In culture will also be possible.

Yama

Yams are produced and eaten extensively In Nigerta. About 15.6 million tons of yams valued at N4 .S billion are procluced annually.

This Is about 74.3 percent of the world total production yet national demand falls 1.62 million tons short of the production figure. As with cassava, current farmers' yields are only 9 t/ha whereas up to 40 tlha Is possible In experimental fields . NRCRl has Identified scarcity of planting matertals, high labour costs. use of unimproved cultlvars, poor agrononUc practices, storage losses and disease as major constraints to yam production.

Major Improvements at !"JRCRI Include the development of the m1n1sett technJque for rapid multiplication of seed yams. By this method, 25gm setts are used as planting matertal for seed yam production, with the result that 1 tonne of seed yams yielded more than 14 tons of planting matertal, reducing the cost of seed yam production from 41k/kg to 3Ok/kg. This technology has opened the way to largescale production of planting matertal by seed yam growers.

Uke cassava, yams are traditionally propagated vegetatively, but unlike cassava, the edible tubers also serve as propagules. This fact alone necessitates alternative means for yam propagation. In vItro propagation from axillary buds Is the most practical route to yam Improvement. Not only do VIne cuttJngs serve as the propagation matertal but also the multJplicatJon rate Is significantly Increased (2,7).

A tremendous amount of vartabllity exists among the various cultivars of yams. About 366 accessions are being preserved In the Institute's germplasIri bank.

ConventJonal breeding for ImproverJ, varletJes, though feasible 11,11), has been fraught with diIllcultJes such as non-flowertng varieties, productJon of only male or female flowers, tnconsistent and sparse flowertng and poor seed set. Therefore yam Improvement over the years has been by selectJon of 'good tubers' by farmers, resulting In well adapted cultivars for different yam nIChes.

But, a means Is urgently needed for effectively combining desirable tratts from the wealth of variability alreacly existing or Improvtng well adapted cuittvars that need alteratJon or addttlon of one characteristic.

A few varieties of yams have been selected at NRCRI based on high yield, early maturity, resistance to disease or pests, storabillty and acceptable culinary qualities.

23

Appllcation ofblotecbnology

The Institute Is currently setting up a tissue culture unit. In uttro culture facllttles will make possible massive propagation of improved/established cultivars of cassava and yarns and Indeed other root and tuber crops fOT d1s1rtbution to farmers. ~arch can then be undertaken to back up enhanced perfonnance of plantlets transferred Jnto soil. tn terms of management practices to ensure high survtvablllty and yield. Merlstem tip culture for the prodUction of pathogen-Cree cassava and yams will also be exploited.

Living germpla'ilJl collections. often the only means of conservation. take up enonnous land areas and are velY costly tn labour and money. There Is also the added risks of losses through diseases, pests. and adverse field mndlttllPs. USIng in vitro facUttles, NRCRl Intends to establish large gennplasm collections of yam, cassava. cocoyarns and other Important root and tuber crops.

NRCRl Is also tnterested tn the use of tnduced mutation for the Improvement of cassava and yams. Mutation br!!edtng-1s a valuable supplement to the techniques available for plant breeders tn developing Improved vaneUes. In fact this technique offers large pOssibilities In vegetatively propagated plants especially where conventional breedtng Is dlfficully. Its main advantage Is the ablllty to change one Qr a few characters of an outstanding or weU-adapted cultlvar without altertng the rematn1ng genotype. Breeding objectiVes for cassava and yam would Include development of well adapted cultlvars. high yield. good tuber shape, earliness to mat urtty. prolonged stora bUlty. modifICation tn plant architecture and eUmtnatton of cyanogenic glucoside as may be applicable.

Mutation can be Induced by the use of lonlztng radiation or chemical mutagens.

Preliminary Investigation Into radiation senSItivity In cassava and yarns show that sprouting and axlliary bud development Is progressively reduced at doses fietween 20 to 40GY [Mbanaso. unpublished). After irradiation of vege,tatlvely propagated plants. a number of vegetatiVe propagation cycles [M I V I-M 1Vn) are necessary to allow the mutated sector to grow and develop tnto nonchimeric shoots [3). Doses Within the range mentioned make aUowance for this foUow-up procedure. Choice of parent material has been shown to be most decisive In any mutation breeding programme (6). Source o! parent material [in VUTO or in vtvo} also Influences radiosensitivity.

AxIllary buds from In vitro plantlets were more sensitive to lonlztng radiation than those from newly Initiated in vtvo shoots tn cassava [Mbanaso, unpublished). GeneraUy yam explants were less senSitive to high radiation doses in vItro than cassava explants (Mbanaso. unpublished). More studies are needed tn this area.

24

REFERENCES

1. Akoroda. M.O .. J.E. Wilson. and H.R. Chheda . 1981. Artificial pollination. pollen viablllty and storage in white yam. In Tropical Root Crops Research Strategies for the 19805. Proc. 1st Triennial Root Crops symposium of IS1RCAB. 8-12 September 1980. Ibadan.

Nlger1a. Teny. E.R.. K..A Oduro and F. Caveness. (Eds). pp 189-194.

2. Arnolin. R.. and Degras L. 1984. In ultro culture. results and prospects for food yams. In: Symposium oC the International Soc. Cor TropIcal Root Crops. 6th. Lima. 1983. Proceedings. Lima International Potato Centre C.I.P. pp. 641-648.

3. 8roertJes. C. 1911. Mutagen treatment and handling of treated material. In: Manual on Mutation 8reedtng 2nd ed. International AtOmic Energy Agency. Vienna. pp 160-166.

4. Cock. J.H .. D. Wholey and J.C. Lozano 1916. A rapid propagation system for cassava. CIAT series EE-20 CIAT Call Columbia.

5. Hahn. S.K. 1984. Progress on root and tuber improvement at IITA In: Symposium of the International Soc. for TropIcal Root Crops.

6th. Lima. 1983. Proceedtngs Ltma International Potato Centre CIP pp.55-58.

6. Mackey. J 1960. Methods of utilizing induced mutation In crop Improvement. Symp. Mutation Plant 8reedlng. Cornell University'.

Ithaca. pp. 336-364.

7. Mantell. S.H .. 5.0. Haque. and A.P. Whitehall. 1919 . A rapid propagatIon system for yams. Yam Virus Project Bulletin!.

Caribbean Agricultural Research and Development l{1stltute.

University Campus. S.L. Augustine. Trinidad.

8. Onwueme. I.C. 1918. The Tropical Tuber Crops. New York: John Wiley and Sons. p.1l1.

9. Roca . W.M .. A Rodriguez. L.F. Patena. R.C. 8arba. and J.C. Toro.

1980. Improvement of a propagation technique for cassava uSing single lear-bud cuttings: a prel1m!nary report. Cassava Newsletter 8 pp . 4路5. CIAT Cali Columbia.

10. Roca, W.M. 1984. Cassava. In: Sharp W.R .. D.A Evans. P.V. Ammiralo. and Y. Yamada Y. (Eds) Handbook of Plant Cell Culture Vol. 2. New York: Macmillan. pp. 269路301.

11 . Sadik. S., and O.U. Okereke . 1975. A new approach to improvement of yam, Dioscorea rotundata. Nature 254 (5496). pp. 134-135.

12. Smith. K.. 8renda J. Biggs. and Kenneth J. Scott. 1986. In vitro propagation of cassava (Manihol esculenla Cranlzl. Plant Cell Tissue and Organ Culture 6. pp. 221 -228.

25

RESEARCH ACTIVITIES OF THE TISSUE CULTURE GROUP AT THE UNIVERSITY OF NIGERIA, NSUKKA e.EA Okezle

The TIssue Culture Group Is part of the enlarged Biotechnology Research Group of the University of Nigeria. Nsukka. The Group consists of two plant phYSiologists and a plant geneticist. Our work has focused principally on yams and consists of the follo~tng: I. Embryo culture. We have for many years ~en raising piantlets through culture of both mature and Immature Dioscorea rotundata embryos either directly or through callus. depending on the way we manipula te our media . The basal media we employ are mainly Murashlge and Skoog . Llnsmaler and Skoog. and Schelnk and Hilderbrandt's medium. We have also had cause to employ White's medium .

Our Interest In embryo culture arose from previous work on morphogeneSiS with emphasis on the tissue culture of yams. which highlighted areas of yam iItlprovement which could not In the short term be achieved through conventional breeding and hybrtdlZation but needed the biotechnological approach .

2.

Anther culture. We have been a ble to raise haploid pla nts through anther cultures.

Culture of other yam explants. We have routinely raised plantlets 3. from both leafy nodal explants In the green house and leafless noda l explants In culture u s ing Dioscorea rotundata. D. cayenensLs and D. bulbifera . Our experience with Internode cultures through which we have also raised plantlets via callus h a s fortified our belief In yam tol1potency and we are extendtng our explant studies to root cultures.

4.

Most of the seeds we use for our embryo cultures have been obtained from a particular area of Imo State (Uturu . Okigwe) where we have nol1ced regular flowertng of D. rotundata species. Because of ~he endemiC nature of flowering In this plant we suspect an Interplay between environmental and chemical factors (probably phytohormones) In the yam flowertng process. Consequently. we have been running a number of expertments involving both photoperiodic studies and the infiuence of growth regulators on flowertng and other related processes of yam plants.

We recognize that meaningful continuation of our work will depend on the level of co-operation and collaboration we get from colleagues working in other parts of Nlgerta. and Afrtca as a whole. on the biotechnology of yams and related crops. We see IfTA as a rallytng body in not only achieving our goal but also providtng leadership In the application of bIotechnology for the Improvement of these crops.

26

THE USE OF BIOTECHNOLOGY FOR IMPROVEMENT OF CASSAVA, YAMS AND PLANTAIN

D.M.Oldop

Use of biotechnology In in llitro transfer of ,ermp1asm Regulations governing the Importation of cassava, yams and plantain Into Kenya demand that the materials should be processed through quarantine. Further, only plants which are established from tissue cultures are eligIble for release.

A primary purpose for the application of tissue culture In plant quarantine Is to mInimize quarantine restrIctions wIthout lowering phytosanltary standards since It Is possIble to Inltlate pest- or dlseasefree plantJets from buds obtaIned from Infected or tnfested mother plants. However. It should be borne In mind that without appropriate therapy and indexing. In vltro culture alone cannot guarantee virus-free daughter plants.

The major fungal and bacterial diseases of plantains, for example. that may be tntercepted durtng the transfer of genetic material as In vltTo culture are listed below. Infected plant matertal from any of these pathogens would readily be Identified since the pathogen would grow and reproduce in the media In ill vitro cultures.

A. FUNGI

1.

2.

3.

4.

5.

6, 7.

8.

9.

Mycosphaerella musicola M. ]igiensis var difformis M. Jli iens is Uredomusae Phylostlctlna musarum Phyllachora musil:ola Haplobasidion musae Mycosphaerella musae Fusarium oxysporum

J

cubense

Leaf spot Sigaloka Black SJgatoka Black leaf streak Rust

Freckle Black cross Malayan leaf spot Speckle Fusarium wllt (race 1.2 and Talwan race)

FungI occurring on the fruit are: 10.

II.

Pyrtcu1arta gri.sea Cercospora hayl

12.

Tranchyspaera fructigena

Pltttng diseaSe DIamond or Brown Spot

Cigar end

27

B. BACTERlA

Pseudoownas

Bacterial Wilt

solanacearum

race 2.

C. NEMATODES 1.

2.

3.

4.

5.

Radopholus slmtlts Pratylenchus coffea Pratylenchus pararobustus Rotylenchulus renifonnts Scutellonema sp.

Of the above路 listed pathogens and nematodes only Mycosphaerella musicola. Fusarium oxysporum J. cubense (Fusarium wUt race 11.

Radopholus slmt1ts and Rotylenchulus reniformts have been reported as occurrtng In Kenya.

The viruses reported on plantain are: 1.

2.

3.

Abaca mosaic Bunchy top Cameroon marbling

None of these viruses has been reported In Kenya. Routine Indexing of tmported germplasm uSing Chenopodium sp. IS carried out to prevent the introduction of these vtruses Into Kenya.

Use of biotechnology In germplum multiplication Rapid mlcropropagatlon of cassava and plantain may be achieved through tISsue culture techniques. The In ui1TO explants are normally of a high standard of health and are readily obtained from both terminal and/or auxlllary buds cultured on an appropriate medium.

Use of biotechnology In germplasm cleaning A valuable germplasm may be Infected with a vtrus. In the past, such germplasm had to be destroyed. However, with the advent of tissue culture techniques, the germplasm can now be saved through c1eantng.

The Infected plant is subjected to heat treatment at 35-37OC for a period of approXImately one month after which virus free buds from the plants are raised through the In ui1TO cultures. As stated above, It Is appropriate to re-Index the daughter plants to ensure freedom from viruses.

Use of biotechnology In &ermplasm conservation

Conservation of cassava and plantain In an ex-situ field genebank can be expensive. Experience at the Plant Quarantine Station, Muguga has shown that the potato plant (Solanum tuberoswn) can be 28

conserved In in vitro conditions provided that the culture medium Is modified for this purpose. The medium for conservation. for example. should contain reduced levels of sugar concentrations with an addition of manitol whiCh .lncreases In osmotic pressure of the medium. The cultures are maintained In reduced Ilght and temperatures. The In vttro plantlets can be malntained under these conditions for a period of 8 to 12 months. after which It ts necessary to transfer the In路vltro plantlets Into the fresh medium. Prellmlnary work with cassava indicates that a similar approach may succeed with cassava and plantains and thus cut down expenses Incurred In maintaining a field gene-bank for these crops.

29

THE PROSPECTS OF IMPROVING YAMS GENETICALLY THROUGH RECOMBINANT DNA TECHNOLOGY G.O路ORUI

Yams are the staple carbohydrate foodstuff for millions in almost all tropical countries. They are cultivated on a large scale In West Africa, Southeast Asia and the Caribbean. Total world production of yams Is estimated at 20 to 25 mill10n tons per year. Nigeria alone produces 70 percent of the world total (l). Cultural practices using yam tubers In rituals In Nigeria suggest that African yams originated and became domesticated along the Niger river basin. In West AfrIca, daily consumption Is from 0.5 to 1.0 kg per person (2): and In Nigeria In particular the white yam. D. rotundata. Is eaten as "fufu". a traditional dish consisting of pounded yam and vegetable soup containlng meat and fish. Even though Nigeria Is the world's largest producer of yams, they are still very bcpensive In the country.

Yams have survived as a tropical staple for many reasons. Their climbing stems permit access to better light In the forest belt with low radiation Intensities: their annual nature coupled with perennial tubers allow them to survive drought (2): their efficient photosynthetic system endows them with the greatest Sink capacity among crop plants 0): their high mOisture (65 to SO percent) and carbohydrate (16 to 33 percent) contents (3) enable them to sprout even when rainS are delayed: and the vine (young plant) is xerophytic. often covered with a waxy bloom (4) thereby mlnlmlzlng transpiration.

ProductlQn constraints

The production of yams as food Is beset with many constraints. In general. Htlle has been done to tmprove yam-cultivation techniques which stU) give low yields of 8 to 15 t/ha In peasant farmers' fields (5) .

Traditional CUltivation Involves tedious manual labor. Young school leavers are therefore not attracted to yam cultivation. Research Into methods of mechanizing planting and harvesting has not produced Significant results because of the large sizes and Var1;).ble shapes of the tubers.

Furthermore, the staking of yam Is not amenable to mechanization.

The planting setts (seed yam) are not readily avallable and are very costly. Large sells weighing 100 to 200g are needed because consumers prefer large tubers. To achieve this, about 30 percent of yams produced by the farmer are reserved for planting, which accounts for about 900 percent ofthe total cost of production (I). Large setls produce luxuriant vegetative growihs which require staking, which In turn has become costly because of deforestation. It Is however In the area of seed yam production that conventional practice has made a breakthrough.

The mlnlsett technique (6) with a multiplication of 40 planting setts from one large (ware) tuber promlses to make planting setts available relatively cheaply. The use of plastic mulch (I) In the minisett 30

technology has further eUmtnated staking and weeding. which were dllflcult processes to mechanize. The mlnlsett technique produces medium sized tubers (I-2kg) which are easier to harvest and transport and so storage losses are reduced. These advantages of the mInIsett technology have stimulated tncreased acceptance of medium sized tubers by yam consumers. Simple planters (8) and harvesters are being developed and tedious labor seems Ukely soon to become a thing of the past In yam cultivation.

The third major constraint Is the tnabUity of the harvested tuber to store well for up to 6 months. Pests. nematodes. 路brulses and sprouttng combtne to reduce the shelf-Ufe of tubers to about 3 months.

Effective storage systems are needed to ensure mInImal post-harvest losses (I). The recently developed thermodynamic envtronments (9. 10) which prolong the shelf-life of tubers have promlstng prospects for commercial yam storage. One factor making effective yam storage necessary Is that yams are an annual crop. But with proper Implementation of the m1n1sett method. It should be possible to raise two crops of tubers yearly. This would make fresh yam tubers available all the year round and would accordtngly minimize the need to store them for many months.

The fourth constraint tn yam production Is that they are attacked by diseases whiCh reduce both the yield and the economlc value of the harvested tubers. No control measures are yet avallable to combat the diseases effectively but IITA has Identified some cultivars which possess high tolerance and resistance to anthracnose (1). Chemical control of yam blodeterloration has been recommended (11).

Yams have been propagated vegetatively for many thousands of years. This has rendered the sexual multipUcaUve apparatus of the tuber so redundant as to be vesUglal. As a result many Important species and culUvars rarely produce flowers and viable seeds; others are completely sterile. Most of the better clones of D. rotundata are plstillates (3). It is also said that cultlvars of D. alala and D. esculenta are mostly tnfertJle. There have been reports of yield trials aimed at selecting cultlvars with high yield and desirable agronomic characters (12). Selection took a long time (1956-1964) because of the slow rate of multipUcation In yam and because of the dllflculty of obtatntng hlghpercentage establishment (13). Up till now. inconsistency of flowertng and difficulty of hand-pollination of yam remain the greatest constratnts to yam Improvement by hybrtdlzatlon 114). Polyloldy also occurs tn yams and It has been suggested as the cause of sterility tn D. a[ata (13). because the high numbers of chromosomes confound sexdetermination and lead to the production of abnormal gametes.

Because of these problems. the chromosomes of yams have not been mapped. neither have the MendeUan genetics of the tubers been studied.

Conventional genetic study on the yam Is thus dllflcult. At lITA, however. efforts have been made to understand the flowertng habit. developing hybridization techniques and improving seed germination tn nursery beds (1).

31

The objectives of the conventional breeding research are to develop high-yielding cultlvars. which are also resistant to diseases; breeding and selection of plants that are erect or semi-erect; selection of tubers wtth tough skln and small rounded or oval shape that would make them amernlble to mechanical harvesting; selection of culUvars with a relatively short growing season; and varieties with a higher protein content while maintaining good culinary qualities. IITA has reported some slgn1ficant progress In their yam breeding research: eUte new genotypes have been Identlfled. viz: 'IDa 291. a high-yielding water yam wtth resistance to necrosiS: and'IDr 131 and 156 which are hlghyielding white yams also wtth resiStam;e to necrosiS (1). These are Important beginnings In the genetic research on yams. However. the genetic complications of the yam chromosomes will prevent the easy application of conventional breeding procedures to the Improvement of the crop. Yam Improvement therefore calls for the application of nonconventtonal genetic procedures. tissue culture techniques and recombinant DNA technology.

Pawpecta of tfuue cultme ID JIIDl tmJll'OftJllent

The application of tissue culture techniques to yarn studies has made some remarkable contributions 路 especially In establishing the protocols for clonal propagation from nodal segments and merlStem tissues. One objective of tiSsue culture research on yam has been to utilize the technique for large-scale clonal propagation In ~rder to provide an allernatlve source of seed yams. In this way farmers may not need to reserve a large part of their harvest to use as seed yam the followtng year. Mantell et al (151 reported that It would be possible to obtain 65.000 seed yarns from a single nodal segment wlthlri 6 months.

Chaturvedl et al. produced about 2.56 million plants from a S1iIg1e-node stem cutting In a year (J 61. IlTA has taken the research further by developing simple procedures which will enable national agrtcultural researchers to handle tissue cullure materials (17). Large-scale clonal propagation of yams from non-tuber segments now seems possible especially for the distribution of IITA elite new genotypes. 'IDa 291. ror 131 and ror 156. whiCh are high-yielding. are resistant to leaf necrosiS and have good cullnary qualities. The distribution of IITA elite culUvars would Significantly Increase yam production In Nlgerta where low-yielding and disease-sensitive cultlvars are stll1 grown by peasant farmers. Further research Is needed to understand the relationship between the performance of cultured nodal explants and the age of the mother plant; the Influence of photopertods on the organogenesis and embryogenesis of cultured yam tissues; and the regeneration of plantlets from cultured yarn ceUs. Potential ofrecomblnant DNA technology ID yam Improvement Recombinant DNA technology has found Immense application In crop Improvement research on many different crops. It has been very highly developed for the manipulation of the genes of plant cells and so 32

can be easlly applied as a routine laboratory experiment on many plants. Interesting experimental results on the structures 路and mechanisms of regulation and expression of plant genes and gene fam!lles have been obta1ned from pea. tomato and potato. Through the agency of the tumor-tnduCIng plasmid of Agrobacterlum tumeJaciens. genes have been experimentally rearranged. and also moved from one plant to another to obta1n transgenic planis with novel characteristics.

Since many yam cultlvars are sterile. recombinant DNA technology appears te> point the way towards the genc:.t1C Improvement of the crop. It Is a rapid procedure because It Is coupled with tissue culture techniques to accelerate the seJectldn of transformed plants.

A number of laboratortes have commenced prellmtnary research on the agroinfectlon of yams with Agrobacterlum (18. 191. The results demonstrate that the plant molecular biology technology Is applicable to the yam (a monocot plant). Thus. those breeding experiments whICh have been dllTlcult to conduct may now be approached through recombinant DNA technology.

In this direction. the following ImpoFtant preliminary st udles need to be carried out on the yam cultlvars: 1.

Study of the susceptlblllty of the species. culttvars and varieties of the yam to agrobacterlal . infection In order to select those most suitable for the recombinant DNA experiments. The elite yam culttvars of IITA would also be used tn this study.

2.

Study of the organogehesls of the selected cultlvars with a view to Isolattng and characterizing' the proteins which are synthesized at speclfic stages of organ and tissue developmenLThls study will use the techniques of tissue/cell culture which IITA has perfected.The protetns which signal the development of specifiC tuber organs wUi be used tn the genetic engtneertng experiments to Identify the genes which control the development of such organs/tissues. Interest will be focused on the genes for tuber initiation and maturation. shoot tnltlatlon. and storage protetn syntheSiS. 3.

Preparation of clones of complementary DNA libraries from mRNAs Isolated from yam tissues at different stages of organogeneSiS. The resultant cDNA libraries will be screened either by abundancy probing or by ustng antibodies raised to the purified protetns.

4.

Preparation of the genomic libraries of the different cultlvars of yam and screening with the purified mRNA of the specifIC genes Isolated tn (31 above. In this way many Important genes of the tuber will be tndentlfled and purified ready for detalled structural studies.

5.

The specific genes as Isolated tn (41 above wUJ be subjected to detalled structural studies tn order to understand their regulation. expression and organization. For the Improvement of the storage protetn for example. the genes would be sequenced and the amtno acid codons. mechanism of regulation and expression modified by slte33

directed mutagenesis In order to Increase the essential amino acid content and quantity of the proteins. The modified genes would then be used to transform yam cells using the disarmed plasmlds of A. tumefactens. The expression of the modified genes In the yarn would be studied, leading ultimately to the rapid production of proteln-rtch yam tubers by tissue culture techniques. My research group Is currently conducting studies on this aspect of yam Improvement.

If the yam tuber can be made proteln-rtch, then It will provide both carbohydrate and proteins In the diets at no extra cost to the consumer. This Is the jusUflcation for the project on the productlon of protein-rich yam tubers through genetic engineering and tissue culture technJques . In the research so far. the cDNA library of the storage protein genes has been prepared and Its structure and expression are being investigated. Progress Is being hampered by the lack of funds to purchase essential equipment and biochemicals.

Thus tissue/cell culture techniques In conjunction with recombinant DNA technology will produce the long expected Improved cullivars of yam tuber.

REFERENCES

Hahn. S.K. . D.S.O . Oslru. M.O . Akoroda. and J.A. Otoo. 1987.

Outlook on Agr1culture. 16. 105路110.

2. Ammlrato. P.V. 1984. Handbook of Plant Cell Culture .VoI.3. 327354 . Amlrato. Evans Sharp and Yamada (Eds).

3. Eka. O.U . 1985. Advances In Yam Research. Codson OSUjl (Ed).

ASUfECH Press. Enugu. Nigerta.

4. Onwueme . I.C. 1978 . The Tropical Tuber Crops. John Wiley, Chichester.

5. Okoli. 0 .0. 1987. Propagation Techniques and Prospects For Breeding of Yams. Paper presented at the '2nd International Symposium on the Yam Tuber. ASlJrECH. Enugu, Nlgeda. 8-10 June.

6. IITA Annual Report 1981.

7. Okol!. 0.0 .. M.C. Igbokwe,.L.S.O Ene andJ.U. Nwokoye. 1982. Rapid multiplication of yam by the 'mInlsett technJque. Research Bulletin No.2 of NRCRI. 8. Akubuo. C.O .. E.U. OdJgboh. and U.C.N Anazodo. 1987. Design of a 2row automatic seed yam planter. Paper presented at the 2nd International SympOSium on the Yam Tuber. ASurECH. Enugu.

Nigeria. 8-10 June.

9 . Osujl, C.O. 1987. The manipulation of carbohydrate metabolism of yam tuber to prolong yam storage and to Induee untformlty of sprouting. Paper presented at the 2nd International Symposium on the Yam Tuber. ASUTECH, Enugu, Nigerta 8-10 June.

10. Ezelke. C.O. 1987.

Effects of cold. ambient and modified environments on storage characteristics of yam tuber. Paper I.

34

11.

12.

13.

14.

15.

16.

17.

18.

19.

presented at the 2nd International Symposium on the Yam Tuber, ASlITECH, Enugu, Nigeria 8-IOJune.

Nwaorgu, O.C., F.!. Onyenobl, and D.J. Wright. 1985. In Advances In Yam Research, Godson OsuJI (Ed) ASurECH Press, Enugu, Nigeria.

279-290.

Edem. E.U. 1975. While yam yleld trials 1957-64. Fed. Dept. of Agric. Research Memo No. 114 .lbadan.Nigeria.

Ene. L.S.O. and 0.0. Okol1. 1985. In Advances In Yam Research.

Godson OsuJI (Ed). ASlJfECH PresS. Enugu. Nigeria. 345-360.

Akoroda. M.a .. J.E. WUson. and H.R. Chheda. 198 J". In Tropical Root Crops. Proc. lsi TrIennial Root Crops Symposium of ISTRC-AB. ibadan. 8-12 Sept. 1980.

E.R Terry. Oduro. KA and Caveness. F. (Edsl.

Mantell. S.H .. S.Q. Haque. and A.P.J. \Vhltehall. 1978. Hortlc. ScI. 53; 95-98.

Chaturvedl. H.C .. A.K. Sharma. M. Sharma. and R.N. Prasad. 1982.

Plant Tissue Culture. Fuglwara. F. (Ed). 687-688. Maruzen. Tokyo ..

UTA Manual Series No.6. 1985.

Schafer. W.. A Gorz. and G. Kalil. 1987. Nature 327: 529-532.

Xinhua. F .. S. QUlquan and J. Xlngcun. 1986. Genetic Manipulation .In Crops Newsletter 2; 52-59.

35

FIELD AND LABORATORY RESEARCH WORK ON CASSAVA IN ZIMBABWE

A.I. Robertson

We started tissue culture work in Zimbabwe in 1978 when I managed to assemble the basic equipment. a flow cabinet. an autoclave. some chemJcals and a constant temperature room. We worked first. battling against incredible infection rates. to set up vlrus-ellmlnation procedures---meristeming-for both strawberry and potato. These are now effective and operating and contributing to Zimbabwe 's high quality in these crops.

In 1982. we felt ready to tackle a tougher crop-cassava. We collected all local land races and imported both seed and 10 selected lines from IITA. When grown out. and pu t into single-row variety trtals. the IITA lines yielded around 30 t/ha which was a big improvement on the local lines which were IO to 20 t/ha.

Since 1982 we have embarked on what are now five cycles of crossing and selection seeking adaptation to our specJf!c condltlons. We have been selecting for tuber shape. canopy shape. resistance to Afrtcan mosaic virus and so on. Recently. many promising lines have been rejected on the basis of a lower tuber-to-total-blomass ratlo-lhat Is. harvest index. We are also looking for high dry-matter content In the tuber.

We have half a dozen promJslng lines which are now at advanced variety trial status on two different locations that we are confident will yield over 40 t/ha/year In ZImbabwean rainfed conditions.

We are aiming to provide a "food securtty" crop for our farmers. one plant which will yield every season In proportion to whatever rains It receives. The reason is that many of our peasants plant hybrid maize tn areas where statistics show that they wUl get nothing at all In one year because of low or wrongly-timed ratnfall.

When we were just about ready to launch the last of these lines. we hit two problems. Firstly, there was a build-up of a systemic fungus.

Colletotrichum. causing severe losses. Secondly. this year and last year. we suddenly had serious African mosaic Virus symptoms--agaln with major losses. Either the heavy viruS infection has overcome the genetic resistance or. we suspect. the resistance may have been lost In our selection procedure since the disease does not always show itself under Zimbabwean condilions. Thus. although the tnfectlon potential Is flown In our susceptible endrows. the disease is not seriously manifested due. we thought. to our rather lower temperature . This has been a serious setback to our programme and means we need an indexing system other than field observation. We are working on a tissue culture solution to this.

36

Nevertheless. the yields are still over 40 t/ha/yr and we will begin demonstrating their potential to government. rural farmers and commercial interests this next rainy season. InCidentally. the crop does best in Zimbabwe if it is harvested after two rainy seasons.

Biotechnology In cassava

Our hope is to carry these promising developments further and we look to modem methods of biotechnology to do this. This Is part of a dream of building a cRUcal mass of DNA and tissue culture scientists. so that we will participate in the coming gene revolution.

The biotechnology aspect of cassava is for genetic improvement through: Ca) (b)

Somaclonal selection and Gene transfer of desirable traits from other species.

To do tWs. we need to crack certatn technical barriers: 1.

As far as I know. no one in the world can produce a plant from a cassava callus.

2.

Similarly. no one can produce a protoplast of cassava that wU\ diVide and grow into an embryo and on to a plant.

3.

Though ClAT was attempting to tnfect young in vitro leaves with Ti-doctored plasmids to provide a route for gene transfer. there is no sign of success yet.

In our laboratory tn the last months. we have made some progress in both areas. somatic embryogenesis from in vitro leaves and embryogenesis from protoplast culture. It would be exclttng if an African laboratory led the way to improvtng what is such a vital crop to so many African farmers.

37

CASSAVA RESEARCH .AND PRODUCTION CONSTRAINTS IN LIBERIA D.D. Wounuab

About 70 percent of the 2 million people of Uberia are engaged In agricultural production. but the country remains an lmporter of its staple food. rtce.

Cassava Is the secllnd most lmportant food crop In the country and serves as a major source of carbohydrates (tubers) and protein (the leaves are widely consumed).

CODStraints 1. High incidence of diseases and pests.

2. Use of local varieties that are Inherently low-yielding and highly

susceptible to diseases and pests.

3. Lack of proper agronomic practices.

4. DUflculties In maintaining germplasm.

In 1978. a root crops program was initiated and In 1982 financial assistance was received from the International Development Research Centre (lORC). Since initiation. the program has identllled and released three vartetles of cassava (CARICASS 1. 2 and 3). which are found to be high-yielding (about 20 t/ha above local strains), resistant to cassava mosaic virus disease and cassava bacterta blight, and tolerant of cassava green spider mite.

Two varieties of sweet potato TIS 2544 and TIS 2532 are welJadapted to Uberta and their cultivation Is gradually spreading.

Tissue euhure facility

The tissue culture unit at the Central Agricultural Research Institute at Monrovta Is an Integral part of the Root and Tuber Program.

Established in 1983, Its basic role has been the handling of tissue culture materials of cassava and sweet potato received from UTA's tissue culture laboratory.

Materials received in vitro are sub-cultured and multlplled for field evaluation. Because of the lack of basic facUlties required for a workable tissue culture unlt, most of the valuable matertals received are lost. In 1978 about 166 accessions of cassava were collected from vartous parts of Liberia. However, this collection has been depleted by disease and pests.

We found that keeping a collection of germplasm under field conditions requires allocation of land every year and Is labor-intensive and therefore very expensive. Despite these constraints and through our collaboration with IITA's tissue culture laboratory which continues to 38

supply us with melistem tip culture which are free from diseases and pests. we have been able to Identify promising varieties of cassava and sweet potato.

At present. two valieties of sweet potato (TIS 2532, TIS 2544) received through this collaboration are well adapted to Liberian conditions and are grown by many fanners. especially In the North.

Future plaDa

1. Training.

2. Allocation of basic eqUipment. Laboratory space Is available but we lack the basic equipment.

3. Conservation of germplasm collection In vitro.

4. Rapid multiplication of Identified valietles for both research and distribution.

39

ROOT CROP TISSUE CULTURE WORK AT THE INSTI'llJTE OF AGRONOMIC RESEARCH, CAMEROON

J. Wutoh

Summary

The tissue culture facillty at the Institute of Agronomic Research (IRA). Cameroon, has Just been established. The main objectives are dlseas~ ellminatJon. rapid multJpUcaUon and germplasm conservation of root crops. cassava. yams. sweet potato and cocoyams. Work has started on testing ditTerent culture media suitable for culturtng of these crops. After testlng different media formulations, especially growth regulators. it was found that the best medium for cocoyam producllon was without growth substances.

40

BIO'IECBNOLOGY AT DTA

LB. FI8cber

Several of the plant breeding objectives having high priOrity In the IITA Strategic Plan are not easUy achieved through conventional breeding. Therefore. during May 1987. the !ITA Board of Trustees approved the development of an enhanced capability within the Institute for biotechnology research. An Important consideration In the Trustees' decision was the need to give JITA the capacity to assess the relevance of future developments In the field of biotechnology for its research programs and to foster links with advanced institutes and laboratOries.

It Is expected that such developments will become Increasingly slgn1flca nt. and that collaboration between breeders. agronomists. tissue culture specialists and molecular biologists will be needed.

IITA contemplates an evolution In Its biotechnology philosophy as new findings prove useful. The emphasis will be on applied biotechnology and not on basic research . The Institute wlll seek the adVice of a well-qualified consultant In developing plans for biotechnology research,and In designing and equipping laboratory facilities .

IITA will promote speCial projects for graduate students and train sclenllsts from nallonal programs. Such training wlll be available to graduate students dOing MSc and PhD thesis research and, as In-~rvtce training. to personnel from national programs already Involved In such work.

IITA will also cooperate with advanced institutions and Invite their scientists to come to IITA In order to validate their experiments under tropical conditions. UTA will thus playa promotional role In speeding up the application of the new techniques. particularly those that could help solve presently Insurmountable genetic problems.

AppUcablUty ofbloteclmoloaln eommodlty improvement research 1. Making crosses where conuenttDnal techniques are rwt Jeaslble (a) Grain legwnes

II). the lITA Grain Legume Improvement Program. reSistance to pod borers (Maruca testllllls) and three pod-sucking Insects has been found' In Vigna uexillata and V. IanCiJolia which are closely related speCies of V. WlgUtculata. Crosses of V. WlgUirulata with these reSistant v#gna species have not been successful. It Is envisaged that wide crosses could be achieved through:

41

fertUlzatlon of ovules iTt IIUro and excision and subsequent iTt vttro culture of embryos: (iI) somatic. or protoplast fusion: and (W) recombinant DNA techniques Involving tntroductlon of foreign DNA. (I)

(b) Roots. tubers and plantain

Utilization of desirable characters from the Wild related species has attracted Increasing attention In cassava and plantaln breeding.

IncompatibUlty between the crosses of cultivated and some Wild species has been encountered. Even In cultivated clones, the tncompatlblllty and shy-flowering which exists limits the choice of parents for breedtng.Prellmlnary work on embry%vule culture of cassava and yams has been started at UTA, but needs to be strengthened through the various manipulations engendered by biotechnology. (c) Rice

Some African wild rice accessions (Oryza glaberrtma. O. barthtO have exhibited higher levels of adaptation to Important African rice stresses. For example, two acceSSIons of o. glaberrtma and O. barthit and one of O. [ongtstamlnata have shown lmmunIty to rICe yellow mottle VIrus (RYMV). On the other hand. all. the O. sattva acceSSions tested so far are either susceptlble or show a low tolerance. Similarly. field observations tend t<J show deSirable levels of early seedling Vigor, drought resistance and other characters among the Afrtcan species. Transferrtng the deSirable traits from African species to the hlgh路yteldlng O. sativa background has not been satisfactory due to sterility problems. Approaches through biotechnology' methods will be highly deSirable tn enhanctng trnnsfer of the desirable tralls from the wild species to the high-yielding O. sativa speCies. [angtstaminata and O.

2. Genetic manipulation iTt tissue and cell cultures UTA is already utillzing tissue culture techniques In the ways deSCribed at the beginning of this statement. At the same tlme. IITA can take advantage of the various manipulations of plants that are engendered through the tissue culture technique. UTA Is handltng no less than four vegetatively propagated crops. Not least Important Is the work In developIng reSIstant culUvars to the black Sigatoka disease tn plantain and bananas through the use of somaclonal variation and iTt vitro screentng techniques. IITA has Identified other opportunities for specillc genetic transformation (gene cloning). The opportunity for the application of such techniques requireS techniques for the regeneration of protoplast. and while such techniques are currently developed for many crops. there Is a need for such work on cassava. plantain. yam and cowpea. Such research wlll not be conducted with UTA biotechnology facllfttes. However, IITA does need to collaborate with other advanced

42

laboratortes In order to focus research In molecular genetics on the problems of its mandated crops for Africa.

3 . Monoclonal antibody techruques Monoclonal antibody techniques provide the ability to conduct and analyze antigen composition of microorganisms for rapid diagnosis and study of disease organlsms such as bact~rta. fungi.. vtruses and plant toxins. The use of such techniques In the detection and Isolation of substratns of viruses associated with pathogenlclty of crops would provide a new avenue for research In the IITA virology unit. It would facilitate more reliable vtrus indexing. which is the cornerstone of IITA clonal germplasm exchange and distribution for International trials and which currently relles only on less efficient serological Indexing.

It Is envisaged that an IITA biotechnology unit w1ll conduct appUed research. relying on collaborative arrangements with approprtate advanced laboratortes for basic research. It Is therefore particularly approprtate thaUhe new unit would playa vital role In. on the one hand. stimulating baSic research on problems relating to food production In Afrtca. and on the other. training of Afrtcan SCientists In appUed areas of biotechnology.

43

TISSUE CULTURE 01" CASSAVA AND YAMS AT DTA S.Y.C. ~

The tissue culture facUlty at IITA was established In 1979. Its objectives were to evaluate and develop" media that are suitable for merlstem and nodal cu)ture of cassava. sweet potate. yams and cocoyams. to distribute virus-free clonal materials and to develop media for germ plasm conservation 01 these crops. Our emphasis at that time was on cassava and sweet potato. In 1981. when we began to distribute virus-free clonal (plantlet) materials to national programs. we faced the problem of low survival rate after transplanting. A training component was therefore added. A three-week tissue culture training course was conducted every year with an average of ten partiCipants per year, Since last year, however. we have concentrated Instead \>n Indlvld ual training.

In 1985, we reviewed the crop priority and placed more emphasis on cassava and yams, More recently. we have started to evaluate and develop some other tissue culture methodology that may assISt breeders to obtain desirable cassava varieties, TIssue culture activity The tissue culture activitieS on root crops can be broadly divided Into four categories: Disease elimination: Rapid multiplication and International distribution of virus-free materials: 3, Germplasm conservation: and 4, Evaluation and development of other tissue culture methods to assist breeding programs In selection of desirable cassava varieties.

L 2,

Disease elimination

Movement of vegetative materials of root crops has posed serious quarantine problems due to the danger of introducing diseases and pests to non-jnfected areas. Merlstem culture coupled with reliable virus indexing method has been effective In disease elimination, particularly viruses which are dIfficult to detect. Media for merlstem culture of cassava, yams, sweet potato and cocoyains were developed. The effICacy in dISease ellmlnatlon Is higher with a combination of heat treatment of mother plants followed by merlstem culture. Over a hundred varieties of cassava. three hundred of yam, more than one thousand of sweet potato and one hundred of cocoyam were regenerated from menstem culture.

44

Rapid multiplication and Internattonal distribution

Media for rapid multiplication uSing node cuttIngS (apical buds and axlllary buds) are well established In these root crops. It was possible to regenerate yam plantlets from tuber dIScs of freshly harvested yam tubers. The In vitro tuberlZation In yams was encouraged when the sucrose concentration In the medla was at 5 percent level. Aerlal tuber formation was also obtalned under tI vUro cultures. These prOvided opportunities for dIStributing virus-free yam clones using m1Crotubers produced from virus-free plantlets. Virus-free cassava clones have been distributed to 39 countries In Africa and to ClAT In Columbia. A proposal for the movemeni of virus-free yams has been approved by the Nigerian Plant Quarantine Service. aDd the mater1als w1ll 900n be ready for dIStribution.

Gennplasm conseroatlon

Conservation of root crop germplasm under field conditions has faced several problems. The most severe ones are the losses due to disease and pest attack, and drought and storage loss. In vUro reducedgrowth storage methods have been developed for these root crops. These methods Include lower incubatIOn temperature. reduced-growth culture medla and a combination of both. We have currently at IITA about one hundred clones of cassava (including ten Mo.nthot sp.). 1.500 clone~ of sweet potato. 300 clones of yam and 150 clones of cocoyam maintained under In vitro reduced-growth conditions. The plantlets can be maintained for 1 to 2 years depending on the varieties and crops.

Other tISsue culture methods

Somaclonal variations and gene transfer are very attractive approaches in developing new crop varieties wtth deSirable traits.

However. it has not been poSSible to regenerate plants from callus cultures of cassava. ThIS Is the major drawback to the application of biotechnology In cassava Jmprovement. We have been able to regenerate roots from callus cultures of cassava. Recently. using In 'vitro leaf grown In liqUid medla. we observed embryogenesIS from the explants. Further studies are being conducted to confirm these findingS wtth di1ferent varieties of cassava. We are also able to ISolate and purlCy protoplast of cassava from In vitro leaf. and micro calIl were obtained. No regeneration has been observed so far.

45

APPLICATION OF NEW VIRUS DETECTION TECHNIQUES AT IITA

G. ThottappUly IIIlcl B.W. Roe8el

Virus diseases may have a disastrous effect on crop yield and threaten the food-producing potential of Afrtca and other parts of the world. Development of resIStant cultlVars Is a basic requirement for effective management o(plant virus diseases. A sound knowledge of each pathogen. Its vartants or strains and their dlstrtbutlon Is a vital prerequisite for reliable breeding for resistance. Virus indexing Is also Important for collection, storage and international exchange of plant materials. Conducting a well-designed survey to gather information on distribution of viruses and their straJns should be one of the first approaches In dealing with a viruS problem. Survey Information will help the IndivIdual researcher to decide which strains of which viruses affect a crop. to detect unknown viruses. and to determine virus incidence. all of which are crucial In developing breeding strategies.

Diagnostic techniques should be sensitive. and reliable and also rapid and easy to use, and preferably capable of detecting vJrus straJns. It Is unrealistic to expect anyone diagnostic technique to meet all these crtterta. bu t It Is also often tmpractlcal to assay a large number of samples by more than one method.

Quarantine regulations. govemJng the tmport and export of plant materials usually do not pennlt. the presence of any virus. However. the Inter-African PhylOSanitary Council and the Nigertan Plant Quarantine Service. the quarantine-governing bodies relevant to UTA. allow improved germplasm to be sent to other countries In Afrtca In vegetative form provided these materials are put In tissue culture form and properly Indexed. National Programs of many Afrtcan countrtes have requested Improved clones of root and tuber crops developed at IITA for local adaptive testing. Sensitive and reliable methods of pathogen detection are the cornerstone for the production and maintenance of pathogen-free germplasm In tissue culture form. The most tmportant factors In the development of sensitive and reliable indexing methods are: 1. The proper Identification of pathogens. especially evaluation of various sensitive methods for detection of viruses and their strains.

2. Evaluation of varIous sensitive methods for detection of viruses and their straIns. The method[s) finally should be easy to handle but sensitive and re\!able.

VIrus lndezlng of regenerated plants

Plants regenerated from merlstem-t1p cultures obviously are not automatically free from pathogens. viruses In particular. Regenerated plants must be tested and shown to be free from virus Infection. This 46

may be the most time-consumIng stage In a dISease elimination scheme.

The regenerated plants are transplanted from tubes to ster1le soU In pots and kept In an isolation room for further growth and monitoring for disease expression. Virus indexing methods may vary depending on the type of virus Involved. and on avatlabUlty of facllltles . Ideally. regenerated plants should be tested for viruses by various methods and these tests should be repeated several times. Virus indexing procedures may take up to one year.

Some methods used for the virus indexing of the plant matertals are: 1. Monitoring for possible occurrence of symptoms over a long period of time.

2. Sap inoculation to test plants.

3. Grafting to suitable indicator plants.

4. Inspection of materials under the electron microscope for possible presence of virus particles In crude JuICe by the slmple "dip method."

5. Inspection of materials under the electron microscope using the immunosorbent electron microscopy method (ISEM,. For thIS method. antiserum Is added to the electron microscope carner !Urns to absorb viruS partIcles specifically. This method gives much greater sensitivity than the dip method. Also. virus particles adsorbed to the carrter fUm may be "decorated" with antibodIes In order to detect and Identify the virus under investigation.

6. Enzyme-linked Irnmunosorbent assay (ELISA!.

BIological diagnostic procedures are sensitive and indicate the infectious nature of the causal agent; however. the .necessary indicator plants require maintenance. screenhouse space and time to grow, while often a serological technique IS stUl required to conflnn the Identity of the virus Involved. Biological diagnostic procedures are not useful In surveys for virus incidence In other countries of concern to IITA's crop Improvement programs, because quarantine regulations prevent the importation of samples. New biochemical and serolOgical diagnostic procedures, which are also sensitive but otten require less labor, space and time, allow field problems to be Investlgated .1n a more thorough manner since more samples can be processed.

In addition to the highly sensitive serological techniques, such as ELISA and ISEM, the "dot-lIlot" immunoassay may be applied. This method seems to be even more sensitive than the others.

Recent developments In biotechnology permit detection of viruses by means of highly spectflc assays such as serological techniques using monoclonal antibodies. Another highly promising technique Is based on nucleic acid hybridization. These modem techniques provide hlgbly effiCient tools for the diagnosis of viruses and vlrolds. The ultimate benefit to be drawn from these activities Is a faster, safer and more effiCient transfer of Improved germplasm In vegetative form.

47

Monoclonal antibodies used In EUSA or dot-blot lmmunoassays. and the so-called Western blot methods are practical biotechnology Inilovations for tmproved dlagnosts of virus dlseases.

Hybrldoma technology provides methods of producing monoclonal antibody preparations of molecular homogeneity.

Monoclonal antibodies have been useful In differentiation of straIns. quantification of viral antigens. and detection and dtagnosls of virus Infection. The ability to dtstlngulsh minor antigenic differences with monoclonal antibodies has made them useful probes to differentiate strains of a virus. Access to them would be very useful to IrrA In order to Identify quickly and reliably several of the viruses and their variants or strains.

Reliable Indexlng methods are particularly Important In the testing of tissue culture materials meant for export and International adaptive testtng.

Visual inspection for virus infection of plants has many ltmltatlons. Reliable Indexlng methods provtde the cornerstone oC all programs leadtng to the production of vtrus-Cree propagation mater1als of clonally propagated crops.

There Is little doubt that monoclonal antibodies can be used to detect the presence oC a coaunon epltype. and that specific antibodies can detect specific serotypes. The superior dlscrtmlnatory capabiUty 'oC monoclonal antibodies compared to polyclonal antisera Is demonstrated by the addltional fact that an unlimited supply of monoclonal antibodies of known quality and specificity can be achieved.

Virus disease indexing will defln1tely be benefittng from the avatlability of a potentially unlimited supply of monoclonal antibodies of known quality and specificity. The ambiguities and limitations oC the present serological indexing ustng conventional. polyclonal antibodies will be eliminated as suitable monoclonal antibodies become available to complement the traditional antisera.

Slanlflcance of monoclonal utlhodlee The control of virus dlseases 15 of utmost Importance to the achievement of Increased food production. For example. the control of African cassava mosaic virus dlsease (CMV) Is of great significance In the cassava-growing areas of Africa and Indta. It Is evtdent that an understandlng of the nature of strains of CMV will be extremely useful In devising an effective strategy for Its control. It will also help to prevent the Introduction of new strains Into areas prevtously free of them. With monocitmal antibodies. when available. It may be possible to detect distinct strains which could not previously be detected.

Complementary DNA technique It Is the expreSSIon of the coat protein genes that has allowed for the extensive compartson of viruses by means of serological techniques.

48

All serological methods are based on the recognition of virus coat protein by antibodies specifically reacting with the coat protein. Only a small portion of viral genome IS Involved In the production of Its coat protein. Recent developments In molecular biology have opened up a range of techniques which Involve all or most of the nucletc acid being examined. Detection techniques using cDNA probes are highly sensitive and will also have Important applications In disease diagnosis.

In brief. cDNA probes are obtained as follows: viral genomtc RNA can be transcrtbed tlf DNA In the presence of labeled precursors via a special enzyme (reverse transcrlptase). The resultant labeled cDNA can be used to detect related viral RNA In plant tissues by an RNA-DNA molecular hybridization assay. The possible health hazards associated with the use of radiolabeled cDNA may be overcome by the development of non-radioactively labeled eDNA. Detection techniques based on the use of cDNA are often more sensitive than serological methods and their use by lITA virologists will matertally Increase the capacity to detect virus Infected plants.

The cDNA probes. and especially non-radioactive ones. are bound to become more popular. There will be increasing use In routine diagnOSIs especially because of the flexibility of being able to deSign the probe to the specific problem. A "broad-spectrum" probe could be developed for quarantine purposes and narrow-spectrum probes for epidemiological studies. UTA Is collaborating on this with scientISts at the U.S. Department of Agriculture laboratory In BeltSVIlle. Maryland USA.

49

BlO11!lCBNOLOGY OF PLANTAIN AND BANANA D. Vu;yt&teke m4 8.IL Balm

Gennplum of JIUsa 1. IITA has received 250 accessions through an intennedlate quarantine. Cathollc Leuven Unlversity. Belgium in collaboration with INIBAB. The gennplasm Is introduced tn tissue culture fonn.

2. Preservation ts In Intra.

3. Exchange IS In vitro.

Ploidy level

GenomJc constitution

Types

Source

Diploid

AA. AB. BE

wild and edible

Calcutta 4.

Plsang L1l1n

TriplOid

AAA

desert. starchy and beer bananas plantain cooking banana

KID 5.

tetraploid

IC2

AAB ABB

Tetraploid

AAAA

Ptsang Kelat Fougamou.

Nzlzi

Rapid multlpllcatJon Multiplication rate: 6-12 Aseptic culture: MedlaMurashlge-5koog media as basal media supplemented with I 11M lAA and 20 ~ SA. Cytoklnln - stimu late massive proliferation.

Mult1pllcatlon ts followed by subdiviSion of the multiple shoots and bud clusters.

The techniques of tissue and cell culture that can be utilized most profitably fall tnto the following broad areas:

50

I. Rapid and massive clonal mulUplicaUons; 2. Production of haploids or homozygous diploids and triplolds through ovule, anther, pollen and endosperm culture respecUvely; 3. Embryo culture for rescuing progeny of difficult crosses; and 4. Generation of somaclones DC tissue culture associated variants.

Each subculture gives propagules of 10-30 every two months. The shoots can be rooted In a basal medium supplemented with I 11M NAA and I j!.M BA. In 2-4 months, many plantlets are produced and transplanted.

EDIbrJO culture Plantain hybJid seeds are surface sterlllzed In 0.75 percentNaOCI solution (30 mJns., without pJior soaking. ZygotiC embryos are cultured on basal Murashlge and Skoog media with macro and micro nutJients at half concentration but full strength Fe-chelate.

This embryo culture/method has been routinely appUed for germination of hybJid seed between plantain and diplOid bananas which Is resistant to black Slgatoka disease.

Somaclonal variation The occurrence of somaclonal variation from tissue culture can be advantageous by introducing novel variabUity such as plant height and disease reSistance, etc. but It can be disadvantageous In in ultra mlcropropagaUon, conservation and exchange of germplasm. In certain culUvars of plantain, male organs are either absent or Incomplete.

Therefore. It Is d1fflcult to make use of the culUvars as male parents In hybridization. A plantain culUvar, "Agbagba", which Is the most popular In NlgeJia Is false hom type lacking male organs. Off-type plants of 60 percent were observed from a total of 965 plants produced by shoot tip culture. Of the variallons observed, the most interesting and useful was the reversion of false hom type to French type at the rate of 2 .6 percent. The latter has complete Influence with male flower organs. ThIs enables breeders to make use of such types as male parents In a crossing program. This also Indicates the possibility of reverting the French type back to false horn type which normally gives larger and more attractive fruits. A study Is being carried out to see tf this reversion Is possible.

In another experiment with cultlvar "Blse Egome 2", which Is a true horn type, 34.6 percent variation Into French type was observed but sUTpJislngly only 3.4 percent was true hom type . These experiments show that the extent of somac1onal variation In plantain seems to be genotype speclflc.

51

MorphOfenellis Induction of embryogenIC tissue

Research Into the regeneration of Musa from single cells through somatIC embryogenesis has concentrated on the use of explants from tISsues with actively dividing cells as these appear to have most embryogenic competence.

Embryogentcally competent tissue obtained by cultUring explants was cultured In medta contalnlng a strong auxin. These sUces of the merlStematiC tissue were excised at a size of 2-3 nun dtameter. Such explants were cultured In liquid Murashlge-Skoog medium containing 3 percent sucrose and 2 mg/l 2,4,5-T. Erleruneyer flasks contalnlng 30 mI medium and 4-6 explants were Incubated on a gyratory shaker under 15 hours Ilght.

Proembryogenlc masses (PEMJ appeared as tiny but protuberant, wllite globules on tbe surface of the expiants within 6-8 weeks to culture. The number of proembryogen1c masses was greatly Increased up to 20 PEM/explant by addition of extra reduced nttrogen to the 2,4,5-T contatnlng medium.

Genntnatton of proembryos Upon the Induction of PEM, somatic embryo maturation Is generally fastened by JOllleJ1ng the auxin concentration or even by omitting auxin In the medium exogenously appUed NAA alone or to combination with BA Invariably resulted In rhtzogenesls. Our tnterpretatlon Is that the rbtzogeneslS In Musa tissue culture Is an alternative morphogenetic pattern or an aberrant form of somatic embryogenesIs. Further expertments are betng carrled out.

52

OUTCOMES

DISCUSSIONS ON THE PRIORITIES Al'ID CONSTRAINTS

The discussions on priorities and constraints In biotechnology research Involved: (a) Sharing of perceptions on the Importance of various suggested foci for research on commodity Improvement of cassava, yam, and plantain In Africa; and (b) Consideration of the posslbUlties of setting and achieving targets In light of international and local resources avatlable for the tasks.

This process brought home to us the great need to improve cassava, the staple food commodity of Africa and especially of Its poorer people; and the equally Imporlant need to Improve Africa's capacity to perform the research In local institutions. On the basts of the dtscussions, the meeting formulated Its recommendations, Approaches to action on urgent Issues In crop Improvement are outlined as follows, under the subheadings of "needs路 and "possible action".

L Cyanide toz:iclty. Can or should the HCN content be reduced or removed?

NEEDS: Although no one would want all cassava to be free of HCN, It would be highly advantageous to have some "sweet" cassava varieties that have less or no HCN. Some populatlons traditionally ferment their cassava, some do not. The HCN Increases under (drought) stress and that Is the very time when the hungry poor reduce processing and cooking time and thus accelerate the slow potsonlng of themselves and their fa!Ililles. Cassava Is a backyard, roadside, "anywhere" crop, and if It were totally free of HCN, much would be lost to rodents and mammals, especially monkeys and baboons. However, In more formal plantings for market or commercial production, HCN-free lines would be benefiCial, particularly those that consumers could identify as such.

The participants were unarlimous In according the Idea very high priority, In that the need could only get worse as population pressure and rising prices pushed more families back Into cassava consumption, POSSIBLE ACTION: Discussion Included three possible approaches to answertng the question.

Mutation breeding based on cobalt-60 Irradiation. (a) Experiments are under way In Ghana and Nigeria along these lines In

55

similar crops. The HCN pathway Is redundant. as far as we know. and any enzyme along It may succumb to random lethal mutation. (b) "Reverse genetics": Identifying key enzymes. e.g. Ilnlmarase. and Interfering with the gene that produces It (Lichtenstein et al.).

(c) Wide crosses bringing In the absense of HeN from wild relatives.

This Is not simple. and would Involve a clearer understanding of the apomlctic-Ilke inheritance (Hahn) pattern In some such crosses already revealed.

2. NematodH. Can resistance to nematodes be found In Manthot or Incorporated from elsewhere?

NEEDS: ResJstance to nematodes Is a desperate need all over AfrIca In many crops. including the key root crops and plantain. Overworked soU Is particularly badly Infested.

POSSIBLE ACTION: It was agreed that a genome map and the use of transposable elements (should cassava have theml and RFLP analysiS were out of the reach of current efforts. However. bIOlogical control In the fonns of fungus and bacteria were being developed (Philippines.

University of Reading and Zimbabwe. perhaps elsewhere) . The need appeared to be great. but the work was percetved as being long-term and low In prtOrity for biotechnology.

3. Protem. improvement. Should we aim to fortify the cassava tuber content with more 'Protein and better protein?

NEEDS: Discussion focused on rising prices that protein (meatl commanded (e .g .â&#x20AC;˘ approxlmately a l5-fold Increase In Nigeria over the past ten yeazs) but that more nutritious vegetables (legumes) are being developed. Should cassava assume the burden for total nutritional needs? Surprtstngly many felt that It could do nothing but good. as cassava for cultural reasons is sometimes all that children receive.

"Kwashiorkor" Is a Ghanaian word and the problem is stili there. More protein In cassava would help. It was certaJnly felt that quantity should Improve. after which quality could be addressed.

POSSIBLE ACTION: discussed:

Two routes. high and low technology. were

(a1 High technology: the transfer of a 'Protein storage gene from. perhaps. 'POtato or a seed storage protein of legumes. The Jmpetus for a breakthrough would thus focus on tiSsue regeneration In cassava. The need was expressed for a protocol for developing plants from callus tissues or. better stili. plants from protoplasts. ZImbabwe claimed to be approaching this goal. ThiS would allow n transformation of already

eHte cassava lines.

Some discussion probed the possible need for regulatory genes and for switching on the protein synthesis. Would one gene be sufficient or Is It a famJly of genes required? (b) Low technology: fermentation route. Both Meyo In Burundi and Senez In Parts have used fungal fennentation (non-sterile) methods In field trials. Possibilities for adaptation (culturally] at village level should be explored. (IITA has a cement mixer based on the ubiquitous 011 drum that could be adapted for nonsterile fermenters. Protein fortification has high priority and we would like to be sure It Is achievable.

4. Vlrua I'eslstance. Do we need another method of providing for African mosaic vJrus resistance?

NEEDS: Mosaic-resistant lines have been developed at UTA The resistance Is not complete but lines derived from IITA do not suffer major losses In the field. However. as the lines are planted farther afield. local adaptability requires further crossing and resistance can be lost through low Infection pressure during selections ( e,g ..

Zimbabwe). If the Infection potential Is too severe. resIStance can be overcome. It Is also true that related crops. particularly yam. suffer from a (potynlrus and also sweet potato. The "gene-In-a-bottle" Is an attractive remedy to have when ongoing breeding Is envisaged. The Importance of fast and reliable virus diagnostic methods and knowledge of pathogenic variations In this continent have also been expressed.

POSSIBLE ACTION: Collaboration with Beachy and others in Europe makes this Option Implementable. although the tissue culture regeneration requirement may not be fully satisfied as yet. Yam could prove a useful "dry run" on Its potulrus. as regeneration and TI transformation have been achieved TOsuj I In Nigeria]. For virus diseases. diagnosis and study of the pathogenic variations are called for. Monoclonal antibody and cDNA technology could be used (ThottappUly and Rossel at IITA) .

On balance It Is believed that the need for this virus-resistance route and reliable dlagnostlc technologies may be highest for South American frog skin disease.

IS. Shy flowedn,.

Can we Induce flowering at will for breeding purposes? The problem of synchronizing male and female flowers was also expressed.

NEEDS: Research (crOSSing) capability should be Improved. which first requires physiological understanding (light Intensity. photoperiod hormone levels. enzyme kinetics of control] of the factors preventing 57

flowering and a cytogenetic approach to discover the reason for uniform 36 chromosomes. Could cassava be an aUopolyplold? Is Brazil harbouring same 18 chromosome diploid ancestors? These are basic questions needing answers to facilitate further breeding and genetic Improvement.

POSSIBLE ACTION: TIssue culture can be used to control Ught Intensity. photoperiod. physiological status and hormone balances. and help us to improve our understanding of the process. Phytotrum studies may also help (ZImbabwe has facility). This Is baslc research to support all other developments. Cobalt -60 irradiation and tn vitro flower Induction were also suggested for the shy-flowering type.

6. MealybUC and green Iplder mite. Can genes be found to discourage these InSects. or Is biologICal control the method of choICe?

NEEDS: Cassava Mealybug Is the Single biggest pest problem.

Biological control Is proving effective. Varieties that have some level of resistance to mealybug and green spider mlte were Identified and being Incorporated Into Improved cassava varieties. Pubescence In the young leaves Is highly correlated with reststance to both pests. Spider mtte needs an appropriate paraslte which Is yet to be tested.

POSSIBLE ACTION: (a) It IS not clear whether genes are avallable. Tbe BT gene wlll not be suitable; Is the trypSin inhibitor gene effective? Are there others? Again. we depend on advances In tissue culture to succeed In transferring any suitable genes: (b) Wide crosses to Introduce pubescence also depend on basIC studies of regeneration. Cytogenetics wlll open the door for future reassessment.

7. Browning. physiological problems and postharvest loss. Can we arrest postharvest deterioration and so extend storage life?

NEEDS: Local experience counsels use of ash to slow brownlng. Does this have to do with "wounds bonds" or adsorbing oxldases? There are no clear Ideas about the possibilities for action. beyond looking at enzymes and genes Involved. polyphenol oxldases and tannic acid production. The Issues hold a low priority because the problems have not been clearly defined. Basic physiological/biochemical studies are needed.

& Blick Sigatoka disease.

Can we control the black Sigatoka disease

In plantain and banana?

NEEDS: African countries are Increasingly facing problems of this disease In plantain and banana production. A genetic source of 58

resistance h as been Identified with the cooking banana. embarked on a breeding program to combat the dtsease.

UTA has

POSSIBLE ACTION: Somaclonal variation and in vitro screening may permit selection of a resistant variety. Basic studies on the fungus and its products and the response of plants to fungus tnfecUon may throw some light on means to tackle the problem.

59

RECOMllolENDATIONS

Participants at the Meeting on the Use of Biotechnology for the Improvement of Cassava. Yams and Plantain tn Afrtca. on 8 and 9 August 1988 at Ibadan. Nlgerta. made the following recommendations.

General recommendations I. Collaboration among dtfferent research workers on the prtorttles Identified below should be fostered by IITA through a network of laboratortes and support for facilities.

2. The network should assemble once a year. consldertng that the present meeting was enormously helpful In exchange of knowledge. problems. prospects. hopes and Ideas.

3. Scientists from selected laboratortes might be Invited to hold semtnars at such network meetmgs.

4. An Informal newsletter should be establJshed for dlssemtnatlon of news of biotechnology actJvJtles In Afrlca. to be wrttten by root crop and plantain biotechnology researchers and coordtnated through lITA. 5. Encouragement should be given to conunerclal1zatJon of rapid multiplication methods as proof L;at biotechnology has practical and useful applications.

Recommendations In spec:lflc an:u ~ UIIIent attention 6. Bastc research. In order. to facilitate traditional breeding and selection and the application of modem biotechnological methods. certain basic research needs should be speedUy fulfUled. They are:

(a) Cytogenetic studies to establish chromosome numbers. plOidy levels and chromosomal vartatlons; (b) Production of haploids and dl-haplolds; (c) Meiotic analysis to evaluate ploidy levels and the degree of heterogeneity of the genome; (d) SuffiCient understanding of the flowertng process In order to enable manipulation of flower production; (e) Tissue culture protocol to dertve plants from callus; (0 Tissue culture protocol to dertve plants from protoplasts.

7. Research targets. (a)

First priorily

acyanogenlc lines. vJrus resistant lines-for both yam poty vJrus and cassava gemenl virus.

(Ill) protein fortification-by DNA route and fermentation route.

(Iv) virus diagnostics-for breeding. movement of germplasm and (t)

(Il)

60

the defln1tlon of pathogenic variatlons. (b) Second priDIity

black Sigatoka disea.se-screen1ng. In vitro selectlon for somaclonal variaUon. (IJ) processing and related deterloratlon problems (browning. tannic aCids-the Identlflcatlon of enzymes Involved and the DNA of their genes).

(WI mycorrhlzaf-tnvesttgatlons of local ISOlates. eco-tolerances and compatlbllity with host plants. (tv) cassava mealybug and green spider mlte-screentng for metabollc blockers oT路antl-feedants and for humoral antibodies.

(I)

(c) Long-tenn prtortty

Nematode resIStance Is highly desirable. but It was recognlzed that genome mapping In these crops followed by RFLP analysis of transposable element locations Is technically not within reach at this time.

61

ANNEX

LIST OF PARTICIPANTS

Elizabeth Achearnpong Department of Botany Unlverslty of Ghana Legon, Accra Ghana

G.O. AdeJare Nigerian Plant Quarantine Services Moor Plantation PMB 5672. Ibadan Nigeria H . Adu-Dapaah International Insltute of Tropical AgrIculture PMB 5320, Ibadan Nigeria

A.M. Almazan International Institute of Tropical Agriculture PMB 5320. Ibadan Nigeria B. Asafo-AdJel International Institute of TropJcal AgrIculture PMB 5320. Ibadan Nigeria K. Bayero

NatIOnal Centre for Genetic Resources and Biotechnology Moor Plantation PMB 5382, Ibadan Nigeria E. Chukwuma International Institute of Tropical Agriculture PMB 5320, Ibadan Nigeria RN.O. Da rku Mlnlstry of Agrtculture Cablnete Tecnlco P.N.S. FAO/ANG/OO8/BfJ PO Box 527, Luanda Angola

65

w. Ezello PO Box 12247, Ibadan Nigeria K. S. Fischer

International Institute of Tropical Agriculture PMB 5320, lbadan Nigeria S.K. Hahn International Institute of Tropical Agriculture PMB 5320, Ibadan NIgeria E.N.A. Mbanaso National Root Crops Research Institute, Umudike PMB 7006, Umuahla Nigeria M. Mgonja InternaUonal Institute of TropIcal Agriculture PMB 5320. Ibadan Nigeria S.Y.C. Ng International Institute of Tropical Agriculture PMB 5320, Ibadan Nigeria Gabriel B. Ogunmola Department of Chemistry Faculty of Science University of Ibadan, Ibadan Nigeria C.EA Okezle Natural Sciences Unit Division of General Studies University of Nigeria, Nsukka Nigeria D .M.Okloga Plant Quarantine Statton, Muguga PO Box 301488, Nairobi Kenya D.S.O. Oslru International Institute of Tropical Agriculture PMB 5320, Ibadan Nigeria

66

G.O. OsUjI Centre for Biotechnology Anambra State University of Technology PMB 01660. Enugu Nigeria A.1. Robertson

Department of Crop Science University of Zimbabwe 44 Quom Avenue Mount Pleasant, Harare Zimbabwe M.B. Sarurn!

National Centre for Genetic Resources and Biotechnology Moor Plantation, Ibadan Nigeria G. Thottapp!l1y IntenIational Institute of Tropical Agriculture PMB 5320, Ibadan Nigeria N. Udensl National Horticultural Research Institute PMB 5432, Ibadan Nigeria D.D . Wounuah Central Agriculture Research Institute Suakoko Bong County PO Box 3929. Monrovia Liberia J. Wutoh lRA-Mesres-Ekona PMB25 Buea, South West Province Cameroon

67