Engler, Dean E. : Dean E. Engler, Research Assistant, U.C., Davis.

Grogan, Raymond G

nologies are only at the developmental stage and , unlike fusion , are not yet routine . The manipulationsdescribed are eminently Protoplast regeneration feasibleand in some cases have been success - fully accomplished . However , we are only at Dean E . Engler the beginning of somatic genetic manipula - G . Grogan Raymond tion of crops , primarilybecausemany impor - tant crop plants behave poorly in culture . For example , corn protoplasts usually cannot di - vide ; soybean callus does not regenerate to plants . Also , too little is known about the Plant cells without walls ( protoplasts ) can fundamental processes of plant development be isolated from leaves by a process of enzy - and gene regulation . Once a foreign gene is matically digesting away the middle lamellae a plant , one faces the next introduced into between cells and the cell walls . Tremen - stage of problems : will the gene express in the of protoplasts can be dously large numbers appropriate organ ; will it cause side - effects isolated from a single leaf ; yields are typically to weaken the plant ? Thesequestionsmust be two to four million protoplasts per gram of addressed and solved before somatic meth - leaf tissue . Development of techniques and ods can be employedto produce better crops . procedures causingisolatedprotoplasts to re - Several aspects of tissue culture are cur - form their walls , proliferate , and regenerate into whole plants is essential for the utiliza - rently being applied to agriculture . For ex - tion of the new genetic technology . ample , the orchid industry now relies almost In recent months we have succeeded in de - exclusively on tissue culture to propagate or - veloping culture media and the methodology Also , tissue chids that are difficult to breed . for regeneration of lettuce protoplasts into culture multiplication can often be used to whole plants . When isolated lettuce proto - eliminate virus contamination in seed stock . plasts are maintained in the right conditions , Vegetative propagation by tissue culture they can be induced to reform their walls and from a single plant might be expected to yield of cells divide to form unorganized clumps identical plants , because all cells would be of ( P - calli ) . These P - calli are transferred to identical genetic constitution , barring very media with the proper balance and concen - rare mutational events . However , quite strik - tration of plant hormones and other ingredi - ing variability has been found in regenerated ents to induce the formation of shoots . The plants . This variation may offer a new source of valuable genetic traits for plant breeding . shoots then are transferred to media for fur - ther growth and eventual root production . Another application of tissue culture is in the increasing use These regenerated plants then can be trans - of haploid plants . Germ a greenhouse for seed produc - planted into cells , usually the immature pollens either en - tion and , finally , the progeny are evaluated closed in ( another culture ) or isolated from and selected for desirable characteristics in the anther ( pollen culture ) are cultured to the field . produce new plants . Sincethese germ cells are haploid , the derived callus or plants are also One would expect all plants regenerated haploid - having only half the chromosome from a single lettuceleaf to be identical , since number of the diploid parent . Such plants their production involves no sexual process . of regenerates , however , re - can be treated with colchicine and made dip - Observations veals the astonishingresult that many of them loid again . During the processes , the plant are different from the source plant and from all loci . Plant breed - becomes homozygous at ers often have to self - pollinate strains for each other . Other researchers have found a many generations to produce â?? true - breeding , similar frequency of variation in potatoes , which has proved to be stable over many gen - pure â?쳌 lines . The anther or pollen culture pro - erations . Although the reasons for genetic vides a quick way to produce homozygous lines in a single step . Finally , somatic genetic variation among regenerates are not fully un - systems based on haploid cultures have the derstood , it may be possible to obtain desir - advantage of allowing isolation of recessive able improvementsin horticultural character - mutants . istics , such as enhanced green color , uniform We believe we can look forward to tissue maturity , and resistance to diseases , because culture making a modest contribution to of this inherent variability of protoplast re - plant breeding and agriculture in the next few generates . years . This contribution might be expectedto The millions of protoplasts that can be cul - of manipu - increaseradically as the capability tured in a single petri dish can be subjected to lating crops in culture improves , gene trans - specificselection pressures that will eliminate fer and cloningtechnologies develop , and the all but the very few tolerant protoplasts . For knowledge of plant growth and development example , many plant - disease - causing organ - increases . isms produce toxins that can be incorporated 18 CALIFORNIA AGRICULTURE , AUGUST 1982 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