HORTSCIENCE 52(9):1278–1282. 2017. doi: 10.21273/HORTSCI12056-17 been considered as an attractive way of propagating Haworthia genus plants. This method facilitates the propagation of plants Establishment of a Rapid and Efficient without seasonal and environment limitations and in a large scale, which are important Micropropagation System factors in the breeding plants for ornamental and landscaping purposes (Kitamura et al., for Succulent Plant Haworthia 2002; Kumari et al., 2016; Preil et al., 1988). Plantlets have been successfully induced in several Haworthia species using explants turgida Haw. from inflorescences (Kaul and Sabharwal, Boling Liu, Hongzhou Fang, Chaorong Meng, Ming Chen, 1972; Majumdar, 1970a; Ogihara, 1979; and Qingdong Chai Ogihara and Tsunewaki, 1978), ovary walls (Majumdar, 1970b), and leaves (Beyl and College of Life Sciences, Qufu Normal University, 273165, Qufu, Shandong, Sharma, 1983; Rogers, 1993b). Growth char- China acteristics in response to various auxins and cytokinins, such as NAA, 2,4-D, and BA Kai Zhang (Kaul and Sabharwal, 1972; Ogihara, 1979; Culaishan Forest Farm of Tai’an City, 271000, Tai’an, Shandong, China Ogihara and Tsunewaki, 1978), have been 1 examined in some Haworthia species. How- Shijuan Liu ever, none of these studies have used TDZ in College of Life Sciences, Qufu Normal University, 273165, Qufu, Shandong, in vitro propagation, although it is an efficient China PGR and is widely used for rapid propagation of plant tissues (Fatima and Anis, 2011; Additional index words. callus, plant growth regulators, propagation, root, shoot Kitamura et al., 2002; Kumari et al., 2016; Abstract. In the present study, the effect of plant growth regulators (PGRs) on callus Murthy et al., 1998). To the best of our regeneration, adventitious shoot differentiation, and root formation of Haworthia turgida propagation knowledge, there has also been Haw. was investigated. The greatest callus induction percentage (95.6%) was achieved no published study on in vitro propagation of with leaf explants inoculated on Murashige and Skoog (MS) medium with 1.0 mg·LL1 6- H. turgida through the leaf explants either. benzyladenine (BA) and 0.1 mg·LL1 1-naphthaleneacetic acid (NAA), and this callus Therefore, the current study aimed to estab- induction medium supplemented with 2.5 mg·LL1 thidiazuron (TDZ) was optimal for lish a rapid and efficient micropropagation callus proliferation. The maximum number of shoots (25.7) was obtained when the callus protocol for H. turgida using the TDZ as was cultured on MS medium supplemented with 1.0 mg·LL1 BA and 0.2 mg·LL1 2,4- a PGR and the leaves as explants. dichlorophenoxyacetic acid (2,4-D). The highest number of roots per shoot (6.2) and highest rooting frequency (82.0%) were obtained when adventitious shoots were Materials and Methods inoculated on MS medium with 0.05 mg·LL1 NAA. Regenerated plantlets were trans- ferred to a mixture of vermiculite and soil and acclimated in a greenhouse. The survival Plant materials. Haworthia turgida plants rate of the transplanted plantlets was about 91.6%. The rate of ex vitro rooting was were donated by the Institute of Botany, 83.3%, indicating that this technique is effective for root induction in H. turgida. This Chinese Academy of Sciences. Plants were study has established a rapid and efficient micropropagation system that can be transplanted in a greenhouse at College of beneficial for commercial cultivation and germplasm conservation of H. turgida. Life Sciences, Qufu Normal University, China in July 2014. Fresh leaves were used as explants, which were washed in running tap water mixed with a few drops of liquid The succulent plant genus Haworthia, shady environmental conditions, require detergent for 30 min. Then, the leaves were belonging to the Liliaceae family, is native minimal maintenance, and have been popu- decontaminated with 70% ethanol for 30 s to South Africa, Namibia, Swaziland, and larly used in various countries as ornamental followed by two to three washes with sterile Mozambique (Beyl and Sharma, 1983; Kaul plant. Some Haworthia species have also water. After decontamination, the explants were and Sabharwal, 1972). Haworthia exhibits been used in miniature gardens, office, and sterilized in 0.1% HgCl2 supplemented with highly variable yet distinct plant and leaf balconies (van Jaarsveld, 1999). Most leafy 0.05% Tween-20 for 5 min, then washed morphology, thus sparking the interest of succulents possess translucent epidermis that with sterilized water for 3–4 times and dried both amateur botanists and succulent plant protects its underlying transparent water with disinfected filter paper. The sterilized collectors. Haworthia has been used in pot storage parenchymatous tissue layer. The leaves were cut into 0.5–1.0 cm pieces for culture and landscaping of open-air rockeries translucent regions of the plant can take the subsequent experiments after removing injured (Barker, 1929). Its species are well adapted to shape of scattered dots, lines, and wide leaf bases using a sterilized blade. streaks. Based on this feature, these have Culture medium and culture conditions. been called window-leaved plants and are The culture medium consisted of Murashige Received for publication 26 Apr. 2017. Accepted mainly observed in the Liliaceae, Compo- and Skoog (1962) basal salts and vitamins as for publication 26 July 2017. sitae, and Mesembryanthemaceae (Krulik, base medium, mixed with 3% (w/v) sucrose This work was funded by the Natural Science 1980). Haworthia turgida hasradialleaveswith and solidified with 0.7% (w/v) plant agar. The Foundation of Shandong Province (ZR2014JL021), a flat cross-section at the leaf apexes and pH of the medium was adjusted to 5.8 using the Science and Technology Planning Project contains several lines forming the special win- 0.1 M NaOH or 0.1 M HCl, and the medium (xkj201403) and the Doctoral Scientific Fund Project dow structure (Egbert and Martin, 2002), and was autoclaved at 121 °C for 20 min. The MS of Qufu Normal University (bsqd20130136). is valued as an important ornamental plant in medium was supplemented with PGRs before We appreciate Byoung Ryong Jeong (Department of Chinese flower markets. pH adjustment and sterilization. All cultures Horticulture, College of Agriculture and Life Sci- Haworthia is self-incompatible and gen- were placed at 24 ± 1 °C under a 14-h ences, Gyeongsang National University, Jinju, m · –2· –1 Korea) for revising this manuscript. erally propagated by leaf cutting. However, photoperiod with 40 mol m s photosyn- Establishment of a Rapid and Efficient Micropropa- this relatively slow process is caused by the thetic photon density quipped with cool-white gation System for Succulent Plant Haworthia turgida low number of offshoots produced by the fluorescent lamps (Philips 40 W tubes). Haw. parent plants (Mycock et al., 1997; Rogers, Callus induction and propagation. The 1Corresponding author. E-mail: [email protected]. 1993a). Consequently, micropropagation has explants were inoculated on a basal MS 1278 HORTSCIENCE VOL. 52(9) SEPTEMBER 2017 PROPAGATION AND TISSUE CULTURE medium with 0, 1.0, 2.0, or 3.0 mg·L–1 BA in inoculated on the MS medium with 0, 0.05, the original units. All data were analyzed combination with 0, 0.1, 0.2, or 0.5 mg·L–1 0.10, 0.15, or 0.20 mg·L–1 NAA for root statistically using a statistical software SPSS NAA (Table 1) based on previous reports formation (Table 4). After 30 d, the fre- (version 19.0; IBM, Armonk, NY). Signifi- (Ogihara, 1979; Ogihara and Tsunewaki, quency of root induction, number of roots cant differences among the treatments were 1978) on H. setata and H. aristata for in- per plantlet, and length of the roots were determined using the Duncan’s multiple duction of callus generation. Then callus was assessed. In each treatment, 24 shoots were range test at P < 0.05. The results were cut into 1–2 cm2 pieces and transferred to used, and each experiment was repeated expressed as the mean ± SE of repeated another medium supplemented with 0, 0.5, thrice. Meanwhile, 60 elongated shoots experiments. 1.0, 1.5, 2.0, 2.5, or 3.0 mg·L–1 TDZ (Table 2) (2–3 cm in length) were wounded gently at for callus proliferation. In each treatment, 15 the bottom of the stem, and then transplanted explants were used, and each experiment was to a mixture of vermiculite and soil (Flower Results and Discussion repeated three times. Fresh callus weight of Nutrition Soil, Deli, Fuzhou, China; 1:1, v/v), each treatment was recorded after 30 d. Effect of NAA and BA on induction of and established in the greenhouse for simulta- callus from the leaf explants. PGRs such as Callus growth rate was calculated as using the neous ex vitro rooting. The rooting percentage following equation: Growth rate = (w – w )/w , auxins and cytokinins are the main regulators t 0 0 was calculated as follows: of plant de- and redifferentiation (Frello where wt and w0 are the final and initial fresh weight of each callus, respectively. The term Rooting percentage et al., 2002; Kordi et al., 2013; Liu et al., ‘‘growth’’ is used to represent callus develop- =ð Number of shoots with roots= 2016; Peeters et al., 1991). Jaco et al. (1993) used BA and NAA for callus induction of ment as evaluated by fresh weight (Ogihara and Total number of shoots plantedÞ · 100 Tsunewaki, 1978), and callus induction ratio sugar beet (Beta vulgaris). In this study, was calculated as follows: various types and concentration of PGRs Well-rooted plantlets were taken out from were selected based on previous studies with Callus induction ratio culture vessels, washed in running tap water some modifications (Kaul and Sabharwal, =ð Number of explanted leaves showing to remove attached agar, transplanted in 1972; Kitamura et al., 2002; Ogihara, 1979; acclimatization boxes (7 · 7 cm) with a mix- Ogihara and Tsunewaki, 1978).