Plant Cell, 1issue and Organ Culture 45: 145-152, 1996. 145 © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Plant regeneration and genetic transformation of Lotus angustissimus

E. N e n z I , F. P u p i l l i l, F. P a o l o c c i I , F. D a m i a n i I , C . A . C e n c i 2 & S. Arc ion i 1. 1 Istituto di Recerche sul Miglioramento Genetico delle Piante Foraggere del CNR, Via dell MadonnaAlta, 130, 06128 Perugia, Italy; 2 Dipartimento di Biologia ed Economia Agro-lndustriale, Via delle Scienze, 208, 33100 Udine, Italy (* requests for offprints)

Received 27 June 1995; accepted in revised form 27 February 1996

Key words: Agrobacterium rhizogenes, callus, shoot organogenesis, tannins

Abstract

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4- dichlorophenoxyacetic acid or c~-naphthaleneacetic acid with kinetin, N 6 -A2-isopentenyladenine or benzyladenine in different combinations and concentrations. Only those calli induced in presence of c~-naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.

Abbreviations: BA - benzyladenine; 2,4-D - 2,4-dichlorophenoxyacetic acid; 1AA - indole-3-acetic acid; NAA - c~-naphthaleneacetic acid; 2iP - N6-A2-isopentenyl-adenine; PA - proanthocyanidins; NOS - nopaline synthase; NPTII - neomycin phosphotransferase; GUS - fl-glucuronidase; CaMV - cauliflower mosaic virus

Introduct ion

The genus Lotus comprises a heterogeneous group of annual and perennial, diploid and tetraploid species which are widely distributed throughout the world. Depending upon the system of classification, from 80 to 200 different species are recognized as belonging to the genus, with the greatest diversity among species found in the Mediterranean basin, reported to be the center of origin for Lotus (Hertzsch, 1959).

The genus Lotus is polymorphic for the presence of foliar proanthocyanidins, also known as condensed tannins, which confer bloat-safety on the forage. Foliar PA could be introduced into Medicago sativa (lucerne, alfalfa) and Trifolium pratense (red clover) to ren- der these important species bloat-safe. Foliar PA also decrease protein loss due to degradation processes in the rumen (Tanner et al., 1994 ). It has been suggest- ed that the production of a bloat-safe alfalfa could be

achieved by somatic cell fusion of this species with oth- er PA-containing forage legumes (Aziz et al., 1990). This approach has been hampered by the necessity to involve in the fusion experiments parents belonging to different genera as no bloat-safe species has been identified in the genus Medicago (Li et al., 1993). Another possibility is to transform lucerne and clover with the genes required for tannin synthesis, but these genes have not been identified so far. The applica- tion of molecular techniques including mutant anal- ysis, and the use of transposable elements to clone genes can be efficiently carried out on autogamous plants with low ploidy level, short regeneration time, small genome size and with established transformation protocols. Therefore to study the genes controlling the synthesis of PA, diploid and PA-positive plants are required. Reports of Lotus species investigated for in vitro responsiveness involve either tetraploid species such as L. corniculatus (Mariotti et al., 1984) and L.

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pedunculatus (Pupilli et al., 1990) or diploid leaf-PA- negative species such as L. tenuis (Piccirilli et al., 1988) and L. japonicus ( Handberg & Stougaard, 1992 ). L. angustissimus satisfies all the requirements for effi- cient analysis, since it exists in diploid and tetraploid forms and contains PA-positive and negative acces- sions. Both chromosomal races are very similar in morphology, though the tetraploid plants may some- times reach a larger size, as a typical case of cryptic chromosomal races (Heyn, 1970).

In this paper, the experimental conditions for plant regeneration from callus cultures and for genetic trans- formation of tannin-positive, diploid and tetraploid accessions of L. angustissimus are reported.

Materials and methods

Plant material

Seeds of 2 accessions of L. angustissimus L., one diploid (2n=2x=12) B-526 (courtesy of Prof. W. E Grant, Mc Gill University, Quebec, Canada) and one tetraploid (2n=4x=24) CPI-113587 (courtesy of Dr P.J. Larkin of CSIRO, Division Plant Industry, Canberra, Australia) were surface-sterilized for 20 min with a mixture of 0.1% (w/v) mercuric chloride and 0.1% (w/v) sodium lauryl sulfate and then for 20 min with a 1.2% (w/v) solution of sodium hypochlorite, and rinsed three times in sterile distilled water. Surface-sterilized seeds were germinated on 1/4 strength MS medium (Murashige & Skoog, 1962) from which growth reg- ulators were omitted and which had been solidified with 0.8% w/v agar (GRFMS), incubated for 2 days in the dark at 4°C and then under growth condition A (23°C-4-1, day fluorescent light tubes, 27 /amol m-2s - j , 12-h photoperiod). Two-week-old seedlings were transferred to Magenta GA-7 culture vessels (6×6× 10 cm) containing 70 ml of GRFMS medium and utilized as source of leaves for callus induction.

Plant material was tested for tannins by crashing young leaves of 5 plants of each accession between two layers of Whatman 3MM chromatography paper and applying vanillin/HC1 solution as reported in Sarkar & Howarth (1976).

Callus induction

Calli were induced from cotyledons, hypocotyls and roots of 10-day-old aseptically grown seedlings, or from leaves and stems excised from sterile 6 - to 7-

week-old plantlets. Cotyledons were sliced in half, hypocotyls, stems and roots cut into segments (8 - 10 mm in length), and leaves scratched on the lower surface were cultured on the agar-solidified (0.8% w/v) medium UM (Uchimiya & Murashige 1974), pH=5.8, sucrose 3% (w/v) and with different concentrations of auxins and cytokinins (Table 1). For each accession, 30 explants per medium were tested, the cultures were incubated under growth conditions A in 10-cm Petri dishes (6 explants/dish) and subcultured weekly. After 2 weeks, callus induction was evaluated with the fol- lowing score:

1. absence of callus; 2. presence of initial callusing on the scratched sur-

face; 3. explant partially differentiated; 4. explant completely differentiated.

Data were submitted to analysis of variance and the mean values of the combinations at each culture medi- um with each accession or explant were further anal- ysed using the Duncan's Multiple Range Test. Callus growth rate was determined by calculating the ratio: callus weight after 4 weeks/callus weight after two weeks.

Plant regeneration

After 1 month of culture, calli were either maintained on dedifferentiation media (Table 1) or transferred to the following regeneration media based on MS salts: MS-1 (NAA 5.37 #M, BA 4.44 #M; Piccirilli et al., 1988), MS-2 (NAA 5.37/aM, BA 1.11/aM), MS-3 (BA 0.89/aM, NH + 5 mM; Swanson & Tomes, 1980), MS- 4 (BA 0.89/aM; Ahuja et al., 1983), MS-5 (IAA 0.57 #M, 2iP 2.98/aM; Mariotti et al., 1984), all supple- mented with 20 g 1-1 of sucrose pH=5.8. The morpho- genetic capacity was evaluated considering 40 explants (10 explants/dish) per medium and per explant type. Shoots which developed on callus were excised when 0.7-2 cm long and transferred for rooting to GRFMS medium into Magenta GA-7 culture vessels maintained under the same environmental conditions used for cal- lus induction. Plantlets were transferred to soil when 10-12 cm tall and maintained under growth condition B (20°C-4-1 day light fluorescent tubes, 216/amol m -2 s -1, 12-h photoperiod), under a polyethylene bag to prevent dessiccation. After 2 weeks, the regenerants were transferred to the glasshouse.

The chromosome number of 5 regenerants per accession was determined in root tips of cuttings after pretreatment at room temperature with 8-

Table 1. Culture media used for callus induction in L. angustissimus.

Culture media Growth regulator (#M) 2,4-D IAA NAA kinetin 2iP BA

UM -0 UM-1 2.26 UM-2 4.52 UM-3 9.05 UM-4 18.10 UM-5 4.52 UM-6 UM-7 UM-8 4.52 UM-9 9.05 UM-10 4.52 UM-I 1 UM-12 UM-13

5.7 11.42

5.37 5.37 5.37

1.16 1.16 1.16 1.16 4.65 1.16 1.16

1.16

0.74 0.74 2.98

1.11

4.44

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hydroxyquinoline (2 mM) for 3 h, fixation with 1:3 acetic acid:ethanol (v/v), hydrolysis in 1N HCI (600C, 10 min) and staining with Feulgen. Chromosome num- bers were counted in 5-6 well spread metaphase cells per slide, and 5 such slides were observed for each plant.

Plant transformation

Aseptically grown plantlets (8 - to 10-week-old) were infected with the A. rhizogenes strain LBA9402 har- bouring the wild-type Ri plasmid 1855 and pBI 121.1 containing the NPTII gene (conferring resistance to kanamycin), under the control of the NOS promoter (Bevan, 1984) and the GUS gene under the CaMV 35S promoter (Jefferson et al., 1987a) between the T-DNA borders. The bacterial strain was grown for two days at 28 o C on solid YMB medium (Hooykaas et al., 1977) supplemented with 50 mg 1-1 kanamycin sulfate. Plant infection was performed by puncturing stems, petioles and leaves with a sterile needle previously dipped in the bacterial culture. Plants were maintained in Magen- ta GA-7 vessels as described under the Plant material section. After one month, adventitious hairy roots were transferred to UM-11 or UM-13 media (Table 1) sup- plemented with kanamycin sulfate (50 mg l - l ) and carbenicillin (1 g 1 -~) to kill the bacterium, and main- tained under growth condition A. Hairy-root derived calli were subcultured at 2-week intervals, and after 8 weeks of culture on UM- I 1 or UM-13 media (Table 1) they started to regenerate. Shoots (1-2 cm high) were

rooted in Magenta GA-7 vessels containing GRFMS with both kanamycin and carbenicillin and transferred to soil, maintained under growth condition B for 3 weeks, and then moved to the glasshouse.

GUS activity was estimated by histochemical stain- ing of newly formed hairy roots and in leaves of regen- erated plants (Jefferson, 1987b). Kanamycin resistance was tested on six transformed and 2 untransformed control plants. Leaves were surface sterilized (10 min in a 10% v/v of commercial bleach, equivalent to 0.6% final concentration of sodium hypochlorite, followed by extensive washing in sterile distilled water), and small pieces of leaf were placed on UM-8 medium with or without 50 mg 1-1 kanamycin. For each plant, and for each medium, 30 explants (10 per Petri dish) were incubated under growth condition A, and subcultured weekly. Callus growth was evaluated as described pre- viously.

Total DNA of transformed and cantrol plants was extracted according to Saghai-Maroof et al. (1984). Ten #g of DNA per plant were digested following the supplier's instructions (New England Biolab), elec- trophoresed on 1% agarose gel, blotted on nylon mem- brane (Hybond N+, Amersham), hybridized with the whole NPTII sequence excised with a BamHI restric- tion from the CaMVNEO plasmid (Fromm et al., 1986). The hybridization was carried out overnight at 65°C with 32p-labelled probes (specific activity >370 kBq per #g of DNA), prepared using a Pharmacia Ready- to-Go DNA labelling kit for the random primer pro-

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Table 2. Callus induction response from hypocotyls of diploid and tetreploid accessions ofL. angustissimus in dif- ferent culture media.

Accession Culture media

UM-2 UM-5 UM-12 Urn-13

B-526

(2n=2 × = 12)

CP1-113587

(2n=4 × =24)

2.52 a 2.43 a 1.72 b 1.25 b

3.01 a 2.85 a 1.60 b 1.15 b

The evaluation was carried out by scoring from 1 (absence of callus) to 4 (explant completely dedifferentiated) after 3 weeks of culture. The values followed by the same letter do not differ forp _< 0.01 using the Duncan's Multiple Range Test.

cedure and washed under high stringency conditions (O.1× SSC, 0.1% SDS; 65°C).

Results and discussion

Tissue culture

The diploid and tetraploid accessions were compared for dedifferentiation by evaluating callus induction from hypocotyls (the most responsive explant) in UM- 2, UM-5, UM-12 and UM-13, that were the most repre- sentative of the different combinations auxin/cytokinin tested. Since no significant differences were found (Table 2) the responses of various explants to dedif- ferentiation media were evaluated by pooling the data of the two accessions (Table 3). After 2 weeks of cul- ture the evaluated efficiency in callus induction was: hypocotyls > leaves > stems > roots > cotyledons. Hypocotyls dedifferentiated on media containing 2,4- D either with kinetin (UM-I, UM-2, UM-3, UM-4, UM-5) or 2iP (UM-8, UM-9).

The most effective media for callus induction from leaves were those containing 2,4-D and 2iP (UM-8, UM-9). When 2,4-D and kinetin were used in a ratio 1:1 (UM-5) an effective callus induction was obtained from all the explants, while the replacement of kinetin with 2iP (UM-10) reduced the dedifferentiation pro- cess.

Callus formation in media with kinetin in combina- tion with IAA (UM-6, UM-7) or NAA (UM-12) was absent and scanty, respectively. The replacement of 2,4-D with NAA, and kinetin with BA, respectively, was unsuccessful and resulted in poor callus produc-

tion, irrespective of the ratio auxin/cytokinin (UM-11 and UM-13).

In L. angustissimus, an efficient callus induction can be reached with a combination of 2,4-D and kinetin or 2iP. This is a common feature of Lotus species: media with 2,4-D at various concentrations have been reported to be effective in L. corniculatus (Mariotti et al., 1984), L. tenuis (Piccirilli et al., 1988), L. peduncu- latus (Pupilli et al., 1990) and L. japonicus (Handberg & Stougaard, 1992).

Callus growth rate was evaluated in those media showing a score for callus induction > 2 in Table 3. It has been observed that UM-8 and UM-9 promoted the fastest callus growth rate for leaves and hypocotyls while UM-5 was the best medium for all the other explants (Table 4).

Plant regeneration

Optimum growth regulator requirements for plant regeneration were markedly different from the growth regulator regime required for callus initiation and growth. The explants cultured in presence of NAA combined with BA or kinetin (UM-11, UM-12, UM- 13) underwent a slow dedifferentiation process with the production of poor, hard and dark green calli that however produced shoots after 4-12 weeks of explant culture. The most morphogenetic medium was UM-13 while no significant differences were found between UM-12 and UM-11. The regeneration fre- quencies (number of regenerating explants/number of total explants cultured) of hypocotyls derived from the tetraploid accessions were 80%, 61% and 55% in UM- 13, UM-12 and UM-11, respectively(I-5 shoots per cal- lus). Hypocotyl-derived-calli were the fastest to regen- erate followed by cotyledon, root, stem and leaf calli, respectively. Shoots were excised from the surface of callus and cultured for 3 weeks in GRFMS where they rooted easily (90% of shoots produced roots). The cal- li induced on media containing 2,4-D (Table 1) and then transferred to the different regeneration media (MS 1-5) did not regenerate. In UM-13 the diploid and tetraploid accessions exhibited the same trend for plants regeneration and the only difference was the percentage of explants which produced shoots. In the tetraploid, 80% of hypocotyl, 60% of cotyledon, 40% of root and 20% of both stem and leaf-derived calli produced shoots. Similarly in the diploid accession, the proportion of initial explants which gave rise to shoots were: 50% of hypocotyls, 20% of cotyledons, 15% of roots and 8% of both stems and leaves. A sim-

Table 3. Callus induction response of L. angustissimus in different media by scoring from 1 (absence of callus) to 4 (completely dedifferentiated explant) after 2 weeks of culture.

Culture Explant media Leaf Stem Cotyledon Hypocotyl Root

UM-0 1.00 n 1.00 n" 1.00 n 1.00 n 1.00 n

UM- 1 2.44 bc 1.73 g 1.72 gh 2.42 bc 1.27 klm

UM-2 1.61 hi 2.00 ef 1.20 lmn 2.65 a 1.55 hij UM-3 2.03 e 1.41 ijk 1.47 ijk 2.62 a 2.00 ef

UM-4 1.56 hij 2.06 e 1.40jk 2.50 ab 1.82 f

UM-5 2.16 de 2.28 cd 2.07 e 2.57 ab 2.40 bc UM-6 1.08 mn 1.00 n 1.00n 1.00 n 1.00 n

UM-7 1.00 n 1.00 n 1.00 n 1.00 n 1.00 n UM-8 2.55 ab 1.46 ijk 1.05 n 2.25 cd 1.63 gh UM-9 2.47 ab 1.57 hi 1.10 mn 2.00 ef 1.55 hij UM-10 1.29 kl 1.46 ijk 1.47 ijk 1.60 hi 1.42 ijk UM-I 1 1.10 mn 1.39 jk 1.09 mn 1.30 kl 1.50 ij

UM-12 1.10mn 1.141mn 1.27 kl 1.35jkl 1.181mn UM-13 1.191mn 1.45 ijk 1.00n 1.00n 1.00n

The values followed by the same letter do not differ forp < 0.01 using the Duncan's Multiple Range Test.

Table 4. Growth evaluation of calli from different explants of L. angustissimus after 4 weeks of culture.

Explant Culture media

UM- 1 UM-2 UM-3 UM-4 UM-5 UM-8 UM-9

Leaf 3.00 e 3.95 d 4.08 cd 5.08 b 5.11 b

Stem 2.00g 2.50 f 2.55 ef

Cotyledons 4.00 d

Hypocotyls 1.79 g 2.25 fg 4.13 cd 4.51 c 3.95 d 5.89 a 4.24 cd

Root 4.00 d

The values followed by the same letter do not differ for p _< 0.01 using the Duncan's Multiple Range Test.

149

ilar s i tuat ion was observed also for other Lotus spp.

(L. corniculatus; L. tenuis) as well as within the genus

Medicago (unpubl ished data, this laboratory).

The regenera t ion o f L. angustissimus occurred via

organogenes is (Fig. 1) and in no case embryo- l ike

structures were observed. All regenerated plants (Fig. 2) were v igorous , set seeds and showed the same

m o r p h o l o g y and c h r o m o s o m e number as seed-der ived

plants.

Transformation

Hairy roots were rapidly formed from stems but not

f rom leaves and petioles. As soon as roots were long

enough ( 1-2 cm) to be excised, 30 hairy roots were test-

ed for G U S activity. This test a l lowed screening tissues which expressed T -DNAs of both plasmids harboured

by the inoculat ing bacter ium f rom those express ing

only the T - D N A of the Ri plasmid. Only 73% of the

hairy roots displayed the typical b lue stain due to the

G U S activi ty and this value represented the percentage

o f co-transformation. Staining o f a var iable intensi ty

150

Fig. 1. Shoots of L. angustissimus originating from callus cultured on UM-I 1 medium. Scale bar =5 mm.

Fig. 3. Southern blot analysis of DNA of plants and plasmids digested with HindlII and probed with: panel (a) the entire NPTII sequence; panel(b) BamHl fragment 31 of the pMP66. Lanes 1, 2, 3, 4 diploid plants; lanes 5, 6, 7, 8 tetraploid plants; lanes 1,2.3,6,7,8 transformed plants; lane 9 plasmid pBIl21.1, lane 10 plasmid pMP66.

Fig. 2. Regenerated plant of L. angustissimus.

was observed and this could be accounted to either a different copy number of the inserted genes or a position effect. Similarly, hairy roots were transferred to UM-13 medium containing kanamycin, and about 70% of them underwent a dedifferentiation into a poor callus. The estimation of co-transformation of Ri and NPTII is thus very similar to that calculated on the basis of GUS activity. After 6 - 8 weeks of culture on dedif- ferentiation medium, from the small amount of callus produced around the hairy roots 1 to 4 shoots were developed on each explant; about 25% of the cultured (91) hairy-roots regenerated shoots which rooted very easily ~ind nearly 90% of plantlets survived after trans- plantation into soil. In total, 60 putative transgenic plants derived from 21 independent transformations events, were grown in glasshouse.

Analysis o f transforrnants

Leaves from six putative transformed plants were tested" for the ability to form callus on antibiotic-

supplemented medium; all of them dedifferentiated and no significant differences in callus growth rate were observed on selective and non-selective medium; conversely, leaves of untransformed plants browned and died in a few days in the presence of kanamycin. A few leaves (3 per plant) of five putative transformed plants were analysed for GUS activity, and all exhibit- ed the characteristic blue color.

The introgression of alien DNA was verified by molecular analysis that is reported in Fig. 3; the DNA of diploid (lanes 1,2,3,4) and tetraploid (lanes 5,6,7,8), transformed (lanes 1,2,3,6,7,8) and untrans- formed (lanes 4,5) plants were digested with HindlII which cut the plasmid inside the T-DNA at the end of the NPTII cassette. Because HindlII cuts the T-DNA only once at the end of the sequence homologous to the NPTII probe, the number of bands observed in transgenic plants represents the number of sites into which T-DNA was inserted in the plant genome. No bands were detected on the control plants (lanes 4,5). The transformed plants showed a variable number of bands (from 3 to 5) with a length ranging from about 8 to 3.5 kbp. The definitive demonstration that alien genes are introgressed into plant genomes is given by their transmission to the progenies. Unfortunate- ly, the transformation withA, rhizogenes binary vector delays some months or years the flowering of transfor-

mants according to the species considered (Damiani & Arcioni, 1991). The transgenic plants of L. angustis- simus have been transplanted into soil two years ago but up to now no flowers have been produced. How- ever, waiting for flowering, molecular evidence of the introgressed NPTII gene is given by the absence of common bands in transformants and plasmid DNA (Fig. 3, lane 9). The same filter shown in Fig. 3a was stripped and re-probed with the Barn HI fragment n. 31 of the MP66 plasmid (Pomponi et al., 1983) encompassing the left border of Ri T1-DNA (Slightom et al. , 1986). With this probe, it is possible to count, as described for NPTII gene, the number of sites into which Ri TI-DNA was introduced in the plant genome (Fig. 3b). This T-DNA was inserted also in multiple copies (4 to 6). Because lanes 1 and 2 and lanes 7 and 8 show identical banding patterns with both probes (Figs 3a, b) we can assume that DNA donor plants derive from the same transformation event.

Conclusions

Callus was induced from different explants on a range of culture media in order to define the opti- mal conditions for plant regenerations from diploid and tetraploid accessions of L. angustissimus. Simi- larly to other forage legumes (Arcioni et al. , 1990), callus induction and proliferation were most rapid in the presence of 2,4-D regardless to the type of explant used. Conversely, NAA induced a slow dedifferenti- ation process, and only a small amount of callus was produced after two weeks of culture, just on the cut sur- face of explants. As a consequence, callus induction and growth was best in media with 4.52 #M 2,4-D for all the explants considered (Tables 3 and 4). With 4.52 #M 2,4-D the increase of kinetin led to higher perfor- mances regardless the type of explant (compare UM-2 and UM-5), the opposite was observed for 2iP (com- pare UM-8 and UM-10). As regards callus growth an interaction has been noted for cytokinin types used at the optimal concentration observed and explant source, in fact 2iP is recommended with leaves and hypocotyls (UM-8) while kinetin with the other explants tested (UM-5). Calli from roots and cotyledons had a growth rate significantly lower than hypocotyls and stems gave the smallest amount of callus.

In the genus Lotus, plant regeneration is achiev- able more easily than in other forage legumes, such as Medicago and Trifolium. Morphogenesis appears to be genotype specific (Mariotti et al., 1984). Howev-

151

er, plants carrying the few genes (Glover & Tomes, 1982) controlling regeneration are largely distributed in populations of many species of this genus. The Lotus species differ in the growth regulators required to induce plant regeneration: L. corniculatus (Niize- ki & Grant, 1971; Mariotti et al., 1984), L. tenuis (Piccirilli et al., 1988) and L. pedunculatus (Pupilli et al., 1990) all require the presence of a cytokinin (BA or 2iP) and an auxin (NAA or IAA); L. japoni- cus (Handberg & Stougaard, 1992) and L. corniculatus (Swanson & Tomes, 1980) are reported to regenerate in presence of low levels of BA only; L. angustissimus produced shoots in presence of NAA combined with BA or 2iP (ratio 1:1 and 1:4) and then behaved similar- ly to L. corniculatus and L. tenuis. The main feature of L. angustissimus that makes this species very different from the others was the absence of morphogenesis in calli induced and grown in presence of 2,4-D. To date, there are many reports of species regenerating in pres- ence of 2,4-D. On the contrary when L. angustissimus was cultured with 2,4-D, abundant callus proliferation was observed but calli did not regenerate. The replace- ment of 2,4-D with NAA considerably decreased the callus growth rate but allowed regeneration of trans- formed plants with only one culture medium. This characteristic could be useful for tagging the genes involved in the synthesis of PA by introgressing trans- posable elements or the T-DNA of Agrobacteria into the diploid tannin-synthetizing plants.

Acknowledgements

We are grateful to Mrs. G. Labozzetta and Mr. G. Carpinelli for the excellent technical assistance and to Mr A. Bolletta for the photographic plates. The research was supported by the National Research Council of Italy, Special Project RAISA, Subproject N.2.

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