RESEARCH ARTICLE Bharat Kumar POUDYAL, Guoqiang DU, Yuxing ZHANG, Jie LIU, Qingchun SHI Studies on browning problem and phenols content on shoots of Yali, Aikansui and Abbe Fetel pears for in vitro culture E Higher Education Press and Springer-Verlag 2008 Abstract Yali is one of the best pear cultivars cultivated extensively in China and other countries. However, mor- tality rates of explants during the initial phase of tissue culture were found to be very high during the summer particularly from the shoot tip explants. A thorough investigation on the browning problem of Yali Aikansui and Abbe Fetel pears was done and their control measures are suggested in this paper. Emphasis has been given to the Yali pear. Shoot explants were collected from the field as well as from the plantlets grown in vitro during different months and observed for browning. The explants were excised and cultured in Murashige and Skoog (MS) solid and liquid medium as needed. The cultures were main- tained in the dark as well as in a 16/8 hours light/dark photoperiod regime as needed and were kept in a growth room at (25 ¡ 2)uC temperature. According to the experi- mental results, Yali was found more severely infected by browning than the other two cultivars. Similarly, it was also found that, the intensity of browning was less during spring and increased with time and reached the maximum during summer. Shoot tips of Yali were found more severely infected by browning than the second node and other nodes. This accelerated the mortality rate up to 81% of shoot explants during the summer months of July and August. Browning was greatly influenced by water-sol- uble polyphenols, more of which were found in the shoot tip of explants collected from the field (significantly higher at P ( 0.05 and P ( 0.01). Total polyphenol contents were found less in the explants from plantlets grown in vitro, and less browning of medium appeared resulting in a lower mortality rate of explants. Collections of explants during early spring (P ( 0.05 and P ( 0.01) and use of other nodes rather shoot tip because explants (P ( 0.05 and P ( 0.01) were found to be better in preventing the browning problem. Finally, for curative measures to con- trol browning, use of ascorbic acid at the rate of 100 mg? L 21 (P ( 0.05 and P ( 0.01), 0.02% polyvinyl pyrrolidone (PVP) in the culture medium (P ( 0.05), 96 hours dark treatment of other nodes (P ( 0.05), and 12 hours cold treatments of explants at 4uC(P ( 0.05 and P ( 0.01) prior to sterilization of explants, were found to be the best methods to control browning and therefore to increase the survival rate of cultured explants of the Yali pear. Keywords Abbe Fetel, Aikansui, Yali, browning prob- lem, explants, in vitro culture, pear, phenols 1 Introduction Propagation of plants by the tissue culture method is very common in many plant species including fruit crops like pear. However, sometimes during in vitro culture of some plant species, the media will become brown and the explants unable to grow further and eventually die. Some explants leach some phenolic substances or second- ary metabolites from cut surfaces, which oxidize later and turn the media brown and is toxic to the explants (Aliyu, 2005). Browning of media is common especially from tree species and mature tissues from the woody species. According to Mager and Harel (1979), in the normal tis- sue, no browning happens, because Polyphenol Oxidase (PPO) and phenolic compounds are separated by a mem- brane structure and if the structure were to be broken browning happens. Likewise, according to Ju et al. (1988), in the normal cells, polyphenolic compounds are in vacuoles, while the PPO is located in the cytoplasm and if compartmentation were to be broken, browning will happen. Although browning of medium is a serious prob- lem of many plant species for tissue culture, it is an important phenomenon in plants. According to Chen et al. (1997), polyphenol has some regulatory effects on Received November 2, 2007; accepted January 3, 2008 Bharat Kumar POUDYAL (*) Department of Agriculture, Fruit Development Directorate, Kirtipur, Kathmandu, Nepal E-mail: poudyal_bharat@yahoo.com Guoqiang DU, Yuxing ZHANG (*), Jie LIU, Qingchun SHI College of Horticulture, Agricultural University of Hebei, Baoding 071001, China E-mail: jonsonzhyx@yahoo.com.cn Front. Agric. China 2008, 2(3): 321–330 DOI 10.1007/s11703-008-0035-1