their economic value (Fraley et al. 1986;. Zambryski et al. 1989). Although most dicotyledonous plants are susceptible to. Agrobacterium, numerous examples ...
Fagopyrurn 10 (1990): 57-61
Susceptibility of buckwheat (Fagopyrum esculentum Moench.) to Agrobacterium tumefaciens and A. rhizogenes M Neskovic, R Vinterhalter, J. MiljuS-Djukic, S. Ninkovic, D. Vinterhalter, V. Jovanovic, J. Knezevic Institute of Botany, Faculty of Science and Institute for Biological Research "S. Stankovic", University of Belgrade, 29. novembra 142, Yu-l1060 Belgrade, Yugoslavia Key words: Agrobacterium tumefaciens, A. rhizogenes, crown gall induction, hairy root induction, Pennquad, Siva
Abstract The susceptibility of buckwheat iFegopyrum esculentum Moench.) to tumour formation by A . tumefaciens and to hairy root formation by A. rhizogenes was investigated. Tumours were incited on stems and leaves of micropropagated plants, or on hypocotyl and cotyledon fragments of aseptically germinated seedlings. Hairy roots were also incited on stems of micropropagated plants. Plantlets and plant parts of the tetraploid cv. 'Pennquad' and the diploid cv. 'Siva' buckwheat responded very well to all A. tumefaciens strains. In all experiments the strain A281 exhibited a remarkable virulence, stronger than that of A348, Ach5 and A6. Hairy roots developed readily on stems, at the site of A. rhizogenes inoculation. Axenic tumour tissues and hairy roots were isolated from primary proliferations and cultured for more than a year on hormone-free media. It has been concluded that buckwheat is very sensitive to Agrobacterium, which could be used as a vector for genetic transformation. Osetljivost heljde (Fagopyrum esculentum Moench.) prema rhizogenes
Agrobacterium tumefaciens
A.
Ispitivana je osetljivost heljde (Fagopyrum esculeatum Moench.) prema Agrobacterium tumefaciens koji obrazuje tumore na biljkama i prema A. rhizogenes koji obrazuje korenove Chairy roots"). Tumori su bili inicirani na stablu i listovima biljaka koje su raznmozavane u kulturi in vitro, ili na otseccima hipokotila i kotiledona izolovanih sa sejanaca koji su klijali u asepticnim uslovima. Korenovi su takodje inicirani na stablu biljaka gajenih in vitro. Biljke i fragmenti biljaka tetraploidnog varieteta 'Penkvad' i diploidne 'Sive' heljde su vrlo dobro reagovali na sve sojeve A . tumefaciens. Soj A281 je u svim eksperimentima pokazao izuzetnu virulenciju, znatno jacu nego sojevi A348, Ach5 i A6 . Korenovi su se razvili na stablu, na mestu inokulacije A. rhizogenes. Od primarnih proliferacija su izolovani aksenicno tumorsko tkivo i korenovi, koji se gaje vise od godinu dana na podlozi bez hormona. Zakljuceno je da je heljda vrlo osetljiva prema infekciji koju izaziva Agrobacterium, koji bi prema tome mogao da se iskoristi kao vektor za geneticku transformaciju ove vrste. Introduction The soil bacteria A . tumefaciens and A. rhizogenes are known to induce crown gall tumours and hairy roots, respectively, by the insertion of a part of the plasmid DNA into
the plant genome. Since the genes located between the ends of the transferred DNA are expressed in plant cells, plasmids carrying this DNA can be used as vectors for genetic engineering. A number of plant species has so far been successfully transformed, and
58
M. Nelkovit, B. Vinterhalter, J. MiljuJ-Djukit, S. Nlnkovie, D. Vinterhalter, V. Jovanovic, J. Knefevjt
transgenic plants of several species are reported to carry new traits that improve their economic value (Fraley et al. 1986; Zambryski et al. 1989). Although most dicotyledonous plants are susceptible to Agrobacterium, numerous examples indicate that tumourigenesis specifically depends both on the type of plasmid harboured by the bacteria, and on the plant genotype. The intended genetic transformation of a species must, therefore, be preceded by investigation of the plants' susceptibility to the vectors. The genus Fagopyrum has been mentioned in recent reviews (De Cleene and De Ley 1976, 1981) as a possible host for A. tumefaciens, but not for A. rhizogenes. The objective of the present work was therefore to investigate the susceptibility of buckwheat to various common strains of these pathogens and to elaborate a procedure for possible plant transformation. This is part of a long term study aimed at introducing unconventional methods in buckwheat breeding.
Material and methods The tetraploid cv. 'Pennquad' and the diploid cv. 'Siva' of buckwheat (Fagopyrum esculentum Moench.) were used in the experiments. Sterile plantlets, obtained either by micropropagation or from aseptically germinated seeds were inoculated with various bacterial strains. The micropropagated clones originated from immature embryos which had developed a shoot-producing callus in culture (Neskovic et al. 1987). Sterile seedlings were raised from surface sterilized seeds, planted in test tubes containing agar solidified half-strength B5 salt medium (Gamborg et al. 1968). The bacterial strains used for inoculation are listed in Table 1. All bacteria were maintained on agar (1.5%) solidified YEB medium. For inoculation of plant material, bacteria were grown overnight at 30°C in a plate culture, or in liquid medium with shaking, to about 5 x lOB cells per ml. The stems of intact micropropagated plants, about 5 em high, were wounded by a sterile
needle and a dense slurry of A. tumefaciens or A. rhizogenes cells was smeared over the wound. For A. tumefaciens inoculation, a variation of the leaf disk method (Horsch et al. 1985) was also employed, .using cotyledon fragments and hypocotyl sections of germinated seedlings, as well as the leaf disks and petioles of micropropagated plants. Three days after inoculation, all plants and plant parts were transferred to hormone-free basal nutrient medium used for buckwheat (Srejovic and Neskovic 1981). Growth of A. tumefaciens was suppressed using carbenicillin, 1 mg ml'? in the first, and 0.5 mg ml" in subsequent subcultures. For the elimination of A. rhizogenes, 0.5 mg ml'? and 0.3 mg ml'? of cefotaxime was used. When tumours developed, the proliferating tissue was excised and cultured on the same hormone-free medium. The root tips about 10 mm long that developed after A. rhizogenes inoculation were excised after 18 days and transferred to Petri dishes containing 10 ml of hormone-free media. All cultures were grown under white fluorescent light (5.0-7.2 W.m- 2 ) , with 16 h light cycles, at 25t2°C. Results and discussion The modified leaf disk procedure produced better results in tumour induction than the inoculation of intact plants, since the plantlets had thin and delicate stems and apparently suffered from mechanical injury. Nevertheless, tumours were induced at the wound sites with all virulent bacterial strains in almost all plants that survived the treatment (Figs. 1-5). In cotyledon and hypocotyl segments, about 90% of explants responded to the . inoculation by forming transformed callus tissue. The A281 strain showed the highest virulence in all experiments, followed by Ach5, A6 and A348. Hypervirulence was evident from the dynamics of tumour formation in cotyledon disks (Fig. 6) and hypocotyl segments (not presented here). After 25 days, tumours incited by the A281 strain were large (Fig. 5) and had approximately double fresh weight (about 60 mg per callus) of those induced by other strains. It was
Susceptibility or buckwheat (Fllgopyrum esoalentum Moench.) to Agrobacterium tumefaciens and A rbizogenes
59
Figs. 1-5. Tumour formation by A. tumefaciens on buckwheat, 3 weeks after inoculation. 1, 2 and 3: Strains A348, A281 and A6, respectively, induced tumours at the site of infection of micropropagated 'Siva' (I, 2) and 'Pennquad' (3) buckwheat plants. 4: Hypocotyl section of a 'Pennquad' seedling with tumours induced by Ach5 strain. 5: Leaf disks of an in vitro multiplied 'Pennquad' plant infected with (from left to right): A136 (avirulent), A348 and A281 strains.
M. N~kovif, B. Vinlerhaller, J. Milju!-Djukif, S. Ninkovif, D. Vinlerhalter, V. Jovanovif, J. Knefevit
60
shown in parallel experiments that the strain A281 was also the most virulent in tobacco, while in KaJanchoe Ach5 tended to be slightly more efficient than the other 3 strains (results not shown here) . Hypervirulence of the A281 strain on legumes has previously been reported (Hood et al. 1987).
Table 1. Bacter ial st rains used for inoculation Strain
Description
A. tuefaciens
AI36
C58 cured of Fi-plasaid, avirulen t
A281
AI36 with pTiBo542, contains succinamopine and mannopine AI36 wi th pTiA6HC, contains octopinc wild type, conta ins octopine wild type, conta ins octop ine
A348
Ach5 A6
A. rhizogencs
15834 13332
ATCC ATCC
Tumour tissues cultured on hormone-free media developed into large autonomous calli. The initial difference in growth rate between the tumours induced by various bacterial strains was lost during the subsequent passages. All calli grew well for up t o 12 months as displayed in an index of growth (W1 -W O)!WO ranging from 6-12. Hairy roots developed at the inoculation site and were indistinguishable from the adventitious roots at the stem base . However, when the tips of transformed roots were transferred to hormone-free media, they formed abundant root colonies (Fig. 7), in contrast to normal roots that immediately died. Out of 73 plants inoculated with A. rhizogenes, 29 (39.7%) formed hairy roots. The strain 15834 produced hairy roots in 22 (75.8%) plants, and strain 13332 in 7 (24.1%) plants that responded to infection. It should be noted that in all experiments controls were run either by exposing plants to the same ' conditions without bacterial inoculation, or by using the avirulent strain A136. Swelling of the tissues, cell
1
I
IIf
A348
Ii
.I. • .••. s:.
c
a.• IC
w
o
5
10
15
20
25
Oap alter Inoculation
Fig. 6. Dynamics of tumour format ion by various A. tumefaciens strains in buckwheat cotyledons; about 50 cotyledons were infected with each bacterial strain.
Fig. 7. Hairy roots produced by A . rhizogenes inoculation on a 'Siva' buckwheat stem and cultured axenically in hormone-free medium for 3 months.
Susceptibility of buckwheat (Fagopyrum esculentum Moencb.) to Agrobacterium tumefaciens and A rbizogenes
proliferation, or root formation did not occur in any control samples. The autonomous growth of axenic callus tissues and roots was taken as preliminary evidence that genetic transformation did occur. Analyses of tumour extracts by paper electrophoresis showed that tissues transformed by Ach5, A6 and A348 produced octopine, while A281-transformed tissue contained mannopine. Tumour DNA hybridized with the corresponding DNA probes in Southern blots. These results will be presented elsewhere. In conclusion, we have shown that inoculation of buckwheat seedlings or excised organs with 4 oncogenic strains of A. tumefaciens resulted in tumour formation in virtually all recipients. Inoculation with A . rhizogenes induced the development of hairy roots. These results show that buckwheat should be added to lists of species that are susceptible to Agrobacterium and that projects for obtaining transgenic plants may be feasible. Acknowledgement . This work was supported by the SFRY-USA Joint Board for Scientific and Technological Cooperation, Grant No. FG-Yu 253, JFP 661. The authors are obliged to Dr.
L. Owens (Beltsville) strains A136, A281 and (Paris) for strains Ach5 Kreft (Ljubljana) for seeds.
61
for A. tumefaciens A348, to Dr. J. Tempe and A6, and to Dr. 1. providing buckwheat
References De Cleene M. and De Ley J. 1976. The host range of crown gall. Bot. Rev. 42: 389-486. De Cleene M. and De Ley J. 1981. The host range of infectious hairy-root Bot. Rev. 47: 147-194. Fraley RT~ Rogers S.O. and Horsch RB. 1986. Genetic transformation in higher plants. CRC Crit. Rev. Plant Sci. 4: 1-46. Oamborg O.L~ Miller RA. and Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exper. Cell Res. 50: 148-151. Hood E.E~ Fraley RT. and Chilton M.D. 1987. Virulence of Agrobacterium tumefaciens strain A281 on legumes. Plant Physiol, 83: 529-534. Horsch R.B~ Fry J.~ Hoffmann N.L~ Eichholtz D., Rogers S.O. and Fraley RT. 1985. A simple and general method for transferring genes into plants. Science 227: 1229-1231. Neskovic M., Vuji~it R. and Budimir S. 1987. Somatic embryogenesis and bud formation from immature (Fagopyrum esculentum embryos of buckwheat Moench.). Plant Cell Reports 6: 423-426. Srejovic V. and Neskovic M. 1981. Regeneration of plants from cotyledon fragments of buckwheat (Fagopyrum esculentum Moench.). Z. Pflanzenphysiol. 104: 37-42. Zambryski P; Tempe J~ Schell J. 1989. Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56: 193-201.
