Of Hibiscus Acetosella Weiw

In Vitro Multiple Shoot Induction and Plant Regeneration from Shoot Apices of Hibiscus acetosella Weiw. ex. Hiern Hamidou F. Sakhanokho

ABSTRACT. Multiple shoots were induced and plants regenerated from shoot apices of Hibiscus acetosella WeIw. ex. Hiern using the growth regulators thidiazuron (N-phenyl-N -1 ,2,3-thidazol-5-ylurea, TDZ) and 6-benzyladenifle (BA). Shoot apices were cultured for 30 days in 16 different media containing various concentrations and combinations of these growth regulators. Multiple shoot induction medium M9 (0.6 1.tM TDZ + 8.9 1iM BA) was one of the best performing media, producing an average of 3.7 shoots per explant. In addition, multiple shoots regenerated on this treatment rooted, elongated and acclimatized readily. Results reported here suggest that both BA and TDZ are involved inproducing multiple shoots from the shoot apex of H. acetosella.

KEYWORDS. Cytokinins, N-phenyl-N -1 ,2,3thidazol-5-ylurea(TDZ), 6-benzyladenine (BA), "red leaf hibiscus"

Hamidou F. Sakhanokho is affiliated with the USDA-ARS, Thad Cochran Southern Hofticultural Laboratory, P.O. Box 287,810 Hwy 26 West, Pdplarville, MS 39470. iThe author wishes to thank Mr. Kermis Myrick, Ms. Lindsey Tanguis, and Ms. Alexandria Goins for their technical assistance. Mention of trade names of commercial products in the publication is solely for the purpose of providing specific information and does not imply recommenda- tion or endorsement by the U.S. Department of Agriculture. Address correspondence to: Hamidou F. Sakhanokho at the above address (E-mail: hsakhanokho @ msastoneville.ars.usda.goV). Journal of Crop Improvement, Vol. 21(2) (#42) 2008 - Available online at http://jcrip.haworthpress.com © 2008 by The Haworth Press, Inc. All rights reserved. - doi:10.1080/15427520701885774 201 202 JOURNAL OF CROP IMPROVEMENT

INTRODUCTION

Hibiscus acetosella Weiw. ex. Hiem, which consists of a red and green variant, is a tetraploid species (2n = 4x = 72) native to western tropical Africa (Menzel and Wilson, 1961). It is sometimes used as a vegetable staple (USDA-ARS, 2007) but mostly as an annual ornamental grown for the attractiveness of its deep burgundy red, maple-like leaves. Also known as "false roselle" or "red leaf hibiscus," H. acetosella shows resistance to insect pests and nematodes (Menzel and Wilson, 1961; Boyd and Cheatham, 2004). Genetic transformation is increasingly used for crop improvement, including ornamentals. Such transformation, however, requires a depend- able regeneration system. A transformation system based on somatic embryogenesis is ideal because it allows for the regeneration from callus of a single cell giving rise to a stably transformed plant. However, many plant species, in particular members of the Malvaceae family such as cotton and hibiscus, are recalcitrant to regeneration via somatic embryogenesis (Trolinder and Xhixian, 1989; Morre et al., 1998; Zapata et al., 1999; Srivatanakul et al., 2000). Shoot apex regeneration is virtually genotype independent and has been used to regenerate many genetically engineered plant species (Gould and Magallanes-Cedono, 1998; Srivatanakul et al., 2001; Gould et al., 2002; Xie and Hong, 2002). Also since its inception (Morel, 1960), the technique of in vitro shoot tip culture has been an invaluable tool that is used for many purposes, including the development of virus-free or indexed plants, safer germplasm exchanges among coun- tries, mass propagation of plants, preservation of endangered species, in vitro mutagenesis, and in vitro screening for salt tolerance. In vitro single shoot regeneration in H. acetosella has been reported (Blackmon and Reynolds, 1982) from leaf and/or cotyledon explants using a modified Nitsch and Nitschs medium (Nitsch and Nitsch 1969), but the success rate was only 40% and only 7% of ihe regenerated plant.. lets formed roots. Production of proembryos and somatic embryos has also been reported in H. acetosella (Reynolds et al., 1980), but no plants were obtained from those embryos. There is no report of multiple shoot induction in H. acetosella so far. Growth regulators such as thidiazuron (N-phenyl-N -1 ,2,3-thidazol-5-ylurea, TDZ) and 6-benzyiadenine (BA) have been shown to induce multiple shoots from various explants (Agrawal et al., 1997; Murch.et al., 2000; Srivatanakul et al., 2000; Sharma et al., 2005). Induction of multiple shoots could help establish a more efficient mass micropropagation system and potentially offer more explant targets for genetic engineering using either particle bombardment Hwnidou F. Sakhanokho 203 or Agrobacterium transformation. The purpose of this study was to establish an efficient and reproducible multiple shoot regeneration system from shoot apices using growth regulators BA and TDZ.

MATERIALS AND METHODS

Induction of Multiple Shoots Shoot apices (2-3 mm) were removed from 5-day-old in vitro grown seedlings by excising and discarding the cotyledons and radicles. The apices were transferred upright with the base into 175 ml (98.5 min 59 mm) vessels containing 50 ml of a matrix of shoot induction media. The 16 (M0-M15) media consisted of MS basal medium containing macro- and micronutrients (Murashige and Skoog, 1962) and vitamins (Gamborg et al., 1968), sucrose 20 g L, myo-inositol 0.2 g L, casein hydrolysate 1 g L, thiamine 1 mg/I, MgCl2 0.75 g L, and combinations of TDZ and 1A (Table 1). The pH was adjusted to 5.8 before addition of the gelling agent (Gelrite 2 g L) and autoclaving for 15 min at 121°C. The cultures were placed into a growth chamber where the temperature was main--2 tained fixed at 28°C with a photoperiod of 16-h light (100 llmol M s- and 8-h dark. After 30 days, number of shoots and root formation were evaluated. The experimental design was a randomized complete block consisting of one H. acetosella species, a 4 x 4 factorial arrangement of BA (0, 4.4, 8.9, and 13.3 RM) and TDZ (0, 0.6, 1. 1, and 2.3 jiM), 15 rep- licates (explants) per medium treatment; and three replications for a total of 45 explants per medium treatmeflt Mean separation (P 0.05) based on Tukeys HSD test and standard error values for each treatment were calculated using SAS (SAS Institute, 2003). Rooting and Acclimatization The regenerated shoots that did not form roots after 30 iaszwere transferred into a rooting medium (HRM or hibiscus rooting medium) developed during the current study. HRM medium consisted of MS basal medium containing macro- and micronutrients (Murashige and Skoog, 1962) and vitamins (Gamborg et al., 1968) supplemented with 0.6 jiM IAA, sucrose 15 g L, and MgCl2 0.75 g L. The pH was adjusted to 5.8 •before addition of the gelling agent (Gelrite 2 g L) and autoclaving for 15 min at 121°C. Callus that formed at the base of the nonrooted explants was gently, scrapped off before transfer of the regenerated shoots to the Im

204 JOURNAL OF CROP IMPROVEMENT

TABLE 1. Effect of BA and TDZ on multiple shoot induction from shoot apex explants of Hibiscus acetosella

Medium TDZ tM BAiM Mean number of shoots/explants

MO 0 0 1.0±0.Oe Ml 0.6 0 3.0 ± 0.2abc M2 1.1 0 2.8 ± 0.2abc M3 2.3 0 2.8 ± 0.4abc M4 0 4.4 1.0 ± 0.Oe M5 0.6 4.4 3.2 ± 0.2a M6 1.1 4.4 3.1 ± 0.3ab M7 2.3 4.4 2.8 ± 0.2abc M8 0 8.9 1.1 ± Ole M9 0.6 8.9 3.7 ± 0.4a Mb 1.1 8.9 3.0 ± 0.3abc Mu 2.3 8.9 2.6 ± 0.2abcd M12 0 13.3 1.3 ± 0.2e M13 0.6 13.3 2.7 ± 0.3abcd M14 1.1 13.3 2.6 ± 0.3abcd M15 2.3 13.3 3.4 ± 0.5a

Means within a column followed by the same letter are not significantly different at the 0.05 rejection level according to Tukeys HSD test. Means are from three independent experiments with 15 explants for each determination. rooting medium. The plants were kept in a growth room where the temperature was maintained at 22°C with a fluorescent light regime of 16-h light -2 (100 .tmol M S-1 )and 8-h dark. Rooted plants were gently washed with tap water to remove any formed callus and residues from the gelling agent and transferred in moistened Jiffy-7 peat pellets (Jiffy Products, Ltd., Canada) and covered with clear plastic bags. After three days, holes were punched in the plastic bags to allow for gradual acclimatization of the plants before transfer into soil in the greenhouse at the end of a two-week period.

RESULTS AND DISCUSSION Multiple Shoot Induction

Multiple shoots were induced and plants regenerated from H. acetosella shoot apices grown for 30 days on media containing various combinations of BA and TDZ. Shoots were induced in all 16 media. TDZ appeared Hamidou F. Sakhanokho 205 to play a crucial role in multiple shoot induction in H. acetosella since excluding this growth regulator (M4, M8, and M12) tended to adversely affect the mean number of shoots/explant (Table 1). BA is reported to be the most widely used cytokinin for multiple shoot induction (Herath et al., 2004). In this study, however, even though BA induced multiple shoot formation, fewer shoots were generally produced in media containing BA alone (M4, M8, and M12) compared with media with TDZ alone, i.e., Ml, M2, and M3 (Table 1). Three media, M5, M9, and M15, were among the top-performing treatments even though these media were not significantly different (P = 0.05) from nine other treatments (Table I). Treatment M9 (0.6 l.1M TDZ + 8.9 tM BA) was of particular interest because multiple shoots regenerated on this medium tended to resemble plants obtained on growth regulator-free medium, with normal leaves and stems (Figures lB and 1C).

FIGURE 1. In vitro shoot apex regeneration in H. acetosella. (A): Earlier and lower leaves sometimes showed signs of hyperhydricity often associated with the use of TDZ, but these signs disappeared as the plants developed and produced more leaves. (B) & (C); Multiple shoots obtained from H. acetosella shoot apex cultured on medium (M9) containing BA and TDZ. (D); Rooted plants after transfer to the rooting medium HAM. (E): Acclimatized plants in Jiffy pellets before transfer to the greenhouse. iF, 206 JOURNAL OF CROP IMPROVEMENT

Root Formation and Plant Acclimatization / After 30 days, the percentage of rooted plants on the various shoot- induction media ranged from 35.6 (M3, Ml 1, M15) to 82.2% (M4) (Table 2). M4 and M9 were the best treatments for root formation. Nonrooted plants were transferred on the rooting medium HRM, which was very effective in inducing roots in plants, including those with abnormal leaves and stems that had not rooted in the different shoot-induction media during the first 30 days. Rapid elongation and rooting were also reported in kenaf shoots regenerated in vitro by Srivatanakul et al. (2000), who suggested that kenaf might contain high auxin levels that are favorable for rooting and might overcome a "carry over" effect of TDZ from multiple shoot-induction medium. An auxin source (0.6 j.tM IAA) was added to the rooting HRM used in the current study. After 30 days on HRM, the percentage of root formation ranged from 62.2% to 100% (Table 2), compared with the 7%

TABLE 2. Root formation in in vitro regenerated shoots of H. acetosel/a cultured on 16 shoot induction media and hibiscus rooting medium (HRM)

Medium TDZ iM BA pM Percentage of root Percentage of root formation formation on HRM medium MO 0 0 55.6 ± 2.1bc 95.2 ± 4.8a Ml 0.6 0 44.4 ± 2.3cde 96.3 ± 3.7a M2 1.1 •0 42.2 ± 2.1cde 96.3 ± 3.7a M3 2.3 0 35.6 ± 2.2de 96.7 ± 3.3a M4 0 4.4 82.2 ± 2.2a 106.6 Oa M5 0.6 4.4 40.0 ± 3.6cde 88.8±0.7ab M6 1.1 4.4 40.0 ±3.8cde 92.5 3.8a M7 2.3 4.4 37.8 ± 2.Ode 89.3 ± 0.4ab M8 0 8.9 46.7 ± 3.8cd 88.4 ± 6.4ab M9 0.6 8.9 66.7 ±3.8a 100.0±Oa Mb 1.1 8.9 46.7 ± 3.8cd 79.5 ± 3.2abc Mill 2.3 8.9 35.6 ± 2.2de 62.2±6.2c M12 0 13.3 40.0 ± 3.7cde 92.5±3.8a M13 0.6 13.3 37.8 ± 2.2de 96.7 ± 3.3a M14 1.1 13.3 46.7 ± 3.8cd 95.8 ± 4.2a M15 2.3 13.1 35.6 ± 2.2de 68.9 ± 5.9bc

Nonrooted plants on every induction medium were transferred to the rooting medium (HRM), and root formation was evaluated after 30 days. Within a column, means followed by the same letter are not significantly different at the 0.05 rejection level according to Tukeys HSD test. Means are from three independent experiments with 15 explants for each determination. Hamidou F. Sakhanokho 207 rooting efficiency reported by Blackmon and Reynolds (1982). Rooted plants were readily acclimatized in Jiffy pellets before transfer to the greenhouse.

CONCLUSIONS

Multiple shoots were successfully induced and regenerated from H. acetosella shoot apices grown for 30 days on media containing various combinations of BA and TDZ. Both BA and TDZ have been shown to induce multiple shoots in various plant species, and the rate of multiple shoot induction obtained in this study is within the range presented in the literature for other species of the Malvaceae family (Agrawal et al., 1997; Morre et al., 1998; Srivatanakul et al., 2000; Banerjee et al., 2003). M9 (0.6 [LM TDZ + 8.9 p.M BA) was one of the best performing treatments for both multiple shoot induction and root formation in H. acetosella, producing normal plants. Shoot transformation in other species (Zhong et a]., 1996; Gould et al., 1998; Zapata et al., 1999) has been achieved using both particle bom- brdment and Agrobacterium-mediated transformation. The results obtained in the current study could establish the basis of a shoot transformation sys- H. acetosella can tem for H. acetosella genotypes. In addition, even though be seed propagated, clonal in vitro propagation offers the potential to pro- duce a large number of plants in a short period of time and also preserve the unique characteristics of the selected plants (Chalupa, 2003).

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