Orton, Thomas J. : Thomas J. Orton, Assistant Professor, Vegetable Crops, U.C., Davis.

Y natively , when using this approach to develop ; . rieties , as well as among cells within the same a new improved variety , a selection scheme plant . Some cells are more responsive than would be devised that would theoretically f others to manipulations that result in embryo find only cells with altered genotypes at loci or organ formation . J . G.Torrey has identi - whose function bears on a desired character , fied the manipulatable cells by the term but which were geneticallyidenticalto the or - meristemoid . In appearance , meristemoids How - iginal tissuedonor in all other respects . of apicalmeristems resemblevery closely cells ever , some of the earliest research papers in or of embryosas found in seeds . Indeed , mer - this area have documented the existence of istemoids are more often isolatable from spontaneous genetic variability in both cul - apical meristems and young embryos . Non - tured cells and corresponding regenerated meristemoid cells sometimes differentiate plants . A useful label , â?? somaclonal â?쳌 varia - into meristemoids in vitro - for example , tion , has recently been advanced for this tobacco stem pith , carrot root phloem , po - phenomenon - â?? soma , â?쳌 occurring in so - leaf mesophyll , and citrus nucellus . Un - tato matic tissues as opposed to sexual progeny , fortunately , the basis for this differentiation and â?? clonal , â?쳌 expressed as differences remains unclear . among and within clones . Meanwhile , the chances of obtaining 400 The cumulative evidence from over plants in cell cultures can be increased by scientific papers shows that somaclonal vari - a few key relationships . Cells of observing ation in cultured cells is more the rule than plants that are in the juvenile phase of devel - the exception . The most common observa - opment , that is , not yet competent to flower , tions are of changesin the number and struc - are more regenerative than those of adult of chromosomes , the subcellularorgan - ture plants . Even among juvenile sources , the elles that carry individual genes . Evidence younger the plant or organ , generally the suggests that these chromosomal changes in - higher their tendency to contain regenerative Date palms were successfullyregenerated crease with culture age and are antagonistic cells . Seasonal climatic requirements of the tissue culture by manipulation of from cell the make - up of the growth medium . to the regeneration process . Regenerated donor plant or organ must be satisfied before plants with altered chromosomal changes cell culture : it must be chilled , grown under opment phase , the auxin is excluded from the often show changes in leaf shape and color , As a appropriate photoperiod , and the like . medium . rule , highly regenerative cell lines can be de - growth rate and habit , and sexual fertility . Aeration is important for embryoor organ rived from a poorly regeneratingtissue by re - Such changes are sometimes seen in regener - development . Anaerobic conditions cause al - ated plants with apparently normal chromo - peatedly selecting for the trait during the cohol accumulationand can lead to intoxica - of subcultures . course some constitution , implying that somaclonal of their tion of the cells and diminution of indivi - Molecular biology may someday furnish variation may extend to the level capacity for organized development . dual genes . methods that would enable selective control Embryos can be produced in darkness , but Somaclonal variation is obviously highly of gene repression and derepression . Per - shoot and roots are more likely to develop in haps , then , it will be possible to achieve ex - undesirable in situations where cell or tissue illuminated cultures . Gro Lux and Cool - pression of totipotential of any plant cell , culture is being used to preserve genetic iden - White fluorescent lamps that emit high levels tity . A limited number or nonmeristemoidand of specific observations whether meristemoid of blue and red light have been most effective . or variety . as a means of expand - regardless of species point to itspossibleuse Low light intensities , about 100 foot - candles ing the pool of desirable genetic variability Toshio Murashige , Professor of Horticulture , as encountered in household illumination , for crop improvement . Examples include al - and Plant Physiologist , Botany and Plant Sciences , Riverside . are preferable duiing the shoot and root dif - tered plant habit and flower type in chrysan - ferentiation steps . However , just before themum and increased yield and diseaseresis - transplanting to soil , plants from tissue tance in regenerated plants of sugarcane and should be hardened by exposure for a two - to potato as compared with the original tissue three - week period to a higher light intensity , ill - donor . Unfortunately , the phenomenon is 1 , OOO foot - candles . Plant regeneration near understood , and we are presently unable to of some species is controlled by photoperiod - direct its manifestations in any way . SomacIonaI variation ism , like their flowering behavior . Thus , for Research conducted recently at Davis has example , kalanchoe cells regenerate better shed some new light on somaclonalvariation . Thomas J . Orton under short days , and walnuts proliferate Using celery as a model organism , we have shoots more readily under long days . of the shownthat variation occurs at the level The temperature in which cells are incu - single gene as well as the chromosome , al - can also be important to plant regenera - bated though the precise nature of the lesions has in vitro cell and successful application of tion . Differencesin day and night optima , or of these not yet been pinpointed . Certain tissue culture technology to crop improve - thermoperiodism , must not be ignored when chromosomal and single gene changes are ment hinges on the ability to regenerate working with plants that differ in origin from For ex - plants of known genetic constitution . transmittable to regenerated plants , and they tropical to temperate and alpine habitats . or tissue culture as a ample , when using cell behave sexually in a predictable fashion . All plant cells probably have totipotential , means of cloning , or amplifying numbers of Somepopulations of regeneratedplants show or the capacity to reproduce entire plants . plants for field or seed production , it is essen - or no chromosomal abnormalities but little Nevertheless , the ease of expression of that tial that regenerated â?? copy â?쳌 plants be geneti - accompanyingvisible alteration , perhaps be - potential differs among plant speciesand va - or identicalto the original . Alter - cally similar cause of observed mixtures of normal with 20 CALIFORNIA AGRICULTURE , AUGUST 1982 0 racil appears to be a useful characteristic for 2 such work . Parent cells growingin culture are to this compound , but vari - highly sensitive ants can be found that are resistant to it , and the difference is easily assayed . We have demonstrated that this difference results from an enzyme deficiencyin the vari - ant cells . They lack an enzyme ( uracil phos - ana - phoribosyltransferase ) that convertsthe log into the compound that actually kills the of cells from two different spe - cells . Strains cies of plants ( diploid Haplopappus gracilk and haploid Daturu innoxiu ) resistant to this analog have been isolated and shown to lack the enzyme . We have studied the effects of several commonly used mutagens on these cells but have not yet found one that effec - tively increases the frequency of azauracil - resistant cells in treated populations . Two possible causes for these results must so far tested be considered . First , the agents might not be mutagenic in cells of these spe - Two plants regenerated from same celery callus culture demonstrate somaclonal variation . cies . Or , second , resistanceto azauracilmight left closely resembles the original . Plant on right has smaller , more divided leaves , Plant on not be the result of real mutations in the gene its growth is much slower . Genetic studies will investigate whether variation resulted and from DNA changes or lingering cultural effects . responsible for the structure of the enzyme but instead might arise from nongenetic causes . In the latter case , mutagenic agents aberrant cells within plants . Approximately would not be expected to affect the frequency 70 percent of plants regeneratedfrom cell cul - of resistant cells . Although we cannot yet Cell mutagens tures of a commercial celery variety and prove that stable azauracil resistance has a grown in three field locations were visibly genetic cause , several characteristics of the normal , while the remaining 30 percent Gary E . Jones resistant cells indicate that this is the case . showed striking differencesin growth rate or Among these traits is the complete lack of habit , leaf shape , color , or flowering be - phosphoribosyltransferase activity af - uracil havior . None of the plants exhibited charac - of plant so - Realizing the full potential ter cells have been cultured for more than two to teristics that could be considered superior matic cell genetic techniques will depend on years without exposure to the analog . the original type . Experiments to assess the development of methods for isolating a wide One feature of our studies showsthat such relative physiological and genetic contribu - variety of cultured cell strains with character - work must be interpreted with care . In many tions to this variation are in progress . of cells in the ori - istics different from those experiments , we demonstrated that several Further results with celery suggest that ginal cultures . To isolate such variant cell potentiallymutagenicagentsappeared at first genotype of the tissue donor , medium consti - strains , techniques well known in microbial to increase the frequency of resistant cells , tution , and culture age are the significantfac - studies will have to be applied to cultured sometimesby as much as 50 - fold . However , tors mediatingsomaclonalvariation , whereas plant cell systems . the vast majority of these resistant popula - differentiated state ( leaf , stem , and the like ) One especiallyimportant method is the ap - tions did not retain the characteristic when and random effects are not important . The plication of mutagens to plant cells to greatly subsequently cultured in medium not con - differences observed among genotypes were increase the frequency of variant strains in loss of the resis - taining the azauracil . Rapid particularly interesting : some lines showed a populations of cells so that they may be more tance strongly indicates that these cells were rapid , progressiveaccumulation of variation easily identifiedand selected . However , work not genetically altered , although we do not ( and loss of ability to regenerate ) , whereas in several laboratories has shown that many know why they initially appeared to be resis - others consistently remained stable ( and able chemicalsthat are potent mutagens in micro - tant . In fact , when careful study of the ini - to regenerate ) . We therefore speculate that a bial systems are only marginally or not at all tially resistant strains was carried out , the fre - of appropriate genotypes and combination effective on cultured plant cells . The search quency of stably resistant cells was not very media may be at least partially effective in for chemicals effective on a wide variety of much increased by the treatment with muta - controlling somaclonal variation . Perhaps it plant cells is therefore an important aspect of gen . Such results demonstrate that detailed will be possible to identify genes responsible plant somatic cell genetics . of resistance to any compound investigation for inhibition or enhancement of variation Testing whether a compound is mutagenic must be performed before conclusionsabout and to transfer them sexually into desired requires that the expression of an easily seen the mutagenic effects of any agent can be backgrounds . Solutions to these problems characteristic be substantially different in claimed , even if stable resistance can be a major block to the applica - will eliminate parent cells than in the variants derived from of a true genetic shown to be the result tion of new molecular and cellular technol - them after treatment with the agent . In our change . ogy in plant breeding and field production . laboratory , we have been attempting to de - GaryE . Jones , Associate Professor , Geneticsand velop such a testing system . Resistance to a Associate Geneticist , Botany and Plant Sciences , ThomasJ . Orton , Assistant Professor , Vegetable U.C . , Riverside . Crops , U.C . , Davis . nucleic acid precursor analog called 6 - azau - CALIFORNIA AGRICULTURE , AUGUST 1982 21