callus clump (Fig. 1D5). Plant regeneration from somatic embryos. B5 vitamins and glucose were beneficial for the development of shoots from somatic embryos ...
In Vitro Cell. Dev. Biol.—Plant 42:148–151, March– April 2006 q 2006 Society for In Vitro Biology 1054-5476/06 $18.00+0.00
DOI: 10.1079/IVP2006748
PLANT REGENERATION OF CHORISPORA BUNGEANA VIA SOMATIC EMBRYOGENESIS JIANHUI WANG1, LIZHE AN1,2*, RUOYU WANG1, DAQUN YANG2, JING SI2, XUANYING FU2, JIANFENG CHANG2, 1
AND
SHIJIAN XU2
Cold and Arid Regions Environment and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, P.R. China 2 School of Life Science, Lanzhou University, Lanzhou 730000, P.R. China (Received 4 May 2005; accepted 27 December 2005; editor S. Guha-Mukherjee)
Summary A method was developed for in vitro regeneration of plants via somatic embryogenesis in Chorispora bungeana, an alpine plant with freeze-tolerance, using cell suspensions initiated from leaf-derived callus. Primary calli were induced from leaves of C. bungeana grown on Murashige and Skoog (MS) media supplemented with 4.0 mg l21 gibberellic acid (GA3), 0.2 mg l21 a-naphthaleneacetic acid (NAA) and 0.2 mg l21 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension culture was initiated by incubating the callus particulates in liquid MS medium supplemented with 1.0 mg l21 kinetin (KT) and 0.2 mg l21 NAA. Individual early cotyledonary-stage somatic embryos isolated from cell suspension developed into whole plants on medium containing high levels of sucrose (60 and 90 g l21), whereas lower sucrose concentrations (0 and 30 g l21) were inhibitory to main root development. On the MS medium with 90 g l21 sucrose, one regenerated plant exhibited hetero-morphologic leaves, while other plants grown on different media showed a transformation from stem to root. Key words: chromosome number; somatic embryo; sucrose concentration; suspension cell culture; tissue culture.
INTRODUCTION
MATERIALS AND METHODS
Chorispora bungeana is a perennial herb belonging to the Brassicaceae family. It is distributed mainly in China, Afghanistan, India, Kashmir, Kazakstan, Kyrgyzstan, Mongolia, Pakistan, Russia, Tajikistan, and Uzbekistan, and is grown in cold alpine grassland, alpine screes, and open slopes (Zhou et al., 2001). We discovered that the C. bungeana plant found next to Glacier No. 1 (43.05N, 86.49E, with a 3600– 3900 m height above sea level) in the source area of Urumqi River in Tianshan Mountain, Xinjiang, China, where the temperature is usually less than 58C in the day and less than 08C at night during the growth period from June to September, is highly variable in leaf morphology (Al-Shehbaz et al., 2000; Zhou et al., 2001). It possesses hetero-morphologic oblong leaves with smooth or serrate leaf margins. The stem, embedded in scree, could develop into new roots in the following year. A tubercle is usually formed at the connection of new and old roots and as a result, the number of tubercles can be used as an indicator of the plant age. Results of our preliminary study showed that C. bungeana was freeze tolerant but its seeds were difficult to germinate. We thought that it might be useful to develop an efficient tissue culture system for high-frequency plant regeneration, which could be used as an important tool for further study of C. bungeana. In this study, we describe an efficient method for regeneration of C. bungeana plants via somatic embryogenesis.
Plant materials. The plants of C. bungeana were collected from Tianshan Mountain in July at blooming stage, and most of them were estimated to be 2–4 years old by tubercle number on the main root. The leaves without serrate margins were selected for callus induction. Callus induction. Leaves were surface-sterilized by immersing in 70% ethanol for 30 –40 s, followed by 15 min in 20% NaOCl, rinsed three times with sterile water, and excess water wiped off with sterile filter paper. The leaves were cut into small pieces (10 mm in length) with the margin removed. Tissues were placed in the culture plates, each with 20 pieces, containing Murashige and Skoog (MS) basal medium supplemented with different concentrations of gibberellic acid (GA3), 2,4-dichlorophenoxyacetic acid (2,4-D), and a-naphthaleneacetic acid (NAA) (Table 1). The medium was also added with 0.1% ascorbic acid and 0.1% polyvinylpyrrolidone (PVP) to avoid browning. Cultures were incubated in a room at 258C under a 16 h photoperiod. The frequency and the number of calluses were evaluated after 8 wk of culture when most calluses grew to a diameter of 2– 4 mm. Somatic embryo induction from suspension culture. Well-grown calluses were crushed into smaller pieces and 5.0 g of callus pieces were transferred to 50 ml of liquid MS basal medium supplemented with 1.0 mg l21 kinetin (KT) and 0.2 mg l21 NAA. Cultures were incubated at 258C on a rotary shaker at 50 rpm in the dark or under light condition with 16 h photoperiod. The medium was changed at weekly intervals and the large calluses were removed by filtering through a steel sieve with the pore size of 1 mm diameter. After 4 wk, cells in the suspension were stained with 0.1% acridine orange fluorescence for 20 min, then examined under a fluorescent microscope. Somatic embryos 1–10 mm long were examined under an optical microscope. Plant regeneration from somatic embryos. Individual somatic embryos at the early cotyledonary stage in the suspension were selected and transferred to culture plates containing different media to investigate the effect of these media on the development of somatic embryos into plants. The media used were MS with different sucrose concentrations (0, 30, 60, and 90 g l21),
*Author to whom correspondence should be addressed: Email lizhean@ lzu.edu.cn
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PLANT REGENERATION FROM CHORISPORA BUNGEANA TABLE 1 THE EFFECTS OF REGULATORS IN THE INDUCTION MEDIUM ON CALLUS FORMATION FROM MATURE LEAVES AFTER 8 WK OF CULTURE Regulators (mg l21) GA3
NAA
2,4-D
Color of explants
4.0 4.0 4.0 4.0 4.0 4.0 4.0 2.0 2.0 0 0 0 0 0
2.0 1.0 0.5 0.2 0 0 0 2.0 1.0 0.5 0.2 0 0 0
2.0 1.0 0.5 0.2 0.5 2.0 0 2.0 1.0 0.5 0.2x 0.2x 2.0x 0x
Yellow Yellow Yellow Greenish yellow Greenish yellow Yellow Greenish yellow Yellow Yellow Mostly bleached Brown Brown Brown Bleached
Callus induction rate (%)y
Number of callus per explants (%)z
31.7 ^ 7.9 28.3 ^ 10.4 71.7 ^ 10.4 98.3 ^ 2.9 21.7 ^ 7.6 15.0 ^ 5.0 0.0 ^ 0.0 5.0 ^ 5.0 6.7 ^ 2.9 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0
4.3 ^ 1.2 2.0 ^ 0.3 2.5 ^ 1.2 5.3 ^ 1.8 2.2 ^ 1.5 1.2 ^ 0.2 0.0 ^ 0.0 1.0 ^ 0.0 1.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0 0.0 ^ 0.0
A drop of CaCl2 solution containing protoplasts was dropped from a height of 500 mm above a slide, and then a drop of acetic acid– alcohol (1:3 v/v) was added to the protoplast. After incubation at 658C for 4 h, the specimen was dehydrated via an alcohol series (70, 80, 95, and 100%, 10 min each), dried at room temperature for 1 min, and immersed in 1.0% aceto-orcein for 10 min. The dispersed chromosomes were observed under a microscope. Meanwhile, the main root tips from regenerated plants were also used for chromosome counting. They were incubated at 08C for 24 h, fixed in acetic acid–ethanol (1:3 v/v) for 3 h, and stained with 1.0% aceto-carmine and then squashed.
RESULTS
x
0.1% ascorbic acid and 0.1% PVP were added in media. Each value represents mean ^ SE of three replicates of 20 explants. z Each value represents mean ^ SE of 20 callus-induced explants or whole number of callus-induced explants (if less than 20). y
1/2MS, 1/2MS þ 1.0 mg l21 NAA, MS þ 2.0 mg l21 NAA, MB, and MBG (Table 2). Each culture plate containing 30 somatic embryos was incubated for 4 wk under 258C and 16 h photoperiod. Well-rooted plantlets were transferred to vermiculite and acclimatized in a greenhouse for 1 wk before transferring to the open field. Chromosome counting. To prepare cells for chromosome counting, protoplasts were isolated from somatic embryos. Ten globular embryos were soaked in 10 ml of enzyme mixture comprising 2.0% Cellulose Onozuka RS (Yakult Honsha, Tokyo, Japan), 2.0% pectinase (Yakult Honsha), 1/2MS salts, and 0.4 M mannitol, with pH 5.8–6.0. The enzyme solution with embryos was transferred to a Petri dish (35 mm dia.), and incubated for 8 h at 258C on a rotary shaker at 60 rpm. Protoplasts were separated from the cell debris by filtering through nylon meshes of 60 mm pore size, and centrifuged at 600 rpm for 5 min. The sediment containing protoplasts was re-suspended in 2 M CaCl2 solution and 700 g l21 sucrose solution was carefully injected into the bottom of the centrifuge tube to form a phase interface between sucrose and CaCl2 solution. After centrifuging at 600 rpm for 5 min, protoplasts formed a white layer at the phase interface. The protoplasts were withdrawn with a syringe.
Callus induction. Results of our preliminary study showed that 0.1% HgCl2 could not be used for surface sterilization of C. bungeana leaves used for callus induction, as it resulted in browning and a high mortality rate of the tissues. In addition, treatment of explants with 20% NaOCl for less than 10 min could not eliminate the bacteria completely. We found that explants grown on medium in the absence of GA3 turned brown or bleached, and were dead after 1 – 2 wk of culture. This problem was not alleviated by adding 0.1% ascorbic acid and 0.1% PVP. In contrast, leaf explants grew well on media containing GA3, and callus was initiated after 8 wk of culture. The highest callus induction rate (98.3%) and the highest number of calli per explant (4.3 ^ 1.2) occurred on medium containing 4.0 mg l21 GA3, 2.0 mg l21 NAA, and 2.0 mg l21 2,4-D (Table 1). After subculture for 4 wk, the callus was friable and yellow in color (Fig. 1A). Somatic embryo induction from suspension culture. The suspension culture (Fig. 1B) grown in the dark formed callus clumps and radicles, and could not develop shoots (Fig. 1E1). This was different from the suspension grown under the 16 h photoperiod, where somatic cell (Fig. 1C1), 2-cell stage (Fig. 1C2), 4-cell stage (Fig. 1C3) and poly-cell proembryos (Fig. 1C4-5) were observed. Somatic embryos at different stages such as globular (Fig. 1D1), heart-shaped (Fig. 1D2), early-cotyledonary (Fig. 1D3) and cotyledonary (Fig. 1D4) were induced in the suspension. We also observed synchronized globular somatic embryo development in one callus clump (Fig. 1D5). Plant regeneration from somatic embryos. B5 vitamins and glucose were beneficial for the development of shoots from somatic embryos, but it had little effect on main root induction (Table 2). The presence of high sucrose concentrations (60 and 90 g l21) in the
TABLE 2 PLANT REGENERATION FROM SOMATIC EMBRYOS AFTER 4 WK OF CULTURE Medium MB (MS salt þ B5 vitamin þ 30 g l21 sucrose) MBG (MS salt þ B5 vitamin þ30 g l21 glucose) MS (0 g l21 sucrose) MS (30 g l21 sucrose) MS (60 g l21 sucrose) MS (90 g l21 sucrose) 1/2MS 1/2MS þ 1.0 mg l21 NAA MS þ 2.0 mg l21 NAA z y x
Shoot induction rate (%)z
Root induction rate (%)z
Number of roots per planty
Length of the main root (cm)x
71.0 ^ 0.2 100.0 ^ 0.0 30 ^ 8.3 63.3 ^ 8.3 100.0 ^ 0.0 100.0 ^ 0.0 61.1 þ 9.6 41.1 ^ 8.4 37.8 ^ 7.7
82.7 ^ 8.1 83.9 ^ 10.0 0.0 ^ 0.0 76.4 100.0 ^ 0.0 100.0 ^ 0.0 78.5 100 100
3.2 ^ 0.5 3.3 ^ 1.2 0.5 ^ 0.5 1.5 ^ 0.5 3.0 ^ 2.3 3.6 ^ 2.2 1.2 ^ 0.8 2.5 þ 1.0 2.2 ^ 1.5
– – – – 3.5 ^ 1.7 4.4 ^ 0.5 – – –
Each value represents mean ^ SE of 3 replicates of 30 embryos. Each value represents mean ^ SE of 20 plants having shoots. Each value represents mean ^ SE of 20 plants having main roots.
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FIG . 1. Plant regeneration of C. bungeana via somatic embryogenesis. A, Leaf-derived primary callus. B, Cell suspension. C, Proembryo development in cell suspension (bar ¼ 20 mm). C1, Embryogenic cell. C2, First division of an embryogenic cell. C3, Second division of an embryogenic cell. C4, C5, Pro-embryo derived from embryogenic cell after several divisions. D, Somatic embryos at different stages of development (bar ¼ 1.0 mm): D1, globular stage; D2, heart stage; D3, torpedo stage; D4, early-cotyledonary stage; D5, cotyledonary stage and D6, synchronized embryos in a callus clump. E: E1, somatic embryo development in the dark; E2, somatic embryo developed in the light. F: F1– F3, conversion of a somatic embryo to shoot. G, Shoot development of plantlets in MS with different sucrose concentration (from left to right, 0 g l21, 30 g l21, 60 g l21, 90 g l21). H, Root development of plantlets in MS with different sucrose concentration (H1–H4, 0 g l21, 30 g l21, 60 g l21, 90 g l21, respectively), and H4 shows a leaf with serrate margin. I: I1–I2, plantlets showing transformation from stem to root. J, Protoplasts isolated form globular embryos. K, Protoplast chromosomes showing 2n ¼ 14 (bar ¼ 1 mm). L, Wild hetero-morphologic leaves. M, Three tubercles on wild root showing it to be 4 years old. N, Regenerated plant transferred to field. O, Root tip cell chromosomes showing 2n ¼ 14 (bar ¼ 1 mm).
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medium was shown to enhance markedly complete plant regeneration from somatic embryos of C. bungeana (Fig. 1G). Root development varied greatly in the presence of different sucrose concentrations (0 – 90 g l21) (Fig. 1H1– 4). On most media, the leaves of regenerated plants had no serrate margins (Fig. 1G, 1H1– 3), but on MS medium with 90 g l21 sucrose, there was a plant showing hetero-morphologic leaves (Fig. 1H4). Regenerated shoots grown on most media could form roots and developed into new plantlets (Fig. 1I1, 2), similar to our observation in the field. When these plantlets were transferred to the open field, the survival rate was 70.5%, and the plants were morphologically normal (Fig. 1N). Chromosome number. To determine the chromosome number of regenerated plants, well-dispersed chromosomes present in protoplasts and root tip cells from regenerants were counted. The chromosome number in both types of cell was 14 (Fig. 1K, O). This number was similar to that of C. tenella, another member of Chorispora (Zhou et al., 2001). There was no detectable variation in chromosome number in somatic embryos, suggesting that protoplasts from somatic embryos are a reliable material for chromosome number determination. DISCUSSION This is the first report describing plant regeneration of C. bungeana from cultured tissues via somatic embryogenesis. Results also show that light may play an important role in the induction and development of somatic embryos in cell suspension. Interestingly, explants did not survive on the medium in the absence of GA3. This is in contrast to the explants which formed callus on GA3-containing medium, indicating the important role GA3 in cell growth of C. bungeana in vitro. The important role of GA3 in plant regeneration from callus or somatic embryos has also been reported previously (Matsuda and Adachi 1996; Atmane et al., 2000; Sagare et al., 2000). However, the mechanism of GA3 action is not clear. In this study, GA3 appears to be important for maintaining the vigorous state of explants. The results of this study also show that sucrose exerts a stimulating effect on plant regeneration of C. bungeana. Sucrose is the most commonly used carbon source in tissue culture. The medium used for callus induction is usually supplemented with 20 g l21 sucrose (Chengalrayan and Gallo-Meagher, 2001; Tonon et al., 2001). The promoting effects of sugars on growth-cultured plantlets have been reported in tobacco (Paul and Stitt, 1993; Ticha et al., 1998), sugar beet (Kotvun and Daie, 1995), potato (Cournac et al., 1991; Sima et al., 2001), orchid (Tokuhara and Mii, 2001). In Morus alba, sucrose at 6% has been shown to be most effective for induction of secondary somatic embryogenesis and cotyledonary embryos (Agarwal et al., 2004). Sagare et al. (2000) also reported the gladiolus tuberization form of somatic embryo-derived plantlets by culturing in media supplemented with either 6% sucrose alone or in combination with ABA, ancymidol, paclobutrazol, or PEG-4000. According to Tokuhara and Mii (2001), a high sucrose concentration may act as an osmotic stress or may inhibit chlorophyll formation to induce embryogenic callus formation. However, Martin (2004) reported that plantlets grown on MS basal medium in the absence of sucrose performed better under field conditions compared to those grown on medium containing sucrose. It was speculated that the addition of a growth regulator in the medium might alter the exogenous sugar demands for in vitro growth of storage organs (Nhut et al., 2004).
Although the level of sucrose in the medium is important to tissue culture, the mechanism whereby sugar concentration affects the growth of plantlets is not clear.
Acknowledgments This study was supported by the ‘Hundred Talents Program’ of Chinese Academy of Sciences, National Nature Science Foundation (90302010), Gansu Province Key Project of Science and Technology, and Gansu Province Biotechnological R&D Project. We also thank Professor Zhongqin Li and Professor Keqin Jiao for their kind help in sampling.
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