AGAMOUS-Like 15

Expression and Maintenance of Embryogenic PotentialIs Enhanced through Constitutive Expression of AGAMOUS-Like151Ellen W.Harding,Weining Tang,Karl W.Nichols2,Donna E.Fernandez,and Sharyn E.Perry* Department of Agronomy,University of Kentucky,1405Veterans Drive,Lexington,Kentucky40546–0312 (E.W.H.,W.T.,S.E.P.);and Department of Botany,University of Wisconsin,430Lincoln Drive,Madison, Wisconsin53706–1381(K.W.N.,D.E.F.)The MADS domain protein AGL15(AGAMOUS-Like15)has been found to preferentially accumulate in angiosperm tissues derived from double fertilization(i.e.the embryo,suspensor,and endosperm)and in apomictic,somatic,and microspore embryos.Localization to the nuclei supports a role in gene regulation during this phase of the life cycle.To test whether AGL15is involved in the promotion and maintenance of embryo identity,the embryogenic potential of transgenic plants that constitutively express AGL15was assessed.Expression of AGL15was found to enhance production of secondary embryos from cultured zygotic embryos,and constitutive expression led to long-term maintenance of development in this mode. Ectopic accumulation of AGL15also promoted somatic embryo formation after germination from the shoot apical meristem of seedlings in culture.These results indicate that AGL15is involved in support of development in an embryonic mode.The MADS domain protein AGL15(AGAMOUS-Like15)preferentially accumulates in a wide variety of tissues that are developing in an embryonic mode, suggesting that it may play an important role during this phase of the higher plant life cycle(Heck et al., 1995;Rounsley et al.,1995;Perry et al.,1996,1999). MADS domain proteins are a family of regulatory factors that share an approximately55-to60-amino acid residue domain(the MADS domain)that medi-ates dimer formation and sequence-specific binding to DNA(for review,see Riechmann and Meyerowitz, 1997).The plant MADS box gene family is relatively large,numbering107in Arabidopsis(Parenicova´et al.,2003).Many members of this group have been shown to play key roles in developmental decisions, as demonstrated by loss-of-function mutations re-sulting in homeotic transformation of organ identity (for review,see Riechmann and Meyerowitz,1997). However,it is not unusual for members of this family to have redundant functions,making a double or even triple mutant combination necessary before a phenotype is observed(Kempin et al.,1995;Liljegren et al.,2000;Pelaz et al.,2000).In cases where functional redundancy exists,ec-topic expression studies can be particularly reveal-ing.For example,the petunia(Petunia hybrida)MADS box gene FBP11produces ectopic ovules on floral organ surfaces when constitutively expressed(Co-lombo et al.,1995),substantiating FBP11’s proposed role as an ovule identity gene.MADS box genes expressed in inflorescence and floral meristems have been studied to the greatest extent,but members of the MADS box family are preferentially expressed in other tissues(Heck et al.,1995;Rounsley et al.,1995; Alvarez-Buylla et al.,2000;Burgeff et al.,2002). AGL15is the only MADS box gene reported to date that is preferentially expressed in developing em-bryos(Heck et al.,1995;Rounsley et al.,1995;Fer-nandez et al.,2000).Although other MADS box genes are expressed in embryos,they are also expressed at similar or higher levels at other stages of plant de-velopment.AGL15accumulates in the nuclei of cells in the embryo beginning very early in development (by the eight-cell stage for Arabidopsis)and remains at relatively high levels throughout morphogenesis and into maturation stage(Perry et al.,1996).The level of AGL15decreases during desiccation.AGL15 accumulates in nuclei of a wide variety of tissues developing in an embryonic mode including apomic-tic,somatic,and microspore embryos,embryonic tis-sue derived from the precocious germination system of Brassica napus,and extra-embryonic organs in mu-tants of Arabidopsis(Perry et al.,1999).AGL15is expressed after germination in Arabidopsis,in the vegetative shoot apical meristem(SAM),and bases of lateral organs such as leaves,cauline leaves,and floral organs(Fernandez et al.,2000).The level of1This work was supported by the National Science Foundation (grant no.IBN–9984274to S.E.P.),by the U.S.Department of Agriculture(grant no.96–35304–3699to D.E.F.),by the University of Wisconsin Graduate School,and by the University of Kentucky. This paper(no.03–06–036)is published with the approval of the Director of the Kentucky Agricultural Experiment Station.2Present address:Third Wave Technologies,Inc.,502S.Rosa Road,Madison,WI53719–1256.*Corresponding author;e-mail sperr2@;fax859–257–7125.Article,publication date,and citation information can be foundlower than found in the embryo(Heck et al.,1995; Fernandez et al.,2000).The presence of AGL15in the nuclei of a wide variety of tissue types developing in an embryonic mode suggests that AGL15may be important for this phase of the life cycle.Other transcriptional regula-tors that are preferentially expressed during embry-ogenesis have been shown to promote somatic em-bryo development when ectopically expressed (Lotan et al.,1998;Stone et al.,2001;Boutilier et al., 2002).In this report,evidence that AGL15also acts to promote development in an embryonic mode is pre-sented.Providing AGL15via a transgene that causes constitutive expression supported maintenance of embryo development in culture for extended periods of time,over6years to date.The effect of AGL15was also examined in genetic backgrounds where somatic embryos readily initiate from the SAM(Mordhorst et al.,1998).AGL15enhanced embryo production from the meristems in these mutant backgrounds and in a wild-type background.RESULTSExpression of AGL15Leads to Extension of thePeriod of Embryonic Development in Culture Previous results have demonstrated that in all cases tested,embryos or embryonic tissue from flow-ering plants accumulate AGL15(Perry et al.,1996, 1999).The correlation is suggestive,but does AGL15 directly contribute to either the establishment or maintenance of the embryonic phase?Plants carrying transgenic constructs consisting of the cauliflower mosaic virus(CaMV)35S promoter driving constitu-tive expression of full-length AGL15(Fernandez et al.,2000)were used to address this question.The full-length transgenes,referred to in this report as MIKC constructs,encode the conserved DNA-binding MADS domain,a short linker region(called an I domain),the weakly conserved K domain,and the unique C-terminal domain.Two forms of the MIKC transgene were used to transform Arabidop-sis:One contains the first three introns,whereas the other represents the cDNA(Fernandez et al.,2000). The transgene with the first three introns generallyprovides higher levels of expression of AGL15.Trans-genic plants expressing full-length AGL15via the35S promoter accumulate AGL15in many tissues,includ-ing those in inflorescence meristems,flowers,and flower buds(Perry et al.,1999;Fernandez et al., 2000),leaves(C.Zhu and S.Perry,unpublished data),and developing seedlings and siliques(S. Perry,unpublished data).When developing embryos were removed from the seed at the green bent coty-ledon stage(10–11d after flowering)and cultured on germination media(GM)without exogenous growth regulators,embryonic foci appeared on the cultured embryos within2to3weeks,as shown in Figure1A. Often,the foci appeared at sites where the embryo had been wounded during isolation,but they also formed at the shoot apex and cotyledons and occa-sionally on the hypocotyl.Often,the root apical mer-istem activated,producing an elongated root(Fig. 1A).As shown in Figure1B,over40%of the embryos cultured from MIKC(with introns)plants showed developing embryonic foci within3weeks after the start of culture.A total of530cultured embryos from two independent transgenic lines expressing moder-ate levels of AGL15(described by Fernandez et al., 2000)were scored for secondary embryo develop-ment.Upon subculturing,over80%of the embryos with embryonic foci continued producingembryonic Figure1.Production and maintenance of embryonic culture tissue (ECT).A,Cultured zygotic embryo carrying the MIKC transgene. Developing embryonic foci are indicated with a blue arrowhead.The developing root of the cultured zygotic embryo is indicated with a red arrowhead.Barϭ1mm.B,Percentage of cultured zygotic embryos that produced embryonic foci.Results shown are means and SE of the mean derived from six to eight experiments.Ws,wt, Wild-type zygotic embryos cultured on GM.MIKC,Zygotic embryos from a hemizygous transgenic plant carrying one copy of the35S: AGL15transgene cultured on GM plus kanamycin.MIKC(kan-r)*, Recalculated percentage of the MIKC embryos that produced sec-ondary embryos estimating that only approximately75%of cultured MIKC zygotic embryos carried the transgene.MIK,Zygotic embryos from a transgenic plant with a transgene consisting of the35S pro-moter driving expression of a truncated form of AGL15lacking the C-terminal domain.C,Percentage of the embryonic foci that contin-ued to develop in embryonic mode after6to7weeks in culture. Results shown are means and SE s from at least four experiments.D, Appearance of the ECT from an MIKC embryo after approximately 1.5years in culture.Barϭ2.5mm.Harding et al.tissue for at least6to7weeks(Fig.1C).At the time of submission of this manuscript,one set of cultured embryos has been producing additional embryonic tissue continuously for over6years.Appearance of the cultured tissue after approximately1.5years is shown in Figure1D.Excised wild-type(Was-silewskija[Ws]ecotype)embryos also produced sec-ondary embryos in culture,but at a lower frequency (18.2%of embryos cultured,se3.7%,873embryos total scored,Fig.1B).Over a2-month period,these foci gradually stopped forming embryonic tissue and began forming leaves(indicated by the presence of trichomes;Fig.1C).The MIKC(with introns)transgenic plants used in these experiments were hemizygous for the trans-gene because the homozygous individuals in these lines are sterile.Therefore,the percentage of MIKC embryos that produce secondary embryos is likely to be an underestimate because25%of the cultured embryos arrest for another reason,i.e.they are kana-mycin sensitive.If the efficiency of production of secondary embryos is recalculated based on the total kanamycin-resistant embryos cultured(estimated as 75%of the total),then over one-half(54.8%,se7.5%) of the cultured embryos showed secondary embry-onic tissue development(Fig.1B,MIKC[kan-r,kana-mycin resistant]).Transgenic plants that expressed a truncated form of AGL15lacking the C-terminal do-main were also generated.These plants expressed a form of AGL15consisting of only the DNA-binding MADS domain,the I-linker domain,and the K do-main.Similar constructs with AGAMOUS and Serum Response Factor sequences produce dominant nega-tive effects(Gauthierrouviere et al.,1993;Mizukami et al.,1996;Belaguli et al.,1999).In general,plants carrying the transgene encoding truncated AGL15, referred to as the MIK transgene,showed phenotypic changes that were opposite of those in plants carry-ing the full-length form of AGL15.For example,the MIKC transgenic plants flower later than wild type (Fernandez et al.,2000),whereas the MIK transgenic plants flower earlier than wild type(K.W.Nichols and D.E.Fernandez,unpublished data).However, MIKC and MIK zygotic embryos progressed through morphogenesis at the same rate as wild-type em-bryos(Fernandez et al.,2000;data not shown).Cul-tured embryos carrying the MIK transgene produced embryonic foci at a very low frequency(Fig.1B,2.4%, se0.7%,714embryos total scored from three inde-pendent MIK transgenic lines).No instances of con-tinued embryonic tissue production were observed after2months.What is the evidence that the MIKC culture tissue was developing in an embryonic mode?The organs produced more closely resembled cotyledons than leaves.The margins of the organs were smooth,and no trichomes were present(Fig.1,A and D).The organs also had a simple vasculature,much like that found in cotyledons from seedlings(compare Fig. 2A,cotyledon from a seedling,with Fig.2C,organ Figure2.Characterization of the ECT.A to D,Cleared organs observed using dark-field opticsto examine vasculature.Cotyledon(A)and leaf(B)from a germinated seedling with the MIKCtransgene.C,Cotyledon-like organ from the em-bryonic cultures.D,Leaf-like organ from theembryonic cultures.E and F,Examples of acti-vated root apical meristems(E)and intact em-bryos(F)occasionally produced from embryoniccultures.G and H,Embryonic-like structures pro-duced in the axils(G)or on the surface(H)oflateral organs.I and J,␤-Glucuronidase(GUS)activity in the ECT carrying a soybean(Glycinemax)␤-conglycinin promoter:GUS construct.K,Sudan red7B staining of ECT indicating accumu-lation of triacylglycerols.L,GUS activity in theECT carrying an AGL15promoter:GUS construct.Arrowheads in B,D,J,K,and L indicatetrichomes.Bars in A to D and J to Lϭ1mm;inE,H,and I,barsϭ0.5mm.AGL15Promotes Embryonic Development in Culturefrom the ECT),rather than the complex network of vasculature found in leaves from seedlings(Fig.2B). During the first few months in culture,leaves with serrated margins,trichomes,and a more complex vasculature were also found at low frequency(Fig. 2D).Besides cotyledon-like organs,what appeared to be fused embryonic axes and root meristems,which occasionally produced an elongating root,were often present(Fig.2,E,F,and I).In addition,whole em-bryos were occasionally observed on the surface of the cotyledon-like organs(Fig.2F).New embryonic structures originated from a variety of regions,in-cluding axils of older organs(Fig.2G)and lamina of cotyledon-like organs(Fig.2H).To test whether the tissues with embryonic features expressed seed-specific programs,a transgene con-sisting of the soybean␤-conglycinin promoter driving expression of the reporter gene GUS(Hirai et al., 1994)was introduced into the MIKC transgenic plants.Developing embryos containing both trans-genes were removed from the seeds at the green bent cotyledon stage and placed into culture.Embryonic foci appeared within3weeks and were stained for GUS activity.As shown in Figure2I,GUS activity was associated with all parts of fused embryonic axes and cotyledons.In cases where leaves were also pro-duced(scored by the presence of trichomes),GUS staining was apparent only in the cotyledon-like or-gans that did not have trichomes and not in organs resembling leaves(Fig.2J).Developing foci also stained intensely with the dye Sudan Red7B(fat red, Fig.2K),which specifically stains neutral lipids (Brundrett et al.,1991),such as the triacylglycerols that accumulate during Arabidopsis embryogenesis (Ogas et al.,1997).Little staining was observed in leaf-like organs produced in culture(Fig.2K).If the culture tissues have embryo identity,rela-tively high levels of AGL15promoter activity might be expected.Immature zygotic embryos that contain a transgene consisting of2.5-kb5Ј-and2.5-kb3Ј-regulatory regions of AGL15driving expression of the reporter gene GUS(Fernandez et al.,2000)were excised and cultured.These embryos,like wild-type embryos,will transiently produce new embryonic tissues in culture.As shown in Figure2L,GUS activ-ity was detected in these tissues,but not in leaf-like organs produced in culture,indicating that AGL15 promoter activity was associated with development in an embryonic mode.The mode of development of the culture tissue was further investigated at a molecular level.Cultures that had been maintained for extended periods(over 5years)were used for these experiments.Reverse transcription(RT)-PCR and gene-specific oligonucle-otide primers were used to compare expression of several embryo markers in the culture tissue with expression in seedlings and siliques.LEC1(LEAFY COTYLEDON1)and LEC2(LEAFY COTYLEDON2) both encode transcriptional regulators that are ex-pressed primarily during embryonic development (Lotan et al.,1998;Stone et al.,2001).As shown in Figure3,both LEC1and LEC2were expressed in the ECT.As expected,expression was also observed in silique tissue,particularly at earlier stages of devel-opment,but not in wild-type seedlings(Fig.3)or in MIKC seedlings(data not shown).In addition,one of the Arabidopsis12S cruciferin seed storage protein genes(AtCRU3)was expressed in the culture tissue and in older stage siliques but not in seedlings(Fig.3).The control,␤-2-tubulin(TUB2;Snustad et al., 1992),was expressed in all tissues.Expression of AGL15Stimulates Production of Embryos from SAMsA group of Arabidopsis mutants with enlarged SAMs showed enhanced production of embryos from the SAM when seedlings were grown in liquid cul-ture media containing2,4-dichlorophenoxyacetic acid(2,4-D;Mordhorst et al.,1998;von Reckling-hausen et al.,2000).This group of mutants includes amp1(altered meristem program;allelic to pt1,cop2,and hpt),clavata1,and clavata3.Increased size of the SAM is correlated with increased frequency of somatic embryo development in this system(Mordhorst et al.,1998).In the amp1background,where the SAM is approximately twice the size of wild type,30%of the seedlings produce secondary embryos(Mordhorst et al.,1998).This finding was intriguing because the very young SAM is one of two places where AGL15 can be detected immunologically after germination in Arabidopsis(Fig.4;Fernandez et al.,2000).Figure 4A shows immunohistochemical localization of AGL15in the SAM of a4-d-old seedling.AGL15was detected in the nuclei of the cells in the SAM and in the young leaf.However,by6d after germination, nuclear localized AGL15could no longer be detected in the SAM and leaves(Fig.4B).Figure3.Expression of embryo-specific genes in the ECT.Gene-specific primers and RT-PCR were used to examine expression of LEAFY COTYLEDON1,LEAFY COTYLEDON2,and a12S cruciferin gene in the ECT and in Ws seedlings and staged silique tissue. Products were analyzed on an agarose gel.Tubulin(TUB2)served as a control.Harding et al.To test whether altering AGL15levels affects pro-duction of somatic embryos in the amp1background,the MIKC and MIK transgenes were introduced into the amp1mutant background via genetic crosses.Ho-mozygous amp1progeny were identified in the F 2generation by screening for seedlings with three cot-yledons,and the transgene was selected by kanamy-cin resistance.Seed was germinated in liquid culture with 2,4-D as described by Mordhorst et al.(1998),with kanamycin present to eliminate any seedlings lacking the MIKC or MIK transgenes.amp1homozy-gous mutants were germinated in media without kanamycin.As shown in Figure 5A,under our culture condi-tions,only 2%(se 0.9%,n ϭ5experiments,891total seedlings scored)of the amp1homozygous seedlings produced new embryos at the SAM.However,when the MIKC transgene was present,27.4%of the amp1seedlings produced new structures at the SAM as shown in Figure 5,B and C (se 2.0%,n ϭ5experi-ments,1,265total kanamycin-resistant seedlings scored).The new growth lacked trichomes and ap-peared to be similar to the shoot meristem embryos reported previously (Mordhorst et al.,1998).Approx-imately another one-third had green smooth meris-tem development (data not shown)that might corre-spond to the embryogenic callus reported by von Recklinghausen et al.(2000).amp1homozygous seed-lings carrying the MIK transgene did not produce embryo-like structures in the culture system (Fig.5A,0.8%,se 0.2%,n ϭ5experiments,1,857total kanamycin-resistant seedlings scored).Typically,the meristem did not develop in these seedlings (Fig.5,D and E).The seedlings callused and looked much like the amp1seedlings,except that the tips of the cotyle-dons often remained green.The MIKC transgene was also able to enhance pro-duction of embryos from the SAM in the absence of the amp1mutation.Although the frequency of pro-duction was somewhat variable,the MIKC seedlings produced meristem-derived embryos at a higher fre-quency on average than Ws wild-type or MIK seed-lings.For MIKC,18.7%(se 8.9%)of seedlings pro-duced embryo structures,compared with 2.8%(se 1.9%)for Ws wild-type seedlings and 1.6%(se 0.8%)for MIK seedlings (Fig.5A).All numbers are means from at least four independent experiments scoring a total of 810to 1,114seedlings.The seedlings bearing embryonic structures were assessed further to confirm that development was occurring in an embryonic mode.MIKC seedlings that produced new structures at the SAM were sep-arated from MIKC seedlings that did not,and RNA was extracted from both sets of seedlings for RT-PCR.As shown in Figure 6A,liquid culture grown seedlings with SAM development showed increased accumulation of LEC1,LEC2,and AtCRU3mRNA relative to seedlings lacking SAM development.Wild-type and MIKC seedlings grown on GM plates did not produce new embryos at the SAM,and none of the embryo markers accumulated in these tissues (Fig.3;data not shown).To examine the effect of AGL15accumulation on early stages of somatic embryo development,expres-sion of AtSERK1was assessed shortly after culture initiation (7d).AtSERK1is expressed in tissues with increased embryogenic competence,and ectopic ex-pression of this gene enhances efficiency of somatic embryo initiation in culture (Hecht et al.,2001).As shown in Figure 6B,amp1seedlings containing the MIKC transgene showed increased accumulation of AtSERK1mRNA compared with amp1seedlings with or without the MIK transgene.AtSERK1was also expressed in the ECT derived from cultured MIKC zygotic embryos (Fig.6B).Because Mordhorst et al.(1998)found that there is a correlation between SAM size and ability to pro-duce SAM somatic embryos in culture,the meristems of MIKC and MIK in wild-type and amp1mutant backgrounds and Ws and amp1were examined.Seed-lings grown on GM plates and mature embryos were cleared in Hoyer’s solution and examined using dif-ferential interference contrast (DIC)optics.As shown in Figure 7,A and B,the meristem of an MIKC seedling was similar in size to that of a wild type (Ws seedling).As shown in Figure 7C,when images of meristems were measured,no significant differences were observed between Ws,MIKC and MIK seed-lings.amp1,as expected,had a larger meristem,but the addition of the AGL15transgenes did not increase meristem size over that found for amp1alone.Like-wise,comparative meristem sizes in mature embryos were similar (data not shown).Differences at the meristem became apparent shortly after initiation of culture in media containing 2,4-D.By 5d in culture,the SAM of amp1with MIKC seedlings was enlarged compared with amp1alone (Fig.8,A and B,respectively).A “collar”of tissue,as described by von Recklinghausen et al.(2000),per-haps derived from the outer SAM,was clearly appar-ent in seedlings carrying the MIKC transgene,aswasFigure 4.Accumulation of AGL15in the shoot apex of seedlings.A,Four-day-old Ws wild-type seedling.B,Six-day-old Ws wild-type seedling.LP,Leaf primordia;St,stipule primordia.Bars ϭ50␮m.AGL15Promotes Embryonic Development in Culturedevelopment at the central region of the SAM.Cell proliferation at the SAM continued,and,by 10d in culture,the SAM of amp1with MIKC was smooth,enlarged,and green in color (data not shown).By 7d after culture,the SAM of amp1seedlings carrying theMIKC transgene was enlarged and showed cytoplas-mically dense protrusions in the central part of the meristem,as revealed by staining of tissue sections (arrowhead,Fig.8C).In amp1,only callus develop-ment was obvious at the meristem (data not shown).The meristem region of amp1seedlings with the MIK transgene had a relatively flat meristem (Fig.8D)or a dome near the apex that did not have any cytoplas-mically dense protrusions (data not shown).DISCUSSIONIn this report,results are presented demonstrating that continuous expression of AGL15driven by the CaMV 35S promoter (MIKC construct)enhances first the production of secondary embryonic tissue from cultured zygotic embryos and then supports the long-term maintenance of the embryonicphase.Figure 5.Embryonic development from the shoot apex of seedlings in culture.A,Percentage of seedlings that showed embryonic devel-opment at the SAM when germinated in liquid media containing 2,4-D.Results shown are means and SE s of four to six independent experiments.amp1,amp1mutant without any transgenes.amp1,MIKC ,amp1mutant containing a transgene consisting of the 35S promoter driving expression of full-length AGL15.amp1,MIK ,amp1mutant containing a transgene consisting of the 35S promoter driving expression of a truncated form of AGL15lacking the C-terminal domain.WS ,wt ,Wild-type Arabidopsis seedlings.MIKC and MIK ,Transgenic Ws seedlings expressing the different forms of AGL15.B to E,SAM development in seedlings germinated in liquid culture containing 2,4-D.B and C,amp1mutant carrying a transgene con-sisting of the 35S promoter driving expression of full-length AGL15.D and E,amp1mutant carrying a transgene consisting of the 35S promoter driving expression of a truncated form of AGL15lacking the C-terminal domain.Blue arrowheads indicate development of embryo-like tissue at the shoot apex;red arrowheads indicate lack of development at the shoot apex.Bar ϭ1mm.Figure 6.Expression of embryo-specific genes in seedlings germi-nated in liquid culture with 2,4-D.A,MIKC seedlings with SAM development (ϩ)were separated from MIKC seedlings lacking SAM development (Ϫ)after 21d in culture and assessed for expression of embryo-specific genes.Gene-specific primers and RT-PCR were used to assess expression of LEAFY COTYLEDON1,LEAFY COTYLE-DON2,and a 12S cruciferin gene in the liquid culture seedlings and in staged 11-to 12-d-old silique tissue.B,amp1liquid culture seedlings with the MIKC or MIK transgene or lacking any transgene were assessed for expression of AtSERK17d after culture.ECT was also assessed for expression of AtSERK1.Tubulin (TUB2)served as a control.RT-PCR products were analyzed on an agarose gel.Harding et al.There are several published reports of induction of Arabidopsis somatic embryo cultures from immature zygotic embryos (Sangwan et al.,1992;Wu et al.,1992;Pillon et al.,1996;Luo and Koop,1997;Ikeda-Iwai et al.,2002)or from leaf protoplasts (O’Neill and Mathias,1993;Luo and Koop,1997).These cultures require the addition of exogenous auxins and,in some cases,cytokinins.Auxin is thought to induce embryogenic competence,but how this occurs is not yet understood (Harada et al.,1998).The synthetic auxin 2,4-D has been found to promote recurrent embryony on the surface of cultured zygotic em-bryos,perhaps by generating cells that recapitulate the current developmental program.However,2,4-D leads to dedifferentiation and formation of embryo-genic callus in the case of the somatic embryos de-rived from the zygotic embryos (Pillon et al.,1996).In the absence of a sufficiently high level of 2,4-D,non-embryogenic callus results (Pillon et al.,1996).Nota-bly,development of secondary embryos in our cul-ture system occurred in the absence of exogenous growth regulators,and continued embryonic devel-opment with MIKC tissues was sustained for exten-sive periods of time.Whereas both wild-type and MIKC transgenic de-veloping embryos gave rise to secondary embryos,MIKC transgenic embryos produced embryonic structures at a significantly higher frequency.Over one-half of cultured MIKC embryos produced sec-ondary embryos;however,only 18%of cultured wild-type embryos showed this development.Trans-genic embryos expressing a truncated form of AGL15lacking the C-terminal domain (MIK construct)were unable to efficiently promote secondary embryo de-velopment.This type of truncated construct has been found to have dominant negative effects for other MADS domain proteins (Gauthierrouviere et al.,1993;Mizukami et al.,1996;Belaguli et al.,1999),possibly by blocking endogenous protein activity ei-ther by formation of nonfunctional heterodimers or occupation of DNA-binding sites.Although the majority of MIKC embryos that be-gan embryonic development continued producing embryonic tissue (cotyledons)over extended periods of time,wild-type cultures shortly switched to gen-erating vegetative tissues (leaves).The wild-type em-bryos contain endogenous AGL15at the time of ex-cision (Perry et al.,1996),which may contribute to competency to promote development ofsecondaryFigure 8.Appearance of the SAM of seedlings in liquid culture containing 2,4-D.A and B,Appearance of the apical region of amp1seedlings with the MIKC transgene (A)or amp1seedlings lacking a transgene (B)after 5d in culture.C and D,Tissue sections (7␮m)stained with toluidine blue O of amp1seedlings containing either the MIKC (C)or the MIK (D)transgene after 7d in culture.Arrows in A and C,Ring of tissue proliferation;black arrowheads,proliferation at the central zone of the SAM.White arrowheads in B and D,Little to no proliferation at the meristem.Bar in A and B ϭ500␮m;bar in C and D ϭ200␮m.Figure 7.Relative size of the SAM of seedlings with and without MIKC and MIK transgenes in wild-type and amp1mutant back-grounds.A and B,Wild-type and MIKC seedlings were cleared and examined under DIC optics.A,Ws wild-type seedling;B,seedling containing a transgene consisting of the 35S promoter driving ex-pression of full-length AGL15.C,Average size (diameter)of the SAM measured in millimeters from pictures of seedlings that were at a final magnification of 125ϫ.Bar ϭmean size with SE ;the number above the bar indicates the number of seedlings photographed and mea-sured.Bar in A and B ϭ50␮m.AGL15Promotes Embryonic Development in Culture。