A45

13. Protoplasts BIO/I'ECHNOLOGY 6 : 5 6 (1988)

Plant regeneration from protoplasts isolated from embryogenic maize cell cultures C.A. RHODES, K.S. LOWE and K.L. RUBY Zoecon Research Institute, Sandoz Crop Protection Corp., Palo Alto, CA 94304-1104, USA Genetic engineering of maize via protoplast technology has been limited due to lack of plant regeneration from maize protoplasts. We have developed a system of protoplast culture that results in high plating efficiency from embryogenic protoplasts and can be followed by plant regeneration. Maize protoplasts were grown on filters directly over a feeder layer of nurse cells in liquid medium. Initial condition and subsequent growth of feeder cells were critical in obtaining a plating efficiency of 10% from protoplasts. Protoplasts were digested from embryogenic cell suspension cultures, and recovered callus retained the morphogenic potential of initial donor cultures. The system has been successfully used with protoplasts of two maize inbreds, one of which is an important commercial line.

PROTOPLASMA 141:64-73 (1987)

Plant cell graft chimeras obtained by co-culture of isolated protoplasts H. B I N D I N G , D A G M A R W I T T , J. M O N Z E R , and R KOLLMANN Botanisches Institut der Universitgit, Kiel, FRG

GUDRUN

MORDHORST

Heterospecific chimeral Solanum nigrum (+) Solarium tuberosum plants were obtained by cell grafting in protoplast co-cultures. Periclinal, sectorial, and mericlinal chimeras have been identified by various morphological and cytological characteristics. Morphogenesis predominantly began in periclinal chimeral organization. Cells of different species have been found to be interconnected by secondary plasmodesmata. Plantlets of all chimeral lines were grown to flowering under tissue culture conditions and some also in the greenhouse. Aspects of organogenesis and interspecific cooperation are discussed.

SCIENTIA HORTICULTURAE 34:85-92 (1988)

Plantlet differentiation from callus protoplasts induced from Citrus embryo TETSUSHI HIDAKA and ICHIRO KAJIURA Okitsu Branch, Fruit Tree Research Station, Okitsu, Shimizu, Shizuoka 424-02, Japan Embryos from young seeds of three Citrus species, 'Washington' navel orange (Citrus sinensis (L.) Osbock), yuko (C. yuko Hort. ex. Tanaka) and ponkan (C. reticulata Blanco), were cultured on a Murashige and Skoog medium with 0.16 M sucrose, 0.5 g1-1 malt extract and 50 p.M kinetin. One of three months after inoculation, whitish friable callus was produced from the hypocotyl region of the embryos. After sub-culture for about 2 years, those callus lines maintained an embryogenic capacity. Protoplasts isolated from the callus produced embryogenic cell aggregates in all media tested. Globular and torpedo-shaped embryoids were also produced in the media

A46 with 1 p.M zeafin or without any growth regulators. Most of them regenerated to entire plantlets after transplanting onto a Murashige and Skoog agar meduim containing 1 ~tM GA 3 and 0.06 M sucrose.

CAN. J. BOT. 63:779-783 (1984)

The isolation and cultivation of protoplasts from cell suspensions of a pantothenate-requiring auxotroph of Datura M I I , M . , S. S E E N I , L . C . F O W K E a n d J. K I N G Department of Biology, University of Saskatchewan, Saskatoon, Sask., S7NOWO Canada Protoplasts isolated from Datura cells requiring pantothenate for growth (Pnl cell line) failed to divide in a medium containing 0.5 M mannitol if the cells from which they were derived had been subcultured in liquid medium more than four times. Division could be reinduced either by increasing the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the growth medium from 1 to 2.5-5 mg/L, by adding low concentrations (0.01-1 mg/L) of benzyladenine to a growth medium containing 1 mg/L of 2,4-D, or by diluting the 0.5 M mannitol in the medium to 0.2 M immediately after isolation of the protoplasts. Thus, the physiological state of Pnl cells seems to change significantly after only a few passages in liquid medium. These changes can be prevented or partially reversed by manipulating the medium in which the cells and protoplasts are cultured. Similar physiological changes did not occur in the case of cultured wild-type (Ph4), adeninerequiring (Adl), or isoleucine-valine-requiring (I-VI) cells of Datura.

CAN. J. BOT. 62:2345-2355 (1983)

Embryogen~se somatique a partir de cultures issues de protoplastes foliaires de Ranunculus sceleratus N. D O R I O N , B. G O D I N and C. B I G O T Service de Physiologie Vdgdtale, Ecole Nationale Sup~rieure d'Horticulture, 4, rue Hardy, 78009 Versailles Cddex, France Following preliminary experiments, studies on isolation and culture of Ranunculus sceleratus L. leaf protoplasts were performed to specify conditions inducing embryogenesis. The best developmental stage for obtaining protoplasts was the second unfolded leaf; protoplasts wre incubated in cellulase Onozuka R 10 (0.1%), Driselase (0.05%), Macerozyme R 10 (0.02%), glucose (8.9%), and Murashige and Skoog major salts (x 0.5) or calcium chloride (220 mg/L), yielding 8 x 105 viable protoplasts per leaf; the best inoculum has 35 x 103 protoplasts/mL, protoplasts were cultured 1-naphthaieneacetic acid (NAA)(3 mg/L), 6-benzylaminopurine (1 mg/L), modified tteller's micronutrients, Morel's vitamins, calcium nitrate (400 mg/L), and glutamine (100 mg/L). Although survival of protoplasts was as high as 80%, only 3% of the cells divide after 3 weeks. After subculturing the suspensions embryogenesis proceeded rapidly. Use of NAA (3 mg/L) or spreading cell cultures on solid medium delays the process. Following induction, cultures become habituated; continuous embryogenesis results from the differentiation of secondary embryos arising directly from primary embryo epidermis. The capacity for embryogenesis, however, diminishes after several subcultures, particularly if media are supplemented with growth regulators. Although growth of many embryos is inhibited, most planets subsequently develop normally. Thus, while we have demonstrated embryogenic capacity in suspension cultures derived from mesophyll protoplasts, direct development of somatic embryos from protoplasts remains to be shown.

A47

C.R. ACAD. SC. PARIS, 303 (III) 10:429 (1986)

Plant cell physiology - Tissue and protoplast culture of Sesbania rostrata JANINA

HANOWER,

GENEVIt~VE

CAS and PHILIPPE

BOUVET

Plants were regenerated from stem derived callus of Sesbania rostrata. Protoplast culture technique for further use in somatic fusion with other Sesbania was developed. Mesophyll protoplasts isolated from greenhouse grown plants of S. rostrata divided and formed callus but were recalcitrant to regeneration. Only pro-embryo- and bud-like formations and rare shoots or roots were observed.

J. PLANT PHYSIOL 126:41-48 (1986)

Plant regeneration from protoplasts of sugarcane (Saccharum officinarum L.) C. SRINIVASAN

and INDRA

K. V A S I L

Department of Botany, University of Florida, Gainesville, FL 32611, USA Embryogenie cell suspensions were established from callus cultures initiated from sections of young leaf tissues of Saccharum officinarum L. (sugarcane). Protoplasm isolated from the cell suspensions were cultured in modified KM or CSK media supplemented with 50 ml coconut milk, 500 mg casein hydrolysate, 1 mg 2,4-D and 0.5 mg BA (per litre). Ceil wall regeneration, sustained cell divisions and colony formation were observed in the same culture medium. Dimethyl sulfoxide (1%) promoted colony formation. Somatic embryos were produced upon transfer of protocolonies to agar-based MS medium plus 1% activated charcoal and 0.25-1 mg/1-1 2,4-D, with or without cytokinins. The embryos were germinated in 0.5 mg/1-1 BA and produced several roots and a coleoptile. When placed on MS medium containing 0.5 mg/l-1 each of BA and fluridone, some of the embryos grew into plantlets with green leaves. Several green plants were grown successfully in soil.

PLANT CELL REPORTS 7 : 5 - 8 (1988)

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon J.W. GROSSER,

F.G. GMITTER,

Jr. a n d J.L. C H A N D L E R

University of Florida, 1FAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA Intergeneric somatic hybrid plants between 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) and 'Flying Dragon' trifoliate orange (Poncirus trifoliata Raf.) were regenerated fonowing protoplast fusion. 'Hamlin' protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with 'Flying Dragon' protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the 'Hamlin' protoplasts with the subsequent expression of the trifoliate leaf character of 'Flying Dragon'. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenitally. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n = 4x = 36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection,

A48 and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.

PLANT CFJJ. REPORTS 7 : 6 7 - 6 9 (1988)

Culture of asparagus protoplasts on porous polypropylene membrane YAN KONG 1 and CHEE-KOK

CHIN2

1China Rice Institute, Hanzou, People's Republic of China; 2Department of Horticulture and Forestry, Cook College, Rutgers University, New Brunswick, NJ 08903, USA A method of using a buoyant porous polypropylene membrane floated on liquid medium to culture protoplasts of Asparagus officinalis L. is described. This method supports very good growth and eliminates the need to periodically replenish culture medium lost to evaporation.

PLANT CELL REPORTS 7 : 5 9 - 6 2 (1988)

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones JULIE A. RUSSELL

and BRENT

H. M c C O W N

Department of Horticulture, University of Wisconsin, Madison, W1 53706, USA Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba x P. grandidentata 'Crandon', NC-5339 and P. nigra 'Betulifolia' x P. trichocarpa, NC-5331) and the Uptight European Aspen (P. treraula 'Erecta') and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrelysate supplement added to the culture medium. The protoplast-dedved cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived ealli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.

PLANT CELL REPORTS 6 : 4 1 4 - 4 1 6 (1987)

Plant regeneration from protoplasts of Dimorphotheca and

Rudbecla'a J.S. A L - A T A B E E

a n d J.B. P O W E R

Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of

Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.

A49 HORTSCIENCE 23(2): 393--395 (1988)

Plantlet regeneration from protoplasts of Petunia alpicola JANE FORD-LOGAN

and K.C. SINK

Department of Horticulare, Michigan State University, East Lansing, MI 48824, USA The isolation, culture, and plant regeneration of protoplusts derived from callus or suspension cultures of Petunia alpicola Smith and Downs was established. Protoplasm formed macrocalli, at 85% plating efficiency, in Murashige and Skoog (MS) liquid medium containing 2,4-D (1.0 mg.liter-1), NAA (0.5 mg.liter-1), BA (0.5 mg.liter-1), and 20% coconut water. Shoot regeneration from macrocalli occurred at 65% frequency on MS containing zeatin (1.0 mg.liter-1), and shoots were readily rooted on either MS containing NAA (0.01 mg.liter-1) or IBA (1.0 mg.liter- 1). However, plantlets failed to grow in artificial planting medium. The results provide the basis for somatic cell genetic studies for a Petunia sp. that has distinctive morphology compared to cultivated petunias. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), N-(phenylmethyl)-lH-purin-6-amine (BA), (E)-2-methyl-4-(1H-purin6-ylamino)-2-buten-l-ol (zeatin), and 1H-in-dole-3-butyric acid (IBA).

PLANT CEIJ. REPORTS 6:470-472 (1987)

Cell regeneration and sustained division of protoplasts from c o t t o n Gossypium hirsutum L. KAMEL SAKA, FRANK R. KATTERMANand JOHN C. THOMAS Forbes Building No. 36, Room 201, University of Arizona, Tucson, AZ 85721, USA Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% ceUulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K3 media ofKao et al., 1974 with 0.9 liM BAP, 5 p.M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5 x 10.5protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.

PLANT CELL REPORTS 6:476-479 (1987)

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (Moench) Voss FAOUZI BEKKAOUI1, PRAVEEN

K. S A X E N A 2 , S T E P H E N M. A T T R E E 2 ,

L A R R Y C. F O W K E 2 a n d D A V I D I. D U N S T A N 1 1Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, Saskatoon, SK, Canada, S7N 0W9; 2Department of Biology, University of Saskatchewan, Saskatoon, SK,

Canada, STN OWO Conditions were standardized for the isolation and culture of protoplasm from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Celldase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme liP- 150 with 0.5M marmitol and 5 mM CaCI2.2H20 produced an average of 4.5 x 106 protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplusts. A density of 105 protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

A50 PLANT CEI.I. REPORTS 6:480--483 (1987)

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca) S . M . A T F R E E l, F. B E K K A O U I 1 , D.I. D U N S T A N 2 a n d L.C. F O W K E 1 1Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N OWO;2 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, Saskatoon, Saskatchewan, Canada, S7N OWO Regeneration of white spruce (Picea glauca somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mg1-1 2,4-D and 1 mgl-I 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarese and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5-10% after 35 days culture.

PLANT CFJJ. REPORTS 6:486-489 (1987)

Regeneration of plants from mesophyll protoplasts of the wild crucifer Eruca sativa Lam. S A M I R R. S I K D A R , G O U R I C H A T T E R J E E , S R A B A N I D A S a n d S . K . SEN Programme in Genetical Research, Bose Institute, P1/12, C.LT. Scheme VII-M, Calcutta 700 054, India Protoplasts isolated from mesophyll ceils of Eruca sativa Lam., cultured on suitable medium, underwent sustained cell divisions to form calli. The plating efficiency was found to be 0.4%. The protoplast-derived ealli subsequently produced plantlets through organogenesis (15.71%) and somatic embryogenesis (11.25%). Regenerated plants exhibited normal appearance. These results indicate potential to introgress desirable traits from this wild crucifer into important oilseed and cole Brassicas by protoplast fusion and hybrid recovery.

PHYSIOLOGIA PLANTARUM 72:374-378 (1988)

Callus formation from cotyledon protoplasts of Pinus oocarpa and Pinus patula E. L A I N ~ , H. D A V I D a n d A . D A V I D Lab. de Biologic et Physiologic vdgdtales, Universit~ de Bordeaux I - Avenue des Facult~s, F-33405, Talence Cedex, France Protoplasts were isolated from cotyledons of 11-day-old seedlings of Pinus oocarpa and P. patula ssp. tecunumanii. The best enzyme combination was Cellulase R10 + Pectolyase Y-23, associated with bovine serum albumin. When cultured at a low density [1.25 x 103 to 5 x 103 protoplast (ml)-1] in a liquid medium, the cells divided. The medium contained glutamine and casein hydrolysate as nitrogen sources, and glucose as osmoticum. Rate of division was increased by supplementing the medium with L-ornithine, putrescine and spermidine. However, the rate remained low, with an absolute division frequency of ca 1%. Dilution allowed colony proliferation and fragmentation, leading to the formation of numerous microcalli that could be transferred to various solid media for further growth.

A51 JOURNAL OF HEREDITY 7 9 : 2 8 - 3 2 (1988)

Genome multiplication in cultured protoplasts of two Nicotiana species H.-C. HUANG

and C.-C. CHEN

Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China Chromosome number and nuclear behavior in mesophyll Protoplasts isolated from haploid plants of Nicotiana plurabaginifolia and Nicotiana sylvestris were investigated during the process of plant regeneration. At the first mitotic division, a great majority of nuclei had the haploid number of chromosomes. Of these, 10.4% in N. plumbaginifalia and 3.5% in N. sylvestris contained diploehromosomes, indicating that endoreduplication occurred. In subsequent divisions, however, only two-chromatid chromosomes were present. One other abnormality observed during the first few days of culture was the formation of di- and multinucleate cells due to failure of eytokinesis and wall formation. Division of the di- and multinucleates was highly synchronized and fusion occurred as the ehromatids of two or more nuclei moved to the same pole at anaphase. As a consequence of the occurrence of these abnormalities, the frequencies of haploids decreased and those of diploids and polyploids increased as culture proceeded. All Protoplast-derived plants were diploid or polyploid and none was haploid.

PLANT SCIENCE 53:263-270 (1987)

Isolation and culture of heterokaryons following fusion of protoplasts from sexually compatible and sexually incompatible Medicago species D.M. GILMOUR,

M.R. DAVEY

and E.C. COCKING

Plant Genetic Manipulation Group, Department of Botany, University Park, Nottingham NG7 2RD, UK Medicagofalcata L. and M. quasifalcata Sinsk. protoplasts were fused with those ofM. sativa L. (alfalfa, lucerne) using polyethylene glycol (PEG). Heterokaryons were identified by their dual fluorescein diacetate-chlorophyll fiuoreseence and selected manually using a micro-pipette system. Fusion products mixed with albino M. sativa nurse protoplast cultures divided when cultured in the dark. Green colonies which appeared amongst the while albino nurse cells following transfer of cultures to the light produced callus tissues which were confirmed as being somatic hybrid in nature by isoenzyme and cytological analyses.

PLANT SCIENCE 5 3 : 1 5 7 - 1 6 0

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.) MOTOYASU

OTANI,

TAKIKO

SHIMADA

and HIROO

NHZEKI

Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi-machi, lshikawa, Japan 921 Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readily isolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N 6 medium and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into ealli. Some of the calli transplanted onto regeneration medium produced roots.

A52

PLANT SCIENCE 53:167-176

Plant regeneration from protoplasts of diploid potato derived from crosses of Solanum tuberosum with wild solanum

species J. M A S S O N l, M . L E C E R F 1 , P. R O U S S E L L E 2, P. P E R E N N E C 2 a n d G . PELLETIER1 1Laboratoire de Biologic Cellulaire, INRA, 78000 Versailles and 2Station d'AmAlioration de la Pomme de terre et des Plantes ~ bulbes, 1NRA, Landerneau BP 5, 29207 Cedex, France A new method has been developed for the obtention of protoplasts of potato (Solarium tuberosum L.) diploid clones and for the regeneration of whole plants. This procedure determined specific conditions of in vitro culture of shoots: the propagation medium, fight intensity and length of illumination period. Four successive media from the protoplast level to the shoot formation have been optimized. This method is suitable for the obtention of calli from 13 diploid potato lines, among 15 tested. Regeneration of plants has been obtained for 10 clones with efficiencies from 0.5 to 4 plants regenerated from 100 plated protoplasts.

14. Somaclonal variation PLANT CELL REPORTS 6 : 4 1 7 - 4 2 2 (1987)

Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaC! tolerant cell lines T.J. M c C O Y Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA Selection of stable, NaC1 tolerant alfalfa (Medicago sativa L.) cell lines was accomplished by a step-up selection procedure, whereby cell lines originally selected for tolerance at 0.5% NaC1 were subsequently selected at 1.0% NaCI. Sodium chloride tolerant cell lines retained tolerance following four subcultures (16 weeks) on control media (0% NaC1). Plants were regenerated from selected NaC1 tolerant cell lines of three initial genotypes, one diploid (2n = 2x = 16) and two tetraploids (2n = 4x = 32). In addition, plants were regenerated from control cell lines maintained on 0% NaC1 media for the same duration. Plants regenerated from NaCI tolerant cell fines were characterized by extensive somaclonal variation compared to plants regenerated from control lines. Morphologically, all plants regenerated from NaC1 tolerant ceil lines are abnormal and many (44.7%) were extreme dwarfs (maximum height of 5 cm). The grossly aberrant phenotypes prevented an in-depth characterization of many of the plants regenerated from NaCt tolerant cell lines. Most plants regenerated from NaC1 tolerant cell lines had unbalanced polyploid chromosome sets with the most extreme cytogenetie variant having 106 chromosomes. In contrast, 98.5% of the plants regenerated from control cell lines were euploid (85% were tetraploid, 15% were octoploid). Isozyme phenotypes of the plants from NaC1 tolerant cell lines were also extensively altered, compared to plants from control cell lines. In vitro NaCI tolerance was maintained following plant regeneration for nine of the 12 regenerants tested. Importantly, whole plant NaC1 tolerance was expressed in two of the seven regenerated plants tested at the whole plant level; however, only one of these plants has flowered and is both male and female sterile; the other plant has never flowered. Although NaCI tolerant alfalfa cell lines are efficiently selected, the extensive somaclonal variation that accompanied the selection was a deterrent to successful recovery of heritable NaC1 tolerance.

A53

PLANT SCIENCE 53:177-182 (1987)

Selection of Citrus limon cell culture variants resistant to the mal secco toxin BARRY NADEL and PINHAS SPIEGEL-ROY Department of Horticulture, Faculty of Agriculture, Hebrew University, Rehovot and Department of Fruit Breeding and Genetics, A.R.O., The Volcani Center, Bet Dagan, Israel Embryogenic cell cultures of Villafranca lemon capable of growth were selected in the presence of toxin produced by the fungus Phoma tracheiphila, the causal agent of Mal secco. Mal secco is a serious tracheomycotic disease of lemon (Citrus limon Burro. f.) and citron (C. medica L.). Rigorous clonal selection with the variant line was conducted for six consecutive subcultures. Viability of cell lines was monitored by the use of fluorescoin diacetate. The stability of the resistance was examined after growth on non-selective medium for three subcultures. The variant line Var. 1.117 showed stable resistance. Cells of the resistant variant line maintained their embryogenic capacity. Callus induced from the resulting somatic embryos also displayed resistance to the toxin.

PLANT SCIENCE 53:191-198 (1987)

Time-course of mitochondrial genome variation in wheat embryogenic somatic tissue cultures CAROLINE

HARTMANN1

, JACQUES

de BUYSER2, YVES HENRY2,

DENIS FALCONET1, BERNARD LEJEUNE1, ABDEL-ALI BENSLIMANE1, FRANCIS QUETIER1 and ANDRI~ RODE 1 1Laboratoire de Biologic Moldculaire Vdgdtale, associd au CNRS (US 1128) B~timent 430 and 2Laboratoire d'Amdlioration des Plantes, associd au CNRS (US 115), B~timent 360, Universitd Paris-XI, F-91405, Orsay, France Mitochondrial (mt) DNA has been isolated from wheat (Triticum aestivum L., cultivax Chinese Spring) plants and embryogenic callus cultures initiated from immature embryos and harvested after various subcultures. Sal 1-restricted mtDNA has been probed with cloned labelled restriction fragments internal to two of the ten sets of recombinationally active repeats found in wheat mtDNA. The resulting hybridization patterns suggest that (1) novel restriction fragments appear in callus culture mtDNA, (2) some of the restriction fragments encompassing a given recombinatory repeat undergo relative quantitative variation (amplification, decrease or loss), (3) this variation is rapidly stabilized during the course of callogenesis.

PLANT SCIENCE 54:83-91 (1988)

Selection of lactose-adapted cells in Vinca minor and Datura innoxia cultures. Location and characterization of [3-Galactosidase and lactase activities O.C. ELAVUMMOOTTIL1, S. D U R E T 1 , A . V A N N E R E A U 1, L. C O S S O N 2 a n d J.C. M E S T R E 1 1Laboratoire de Biologie Cellulaire and 2Laboratoire de Botanique et Phytochimie, Facultd de Pharmacie, rue J.-B. Cldment, 92296 Chdtenay-Malabry Cedex, France Lactose-adapted cells were obtained from Datura innoxia sucrose growing caUi cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the

A54 homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous populations in D. innoxia cultures. In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown coils as well in Datura lactose-adapted ceils cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for the ceils; it was released into the culture medium and not metabolized in Vinca cdtures while it was metabolized in Datura cultures at the end of the culture period.

PLANT CF.IL REPORTS 7 : 8 3 - 8 7 (1988)

The characterization of herbicide tolerant plants in B r a s s i c a napus L. after in vitro selection of microspores and protoplasts ERIC B. SWANSON, MARC P. COUMANS, GERRY L. BROWN, JAYANTI D. PATEL and W.D. BEVERSDORF Department of Plant Biology, Allelix Inc., 6850 Goreway Drive, Mississauga, Ontario, Canada, IMV 1P1 Brassica napus L. (cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid pretoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected fines produced selfed progency which were tolerant to from 10-100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progency of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the ALIAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.

PLANT SCIENCE 5 4 : 5 5 - 6 3 (1988)

Glyphosate-tolerance in C a t h a r a n t h u s r o s e u s R.C. CRESSWELL, M.W. FOWLER and A.H. SCRAGG Wolfson Institute of Biotechnology, The University, Sheffield, $10 2TN, UK Cultured cells of Catharanthus roseus were selected by a stepwise procedure for tolerance to the herbicide glyphosate. The selected cells were found to contain levels of extractable 5-enolpyruvylshikimic acid-3-phosphate (EPSP) synthase activity significantly greater than those found in non-selected cells. EPSP synthases from glyphosate-tolerant and non-selected cells were both inhibited by glyphosate. The glyphosate-tolerant cells accumulated less shilcimic acid and/or shikimic acid-3-phosphate when treated with glyphosate than did non-selected cells. There was no over accumulation of aromatic amino acids in the glyphosate-tolerant C. roseus. Treatment of

A55 non-selected cells with aromatic amino acid supplements reversed, partially, the effects of 1 mM glyphosate but did not prevent, markedly, growth inhibition caused by 10 mM glyphosate. Production of the secondary metabolites ajmalieine and serpentine, originating from tryptophan, by the selected and non-selected cells was low.

PLANT SCIENCE 55:159-167 (1988)

In vitro culture and the incidence of somaclonal variation in regenerated plants of Trifolium pratense L.) HONG WANG and F.B. HOLL Department of Plant Science, University of British Columbia, Vancouver, B.C., V6T 2A2, Canada Red clover (Trifolium pratense L.) cvs 'Altaswede' (2n = 2x = 14) and 'Norseman' (2n = 4x = 28) have been used to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. After callus induction shoots were induced both when ealli on L2 medium containing 2 mg 1-1 2,4-dichlorophenoxy acetic acid (2,4-D), 2 mg 1-1 6-benzylaminopurine (BA) and 2 mg 1-1 6-amino-purine (AP) were subcultured on media containing naphthalene acetic acid (NAA) (0.05 mg 1-1) and kinetin (KIN) (0.05 or 0.5 mg 1-1) and when embryogenie calli were cultured and subcultured on L2 medium containing 0.002 mg 1-1 4-amino-3,5,6-trichloropieolinic acid (PIC) and 0.2 mg 1-1 BA. Shoot tip cultures were also established to induce multiple shoots for regeneration of plants via organogenesis. Regenemnts from different regeneration pathways were evaluated for chromosome number stability, morphology and several biochemical traits. Regenerated plants showed stable isozyme banding patterns for malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase, as well as their nodule leghemoglobin profiles. Variations were detected in the chromosome number of some regenerants as well as in leaflet length-to-width ratio and leaflet number. Factors related to the incidence of somaclonal variation are discussed.

J. PLANT PHYSIOL. 121:131-139 (1985)

Variation in nuclear DNA content in Pinus taeda L. tissue cultures of diploid origin MICHAEL

H. RENFROE

and GRAEME

P. B E R L Y N

School of Forestry and Environmental Studies, Yale University New Haven, CT 06511, USA Despite prolonged efforts to obtain plant regeneration from pine callus cultures, little attention has been given to the genetic condition of callus cultures. We measured nuclear DNA content in loblolly pine (Pinus taeda L.) callus during six months of continuous culture. Absolute DNA content was measured quantitatively using microspectrophotometry in conjunction with an internal standard. Twenty to thi_~y percent of the cells in the cultures contained nuclear DNA contents in excess of the 4C amount with the remainder having the 2C-4C amount, equivalent to cells in a diploid cell cycle. Cells with increased DNA contents arose early and were present throughout the culture period. Examination of occasional shoot medstems that formed on 'callus-induction' medium revealed that shoots formed which contained cells with abnormal nuclear DNA content as compared with controls. Because polyploidy is deleterious to pines, these results demonstrate the importance of screening tissue cultures for genetic stability in an in vitro propagation program.

A56 J. PLANT PHYSIOL. 121:97-101 (1985)

Mature Phenotype in Hemerocallis plantlets fortuitously generated in vitro M I N D Y S. FITTER and A.D. KRIKORIAN Department of Biochemistry, Division of Biological Sciences State University of New York at Stony Brook, Stony Brook, NY 11794, USA Daylily planflets generated on semi-solid media from morphogenetieally competent cells or morphogenetically competent cells regenerated from Protoplasts can give rise in aseptic culture to plantlets with a mature phenotype. The individual leaves of these planflets open to the extreme base so that no enciroling leaf sheath is present. This permits the overlapping bases and leaves to assume an open fan-like arrangement. The occurrence of fans correlates with exceptionally tightly sealed culture vessels and experiments to date suggest a gaseous component is associated with this change of growth form. It has not been possible to fix the mature growth mode, however, and new leaf growth assumes the more normal juvenile phenotype when the gaseous environment is altered by admitting or exposure to room air.

CROP SCI. 28:363-369 (1988)

Genetic and eytogenetic variation in plants regenerated from organogenic and friable, embryogenie tissue cultures of maize C.L. ARMSTRONG 1 and R.L. PHILLIPS 2 1Agrigenetics Advanced Science Co., 5649 Buckeye Rd. Madison W153716; 2Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA Recent advances in maize (Zea mays L.) tissue culture methods permit reproducible establishment of rapid-growing, friable, embryogenic (Type-IF) cultures from some genotypes. Although Type-lI cultures have replaced the more common Type-I (relatively compact, organogenic) cultures for many applications, little information is available concerning somaclonal variation in plants regenerated from Type-II cultures. This study was conducted to determine the relative frequency of genetic and cytogenetic variants in plants regenerated from Type-I compared to Type-II maize tissue cultures. Type-I and Type-II cultures were initiated from immature embryos of the inbred line A188, A188-BC6 lines containing various genetic markers, and segregating progenies of an A188/B73 genetic background. Plants were regenerated 16 and 36 wk after culture initiation and were analyzed for pollen sterility, cytological abnormalities, and phenotypic variants in progency generations. After 16 wk in culture, 33.3 and 20.8% of the regenerants from Type-II and Type-I cukures, respectively, possessed at least one abnormality. These values increased to 37.2 (Type-II) and 24.2% (Type-I) after 36 wk in culture. As expected, the frequency of chimeric plants was lower from Type-II than from Type-I cultures: however, culture age had a larger effect on the frequency of chimeric plants than culture type. Although reduced compared to Type-I cultures, the frequency of chimerism in regenerants from Type-II cultures was still quite high, ranging from 9.4 to 50.0% of the variant regenerants. The results demonstrate that genetic and cytogenetic abnormalities are frequently recovered in plants regenerated from both organogenie and friable, embryogenic maize tissue cultures.

A57

15. Somatic embryogenesis JOURNAL OF EXPERIMENTAL BOTANY 39 (199): 263-270 (1988)

Inhibition of somatic embryogenesis in tissue cultures of Medicago sativa by aminoethoxyvinylglycine, amino-oxyacetic acid, 2,4-Dinitrophenol and salicylic acid at concentrations which do not inhibit ethylene biosynthesis and growth ELTJO

G.M. MEIJER

and DANIEL

C.W. BROWN

Genetic Engineering Section, Plant Research Centre, Agriculture Canada, Ottawa, Ontario, KIA 0C6, Canada The effects of aminoethoxyvinylglycine (AVG), amino-oxyacetie acid (AOA), 2,4-dinitrophenol (DNP) and salicylic acid (SA) on ethylene production, tissue proliferation and somatic embryogenesis in a recently developed rapid in vitro regeneration system of Medicago sativa L. were examined. Contrary to numerous publications, AVG, AOA and DNP did not affect the rate of ethylene biosynthesis, while SA even eansed an increase in ethylene production. All four compounds were, however, potent inhibitors of somatic embryo formation in the M. sativa tissue cultures, even at concentrations which did not affect tissue growth. Generally, a 5-d exposure to the inhibitors reduced the number and quality of somatic embryos obtained. It is suggested that the inhibitors may not reach the site of action of enzymes involved in ethylene biosynthesis and may possibly block other biosynthetic pathways which are of crucial importance to somatic embryo development. The results indicate that a delicate differentiation process like somatic embryogenesis is very sensitive to metabolic perturbances. The results are also discussed in the light of other known effects of these four compounds on higher plants.

PHYTOMORPHOLOGY 36 (3,4): 315-324 (1986)

In vitro studies on Achras sapota (Chikoo) I. Callus and early embryogenesis SARO~ SACHDEVA

and P.N. MEHRA

Department of Botany, Punjab University, Chandigarh 160014, India Callus was initiated from various parts of the in vitro raised seedlings ofAchras sapota Linn. on Nitsch's medium supplemented with different growth substances. Of the various combinations tested, BN + CW (15%) + NAA (4 ppm) + Kn (2 ppm) or the CNK medium was established as the optimal for callus induction and growth from the root, hypocotyl, stem and shoot tip explants, while BN+CW (15%) + 2.4-D (4 ppm) + Kn (2 ppm) or CDK medium proved optimal for the leaf cotyledon, embryo and endosperm explants. The ealli were heterogeneous in nature. Cytological analysis of calli made after two and four subcultures of one month duration each showed the presence predominantly of diploid cells, though polyploid and aneuploid cells were also noticed. Differentiation of roots and shoots occurred in all the calli except those from root and endosperm. The explants cultured on BW + CW (15%) + NAA (4 ppm) + Kn (0.4 ppm) developed masses of embryoids directly on the surface. The calli induced on CNK or CDK media on transfer to media containing BAP (2-4 ppm) in place of kinetin became nodular and differentiated early stages of embryoids in great numbers. Embryoidal initials were invariably free from the callus tissue and were densely packed with globular starch grains. Earlier stages of their division up to the formation of globoid and heart-shaped embryoids have been noticed. The embryoid initials were mostly diploid but for a few aneuploid and polyploid ones.

A58

PLANT CELL REPORTS 6:484--485 (1987)

Somatic embryogenesis and plant regeneration in callus culture derived from immature seeds and mature zygotic

embryos of Dysosma pleiantha (Hance) Woodson MENG-JIN

CHUANG

and WEI-CHIN

CHANG

Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China Callus was induced on the wounded immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson (Berberidaceae) on a medium based on Murashige and Skoog's (1962) formula supplemented with 1 mg/l 2,4-dichlorophenoxy-acetie acid (2,4-D). Spontaneous embryoid formation occurred on the media containing low concentrations of 2,4-D (0.1-0.5 mg/1). These embryoids germinated in either MS or B5 medium containing 1 mg/l Nt-benzyl adenine and 1 mg/l gibberellie acid. The regenerated planflets were successfully transferred to soil.

J. PLANT PHYSIOL. 121:111-118 (1985)

Efficient plant regeneration by somatic embryogenesis from root callus tissues of rice (Oryza sativa L.) TOSHINORIABE and YUZO FUTSUHARA Laboratory of Theory of Agronomy and Plant Breeding, Faculty of Agriculture, University of Nagoya, Furocho, Chikusaku, Nagoya, 464 Japan Embryogenic callus which was compact and white to pale-yellow in appearance was obtained from root sections of dee (Oryza sativa L.) cultured on Murashige and Skoog's (1962) medium supplemented with 3 mg1-1 2,4-dichlorophenoxyacetic acid (2,4-D). Embryo-like structures were formed by the callus during one to several subcultures on a medium with no or reduced 2,4-D and supplemented with kinetin. Well formed bipolar somatic embryoids with a plumule and radicle axis surrounded by a scutellum could be easily isolated from the tissue mass. Some aberrant somatic embryoids were also observed, Embryogenic capacity of the rice root cultures was very high, resulting in the regeneration of many plantlets from the two varieties tested. Regenerated plantlets were phenotypically similar to seed derived plants. Plant type, grain filling and other morphological characteristics were normal and uniform.

PLANT CELL REPORTS 7:144-147 (1988)

Somatic embryogenesis and plant regeneration in two-year old cultures of Zea diploperennis B. SWEDLUND

and R.D. LOCY

NPI, 417 Wakara Way, Salt Lake City, UT 84108, USA Immature embryos and immature leaf tissues were used to establish embryogenie cultures of Zea diploperennis. Callus was induced on media containing MS salts and vitamins, sucrose (2% for leaves, 6% for embryos), 5% coconut milk and 1-6 rag/1 2,4-D. Embryogenie callus was maintained by subeulturing on media containing MS salts and vitamins, 2% sucrose 500 mg/l casein hydrolysate and 1 rag/1 2,4-D. Regeneration occurred when the 2,4-D level was reduced to 0.25 mg/l. Kinetin added at 025 mg/l further stimulated regeneration, Root tip squashes on 10 plants regenerated after 2 years in culture indicated a normal 2n=20 chromosome number.

A59 PLANT CEIJ. REPORTS 7:23-25 (1988)

Plant regeneration via somatic embryogenesis in the seeded diploid banana M u s a ornata Roxb. SANDRA

S. C R O N A U E R - M I T R A

and A.D. KRIKORIAN

Department of Biochemistry, Division of Biological Sciences, State University of New York, Stony Brook, NY 11794, USA Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mgil) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described.

PLANT SCIENCE 54:157-164 (1988)

Characterization of a temperature-sensitive carrot cell mutant impaired in somatic embryogenesis F. L O S C H I A V O 1 , 2, G. G I U L I A N O 1 and Z . R . S U N G 3

l lnstituto di Mutagenesi e Differenziamento CNR, Via Svezia 10, 56100 Pisa; 21nstituto Internazionale di Genetica e Biofisica, CNR, C.P. 3061, 1-80100 Napoli (Italy); 3Department of Genetics, University of California, Berkeley, CA 94720, USA Among the various carrot cell mutants, temperature-sensitive for embryogenesis, line ts59, blocked at the globular stage, was characterized. A biological characterization indicates that the function missing or altered in ts59 is necessary two times during development: the first, 5 days after the onset of embryogenesis and the second, at the transition between heart- and torpedo shaped embryos. Fusion experiments of protoplasts of ts59 with a wild-type line, inactivated with iodoacetate, indicate dominance of the ts59 mutation. A bioehemieal analysis was carried out of various embryonic markers of tsS9 and compared with wild-type. A remarkable alteration was found in the pattern of heat-shock proteins whose expression was shown to vary during development.

J. PLANT PHYSIOL. 121:149-158 (1985)

Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce) INGER

HAKMAN

and SARA VON ARNOLD

Institute of Physiological Botany, University of Uppsala, Box 540, S-75121 Uppsala, Sweden Embryogenic callus was produced from immature zybotic embryos of Picea abies cultured on a defined medium supplemented with 2,4-dichlorophenoxyaectic acid (10-5 M) and a cytokinin (10-6-10 -5 M). Subsequently numerous somatic embryos developed from the callus. Upon subculture the somatic embryos could be stimulated to develop further into plantlets with cotyledons, a hypocotyl and a root. Under suitable conditions 24% of the calli produced plantlets, and up to 25 plantlets were formed in a single callus, in addition to numerous somatic embryos of smaller sizes. Planflet formation was followed both in living and sectioned materials and showed close similarity to zygotic embryogeny.

A60 J. PLANT PHYSIOL. 121:159-169 (1985)

Plant regeneration by somatic embryogenesis from callus initiated from immature embryos and immature inflorescences of Hordeum vulgare M.R. THOMAS and K.J. SCOTT Department of Biochemistry, University of Queensland, St. Lucia, Qld, 4067, Australia Callus cultures were obtained from immature embryos and immature inflorescences of Hordeum vulgare. Embryogenie callus was produced from both types of explant tissue and was affected by cultivar, size of explant and media components. The highest number of immature embryos (34.3%) and immature inflorescences (39.1%) formed embryogenic callus in the presence of culture medium containing 10 IxM 2,4 dichlorophenoxyacetic acid. The majority of embryoids developed with a leafy structure in place of a scutelhtm. One to many shoot meristems were arranged along the base of this leafy structure. Morphologically normal green plants with a chromosome number of 2n = 14 were regenerated from both inflorescence and embryo cultures. Regenerated albino plantlets had small undifferentiated plastids containing large plastid DNA nucleoids. Also described is pistil development from embryogenie callus initiated from immature embryos of the cultivar Prior.

PHYSIOL PLANTARUM 69:591-596 (1987)

A novel system for rapid high frequency somatic embryogenesis in Medieago sativa ELTJO G.M. MEIJER and DANIEL C.W. BROWN Genetic Engineering Section, Plant Research Centre, Agriculture Canada, Ottawa, Ontario, Canada A novel, genotype dependent system for rapid high frequency somatic embryogenesis in

Medicago sativa L. was. developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g 1-1 cascein hydrolysate, 9 IxM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 I/M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 IxM 2,4-D and 4,7 p.M kinefin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogertic and very/ittle callus proliferation occurred when this method was used. Selected clones from three ssp.falcata-based M. sativa cultivars showed a response very similar to the highly regenerablefalcata clone FI.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa, with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.