Hort. Environ. Biotechnol. 53(4):316-319. 2012. DOI 10.1007/s13580-012-0081-8
Research Report
Anatomical Observations of Adventitious Bud Regeneration from Leaf Explants of Ziziphus jujube Mill. ‘Huizao’ *
Chunhua Ma, Xia Ye, Yanhui Chen, Jiancan Feng , Xiaoli Shang, Jidong Li, Yingxia Wu, and Jianbin Hu College of Horticulture, Henan Agricultural University, 95 Wenhua Road, 450002 Zhengzhou, People’s Republic of China *Corresponding author:
[email protected],
[email protected]
Received September 28, 2011 / Revised July 7, 2012 / Accepted July 20, 2012 GKorean Society for Horticultural Science and Springer 2012
Abstract. The histological process of adventitious shoot regeneration from the leaf explants of Zizyphus jujuba ‘Huizao’ was reported in this study. This is the first report on the detailed histological process of direct shoot regeneration from leaf explants in Z. jujube. Shoot regeneration was obtained from woody plant medium (WPM) supplemented with 2.27 ȝM thidiazuron (TDZ), 1.07 ȝM Į-naphthalene-acetic acid (NAA) and 2.94 ȝM silver nitrate (AgNO3) for 10 days in the dark followed by 3 weeks at a 14 hours photoperiod. The adventitious buds mostly formed from leaf veins and petioles, and the further histological studies revealed that there were multiple vascular bundles around leaf veins and the adaxial side of explants, and the adventitious buds directly originated from the parenchymatous cells around the vascular bundles without the intervening callus phase. After 3 days of culture, the parenchymatous cells started dividing and meristemoids formed thereafter. The meristematic cells continued division and subsequently gave rise to bud primordia. Well-developed shoot buds through direct organogenesis was achieved after 20 days of culture. Additional key words: histology, jujube, morphogenesis, organogensis
Introduction Jujube (Ziziphus jujuba Mill.), a native fruit tree of China, is one of the world's major fruit crops and is cultivated in India, Russia, the Middle East, southern Europe, and especially China. It has a highly economic value in food, especially in medicine (Liu, 2008). Jujube fruits are the important agriculture exports in China and rich in nutritional value. Jujube flowers have a long flowering phase and can produce high-quality honey. Tissue culture technique has been used in jujube in order to rapidly propagate elite cultivars or establish genetic transformation system. To date, successful plant regeneration has been reported in Z. jujuba from stem (Mathur et al., 1995) and leaf (Gu et al., 2005) explants. In our previous studies, several factors influencing efficiency of shoot regeneration in Z. jujuba were elucidated (Feng et al., 2009, 2010). However, genetic instability is commonly encountered in the tissue culture, and a callus intermediate phase, which has a higher frequency of somaclonal variation and the genetic instability, is generally undesirable for the regeneration of elite material (Che et al., 2007; Pereira et al., 2000). Hence, it is necessary to reduce the callusing stage, clarify the critical events during shoot
formation and understand how and where adventitious bud is originated and formed, and whether somatic embryo is induced in the shoot regeneration of jujube. Clarification of this process will facilitate improvement of the efficiency of the regeneration of jujube, and aiding the further genetic transformation. In this study, the definition of morphogenic pathway of adventitious buds was investigated, and the histological characterization of regeneration of Zizyphus jujuba ‘Huizao’ was clarified at details.
Material and Method 3ODQW 0DWHULDO DQG &XOWXUH &RQGLWLRQ In vitro jujube plants were maintained on Murashige and Skoog (MS) (1962) medium supplemented with 6.66 ȝM 6-benzylaminopurine (6-BA), 0.57 ȝM indoleacetic acid (IAA) and 5.77 ȝM gibberellic acid (GA3), and sub-cultured on fresh medium every four weeks (Feng et al., 2010). Expanded leaves excised from the middle portion of three -week-old shoots of ‘Huizao' plantlets were used as explants for adventitious bud induction, which were wounded by three cuts transversely to the midrib, and placed with the abaxial surface contacting the medium. The materials were
Hort. Environ. Biotechnol. 53(4):316-319. 2012.
cultured at 26 ± 2°C with a 14/10 h (light/dark) photoperiod provided by cool-white fluorescent tubes at a photon flux -2 -1 density of 30 ȝmolm s . $GYHQWLWLRXV %XG ,QGXFWLRQ Leaf explants were wounded by three transverse cuts on the midrib without full separation of the blades, and placed the abaxial surface contacting the woody plant medium (WPM) supplemented with 2.27 ȝM thidiazuron (TDZ), 1.07 ȝM Į-naphthalene-acetic acid (NAA), 2.94 ȝM silver nitrate (AgNO3), 40 gL-1 sucrose, and 6.5 gL-1 agar (differentiation medium) (Feng et al., 2010). After incubation in darkness for ten days, cultures were moved into growth room under the same photoperiod and temperature as described above. +LVWRORJLFDO $QDO\VLV To obtain histological confirmation of the initiation and development of the adventitious buds, the leaf explants cultured on the differentiation medium were sampled from 0-30 day at every 2-3 days. Samples were fixed in FAA solution (formalin/acetic acid/50% ethanol (1:1:18 by vol.; FAA) for 2 h, washed in 50% ethanol, 30% ethanol and distilled- water in succession for 30 min in each step. Following this, samples were dehydrated in an ethanolic graded series (30, 50, 75, 85, 95, 100%), and then embedded in paraffin wax for 48 h. Sections were cut (10 ȝm) with a rotary microtome (LEICA RM2235), fixed to microscope slides and affixed by heating (Laparra, 1997). Slides were stained with Heidenhain’s hematoxylin and then covered with the cover slips after one to two drops of neutral balsam were added. The slides were examined under a light microscope (OLYMPUS BX51).
Results $GYHQWLWLRXV %XG ,QGXFWLRQ After 3 days of culture, the explants became curly and thicker, especially the cut regions (Fig. 1A), and then turned yellow gradually. After the first 10 days of culture in the dark, the leaf explants were transferred to light conditions to induce adventitious shoot formation. By day 11 of culture, a few small white callus-like protuberances (Fig. 1B) were formed in the cut regions. The leaf explants and some of those white protuberances turned green after 14 days of culture (Fig. 1C). After 14-17 days in culture, obvious shoot buds at various developmental stages were observed on both the abaxial and adaxial surface of the explants along the midrib. About 4 days later, green leaves emerged from the shoot buds (Figs. 1D, 1E, and 1F). No obvious callus was observed in the explants during in vitro culture. According
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Fig. 1. Shoot formation by organogenesis on leaf explants of Z. jujuba. (A) Leaf explant with visible morphological changes after 3 days in culture (Bar = 1 mm). (B, C) Protuberances on the midrib surface of the leaf explants (Bar = 0.5 and 1 mm). (D, E, and F) Regenerated plantlet (Bar = 0.5, 2, and 2 mm).
to our results, it took 18-20 days for shoot regeneration after inoculation which reflecting that leaves of Z. jujuba have great potential for shoot regeneration. $GYHQWLWLRXV %XG )RUPDWLRQ IURP /HDI 9HLQV DQG 3HWLROHV It was clearly shown that the adventitious buds were mainly originated from leaf veins and petioles of in the culture of Z. jujuba (Fig. 2). Callus-like protuberances were firstly observed on the petioles after three days of culture and on the leaf veins after eleven days of culture, respectively, and then adventitious buds were also formed from the leaf veins and petioles (Fig. 2). There were no organogenic callus and shoot formation in the leaf segments in the culture of Z. jujuba. +LVWRORJLFDO &KDUDFWHUL]DWLRQ RI 5HJHQHUDWLRQ Transverse sections of the leaves revealed that leaf explant consisted of epidermis, subepidermis, vascular tissue and a large number of parenchyma cells around the vascular bundles. The vascular bundles arranged in a succession were open to the adaxial side of the explants (Fig. 3A). The first histological change was observed in the leaf explants after 3
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Chunhua Ma, Xia Ye, Yanhui Chen, Jiancan Feng, Xiaoli Shang, Jidong Li, Yingxia Wu, and Jianbin Hu
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Fig. 2. Adventitious bud formation from the leaf veins and petioles of Z. jujuba. (A) Day 3 (Bar = 2 mm), (B) Day 11 (Bar = 1 mm), (C) Day 14 (Bar = 2 mm), (D) Day 19 (Bar = 1 mm), (E) Day 24 (Bar = 1 mm), (F) Day 34 (Bar = 2 mm).
days of incubation (Figs. 3B and 3C). At this stage, several kinds of cells i.e. palisade cells, spongy parenchyma, cambial cells of main veins and those parenchyma cells adjacent to vascular bundles, were characterized by a dense cytoplasm and a large, prominent nucleus in the middle of cells, and showed an increase in size. Also, some of those cells adjacent to vascular bundles started initial division while the cells further apart with vascular bundles remained unchanged. The second phase was characterized by the emergence of meristems after 5-11 days of culture. At this phase, parenchyma cells adjacent to the vascular bundles continued to divideand nucleus and division were emerged in those cells further apart from the vascular bundles (Fig. 3D). Due to successive dedifferentiation and division of parenchymatic cells in all directions, meristemoids composed of small tightly packed cells appeared in these regions of the explants on the 11th day (Fig. 3E). Those meristemoid cells were found to be much smaller than the surrounding cells in term of densely stained cytoplasms with small vacuoles, a large and prominent centrally positioned nucleus, and a high nucleus -to-cell area ratio. They were easily distinguished from the surrounding cells with flattened features and large vacuoles. Formation of primordia and adventitious shoots was the characteristics of the third phase which was observed 14-20 days in culture. At this stage, the meristematic zones show-
Fig. 3. Histological aspects of adventitious shoots developed from leaf explants of Z. jujuba. (A) Transverse section of leaf explant showing epidermis, subepidermis, vascular tissue and a large number of parenchyma cells (Bar = 50 ˩m). (B, C) After 3 days in culture, cells in leaf explants, i.e. palisade cell, spongy parenchyma, cambial cells of main veins and those parenchyma cells adjacent to vascular bundles showed nucleus and some parenchyma cells underwent division (Bar = 100 and 50 ˩m). (D) Division of parenchyma cells farther apart with vascular bundles between 5 and 11 days (Bar = 50 ˩m). (E) Meristematic zones (MZ) originating from parenchymatous region (Bar = 20 ˩m). (F, G) Single and multiple bud primordia with leaf primordum (LP), apical meristem (AM) (Bar = 10 and 50 ˩m). (H) Single or multiple well-developed adventitious bud with leaves (L), apical meristem and vascular connections (V) (Bar = 50 ˩m).
Hort. Environ. Biotechnol. 53(4):316-319. 2012.
ed intense dedifferentiation ability. The meristematic cells divided constantly and gave rise to single (Fig. 3F) or multiple (Fig. 3G) bud primordia with obvious leaf primordia and apical meristems without an intervening callus phase. After 20 days of incubation in differentiation medium, shoot buds with apical meristems, leaves and visible vasculars connected to the explants were observed (Fig. 3H). At the same time, some callus structures were formed on the midvein or on lateral leaf veins, however, no further development was observed. In addition, the formation of adventitious buds was asynchronous and continuous throughout the whole culture period. For example, some adventitious buds have already well developed after 20 days of culture, while the parenchymatous cells adjacent to vascular tissues differentiated continuously and gave rise to new meristemoids, and a few of them failed to develop into normal shoots.
Discussion Plant regeneration could occur through organogenesis and/or somatic embryogenesis directly or indirectly (Phillips, 2004; Subotic, 2007). In this study, the adventitious shoots of jujube were differentiated from the meristematic areas formed from parenchymatous cells around the vascular bundles without an intermediate callus phase. Although the callus structures occasionally formed on the midvein or on lateral leaf veins, almost all meristemoid in the callus could not produce normal shoots based on the histological observations. Hence, the adventitious shoot regeneration from in vitro leaf explants of Z. jujuba ‘Huizao’ via direct shoot organogenesis could be confirmed. The pattern of shoot bud origin and development was very similar to other plant species, such as Indian spinach (Mitra and Mukherjee, 2001) and Spilanthes acmella (Saritha and Naidu, 2008). In general, the cells located in leaf epidermal regions are the sources of organogenesis, which have been reported in Santalum album L. (Mujib, 2005) and Aloysia polystachya (Burdyn et al., 2006). However, we found that the formation of adventitious buds mainly occurred around leaf veins and petioles explants, especially the regions close to the base of the leaf instead of the leaf lamina. Similar findings were reported in Stevia rebaudiana (Sreedhar et al., 2008). The further histological results indicated that there were multiple vascular bundles around leaf veins and the adaxial side of explants (Fig. 3A), which were essential elements for nutrient transport and intra-net communication. After a few days of culture, cells adjacent to vascular bundles firstly increased in size and then started initial division while the cells further apart with vascular bundles remained unchanged,
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which may explain why callus-like protuberances were firstly observed on the leaf veins and petioles, and leaf explants wounded along the midribs and the adaxial side of explants were more regenerative than other explants in the culture of Z. jujube. Acknowledgement: The research was supported by Innovation Scientists and Technicians Troop Construction Projects of Zhengzhou City (No: 10LJRC179).
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