Murashige, Toshio : Toshio Murashige, Professor of Horticulture, and Plant Physiologist, Botany and Plant Sciences, Riverside.

Regeneration of plants Toshio Murashige Reporting his pioneering experiments on plant cell culture to the German Academy of Sciencein 1902 , G . Haberlandt predictedthat someday â?? in this way one could successfully cultivate embryos from vegetative cells . â?쳌 IAA and kinetin been at his disposal , Had Haberlandt might have realized his predic - tion , and there is no telling how much further plant cell culture might be today . Widespread success with plant cell cultures into protoplast regeneration media , thus al - was made possible when the plant hormones lowing the few resistant cells to grow into auxin and cytokinin were discovered and of sensitive calli , while killing the millions when F . Skoog and C . 0 . Miller revealed in cells . In lettuce this technique has been used 1957 that regeneration of shoots and roots in to select cells capable of survivingexposureto cultured cellscould be manipulated simplyby oxalic acid ( the toxin produced by the lettuce of these hormones in varying the proportions drop organism ) and ethylene gas ( the chemi - the nutrient medium . cal responsible for injury resulting in russet Genetic engineering of crop plants must spotting ) . Selection pressures also can be ap - usually begin with single cells or protoplasts plied to select for characteristics other than as the objects of molecular manipulations . disease resistance . Cold and heat tolerance The effort must culminate with reconstituted could be selected for , as well as tolerance to plants . Regeneration of plants from isolated chemicals such as salts and herbicides . cells currently follows one of two pathways . We have used this selection pressure tech - In the first , plants are obtained through a se - nique to identify the few lettuce protoclones quence of shoot formation followed by root - capable of proliferating on media containing ing of the shoot . In the other , embryos - that greatly reduced calcium levels . We hope that is , structures with simultaneously differenti - plants regenerated from these protoclones ated shoots and roots - are initiated . will be resistant to tipburn , a disease caused A series of nutrient formulations is usually by a deficiency of calcium in the interior tis - required by eitherpath . In the method of sep - of head lettuce . sues arate shoot - and root - forming steps , a criti - Crops have been improved over the years cal cell mass , or callus , is prerequisite to any by crossing plants with desirable traits , but An auxin and sometimes organ formation . the parent plants must be sexually compati - also a cytokinin must be provided for callus ble . The use of protoplast fusion can remove development . When transferred to a medium this restriction . Protoplasts from any two containing a relatively high level of cytokinin plants , regardless of species , can be fused . In and a low level of auxin , the callusdifferenti - many cases it has been possible to regenerate ates shoots . Shoots of suitablesize are separ - fused protoplasts into whole plants , resulting ated and recultured in still another medium , in â?? somatic hybrids â?쳌 or asexual crosses . In one lacking cytokinin but containing some theory , this technique opens up the potential freshly Above : Micrograph of auxin , to generate roots . Additional supple - isolated lettuce leaf protoplasts â?? gene pool â?쳌 of a plant to every other bio - ments , such as adenine and tyrosine , may containing bright green logical organism . In lettuce , this means that chloroplasts . enhance the shoot initiation step . Similarly , we can now tap the vast resources of wild let - rooting may be improved by reducing the tuce species that contain desirable charac - Left : Protoplast - derivedcalli giving as salts and by including cofactors , such as genes for disease resistance , teristics , such rise to lettuce plants . phloroglucinol and caffeic acid . but that have previously been unavailable to Above right : Lettuce plants ready In the embryo method , the cells are first breeders because of sexual incompatibility . Each plant for transplanting . stimulated to divide and induced , or other - Crop improvement with this new method - originated from one protoplast . wise prepared for eventual development into . ology seems promising . Field testing and embryos . The induced cells are then allowed selection for desirable characteristicsin head to proceed with embryo formation . The me - lettuceplants derived from protoplasts will be dium for the cell divisionand induction phase 1982 . done for the first time in as 2 , 4 - D ; a often contains an auxin , such Dean E . Engler , Research Assistant , and - Ray - high level of nitrogen , preferably NH t ; and mond G . Grogan , Professor , Plant Pathology , U.C . , Davis . sufficient potassium . For the embryo devel - CALIFORNIA AGRICULTURE , AUGUST1982 19 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