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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.2 pp.45-50
DOI : https://doi.org/10.14397/jals.2020.54.2.45

Shoot Regeneration from Leaf and Shoot Derived Callus of Hybrid Poplar(Populus alba × P. glandulosa) Clone

Eun-ji Choi1†, Hak-Gon Kim2†, Seong-Hyeon Yong1, Yu-Won Seol1, Dong-Jin Park3, Myung-Suk Choi1*
1Division of Environmental Forest Secience(Institute of Agriculture of Life Science), Gyeongsang National University, Jinju, 52828, South Korea
2Forest Research Department, Gyeongsangnam-do Forest Environment Research Institute, Jinju, 52615, South Korea
3Dept. of seed and seedling management, National Forest Seed and Variety Center, Chungju 27495, South Korea

These authors contributed equally to this work.


*Corresponding author: Myung-Suk Choi Tel: +82-55-772-1856 Fax: +82-55-772-1859 E-mail: mschoi@gnu.ac.kr
January 6, 2020 January 9, 2020 January 10, 2020

Abstract


Plant regeneration from stems and leaves was carried out for the growth of biomass, hybrid poplar(Populus alba × P. glandulosa)clone, which has various uses for plant purification. Callus was well induced when stem and leaf tissues were cultured in 1.0 mg/L 2,4-D containing MS medium. Shoot regeneration was best induced by zeatin among growth regulators, and Plant regeneration was more regenerated in leaf-derived callus than in stem-derived callus. The growth of regenerated shoots at high and low concentrations of zeatin was similar to that of the control at low concentrations. As the concentration of growth regulators increased, the growth of regenerated shoots showed a big difference among individuals. Hybrid poplar showed color variation of plant stem in medium containing high concentration of growth regulator. Regenerated individuals were in vitro rooted in MS medium containing 0.5 mg/L IBA after 2 weeks of culture. and transferred to the greenhouse for acclimatization. The study is believed to be widely used for the induction of in vitro variants through organogenesis.



초록


    INTRODUCTION

    In vitro technology is a good way to solve plant material problems with production costs and special characteristics (Ahuja, 1987). In addition, poplar is a biotechnology model system and has been widely used for micropropagation, gene transformation, and the like (Chun, 1987;Kang & Chun, 1997). Plant regeneration is a basic technology of biotechnology and is widely used for plant propagation, genetic transformation, and genetic conservation (Sul & Shin, 1997;Kang & Chun, 1997).

    Poplar in vitro culture studies have been intensive since 1980 (Chun et al., 1988). Poplar has been used as an in vitro model woody species that exhibits developmental plasticity similar to tobacco, providing a key foundation for biotechnology applications. in vitro propagation through organogenesis in poplar has many research cases (Coleman & Ernst 1990;Noh & Minocha 1986;Park & Son 1988). Most of the plants were regenerated by cytokinin treatment such as 0.2-1.0 mg/L BA or 2.0-5.0 mg/L zeatin.

    Poplar shoot regeneration can be largely divided into regeneration from organs and tissues and plant regeneration from callus (Sugimoto & Meyerowitz, 2013). Callus, an undifferentiated tissue, is derived from various tissues such as stems, leaves, roots, embryos, pollen or protoplasts, from which plant differentiation is re-differentiated in two ways. First, there is organ formation in which stem primodia are formed directly from callus, and second, somatic embryogenesis by embryonic development. In vitro shoot regeneration studies were performed in callus cultures of triploid quaking aspen (Winton, 1968). Wolter (1968) induced stem regeneration from the callus of Populus tremuloides in MS medium supplemented with 0.2–0.5 mg/L BA and induced in vitro rooting.

    Hyunsasi (Populus alba × P. glandulosa) is a hybrid poplar breeded in Korea. Poplars originally consisted of male and female trees, but sometimes in some hybrid poplars, purple and yellow flowers appear at the same time. Hyeonsasi is a fast-growing species that contributed greatly to short-term greening during national greening. However, in the process of mass distribution during the greening period, many afforestation areas failed to succeed due to the dissemination of defective varieties and the selection of proper lands(Son et al., 1995).

    In addition, as the current planting planted during the greening period grew into mature trees in the 1980s, a large amount of seeds were lost every year in the female trees, and these seeds were scattered like snowflakes as a means of breeding. P. alba × P. glandulosa clone Bonghwa 1 was selected to completely solve these shortcomings. This clone should be planted as environmental trees such as roadside trees, park trees and green trees at village cold places.

    However, there is no information on resistance to stress, such as biomass growth and disease resistance. In recent years, poplar has been newly recognized for its value as a carbon absorber in response to climate change and as an alternative energy source such as bioethanol. In line with this trend, poplar breeding should be newly made using various native species of Korea and over 160 clones. However, as mentioned earlier, P. alba x P. glandulosa clone Bonwha1, which is very suitable as a bioenergy species, has not been studied for growth and development of disease-resistant individuals.

    Hyeonsasi clone Bonwha1 is a selection entity and there is no breeding group for subsequent breeding. Therefore, in vitro culture is effective to induce a variety of variants. In other words, it is good to induce various variants by inducing callus from the tissue of P. alba x P. glandulosa clone Bonwha1 and re-differentiating it. Genetic modification requires the creation of various variants, but this cannot be expected with asexual breeding such as traditional cutting. Also, among the in vitro regeneration methods, stem regeneration from organs, such as the side, has also been reported to have a very low incidence of variants (Ahuja, 1983). Thus, plant regeneration from callus is a way to increase genetic diversity. This study investigated the optimal growth regulator concentration to induce callus from the cambium and leaf of Bonwha1 and to establish the conditions of regeneration.

    MATERIALS AND METHODS

    1. Materials and surface sterilization

    The materials used in this study were collected from February 7th year of hybrid poplar(P. alba x P. glandulosa clone Bonwha1) planted in the Forest Bioresources Department, National Institute of Forest Science(NiFos). The collected explants were cut into 2-3 cm size with eyes attached and surface sterilized three times for 5 seconds in 70% ethanol for 30 seconds and 3% NaClO. The surface sterilized tissues were washed several times with sterile water and then transferred to 1/2MS medium (Murashige & Skoog, 1962.) and cultured for 4 weeks to establish in vitro plants.

    2. Callus induction and proliferation

    In the case of leaves, the third leaf was cut from the apex of the poplar cultured in the case of the leaf, and the leaf was cut into 2-3 cm size using a scalpel, and the stem was cut by 3 cm. A wound was made with a pin to induce callus in the leaves and stems, and then healed and cultured in MS medium containing 1.0 mg/L 2,4-D.

    Induced callus (2-3 cm) was isolated from the leaf surface and transferred to MS medium containing 1.0 mg/L 2,4-D to grow in the dark condition. Callus was subcultured on the same medium every four weeks.

    3. Regeneration of plants from callus

    Induced callus was cut into light yellow friable callus to 0.5 cm size and cultured in 1/2MS medium containing various cytokinin. As a growth regulator added to the medium, cytokinins such as BAP, Kinetin, Zeatin, and 2ip were treated with 2 mg/L and 1.0 mg/L 2,4-D + 0.2 mg/L BAP. Plant regeneration was determined under the light microscope (x50) and measured shoot primodia induced in callus .

    4. Growth of regenerated plants

    Growth measurements of regenerated plants were performed in plants treated with 2 mg/L of cytokinins, such as BAP, Kinetin, Zeatin, and 2ip, and in plants incubated with 1.0 mg/L 2,4-D+0.2 mg/L BAP in the treatment group. Regenerated plants were cultured for 4 weeks in a culture room of 25±2°C., 2,000 lux, 16 hours light, 8 hours dark, and growth was measured.

    5. Induction of variants and measurement

    Throughout the previous study, regenerated individuals had a red color near the root, which was not good for later growth(data not shown). In this study, we counted white individuals as variants. In order to measure the degree of variant induction according to the growth regulator concentration, 1-9 mg/L of zeatin was treated, and stem growth was measured 4 weeks later. This experiment was repeated 5 times and averaged.

    6. In vitro rooting and acclimatation

    Regenerated plants were transferred to MS medium containing IBA (0.5-2 mg/L). In vitro plants were taken out of the culture bottle, the medium was removed, washed with water, transferred to a plastic pot containing sterile artificial soil (vermiculite: peatmos/1: 3, w/w) and acclimatized to the greenhouse.

    RESULTS AND DISCUSSION

    1. Callus induction

    Callus was induced from leaves and stems of P. alba x P. glandulosa clone Bonwha1 (Fig. 1). Callus was induced from both stem and leaf in MS medium containing 1.0 mg/L 2,4-D. Callus induction rate was higher in the stem than in the leaves. Callus was also induced more quickly in the stem than in the leaves. Plant growth regulators suitable for inducing callus are 2,4-D. Jafari et al. (1995) also induced P. nigra callus induction in N6 medium containing 0.1-1.0 mg/L of 2,4-D.

    2. Shoot regeneration from callus

    Plant regeneration from leaf and stem-derived callus was observed (Fig. 1A-J). Plant regeneration has resulted in more shoots with leaf-derived callus than with callus in stems. After 7 days of incubation, the stem surface swelled(Fig. 1A), and after 14 days, callus was formed(Fig. 1B), and then organogenesis occurred in friable callus(Fig. 1C). The developing primodia grew vigorously (Fig. 1D) and then formed a complete plant(Fig. 1E). Callus also formed on the leaf surface like stems in the leaves(Fig. 1F, G), after which the callus tended to be the callus of the stems(Fig. 1I-J).

    Among the growth regulators, zeatin showed the highest regeneration rate of 62%, followed by 2ip and kinetin (Fig. 2). Shoot regeneration was lowest at 1.0 mg/L 2,4-D + 0.2 mg/L BAP. The growth of regenerated shoot in leaf-derived callus was better than regenerated from stems.

    Many Populus have been regenerated at various parts such as arthropods, intercalates, leaves and roots (Coleman & Ernst 1990). The use of appropriate growth regulators is very important for shoot regeneration from poplars. Jafari et al. (1995) induced shoot regeneration from P. nigra callus in MS medium containing 2.5 mg/L BA and 0.2 mg/L NAA. Kim et al. (2014) also found that The regeneration of P. davidiana was greatly influenced by the concentration and the optimal concentration of BA and NAA. In addition to appropriate plant growth regulators, plant regeneration from poplar species has also been reported to aid in the physical treatment of pin surface stimulation (Park & Son 1988).

    2.1. Growth of shoots regenerated from leaf and stem derived calli

    Shoot regenerated from callus of leaves and stems were planted in MS medium containing five growth regulators and shoot growth was examined (Fig. 3). The best growth regulator for regenerated shoot growth was zeatin. The longest of the regenerated shoots reached 7.2 cm in 2 months of the zeatin treatment. Other growth regulators grew to 2 cm for 2ip, 4.4 cm for kinetin, 2.5 cm for BAP, and 1.2 cm for 2,4-D 0.2 BAP.

    Growth regulator types and concentrations had a significant effect on the growth of regenerated shoot (Fig. 4). In the case of zeatin and 2ip treatment, the shoot elongated at low concentration, but the shoot length rapidly decreased afterwards. However, treatment with kinetin, BAP, 2,4-D and BAP increased shoot elongation at higher concentrations.

    The addition of cytokinin is known to be good for the induction of multiple shoot but not for the growth of shoot. In the present study, the addition of cytokinin was good for the induction of multiple shoot in replanted seedlings, but inhibited growth at high concentration. Rowland & Ogden (1992) also indicated that the addition of cytokinin was important for stem regeneration and plant growth of blubbery.

    2.2. Growth rates of regenerated and cutting propagated plants

    Shoot growth was investigated at two concentrations of zeatin, the highest regeneration rate in stem callus-derived callus (Fig. 5A). In the MS medium containing 2.0 mg/L zeatin, shoot growth was lower than other treatments until 14 days, but the shoot growth was the best after that. On the other hand, 9 mg/L zeatin treatment showed good shoot growth before 14 days but slow shoot growth afterwards. However, the cutting propagated shoots did not grow better than the zeatin treatments.

    Shoot growth of regenerated from stem-derived callus was different from callus derived from leaves (Fig. 5B). Stem growth in the 2 mg/L zeatin treatment did not grow until 7 days and began to increase rapidly after 7 days. But after 14 days, growth appeared slow. On the other hand, stems cultured in 9 mg/L zeatin treatment showed better growth up to 10 days of cultivation than 2 mg/L zeatin treatment, but there was no growth after the cutting propagated shoots and 2 mg/L zeatin treatment.

    As a result, there was little difference in growth between in vitro regenerated plants and extracellular seedlings. There are few biochemical and morphological studies between in vitro regenerated plants and extracellular seedlings. Shahzad et al (2007) found that the total soluble protein profiles of in vitro regenerated plants, extracellular seedlings, and seeds were nearly identical. Mitosis studies reported normal lines were examined by electron microscopy of the pores of tracheal developmental variants. The stoma shape in vitrified leaves was circular and expanded to larger stoma than cutting propagated plants.

    2.3. Variation of regenerated plants

    The incidence of variants differed according to the growth regulator type and concentration (Fig. 6). As the concentrations of growth regulators zeatin, BAP, 2ip and kinetin increased, the variation number increased. Kang and Hall (1996) studied morphological variations of in vitro cultured hybrid aspens (P. alba x P. grandidentata cv. Crandon and cv. Hansen). The morphological differences of each chromosomal movements and numbers (2x=16).

    Anthocyanin is one of the eight flavonoids contained in poplar (Tsai et al. 2006), and the incidence and concentration of flavonoids vary between Populus species and clones (Greenaway et al. 1992, Donaldson et al. 2006). Anthocyanins accumulate in response to stress such as wounds, pathogen attack, nitrogen deficiency and ultraviolet radiation (Osier & Lindroth 2004, Miranda et al. 2007, Mellway et al. 2009).

    2.4. In vitro rooting and acclimatization of regenerated plant

    Microshoots regenerated from callus were rooted in MS basal medium or MS medium containing IBA (0.5-2 mg/L)(Fig. 7). In vitro root induced 7 days after cultivation in MS medium containing 0.5-2 mg/L IBA. An average of five roots developed per treatement, growing 11 cm after 11 weeks. The best IBA concentration for rooting was 1 mg/L IBA. In vitro plants were removed from the culture bottles, the medium was removed, washed with water, transferred to a plastic pot containing sterile artificial soil (vermiculite: peatmos/1:3, w/w) and acclimatized in a greenhouse. As a result, more than 95% of the purified seedlings survived.

    This study is expected to greatly contribute to the mass growth of P. alba x P. glandulosa clone Bonwha1, which is widely used as a bioenergy and plant purification species.

    Acknowledgments

    This study was conducted as part of the Multiple stress tolerance assessment and customized mapping of domestic major afforestation tree species to prepare for climate change (2017 R1D1A1B04036320).

    Figure

    JALS-54-2-45_F1.gif

    Shoot regeneration of P. alba x P. glandulosa clone Bonwha1.

    1A-E:shoot regeneration from stem derived callus and 1F-J:shoot regeneration from leaf derived callus

    JALS-54-2-45_F2.gif

    Shoot regeneration on P. alba x P. glandulosa clone Bonwha1 callus in MS medium with 2 mg/l cytokinins and 2,4-D+BAP treatment.

    JALS-54-2-45_F3.gif

    Shoot growth of regenerated plants in MS medium with 2 mg/l cytokinins and 2,4-D+BAP treatment.

    JALS-54-2-45_F4.gif

    Shoot growth of plantlet regenerated from leaf and stem derived callus by growth regulators.

    JALS-54-2-45_F5.gif

    Shoot growth of plantlet regenerated from leaf (left) stem(right) derived callus on zeatin containing medium.

    JALS-54-2-45_F6.gif

    Variation of shoots regenerated in various growth regulators treatment.

    JALS-54-2-45_F7.gif

    In vitro rooting (left) and acclimatization of regenerated plant(right).

    Table

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