PROBLEMS IN USING INDUCED MUTATIONS IN VEGETATIVELYPROP AGA TED PLANTS

K.Mikaelsen*

ABSTRAK - ABSTRACT

PROBLEMS IN USING INDUCED MUTATIONS IN VEGETATIVELY PROPAGATED

PLANTS. Conventional breeding techniques are difficult to apply in most vegetativdy propaga­ted plant species. The mutation breeding is therefore very important in these plant species, butpractical results have been difficult to obtain due to the problems in recovering the mutatedcells in the plant. The importance of various techniques are discussed and the advantage develop­ing various tissue culture techniques have been described.

MASALAH DALAM PENGGUNAAN MUTASI BUATAN PADA TANAMAN YANG

DIBIAK SECARA VEGETATIF. Teknik pemuliaan secara konvensional umumnya sukarditerapkan pada spesies tanaman yang dibiaksecara vegetatif. Oleh karena itu teknik pemuliaanmutasi sangat penting bagi spesies tanaman yang demikian, tetapi mutan yang dapat digunakandalam praktek sukar diperoleh karena masalah kembalinya sd yang termutasi dalam tanamanke bentuk normalnya. Dalam makalah ini dibahas keunggulan berbagai teknik mutasi buatanclan keuntungan pengem bangan berbagai teknik kul tur jaringan.

INTRODUCTION

A large number of plant species are propagated asexually (or vegetatively) suchas many root and tuber crops, sugarcane, fruits, and other three species and shrubsand many ornamentals.

Cross-breeding is often limited by specific problems in most vegetativelypropagated plants (VPP). Many VPP have a rather long vegetative phase before goinginto sexual reproduction. In addition, these plants are generally highly heterozygous,which causes complicated segregations and makes the detection of a useful recom­binant very difficult. 'This problem is further enhanced by frequently polyploidiin such plants. All these factors make cross breeding very difficult and timeconsuming. Furthermore, incompatibility and other cross barriers, apomixis andsterility exist quite often and hinder the plant breeder in making use of conventionalcross-breeding.

* Expert, UNDP Project INS/78/074

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MUTATION BREEDING OF VEGETATIVELY PROPAGATEDPLANTS'

It is, therefore, quite clear that mutation breeding techniques are an attractivealternative in breeding of VPP. The most promising aspect of mutation inductionin VPP compared to cross-breeding methods is the ability to change only a fewcharacters of an otherwise good cultivar without altering significantly the originalwell established genotype. Mutation breeding, therefore, must be considered as

the obvious means to perfect the leading cultivars and as a possible sh~r_tcut forinducing desired genetic alterations in outstanding cultivars. Obviously, mutationsare the only means for producing variability in sterile VPP and in obligate apomicts.Further, mutations can be useful to break apomixis, to overcome self-sterility andcross barriers as well as to uncover rearrange chimeras.

In spite of these many advantages of mutation breeding techniques in VPP theresults have been rather small in comparing with seed propagated plants such asrice, barley. and many others crops. The main problem has been the recovery ofmutants in VPP. Normally a shoot, or any other multicellular organ are treated formutation induction and the desired mutations has occured in one cell. The chances

of such a mutated cell growing into a sector or layer and be able to manifest itself

will depend on l!s .position within the apex as well as its growth rate as comparedwith the surrounding cells or tissue layers. It is obvious that mutated cells will beeasily hidden and not be visible in such multicelledar tissues and special methodsare required to unciver such mutated cells.

The first requirement is, therefore, that mutagen treated (unirradiated)material has to be propagated to permit the formation of periclinallayers beforeselection can be applied. Very often repeated propagations are necessary forobtaining large sectors of mutated tissue from which a maximum number ofmutants can be recovered. In fruit trees, for example, (Figure 1) the best conditionsare found in the axillary meristems of the basal leaf primordia of the dormant buds.(The primordia to be used for treatment should consist of as few cells as possible).Experience shows that the buds derived from leaves 4 - 8 (approximately) of theprimary shoot are those that exhibit the highest frequency of relatively broadmutated sectors in the second vegetative generation. Measures have to be taken(by pruning) to force these buds into growth. Several authors have reported betterresults after irradiating buds that have just started growth than with buds in deepdormancy. If growing plants are irradiated, decapitation of the main shoots willforce new buds to develop through regeneration and thereby increase the chancefor recovering a larger number of mutants.

Many of the complications described above could be eliminated if chimeraformation could be avoided. One of the methods available is the adventitious bud

techniques which was developped by BROERTJES and collaborators at Wageningen(1). This technique is based on the phenomenon that the apex of the adventitionsbuds, such as may be formed the base of the petiole of detached leaves, originatesfrom only one (epidermal) cell. Consequently, adventitious plantlets either arecompletely normal or are complete, solide mutants. In other words, chimeraformation does not take place. This adventitious bud technique offers a great

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advantage for practical mutation breeding and has given many good results particu'larly in ornamental plant (Saintpaulia, Streptocarpus, Achimenes, Begonia, Liliwrliand many others).

Many plant species can be propagated this way. BROERTJES ~~. (1) listother 350 of such species. Many species of economically important plant familieshave not been tested for developing the adventitious bud techniques and may notalways develop adventitious buds from only one cell. In addition, the condition ofthe mother plant, the age of leaves, environmental conditions during and afterrooting and the auxin-cytokinin balance appear to influence the formation anddifferentiation of adventitious buds.

POTENTIALS OF TISSUE CULTURE TECHNIQUES IN BREEDINGOF VPP

The modern developments of culturing somatic plant cells should also beinvestigated for use in VPP in this respect tissue culture technology holds muchpromise. Recent advances in the field of plant protoplast, cell, tissue, and organculture (embryo, anther) have, transferred this area if fundamental research intoone that is dynamic and promising also for obtaining further advances in cropimprovements programmes. I believe the potentials of this technology can bepowerfull tool also in the VPP.

During the last 30 years, the culture of ovule, ovary, and embryo has beenemployed to overcome sterility. incomtability and dormancy; to induce poly·embryony; and to succesfully hybridize various crops.

By meristem culture, large numbers of horticulturally important plants havebeen commercially propagated and freed from pathogens.

These established techniques have played an important role in wide hybridi­zation and clonal propagation programmes and will, no doubt, continue to contri­bute to future demands. However, some of the recent advances in the area of plantprotoplast, cell, tissue, and organ culture have attracted international attentionbecause of their significance in and far-reaching implications for agriculturalresearch and crop improvement programmes.

It is expected that the following techniques will playa significant role, notonly in the improvement of existing crops but also in synthesizing new plants :

1. Wide hybridization in crops through in vitro pollination and fertilization.2. Production of haploid and homozygous plant from exised anthers, isolated

pollen and by chromosome elimination.3. Somatic hybridization and genetic engineering through the fusion of protoplasts

and the uptake of DNA (Figure 2).4. Induction of genetic variability in crops (mutants, and various ploidy levels)

through protoplast and cell culture and selection in these cultures for resistanceto diseases, salinity, water, and temperature stresses and possibly many otherimportant characteristics.I expext that the VPP will have great potentials for benefits from such

techniques.

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REFERENCES

1. BROERTJES, C:; HACCIUS, B., and WEIDLICH, S., Adventitions bud for:mation on isolated leaves and its sigriificance for mutation breeding,

Euphytica 17 (1968) 321.

2. DONINI, B., '1nduction and isolation of somatic mutations in vegetativelypropagated plants", Improvement of Vegetatively Propagated PlantThrough Induced Mutations (Technical Reports Series No. 173), IAEA,Vienna (1975)35.

3. BAlAl, Y.P .s., Potential of protoplast culture works in agriculture, Euphytica23 (1974)633.

4. CARLSON, P .s., SMITH, H.H.,and FEARING, R.D., "Parasexual interspecificplat hybridization", Manual on Mutation Breeding, 2nd Ed. (TechnicalReports Series No. 119), IAEA, Vienna (1977) 206.

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DlSKUSI

S. SUTRISNO :

You have mentioned that atomic radiation may induce mutation on VPP (Vegeta­tively Propagated Plants). But at the same time radiation also destroy the biologicalsystem such as enzymatic system as well as propagation system. If we irradiatepotato it will delay sprouting. I don't know wether it is a mutation or not. May beyou have some explanation why radiation can delay the sprouting of potatoespecially on propagation system.

K. MIKAELSEN :

The mutations induced by ionizing radiations are caused by the effects of andchanges in DNA in the chromosomes. If too much irradiation is hitting the cellnuclei chromosomes damages occur which may kill the cells or tissue or causeserious damages leading to abnormal growth or development. Therefore. it is veryimportant in VPP to get rid of all the abnormal growth and development and givecells or tissue layers with small effects having viable mutations to develop so unsefulmutations can be selected from the induced variability.

M. !SA D. :

I am interested in your paper, because induced mutations in VPP is a promizingmethod for new clo~ perennial plants not only for the high crop but also facedfrom pathogens. Your paper is only a review, would you like to tell me exampleof plant which has been succeeded well may be merlstem culture, and what kind

of poblems has to be overcome in carrying out this method~ The main problem isthe recovery of mutants in VPP.' What kind of important Ichemicals has to be used~for the recovery of mutants.

K. MIKAELSEN :

One of the best examples of useful mutants in VPP is the dwarf-type trees in somefruit-tree crops as apple, cherry, olive, and peach. These smaller tree types are easierto harvest and higher yields can be obtained as a larger number of trees can beplanted per unit area. These mutant types have been obtained with cub-backmehod as well as with adventious bud and meristem tissue culture techniques.

Anther example of the successful use of meristem-protoplast fusion techniques isthe species hybrid mentioned in my paper (Fig. 2b). The problem to overcome inusing in vitro techniques are obtaining tissue differensiation and the recovery ofcomplete plantlets. The chemicals needed for recovery ofplantlets may differ fromspecies to species.

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