Morphological Alterations and Root Nodule Formation in ...

Annals of Botany 81 : 355–362, 1998

Morphological Alterations and Root Nodule Formation in Agrobacterium rhizogenes-mediated Transgenic Hairy Roots of Peanut (Arachis hypogaea L.) Y O K O A K A S A KA*, M A S A H I R O M I I* and H I R O Y U K I D A I M O N†‡ * Faculty of Horticulture, Chiba Uniersity, Matsudo, Chiba 271, Japan and † College of Agriculture, Osaka Prefecture Uniersity, Sakai, Osaka 593, Japan Received : 21 August 1997

Returned for revision : 24 September 1997

Accepted : 27 October 1997

Transformed hairy roots were induced at the excised site of the epicotyl of dry mature seed of a Spanish type peanut (Arachis hypogaea) cv. Java 13 2 weeks after inoculation with a wild type strain of Agrobacterium rhizogenes, MAFF02-10266. Composite plants consisting of transformed roots with non-transformed shoots were cultured using pouches. Forty days after inoculation, the composite plant showed a root system with abundant root mass, more lateral branching and high fractal dimension compared to the control. No differences were observed in production of rosette-type root hairs or the cross sectional structure between transformed and non-transformed roots. The inoculation of Bradyrhizobium sp. A2R1 strain to the composite plants led to the induction of transformed root nodules. These transformed root nodules showed production of leghaemoglobin in the bacterial zone and nitrogenase activity as assayed by C H reduction, and exhibited enlargement of the nodule cortex region and de noo root # # formation from the nodule cortex. # 1998 Annals of Botany Company Key words : Agrobacterium rhizogenes, Arachis hypogaea L., composite plant, peanut, transformation, root nodule.

INTRODUCTION Virulent strains of Agrobacterium tumefaciens and A. rhizogenes infect a wide range of dicotyledonous plant species. The Ti (tumour-inducing) plasmid of A. tumefaciens is responsible for development of crown gall tumours on appropriate host plants, and A. rhizogenes carrying the Ri (root-inducing) plasmid is the causative agent for hairy root disease (Chilton et al., 1982 ; Weising and Kahl, 1996). In both cases the T-DNA region of the Ti or Ri plasmid is transferred into the nucleus of the host plant and is stably integrated into the nuclear genome. Ri T-DNA of A. rhizogenes alters phytohormonal metabolism, for example synthesis of auxin in the host plant, and hairy roots are induced at the inoculation sites. Transgenic plants regenerated from hairy roots usually exhibit various phenotypes such as wrinkled leaves, short internodes, reduced apical dominance and abundant root development in several plant species (Tepfer, 1983 ; Handa, 1994 ; Otani et al., 1996 ; Godo et al., 1997). Hairy rootderived plants of Rudbeckia hirta also exhibited morphological alterations such as small flowers, dwarfing and abundant root mass with extensive lateral branching (Daimon and Mii, 1995). The alterations in root growth and development could be especially important for improvement of root systems in crop breeding. In leguminous crops, modification of root systems by A. rhizogenes-mediated transformation may also improve root nodule formation and nitrogen fixation ability. However, a detailed comparison of transformed and non‡ For correspondence at : Department of Plant Science, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 593, Japan. e-mail daimon!plant.osakafuju.ac.jp

0305-7364}98}02035508 $25.00}0

transformed roots studying morphology, structure and nodulation has not been made due to difficulty in regenerating plants from hairy roots in most leguminous species. As an alternative strategy for evaluating the character of transformed roots, a composite plant, which was generated by inducing transformed roots on non-transformed shoots, has been used for analyses of gene expression regarding infection of rhizobia and nitrogen fixation in some legumes such as Lotus corniculatus (Stougaard et al., 1986 ; Hansen et al., 1989), Trifolium repens (Diaz et al., 1989) and Vicia hirsuta (Quandt, Puhler and Broer, 1993). We report here the production of composite plants consisting of A. rhizogenes-mediated transformed roots with non-transformed shoots in peanut (Arachis hypogaea L.), an important economic oil-seed crop and a source of protein, food and feed, and present a morphological evaluation of the root system, including lateral branching and root nodule formation. MATERIALS AND METHODS Plant materials and bacterial strains Seeds of Arachis hypogaea L. cv. Java 13 were obtained from Chiba Prefectural Agricultural Experimental Station (Chiba, Japan). The embryonic axes were removed from mature dry seeds, surface-sterilized for 5 min with sodium hypochlorite solution (1 % active chlorine) containing a drop of Tween 20 and rinsed three times with sterile deionized water. The apical portion containing the upper one-third of the axis was excised according to the method of Baker et al. (1995) and used as an explant for co-cultivation with Agrobacterium rhizogenes. A wild-type strain of A.

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Akasaka et al.—Morphological Alteration and Nodulation on Peanut Hairy Roots

rhizogenes, MAFF-02-10266 (mikimopine type), was isolated from hairy roots of melon plants grown in a glasshouse at Chiba, Japan (Daimon, Fukami and Mii, 1990). Inoculation conditions The bacterial culture used for the inoculation was prepared by culturing a bacterial colony in 20 ml of liquid YEP medium (Chilton et al., 1974) in the dark at 25 °C for 24 h at 200 revs min−". The bacteria were then collected by centrifugation and resuspended in liquid MS basal medium (Murashige and Skoog, 1962) supplemented with 10 mg l−" acetosyringone at a density of 1¬10) cells ml−". The cut surface of the epicotyl was inoculated by bringing it into contact with filter paper wetted with the bacterial suspension. As a control, MS basal medium containing no bacteria was used to wet the filter paper. The experiment was conducted with 60 explants for each treatment. Establishment of composite plants After 15 min of infection with bacterial cultures, the explants were transferred onto 4 g l−" gellan gum-solidified 1}2 MS medium (in which inorganic salts of MS basal medium were halved) supplemented with Vitamin B5 (Gamborg, Miller and Ojima, 1968), 30 g l−" sucrose and 10 mg l−" acetosyringone. After 24 h of co-cultivation in the dark at 25 °C, explants were transferred to the same medium without acetosyringone but containing 200 mg l−" cefotaxime (Hoechst Japan Ltd) and subcultured at 7 d intervals to eliminate the bacteria. The cultures were incubated under a 16 h photoperiod provided by cool white fluorescent lights at 60 µmol photons m−# s−" at 25 °C. After the third subculture on cefotaxime-containing medium, those explants with 5–8 cm long roots emerging from the inoculated site were rinsed in sterile deionized water containing cefotaxime. Half of the explants with roots were transferred to pouches (17±8¬16±5 cm, Mega International, USA), each containing 20 ml of Broughton and Dilworth (1971) (BD) medium ; the others were transferred to the plant box (Magenta Inc., USA) containing 4 g l−" gellan gum-solidified 1}2 MS medium supplemented with B5 vitamins and 30 g l−" sucrose. The pouches were arranged in a rack to keep the rooting zone dark. The rack with pouches and the plant boxes were incubated under the same conditions used to eliminate bacteria. Confirmation of transformation Transformation of the roots induced after infection with A. rhizogenes was confirmed by analysis of both opine and PCR products. Mikimopine, a specific opine produced by the transgenic tissue with the bacterial strain used (Isogai et al., 1988) was analysed in extracts prepared from roots of composite plants by high voltage paper electrophoresis at constant voltage of 450 V for 2 h in running buffer (5 % formic acid, 15 % acetic acid, 80 % deionized water) according to the procedures described by Isogai et al. (1988). For PCR analysis, total DNA of the roots of composite plants was extracted by a sodium dodecyl sulfate

method as described by Honda and Hirai (1990). For the detection of rolC gene, PCR was performed using two oligonucleotide primers (1724C–1724D) (Kiyokawa et al., 1992) with 40 cycles of the following sequential thermal treatments : 1 min at 94 °C, 1±5 min at 55 °C and 2 min at 73 °C. Amplified DNAs were detected by ethidium bromide staining after 1 % agarose gel electrophoresis. Fractal analysis of the root system of the composite plant The profile of the root system of the composite plant as it appeared on the pouch surface was photocopied after 40 d of culture. The photocopied root system was digitized with an image scanner (ScanJet II cx, Hewlett Packard Inc., USA) using a personal computer (Power Macintosh 7100}80AV, Apple Computer Inc., USA). The processed root image was used to analyse the fractal dimension. The fractal dimension, D value, ranging from 0±35 to 5±6 mm in length of a side of a pixel as scaling factor was measured according to the method described by Tatsumi, Yamauchi and Kono (1989). The experiment was conducted with five composite plants. The number of branch roots formed within 5 cm above the newest (i.e. the lowermost branch root) was counted for each primary branch root. For each composite plant, at least five primary roots were evaluated. Root nodule formation on the composite plant After 14 d of culture in the plant box, the composite plants were transferred to pots (7 cm in diameter, 10 cm in depth) filled with sterilized vermiculite, and grown under a 16 h photoperiod at 25 °C. Twenty ml of BD medium was poured into each pot at 5 d intervals. Three days after transfer, each pot was inoculated with 1 ml suspension culture of Bradyrhizobium sp. strain A2R1. The bacteria used for the inoculation had been cultured in 20 ml of YMB (Somasegaran and Hoben, 1985) medium in the dark at 25 °C for 7 d at 200 revs min−", and the density adjusted to 1¬10) cells ml−" just before inoculation. Nitrogenase activity of the composite plant was measured by the acetylene reduction method according to Hardy et al. (1968). The experiment comprised eight pots for each treatment. Light microscopy Root and nodule samples of the composite plant were fixed with 3 % (w}v) paraformaldehyde and 2 % (v}v) glutaraldehyde in 0±2  cacodylate buffer at pH 7±2, dehydrated through a graded ethanol series, and infiltrated and embedded in Technovit 7100 (Heraeus Kulzer, Germany). Embedded samples were sectioned to 8 µm on a microtome (Yamatokoki Inc., Japan), stained with 0±05 % toluidine blue, and viewed and photographed with a light microscope (IX70, Olympus Inc., Japan). RESULTS AND DISCUSSION Production of the composite plant Ten to 14 d after inoculation with A. rhizogenes, small outgrowths followed by root induction occurred at the cut


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T     1. Effect of inoculation with A. rhizogenes MAFF02-10266 strain on root formation at the base of epicotyl explant after 21 d culture Treatment

% of explants showing root formation

Number of roots per explant

Control Inoculation t-test

90±0 61±7 **

3±6 1±8 **

Values are means of 60 explants. ** P ! 0±01.

F. 2. Electrophoretic analysis of mikimopine production (A) and PCR analysis of integrated Ri T-DNA (B) in A. rhizogenes-induced roots of composite plants in peanut cv. Java 13. A, Lane 1, mikimopine marker ; lane 2, histidine marker ; lanes 3, 4 and 5, A. rhizogenesinduced roots ; lanes 6 and 7, control roots. The arrow indicates mikimopine. Note the absence of mikimopine in the control roots. B, Lane 1, pHY marker ; lane 2, bacterial DNA as a control (strain MAFF-02-10266) ; lanes 3, 4 and 5, DNA of A. rhizogenes-induced roots ; lanes 6 and 7, DNA of control roots. The arrow indicates the amplified bands of rol genes. F. 1. Composite plant produced by inducing transgenic adventitious roots at the base of epicotyl explant 30 d after inoculation with A. rhizogenes strain MAFF-02-10266 in peanut cv. Java 13. A, Composite plant with A. rhizogenes-induced roots. B, Control plant. Bars ¯ 30 mm.

surface of the epicotyl explant. The frequencies of root formation 21 d after inoculation were 62 % in explants inoculated with A. rhizogenes and 90 % in control explants. The number of roots per explant in the control was greater than that in explants inoculated with bacteria (Table 1). Hairy root phenotype is characterized by more rapid growth and more branching than non-transformed roots in many plant species (Tepfer, 1983 ; Porter, 1991 ; Handa, 1994 ; Daimon and Mii, 1995). In the present study, the growth rate of roots in the inoculated explant was higher than that in the control, and the phenotypes of roots of the

inoculated explants were remarkably different from the control, i.e. the inoculated explants had a thinner primary adventitious root and produced more lateral branching roots (Fig. 1). Non-transformed roots, which were characterized by slow growth and less branching, were rarely formed in the inoculated explants. Production of mikimopine and integration of Ri T-DNA as evidence of transformation were found in all of the proliferated roots with vigorous lateral branching (Fig. 2), but not in the roots of the inoculated explant which had a similar phenotype to the control. Thus, the composite plants consisting of transformed (hairy) roots formed on non-transformed shoots were generated, and they were used to evaluate the root system and root nodule formation. Any explants with normal root phenotypes were not used for the subsequent experiments.

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Akasaka et al.—Morphological Alteration and Nodulation on Peanut Hairy Roots T     2. Effect of inoculation with A. rhizogenes MAFF02-10266 strain on fractal dimension and number of branch roots on composite plants after 40 of culture

Treatment

Fractal dimension (D value)

Number of branch roots within 5 cm root

Control Inoculation t-test

1±21 1±44 **

22±1 30±4 **

Values are means of at least five primary roots per composite plant using five plants. ** P ! 0±01.

F. 3. Comparison of root system profiles between composite plant (A) grown in a pouch 40 d after inoculation with A. rhizogenes and control plant (B) of peanut cv. Java 13. C and D, Digitized images of A and B, respectively. Bars ¯ 40 mm.

Adventitious roots, including both transformed and nontransformed roots, emerged 5–7 d later in inoculated plants than in control explants. In A. rhizogenes-mediated transformation on Erica¬darleyensis, a similar delay in root induction in inoculated plants was reported (Viemont and Lambert, 1994). It is possible that the delay was caused by substances secreted by the bacteria. The effect of factors such as the medium being used for bacterial culture and inoculation with bacteria harbouring disarmed plasmid on the root emergence from epicotyl explants should be investigated to clarify the reason for this delay. Ealuation of the root system of the composite plant Morphological alterations of transformed roots by inoculation with A. rhizogenes have been reported in several plant species such as Antirrhinum majus (Handa, 1994), Nicotiana tabacum (Tepfer, 1983), Nierembergia scoparia (Godo et al., 1997) and Rudbeckia hirta (Daimon and Mii, 1995). However, little information is available on root branching. In the present study, phenotypes of the transformed roots of peanut were quantitatively evaluated by fractal analysis and by counting the number of branches. Figure 3 A and B shows the root system of the composite plant as it appeared on the surface of the pouch 7 d after transfer, i.e. 40 d after inoculation with A. rhizogenes. Composite plants possessed abundant, intricate root mass with more lateral branching compared to the control. Remarkable differences in the root system were observed

F. 4.Occurrence of rosette-type root hairs at the base of the branch root formed on the induced primary roots of (A) transformed and (B) non-transformed root. Bars ¯ 2 mm.

between transformed and non-transformed roots by digitizing the root system on the image processor (Fig. 3 C and D). It has been demonstrated that the profile of the root system is fractal in several crops, and the fractal dimension, D value, was considered an indicator of the intricacy of the root system (Tatsumi et al., 1989 ; Bahman et al., 1993). In peanut, the D value of the root system of the composite plant was significantly higher than that in the control (Table 2). The number of branch roots per unit area in the


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composite plant was significantly larger than that in the control. These results clearly indicate the intricacy of peanut hairy roots induced by A. rhizogenes compared to normal roots. Morphology of the transformed root Peanut lacks root hairs on the surface of the tap root because the surface layers are shed, but it has rosette-type root hairs up to 4 mm long at the base of lateral roots (Yarbrough, 1949 ; Meisner and Karnok, 1991 ; Uheda, Akasaka and Daimon, 1997). The detailed process of infection by rhizobia in peanut is not known at present, although rosette-type root hairs might be involved in early stages of infection (Chandler, 1978 ; Nambiar et al., 1983 ; Boogerd and van Rossum, 1997). In the present study, the rosette-type root hairs were observed at the base of the branch roots of both transformed and non-transformed primary roots (Fig. 4). Histological observation also showed that transformed roots had similar traits to non-transformed roots, i.e. both roots did not exhibit shedding of surface layers but developed epidermal cells, cortex and central stele delimited by an endodermis (Fig. 5). The morphological similarity between the two categories of roots may be due to the fact that both transformed and non-transformed roots induced in the present experiment were not tap roots and their laterals but adventitious roots formed at the epicotyl of the explant. F. 5. Cross section of the induced primary root of composite plant (A) and that of non-transformed root (B) after 40 d of culture in a pouch. Note the close similarity between these two roots. Bars ¯ 500 µm.

Nodulation of the hairy root system Forty days after inoculation with Bradyrhizobium sp. A2R1 strain, root nodules were observed in both the

F. 6. Formation of root nodules at the branching sites of roots in the composite (A) and control (B) plant. The arrows indicate nodules. Bars ¯ 10 mm.

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F. 7. Microscopic observation of the root nodules formed on the transformed roots of the composite plant of peanut cv. Java 13 after 40 d of culture in a pot. A and B, Normal phenotype of root nodule. C and D, Enlargement of nodule cortex cells. An asterisk indicates outgrowth. E and F, Root emergence from surface of the root nodule. An asterisk indicates the new emergence root. A, C and E, Bars ¯ 2 mm ; B, D and F, Bars ¯ 1 mm.

transformed and non-transformed root systems. All nodules in the transformed root system were only formed at the branching site of roots as occurs in the normal root system of peanut (Fig. 6 A and B). Red pigmentation of leghaemoglobin was observed in the bacterial region of the nodules. Similar levels of nitrogenase activity, measured as acetylene reduction, were found in nodules from nontransformed and transformed roots (25³6 and 23³11 nmol C H per plant h−", respectively). # % Although peanut generally has determinate type nodules (Fig. 7 A and B), some nodules produced on the transformed

roots exhibited abnormal phenotypes such as enlargement of the nodule cortex region (Fig. 7 C and D) and de noo root formation from the nodule cortex (Fig. 7 E and F). Similar morphological alterations of A. rhizogenes-mediated transformed root nodules in soybean were reported by Bond and Gresshoff (1993). They found the presence of multiple active meristems in the nodule cortex and showed that those nodules were elongated and branched and similar to those known as indeterminate root nodules found in pea and alfalfa. As incorporation of Ri T-DNA into the plant genome might cause a change in auxin metabolism, the


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the critical understanding of the structure and function of transformed roots in other plant species for which it is difficult to regenerate the whole plant. A C K N O W L E D G E M E N TS We thank Dr Hazel Wetzstein (University of Georgia, Athens, GA, USA) for her helpful suggestions in this study, Dr S. Kiyokawa for supplying the primers used in PCR analysis and the Chiba Prefectural Agricultural Experimental Station for supplying the seeds of peanut cv. Java 13. This work was supported in part by Grant-in-aid No. 08660019 to H. D. from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED

F. 8. PCR analysis of integrated Ri T-DNA in A. rhizogenes-induced roots of composite plants in peanut cv. Java 13. Lane 1, Øx174}HaeIII digest ; lane 2, bacterial DNA as a positive control (strain MAFF-0210266) ; lane 3, DNA of the nodule on control plant ; lane 4, DNA of transformed nodule on composite plant. Arrow indicates the amplified bands of rol genes.

endogenous phytohormonal imbalance would stimulate the nodule cortex to enlarge and differentiate into de noo root formation. In this study, we could select composite plants consisting of transformed roots formed on non-transformed shoots by remarkable characteristics of the roots such as rapid growth and extensive lateral branching 3 weeks after inoculation with A. rhizogenes. In a pouch experiment, emergence of new roots from the base of the inoculated explants after transfer to the pouch was not found during the experiment. Therefore, it can be concluded that the transformed root systems of the composite plants would be appropriately evaluated in the present study. In the nodulation experiment, however, it is possible that the composite plants might produce non-transformed roots during pot culture due to the difficulty in observing the root system. In the present study, therefore, all of the nodules which showed de noo root formation from the cortex region were confirmed as transformed root nodules by PCR analysis (Fig. 8) suggesting that all of the composite plants analysed in the present study possessed only transformed roots. To distinguish the non-transformed roots which could unexpectedly appear on composite plants, it is beneficial to establish hairy roots with assayable marker genes such as β-glucronidase (gus), luciferase (luc) and green-fluorescent protein (gfp). The best way to exclude the possibility of contaminated non-transformed roots is, of course, the establishment of transgenic plants of peanut with A. rhizogenes. However, there has been no report of successful plant regeneration from peanut hairy roots. Therefore, the composite plant described here could help in

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