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Regeneration and Application: From Suspension Cultured-Derived Inflorescences of Vetiveria zizanioides (L.) Nash to Selection of

Herbicide-Resistant Cell

Nitsri Sangduen and Somporn Prasertsongskun* Department of Genetics, Faculty of Science, Kasetsart University

Bangkok 10900, Thailand Email: <fscinss@ku.ac.th>

Abstract

Cell-suspension culture of the ‘Surat Thani’ ecotype of vetiver (Vetiveria

zizanioides (L.) Nash), syn. Chrysopogon zizanioides (L.) Roberty was initiated from embryogenic region of callus derived from inflorescences in the modified MS medium supplemented with 10-15 μM 2,4-dichlorophenoxy acetic acid (2,4–D). Two types of callus proliferation namely, embryogenic (E) and non-embryogenic (NE) calli were obtained. Both light and scanning electron micrographs were employed to distinguish the surface details as well as cell-shaped of E and NE calli. Light micrograph of E callus was compact, nodular, knobs, white or creamy. NE callus was soft, friable, unorganized light-stained cells, translucent, watery and light yellow. Scanning electron micrographs indicated distinct morphology of cell – shaped. NE cell-shaped was long – like tubular and loosely arranged cells. E callus comprised nodular and knobby, quite deep embedded and tightly packed cell was similar to the typical E callus. These results suggested that the appropriate suspension-cultured medium was modified liquid N6 medium supplemented with 10 mM proline and 2% initial cell density. The regeneration process comprised modified MS medium without 2,4-D plus 2-day dehydration-treated and then culturing for five weeks. The percentage of regenerated plant was 14.5 Exponentially growing cell suspension were sub-cultured with a selection medium containing glufosinate (ammonium DL-homoalanin-4 yl (methyl) phosphinate). The glufosinate-resistant cells grew in a medium containing 5x10-5 M glufosinate was selected by a stepwise selection, and its I50 value was 4.2x10-5M. The selected cells were 170-fold resistant compared with the susceptible cells and retained its capability up to five months. The cell selection system could be used to obtain resistant cell, e.g. herbicide, salinity, acidity and drought.

Keywords: Callus, scanning electron micrographs, cell suspension, regeneration, selection, glufosinate, Vetiveria/Chrysopogon.

Introduction

Vetiver (Vetiveria zizanioides (L.) Nash), syn. Chrysopogon zizanioides (L.) Roberty, a graminaceous plant, seems to have originated in the area from India to Vietnam, has been extensively used for soil and water management. * Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand Email: <psomporn@bunga.pn.psu.ac.th>

It is called the lowland vetiver (Faek Hom in Thai). Its fragrant roots, from which is extracted the essential ‘oil of vetiver’, have been used for centuries for mats and perfumes. Based on an overlap of genetic and morphological data, Veldkamp (1999) combined Vetiveria and Chrysopogon under Chrysopogon. Although this has led to the recognition of Chrysopogon zizanioides (L.) Roberty as a proper classification for Vetiveria zizanioides (L.) Nash, in this paper we will continue to use both names for clarity.

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Tissue culture techniques are used for the mass propagation of novel plants. Cell suspension cultures facilitate the isolation of protoplast, transformation, in vitro selection, physiological and biochemical studies (Ozawa, et al. 1996). Plant regeneration is a critical step in the success of any crop improvement program entailing tissue culture techniques. It can be achieved in two ways: through organogenesis or somatic embryogenesis. Somatic embryo-genesis is thought to be the most common method of plant regeneration in the Gramineae, as reported for several species (Vasil 1998). The detailed characterization of embryogenic tissue cultures highlights the need to use explants from immature organs, such as embryos, leaves and inflorescences, that contain undifferentiated cells (Vasil and Vasil 1981). There were some reports of successful regeneration of vetiver using leaves (Mucciarelli, et al. 1993), inflorescences (Keshavachandran and Khader 1997; Na Nakorn, et al. 1998; Prasertsongskun 2003; Giang 2007) via callus culture. Improvement in vetiver plant, such as herbicide resistance and quality, has been generally achieved via cell selection (Prasertsongskun, et al. 2002). We are unaware of any reports on morphological character of embryogenic and non-embryo-genic calli of vetiver. The project aimed at obtaining: (i) the morphological character of embryogenic and non-embryogenic calli, (ii) the appropriate medium and culture condition for inflores- cence-derived suspension cultured cell and plant regeneration of Vetiveria zizanioides (L.) Nash ‘Surat Thani’ ecotype, and (iii) the vetiver suspension-cultured cell resistant to glufosinate via selection system.

Materials and Methods

Vetiveria zizanioides (L.) Nash, ‘Surat Thani’ ecotype was used as a model plant for tissue culture and genetic manipulation of plant cells. Callus Induction and Proliferation

Young inflorescences of vetiver (Vetiveria

zizanioides (L.) Nash), ‘Surat Thani’ ecotype)

were excised and surface sterilized. Aseptic inflorescences were cultured on MS basal medium (Murashige and Skoog 1962) supplemented with 15 μM 2,4-D (2,4- dichlorophenoxyacetic acid) for 3-4 weeks. Then transferred to modified MS medium containing 10 μM 2, 4-D for another 2 weeks and maintained at 25 ± 2oC under 16 hr light. Embryogenic (E) and non-embryogenic (NE) calli were observed and photographed using light and scanning electron microscope.

For scanning electron microscope observation, E and NE calli were prefixed in a mixture fixative solution (3% gluta-aldehyde, 1.5 % paraformaldehyde and 0.1 M cacodylate buffer), gently shaking at room temperature for 5 hr, refrigerated overnight, rinsed 3-4 times in phosphate buffer, dehydrated in an ethanol series (30-100 % v/v) and critical point dried. Dried specimens were coated with gold-palladium alloy and examined under a Joel JSM 5000 LV scanning electron microscope (SEM).

Establishment of Cell Suspension Culture

E callus was dissected and used to

initiate cell in liquid MS and N6 media for comparison. Various initial packed cell volumes (PCV) at 1, 2, 3 and 4 percent inoculum cell density were experimented. Then the appropriate PCV of calli and medium were used for further experiment with various proline concentrations (0, 5, 10, 15 and 20 mM). The effective proline concentration for growth of secondary calli was chosen. Cultures were placed on a gyratory shaker at 110 rpm in light for 2 weeks and weekly sub-cultured at 1:3 dilution with fresh medium for 5 passages. Growth curve was constructed from PCV measurement at two-day interval, by centrifugation 5 ml suspension at 2000 rpm for 5 min. An average of three replicates were recorded on cell viability and micro-calli separation from mother calli.

Cell viability was estimated by staining cell with FDA (fluorescein diacetate) and Evans blue. The percentage of living and dead cells stained with 0.01 % FDA and 0.1 % Evans blue were recorded.

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Microcalli arising from modified liquid N6 medium with 10 mM proline were prefixed and followed the same procedure as in calli proliferation for SEM observation.

Establishment Plant Regeneration from Cell Suspension

One-month-old cell suspension culture

derived from inflorescences was cultured on a suitable callus induction medium proliferated a soft friable calli. The micro-colonies, sized around 0.4-0.5 mm, were screened for regeneration process. They were used in two experiments. In the first experiment, 0.5 mm microcalli were cultured immediately on regeneration medium without 2, 4-D. In the second experiment, 0.5 mm. microcalli were dehydrated 1, 2, 3, 4 and 5 days for 5 weeks. They were then transferred to the regeneration medium and the percentage of plantlet formation was recorded. The effect of sorbitol and mannitol concentrations on regenerated plant was observed.

Application: Selection of Resistant Cells to Glufosinate

Glufosinate treatment and cell growth

study: Glufosinate (99.1% purity) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) was used. After dissolving the glufosinate in distilled water, it was sterilized with a membrane filtration and added to the culture medium at various concentrations. Cell growth rates were determined by measuring PCV after centrifugation of 5 ml cell suspension at 760 × g for 5 min. Three replicates of each treatment were recorded.

Effect of Glufosinate on Growth of Suspension-cultured Cells

Cell suspension derived from vetiver calli

were first tested for their responses to glufosinate in the modified liquid N6 medium (supplemented with 10 μM 2,4-D and 10 mM proline) containing 0, 10-8, 10 -7,10 -6,10-5,10-4 and 10 -3 M glufosinate. Effects of glufosinate on growth, viability and dose-responded curve of glufosinate-treated cells at six days were reported.

Results and Discussion

Callus Initiation and Proliferation After four-week culturing, callus initiation was visible and a number of small protuber- ances was observed on the surface of the spikelets (Fig. 1A). The initial callus tissue became larger, keeping their smooth epidermal surfaces and forming yellow, white or whitish yellow nodule-like structures. Large masses of inflorescence-derived callus developed into two distinct callus types (Fig. 1B). They were identified as non-embryogenic (NE) callus characterized by translucent fast growing, friable, watery and non-embryogenic structures and embryogenic (E) callus was compact, nodular-like knobby, yellow, white or whitish yellow. NE callus grew rapidly covering a large portion of callus. By applying squash technique, E cells were uniformly stained and small cells with dense cytoplasm and NE cells were light-stained and long-like tubular cells. Observation using a scanning electron micro-scopes, the portion with compact and knobby/ nodular structures contained tightly packed cells and deeply embedded cells similar to the typical embryogenic callus (Fig. 1C). The portions containing long-like tubular and loosely arranged cells were designated as non- embryogenic callus (Fig. 1D). These findings are similar to what described earlier for indica rice (Nabors, et al. 1983) and aromatic rice cv. ‘Khao Dok Mali 105’ (Klamsomboon, 1997; and Sangduen and Klamsomboon, 2000). However, SEM observation of morphological structure revealed what we have described by light microscope. Initiation of Cell Suspension Culture Initial calli started to rapidly divide and increasing when transferred to solid MS medium containing 10 μM 2,4-D comparison with 0 μM 2, 4-D (Fig. 2A). Two types of calli, E and NE, were observed and looked like the above-mentioned calli. Proliferating calli (compact) from the second subculture passage were used for cell suspension cultures. Of the two nutrient media (liquid MS and modified N6 media) evaluated, the optimal

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cell-proliferation was observed in liquid MS medium as well as modified N6 medium (Fig. 2A and 2B). 2,4-D appeared to play a major role on cell suspension initiation in the two media. Newly initiated, the large cell clump in liquid MS medium was observed. Cell suspension growth was recorded by measuring PCV using graduated centrifuge tube. The growth curve (Figs. 2C and 2D) based on the PCV, it comprised two days of initial lag phase and 2-6 days of exponential phase in both media. The effect of proline was obtained , with 10 mM proline, it initiated a distinct growth curve and also enhanced prolific growth of secondary calli as well as separation of microcalli from mother calli (Fig. 3A and 3C). Without proline, it promoted poor initiation of secondary calli, unsustained growth of secondary calli and hardly separated of microcalli from mother calli (Fig. 3B). There were quite a number of reports on roles of proline in plant tissue culture e.g. embryo-genesis promotion in corn (Armstrong and Green, 1985; Vasil and Vasil, 1986), suspension initiation in indica rice (Ella and Zapata, 1993; Klamsomboon 1997 and Sangduen and Klamsomboon, 2002 ) and plant regeneration (Chowdhary, et al. 1993) and also acted as antioxidants to protect cell from oxidative stress (Bors, et al. 1989 ; Flores, et al. 1989). Collectively, our results and those of another studies (Ella and Zapata 1993); Klamsomboon 1997 and Sangduen and Klamsomboon 2002) suggested an important role of proline on prolific growth of secondary calli as well as separation of microcalli from mother calli leading to an established vetiver cell suspension culture for further stress selection experiment. Plant Regeneration from Cell Suspension Suspension culture of the normal cells proliferated a soft friable calli within one month. The size of colonies in modified MS medium with 10 μM 2, 4-D was larger than those in 0, 5, 15 and 20 μM 2, 4-D (Table 1 and Fig. 4). Based on this study, the solid MS medium supplemented with 10 μM 2, 4-D was optimal for induction of small colonies

from cell suspension culture. The dehydration process seemed to promote plant regeneration. After five weeks of transferring small colonies to the regeneration medium with a 2-day-dehydration treatment, the percentage of regenerated plant was 14.5. On the others, after 0, 1, 3, 4 and 5-day-dehydration, there was no sign of regeneration (Table 2). The effects of sorbitol and mannitol in the regeneration process were also tested. The concentration of 0.1 M sorbitol gave higher percentage of plantlets (80 %) whereas 0.1 M mannitol also promoted plantlets (2.5%) (Table 3). Mucciarelli, et al. (1993) reported callus induction and plant regeneration in Vetiveria zizanioides from young basal leaf was obtained via somatic embryogenesis. The maximal viability obtained was 84.1 % while culturing in modified liquid N6 medium supplemented with 10-8 M glufosinate and its viability decreased to 17.1 % at 10-3 M glufosinate (Table 4 ) Based on our results we can postulate that V. zizanioides, in vitro plant regeneration can occur via somatic embryogenesis. We believe that a biotechnological approach for using V. zizanioides could overcome some of the problems found in its current exploitation. Even though it is highly unlikely to produce high levels of essential oils, cell lines cultured in liquid media or immobilized in suitable substrata could be potentially used for in vitro production / transformation of useful secondary compounds. Regenerated plants could be manipulated in vitro to produce beneficial variants, such as higher protein content in the leaves or higher vetiver oil content in the roots (Mucciarelli, et al. 1993).

Selection of Glufosinate-resistant Cells. To select glufosinate-resistant cell, normal

cells were initially cultured in modified N6 medium supplemented with 3.2 x 10-6 M glufosinate (I50, Fig. 5B) and growth rate was inhibited at the early selection stage (3-week). At 6-week culturing, growth rate was elevated the same level as those of normal cells. Then transferring these selected cells to a higher glufosinate-containing medium and the glufo-sinate-resistant cells were selected at 10-5M. Culturing them in 10-5M glufosinate-contain-

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ing medium, the growth response was rather low around 5-week while the elevation of its response to the same level of normal cells in 3.2 x 10-6 M glufosinate after eleven weeks was obtained (Fig. 5A). This may indicate a period of time was required for adjusting themselves to a stress condition.

The cells grown at 10-5 M glufosinate transferred to 5x10-5M glufosinate-containing medium, then growth rate of cell-recovered was elevated to the same level as normal cell around six weeks (Fig. 6A). These normal cells cultured in modified N6 medium without and with 5x10-5 M glufosinate comparing to the resistant cells in 5x10-5 M glufosinate were also shown (Fig. 6B). Growth curve of normal cells in glufosinate-free medium and resistant cells in 5x10-5 M glufosinate-containing medium were almost exactly the same profile, PCV was increased up to 3 ml within 6-day culturing (Fig. 6B), while normal cells in 5x10-5 M glufosinate were uniform at 2 ml PCV from 0 to 6 days) (Fig. 6B). Culturing both normal and resistant cells in modified N6 medium varied glufosinate concentration were shown (Fig. 6C). Fifty percent growth inhibition (I50) of normal cells and resistant cells were 2.5x10-7 M and 4.2x10-5M, respectively (Fig. 6C). Based on its I50 value, resistant cell was 170-fold resistant to glufosinate those of the normal cell. The glufosinate-resistant cells after being cultured in glufosinate-free medium then transferring to glufosinate containing medium they could retain their resistance with slightly changeable – for five months (Fig. 6 D). They seemed to be stable and unaffected by the continuous selective pressure of herbicide. Glufosinate resistant cells have been reported in some species, i.e. soybean (Pornprom, et al. 2000), vetiver (Praertsongskun, et al. 2002) and mungbean (Pengnual and Pornprom 2005). Selection for herbicide-resistant in plant could be accomplished by conventional breeding and biotechnological techniques. Cell suspension and tissue culture represent major advantages and being used as selection system of herbicide -resistant plant. There were several reports on selection of herbicide-resistant cell cultured, i.e., imazethapye-resistant soybean cell (Taregyen et al. 2001). Glufosinate-resistant mechanism reported was elaborated as follows:

(1) glutamine synthase overproduction, detoxification of herbicide and / or recovery from decreasing absorption,

(2) increased glutamine synthase activity and its decrease sensitivity to the herbicide (Prasertsongskun, et al. 2002) and

(3) altered at the target size as glutamine synthase enzyme conferring less sensitivity to glufosinate (Pengnual and Pornprom 2005).

References

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Establishment and maintenance of friable embryogenic maize callus and the involvement of L-proline. Planta 1644: 207-14.

Bors, W.; Langebartels, C.; Michel, C; and Sanderman, H. 1989. Polyamine as radical scarvengers and protectants against ozone. Phytochem. 28: 1589-95.

Chowdhary, C.W.; Tyagi, A.K.; Maheswari, N.; and Maheswari, S.C. 1993. Effect of L-proline and L-tryptophane on somatic embryogenesis and plant regeneration of rice (Oryza sativa L. cv. Pusa 169). Plant Cell Tiss. Org. Cult. 32: 357-61.

Ella, E.S.; and Zapata, F.J. 1993. Suspension initiation in indica rice requires proline. IRRN 18: 17-18.

Flores, H.E.; Protacio, C.M.; and Signs, M.W. 1989. Primary and secondary metabolism of polyamines in plants. Rec. Adv. Phytochem. 23: 329-93.

Giang, H.T. 2007. The in vitro regeneration of vetiver (Vetiveria zizanioides (L.) Nash) using thin cell layer culture of inflorescences and selection for salt tolerant callusclones. <http://www.vetiver.com/ VNN invitrio r-pdf>

Keshavachandran, R.; and Khader, M.A.1997. Growth and regeneration of vetiver (Vetiveria zizanioides (L.) Nash) callus tissue under varied nutritional status. In Biotechnology of Spices, Medicinal and Aromatic Plants (eds. Edison, S., Ramana, K.V., Sasikumar, B. and Badu, K.N.) pp 60-64,. Proc. Nat. Sem. Biotechnol. Spices Arom. Plts. Calicut, India.

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Klamsomboon, P. 1997. Development of the proper medium for the high plating efficiency in indica rice protoplast and cell suspension culture. Ph.D. Thesis, Kasetsart University, Bangkok, Thailand.

Mucciarelli, M.; Gallino, M.; Scannerini, S.; and Maffei, M.1993. Callus induction and plant regeneration in Vetiveria zizanioides. Plant Cell Tiss. Org. Cult.35: 267-71.

Murashige, T.; and Skoog, F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue culture. Physiol. Plant. 15: 473-97.

Na Nakorn, M.; Surawattananon, S.; Wong-wattana, C.; Namwongprom, K.; and Suwannachitr, S. 1998. In vitro induction of salt tolerance in vetiver grass (Vetiveria zizanioides Nash). J. Weed Sci. Tech. 43: 134-37.

Nabors, M.W.; Heyeer, J.W.; Dukes, T.A.; and De Mott, K.J. 1983. Long duration high frequency plant regeneration from cereal tissue cultures. Planta 15: 385-91.

Ozawa, K.; Liang, D.H.; and Komamine, A. 1996. High frequency somatic embryo-genesis from small suspension-cultured clusters of cells of interspecific hybrid of Oryza. Plant Cell Tiss. Org. Cult. 46: 157-59.

Pengnual, A.; and Pornprom, T. 2005. Alteration of glutamine synthase activity and ammonia accumulation of glufosinate- resistant in a selected mungbean cell line. Agric. Sci. J. 36 (5-6): 223-31.

Pornprom, T.; Surawattananon S.; and Srinives, P. 2000. Ammonia accumulation as an index of glufosinate-tolerant soybean cell lines. Pestic. Biochem. Physiol. 68: 102-106

Prasertsongskun S.; Sangduen N.; Suwanwong S.; Santisopasri V.; and Matsumoto, H. 2002. Increased activity and reduced sensitivity of glutamine synthetase in glufosinate-resistant vetiver (Vetiveria zizanioides Nash) cells. Weed Biology and Management 2: 171-76.

Prasertsongskun, S. 2003. Plant regeneration from callus culture of vetiver (Vetiveria zizanioides Nash). Songklanakarin J. Sci. Technol. 25: 637-42.

Sangduen, N.; and Klamsomboon, P. 2000. Histological and scanning electron observations on embryogenic and non-

embryogenic calli of aromalic Thai rice (Oryza sativa L. cv. Khao Dawk Mali 105). Kasetsart J. (Nat. Sci.) 35: 427-32.

Sangduen, N.; and Klamsomboon, P. 2002. Arginine enhancement of cell dissociation in suspension culture of aromatic rice cells (Oryza sativa L. var. Khao Dawk Mali 105). Kasetsart J. (Nat. Sci.) 36: 353-60.

Taregyen, M.R; Mortimer, A.M.; and Putawin, P.D. 2001. Selection for resistance to herbicide imazethapyr in somaclones of soybean. Weed Res. 41: 143-54.

Vasil, V.; and Vasil, I.K. 1986. Plant regeneration from friable embryogenic callus and cell suspension cultures of Zea mays L. J. Plt. Physiol. 124: 399-408.

Vasil, I.K. 1998. Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology 6: 387-402.

Vasil, V.; and Vasil, I.K. 1981. Somatic embryogenesis and plant regeneration from tissue cultures of Pennisetum americanum x P. purpureum hybrid. Amer. J. Bot. 68: 864-72.

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Fig.1. Calli-derived inflorescences of Vetiveria zizanioides (L.) Nash, ‘Surat Thani’ ecotype,

cultured on modified MS medium: (A) supplemented with 15 μM 2,4-D for three weeks, (B) Two types of calli were distinguished, embryogenic (E) and non-embryogenic (NE) (bar = 500 μM), (C) scanning electron micrographs (SEM) of E callus comprised of nodular, compact and deeply embedded cell, and (D) SEM of NE callus comprised long-like tubular and loosely arranged cells.

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Fig.2. Growth response of suspension-cultured vetiver cells. A - Cultured in modified liquid MS medium containing various concentrations of 2,4-D; B - Cultured in modified liquid N6 medium containing various concentrations of 2,4-D

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Fig. 2. Growth response of suspension-cultured vetiver cells (con’t.). (C) cultured at various proline concentrations in modified liquid N6 medium supplemented with 10 μM 2,4-D, and (D) cultured in modified liquid N6 medium supplemented with 10 μM 2,4-D and 10 mM proline and applied various initial cell density percentage.

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Fig. 3. Suspension cultured-derived inflorescences of Vetiveria zizanioides (L.) Nash, ‘Surat Thani’ ecotype in modified liquid N6 medium, (A) with 10 mM praline showed prolific growth of secondary calli (arrow), (B) without praline showed poor initiation of secondary calli (bar = 30 μM), and (C) scanning electron micrograph of cell suspension in the presence of 10 mM proline showed separation of microcalli (arrow) from mother cells (MC).

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Fig. 4. Suspension-cultured of vetiver after one-month plating on modified MS medium: (A) normal vetiver calli without 2.4-D, (B) normal vetiver calli with 10 μM 2,4-D, (C) glufosinate-resistant calli with 2,4-D, and (D) regenerated plant after 2-day dehydration and maintained for 5-weeks in MS medium without 2,4-D.

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Cells in 3.2 × 10-6 M glufosinate

Cells in 10-5 M glufosinate

A

Normal cells

Cells in 3.2 × 10-6 M glufosinate

B Fig. 5. Growth response of suspension-cultured cells of Vetiveria zizanioides (L.) Nash, ‘Surat Thani’ ecotype in modified liquid N6 medium supplemented with 10-5 M glufosinate in a step-wise culture. Packed cell volume was measured at 2-day intervals and subcultured at 6-day intervals: (A) supplemented with 10-5 M glufosinate in a step-wise culture, supplemented with 3.2 × 10-6 M glufosinate in a step-wise culture, and (B) dose-response curve of glufosinate-treated and suspension-cultured cells at 6-days after treatment indicated I50

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Resistant cells in 10-5 M glufosinate Resistant cells in 5 × 10-5 M glufosinate Normal cells in glufosinate-free medium

Normal cells in 5 ×10-5 M glufosinate

BB

cc

C D Fig. 6. Growth response of suspension-cultured vetiver cells in modified liquid N6 medium. Packed cell volume was measured at 2-day intervals and subcultured at 6-day intervals, (A) supplemented with 5 × 10-5 M glufosinate, (B) comparison between normal and resistant cells in 5 × 10-5 M glufosinate and in glufosinate-free medium, (C) normal and resistant cells at 7-day after various glufosinate-treated concentration indicated I50 of normal cells and resistant cells, and (D) 10-5 M glufosinate resistant cells after culturing in a glufosinate -free N6 medium for 5 months after selection.

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Table 1. Effect of 2,4-D concentrations on small colonies formation from cell suspension-derived callus of Vetiveria zizanioides Nash ‘Surat Thani’ ecotype (normal cells).

2,4-D (μM) The size of small colonies(mm)

__________________________________________________________________________________

0 < 0.1 5 0.10-0.25

10 0.25-0.45 15 0.10-0.15 20 0.10-0.15

Table 2. Water content of callus on plantlet regeneration in normal vetiver cells.

Dehydration (days) Number of plantlets (%) ± S.E. 0 0.00 ± 0.00 1 0.00 ± 0.00

14.50 ± 3.50 2 0.00 ± 0.00 3 0.00 ± 0.00 4 0.00 ± 0.00

Table 3. Effect of sorbitol or mannitol concentrations in regeneration medium on vetiver plantlet

formation.

Treatment Number of plantlets (%) ± S.E. Control (no alcohol sugar) 0.00 ± 0.00 0.05 M sorbitol 0.00 ± 0.00 0.10 M sorbitol 8.00 ± 2.80 0.30 M sorbitol 1.50 ± 2.10 0.50 M sorbitol 0.00 ± 0.00 0.05 M mannitol 0.00 ± 0.00 0.10 M mannitol 2.25 ± 3.50 0.30 M mannitol 0.00 ± 0.00 0.50 M mannitol 0.00 ± 0.00 Table 4. Effect of glufosinate concentration on growth and viability of suspension-cultured vetiver

cells.* Glufosinate concentration Cell growth Cell growth Viable cell (%) (M) (ml/5ml) (%) (mean ± SE) (mean ± S.E.) 0 0.47 ± 0.03 100.0 88.6 ± 7.40 10-8 0.42 ± 0.03 89.4 84.1 ± 20.40 10-7 0.35 ± 0.05 74.5 76.3 ± 8.90 10-6 0.30 ± 0.05 63.8 72.0 ± 10.70 10-5 0.19 ± 0.02 36.2 54.2 ± 4.70 10-4 0.14 ± 0.01 27.7 30.6 ± 22.80 10-3 0.07 ± 0.03 12.8 17.1 ± 6.20 * Values represent the mean of 3 replications.

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