Leaves of 47,000 Lotus japonicus plants were screened using a butanol:HCl histochemical test to select “gain of function” mutants. These plants were progeny from L. japonicus lines which were transformed with T-DNA constructs containing either the maize Ds or Ac transposon (Thykjaer et al., 1995). Among 21 putative leaf tannin mutants, five (tan1-5) were characterized for synthesis of condensed tannins, leucocyanidin reductase activity and the presence of Ac and the selectable marker gene, nptII . A range of leaf tannin content among other Lotus species was also characterized

Auser, P

Coulman, B

Gruber, M Y

Muir, A D

Skadhauge, B

Stougaard, J

Thomsen, K K

Wettstein, D von

English

University of Kentucky

University of Kentucky UKnowledge IGC Proceedings (1997-2023) XVIII IGC (1997) Manitoba & Saskatchewan Induction of Mutants with Ectopic Expression of Condensed Tannins M Y. Gruber Agriculture & Agrifood Canada B Skadhauge Carlsberg Research Centre P Auser Agriculture & Agrifood Canada A D. Muir Carlsberg Research Centre K K. Thomsen University of Aarhus See next page for additional authors Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Agricultural Science Commons, Agronomy and Crop Sciences Commons, Plant Biology Commons, Plant Pathology Commons, Soil Science Commons, and the Weed Science Commons This document is available at https://uknowledge.uky.edu/igc/1997/session8/3 <>Grasslands 2000</> This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in IGC Proceedings (1997-2023) by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Presenter Information M Y. Gruber, B Skadhauge, P Auser, A D. Muir, K K. Thomsen, J Stougaard, B Coulman, and D von Wettstein This event is available at UKnowledge: https://uknowledge.uky.edu/igc/1997/session8/3 Session 8 - Tannins: Plant Breeding and Animal Effects 8-31ABSTRACTLeaves of 47,000 Lotus japonicus plants were screened using abutanol:HCl histochemical test to select “gain of function” mutants. Theseplants were progeny from L. japonicus lines which were transformedwith T-DNA constructs containing either the maize Ds or Ac transposon(Thykjaer et al., 1995). Among 21 putative leaf tannin mutants, five(tan1-5) were characterized for synthesis of condensed tannins,leucocyanidin reductase activity and the presence of Ac and the selectablemarker gene, nptII . A range of leaf tannin content among other Lotusspecies was also characterized.KEYWORDScondensed tannins, Lotus japonicus, mutants, transgenic plants, T-DNAand Tn taggingINTRODUCTIONCondensed tannins are important plant secondary metabolites with foamreducing, dietary protein protecting, insect resistant and antioxidantproperties. As such, their synthesis in relevant plant organs is of particularinterest for the industrial and agricultural use of legume crop plants.Particularly, it is an important breeding aim to express the condensedtannin pathway ectopically in leaves of the world’s major forage legumes,alfalfa and white clover, as a means of eliminating pasture bloat andimproving rumen protein-bypass and insect resistance. In an exploratoryinvestigation, we have developed a system to obtain flower and leaftannin mutants using Lotus japonicus transformed with maizetransposable elements and a histochemical test for condensed tannins.The characterization of several mutants with leaf tannins is presented.MATERIALS AND METHODSScreening for leaf tannin mutants: Leaves of 32 species of Lotus wereassayed for condensed tannin content using a modifiedbutanol:HCl:PVPP assay (Skadhauge et al., 1997) to determine candidatespecies for mutant development. Floral, leaf, stem and root tissues ofthe selected species, L. japonicus, were also tested for tannin content. L.japonicus lines were transformed with Agrobacterium tumefaciens binaryvectors and selected as T-DNA lines (containing a disabled maizetransposable element) or Ac excision lines (containing an active maizeAc transposable element) (Thykjaer et al., 1995). Approximately 47,000self pollinated progeny seeds from 1230 independent transformed lineswere sown in flats and grown in greenhouses. A subset of seedlings wascultivated under field conditions as outlined below. Seed grown formutant selection from T-DNA insertion lines originated from the 2ndgeneration of self-pollinated transgenic plants (T2). Seed from Ac linesarose from T3 and T4 transgenic plants selected for transposon excision(Thykjaer et al., 1995). Two leaves per progeny from 7-10 week oldplants and two flag petals per progeny from 3.5 month old plants weretested histochemically for variation in tannin content (Skadhauge et al.,1997). Lines were also assessed for variation in morphology. It wasmost efficient to test leaves for tannins in the greenhouse prior totransplanting, but f lowers were easily collected from field plots. Variantswhich gave similar results repeatedly were designated mutants andcultivated in the greenhouse. Subsequently, several tannin mutants weretested genetically and biochemically as follows. Monomeric and dimericflavonoids were isolated from mutants as an ethyl acetate fractionID NO. 1948INDUCTION OF MUTANTS WITH ECTOPIC EXPRESSION OF CONDENSEDTANNINS1M.Y. Gruber, 2B. Skadhauge, 1P. Auser, 1A.D. Muir, 2K.K. Thomsen,3J. Stougaard, 1B. Coulman and 4D. vonWettstein1Agriculture & Agrifood Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada2Carlsberg Research Centre, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Denmark3Dept. of Molecular and Structural Biology, University of Aarhus, Aarhus C8000, Denmark4Dept. of Crop and Soil Science, Washington State University, Pullman, WA 99164-6420, USAAAFC publication 1253according to Koupai-Abyazani et al. (1992), separated by HPLC on auBondapak phenyl column (30 cm x 3.9 cm, Waters Assoc.) (2 to 10%v/v linear acetic acid gradient, 60 min, 2 ml.min-1 flow rate) and detectedby UV absorption. Condensed tannins were purified from the remainingaqueous fraction by elution from a Sephadex LH20 column (15 cm x 4cm, Pharmacia) using 75% acetone (Koupai-Abyazani et al., 1992), andtheir molecular weights determined according to Koupai-Abyazani etal. (1993). Southern blot hybridization with Ac and nptII genes wasperformed according to standard molecular biology protocols (Sambrooket al., 1989). Condensed tannin content was determined for self pollinatedS1 and S2 generations of selected mutants by the butanol:HCL:PVPPmethod above. A transgenic field trial (#94-ACS1-LOJ01-SK01-01) wasundertaken at the AAFC Saskatoon Research Centre farm.Approximately 6,500 seedlings from 76 independently transformed lineswere manually transplanted to the field, two months after germination.The row/plot design was equally useful for manual harvesting of seedfrom individual plants, manual harvesting of bulked seed from each lineor mechanical harvesting of bulked seed. A misting system was devisedto reduce excessive seed shattering under dry prairie conditions. A blackpolyethylene woven ground cloth protected the plot from weed growthand aided in the harvest of valuable seed from any shattered pods. Eachtransgenic line was covered by a translucent row crop cover cloth toprotect against insect and wind damage and to prevent cross pollinationamong the 76 transgenic lines and with other Lotus species planted onthe farm. Plant lines were monitored for insect damage and growth.Since L. japonicus flowers indeterminantly, ripe (brown) pods weremanually harvested in several rounds from 4-5 month old field plants,then stored in paper bags for up to 5 months at temperatures rangingfrom 10-20o C until seed was cleaned. At the end of the season (September30, 1994), the forage from each plant was harvested manually and placedinto paper bags. Plot condition, the number of nonflowering plants andtop growth, (forage length and weight of 15 plants) were recorded foreach line. All remaining green pods were collected manually from thecut forage, stored in a cool forage shed in paper bags and dried at 50o Cin a forced air forage dryer. Seeds were removed manually from drypods, weighed, and then packaged as green or ripe seeds (from brownpods) into paper envelopes, and stored at 4o C. Seed weight andgermination rate were compared among 10 lines. Seed was germinatedby treatment for two minutes in conc. H2SO4 to promote imbibition,followed by several washes in sterile water placement on damp filterpaper for 5 days.RESULTS AND DISCUSSIONA range of leaf tannin concentrations were present among the 32 Lotusspecies tested (Table 1). Diploid L. japonicus was identified as a goodcandidate for development of mutant legume populations with alteredtannin content. Flowers of this species contained large amounts of tannin;levels in leaves were undetectable (Table 2). Subsequently, progeny linesof L. japonicus transformed with either an active Ac transposon or adisabled transposon (Thykjaer et al., 1995) were screened for variationin tannin content and growth traits. The design and nature of the fieldtrial enabled us to recover and sort all seed from ripe, unripe and shatteredpods with reduced risk of plant loss through early freezing damage, animportant consideration when undertaking a field increase of suchSession 8 - Tannins: Plant Breeding and Animal Effects 8-32Table 1Leaf Tannin Variation Among Lotus SpeciesLotus Species Name %FW Tannin (+/- SD)L. purshianus (Benth.) Clem.& Clem. 4.1 (0.3)L. palestinus (Boiss.) Bornum 0.5 (0.3)L. palustris Willd 3.4 (0.7)L. arabicus L. 0.4 (0.3)L. angustissimus 3.2 (0.7)L. peregrinus L. 0.4 (0.2)L. corniculatus var. Hirsutus 2.5 (0.3)L. decumbens 0.4 (0.2)L. parviflorus Desf. 2.4 (0.7)L. campylocladus Webb & Berth 0.3 (0.1)L. hispidus 2.3 (0.5)L. jacobeus L. 0.3 (0.2)L. creticus L. 1.9 (0.5)L. hirsutus 0.2 (0.2)L. scoparius (Nutt.) Ottley 1.5 (0.7)L. cytisoides 0.2 (0.2)L. caucasicus Kupr. 1.2 (0.8)L. krylovii Schischk and Serg. 0.1 (0.2)L. discolor 1.0 (0.6)L. schoelleri Schweinf. 0.0 (0.0)L. macroccanus Ball 1.0 (0.9)L. borbasii Ujhelyi 0.0 (0.0)L. conjugatus L. 0.9 (0.3)L. burttii Sz. Borsos 0.0 (0.0)L. ornithododiodes L. 0.8 (0.3)L. cruentus Court. 0.0 (0.0)L. sulphareus 0.7 (0.4)L. edulis L. 0.0 (0.0)L. siliquosis L. 0.6 (0.5)L. filicaulis Dur. 0.0 (0.0)L. carmeli Boiss. 0.6 (0.02)L. japonicus (Regel) Larsen 0.0 (0.0)valuable germplasm in Canada. Although seed was bulked from eachline, the design could be used easily to collect seed from individualplants. Germination rates of seed from fully ripe pods were consistently>80% (measured on 10 lines). When hard seeds which did not imbibewere subsequently scarified with a scalpel and incubated for a further 5days, germination rates rose to >95% (measured on 3 lines). Seedlingsgrown from the ripened field seed were very vigorous. Seed from greenpods germinated inconsistently at a frequency ranging from 3-30 %(measured on 8 lines). Seed stocks are maintained at the University ofAarhus. Several plants in one line had a dried brown curled leaf at node4. Three lines had dwarf plants. In total, 23 plants had detectable levelsof leaf tannin and were designated tan mutants. One line had a plantwith a pleiotrophic mutation leading to very small flowers, hairy leavesand leaf tannins (tan-1). With the exception of tan- 1, all tan mutantsresembled untransformed L. japonicus morphology. Flower variantswithout tannins were not observed under either greenhouse or fieldscreening conditions. Condensed tannin levels in field-grown flowerswere generally much higher than in greenhouse grown, but the levelswere more variable. Conditions across the field plot varied and wereless easy to control compared with greenhouse growth. Hence, selectionof variants with subtle genetic differences in tannin expression wasimpossible in the field trial. Five mutants (tan1-5) were chosen for furtheranalysis (Table 2). These mutants all have the requisite T- DNA/transposon insertion elements. Tan-1 was the only one selected after Acexcision selection, i.e. from the T4 generation; the rest were selectedfrom lines with disabled transposons from the T2 generation (Table 1).Leucocyanidin reductase (LCR), the first enzyme in the flavonoidpathway uniquely committed to condensed tannin biosynthesis, waseasily detected in the leaves of the five mutant genotypes. LCR activitycorrelates well with the level of leaf tannin. The leaf tannin content oftan1 was uaually stable in plants of two subsequent generations (S1 andS2) after self pollination, although the tannin phenotype had reverted in7% of the S1 plants (Table 1). Phenotype reversion occurs on average in10-15% of progeny in L. japonicus Ac excision lines (Thykaer et al.,1995). In contrast, leaf tannins could only be detected in 4-8% of the S1progeny of tan-2 to tan-5. This low heritability improved slightly in theS2 generation, such that 7-11% had detectable leaf tannins. The maskingof the mutant phenotype in tan2-5 was not due to transposition of thestable Ds element through the activity of a native L. japonicus transposase(data not shown). These plants are now being backcrossed tountransformed L. japonicus to try and improve tan2-5 expression and tostabilize the mutations. The flavonoid content and morphology of tan-1was compared with L. angistissimus, a species with morphology unlikeL. japonicus. Tan-1 and L. angustissimus look very similar with smallhairy leaves, short internodes and small flowers. Untransformed L.japonicus has large flowers, large smooth leaves and longer internodes.The leaf flavonoid profile of tan-1 and its S2 progeny appeared morelike L. angustissimus than like untransformed L. japonicus. Preliminarymolecular weight determinations on purified condensed tannin polymerfractions from tan-1 and L. angustissimus were also similar. We considertan-1 to be a pleitrophic mutant, since both morphology and tannincontent were altered. At this point, it is not clear whether the tan1-5phenotypes arose directly from transgene insertion events or,alternatively, from some other mutation event. For example, recentobservations of the generation and disappearance of the tan-1 phenotypein untransformed L. japonicus and untransformed L. angistissimus(Gruber, unpublished) lend support to a mechanism involving a naturaltransposon system in Lotus species. However, the suppression of thetan2-5 phenotype, followed by improvement of expression duringsubsequent generations, parallels what has been observed with transgenesin Arabidopsis thaliani (Mittelsten Scheid et al., 1991; Dehio and Schell,1994).REFERENCESDehio, C. and J. Schell. 1994. Identification of plant genetic lociinvolved in a posttranscriptional mechanism for meiotically reversibletransgene silencing. PNAS (USA) 91: 5538-5542.Koupai-Abyazani, M.R., J. McCallum and B.A. Bohm. 1992.Identification of the constituent flavanoid units in sainfoinproanthocyanidins by reverse-phase liquid chromatography. J. Chromat.594: 117-123.Koupai-Abyazani, M.R., A.D. Muir, B.A. Bohm, G.H.N. Towers andM.Y. Gruber. 1993. The proanthocyanidin polymers in some speciesof Onobrychis. Phytochemistry 34: 113-117.Millelsten Scheid, O., J. Paszkowski and I. Potrykus. 1991. Reversibleinactivation of a transgene in Arabidopsis thaliana. Mol. Gen. Genet.228: 104-112.Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning.A laboratory manual. Cold Spring Harbour Laboratory Press, New York.Skadhauge, B., M.Y. Gruber, K.K. Thomsen and D. von Wettstein.1997. Leucocyanidin reductase activity and accumulation ofproanthocyanidins in developing legume tissues. Am. J. Botany. In press.Thykaer, T., J. Stiller, K. Handberg, J. Jones and J. Stougaard. 1995.The maize transposable element Ac is mobile in the legume Lotusjaponicus. Plant Mol. Biol 27: 981-993.Table 2Leaf Tannin Mutant Biochemistry and Genetics (tan1-5)S0 Genotype Tannin Content in S0 Generation LCR Activity           Segregating Plants(screening (ug.mg-1FW) (leaves)             with Leaf Tannin (%)generation)  Flowers Leaves Stems (DPM.10-1.mg-1FW.h-1) S1 S2Untransformed 188 N.D. 6 N.D. - -tan-1 (T4) 137 118 5 16.8 93.2 100.0tan-2 (T2) 222 59 9 9.3 8.5 11.3tan-3 (T2) 172 37 7 5.1 6.8 9.8tan-4 (T2) 183 25 5 1.6 3.7 7.3tan-5 (T2) 196 12 8 2.1 3.9 6.9

Induction of Mutants with Ectopic Expression of Condensed Tannins

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Induction of Mutants with Ectopic Expression of Condensed Tannins

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