6
Transgenic Research 2, 330-335 (1993) Introduction of hygromycin resistance in Lotus spp. through Agrobacterium rhizogenes transformation FRANCESCO DAMIANI*, ELENA NENZ, FRANCESCO PAOLOCCI andSERGIO ARCIONI Istituto Miglioramento Genetico delle Piante Foraggere CNR, Via Madonna A lta, 130 06128 Perugia, Italy (Fax: +39 75 500 5228) Received 15 December 1992; accepted i8 January 1993 The species Lotus corniculatus and L. tenuis were transformed with an Agrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation. Progeny analysis of the hygromycin resistance indicated this to be a single Mendelian trait in test plants. The transformed plants will be utilized in somatic hybridization experiments with lucerne for producing non-bloating genotypes with condensed tannins in leaves. Keywords: Lotus; Agrobacterium rhizogenes; transformation; hygromycin resistance; tannins Introduction The genus Lotus includes important forage species. The most important, L. corniculatus, gives a bloat-safe forage, owing to the presence of condensed tannins in the leaves. These compounds react with soluble plant and animal proteins, forming insoluble complexes (Jones and Mangan, 1977) which prevent protein degradation in the rumen and hamper the formation of bloating products. Tannins are present in the leaves of some species of this genus ( L. corniculatus, L. pedunculatus and L. angustis- simus); in other species they are present in the nervature (L. tenuis). The presence of condensed tannins is probably an adaptive character, as observed by Ross and Jones (1983) who found polymorphism in accessions of L. corniculatus collected from different geographical areas. The presence of this wide variability and the possibility of intercrossing or hybridizing through protoplast fusion provide a great opportunity to further understanding the genetical control of the synthesis of condensed tannins. The isolation of the genes responsible for the presence of tannins would permit the transfer of this trait to some other more important and widespread forage legumes such as Medicago sativa (lucerne or alfalfa). At present, this goal is being pursued through somatic hybridization *To whom correspondence should be addressed. 0962-8819 1993 Chapman & Hall between lucerne and a non-bloating Lotus species. The availability in the latter species of modified genotypes carrying marker traits could be utilized both in somatic hybridization programmes (Aziz et al., 1990) and for keeping track of particular genotypic combinations obtained from the hybridization of species expressing and not expressing this trait. Tissue culture and plant regeneration have been accomplished in various species of Lotus (Arcioni et aL., 1988; Piccirilli et al., 1988; Pupilli et al., 1990). Trans- formed plants have already been obtained in L. cornicu- latus (Jensen et al., 1986; Petit et al., 1987), but less information is available for the other species. This paper deals with the transformation of the species Lotus (L. tenuis and L. corniculatus) through an A. rhizogenes binary vector carrying the gene for hygromycin resistance and with the transmission of the introgressed trait to the progeny. Materials and methods Seeds of Lotus tenuis, an ecotype from central Italy, and Lotus corniculatus cv. 'Franco' were surface sterilized with a mixture of 0.1% (w/v) mercuric chloride plus 0.1% (w/ v) sodium lauryl sulphate (20 min), subsequently with 20% (v/v) 'Linda clor' (commercial bleach containing 6% sodium hypochlorite) for 20 min, then rinsed 5 times with

Introduction of hygromycin resistance inLotus spp. throughAgrobacterium rhizogenes transformation

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Page 1: Introduction of hygromycin resistance inLotus spp. throughAgrobacterium rhizogenes transformation

Transgenic Research 2, 330-335 (1993)

Introduction of hygromycin resistance in Lotus spp. through Agrobacterium rhizogenes transformation

F R A N C E S C O D A M I A N I * , E L E N A N E N Z , F R A N C E S C O P A O L O C C I a n d S E R G I O A R C I O N I

Istituto Miglioramento Genetico delle Piante Foraggere CNR, Via Madonna A lta, 130 06128 Perugia, Italy (Fax: +39 75 500 5228)

Received 15 December 1992; accepted i8 January 1993

The species Lotus corniculatus and L. tenuis were transformed with an Agrobacterium rhizogenes binary vector, conferring resistance to the antibiotic hygromycin. Transgenic plants recovered from both species were tested for the ability of leaf-derived calluses to grow in a hygromycin-supplemented medium. Molecular analysis showed the integration of the Ri T-DNA and of the gene for hygromycin resistance, with a high frequency of co-transformation. Progeny analysis of the hygromycin resistance indicated this to be a single Mendelian trait in test plants. The transformed plants will be utilized in somatic hybridization experiments with lucerne for producing non-bloating genotypes with condensed tannins in leaves.

Keywords: Lotus; Agrobacterium rhizogenes; transformation; hygromycin resistance; tannins

Introduction

The genus Lotus includes important forage species. The most important, L. corniculatus, gives a bloat-safe forage, owing to the presence of condensed tannins in the leaves. These compounds react with soluble plant and animal proteins, forming insoluble complexes (Jones and Mangan, 1977) which prevent protein degradation in t h e rumen and hamper the formation of bloating products.

Tannins are present in the leaves of some species of this genus ( L. corniculatus, L. pedunculatus and L. angustis- simus); in other species they are present in the nervature (L. tenuis). The presence of condensed tannins is probably an adaptive character, as observed by Ross and Jones (1983) who found polymorphism in accessions of L. corniculatus collected from different geographical areas. The presence of this wide variability and the possibility of intercrossing or hybridizing through protoplast fusion provide a great opportunity to further understanding the genetical control of the synthesis of condensed tannins. The isolation of the genes responsible for the presence of tannins would permit the transfer of this trait to some other more important and widespread forage legumes such as Medicago sativa (lucerne or alfalfa). At present, this goal is being pursued through somatic hybridization

*To whom correspondence should be addressed.

0962-8819 �9 1993 Chapman & Hall

between lucerne and a non-bloating Lotus species. The availability in the latter species of modified genotypes carrying marker traits could be utilized both in somatic hybridization programmes (Aziz et al., 1990) and for keeping track of particular genotypic combinations obtained from the hybridization of species expressing and not expressing this trait.

Tissue culture and plant regeneration have been accomplished in various species of Lotus (Arcioni et aL., 1988; Piccirilli et al., 1988; Pupilli et al., 1990). Trans- formed plants have already been obtained in L. cornicu- latus (Jensen et al., 1986; Petit et al., 1987), but less information is available for the other species.

This paper deals with the transformation of the species Lotus (L. tenuis and L. corniculatus) through an A. rhizogenes binary vector carrying the gene for hygromycin resistance and with the transmission of the introgressed trait to the progeny.

Materials and methods

Seeds of Lotus tenuis, an ecotype from central Italy, and Lotus corniculatus cv. 'Franco' were surface sterilized with a mixture of 0.1% (w/v) mercuric chloride plus 0.1% (w/ v) sodium lauryl sulphate (20 min), subsequently with 20% (v/v) 'Linda clor' (commercial bleach containing 6% sodium hypochlorite) for 20 min, then rinsed 5 times with

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Agrobacter ium transformation in Lotus spp.

sterile distilled water and placed on hormone-free MS medium (Murashige and Skoog, 1962) with 0.8% agar (MSO). Seeds and plantlets were incubated at 23 + I*C under fluorescent light (27 ~tE s -1 m -2, 12 h photoperiod) and subcultured every 4 weeks. Plantlets (8-10 weeks old) were infected with A. rhizogenes strain LBA9402, kindly provided by J. Hamill (Hamill et al., 1987), harbouring the wild type Ri plasmid 1855, and plasmid AGS125 (kana- mycin-resistant) containing, between the A. tumefaciens borders, the HPT gene (hygromycin phosphotransferase) under the control of the nos promoter (van den Elzen et a/., 1985). The bacterial strain was grown for two days (28* C) in solid YMB medium (Hooykaas et al., 1977) supplemented with 50 mg 1-1 kanamycin sulphate. In- fection was performed by puncturing stems, petioles and leaves with a sterile needle previously dipped in the bacterial culture. After one month, adventitious hairy roots were explanted and dedifferentiated on agar-solidi- fled MS medium supplemented with NAA (1 mg 1-1), 6-BAP (1 mg 1-1), hygromycin (20 ~tg m1-1) and carbeni- cillin (1 mgm1-1) to kill the bacterium. Single, root- derived calluses were subcultured at 2-week intervals and calluses 5-6 weeks old regenerated shoots only 3 weeks after being transferred to the same MS medium but without antibiotics (MSN1).

Shoots (1-2 cm high) were transferred to sterile vessels with hormone-free MS medium and after rooting, to jiffy pots in a growth cabinet (216 ~tE sec -1 m -2, 20+ 1 ~ C, 12 h photoperiod, 80% relative humidity). After 2-3 weeks, 10 plants per species were transferred to green- house,

For each species, transformed and non-transformed plants were tested for hygromycin resistance. Leaves of each plant were surface sterilized (10 min in a 10% v/v of commercial bleach 'Linda Chlor' followed by extensive washings with sterile distilled water), transversally dissected and induced to callus either on MSN1 medium with 20 mg 1-1 hygromycin (MSNlhy) or in the same medium without the antibiotic. For each plant and callus induction medium, 20 explants (5 per Petri dish) were incubated, subculturing every two weeks. Callus growth was evaluated by weighing calluses after one month of culture. Data were submitted to analysis of variance.

Total DNA of transformed and control plants of each species was extracted according to Della Porta et al. (1983) and further purified with a CrAB (hexadecyl- trimethylammonium bromide) treatment followed by a chloroform-octanol extraction and isopropanol precipita- tion (Murray and Thompson, 1980). Ten ~tg of DNA per plant were restricted according to the manufacturer's recommendations (Boehringher Mannheim), electrophor- esed on a 1% agarose gel, blotted on a nitrocellulose filter and hybridized with nick-translated probes (specific activity > 370 kBq per ~tg of DNA), as described by Maniatis et al. (1982), and washed at high stringency

331

conditions (0.1% SSC, 0.1% SDS; 65* C). Plant DNA was probed for the HPT gene with the

entire AGS125 plasmid and for T l and T r T-DNA of pRi 1855 with plasmids MP66 and MP17, respectively (Pomponi et al., 1983). Plasmids were isolated by alkaline lysis followed by CsC1 centrifugation (344000 • g), as described by Maniatis et al. (1982).

Progeny analysis was performed on four transformed plants of L. corniculatus and 3 of L. tenuis. As relatively high amount of seeds were needed, manual crosses were not performed but each transformed plant was put in the middle of about 50 non-transformed plants and the plants allowed to pollinate freely. The seeds harvested from each plant and those of the starting populations were sterilized (as described previously) and maintained in MSO. After germination, seedlings were transferred to sterile vessels containing MS0 supplemented with 20 mg 1-1 of hygro- mycin. After 1 and 2 months the surviving plants were scored and x 2 analysis was performed.

Results and discussion

Transformation and plant regeneration

Hairy roots easily formed in all wounded sites, including leaves and petioles: As soon as roots were long enough to be excised, they were transferred to the callus-inducing selective medium: around 80% of them dedifferentiated to callus, indicating a high frequency of co-transformation with the two plasmids. Callus induction was preferred to direct regeneration for two reasons: (1) the plant material had already been tested for its ability to regenerate after a callus phase (Arcioni et al., 1988), so it was not necessary to reset the medium and the conditions for plant re- generation and (2) for our experiment, it was necessary to select explants for their ability to grow on hygromycin- supplemented media, and this was not possible if they had been regenerated directly. In fact, after a short callus phase, regeneration took place in the same dedifferen- tiating medium only when devoid of hygromycin. This behaviour has often been observed in forage legumes transformed both with A. tumefaciens and A. rhizogenes, carrying resistance both to kanamycin and hygromycin (Shahin etal., 1986; Damiani and Arcioni, 1991; Pezzotti et al., 1991). Plant development, root formation and soil transfer were easily achieved, confirming that both these species are particularly suitable for in vitro culture and transformation.

Analysis of transformants

All the putative transformed plants were able to form callus on antibiotic-supplemented medium, and no signifi- cant differences in callus weight were observed on selec- tive and non-selective medium; on the contrary, leaves of untransformed plants browned and died in a few days in the presence of hygromycin (Fig. 1).

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332 Damiani et al.

Fig. 1. Callus growth of leaf derived calluses of L. tenuis transformed (right column) and non-transformed (left column) plants in MSN1 medium supplemented with 20 mg 1-1 of hygromycin (bottom) and on antibiotic-free medium (top).

The introgression of alien DNA was verified by molecular analysis. DNA of two transformed plants per species, of two non-transformed controls (one per species) and of the AGS125 plasmid were restricted with Bam HI and Eco RI (Fig. 2a). Plasmid AGS125 restricted with these two enzymes showed 4 bands. The DNA comprised between the A. tumefaciens borders and then introgressed into the host plant genome was about 4.4 kb in length (Fig. 2b). When this DNA was restricted with Eco RI and Barn HI, it released an internal 2.6 kb fragment (van den Elzen et al., 1985).

The Southern blot hybridization showed the expected 2.6 kb band and an extra band of about 800 bp for all the tested plants of the two species. No band was detected in DNA extracted from untransformed plants (Fig. 2). The use of the whole AGS125 plasmid as a probe allowed us to establish if the hybridization signals are the result of the T-DNA introgression or of bacterial contamination. The presence in the transformed plants of the 800 bp band, which is absent from the restriction pattern of the plasmid, and the absence of the 1.9 and 1.4 kb band, typical of the plasmid, demonstrated that the T-DNA had integrated into all the plants.

The hybridization of total DNA, restricted with Eco RI, with plasmids MP66 and MP17 also showed the presence in the same transformed plants, of the Ri T-DNA, consisting of two stretches (TI and Tr) that were both present in all the transformed plants (Figs 3 and 4).

The effect of Ri DNA on the plant morphology was evident as it showed reduced internode length and leaf size, leaf wrinkling, a more prostrate growth habit and a shallow and fibrous root system. A particular behaviour was observed for flowering: L. corniculatus plants

Fig. 2. Southern blot analysis of DNA restricted with Eco RI and Bam HI and hybridized with pAGS125. (a) lane 1 and 2 contain k marker and DNA of pAGS125; lanes 3 and 6 contain two untransformed controls, one per species; lanes 4-5 have been loaded with L. corniculatus and lanes 7-8 with L. tenuis transformed plants. (b) schematic diagram of the inserted T-DNA.

regularly flowered and set seed, L. tenuis transformants flowered and set seed only one year later.

Progeny analysis

Seeds harvested from the transformed plants were germinated in vitro and plantlets grown on media supplemented with 20 mg 1-1 of hygromycin (Fig. 5). Progeny of untransformed plants developed neither leaves nor roots in the selective medium and after a few days browned and died; some of the seeds from the transformed plants behaved as the control ones, while others developed a normal epigeal system but showed slower root growth, as observed in other species (Nehra et

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Agrobacterium t rans format ion in Lotus spp. 333

Fig. 3. Southern blot analysis of DNA restricted with Eco RI and hybridized with pMP66. In lane 1 the plasmid used as probe, in lanes 2 and 3 L. corniculatus control and transformed plants respectively, in lanes 4 and 5 L. tenuis transformed and control plants, respectively.

Fig. 4. Southern blot analysis of DNA restricted with Eco RI and hybridized with pMP17. In lane 1 the plasmid used as probe, in lanes 2 and 3 L. corniculatus control and transformed plants respectively, in lanes 4, 5 and 6 two L. tenuis transformed and one control plants, respectively.

Fig. 5. L. corniculatus plantlets after one month of growth on hygromycin-supplemented medium; top) control seedlings on non-selective medium, middle) seedlings of transformed plants and bottom) control seedlings on selective medium.

a/., 1990). About half of the .plants survived on the selective medium, indicating that the hygromycin resistance trait was regularly transmitted to the progeny. Offspring segregation was examined with the aid of x 2 analysis (Table 1) by testing for the expected 1:1 ratio. The results of this test indicate that for all plants, with the exception of plant A of L. corniculatus, where the ratio is 2:1 (probably due to a certain degree of selfing), the 1:1 ratio fits best with the observed value. It thus indicates that the introgressed DNA behaves as a single Mendelian locus.

Some of the hygromycin-resistant L. tenuis progeny were transferred to soil and after a few months were tested for their ability to form callus from leaves culture on MSNlhy. All plants were able to grow in the presence of antibiotic. Southern blot analysis of their DNA, cleaved and electrophoresed as those of the mother plants, was performed, hybridizing with the hygromycin coding region of the AGS125 plasmid and with the entire MP66 plasmid containing the genes (rolA, rolB, rolC) responsible for the insurgence of the hairy root syndrome and of the subsequent plant morphological alterations (Schmtilling et a/., 1988). Results of this analysis showed the presence in all plants of the hygromycin-coding region (Fig. 6). However only 14 plants out of 20 showed also the presence of the Ri T-DNA (Fig. 7). Although the low number of scored plants does not permit general

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334 Damiani et al.

Table 1. X 2 analysis of open pollinated progeny of transformed Lotus corniculatus and Lotus tenuis plants resistant (R) and not resistant (Nr) to hygromycin

Plant Offspring P for segregation ratios

R Nr 1:1 2:1

L. corniculatus A 25 12 < 0.05 0.9-0.5 C 67 56 0.5-0.1 < 0.005 6 21 20 0.995 < 0.05 8 14 12 0.5-0.1 0.5-0.1

L. tenuis 2 28 38 0.3-0.1 < 0.005 7 30 32 0.9-0.7 < 0.005 8 25 25 > 0.9 < 0.01

conclusions to be drawn, these results could indicate that the T - D N A regions of the two different plasmids harboured on the same Agrobacterium strain segregated as independent loci.

Conclusions

Hairy root formation, regeneration and rooting of trans- formants were easily achieved in the two Lotus species, confirming that these species are well suited for in vitro culture and molecular manipulation (Arcioni et aL, 1988). The antibiotic used as a reporter shows its ability to allow screening of transformants even at low doses and is suitable for the selection of somatic hybrids between Lotus and other forage legumes. The availability of hygromycin- resistant Lotus plants allows for experiments of asym- metric somatic hybridization between irradiated Lotus protoplasts and protoplasts of a bloating species, such as lucerne, which are hygromycin-sensitive (unpublished data from our laboratory), and the recovery of hybrids on

a hygromycin-supplemented medium. The need to regenerate plants in antibiotic-free medium

is a feature common to many species and often observed when hpt is the reporter gene (Horn et al., 1988; Shimamoto et al., 1989). This is one of the reasons for using A. rhizogenes instead of A. tumefaciens as the transformation agent. In fact, the A. rhizogenes allows for the visual selection of transformation events, precluding the possibility of regenerating false 'transformed' plants, escaped from the antibiotic selection as a consequence of the detoxifying effect of neighbouring transformed cells on untransformed ones. With regard to the problems of morphological alteration induced by the hairy root transformation, it must be noted that unless the induced aberrations affect flower morphology and plant fertility, it is possible to select progeny which (through meiotic segregation) contain only the desired T-DNA, eliminating the deleterious alterations induced by the rol genes (Tepfer, 1990; Hatamoto, 1991).

Fig. 6. Southern blot analysis of L. tenuis transformed progenies. DNA of seven different plants (lanes 1-7) and of the plasmid AGS125 (lane 8) restricted with EcoRI and Barn Hi and hybridized with the Eco RI-Bam HI fragment of the pAGS125 (see Fig. 2b).

Fig. 7. Southern blot analysis of L. tenuis transformed progenies. DNA of seven different plants (lanes 1-7) and of the plasmid MP66 (lane 8) restricted with Eco RI and hybridized with pMP66.

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Agrobac te r ium transformation in Lotus spp.

Acknowledgements

We are grateful to Dr J.D. Hamill, Dr J.R. Bedbrook and Prof. P. Costantino who provided plasmids for this study. Special thanks are due to Dr Y. Zafar for useful comments, to Mr S Martini for his help on tissue culture work and to Mr A. Bolletta for the photographic plates. Research supported by National Research Council of Italy, Special Project RAISA, Subproject N.2, Paper N. 185.

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