Efficient callus formation and plant regeneration of goosegrass ...

Plant Cell Rep (2003) 21:503–510 DOI 10.1007/s00299-002-0549-6

CELL BIOLOGY AND MORPHOGENESIS

A. I. Yemets · L. A. Klimkina · L. V. Tarassenko Y. B. Blume

Efficient callus formation and plant regeneration of goosegrass [Eleusine indica (L.) Gaertn.] Received: 12 November 2001 / Revised: 10 October 2002 / Accepted: 11 October 2002 / Published online: 14 December 2002 © Springer-Verlag 2002

Abstract Efficient methods in totipotent callus formation, cell suspension culture establishment and wholeplant regeneration have been developed for the goosegrass [Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1–2 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l–1 glycine, 100 mg l–1 asparagine, 100 mg l–1 casein hydrolysate, 30 g l–1 sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l–1 kinetin (Kn) and 0.1 mg l–1 indole-3acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N6 mineral salts with an additional 0.2 M KCl and 0.1 M CaCl2 (pH 5.4–5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l–1 2,4-D and 0.2 mg l–1 Kn.

Communicated by K. Glimelius A.I. Yemets (✉) · L.A. Klimkina · L.V. Tarassenko · Y.B. Blume Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Acad. Zabolotny str., 148, 03143, Kiev, Ukraine e-mail: [email protected]

Keywords Eleusine indica · Somatic embryogenesis · Organogenesis · Dinitroaniline herbicides · Cross resistance · Protoplasts Abbreviations Asp: Asparagine · BA: 6-Benzylaminopurine · 2,4-D: 2,4-Dichlorophenoxyacetic acid · DNH: Dinitroaniline herbicides · Gly: Glycine · IAA: Indole-3-acetic acid · Kn: Kinetin

Introduction Goosegrass [Eleusine indica (L.) Gaertn.] is one of the most widespread weeds occurring in croplands. To date, the dinitroaniline herbicides (for example, trifluralin, oryzalin, pendimethalin, ethalfluralin) have been widely used to control goosegrass in arable crops (Smeda and Vaughn 1994; Vaughn 2000). The dinitroaniline herbicides target tubulin, which is the principle constituent protein of microtubules (MTs) (Vaughn 2000). The general mechanism of DNH is to alter the ability of tubulin to polymerise into MTs, and the inhibition of this polymerisation process results in the eventual loss of all MTs (Smeda and Vaughn 1994; Vaughn 2000). MTs are crucial for such cellular processes as cell-shape establishment, chromosome movement during mitosis and cell-plate formation (Smeda and Vaughn 1994; Vaughn 2000). Repeated application of DNH on field-grown crops is considered to be the causal factor for the appearance of resistant (R) biotypes of E. indica from susceptible (S) ones (Mudge et al. 1984). The highly resistant biotype has a 1,000–10,000-fold higher resistance to trifluralin than the S-biotype and also exhibits cross-resistance to another DNH as well as to the phosphorothioamide herbicide amiprophosmethyl (APM) (Smeda and Vaughn 1994). Genetic segregation analyses on F1 hybrids and F2 and F3 generations indicated that the DNH-resistance phenotype is inherited as a single, recessive nuclear gene (Zeng and Baird 1997). Moreover, both Cronin et al.

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(1993) and Yamamoto et al. (1998) proved independently that resistance to dinitroanilines in the R-biotype of E. indica is related with α-tubulin (TUA1) missense mutation, which converts Thr-239 to Ile in the R-biotype. Since DNH are still commonly used in agriculture, the production of dinitroaniline-resistant crops may be of practical interest (Baird et al. 2000). One of the ways to produce such plants is to transfer the trait of DNH resistance from corresponding sources (mutants) to recipients using cellular (Yemets et al. 1997; Yemets and Blume 1999) or genetic engineering (Antony et al. 1998) techniques. To date, it has been impossible to obtain genetically modified regenerated monocot plants with resistance to DNH due to peculiarities of the autoregulatory mechanisms of α- and β-tubulin co-expression, which is probably suppressed by transformation with exogenous tubulin genes, especially under powerful promoters (Yemets and Blume 1999). Taking into account the importance of cellular engineering methods for the transfer of the desirable traits, we have established in vitro culture system for the DNH-resistant biotypes of E. indica. Such cultures can be suitable sources for the transfer of DNH resistance into different cereals by, for example, somatic hybridisation or micronucleation. In vitro cultures of such biotypes would also be useful material for cell biology studies. For example, the embryogenic goosegrass model developed here could be used to investigate: (1) the tissue specificity of tubulin gene expression, especially of mutant α-tubulin, during differentiation and mechanisms involved in the regulation of tissue-specific expression of mutant tubulin; (2) the possibility to produce mini-chromosomes (via microprotoplasting) for further creation of artificial chromosomes with tubulin genes; (3) peculiarities of microtubular organisation in the processes of differentiation and embryogenesis (using specific monoclonal antibodies produced against altered tubulin). As new crop varieties of this weedy species have been recently developed in the Ukraine, these newly developed protocols for cell and tissue culture establishment, as well for protoplast isolation, may also form the practical basis for further in vitro manipulations with these new E. indica varieties. These procedures may be first step in the biotechnological development of herbicideresistant goosegrass crops as well as an approach for improving such closely related agricultural species as finger millet, E. coracana. In this paper we report the development of methods for inducing totipotent calli in vitro, resulting in our obtaining cell suspension cultures and regenerating R- and S-biotype plants. We also describe the peculiarities of the regeneration processes of E. indica via somatic embryogenesis and organogenesis under in vitro conditions by presenting data from a histological analysis. Our results revealed the preservation of resistance to trifluralin and cross-resistance to APM in established totipotent calli of the E. indica R-biotype. We also present our elaborated method for goosegrass protoplast isolation.

Materials and methods Plant material Eleusine indica seeds of DNH-resistant biotype RGG and susceptible biotype SGG were obtained from the American Cyanamid Co. (New Jersey), and DNH-resistant biotypes MSC-R and MGAR and susceptible biotypes ASC-S and GTN-S were kindly provided by Prof. W.V. Baird (Clemson University, S.C.). Coleoptiles, mesocotyles and young leaves from seedlings of both R- and S-biotypes were used as initial explants for callus induction. Herbicides Trifluralin [2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine] was obtained from Dr. L. Guse (DowElanco, Greenfield, USA). APM, also known as Bay NTN 6867, was obtained from Dr. J.R. Bloomberg (Agriculture Division, Miles, Kansas City, Kan.). The preparation of the herbicide stock solutions and their addition into media were carried out as described by Yemets et al. (2000). Seed sterilisation and germination Seeds were surface-sterilised for 7 min in 70% (v/v) ethanol, followed by 10 min in hypochlorite, and then washed twice for 20 min each time in sterilised distilled water and once for 10 min in media-A (a combination of different mineral salts; W.V. Baird, personal communication). The seeds were then germinated under sterile conditions on filter papers moistened with medium-A in petri dishes. The dishes were incubated in darkness for the initial 12 h at 36–37°C and then at 25°C for 3–5 days. Basal medium and incubation conditions The basal medium used in this investigation consisted of N6 basal salts (Chu et al. 1975) and B5 vitamins (Gamborg et al. 1968) supplemented with 30 g l–1 sucrose. Except for the cell suspension medium, all media were solidified with 6 g l–1 agar. The pH of all media was adjusted to 5.7 before autoclaving. All cultures were incubated in controlled environment chambers at 25±1°C. Callus culture The following phytohormones and medium supplements were tested for callus induction: (1) 2,4-D at concentrations of 1–6 mg l–1 and 30 g l–1 sucrose; (2)1 mg l–1 2,4-D, 0–0.1 mg l–1 Kn and different organic supplements (Table 1). After induction, the calli were maintained on the same media or on media with reduced 2,4-D (from 2.5 mg l–1 to 2 mg l–1, or from 2 mg l–1 to 1–1.5 mg l–1), depending upon the biotype. Calli were incubated in darkness. Cell suspension cultures To establish suspension cell cultures, we transferred callus, at early stages of formation, to liquid basal medium supplemented with 1–2 mg l–1 2,4-D, 2 mg l–1 Gly, 100 mg l–1 Asp and 100 mg l–1 casein hydrolysate. Suspension cultures were incubated in 250-ml Erlenmeyer flasks containing 80 ml of liquid medium on a shaker at 180 rev min–1. Development of morphogenic structures Calli with morphogenic structures at early developmental stages were transferred either directly to development medium, or first to

505 Table 1 Media for callus induction from Eleusine indica explants Media

Mineral salts, vitamins, sucrose

2.4-D (mg l–1)

Kinetin (mg l–1)

Glycine (mg l–1)

Asparagine (mg l–1)

Casein hydrolysate (mg l–1)

A B C D E F G H I J K L M N

N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3% N6+B5, 3%

1 1 1 1 1 1 1 1 1 1 1 1 1 1

– – – – – – – 0.1 0.05 0.05 0.05 0.05 0.05 0.05

– 2 – – 2 2 2 – – 2 2 2 2 –

– – 150 – – 150 100 – – – 150 – 100 150

– – – 250 250 – 100 – – – – 250 100 –

induction medium with the 2,4-D concentration reduced to onehalf for 1–2 weeks prior to transfer to the development medium. The development media consisted of basal medium but only with MS salts (Murashige and Skoog 1962) supplemented with 1 mg l–1 Kn or BA and 0.1 mg l–1 IAA. Calli transferred onto these media were incubated under a 12/12-h (light/dark) photoperiod. Plant regeneration Rooted plantlets were isolated from the development medium and subcultured on a hormone-free MS medium under 14/10-h (light/dark) photoperiod. Plantlets that developed well-developed roots were subsequently transferred to soil for further growth. Histological analysis Nodular morphogenic calli at various stages of development were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2) at room temperature for 12 h, washed in distilled water, dehydrated in a graded series of ethanol and then xylol-substituted (in place of the ethanol). The specimens were embedded in paraffin, and serial sections (5–15 µm) were cut on a rotary microtome. The sections were stained with Shiff’s dye, counterstained with a 1% Fast Green FCF (Sigma, St. Louis, Mo.) solution in ethanol and examined under the optic microscope. Initial screening of cell lines for herbicide resistance The capacities of the resultant callus lines for resistance to DNH and cross-resistance to phosphorothioamidates were evaluated. Callus lines from the resistant (MGA-R) and susceptible (GTN-S) biotypes were incubated on induction media supplemented with 0.1–100 µM trifluralin or APM as described earlier (Yemets et al. 2000). The comparative growth rates of calli were calculated by the following formula:

where W=the comparative growth rate of callus, M=4-week-old callus weight and m=initial callus weight. The experiments were repeated three times, and mean values were defined for each of the points.

Protoplast isolation Mesophyll tissue, callus and embryogenic cell suspension culture were used for refining the method of protoplast isolation from E. indica. We tested 12 different combinations of enzymes dissolved in six different osmotic media. The enzymes cellulase Onozuka R-10 (Serva, Germany), driselase (Sigma), pectolyase Y-23 (Kikkoman, Japan) and hemicellulase (Sigma) in various concentrations and combinations were used. Osmotic media were prepared on the basis of sucrose and mannitol or MS and N6 mineral salts with the addition of KCl and CaCl2 for osmotic maintenance. The protoplasts were isolated and purified using the following steps: 1. Suspension culture cells (5–10 ml) were pelleted by centrifugation at 800 rpm for 5 min. 2. Cells were placed in 10 ml of a filter-sterilised enzyme solution in a petri dish and incubated for 18–20 h on an orbital shaker, 100 rpm, at 25°C, in the dark. The enzyme solution consisted of 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N6 mineral salts supplemented with 0.2 M KCl, 0.1 M CaCl2, pH 5.4–5.5. 3. Protoplasts were separated from undigested cell clumps by filtering through a 60- to 70-µm sterile nylon mesh into a centrifuge tube and washed with 3–4 ml of washing solution (WS; 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose). 4. Protoplasts were precipitated by centrifugation at 600–800 rpm for 5 min. After removal of the supernatant, 2–3 ml WS was added. 5. Protoplasts were teased carefully onto the surface of a 3- to -4ml aliquot of 20% sucrose solution in a fresh centrifuge tube and centrifuged at 600–800 rpm for 5 min. 6. A white band of protoplasts that formed after at the interface of the sucrose and W5 solution is removed diluted to 5–10 ml in WS (v/v) for cell counting. The purified protoplasts were cultivated in KM8p (Kao and Michayluk 1975) liquid medium supplemented with 2 mg l–1 2,4-D and 0.2 mg l–1 Kn. Regeneration and cell division were visible approximately 7–10 days later. After 30 days of cultivation microcalli were embedded into solid KM8p medium for further plant regeneration.

506 Table 2 Influence of 2,4-D on callusogenesis of E. indica biotypes 2,4-D (mg l–1)

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

RGG

SGG

MSC-R

ASC-S

Intensity of callusogenesisa

Type of callusb

Intensity of callusogenesisa

Type of callusb

Intensity of Type of callusogenesisa callusb

Intensity of Type of callusogenesisa callusb

+ + + ++ + + ++ ++ + + +

White, – Cream-coloured, – Cream, – Cream, s/a Cream, – Cream, – Cream, s/a Yellow, rl Yellow, rl Yellow, rl Yellow, rl

++++ +++ ++ ++ +++ ++ ++ ++ ++ ++ ++

White, s White, s Cream, s Cream, s Cream, s/a Cream, s/a Cream, s/a Cream, rl Yellow, rl Yellow, rl Yellow, rl

++++ +++ +++ +++ ++ ++ ++ ++ – – –

+ + ++ ++ ++ + + + – – –

White, s Cream, s Cream, s Cream, s Yellow, s Brown, s Brown, – Brown, rl

Cream, – Cream, – Cream, – Cream, s Cream, s Cream, – Yellow, – Brown, –

++++, No fewer than 50% of explants with colonies having diameters of 7–8 mm and more; +++, no fewer than 25% of explants with colonies having diameters of 8–10 mm; ++, fewer than 25% of explants with colonies having diameters of 4–6 mm; +, single

cases of callusogenesis, very small and dying colonies; –, not investigated b Type of callus: s structured, a amorphous, s/a partially structured; –, difficult to establish the type of callus; rl with root-like structures

Results and discussion

ASC-S biotype explants did not form calli on media B-E, G, J-M. Explants from MSC-R, MGA-R and GTN-S biotypes formed friable, white, cream- or yellow-coloured type II calli with high viability on some media (Table 3). We found that Kn, at a very low concentration (0.05 mg l–1), in combination with 1 mg l–1 2,4-D and in some cases with casein hydrolysate, glycine and/or asparagine positively influenced callus production in the MSC-R (M-medium), MGA-R (L- and M-media) and GTN-S (I-medium) biotypes (Table 3). Nevertheless, we conclude that the most suitable medium for callus formation in all three of these biotypes is the G-medium, which contained basal medium supplemented with 1 mg l–1 2,4-D, 2 mg l–1 Gly, 100 mg l–1 Asp and 100 mg l–1 casein hydrolysate. For the MGA-R and GTN-S biotypes, the F-medium is also suitable (identical to the G-medium but without casein) as is the E-medium for MSC-R. Results from our experiments showed that the presence of Kn did not significantly increase callus production in any of the E. indica biotypes. Figure 1A shows a typical example of friable, white type II callus. We also obtained homogeneous, dispersed suspension cultures of four E. indica biotypes (MSC-R, MGA-R, GTN-S and ASC-S) on liquid medium supplemented with 1–2 mg l–1 2,4-D, 2 mg l–1 Gly, 100 mg l–1 Asp and 100 mg l–1 casein hydrolysate. Cell suspension cultures consisted of individual cells and small clumps/microcalli (Fig. 1B). Some of the cells could be differentiated morphologically. These cells had a lengthy, curved or spirallike form, but most individual cells and cells of microcalli were round, sometimes with small ledges, which is typical for suspension culture cells.

a

Establishment of calli and suspension cultures Calli were produced primarily from coleoptile and mesocotyle explants after 2–3 weeks in incubation media. Root-like structures developed from calli cultured on media supplemented with high levels of 2,4-D (4.5 mg l–1 and higher), while calli cultured on media with reduced levels of 2,4-D varied in colour (white, cream, yellow, brown) and structure (structured, partially structured, amorphous) (Table 2). For example, the SGG and MSC-R biotypes formed white, well-structured calli on media supplemented with 1–2 mg l–1 2,4-D and, in general, they expressed efficient callus induction and high rates of growth. Callus induction in the RGG and ASC-S biotypes was weaker than that in the SGG and MSC-R lines. The most suitable concentrations of 2,4-D for callus formation in these lines were 2.5–4 mg l–1 for the former two and 2–3 mg l–1 for the latter two. However, for further maintenance of viable calli it was necessary to decrease the 2,4-D concentration to 1–2 mg l–1. We conclude that the N6-based media are suitable for callus formation in E. indica and that each genotype has a different callus induction potential. Organic supplements such as some amino acids (Gly, Asp, glutamine, proline), casein hydrolysate or yeast extract can positively influence callus growth of some cereals (Torres et al. 1989). Taken further, in some species the addition of cytokinins, in particular Kn, at low levels to medium containing 2,4-D can significantly increase the percentage of callus formation (Sorghum for example; Gendy et al. 1996), or it can simply be necessary for callus establishment (palmarosa grass for example; Patnaik et al. 1997). We have also studied the influence of Kn in combination with 2,4-D and other organic supplements on callus formation in E. indica (Table 3).

507 Table 3 Influence of kinetin and specific organic compounds on callus formation of E. indica biotypes Medium

A (12,4-D) B (12,4-D,Gly) C (12,4-D,Asp) D (12,4-D,Casein) E (12,4-D,Gly,Casein) F (12,4-D,Gly,Asp) G (12,4-D,Gly,Asp,Casein) H (12,4-D,0.1Kn) I (12,4-D,0.05Kn) J (12.4-D,0.05Kn,Gly) K (12,4-D,0.05Kn,Gly,Asp) L (12,4-D,0.05Kn,Gly,Casein) M (12,4-D,0.05Kn,Gly,Asp,Casein) N (12,4-D,0.05Kn,Asp)

MSC-R

ASC-S

MGA-R

GTN-S

Intensity of callus formationa

Type of callusb

Intensity of callusa

Type of callusb

Intensity of callusa

Type of callusb

Intensity of callusa

Type of callusb

++ ++ ++ + ++++ +++ ++++ + ++ ++ ++ ++ +++ ++

cy/n cy/n cy/n yb/p w/f wcy/f w/f yb/p cy/n cy/n cy/n cy/n wcy/f cy/n

+ – – – – + – + + – – – – +

yb/p – – – – yb/p – yb/p yb/p – – – – yb/p

+ + +++ ++ +++ ++++ ++++ + ++ + ++ +++ +++ ++

yb/p yb/p wcy/f cy/n wcy/f w/f w/f yb/p cy/n yb/p cy/n wcy/f wcy/f cy/n

+ + ++ ++ ++ ++++ ++++ + +++ + ++ ++ ++ +

yb/p yb/p cy/n cy/n cy/n w/f w/f yb/p wcy/f yb/p cy/n cy/n cy/n yb/p

a Intensity of callus formation: ++++, +++, ++, +, as described in Table 2; –, callus formation was absent b Type of callus: w/f white, friable callus colonies formed; wcy/f white, cream-coloured or yellow, friable callus colonies formed;

cy/n cream-colored or yellow, non-friable callus colonies formed; yb/p yellow or brown callus poorly formed; –, callus formation was absent

Morphogenesis and plant regeneration from calli

Histological data

After the explants had been incubated for 10–14 days in darkness in induction media with the 2,4-D concentration reduced to one-half, white, dense type I calli with well-organised structures that resembled embryos or small leaves appeared (Fig. 1C). These colonies turned green within 2–3 days following their transfer to light. Among these greenish cultures, typical signs of embryogenesis and organogenesis were observed, the presence of which was subsequently confirmed by histological analysis. For plant regeneration, embryogenic and organogenic calli were transferred to the development media. Plant regeneration occurred more quickly and intensively in callus colonies first subjected to a intermediate step involving culture in induction media with a decreased 2,4-D concentration than in those transferred directly to the development media. Kn was observed to more potent than BA in combination with IAA for plant development of E. indica. For example, after only 20 days of culture the totipotent calli regenerated into plantlets, while in BA-supplemented medium, about 40 days were needed. Sivadas et al. (1990) demonstrated that Kn had a promotive effect on somatic embryogenesis and plant regeneration in finger millet, E. coracana, however, Pius et al. (1994) showed that regeneration of E. coracana embryogenic callus into plantlets could occur on medium lacking phytohormones. We therefore transferred the regenerated and rooted E. indica plants (Fig. 2A) to hormonefree medium for further growth and development.

Histological investigations were conducted on two type I callus types of E. indica: (1) white nodular, folded calli (Fig. 1C) and (2) more advanced greenish callus masses with visible green shoot tips and sometimes with a delicate white net of slender roots and/or numerous darkgreen leaves (Fig. 1D). The callus mass was composed of large vacuolated, irregular-shaped cells and many scattered clusters, or a few lamina, composed of small spherical cells with dense cytoplasm and distinct nuclei (Fig. 1E–G). These latter cell aggregates actively proliferated. As a result, almost all regions of the callus contained some morphogenic or embryo-like structures. Both bipolar embryogenic structures (Fig. 1E) and unipolar organogenic structures with vascular strains connected to the callus tissue (Fig. 1F, G) were found on E. indica type I callus sections. The morphogenetic processes in E. indica calli, simultaneously expressed through somatic embryogenesis and organogenesis, resulted in shoot, root and bud formation. This situation is not rare for cereals (Wernicke et al. 1982; Vasil 1988; Bhaskaran and Smith 1990; Welter et al. 1995). In fact, somatic embryogenesis (Eapen and George 1989), direct shoot morphogenesis (Rangan 1976) and multiple bud formation (Wakizuka and Yamaguchi 1987) have been shown for another Eleusine species (E. coracana). However, for E. coracana, these morphogenetic pathways were achieved in different callus cultures and with different cultural procedures. An interesting point is that in E. indica separate organ (roots or leaves) and bud induction was present not only on the callus surface but also inside the callus masses. As such, the developing structures had to push and destroy surrounding cell layers for their growth on and through the callus surface.

508 Fig. 1 A Friable type II callus. Bar: 1 mm. B Embryogenic cell clumps in the cell suspension culture. Bar: 25 µm. C Folded embryogenic and bud-forming type I callus with somatic embryo (arrow). Bar: 2 mm. D Type I callus with shoots and leaves on the callus surface. Bar: 2.5 mm. E Callus section showing bipolar embryogenic structures. Bar: 100 µm. F, G Callus sections showing organogenic buds with vascular strains connecting to the callus tissue. Bar: (F): 230 µm, bar (G): 160 µm. H Section of advanced type I callus with organogenesis (o), several transversal section of leaf (l) and new morphogenic structures. Bar: 140 µm

Initial screening of the cell lines obtained for trifluralin resistance and cross-resistance to APM For estimating resistance to DNH (namely to trifluralin) in the calli, we compared the growth rate of MGA-R and GTN-S calli, which were supposed to be respectively resistant and susceptible (Fig. 3A). Significant differences in the dynamics of the growth rate with increasing trifluralin concentrations were found between callus line

MGA-R and callus line GTN-S. The LD50 of the growth rate of GTN-S callus was approximately 0.26 µM trifluralin, whereas the LD50 for line MGA-R obtained from the resistant biotype explants exhibited 26 µM trifluralin. Therefore, we can conclude that the established callus line MGA-R has a 100-fold higher resistance than line GTN-S obtained from the susceptible biotype. Furthermore, at the lethal concentration for the susceptible line (50 µM and 100 µM), specific individual callus colonies

509

Fig. 2 A In vitro regeneration of Eleusine indica plants. Bar: 20 mm. B Potted in vivo E. indica plants. Bar: 45 mm

Fig. 4 Isolated E. indica protoplasts. Bar: 10 µm

of the MGA-R line remained viable, and the growth rate means at these concentrations were about 30% and 12%, respectively. Our results correspond with the results of an analysis on the dinitroaniline resistance of R- and S-biotypes of E. indica (Vaughn et al. 1987), where a 100-fold increase in resistance to dinitroaniline was also shown when seeds of both are imbibed and germinated in direct contact with herbicide. Callus obtained from a resistant line was cross-resistant to a structurally nonrelated phosphorotioamidate herbicide, APM. The LD50 of the MGA-R callus was 32 µM and for the GTN-S callus line, 0.9 µM, on media with APM. Thus, callus of the R-biotype possessed a 36-fold greater resistance to APM than callus of the S-biotype, which can be explained by the existence of a common site for α-tubulin interaction with APM and DNH in non-resistant plants (Nyporko et al. 2002). Therefore, in spite of the tissue-specific character of expression of the mutant α-tubulin isoform in goosegrass (it expresses especially in roots of the R-biotype) (Yamamoto et al. 1998), a non-differentiated cell line of resistant plant E. indica also exhibits the resistance to herbicides trifluralin and APM – i.e. it keeps the ability to express the mutant tubulin gene. This is very important for further research goals. Protoplast isolation and cultivation

Fig. 3 A Growth rates of calli (%) of the MGA-R and GTN-S biotypes on callus media containing various concentrations of trifluralin. B Growth rates of calli (%) of MGA-R and GTN-S biotypes on callus media containing various concentrations of APM

Mesophyll tissue, callus and embryogenic cell suspension culture were used to refine the method of protoplast isolation from E. indica. Tissues and the cell suspension were digested in 12 various enzyme solutions dissolved in six different osmotic media in various combinations. Osmotic media were prepared on the basis of sucrose and mannitol or MS and N6 mineral salts with the addition of KCl and CaCl2 for osmotic maintenance. It was found that the enzyme solution containing 1.5% cellulase

510

Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N6 mineral salts supplemented with 0.2 M KCl, 0.1 M CaCl2 (pH 5.4–5.5) was the most suitable for protoplast isolation. Embryogenic cell suspension culture was better a source of E. indica protoplast isolation than callus or mesophyll tissue. Our results confirm the results of Bhojwani and Razdan (1996) that embryogenic cell suspension is the most ideal tissue type for cereal protoplast isolation. The protoplast yield from the embryogenic cell suspension was 2–3×106 protoplasts. The protoplasts (Fig. 4) were then cultivated in KM8p (Kao and Michayluk 1975) liquid medium supplemented with 2 mg l–1 2,4-D and 0.2 mg l–1 Kn. Regeneration and cell division took place 7–10 days later, and microcolonies had formed within 30 days of cultivation. The microcalli were then embedded into solid KM8p medium for further plant regeneration. The E. indica protoplasts that were isolated as well as the cell and tissue cultures can be used in further biotechnological and cell biological experiments. Acknowledgements The authors wish to express their thanks to Prof. W.V. Baird (Clemson University, South Carolina, USA) for reading the manuscript and improving its English version. We would like to express our special gratitude to Prof. Emeritus C.-Y. Hu (Wm. Paterson University, New Jersey, USA) for his positive criticism and also for improving English version of the manuscript.

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