Introduction
Pear (Pyrus spp.) is an important horticultural germplasm that is grown worldwide. Generally, fruit tree germplasms, including those of pear, are preserved in field banks, but this approach is expensive and the materials may be exposed to pathogens; therefore, a rapid, efficient shoot tip culture method for in vitro propagation is desirable (Thakur et al., 2008). With shoot tip culture, the terminal 0.1-1.0 mm of a shoot including the meristem (0.05-0.1 mm), together with primordial and developing leaves and adjacent stem tissue, is cultured (Pollard and Walker, 1990). Plants that are diseasefree and have high metabolic activity and genetic stability can be produced using this technique because plantlet production from adventitious organs can be avoided. Micropropagation of pear was first achieved in 1979 using the pear rootstock ‘OHxF51’ (Cheng, 1979) and the scion variety ‘Bartlett’ (Lane, 1979). Since these first reports, significant progress has been made in various areas of in vitro pear culture (Singha, 1986; Hutchinson and Zimmerman, 1989; Chevreau et al., 1992). Of the methods for long-term ex situ conservation of clonally propagated germplasm, cryopreservation of shoot tips is the most reliable and cost- and space-effective option (Condello et al., 2009). Cryopreservation of pear shoot tips propagated in vitro has been successful (Scottez et al., 1992; Niino and Sakai, 1992; Suzuki et al., 1997; Reed et al., 1998; Chang and Reed, 2001). To implement cryopreservation, the establishment of an efficient micropropagation system is essential. Micropropagation methods are also a prerequisite for exploiting somaclonal variations and induced mutations and for the development of transgenic plants (Yeo and Reed, 1995; Bell and Reed, 2002; Kang et al., 2011).
In this study, we attempted to standardize a protocol for in vitro shoot formation, proliferation, rooting, and ex vitro acclimatization of three pear genotypes.
Materials and Methods
Plant material
The starting material for establishing pear (Pyrus spp.) in vitro culture consisted mostly of shoot tips from grafted plants maintained at the National Institute of Horticultural and Herbal Science of the Rural Development Administration (NIHH, RDA; Jeonju, Korea). Current-season shoots (12-15 cm) were excised and 2-3 mm-long shoot tips were prepared. The explants were surface sterilized with 2.5% sodium hypochlorite for 15 min and rinsed several times in sterilized water for 20 min before the shoot tips were excised. Shoot tip cultures of three pear (Pyrus spp.) genotypes, BaeYun No. 3, Bartlett, and Hwanggeum, were established and propagated in vitro. In these three genotypes, regeneration rates were much higher than other genotypes we tried. The basal medium used for all of the trials was Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% sucrose and adjusted to a pH of 5.7 before autoclaving at 121℃ for 15 min. All cultures were conducted under white fluorescent light (2000 lux) with a 16-h day photoperiod at 25℃ and a light intensity of 40 μmol/m2/s provided by cool white fluorescent tubes.
Shoot formation and proliferation
In vitro propagated cultures, obtained from single axillary buds of pear genotypes, were established according to Caboni et al. (1999). Five different combinations of plant growth regulator treatments were added to MS medium containing 3% sucrose for shoot induction. After six weeks in culture, nodal segments (> 0.5 cm) of the established shoots were subcultured for shoot multiplication on MS medium containing 3% sucrose and various concentrations of N6-benzyladenine (BA) and indole-3-butyric acid (IBA). Shoots in the proliferation phase were subcultured at three-week intervals. To evaluate the influence of plant growth regulator treatment on shoot multiplication, both shoot number and shoot length were investigated.
Rooting and ex vitro acclimatization
To induce rooting, shoots raised in vitro (2.0-2.5 cm) were cultured on half-strength MS medium supplemented with 3% sucrose and various concentrations of 1-naphthaleneacetic acid (NAA) or IBA. To investigate the effect of a single application on root initiation, NAA (0.1 or 0.2 mg/L) or IBA (1.2 or 2.5 mg/L) was applied, and to test the effect of combined application on in vitro rooting, three plant growth regulator combinations were applied. The influence of phloroglucinol (PG) on in vitro rooting of microshoots was also studied using concentrations of 0, 75, 150, and 300 mg/L. In all of the rooting experiments, the percent in vitro rooting, number of roots per shoot, and root length were observed. Data were collected 30 days after beginning the root induction cultivation
Plantlets grown in vitro with well-developed roots were removed after 30 days, washed thoroughly with running tap water to remove residual culture medium, planted in plastic pots containing 2:1 (v/v) sand:soil mixture followed by 1:1 (v/v) sand:perlite mixture, and placed in an environmentally controlled mist house under natural photoperiod conditions. After two weeks, the acclimatized plants were successfully transferred and established in a greenhouse.
Statistical analysis
Each treatment was repeated in three replicated experiments with 20 explants per treatment. All data were analyzed using analysis of variance (ANOVA) and means were separated using the Duncan’s multiple range test (DMRT) at P ≤ 0.05 (R ver. 3.1.3).
Results and Discussion
Effects of plant growth regulators on shoot induction and proliferation
Explant establishment and the shoot proliferation technique were standardized for three pear genotypes. Table 1 shows the effects of various plant growth regulator combinations on adventitious shoot formation. Shoots from the established cultures were cultured on MS medium supplemented with various concentrations of N6-benzyladenine (BA), indole-3-butyric acid (IBA), gibberellic acid (GA3), and 1-naphthaleneacetic acid (NAA). The combination of 1.0 mg/L IBA and 0.1 mg/L GA3 induced the highest shoot formation rates from shoot tips of all three cultivars (Fig. 1A). In this medium, the shoot formation rate ranged from 90.5% (Hwanggeum) to 100% (BaeYun No. 3, Bartlett). Shoot tip and meristem culture have been used to produce pathogen-free plants following thermotherapy or chemotherapy (Reed, 1995). A major advantage of working with shoot tips or meristems is the potential to exclude pathogenic organisms present in the donor plant. A second advantage is the genetic stability inherent in the technique because plantlet production from adventitious organs can be avoided (Murashige, 1974; Ancora et al., 1981; Chevreau et al., 1992).
Table 1.zM1: BA 1.0 mg/L+GA3 0.1 mg/L, M2: BA 2 mg/L+IBA 0.1 mg/L, M3: BA 3 mg/L+IBA 0.1 mg/L, M4: BA 3 mg/L+NAA 0.1 mg/L, M5: BA 1 mg/L+GA3 0.1 mg/L+IBA 0.1 mg/L.yMean separation within columns by Duncan’s multiple range test at 5% level by R project (R version 3.0.1) for statistical computing.
Fig. 1.Diverse stages of plant regeneration from shoot tips of pear (Pyrus spp.). genotypes. A, Adventitious shoot formation six weeks after shoot tip establishment; B, Proliferation of shoots three weeks after subculture in medium with BA 2.0 mg/L + IBA 0.2 mg/L; C, Rooting of microshoots 30 days after establishment on root induction medium a) NAA 0.2 mg/L + IBA 0.2 mg/L, b) NAA 0.1 mg/L + IBA 0.1 mg/L, c) IBA 2.5 mg/L; D, Acclimatized plantlets in greenhouse.
Both the shoot number per culture and shoot length increased markedly in all three genotypes with the combination of 2.0 mg/L BA and 0.2 mg/L IBA (Table 2). During axillary shoot proliferation, cytokinins are used to overcome the apical dominance of shoots to enhance the branching of lateral buds from leaf axils (Hu and Wang, 1983). The effective concentration of cytokinins required to reverse apical dominance varies with the culture system (Anirudh and Kanwar, 2008a). A positive correlation between BA level and shoot multiplication in pear was reported (Dwivedi and Bris, 1999) and a correlation between low auxin levels and shoot proliferation has been reported (Bhojwani et al., 1984; Dwivedi and Bris, 1999). Finding the best plant growth regulator combination for micropropagation of a crop is very difficult. In this study, we found that the combination of 2.0 mg/L BA and 0.2 mg/L IBA was ideal for shoot proliferation (Fig. 1B). One of the possible roles of auxin during shoot proliferation is to nullify the suppressive effects of high cytokinin concentrations on axillary shoot elongation thereby restoring normal shoot growth (Lundergan and Janick, 1980).
Table 2.zN, number of shoots per culture, yL, Shoot length, xMean separation within columns by Duncan’s multiple range test at 5% level by R project (R version 3.0.1) for statistical computing.
Browning of explants cultured in vitro is also a serious problem that impairs the micropropagation of woody plants (Anirudh and Kanwar, 2008a). Pear is very sensitive to phenolic compounds from explants during in vitro culture. A short subculture interval is critical for pear micropropagation. In this study, pear plantlets were subcultured at three-week intervals.
Effects of plant growth regulators and phloroglucinol (PG) on rooting
As shown in Table 3, the combination of NAA and IBA resulted in a significant increase in rooting frequency over NAA or IBA alone. Maximum rooting (68.9%) in BaeYun No. 3 was obtained on the medium with the combination of NAA and IBA (0.2 mg/L each) (Fig. 1C), i.e., the rooting ratio improved only at lower auxin levels (0.2 mg/L each). The improved rooting with NAA and IBA in combination, as compared to NAA or IBA alone, may be due to the complementary effect of NAA and IBA. In vitro adventitious root formation can be induced easily in many herbaceous species, but it is very difficult in most woody species. In particular, rooting pears in vitro has proven difficult and scion cultivars are more difficult to root than rootstock cultivars (Bhojwani et al., 1984). Nevertheless, the induction of roots on micropropagated pears in vitro has been reported (Reed 1995; Bell and Reed, 2002; Anirudh and Kanwar, 2008b), although the rooting responses were poor. The ability of plant tissues to form adventitious roots depends on the interactions of many endogenous and exogenous factors. Torrey (1976) reviewed the role of auxins in root development and stated that that auxins have been established as the main factors involved in root formation.
Table 3.zMean separation within columns by Duncan’s multiple range test at 5% level by R project (R version 3.0.1) for statistical computing.
The rooting behaviors of the three pear genotypes were affected significantly by the PG concentration in the rooting medium. As shown in Table 4, PG stimulated the rooting response, i.e., the rooting ratio, root number per culture, and root length. The rooting ratio in BaeYun No. 3 improved to 91.8% with 150 mg/L PG. The number of roots per microshoot also increased with the PG concentration. The most roots (3.15) were observed with 150 mg/L PG in the rooting medium for Bartlett. The differences in root number and length between 75 and 150 mg/L PG were not significant in all three genotypes (Table 4) so supplementation with 75 mg/L PG was suitable for rooting medium. The stimulation or inhibition of root initiation by phenolic compounds like PG is due to their interaction with auxins (Nemeth, 1986). James and Thurbon (1979) reported that the addition of PG to the rooting medium improved the rooting percentage in the M9 apple rootstock. James and Thurbon (1981) also showed that increased levels of endogenous PG in the microshoot favor root initiation during the auxin-sensitive phase and have synergistic effects with auxin. In contrast, Zimmerman and Broome (1981) reported that PG stimulated rooting in only one of the eight apple cultivars examined. In this study, we found stimulatory effects of increasing PG concentration on the rooting response, i.e., increases in the percent in vitro rooting, root number per culture, and root length. Most well-rooted plantlets survived on 2:1 (v/v) sand:soil mixture followed by 1:1 (v/v) sand:perlite mixture (Fig. 1D). The surviving plants were hardened in a normal greenhouse.
Table 4.zN, root number per culture; yL, root length, xMean separation within columns by Duncan’s multiple range test at 5% level by R project (R version 3.0.1) for statistical computing.
Micropropagation under controlled conditions could be an alternative to the field production of pear cultivars because more plants can be obtained in less time with guaranteed genetic stability and phytosanitary quality (Vengadesan et al., 2002; Martin, 2003), which are prerequisite conditions for a mid- to long-term germplasm preservation system for pear germplasm.
참고문헌
- Ancora, G., M.L. Belli-Donini and L. Cozzo. 1981. Globe artichoke plants obtained from shoot apices through rapid in vitro micropropagation. Sci. Hort. 14:207-213. https://doi.org/10.1016/0304-4238(81)90014-5
- Anirudh, T. and J.S. Kanwar. 2008a. Micropropagation of 'wild pear' Pyrus pyrifolia (Burm F.) Nakai I. Explant establishment and shoot multiplication. Not. Bot. Hort. Agrobot. Cluj. 36(1):103-108.
- Anirudh, T. and J.S. Kanwar. 2008b. Micropropagation of 'wild pear' Pyrus pyrifolia (Burm F.) Nakai II. Induction of rooting. Not. Bot. Hort. Agrobot. Cluj. 36(2):104-111.
- Bell, R.L. and B.M. Reed. 2002. In vitro tissue culture of pear: Advances in techniques for micropropagation and germplasm preservation. Proc. 8th IS on pear. L. Corelli-Grappadelli et al. (eds.), Acta Hort. 596. ISHS. pp. 412-418.
- Bhojwani, S.S., K. Mullins and D. Cohen. 1984. In vitro propagation of Pyrus pyrifolia. Scientia Hort. 23:247-254. https://doi.org/10.1016/0304-4238(84)90068-2
- Caboni, E., M.G. Tonelli, P. Lauri, S. D’Angeli and C. Damiano. 1999. In vitro shoot regeneration from leaves of wild pear. Plant Cell Tiss. Organ Cult. 59:1-7. https://doi.org/10.1023/A:1006351214343
- Chang, Y. and B.M. Reed. 2001. Preculture conditions influence cold hardiness and regrowth of Pyrus cordata shoot tips after cryopreservation. HortScience 36:1329-1333.
- Cheng, T.V. 1979. Micropropagation of clonal fruit tree root-stocks. Compact Fruit Trees 12:127-137.
- Chevreau, E., B. Thibault and Y. Arnaud. 1992. Micropropagation of pear (Pyrus communis L.): In Bajaj, Y.P.S. (ed.), Biotechnology of Perennial Fruit Crops, C.A.B. Intl. Wallingford, UK. pp. 263-276.
- Condello, E., M.A. Palombi, M.G. Tonelli, C. Damiano and E. Caboni. 2009. Genetic stability of wild pear (Pyrus pyraster, Burgsd) after cryopreservation by encapsulation dehydration. Agricultural and Food Science 18:136-143. https://doi.org/10.2137/145960609789267533
- Dwivedi, S.K. and L.D. Bris. 1999. In vitro propagation of low-chill pear cv. Gola. Indian J. Hort. 56:189-193.
- Hu, C.Y. and P.J. Wang. 1983. Meristem, shoot-tip and bud culture: In Evans, D.A., W.R. Sharp, P.V. Ammirato and Y. Yamada (eds.), Handbook of Plant Cell Culture, Macmillan, New York, USA. pp. 177-277.
- Hutchinson, J.F. and R.H. Zimmerman. 1989. Tissue culture of temperate fruit and nut trees. Hort. Rev. 9:273-349.
- James, D.J. and I.J. Thurbon. 1979. Rapid in vitro rooting of the apple roostock M9. J. of Hort. Sci. 54:309-311.
- James, D.J. and I.J. Thurbon. 1981. Phenolic compounds and other factors controlling rhizogenesis in vitro in the apple rootstocks M9 and M26. Zeitschrift für Pflanzenphysiologie. 105:11-20. https://doi.org/10.1016/S0044-328X(81)80003-7
- Kang, C.H., B.S. Han, S.G. Han, S.H. Kwon and Y.J. Song. 2011. Selection of suitable varieties of carnation (Dianthus caryophyllus L.) and optimization of culture conditions for efficient tissue culture. Korean J. Plant Res. 24:121-129 https://doi.org/10.7732/kjpr.2011.24.2.121
- Lane, D.W. 1979. Regeneration of pear plants from shoot meristem-tips. Plant Sci. Lett. 16:337-342. https://doi.org/10.1016/0304-4211(79)90046-4
- Lundergan, C.A. and J. Janick. 1980. Regulation of apple shoot proliferation and growth in vitro. Hort. Res. 20: 19-24.
- Martin, P. 2003. Plant regeneration through direct somatic embryogenesis on seed coat explants of cashew (Anacardium occidentale L.). Sci. Hort. 98:299-304. https://doi.org/10.1016/S0304-4238(03)00005-0
- Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
- Murashige, T. 1974. Plant propagation through tissue culture. Ann. Rev. Plant Physiol. 25:135-166. https://doi.org/10.1146/annurev.pp.25.060174.001031
- Nemeth, G. 1986. Induction of rooting: In Bajaj, Y.P.S. (ed.), Biotechnology in Agriculture and Forestry-Trees I. Springer-Verlag, Berlin, Germany. pp. 49-64.
- Niino, T. and A. Sakai. 1992. Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry. Plant Sci. 87:199-206. https://doi.org/10.1016/0168-9452(92)90151-B
- Pollard, J.W. and J.M. Walker. 1990. Plant cell and tissue culture: In Walker, J.M. (ed.), Methods in Molecular Biology, New Jersey (USA). pp. 81-91.
- Reed, B.M., J. Denoma, J. Luo, Y.J. Chang, and L. Towill. 1998. Cryopreservation and long-term storage of pear germplasm. In Vitro Cell Dev. Biol. Plant 34:256-260. https://doi.org/10.1007/BF02822718
- Reed, B.M. 1995. Screening Pyrus germplasm for in vitro rooting response. HortScience 30:1292-1294.
- Scottez, C., E. Chevreau, N. Godard, Y. Arnaud, M. Duron and J. Dereuddre. 1992. Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation-dehydration. Cryobiology 29:691-700. https://doi.org/10.1016/0011-2240(92)90073-B
- Singha, S. 1986. Pear (Pyrus communis): In Bajaj, Y.P.S. (ed.), Biotechnology in Agriculture and Forestry. Vol. I. Springer-Verlag, New York, USA. pp. 198-206.
- Suzuki, M., T. Niino, T. Akihama, and S. Oka. 1997. Shoot formation and plant regeneration of vegetative pear buds cryopreserved at −150℃. J. Japan. Soc. Hort. Sci. 66:29-34. https://doi.org/10.2503/jjshs.66.29
- Thakur, A., R.P.S. Dalal, and D. Navjot. 2008. Micropropagation of pear (Pyrus spp.): A review. Agric. Rev. 29:260-270.
- Torrey, J.G. 1976. Root hormones and plant growth. Ann. Rev. Plant Physiol. 27:435-459. https://doi.org/10.1146/annurev.pp.27.060176.002251
- Vengadesan, G., A. Ganapathi, S. Amutha and N. Selvaraj. 2002. In vitro propagation of Acacia species-a review. Plant Sci. 163:663-671. https://doi.org/10.1016/S0168-9452(02)00144-9
- Yeo, D.Y. and R.M. Reed. 1995. Micropropagation of three Pyrus rootstocks. HortScience 30:620-623.
- Zimmerman, R.H. and O.C. Broome. 1981. Phloroglucinol and in vitro rooting of apple cultivar cuttings. J. Ame. Soc. Hort. Sci. 106:648-652.
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