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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (1) : 60-66     DOI: 10.1007/s11703-009-0009-y
Adventitious shoot regeneration from the leaves of in vitro grown ‘Zhongli 1’ pear (Pyrus spp.)
Jie LIU1, Yuxing ZHANG1(), Jing QI1, Xi ZHANG2, Bharat Kumar POUDYAL3, Zhan JIAO4
1. College of Horticulture, Agricultural University of Hebei, Baoding 071001, China; 2. College of Life Science, Agricultural University of Hebei, Baoding 071001, China; 3. District Agriculture Development Office, Bhojpur, Koshi Zone, Nepal; 4. Department of Pharmaceutical Engineering, The Chemical and Pharmaceutical College of Hebei, Shijiazhuang 050026, China
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The pear (Pyrus spp.) is one of the most important temperate fruit crops. The technique of adventitious shoot regeneration from leaves is considered to be one of the shortcuts in the research on pear genetic modification and cellular engineering, which, however, has not been widely used. As the regeneration frequency of pear leaves is usually very low, the research on adventitious shoot regeneration from pear leaves is eagerly needed. In this experiment, the factors affecting shoot and bud regeneration from the leaves of ‘Zhongli 1’ pear were studied, and an efficient protocol for shoot regeneration was established. The results showed that different types of basic media, different combinations of plant growth regulators, leaf placement on medium, periods of dark culture and the use of silver nitrate (AgNO3) on culture media all significantly affected the adventitious shoot regeneration frequency of ‘Zhongli 1’ pear. The details are as follows: (1) Among three kinds of basic media, NN69 was better for ‘Zhongli 1’ shoot regeneration, followed by half (?) MS, while full MS had no effect on shoot regeneration; (2) Thidiazuron (TDZ) was better than 6-benzylaminopurine (6-BA) for ‘Zhongli 1’ regeneration, with an optimal concentration of 1.5 mg·L-1, and the regeneration rate under this concentration could reach 85%, with 2.72 buds per leaf. 0.5 mg·L-1 indole-3-butyric acid (IBA), which induced a higher regeneration frequency, was a better choice for pear regeneration compared with 0.3 mg·L-1 naphthaleneacetic acid (NAA). Among the different combinations of plant growth regulators, TDZ + IBA was better for inducing high regeneration frequency; (3) The abaxial surface of leaves touching the medium was beneficial for leaves to uptake nutrients from the medium, and because of that, the regeneration frequency of leaves was significantly higher than that of leaves touching the medium with their adaxial surfaces (obverse side of leaf); (4) Dark culture was necessary for bud regeneration, and the best duration for dark culture of ‘Zhongli 1’ pear was 21 days; (5) The addition of 1.0 mg·L-1 AgNO3 into the culture medium could promote adventitious shoot regeneration significantly. A high adventitious shoot regeneration frequency was obtained in this research, which will be beneficial for further research on efficient and stable in vitro plant regeneration systems and genetic modification of pear.

Keywords ‘Zhongli 1’ pear      adventitious shoot regeneration      growth regulators      tissue culture      in vitro     
Corresponding Authors: ZHANG Yuxing,   
Issue Date: 05 March 2009
URL:     OR
basic mediumMSMS, 1/2MS and NN69MS
growth regulators
TDZ0.5, 1.0, 1.5 and 2.0
BA13.0, 4.0, 5.0 and 6.00.5
AgNO30 and 1.0
Tab.1  Culture media and their compositions used during shoot proliferation and adventitious shoot regeneration
Fig.1  Procedures of the adventitious shoot regeneration from leaves
Note: (a), (b), (c), (d), (e) and (f) represent formation of white callus from the cut surface of leaves, initial stage of adventitious shoot regeneration, adventitious shoots from leaves, elongation of adventitious shoots on leaves, many adventitious shoots from 1 leaf, and elongated shoots in SEM, respectively.
basic mediumhormone concentration/mg·L-1regeneration frequency/%shoot number per leaf
MSTDZ 1.0 mg·L-1+IBA 0.5 mg·L-10c0b
MSTDZ 2.0 mg·L-1+IBA 0.5 mg·L-11.67c0.02b
Tab.2  Different types of media and their effects on adventitious shoot regeneration
Fig.2  Vitrification of adventitious shoots
combination numbercombination of plant growth regulator /mg·L-1regeneration frequency /%shoot number per leaf
Tab.3  Effects of different plant growth regulator combinations on bud regeneration of leaves
ways that leaves put on mediumregeneration frequency/%shoot number per leaf
abaxial surface (leaf back) touching medium56.67a1.55a
adaxial surface (obverse side of leaf) touching medium28.33b0.48b
Tab.4  Effects of leaf placement ways on medium on adventitious shoot regeneration
dark period /dregeneration frequency /%shoot number per leaf
Tab.5  Effects of different dark culture periods on adventitious shoot regeneration
mediumAgNO3/mg·L-1regeneration frequency/%shoot number per leaf
NN69+TDZ 1.0 mg·L-1+IBA 0.5 mg·L-1-56.67a1.55a
NN69+TDZ 1.0 mg·L-1+NAA 0.3 mg·L-1-45.00a0.57a
NN69+BA 4.0 mg·L-1+NAA0.3 mg·L-1-20.00a0.23a
NN69+BA4.0 mg·L-1+IBA0.5 mg·L-1-40.00a0.62a
Tab.6  Effects of AgNO on adventitious shoot regeneration
1 Caboni E, Tonelli M G, Lauri P (1999). In vitro shoot regeneration from leaves of wild pear. Plant Cell, Tissue and Organ Culture , 59(1): 1-7
doi: 10.1023/A:1006351214343
2 Cao X, Chai M L (2005). Adventitious shoot regeneration from leaves of pear cultivars belonging to Pyrus pyrifolia. Journal of Fruit Science, 22 (5): 557-560 (in Chinese)
3 Eng C P, Swee H T, Chiang S (1996). Interactive role of ethylene and polyamines on shoot regenerability of Chinese Kale (Brassica alboglabra Bailey) in vitro. Plant Physiol , 149: 138-148
4 Han Q F, Xu L F, Ma F W, Wang J Z, Ren X L (2002). Adventitious bud regeneration from petiole of pear (Pyrus). Acta Bot Boreal –Occident Sin , 22(6): 1485-1488 (in Chinese)
5 Jun J, Yae B, Yang M (1996). Influence of cultivar, light condition and pretreatment on adventitious shoot regeneration from leaves, internodes and petioles of Malus domestica Borkh in vitro. Journal of the Korean Society for Horticultural Science , 37(5): 700-703
6 Lane W D, Iketani H, Hayashi T (1998). Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia). Plant Cell, Tissue and Organ Culture , 54(1): 9-14
doi: 10.1023/A:1006032707849
7 Leblay C, Chevreau E, Raboin L M (1991). Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communs L.). Plant Cell Tissue and Organ Culture , 25(2): 99-105
8 Li X G, Yang J, Wang L (2006). A new early and high quality pear cultivar—“Zhongli 1”. Journal of Fruit Science , 23(4): 648-649 (in Chinese)
9 Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant , 15: 473-497
10 Nitsch J P, Nitsch C (1969). Haploid plants from pollen grains. Science , 163: 85-87
11 Poudyal B K (2007). Regeneration of adventitious shoots from the leaves and stems of some Pyrus spp. grown in vitro and transformation of CpTI gene for insect pest resistance, using biological vector: Agrobacterium tumefaciens.Dissertation for the Doctoral Degree. Baoding: Agricultural University of Hebei
12 Poudyal B K, Zhang Y X and Du G Q (2008). Adventitious shoot regeneration from the leaves of some pear varieties (Pyrus Spp.) grown in vitro. Frontiers of Agriculture in China . 2(1):82-92 .
13 Sun Q R, (2000). Regeneration of adventitious shoots from leaves of Pyrus pyrifolia Nak. cv. Jinhua. Deciduous Fruits , (3): 8-10 (in Chinese)
14 Sun Q R, Fan S H, Liu Q Z (2003). High frequency shoot regeneration from giant dunkpear leaves in vitro. Shandong Agricultural Sciences , (2): 10-12 (in Chinese)
15 Sun Q R, Liu Q Z, Zhao H J, Liu P (2004). The study on factors affecting high frequency shoot regeneration from in vitro leaves in pear. Chinese Agricultural Science Bulletin , 20(4): 99-100 , 107 (in Chinese)
16 Sun Q R, Sun H Y, Zheng H J (1998). The promotion of ethylene inhibitor AgNO3 on buds regeneration from pear leaves (Pyrus Spp.) grown in vitro. Deciduous fruits , (4): 1-2 (in Chinese)
17 Tang H R, Liu C Q, Luo Y (2005). Adventitious bud regeneration from in vitro leaves of Pyrus ussuriensis cv. Manyuanxiang. Journal of Fruit Science , 22(6): 706-708 (in Chinese)
18 Xu L F, Ma F W, Wang Z Z, Ren X L (2002). Regeneration of adventitious bud from leaves of ‘Bayuehong’ pear. Journal of Northwest Sci-Tech Univ of Agri and For , 30(4): 73-75 (in Chinese)
19 Xu L F, Ma Z Z, Ren X L, Cao X Y (2002). Leaf culture and plantlet regeneration of pear (Pyrus). Acta Horticulturae Sinica , 29(4): 367-368 (in Chinese)
20 Zhang P, Ling D H (1995). Enhancement of plant regeneration rate of Brassica parachinensis cultured in vitro. Acta Botanica Sinica , 37(11): 902–908 (in Chinese)
21 Zhao H B, Xu L F, Ma F W, Liang D, Fu X C (2007). Leaf culture and plantlet regeneration of pear scion cultivar ‘Dangshansu’. Acta Agriculturae Boreali-occidentalis Sinica , 16(2): 153-157 (in Chinese)
22 Zhou L L, Cai B H, Qiao Y S, Wang S H, Zhang Z (2007). Effects of different treatment on adventitious bud regeneration from in vitro leaves of Pyrus pyrifolia ‘Hosui’. Journal of Nanjing Agricultural University , 30(2): 34-38 (in Chinese)
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[3] ZHANG Yuxing, DU Guoqiang, POUDYAL Bharat Kumar. Adventitious shoot regeneration from the leaves of some pear varieties ( spp.) grown [J]. Front. Agric. China, 2008, 2(1): 82-92.
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