Acta Bot. Neerl. 497-505 41(4), December 1992, p.

Meetings of the Royal Botanical Society of

The Netherlands

MEETING OF THE SECTION FOR SYSTEMATICS AND

GEOGRAPHY ON 24 APRIL 1992

the variation these is Significance of Cytological Characters in cytological among higher taxa not yet clear. Systematic and Evolutionary Studies of

Crassulaceae Chloroplast DNA Variationand Henk ’t Hart. Departmentof Plant Ecology and in the Evolutionary Biology, University of Utrecht, Evolutionary Relationships Padualaan 8, 3584 CH Utrecht, The Netherlands

R.C.H.J. van Ham. Department of Plant Ecology Cytologically the family Crassulaceae is probably the and Evolutionary Biology, Padualaan 8, 3584 CH most diverse group of angiosperms. Chromosome Utrecht, The Netherlands numbers range from 2n = 8 to In = 640, and basic

numbers from = = and The x 4 to x 37 more. Evolutionary relationships within the Crassulaceae

Sedum especially is extremely variable. The 54 were investigated at various taxonomic levels. To

European species for instance, comprise about 140 investigate characters independentfrom those thus far

different cytotypes. used ('t Hart, H. (1991): Flora Mediterranea 1: 31-61)

Chromosomes of Crassulaceae are generally very we studied variation in the conservatively evolving

less than 2 but variation in chromo- restriction-site small, usually pm, chloroplast genome by comparative some size is nevertheless considerable. Chromosome mapping. As a basis for this study we generated a

10-fold well of length may vary among species as as complete clone bank the chloroplast DNA among chromosomes of a single karyotype. The (cpDNA) of the European album L. from

of DNA nucleus is which clones used in amount per proportional to were as homologous probes chromosome number and chromosome length. species comparisons (van Ham, R.C.H.J. et al. 1992.

2C values low in Biochem. Ecol. 20: Although are extremely many species Syst. 243-253),

(0-5 pg and less), 40-fold differences have been CpDNA restriction-site variation in Sedum series

observed among species. Rupestria revealed new insights in the evolution of the

to Cytological variation in Sedum is due autopoly- seven species of this comparium, i.e. a different basal

trans- ploidy, descending dysploidy (Robertsonian branching and a more complicatedreticulate terminal locations), and amphiploidy, and in most cases the branchingpattern includingone taxon (S. montanum

direction of the transformations of the of A cytological ssp. montanum) putative triple hybrid origin. characters can be determined unequivocally. Further- preliminary analysis of restriction-site variation at more, variation in 2C values is often correlated with the familial level provided evidence for a basal split in life-form or adaptations to more extreme habitats. In the family between a Crassula-lineage and a Sedum- small variation is Within the infrageneric groups, cytological lineage. Crassula-lineage, the divergence

often a powerful tool for reconstructing phylogenies. between Crassula and the disputed genus Tillaea must

For instance, in Sedum series Rupestria the phylogeny, have taken place relatively early in the evolutionary based on cytological variation, fully agrees with a history of the family. The evolution of the Sedum-

states cladogrambased on 240 different characters of lineage is much more diverse and complicated. The

104 morphologicalcharacters. analyses suggest that (a) the classical subfamilies

Among the European infrageneric taxa of Sedum, Kalanchoideae, Cotyledonoideae,

different levels of chromosomal evolution can be and Echeverioideae are terminal taxa of the Sedum-

distinguished. Groups either have a single basic lineage; (b) there is a close, possibly monophyletic, chromosome number or their basic number varies. relationship between the Kalanchoideae and Cotyle-

Some groups have low primary basic numbers ranging donoideae; (c) the genus Sedum, is paraphyletic and

from x = 5 to x = 13, whereas others comprise possibly comprises three major lineages, each ofwhich primary as well as secondary basic numbers (from has members on more than onecontinent. Finally, a

= 6 to = have 14 of characters x x 37), or only secondary (x= or parsimony analysis morphological

basic numbers. similar of showed little with the higher) A pattern cyto- congruence cpDNA phylogeny,

contributed the logical variation can be observed among genera and indicating that parallelism greatly to

subfamilies. However, the evolutionary significance of phenotypic diversification in the Crassulaceae.

497 498 MEETINGS

Of the Crassulaceae for the of Rosularia (Crassulaceae) —A Systematic investigated presence

alkaloids, Sedum acre has received most attention. Study Some 20 piperidine alkaloids have been reported Urs Eggli. Stadtische Sukkulenten-Sammlung, for this species alone (Hegnauer, R. (1989): Chemo- Mythenquai88, CH-8002 Zurich, Switzerland taxononie der Planzen, 8, Birkhauser, Basel). S. acre

has retained combination of charac- Rosularia (De Candolle) Stapf 1923, based on a morphological

Umbilicus sect. Rosularia De Candolle 1828, was mono- ters, which are considered to be primitive within the

graphed by the author (Eggli, U. (1988): Bradleya 6: Crassulaceae (’t Hart, H. & Koek-Noorman, J.

Taxon (Suppl.) 1-120) and counts 36 taxa (26 species (1989): 38:535). Because a similar combination

S. including one described after the completion of the of primitive characters occurrs in S. aetnense, monographand 10 infraspecific taxa). anglicum, S. brissemoreti, S. farinosum. S. fusiforme,

to in most The genus is closely related Sedum S. lancerottense,S. melanantherum, and S. nudum.we characters and is accordingly placed in subfamily investigated these species for the presence of alkaloids.

Sedoideae, at variance with Bergers’ treatment. It A number of pyrrolidine and piperidine alkaloids,

differs from Sedum. in that its leaves are always rosu- some of which have not been reported as natural late, its petals are sympetaly (petals united for one- constituents, were detected in the leafy parts. tenth to three-quarters of their length), and its carpels Thedistribution ofthe alkaloids agrees perfectly well remain erect even at fruiting time. The pollen is tri- with the infragenericclassification ofthe Europeanand

colporate and may be without ornamentation, or Macaronesian Sedum species (’t Hart, H. (1991): Flora rugulate-striate; the seeds are of the bipapillate type Mediterranea 1:31),except for S. farinosum ofS. series

and are longitudinally striate to costate. Cytologi- Macaronesia. S. farinosum should be separated from

the falls into dissimilar the other of S. series i.e. S. cally, genus two widely groups, species Macaronesia,

7 either with x= or x = 9. brissemoreti, S. fusiforme. S. lancerottense. and S.

The is classified into four sections the base based its alkaloids genus on nudum, on composition, ecological

of comparative morphology and cytology. Section preference,polyploidy level, colour ofthe , and

Rosularia is widespread in the Near and Middle East its hybridizationpattern.

(from Crete to Afghanistan), has x=9, and the only Primitive as well as advanced morphological

known which character- characters in the which polyploids are tetraploids are are present acre-group, com- istic for three Turkish subspecies of the widespread prises S. ursi, S. alpestre, S. grisebachii, S. laconicum,

R. . S. annuum. S. borissovae. S. tuberiferum, S. urvillei, S.

Section Sempervivella ( = Setnpervivella as separate apoleipon. S. multiceps. S. sexangulare, S. samium,

genus; but also including Sedum hirsutum)) is widely and S. litoreum in addition to S. acre itself. Except for

to distributed in the western Mediterranean,North Africa, S. acre, these species were found contain only

Ethiopia, and the Hindukush-Himalaya region; its pyrrolidines and 2-monosubstituted piperidines ofthe largely disjunct area is interpreted as being relictic. pelletierine-type. A good concordance was observed

The section is cytologically heterogeneous,but at least between the composition of alkaloids and the some taxa also have x = 9. hybridizationpattern.

Section is distributed in the In the of alkaloids in the Ornithogalopsis contrast to presence more

Inner Himalaya-Hindukush area and in South Asia. primitive Sedum species, pyrrolidine and piperidine

in of series This is the least well-known section; the basic chromo- alkaloids were absent species Sedum

to be = and all and some number seems x 7, specimens Rupestria, , Greenovia, Jovibarba, investigatedweretetraploid. Sempervivum, which all share the same combi-

Section Chrysanthae has a relatively narrow distri- nation ofadvanced characters. These results indicate a bution in the mountains of Turkey and immediately correlation between the distribution of alkaloids and adjacent regions of Iran, Iraq and Soviet Armenia. the major evolutionary trends in the European

= of alkaloids The base number is x 7, with predominant poly- Crassulaceae. Whether the occurrence

to 16 taxa taxa ploidy (up x ) in all investigated; all are may be considered a primitive character in other

neoendemics. of Sedoideae well is clear. interpreted as groups as not yet quite

Alkaloids of some European Sedoideaeand Systematics of Monanthes (Crassulaceae) Sempervivoideae Reto Nyffeler. Institut fur Systematische Botanik, J.F, Stevens, H. t’ Hart,* H. Hendriks and Th. M. Universitat Zurich, Zollikerstrasse 107, CH-8008 Malingre. Department ofPharmacognosy, University Zurich, Switzerland. ofGroningen.Ant. Deusinglaan2,9713 AW

Groningen and ’DepartmentofPlant Ecology Monanthes is a small genus of 10 species and one

and EvolutionaryBiology, University ofUtrecht, heterotypic subspecies. Itis mainlycharacterized by its

Padualaan 8, 3584 CH Utrecht, The Netherlands enlarged,petaloid nectaries. Furthermore Monanthes MEETINGS 499

is remarkable for its partly extensive development of A. gorgoneum (Cape Verde Islands), A. holochrysum bladder-cell idioblasts. Except for two species (one () and A. leucoblepharum (Yemen). occurs in the Atlas Mountains of Morocco, and oneis They have almost identical RFLP patterns. CpDNA- restricted few localities the all these is that to a on SalvageIslands), divergence among species comparable to

A. taxa are confined to the Canary Islands. The most of two populations of spathulatum from different useful characters for the distinction of the recognized islands. It can be concluded that the aforementioned

taxa proved to be growthform,origin of inflorescence species have diverged recently and certainly do not

Aeonium (vegetative axes determinate or indeterminate),petal represent the ancestral forms in as suggested

shape, nectary shape, and the size and distribution of by Lems (Lems, K. (1960): Ecology 41: 1-17). Lems

bladder-cells and glandular hairs. Based on their came to this conclusion on the basis of growth form

growth form and supported by several other charac- and geographicaldistribution. Our results are also in

ters, the perennial members of Monanthes are easily conflict with Liu (Liu, H.-Y. (1989): Nat. Mus. Nat.

assigned to one of three sections. Distribution Sci. 1) who even classified the three species in two

patterns and ecologicalpreferences support this classi- different sections.

fication. Section Monanthes includes M. polyphylla CpDNA of Greenovia aurea does not differ signifi-

polyphylla and M. M. from that of Aeonium. The F values ssp. polyphylla ssp. amydros, cantly vary

muralis; Section Petrophyllea includes M. atlantica, between 0-92 and 0-96 and are comparable to the F

M. lowei. M. brachycaulos, M. minima, M. pollensand values ofcpDNA within Aeonium.These cpDNA data

M. icterica (annual; included here for its floral mor- suggestthat Greenovia should be included in Aeonium.

phology); Section Sedoidea includes M. anagensis and

M. laxiflora. the morphologicaldiversity found in this Variationand EvolutionofCrassulacean small genus facilitates speculations on possible path-

of the evolution. floral Acid Metabolism in Sedum and Aeonium ways Several, especially

characters support the assumption that the members E.A.H. Pilon-Smits,H. ’t Hart and J. van Brederode.

of Section Monanthes and Section Sedoidea are Department of Botany, Ecology and Evolutionary

advanced relative to those of Section Petrophyllea Biology, Padualaan 8, 3584 CH, Utrecht, The

(which includes character states generally referred Netherlands

to groups thought to be rather primitive in the Crassulacean acid metabolism is a photosynthetic Crassulaceae). The same data makes obvious that the

pathway which can be considered as anadaptation to derived have different within two groups origins arid environments. The CAM pathway has evolved Section Petrophyllea. M. atlantica (often treated from the ‘normal’ C3 pathway, and has developed under Sedum (S. surculosum ) most probably relates families. independently in many plant The main sub- Monanthes with taxa actually referred to Rosularia ject of our investigations was the evolution ofCAM in (especially R. jaccardiana). Sedum and Aeonium. Using several parameters, the

variation of CAM within these genera was analysed

Evolutionary Relationships in Aeonium and evolutionary models are proposed. Analysis of the variation of CAM in Sedum showed (Crassulaceae) a distinct difference between the Mexican (obligate T.H.M. Mes, H. ’t Hart and J.v. Brederode. CAM) and the European (facultative CAM) species. Department of Botanical Ecology and Evolutionary The variation in CAM and Sedum agreed to some Biology, Padualaan 8, 3584 CH Utrecht. extent with the major evolutionary trends within the

The is confined the from the advanced genus Aeonium largely to Canary genus. Species more groups gener-

Islands, Thirty-two of the 37 species and subspecies ally had a higher CAM activity than less specialized

occur on the Canary Islands; two species occur on species, even though certain members of the latter

Madeira,oneon the Cape Verde Islands and Morocco group have developed a high CAM activity in the dry

Aeonium and two arefound in East Africa and Arabia. climate of Southern Madeira. Consequently, CAM

is generally considered to be a young genus in which may have evolved morethan oncein this genus and we

the variety of growth forms is a result of adaptive assume that all Sedum taxa have the ability to develop

radiation. Restriction patterns of chloroplast DNA CAM.

(cpDNA) were used to study evolutionary relation- The large variability of CAM in Aeonium appeared

12 Aeonium be correlated with the habitat of the ships among species. CpDNA was to species (strong

12 digested with restriction enzymes recognizing six CAM in arid habitats), as well aswith the growthform

base-pair sites. Divergence ofcpDNA in Aeonium is and taxonomic classification into sections. From these

low. The fraction of shared results conclude thatCAM evolved in the very (F) fragments (frag- we probably

ments ofidentical size) varies between 0-92 and 0-99. monophyletic genus Aeonium parallel with the mor-

from Remarkably, three of the geographicallymost separ- phologicaldifferentiation, small undifferentiated

ated be related: Sedum- like little CAM, via species appear to especially closely species, displaying or no 500 MEETINGS

rosettes and/or candelabrum-shapedforms with inter- Bryophyllum have drooping flowers, of the mediate or strong CAM, to large shrubby species dis- section Kalanchoë have upright flowers. The genus playing strong CAM activity. As a possiblescenario we exhibits considerable genetic variation. This variation

the succession of Aeonium should enable breeders obtain further propose following events. plant to a

evolved from an -like ancestor, which in improved assortment for both pot plants and cut turn was derived from a Sedum- like progenitor, prob- flowers. For evaluation of important characters of ably resembling the present-day S. gattefossei from wild species, 116 species and 47 F 1-hybrids were

Morocco. The diversity ofpresent-day species rapidly studied. The most important attributes measured

evolved from this ancestor throughadaptive radiation were: mean reaction time (number of days from the

(comparableto Darwin’s finches), with similar species beginning ofshort-day treatment to flowering), stem occupying comparable niches on different islands. length and endurance ofindividual flowers. Minimum

reaction time found in K. was rotundifolia ssp. Genetic Variationand Interspecific strictifolia (58 days). Stem length varied from 5 cm

in K. guignardii to 95 cm in K. velutina. Endurance Hybridization in Kalanchoë of flowers varied from 4 days in K. afzeliana, K. G.H. Kroon. Centre for Plant Breeding and

densiflora, K. gracilipes, K. lateritia and K. serrata to Reproduction Research (CPRO-DLO), P.O. Box 16, 36 days in K. blossfeldiana. Ten species were selected 6700 AA Wageningen,The Netherlands

for interspecific diallelic hybridization.Only a limited

The = genus Kalanchoë Adanson {In 34,68,102,136)is number of hybrids was obtained. According to our

divided in the sections and results Kitchingia, Bryophyllum crossing barriers are not related with the

Kalanchoë. It contains 125 of which current c. 150 species, are systematic classification ofthe genus but seem

(cyto)taxonomicallydetermined (Baldwin,J.T.(1938): to depend on specific combinations of species used.

Am. J. Bol. 25: 572-579; Flamet, R. & Mamier- Nature and location of crossing barriers have been

Lapostolle, J. (1964): Arch. Mus. Hist. Nal. (Ser. 7) studied. Promising hybrids, partly with restored fer-

8: 1-110). Species of the sections Kitchingia and tility will be released to commercial plant breeders.

MEETING OF THE SECTION FOR FERTILIZATION RESEARCH IN PLANTS

ON 21 FEBRUARY 1992

Cytological Analysis of Apomixis and as estimated from homogeneity in their offspring.

Material was collected from greenhouse plants, Sexuality in Poapratensis L. grown cultured at 18°C, 2-11 days after pollination, and T. Naumova,* M.T.M. Willemse§and A.P.M. den fixed in FAA and stored in ethanol 70%. Nijs{. *Komarov Botanical Institute, St Petersburg, Ovules dissected from were the ovaries, embryo sacs Russia, JCPRO-DLO, Wageningen,The Netherlands were taken out from the ovules by hand with fine and §Department of Plant Cytology and Morphology, needles under binocular microscopes. Embryo sacs WageningenAgricultural University, The Netherlands

were then stained for 10 min with the DNA-specific

5 Interest in apomixis has recently increased in breed- fluorochrome (DAPI) (10 m DAPI buffered (pH =

and 4 embedded in and ing.biotechnological geneticengineeringprograms. 0) solution), glycerin-gelatin

Therefore it isimperative that time-saving and informa- examined and photographed under phase-contrast tive techniques are developed for screening wild and and interference contrast microscopes. A microcyto-

well cultivated species for apomixis as as for deter- photometerwith a Phloem incident UV light with an mining the degree of apomixis and estimating ploidy excitation wave length of 365 nm was used to measure levels ofendosperm and embryo cells. Classical cyto- the amount ofDNA in the intact nuclei ofendosperm,

not logicaltechniquesare reallyuseful for investigating embryo and diploid nucellus cells. Excitation wave numerous ovules and embryo sacs or for estimating lengthwas 365 nm, emission wave length was 470 nm. ploidy levels. Methods for complete embryo-sac Measurements were taken from at least 10 different

have been in Russia. These of 16 at 240. investigation developed areas x 16pm a magnificationof x techniques enable the study ofnumerous embryo sacs, The analysis of complete ovules and embryo sacs the identification of polyembryony, the cytological indicated that most of the Poa pratensis genotypes analysis of the endosperm and the estimation of studied, display a tendency to apomictic seed repro-

there differences in embryo-sac fertility. duction but are degree of amphi-

Twelve Poa pratensis genotypes were investigated mixis and apomixis: (a) predominant apomictic seed

afterself- and open-pollinationand reciprocal crosses development; (b) apomictic and amphimictic repro-

and included in a research programme of CPRO- duction more or less in balance; (c) amphimixis pre-

DLO. Some genotypes showed a tendency toapomixis dominant. Viable seeds in P. pratensis result for MEETINGS 501

after double fertilization well from and via The amphimixis as as apomictic tendency progeny testing. parthenogeneticembryo developmentand endosperm majority of the plants contained chromosome formation after single fertilization. Embryo develop- numbers as high as or higher than the parent, but ment without endosperm is necessarily the result of several plants with reduced chromosome numbers

not viable in diploidparthenogenesis. These seeds are were selected. Some of these reproduced (mainly) via nature but abort. The failure of endosperm develop- apomixis as evidenced by the homogeneity of their

in the absence of fertilization is the main As their considered ment reason progeny. ploidy was still too high, for seed in of and the extraction degeneration case apospory parth- twin-seedling procedure was repeated. enogenesis. A tendency to normal (1* genome) and Twin percentages of up to 20% were observed, and aposporic (2.x) embryo-sac formation in the same plant there was no relation between twin percentage of the

observed in P. and and that of selected was many pratensis genotypes con- parent offspring plants. firmed the which by cytophotometric measurements, Selected deviatingtwin-halves are being evaluated

evidence of and This it is gave 3x 5.v endosperms. technique and anticipated that crosses between contrasting could give quantitative data on the prevalence of apo- low chromosome number genotypes could be made by

and amphimixis in individual genotypes in a breeding 1993. programme.

Promoter Analysis of the Pollen-Specific

to find the Genetic Back-Ground Attempts Gene NTP303 from Tobacco of Apomixis in Poa pratensis L. K.A.P. Weterings, Department ofExperimental

A.P.M. den Nijs. Centre for Plant Breeding and Botany, Research Group Molecular Plant

ReproductionResearch, CPRO-DLO, P.O. Box 16, Physiology, University ofNijmegen,Toernooiveld,

6700 AA Wageningen,The Netherlands 6525 ED Nijmegen,The Netherlands

L. Poa pratensis (Kentucky bluegrass) is one of the The isolation of a pollen-specific cDNA clone

used both in from tobacco most important temperate grasses (NTPc303) was reported previously

for fodder for lawns. pastures, and Most cultivars are (Weterings el al. (1992): Plant Mol. Biol (in press)).

= with = The of the highly polyploid aneuploids (2« 36-82, x 7) highly specific expression pattern gene which reproduce mainly via aposporous apomixis. NTP303 as determined by northern blot analysis has

This method of asexual seed production (agamospory) been corroborated by in-situ localization using con- maintains the genotype of the maternal parent despite focal laser scanning microscopy. Transcription of the the fact that pollinationand fertilization ofthe endo- NTP303 gene starts late during development of the

is for seed and continues tube sperm required development(pseudogamy). pollen grain duringpollen growth.

Plant face difficulties in No be detected in breeders, however, generating homologous transcripts can vege-

and exploitinggenetic variation forthe selection ofnew tative or other tissue. Combined with the fact improved cultivars. that NTP303 is expressed in a broad varietyof pollen

accessions of the facul- these results that NTP303 codes for Although many species are species, suggest an tative both and via function in Nucleotide apomictic (i.e. reproduce sexually indispensable pollen. sequence apomixis, dependingon genotype and environment), analysis and database searches however, have not selection ofcultivars has favoured a with function for this strongly very high come up a possible gene.

degreeofapomixis. Crosses with sexual types generally Homologouspollen-specific cDNA clones have been

lead loss of which is however: LAT51 from to apomixis, only very erratically isolated, tomato(S. McComick)

recovered. The genetic basis of apomixis in Poa and Bp 10 from Brassica pollen (S. Fabijanski) (pers, pratensis is not known, whereas in someother species a comm.).

relatively simple hereditary basis is postulated, one The highand very specific expression characteristics

for this the chromosome numbers of of NTP303 make this suitable for reason being high gene a object study-

available CPRO-DLO started the mechanisms involved in tissue- genotypes. a project ing regulation

several obtain with clone years ago to contrasting genotypes specific gene expression. A genomic (NTPg303), a lower ploidy level to be crossed for genetic analysis. homologous to NTPc303 was therefore isolated. The

Twin these in restriction of the clone when seedlingswereextracted; occur relatively map NTPg303, com-

high frequency in this and other facultative apomictic pared to the NTPc303 map suggests an intronless

species. Twin percentages of up to 8% were found. gene. This suggestion is confirmed by the preliminary

About one-tenth of these twins were visibly non- sequence results.

identical. Of these the less vigorous halves were Using the 5' codingregion of the NTPg303 clone,it evaluated, as these individuals may have developed has been determined that three copies of NTP303 are

from reduced in the oftobacco. Also a non-fertilized, sexual, numerically present genome homologous

Chromosome have been found in the of embryo-sac (haploid parthenogenesis). genes genomes Petunia,

estimated via flow numbers were cytometry (FCM) tomato and Arabidopsis. 502 MEETINGS

In order be able the of TATA of LAT52 from to tostudy promoter a gene, region (a pollen-specific gene

deter- 10 first the transcription site of that gene has to be tomato) and Bp (a pollen-specific gene from mined. The of the of of Nucleotide of the first region start transcription Brassica). sequence analysis

NTP303 has been determined northern blot 500 of the of the roughly by bp upstream coding region gene analysis using two different probes: F9-3 containingthe has resulted in the identification of three coremotifs

5'region —700to — 77;F9-32containing— 700 to—199 of the c/.v-acting element conferring pollen speci-

(relative to the start ATG). As F9-3 could and F9-32 ficity (GTGG or GTGA). An extended region sur- could not generatea hybridizationsignal, the transcrip- rounding one ofthese motifs was 100% homologous tion-start site was determined to lie between positions to the pollenbox (TGTGGTT) and 52/56-box

—77 and —199. An RNase F9-3 D. Twell. protectionassay using (TGTGGTTAATATA) as postulated by as a probefurther delineated the transcription-start site The functionalityof these regions will be determined to position — 152 (relative to the start ATG), in future research, targetting these regions for muta-

Interestingly the TATA box most proximate to the genesis and subsequent transformation of pollen transcription-start site is highly homologous to the and/or plants.

MEETING OF THE NETHERLANDS SOCIETY FOR PLANT CELL AND TISSUE

CULTURE SPRING 1992

Anther and Microspore Culture of ‘Re-invigoration’ of Rose Rootstocks

Hordeum vulgare cv. Igri Following Micropropagation

D P. S. Hoekstra, M.H. van Zujderveld,J.D. Louwerse, de Vries and Lidwien A.M. Dubois. DLO-

F.L. F. Olsen,* F. Heidekamp and van der Mark. Centre for Plant Breeding and Reproduction

Center for Phytotechnology RUL-TNO, Research (CPRO-DLO) P.O. Box 16, 6700 AA

Wassenaarseweg 64,2333 AL Leiden, The Wageningen,The Netherlands

Netherlands and ‘Department ofPhysiology,

Carlsberg Laboratory and Carlsberg Research In the developmentofwoody plants from seed there is

Laboratory, 10, Gl. Carlsberg Vej, 2500 Copenhagen ajuvenilephasewhich,dependingon the species, varies

Valby, Denmark from several weeks to 30-40 years. When juvenile,

flowering cannotoccurunder normal conditions. Once

The influence of environmental and culture changes the ability toflower is achieved, the plant is considered was investigated on both anther culture and micro- adult orsexually mature.Before transition tomaturity,

culture of The decreases in The spore barley cv. Igri. highest regener- vigourgradually most species. reverse

ation for both culture obtained in which the is termed frequency systems was process, plant regainsjuvenility when at least 50% of the microspore populationwas ‘rejuvenation’; when only juvenilevigour is restored, in the mid-late to late uninucleate stage, when the this is called ‘re-invigoration’. Restoration of vigour

anthers were pretreated for 4 days on mannitol and bringswith it improvedadventitious root formation in when culture with recalcitrant was performed oxygen supply at some woody crops. regular intervals. Furthermore, the supplement of In the Dutch rose industry on artificial substrates, vitamins and casein hydrolysate in the culture medium scion varieties are propagated onto clonal rootstocks

the whereas such method in which both the scion improved microspore culture, sup- by cutting-grafting, a plementshowed no or negative effect on the culture of and the rootstock are shoot internodes. Normally

anthers. stock internodes form adventitious root in 2-3 weeks

Anther culture is not as laborious as microspore time. Newlyintroduced clonal R. canina Tnermis’, that

culture, but turned out to be at least five-fold less would be otherwise promising stocks, are notoriously

efficient. When mechanically-isolated microspores recalcitrant. The possible re-invigorationfollowing 24 werecultured, under the conditions found to be opti- months of micropropagation of six clonal rose root- mal in the present study, ameanof 12-4 green plantsper stocks, includingtwo Tnermis’ selections, was studied

anther was obtained. Consequently, microspore cul- with regard tothe rooting ofsoftwood cuttings, which ture does meet the requirementof high regeneration is comparableto cutting-grafting. frequency thus enabling application. Experiments Under optimal conditions, cuttings of micro-

aimed at stable transformation through particle propagated source plants had heavier roots, more bombardment of in total microspores, are progress. roots per cutting, larger root length, larger MEETINGS 503

specific root length, and longeraxillary sprouts than react to situations in which the cytochrome pathway cuttings from source plants that were continuously functions less or not at all.

grown in the greenhouse. In general, re-invigoration This subject was studied by growing batch culture

stocks recalcitrant of Petunia in the of was more conspicuous as were more suspensions hybrida presence under normal but also the substances that the conditions, well-rooting suppress cytochrome pathway.

‘Multic’ was evidently reinvigorated. Re-invigoration Chloramphenicol(CAP) is an inhibitor ofthe mito-

following sustained micropropagation may be con- chondrial protein-synthesis and therefore has aneffect

ducive to cutting-grafting in other recalcitrant rose on a.o. cytochrome oxidase and ATP-synthase. stocks. Although the activity ofcytochromeoxidase decreased,

thecytochromepathway mediated respirationofCAP-

treated cells did not reveal much difference to that of 2,4-D as a Signal Molecule in Plant-Cell control cells duringthe batch cycle.Theemploymentof Culture the alternative pathway increased significantly com- K.B. Koens, F.T. Nicoloso, Th. B van Vliet, F. van to control cells. The cells showed little pared very orno Iren and J.W. Kijne. Institute of Molecular Plant growth. Sciences, Leiden University, Nonnensteeg 3, 2311 VJ Growth of cells cultivated in the presence of Anti- The Netherlands Leiden, A mycin (AA), an inhibitor of the cytochrome

pathway mediated respiration showed a rather long 2,4-Dichlorophenoxyacetic acid (2,4-D) is used as a lag-phase of about I week. After that, dry weight mitogenin plant-cell and tissue cultures. Its function is increased somewhat slower than in control cells and of that of a single molecule, not a nutrient.

ended up at about2/3 ofcontrol cells. In our laboratory, the role of 2,4-D in plant-cell

The AA-treated cells showed an immediate decrease division was studied using suspension cultures of in activity ofthe cytochrome and the tobacco cells. In standard LS-medium, cell division pathway engage- ofthe alternative The ment pathwaywas high. presence eventually stopped because of a concurrent lack of

of AA does not prevent the cytochrome pathway to 2,4-D and phosphate. 2,4-D limitation appeared to 10 increase again after days. However, it seems unlikely result in accumulation of cells in the G2-phase of the that the cytochromal respiration is responsible for all cell cycle. Addition of auxin (2,4-D or NAA) induced ATP-formation and growth in the AA-treated cells; cells to continue the cell cycle, whereas addition of probably the alternative respiration takes part too. cytokinin (kinetin or BAP) had no such effect.

Itremains uncertain whether plant cells cangrow on The total amount of free 2,4-D remained constant alternative respiration alone. Nevertheless the results during the growthperiod of the cells, but the distri- indicate that the alternative pathwaycontributes tothe bution of 2,4-D among cells and medium changed. physiological flexibility ofthe cells. Apparently, 2,4-D is not lost due to breakdown or

conjugation.2,4-D transport might be a regulatory

factor in control ofplant-cell division. The first indi- A Dramatic Influenceof Embedding in of cations for a reduced uptake 2,4-D by tobacco cells Calcium Alginate on Protoplast Culturein grown under phosphate-limiting conditions were Beet (Beta vulgaris L.). found. The site of action of 2,4-D has yet to be Robert D. Hall, C. Pedersen and F.A. Krens. DLO- determined. Centre for Plant Breeding and Reproduction

Research (CPRO-DLO), Department ofCell Cells The Flexibility ofPlant in Tissue Biology, P.O. Box 16, 6700AA Wageningen,The

Culture—Respiration and Growth Netherlands

Willie A.M. van Emmerik, Anneke M. Wagner and Plating efficiencies in protoplast cultures of Beta Linus H.W. van der Plas.* Department of vulgaris L. (sugar beet, fodder beet) are generally low. Physiology and Biochemistryof Plants, Vrije In relation to our cybridization programme for Beta Universiteit, De Boelelaan 1087, 1081 HV,

spp. we investigated a number ofmodifications to the Amsterdam,The Netherlands and ‘Departmentof to basic protoplast culture protocol identify means to Plant Physiology,Agricultural University in-vitro improve responses. Of these, the embedding Wageningen, Arboretumlaan 4, 6703 BD, of mesophyllprotoplasts in thin, 1% calcium alginate Wageningen, The Netherlands discs resulted in the most dramatic improvement in

Plant cells the cell in culture in standard possess two respiratory pathways: cyto- development, comparison to

chrome pathway which is used preferentially, and the liquid medium. Many more cells remained viable

alternative, cyanide-resistant pathway. Respiration longer, resynthesized a cell wall and retained visible

little The via the latterprovides energy (ATP) comparedto cytoplasmic activity. greatest influence was,

respiration via the cytochrome pathway. The presence however, on cell division. In alginate cultures this

of the alternative pathway possibly enables the cell to occurred much earlier (after 2 days in contrast to 504 MEETINGS

7 2+ days in liquid medium), and at a significantly higher Imaging of Cytosolic Ca in Embryogenic

frequency (10-200-fold, dependenton genotype and Plant Cells by Confocal Microscopy tissue source) and lead to the formation of colonies A.C.J. Timmers and J.H.N. Schel. Agricultural which required transfer to fresh medium in half the University, Department of Plant Cytology and usual time (18 days instead of 35 days). The re- Morphology, Arboretumlaan 4, 6703 BD generationcapacity of the friable calli obtained from Wageningen, The Netherlands alginate cultures also appeared to be significantly

Confocal enhanced although this requires confirmation. At- laser scanning microscopy (CLSM) is a

for tempts to identify the critical feature(s)of the alginate powerful tool studying the distribution of free

2+ protocol which bring about these effects have lead to cytosolic Ca in multicellular plant systems. In a

CLSM with the conclusion that the alginatemust be solidified and equipped an argon or argon-krypton

2+ the cells must be embedded therein in order to realize laser, fluo-3 is a suitable Ca probe. We used fluo-3 to

2+ the stimulatory influence of the technique. Agarose examine the distribution of free cytosolic Ca during

not is a suitable substitute for alginate in these plant embryogenesis.

carrot experiments. Somatic embryogenesis of is a well-known

In These results, and those reported for other systems, model system. this system, embryos can easily be

obtained in numbers the culture lead to the strong recommendation to test alginate vast by changing embeddingofprotoplasts for all recalcitrant species in medium fromauxin-containingto auxin-free medium.

order the cell-division Embryos arise from single cells in the to improve response. present per-

of and iphery pro-embryogenic masses (perns) pro-

ceed through the succeeding stages ofembrogenesis: HormoneSensitivity During the Successive globular,heart shaped and torpedo shaped.

Phases of Rooting in Malus Microcuttings We were unable toload fluo-3 into carrot cells in its

Geert-Jan de Klerk, Jolanda and Marina form low Therefore ter Brugge AM or at pH. we developed a

Keppel. COWT, P.O. Box 85, 2160 AB Lisse, The protocol which included the use of 0-1 % digitonin to

Netherlands permeabilize the plasma membrane (Fiskum, G.

(1985): Cell Calcium 6:25-37). It appeared that carrot

Regeneration of roots occurs in three phases, namely somatic embryogenesis coincides with a rise in the

dedifferentation,differentiation and outgrowth. We 2+ level offree cytosolic Ca . In embryos from the heart examined the timingof these phases in microcuttings to the torpedoshaped stage,a conspicuous signal was of Malus ‘Jork’ rooted 20°C with indole-3- at 1 pM present in the protoderm, Intracellularly, the highest butyric acid (IBA). We assumed that the differentiation signal was observed in nuclei. is inhibited Pulses with 2 6- phase by cytokinin. pM As embryogeneic cells normally develop into com- benzylaminopurine(BAP) from 24 to48 h or from 48 to plete somatic embryos after dye loading (Timmers, 72 h strongly inhibited rooting, whereas 24-h pulses at A.C.J., Reiss, H.-D., Schell, J.H.N. (1991): Cell

other times had a much reduced effect. We therefore Calcium 12: 515-521), weconclude that this procedure conclude that the differentiation phaselies between 24 can be used to study the complete developmental and 72 h after excision of the microcuttings. Pulses process of somatic embryo formation by intravital with IBA given during the differentiation to phase microscopy. microcuttings cultured on medium without hormones stimulated root formation. The promotive effect of A Model-System for Studying Cold- IBA distinct of BAP was not as as the inhibitory effect Induced Sweetening in Potato and was only observed in pulses with a high concen- M. Manuela Gouveia, Pieternel A M. Claassen, tration of auxin (3 pM). We conclude that the shoots Hans Mooibroek and Henk J. Huizing. ATO-DLO have low-auxin requirement during the dedifferenti- Agrotechnological Research Institute, P.O. Box 17, ation phase to sustain competence, and high-auxin 6700 AA Wageningen, The Netherlands the differentiation requirementduring phase to pro- mote rooting. In discs excised from microcuttings Cold-storage-induced sweetening is of considerable

at and cultured 25”C, the differentiation phase was economic importance for the potato processing somewhat advanced. In discs rooted under the same industry. During frying, the Maillard reaction

has between conditions, microscopical analysis previously reducing sugars and amino acids causes un-

shown the that first cell divisions occur after 48 h. acceptable levels of browning. The susceptibility of

Thus, the differentiation phase coincides with the potato tubers to low temperature is dependent on first cell divisions. We also examined the effect of tuber maturity, environmental factors during tuber

2,4-dichlorophenoxyacetic acid (2,4-D). This syn- growthand, above all, varietal characteristics. thetic auxin strongly inhibited the outgrowth of roots, Cold-induced sweetening has been correlated with but the not dedifferentiation and differentiation cold-labilityofkey glycolytic enzymes; ATP-phospho- phases. fructokinase (PFK) and/or PPi-phosphofructokinase MEETINGS 505

T (PFP). oestablish the contributions ofthese enzymes, Initiationand Establishmentof transgenic plants were produced in which the corre- Morphogenic Cell Suspensions of Barley had been altered. The of sponding genes investigation (Hordcum vulgare) the first group of transformants, derived from a N.J. Ruys, K.J. Van Dam, A. Taal and F. Van Der readily transformable, diploid genotype (1024-2) Mark. Centre for Phytotechnology RUL-TNO, expressing antisense PFPfi, is now in progress, as Department of Molecular Plant Biotechnology, well as transformations of cold-sensitive target cv. Wassenaarseweg 64, 2333 AL Leiden, The Saturna. Netherlands Until now, the success of selecting improved geno-

with reduced The main aim of research efficient types a cold-susceptibility depended on our was to developan

cold-storage experiments of field-growntubers. How- system for transformation and plant regenerationfrom ever, this procedure is time- and space-consuming and barley cell suspensions.

be affected seasonal and environmental fac- Two methods for initiation may by cell-suspension were

tors. Therefore, the use of microtubers produced by compared for sevenbarley genotypes. Callus was in-

duced immature in-vitro grown plantlets offers an attractive alterna- on embryos and, upon subculturing,

tive. However, striking differences were observed fast-growing friable callus was selected. This selected

between different genotypes for supplementsrequired callus was used to initiate cell suspensions. For the

for optimal microtuber production. Supplements second initiation method, immature embryos were

(and their concentrations) applied to the standard directlycultured in liquidmedium.

MS-medium were: sucrose (2-8%), jasmonic acid (JA, An established cell suspension should (a)consist of

BAP 0 01-100 pM), (benzylaminopurine) or kinetin small cell aggregates, (b) have a good growthrate and

have A (10-2-5 mg U 1 ), agar (0-8%) or Gelrite (0-2%). For (c) morphogenic capacity. very important pre-

the for the of normal fertile diploid genotype: 6% sucrose, 1 pM JA, plus requisite regeneration plants

1 mgl 1 BAP were optimal and for cv. Saturna: 6% from cultivated cells is the diploid status of the cells.

> I 1 BAP. the the level of nuclei isolated from the sucrose, plus mg 1 Surprisingly, Therefore, ploidy

tuber-inducing substance jasmonic acid did stimu- suspension cells was determined by flow cytometry.

late microtuber formation in the With both initiation methods able diploid genotype, we were to gener-

but not in cv. Saturna. Kinetin did not stimulate ate fast-growing cell suspensions consisting of small

microtuber formation in 1024-2, whereas in cv. aggregates. Ifa suspensionwas initiated directly using

immature months to establish Saturna a positive effect was only observed in embryos it took 2-3 a

Gelrite-solidified media. Under the optimized homogeneous cell suspension; with callus it took at

conditions, more than 90% of explants formed least 5 months. At this moment we have established

microtubers in c. 4 weeks. In addition, it was cell suspensions of four different genotypes.

demonstrated that cv. Kennebec microtubers The ploidy level of the cell cultures seemed to be

showed sweetening during storage at low tem- dependent on the initiation method, tissue-culture

perature. Glucose and fructose concentrations time and the genotype.

increased almost 10-fold to l-0g lOOg 1 fwt. in For one genotype we were able to establish cell

about 1 month of storage at 2°C (Claassen el al. cultures with a high regenerationcapacity. Approxi-

Potato Res. Similar results 50 cells (1992): (in press)). mately green plants g 1 suspension were

have been reported for field-grown tubers of this obtained. All regenerants were diploid and morpho-

cultivar. logical normal. The first floweringplants were fertile.