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In Vitro Cell. Dev. Biol.Plant 42:354358, July August 2006 q 2006 Society for In Vitro Biology 1054-5476/06 $18.00+0.

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DOI: 10.1079/IVP2006783

ADVENTITIOUS SHOOT REGENERATION OF SCOTCH SPEARMINT (MENTHA 3 GRACILIS SOLE)


CHARLESON R. POOVAIAH, STEPHEN C. WELLER,
AND

MATTHEW A. JENKS*

Department of Horticulture and Landscape Architecture, Purdue University, 625 Agriculture Mall Drive, West Lafayette, IN 47907-2010
(Received 26 September 2005; accepted 11 May 2006; editor T. J. Jones)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Mentha gracilis Sole (scotch spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 mM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, at TDZ 4.5 mM and 10% (v/v) coconut water and solidied with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, 2.0% sucrose, and 0.054 mM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the rst report describing adventitious shoot regeneration of scotch spearmint. Key words: cytokinins; internodes; micropropagation; thidiazuron.

Introduction Mints are members of the genus Mentha (Lamiaceae) and are cultivated for the essential oils they produce in glandular trichomes on both leaves and stems. These essential oils are used commercially as food additives, avoring agents, and in cosmetics, pharmaceuticals, and perfumery. The mints that are mainly grown for the essential oils are peppermint (Mentha piperita L.) and various spearmints. The economically important spearmints are native spearmint (Mentha spicata L.) and scotch spearmint (Mentha gracilis Sole). Scotch spearmint is derived from a cross between eld mint (Mentha arvensis L.) and native spearmint and has organoleptic properties slightly different to that of native spearmint. Carvone oil is 60 70% of the total oil extracted from both native and scotch spearmint, but scotch spearmint has 20% limonene of the total oil and native spearmint has only 8% limonene of total oil extracted. Scotch spearmint oil also has a menthone content of up to 2% (of total oil content), whereas native spearmint does not produce menthone (Bircout et al., 1978; Bhat et al., 2002). A major constraint in scotch spearmint production is weeds, which compete for light, water, and nutrients, and often form a part of the herbage of mechanically harvested spearmint. Weeds impart an off-avor to the oil and decrease its value. Production of scotch spearmint can also be signicantly reduced by diseases such as rhizome rot (Rhizoctonia solani Kuhn.) (Skotland, 1979; Skotland and Traquair, 1982), septoria leaf spot (Septoria menthae Oudem.) (Green, 1961) and verticillium wilt (Verticillium dahliae Kleb.), and insects such as mint ea beetle (Longitarsus waterhousei Kutschera) and mint bud
*Author to whom correspondence should be addressed: Email jenksm@ purdue.edu

mite (Tarsonemus spp.). Technical advances in genetic engineering make biotechnological approaches a viable option for improving spearmint pest resistance. Genes likely to be transferred into scotch spearmint are those known to convey resistance to herbicides such as the Bar gene encoding glufosinate resistance (Li et al., 2001), the gene conferring resistance to lepidopteran insects, CRY (Perlak et al., 1993), the gene conferring tolerance to verticillium wilt, VET1 (Veronese et al., 2003), and the osmotin gene that encodes a protein with anti-fungal activity (Liu et al., 1994). However, before gene transfer technology can be applied to the genetic improvement of spearmint, an efcient in vitro adventitious shoot regeneration system is needed. Previous attempts to regenerate scotch spearmint have been unsuccessful (Van Eck and Kitto, 1992; Berry et al., 1996). We describe here the rst report of efcient in vitro adventitious shoot regeneration in scotch spearmint.

Materials and Methods


Stock plant culture. Stock cultures of scotch spearmint (Mentha gracilis Sole) [syn. M. cardiaca (S.F.Gray) Bak] (USDA and NRCS, 2004) were obtained from shoot tips derived from stolons of greenhouse-grown plants, originally obtained from USDAARS National Clonal Repository, Corvallis, OR. Stolon pieces with nodes were surface-sterilized by immersing them in 70% v/v ethanol for 1 min and then in a 20% solution of Cloroxw (1.05% w/v sodium hypochlorite) with 3 drops per liter of Tween 20 for 20 min. The explants were then rinsed 7 times with sterile, deionized water. The sterilized stolon pieces were placed in Magenta (GA7) boxes (Bio-world, Dublin, OH) on stock plant medium containing MS salts (Murashige and Skoog, 1962), 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, 2.0% sucrose, and 7.5 g l21 of agar (Sigma-Aldrich Chemical Co., St Louis, MO), and supplemented with 0.054 mM a-naphthalene acetic acid (NAA). The pH of medium was adjusted to 5.8 (with KOH 0.01 N) before adding agar. The media was autoclaved for 20 min at 1218C. Stock plants were re-cultured

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REGENERATION OF SCOTCH SPEARMINT from shoot tips on culture medium every 3 wk and maintained under 16-h photoperiod provided by cool white uorescent light (25 mmol m22 s21) at 27 ^ 18C. Explants. Stem internodal explants of 34 mm length were taken from the rst and second internode from the top of in vitro cultured, 2-wk-old, unbranched stock plants. Explants from fully expanded leaves (with petioles but lacking axillary buds) were excised from in vitro grown plantlets. The edges of leaves were trimmed on three sides leaving a piece of the petiole with the explant, as described by Li et al. (1999). These explants were placed on the surface of a regeneration medium consisting of MS basal salts, 2.0% (w/v) sucrose, 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, 10% (v/v) coconut water, and phytagel 2.0 g l21 (Sigma-Aldrich Chemical Co.) (referred to as a basal medium) and cytokinins: thidiazuron (TDZ) at 2.3, 4.5, 9.1, 18.2, 36.3, 72.6, 145.3 mM, zeatin (ZT) at 2.3, 4.6, 9.1, 18.2, 36.5, 73.0, 146.0 mM, kinetin (KIN) 2.3, 4.6, 9.3, 18.6, 37.2, 74.3, 148.7 mM, or benzylaminopurine (BA) alone at 2.2, 4.4, 8.9, 17.8, 35.5, 71.0, 142.1 mM. Coconut water was shown to promote greater adventitious shoot regeneration from native spearmint leaf explants (Li et al., 1999). Coconut water was taken from ripe brown coconuts purchased from the market and ltered through four layers of cheesecloth, and then through two layers of Whatman No.1 lter paper. The ltered coconut water was stored at 208C until use. The pH in all experimental media was adjusted to 5.8 and the media were autoclaved at 1218C for 20 min. In all shoot regeneration experiments, explants were rst placed in the dark at 258C for 3wk before being moved (without subculture) to light [16-h photoperiod provided by cool white uorescent light (25 mmol m22 s21) at 27 ^ 18C]. Experimental analysis. A completely randomized block design with four replications per treatment for each explant type was used in the study. In all experiments, each treatment consisted of four replicate Petri dishes with 18 explants per dish. To note, all data sets presented here were highly consistent with preliminary studies of scotch spearmint regeneration used to set the growth regulator concentration ranges tested here. Rooting of regenerated shoots. In vitro regenerated shoots, c. 2.0 cm long, were excised from the callus and placed in GA7 boxes on the same medium as used for stock plant culture. Environmental conditions for rooting were the same as used for stock plant cultures. Acclimatization and growth in greenhouse. Plantlets with well-developed root systems (c. 5 cm long) and shoots (710 cm long) were removed from GA7 boxes and planted in pre-watered cell packs in ats lled with Metromix360 medium (SUNGRO Horticulture Inc, Bellevue, WA). The ats were covered with plastic domes to maintain 100% humidity for 2 d. Humidity was then decreased gradually by removing the plastic dome for varying periods of time every day with complete removal after 1 wk. Plants were transplanted into individual 4-in. plastic pots containing Metromix360 medium after 3 wk growth in ats.

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Results Effect of cytokinins on callus initiation and shoot regeneration. Experiments were conducted to evaluate the effect of cytokinins on the regeneration efciency on scotch spearmint leaf and internode explants. After fresh-cut explant cultures were placed in darkness, callus was rst observed in 2 4 d on internode explants and 6 8 d on leaf explants, on all individual treatments (but not on cytokinin-free media). Callus formed only on the cut surfaces. Internode explants initiated callus rst on the proximal side and 2 d later on the distal side, whereas leaf explants initiated callus rst on the cut end of the petiole and 5 d later on the cut blades. Callus formation on internode explants was clearly the greatest (based on visual appearance of callus) on a basal medium supplemented with 4.5 mM TDZ, while leaf explant callus formation was greatest on a medium with 9.1 mM TDZ supplementation (data not shown). Callus of internodal explants (Fig. 1A) formed meristemoidal regions in c. 18 d on media supplemented with TDZ at 72.6 mM or less and ZT at 146.0 mM or less, whereas callus differentiated into meristemoidal regions in 21 d on media supplemented with BA at

35.5 mM or KIN at 37.2 mM or less (Fig. 1A). These meristemoidal regions emerging on the top surface of the initial callus, were smooth and compact, and represented areas from which adventitious shoots then arose. Adventitious shoots with leaf primordia were rst observed elongating from meristemoidal regions of callus in c. 21 d in the dark on media supplemented with TDZ at 36.3 mM or below (Fig. 1B). Internode explants cultured on a basal medium supplemented with 4.5 mM TDZ had the maximum proportion of explants producing shoots (85%) and also the highest number of shoots per explant (28.5) (Table 1, Fig. 1C). Shoots also arose from explants on a basal medium supplemented with TDZ from 18.2 to 72.6 mM after 21 d, but these did not elongate normally and were usually malformed having fused leaves and fasciated stems and leaves (data not shown). Shoots produced on BA-, KIN-, and ZT-supplemented basal media did not show these malformations. Callus on leaf explants formed visible meristemoidal regions 4 d after being removed from the 21 d dark treatment and being placed in light (25 d), on a basal medium supplemented with TDZ at 72.6 mM or less and ZT at 36.5 mM or less, while meristemoidal regions formed in 28 d on leaf explants on a basal medium with BA at 35.5 mM or KIN at 37.2 mM or less. Meristemoidal regions were not formed on media if supplemented with cytokinins at higher concentrations. Adventitious shoots (having visible primordial leaves) were rst observed on leaf explant meristemoidal regions in c. 32 d on a basal medium with TDZ at 72.6 mM or less. For leaf explants, a basal medium supplemented with TDZ at 9.1 mM had the highest regeneration efciency (79% of explants having shoots) among the cytokinins tested, and also produced the maximum number of shoots per explant (11.1) (Table 1). Shoot malformations such as those described for internode cultures on TDZ at 18.2 mM or below were not observed on leaf explants with any concentration of the cytokinins tested. Root formation was rst observed on internodal explants on BA from 4.4 to 17.8 mM and KIN from 4.6 to 9.3 mM supplementation after 4 wk from initial culture. No root formation was observed on internodal explants cultured on TDZ and ZT media; however, internode explants on media without growth regulators initiated roots in c. 6 d in the dark. Shoots from leaf explants did not initiate roots on a basal medium either with or without growth regulator supplementation. Rooting of regenerated shoots in vitro and acclimatization of the in vitro produced plantlets to the greenhouse environment. Shoots c. 2.0 cm long were excised and transferred to a rooting medium in GA7 boxes (Fig. 1D) where explants initiated roots in 4 6 d and developed a well branched root system within 2 wk (Fig. 1E). The rooted plants were transplanted to the greenhouse and acclimated, where all transplants grew vigorously (Fig. 1F). Discussion We describe the development of an efcient in vitro adventitious shoot regeneration protocol for scotch spearmint. While previous reports have described successful in vitro regeneration for peppermint and native spearmint, this is the rst report for in vitro adventitious shoot regeneration of scotch spearmint. Four cytokinins were tested for their ability to induce shoots on scotch spearmint leaf and internodal explants. As reported for other mint species (Shasany et al., 1998; Bhat et al., 2002), our results show that internodal explants regenerated more adventitious shoots in a

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POOVAIAH ET AL.

FIG . 1. Regeneration of scotch spearmint. Internodal explants cultured on a basal medium supplemented with 4.5 mM TDZ. A, Primary callus formed at both sides of the explant after 10 d in the dark (bar 1 mm); B, Shoot initiation in 24-d-old explants (since initial culture) 3 d after transfer to light (bar 1 mm); C, Elongated shoots at 28 d after initial culture (7 d after transfer to light) (bar 2 mm); D, 32-d-old plantlet excised from the callus and placed on a medium for rooting (bar 1 cm); E, Root production by 45-d-old plantlet after 1 wk on a rooting medium (bar 2 cm); F, 60-d-old plant growing vigorously in plastic pot (bar 3 cm).

shorter time than leaf explants, and there were major differences both in the effective concentrations and the types of cytokinins for the explant types tested. In shoot formation from internodal explants, primary callus arose initially from internode cut surfaces within 2 4 d. Callus continued to enlarge until distinctly compact and mounding structures began

to form above the callus mass. These regions subsequently differentiated into meristemoidal regions from which shoots arose. Meristemoidal regions were rst visible on internode explants in c. 18 d, with the rst visible shoots appearing on meristemoidal regions in c. 23 d. The meristemoidal regions often gave rise to more than one shoot, with a primary shoot forming rst, followed 3 5 d

REGENERATION OF SCOTCH SPEARMINT TABLE 1 EFFECT OF INDIVIDUAL CYTOKININS ON IN VITRO ADVENTITIOUS SHOOT REGENERATION OF SCOTCH SPEARMINT Thidiazuron Explant type Leaf Conc. mM 0.0 2.3 4.5 9.1 18.2 36.3 72.6 145.3 0.0 2.3 4.5 9.1 18.2 36.3 72.6 145.3 Shoots/explant (%) 0.0 ^ 0.0 1.4 ^ 0.2 3.8 ^ 0.4 11.1 ^ 0.8 5.2 ^ 0.7 0.7 ^ 0.2 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 9.8 ^ 0.9 28.5 ^ 1.6 15.1 ^ 1.2 7.7 ^ 1.0 1.0 ^ 0.3 0.0 ^ 0.0 0.0 ^ 0.0 (0) (49) (71) (79) (54) (27) (0) (0) (0) (64) (85) (21) (49) (11) (0) (0)
a

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Benzyladenine Conc. mM 0.0 2.2 4.4 8.9 17.8 35.5 71.0 142.1 0.0 2.2 4.4 8.9 17.8 35.5 71.0 142.1 Shoots/explant (%) 0.0 ^ 0.0 0.0 ^ 0.0 1.8 ^ 0.2 3.8 ^ 0.4 1.3 ^ 0.2 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 1.5 ^ 0.3 3.4 ^ 0.5 11.2 ^ 1.1 2.8 ^ 0.5 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 (0) (0) (55) (67) (38) (0) (0) (0) (0) (26) (45) (59) (34) (0) (0) (0)
a

Zeatin Conc. mM 0.0 2.3 4.6 9.1 18.2 36.5 73.0 146.0 0.0 2.3 4.6 9.1 18.2 36.5 73.0 146.0 Shoots/explant (%) 0.0 ^ 0.0 0.0 ^ 0.0 2.5 ^ 0.4 5.7 ^ 0.7 4.0 ^ 0.6 0.9 ^ 0.2 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.6 ^ 0.2 1.0 ^ 0.3 5.0 ^ 0.5 3.4 ^ 0.6 1.3 ^ 0.3 0.0 ^ 0.0 0.0 ^ 0.0 (0) (0) (35) (51) (37) (24) (0) (0) (0) (9) (16) (66) (37) (20) (0) (0)
a

Kinetin Conc. mM 0.0 2.3 4.6 9.3 18.6 37.2 74.3 148.7 0.0 2.3 4.6 9.3 18.6 37.2 74.3 148.7 Shoots/explant (%)a 0.0 ^ 0.0 0.0 ^ 0.0 0.8 ^ 0.2 2.9 ^ 0.5 1.1 ^ 0.3 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 1.5 ^ 0.4 3.5 ^ 0.5 1.6 ^ 0.3 0.2 ^ 0.1 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 (0) (0) (16) (33) (24) (0) (0) (0) (0) (21) (38) (27) (11) (0) (0) (0)

Internode

a Values represent the mean number of shoots produced per explant (^SE) on a basal medium supplemented with individual cytokinins at different concentrations, and percent (%) of explants producing shoots.

later by up to four secondary shoots. It is unclear whether the secondary shoots arose adventitiously from undifferentiated tissue at the base of the primary shoot due to changes in the local hormonal balance caused by the rst shoot, or whether the secondary shoots arose from meristems present before the primary shoot emerged. A basal medium supplemented with 4.5 mM TDZ produced the highest regeneration efciency, both in percentage of explants (85%) and shoots per explant (28.5). TDZ at all concentrations except 145.3 mM, promoted adventitious shoot initiation in scotch spearmint, but TDZ at 18.2 mM or greater suppressed continued shoot development and prevented normal elongation while causing other shoot malformations. Similar results with TDZ on explants from woody plant species and native spearmint were found (Huetteman and Preece, 1993; Li et al., 1999). Native spearmint was shown to require both TDZ and ZT to produce elongated adventitious shoots from internodal explants (Pooviah et al., 2006). Peppermint (Mentha piperita) shoot initiation and elongation from leaf explants was maximized using only TDZ at 8.4 mM (Niu et al., 1998), which is similar to scotch spearmint, where maximum shoot initiation and shoot elongation occurred on a medium supplemented with TDZ at 4.5 mM. It is interesting to note that shoot elongation in native spearmint is inhibited by all TDZ treatments, but elongation is not inhibited in the two hybrid offsprings (peppermint and scotch spearmint). It is unclear what genetic differences exist between the two mint hybrids and their native spearmint parent that cause this differential response to TDZ treatments. Our results showing production of roots by internodal explants on media without growth regulators suggests that Mentha may produce a high level of endogenous auxin, since high auxin concentrations are known to stimulate rooting. Root formation by scotch spearmint explants on a medium containing TDZ and ZT was inhibited at all concentrations, while BA at 17.8 mM or greater and KIN at 9.3 mM

or greater suppressed root formation, possibly by counteracting the effect of endogenous auxins. The highest adventitious shoot regeneration of scotch spearmint was obtained on a medium supplemented with MS basal salts, 2.0% (w/v) sucrose, 100 mg l21 myo-inositol, 0.4 mg l21 thiamine-HCl, 10% (v/v) coconut water, 0.2% (w/v) phytagel, and TDZ at 4.5 mM. This research developed an efcient shoot regeneration protocol for scotch spearmint that will be useful in future research to transform scotch spearmint for valuable stress resistant and yield-increase traits. Acknowledgment
The authors would like to thank the Mint Industry Research Council for their support.

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