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Efficient Somatic Embryogenesis and Plant Regeneration from Immature

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Efficient Somatic Embryogenesis and Plant Regeneration from Immature HORTSCIENCE 49(12):1558–1562. 2014. Reserve in Hubei Province, China (Seed of the species mature in September in Hubei Province.). The immature seeds were washed Efficient Somatic Embryogenesis and in 250 mL water containing two drops of Tween-20 and then surface-disinfected in Plant Regeneration from Immature 75% (v/v) ethanol for 1 min. They were soaked in 0.1% (w/v) HgCl2 solution plus Embryos of Tapiscia sinensis Oliv., two drops of Tween-20 per 100 mL for 10 min followed by washing five times with sterile water and soaked in sterile water an Endemic and Endangered Species overnight at 4 °C to soften the seedcoats. The hard seedcoats were removed mechan- in China ically. The decoated seeds were rinsed five Yuyu Wang, Faju Chen, Yubing Wang, Xiaoling Li, and Hongwei Liang1 times with sterile water then disinfected with 0.1% (w/v) HgCl2 solution for 5 min and Biotechnology Research Center, Hubei Key Laboratory of Natural Products rinsed with sterile water five times, and then Research and Development, China Three Gorges University, Yichang, the immature embryo of the decoated seeds Hubei, China 443002 was placed on the culture medium. The cultures were incubated at 25 ± 2 °C under Additional index words. Tapiscia sinensis Oliv., immature embryo, somatic embryogenesis, a 16/8-h (light/dark) photoperiod with illu- histocytology mination by white fluorescent light with an intensity of 50 mmol m–2 s–1. Abstract. High-frequency somatic embryogenesis and plant regeneration were achieved · · Callus induction. The immature embryo from immature cotyledonary-stage embryos in the endangered plant, Tapiscia sinensis explants were cultured on MS basal medium Oliv. Plant growth regulators with different concentrations and combinations on supplemented with 2,4-D and in combination embryogenesis capacity were studied. The optimal explants for in vitro somatic with activated charcoal to induce embryo- embryogenesis were immature embryos in T. sinensis. A high callus induction rate of genic calli. Different concentrations of 2,4-D 100% was achieved on Murashige and Skoog (MS) basal medium supplemented with 1.0 –1 L (0.1, 0.5, 1.0, and 2.0 mg L ) and activated mg Ll 1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5% (w/v) activated charcoal. · · charcoal (0%, 0.05%, 0.1%, 0.5%, and 1.0% Alternatively, a high induction rate (96.16%) of somatic embryogenesis was obtained on w/v) were tested for their effect on the initial MS basal medium supplemented with the combination of 0.05 mg·LL1 a-naphthaleneacetic L callus induction. The rate of callus induction acid (NAA) and 0.2 mg L 1 6-benzylaminopurine (6-BA), and somatic embryos proliferated · was measured after 20 d. Eight petri dishes fastest on the mentioned medium supplemented with 0.5% (w/v) activated charcoal and 3% for each treatment combination were pre- (w/v) sucrose, inoculation of explants proliferating 21 times in the 23-day subculture. Of the pared. On average, 30 explants were cultured 100 plantlets transferred to field after the acclimation, 95 (95%) survived. Based on the to each petri dish, and each treatment was histocytological observations, the development of somatic embryos was similar to that of replicated three times. zygotic embryos. There were two accumulation peaks of starch grains in the embryogenic Differentiation and induction of somatic calli and in the globular-stage embryos, both closely related to the energy supply, and the embryos. Calli, induced from explants after embryoids were of multicelluar origin. cultured for 20 d, were transferred to new MS culture medium with 6-BA (0.1, 0.2, 0.5, and 1.0 mg·L–1) and NAA (0, 0.01, 0.05, and 0.1 Tapiscia sinensis Oliv., one of the rare fruit maturation (Liu et al., 2008). Moreover, it –1 species in China, belongs to the genus Staph- has been subject to tremendous human distur- mg·L ) and different concentrations of su- ylea, family Staphyleaceae. It has not only bance and thus has become an endangered crose (0.00%, 1.50%, 3.00%, 6.00%, and significant scientific value for the investiga- species. Considering its rarity and important 9.00%) to develop somatic embryogenesis. tion of the origin of the semitropical flora of value, it has been listed in the Chinese Plants Each treatment consisted of five petri dishes China, and the phylogenesis of the family Red Cover Book as a priority plant to promote and 20 calli were inoculated to each petri Staphyleaceae for its characteristics of the its conservation in China (Fu, 1992). Some dish, and all treatments were repeated three ancient origin, but also a promising ornamen- researches were also performed on seedling times. After 19-d culture, somatic embryos tal species because of its beautiful flowers and the process of seed germination of Tapiscia emerged and the embryogenesis rate was and tree form. It is also an excellent plant in sinensis (Han, 2010; Zhou and Duan, 2008). counted. Then, the somatic embryos were afforestation because of its rapid growth However, no efficient in vitro plant regenera- cultured on the growth regulator-free MS habit. This species has a very narrow dis- tion system of T. sinensis has been reported. culture medium to allow the development of tribution range in southwestern China and Hence, there is a demand for the develop- plantlets. The induction frequency of somatic regenerate natural community by seedling. ment of a conservation strategy through micro- embryogenesis was calculated as follows: the However, natural regeneration capacity of propagation to prevent extinction. Somatic number of explants that induced somatic em- the species is weak because of its low seed embryogenesis is an alternative method for bryos divided by the total number of explants. setting rate and poor seedling viability under mass propagation and production of synthetic The growth of embryogenic callus was natural conditions (Sun and Liu, 2004). The seeds (Cheruvathur et al., 2013; Levin et al., also evaluated. After weighing, the calli was species takes 17 months to complete the sexual 1988; Tejavathi et al., 2007). Therefore, the cultured on embryo induction medium (which reproductive cycle from zygote initiation to aim of the present investigation was to establish was screened out form the previous step) a reproducible, affordable, and efficient in vitro supplemented with different concentrations plant regeneration protocol from immature of glucose, and the inoculum was weighted again after 23-d culture. Multiplication rate Received for publication 2 Mar. 2014. Accepted embryos of T. sinensis through somatic em- bryogenesis. was counted and presented as the mean ± SD. for publication 27 Aug. 2014. Each treatment consisted of five petri dishes The authors gratefully acknowledge the financial and 0.85 to 0.90 g calli were inoculated to each support of the ‘‘Five-twelfth’’ National Science and Technology Support Program (2013BAD03B03) Materials and Methods petri dish, and all experiments were repeated and of Nature Science Foundation of Hubei Prov- three times. ince (2012FFB03806). Materials and culture conditions. Imma- The basal culture medium used for somatic 1To whom reprint requests should be addressed; ture seeds of T. sinensis were collected in embryogenesis induction was MS medium e-mail [email protected]. late May 2008 from the Shennongjia Nature (Murashige and Skoog, 1962) supplemented 1558 HORTSCIENCE VOL. 49(12) DECEMBER 2014 PROPAGATION AND TISSUE CULTURE with 3% (w/v) sucrose and solidified by 0.8% (w/v) agar (National Pharmaceutical Group Corporation, Shanghai, China). The medium was adjusted to pH 6.0 before autoclaving at 0.11 MPa and 121 °C for 20 min. Histology. Embryogenic tissues were col- lected during embryonic development of somatic embryos and were fixed in FAA solution (formalin:acetic acid:absolute etha- nol:distilled water of 5:5:45:45, v/v/v/v) for 24 h at room temperature for fixation, then dehydrated in a graded ethanol series (70%, 85%, 95%, and 100%, v/v) and embedded in paraffin. Sections were cut 6 to 8 mm thin with a rotary microtome, mounted onto glass Fig. 1. Various calli from immature embryos of Tapiscia sinensis.(A) Callus from immature embryos. (B) slides, and then stained with Periodic acid- Callus turned from white to black (· 8). Schiff. At the end, they were observed and photographed with a photomicroscope (Nikon 80i, Japan). Table 1. Effect of 2,4-D concentrations on callus induction in Tapiscia sinensis.z Statistical analysis. The induction per- Concentrations of Induction frequency Transformation the centages were transformed into arcsine values 2,4-D (mg·L–1) of callus (%) percentages in arcsin values before analysis, and then data were analyzed 0.00 0.00 0.00 ± 0.00 by analysis of variance to detect significant 0.10 0.00 0.00 ± 0.00 differences between means using SPSS V11.5 0.50 33.34 0.34 ± 0.03 c software (IBM, Chicago, IL). Means differing 1.00 64.67 0.70 ± 0.02 a significantly were compared using Duncan’s 2.00 50.20 0.53 ± 0.01 b multiple range test at the 5% probability level. zExplants of immature embryos were cultured on Murashige and Skoog basal medium supplemented with Variability around the mean was represented the different concentrations of 2,4-D. Data generated after 20 d in culture and were presented as the mean ± as ± SD. SD. Different letters indicated that the values were significantly different at P < 0.05 according to Duncan’s multiple range test. 2,4-D = 2,4-dichlorophenoxyacetic acid. Results Callus induction. Explants of immature embryos, cultured on the growth regulator- Table 2. Effect of active charcoal on callus induction from explants of immature embryos of Tapiscia sinensis.z free culture medium or the medium con- –1 Concn of 2,4-D Concentrations of active Induction frequency Transformation the taining 0.1 mg·L 2, 4-D, largely formed –1 seedlings rather than calli.
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