114 SouthAfrican Journal of Science98, March/April2002 Gommentary

reveals slight heterochromatin differ- lnducing sex change and ences,"'" which is itself due to differential methylation."''n Therefore, it is distinctly organogenesis from tissue possible that methylation controls sex determination. culture in the endangered African Methylation and accompanying hetero- chromatin can be removed by various cycad Encephalartos woodii f actors - such as temper attJte,'5''6 lighft ,27 osmotic stress,28or hormones2e-31- result- (Gycadales, Zamiaceael ing in sex change.3''33Sex change occurs only in organisms that have (virtually) indistinguishable sex chromosomes, indi- Root Gorelicku" and Roy Osborneb cating that incipient sex chromosomes are formed by slight differences in methylation. Differential methylation is I F INCIPIENT SEX CHROMOSOME DIFFEREN- Fourth, if backcrossing is the only realistic evolutionarily the first difference between I tiutiott is caused by differential methy- approach to conservation, then it is pref- females and males3nand is the likely cause I lation between females and males, then erableto use E.woodiiasthe female parent of reported sex changes in cycads. methylating or demethylating cytosine because of maternal inheritance of nucleotides may induce sex change. chloroplast genomes. Fifth, induced sex Methylation may also stimulate regeneration Application of theory to sex change of roots and shoots from tissue culture callus change may assist in the conservation of in cycads and increase genetic variation via greater other dioecious plants. Sixth, sex change Although sex-specific differential mutation. We propose using these methods provides fundamental insight into the methylation or heterochromatin has not for conserving Encephalartos woodii, for biology of sex determination. We propose been examined in cycads, lack of identifi- which only a single male clone exists, sex a method for inducing sex change in able sex chromosomesr and occasional change has neverbeen induced" and regenera- cycads and regeneration of roots and induction of sex change via environmen- tion from callus tissue has not been accom- shoots from callus, including discussion plished. tal shocks35suggest that sex determina- of the pitfalls. tion in cycads is due to differential Definitive for The conservation status of the African methylation. evidence Theory of sex demethylation cycad Encephalartos woodii Sander is bleak. determination differential determining This article is predicated on the hypoth- sex should be sought in cycads, especially All extant plants are derived from a single in individuals male clone.' Although vegetative propa- esisthat, inboth females and males, incip- in populations which some gation is readily accomplished from off- ient sex chromosomes arose as a different have undergone sex changes, using tech- sets and more recently from leaf cuttings, methylation pattern on one of two niques such as chromomycin staining," genetic diversity is zero. homologous autosomes and the altered high-performance liquid chromatogra- Osborne' proposed two conservation methylation was of regulatory genetic phy (HPL C),uu't'or bisulphide sequencing strategies for E. woodii: (1) repeated back- elements that control sex hormone pro- of promoters of genes that regulate crossing of the existing male with females duction. Sexual differentiation in plants is hormone levels."tt of a closely related species,and (2) chemi- regulated by sex hormones.s Cytosine nu- We propose applying demethylating cally or environmentally inducing sex cleotides are methylated via replacement compounds to cycad cells in tissue change in tissue-cultured plantlets from of the hydrogen at the C-5 position by a culture, an approach that has resulted in the existing male. The first approach methyl group and are highly heritable.oz sex change in at least one angiosperm resulted in an F1 generation of Methylation causes sex by blocking bind- species.T S-azacytidine, and S-aza- -deoxycytidine Encephalartosnatalensis x E. woodii in the ing sites for enzymes that mediate tran- 2' demethylate CpG 1980s and recently F, seedlings of (E. scription of sex hormones.&tt dinucleotides in most eukaryotes,42-+6 natalensis x E. woodii) x E. woodii (pers. Methylation suppressestranscription in while L-ethionine and dihydroxy- obs.). We focus on sex change because it is several ways. Heterochromatic proteins propyladenine demethylate cytosines in the least well understood of the two bind to methylated cytosines, occupying CpNpG trinucleotides of plants (N can be approaches and has potentially the protein binding sites.t2'13Methylation any nucleotide base).nt greater conservation benefits. alters interactions of histones with Although demethylating agents have There are potentially six benefits to promoter regions by stimulating histone not been used to alter the sex of cycads, inducing sex change in Encephalartos deacetylation.la-16Bound heterochro- we propose that altering methylation of zaoodii.First, our proposed method of sex matic proteins and histones are called tissue-cultured cells of Encephalartos change promotes mutation, thereby heterochromatin. Transcription is also ruoodii could yield female plants. We increasing genetic variation. Second, suppressed because methyl groups are suggest trying this procedure first on rela- there would be increased epigenetic vari- bulky and hydrophobic, thereby chang- tively common species of Encephalartos, ation due to meiotic recombination.3 ing DNA conformation and blocking such as E. natalensis.Tissue-cultured Third, female plants produce mega- binding sites,17sometimes converting the angiosperms have survived exposure to gametophytes and zygotes providing the normally right-handed DNA helix to S-azacytidine, although with lower viabil- cells of choice for cycad tissue culture.n left-handed.18'1e ity and higher mutation rates.nn'n8 aDepartment Dioecious seed plants generally have Measuring methylation levels using of Biology,Arizona State University,Tempe, 4285287-1501, U.S.A. putative sex chromosomes that are indis- HPLC or bisulphide sequencing before bP.O. Box244, Burpengary,Queensland 4505, Australia. tinguishable under a light microscope.2O and after application of demethylating -Author for corresoondence.E-mail: [email protected] Close inspection, howevel sometimes compounds would provide a means for Gommentary SouthAfrican Journal of Science98, March/April2002

determining appropriate amounts of (reviewed in ref. 54). Tissue culture of tion of downstream regions of genes may these chemicals to use in subsequent stem, root, and leaf material from several also affect regulation.ut Our best hope is experiments. The quantity of demethyl- species of Encephalartoshas given rise to for selective methylation and demethyla- ating compounds could be adjusted so callus.tn'tt Cell cultures from zygotes of tion o1, less elegantly, for many random that the resulting diminution of methyl- two species of Encephalarloshave been attempts in the hope that one explant will ation matches that found in cycads of the partially successful: shoots were formed, be successfully regenerated. requisite sex, although we recognize that roots were not, and no plants matured.56 The proposed applications of methyla- 'wrong' the methyl groups might get Recent work using tissue from mega- ting and demethylating agents serve a stripped away.nt gametophytes and zygotes of the Mexi- dual purpose: sex change and regenera- If sex determination in Encephnlartos can cycads Ceratozamiahildae,C. mexicana, tion of roots and shoots. The optimal znoodii is controlled by differential Zamia fischeri, Z. t'urfuracea, Z. pumila and amounts of these chemicals may, how- methylation, then there is an a priori 50% particularly Dioon edule,has been encour- eve(, be different for each function. The chance that a male-to-female change can aging, producing plants that can be targeted loci for methylation may also be be induced via demethylation. The odds grown in soil.a'57'58Tissue culture of different. may be loweq, howeveq, because nearly all megagametophyte and zygote explants sex changes reported in Encephalartos probably leads to more successful regen- Problems following sex change and have been from female-to-male (re- eration of entire plants because global regeneration viewed in ref. 35). If females of E. woodii demethylation and de noao methyla- Even if male-to-female sex change can have more heavily methylated promoters tion may be a prerequisite for proper be induced and viable sexual offspring of sex determining genes than males, sex development, as with most eukaryote formed in E. woodii, there will still be change of the extant male clone can be embryos.5e-6iAn important but as yet no genetic variability in the species. attempted via methylation. Addition of unattained objective is to take leaf, stem, There will be only epigenetic varia- methyl groups to cytosine nucleotides or root explants of Encephalartos and tion due to different methylation pat- may be possible by treating tissue cultures induce shoot and root formation from terns. It is noteworthy that F, and F, back- with S-methyl deoxcytidine. This calli. This is necessary tor E. woodii, in crosses of E. natalensis x E. woodii have approach has been attempted with which neither megagametophytes nor been raised. mammalian cells, where the dose of zy gotespresently exist. Another incentive Methylation-induced mutations, S-methyl deoxcytidine was adjusted so for inducing sex change of the extant male although often deleterious, could be of that half the cell line survived.4e-s1How- E. woodii clone is that megagametophytic conservation benefit for E. woodii because eve4 much less experimental work has and zygotic cells can then be obtained, the surviving mutants increase genetic been done with methylation than with cultured, and treated with demethylating variability of the population.u' Methy- demethylation. or methylating agents. lation creates a tension between in- Ideally, the specific nucleotides respon- Although regeneration of tissue cul- creased genetic variation and decreased sible for regulation of sex hormones and tured E. woodii has not yet been accom- viability.66'67The balance between these sex should be identified, an assessment plished, our theoretical framework of sex two will determine the conservation im- made of whether their regulation is con- determination via methylation may plications of the proposed methylation/ trolled by methylation, and then only provide the method by which differentia- demethylation induced sex change of E. those specific nucleotides should be selec- tion of callus tissue can be induced.6' woodii. The best possible outcome is that tively methylated and/or demethylated Ontogenetic regulation of all plant and viable sex-changed females will emerge in an attempt to change sex and regener- animal tissues is largely controlled by from this protocol carrying a large num- ate roots and shoots from callus. Selective demethylation, although many of the ber of mutations. It is not necessary that methylation can be attempted using methylated genes controlling regulation all these mutations appear on a single methylated single-stranded oligonucleo- have not yet been identified. Until noW female; the genetic variation of the entire tides,52'53a technique that is not yet fully regeneration of tissue-cultured roots population is critical. A successful proto- developed. Until then, attempts should and shoots relied on the application of col for tissue culture of E. ruoodii wlll be made by randomly methylating and plant hormones, which work largely produce many sex-changedmutants tobe demethylating the genome, although by demethylating chromosomal cyto- grown into explants with (collectively) as most such attempts are likely to be unsuc- sines."'3t'63Until we know which genes to much genetic variation as possible, cessful. demethylate and how to demethylate thereby reducing the probability of them selectively, we propose skipping the extinction. Regeneration of roots and shoots intermediary of plant hormones and in- Cycads have maternally inherited from tissue culture callus stead applying S-azacytidine to E. woodii chloroplasts6s (and mitochondria?). The Even if the sex of E. woodii can be callus to induce root and shoot regenera- main disadvantage of backcrossing the changed with demethylating or methyla- tion.6a We also suggest first applying existing male E. woodii with females of ting chemicals, other problems need to be S-methyl deoxcytidine to the callus to other Encephalarfosspecies is that these addressedbefore any protocol canbe con- simulate early embryonic de noao non-Mendelian organelle genomes from sidered effective, such as growing sex- methylation. the male parent are irrevocably lost from changed tissue cultures up to full-sized As with sex change, we expect that all backcrosses.utA preferred option cone-bearing plants, difficulties relating regeneration from callus will usually be would be to backcross an induced female to small effective population size, and unsuccessful because of the randomness E. woodii with males of a closely related evolutionary loss of organelle genomes. of methylation and demethylation when species so that the E. woodii organelle Early attempts to propagate cycads applying compounds like S-azacytidine. genomes are preserved, providing addi- from tissue culture, including Encepha- Although regulation is largely controlled tional impetus for inducing sex change in lartos species, met with limited success by methylation of promoters, methyla- E. woodii. 116 SouthAfrican Journal ol Science98, March/April2O02 Gommentary

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