Indian Journal of Experimental Biology Vol. 49, July 2011, pp. 540-546

Combined effect of dark and wounding on regeneration potential of cordata Thunb.

Y. Wen Xu 1, Jian Wei Zeng 1, Yu Ting Zou 1, Amjad M Husaini 2, Ru Yu Yao 1, De Gang Wu 1 & Wei Wu 1* 1Agronomy College, Agricultural University, Ya’an 625014, PR China 2 Division of Breeding and Genetics, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, Jammu & Kashmir 191 121, India

Received 8 November 2010; revised 7 April 2011

Houttuynia cordata is one of the most potential medicinal and edible wild whose resources have decreased sharply due to excessive exploitation. Besides its slow agamic propagation, problems of browning and non-dedifferentiation have prevented the application of micropropagation in H. cordata . Through 4 week pre-culture in darkness and wounding after 1 week pre-culture, the browning rate of explants decreased significantly and resulted in efficient regeneration (20.64 ± 5.94 adventitious buds per explant) on the induction medium. The protocol shall facilitate conservation and commercial cultivation of the endangered species.

Keywords: Dark, , Regeneration, Wounding

Houttuynia cordata Thunb. is a pungent, heart-like resources in China cannot meet such huge demands, leafed perennial herb native to many Asian countries. because of slow agamic propagation by stoloniferous It is a single species of the genus Houttuynia in . Therefore, it is necessary to establish an . In Chinese known as ‘Yuxingcao’, efficient regeneration system for its conservation and which means ‘producing unique fishy smell’, H. commercial cultivation. cordata has been identified as one of the most Leaves are usually considered as efficient explants potential medical and edible wild plant resources in for high-frequency propagation in vitro 9-13 . In order to 1 China . Its young , including the aerial stems, develop an efficient regeneration system for H. leaves, and underground stems, are all very popular cordata , leaf as explant was used and attempted to wild vegetable, and its mature plants are also adjust hormone types and concentrations in the commonly used as a kind of traditional medical herb induction medium 14 but failed to achieve the desired 2 3 4 5 6 in China , Korea , India , Vietnam and Thailand . H. results because of heavy browning and non- cordata contains six major effective chemical dedifferentiation of the explants. components viz. essential oils, flavonoids, alkaloids, Browning process is associated with the oxidation fatty acids, sterols and polyphenolic acids, and these of phenolic compounds catalyzed by polyphenol compounds exhibit a variety of pharmacological oxidase (PPO), resulting in the formation of quinines activities like anti-cancer, anti-oxidative, anti- which are highly reactive and toxic to plant tissue. hypertension, anti-inflammatory, anti-mutagenic, anti- Explants’ browning is generally observed in other bacterial, and anti-purulent. It is effective in treating phenolic-rich plants 15-23 . PPO is a light-inducible pneumonia, severe acute respiratory syndrome, enzyme 15,16 . Thus it is hypothesized that dark human immunodeficiency virus and influenza virus treatment may arrest its enzymatic activity. and refractory hemoptysis 6-8. Due to these medicinal It is also well known that wounding can improve uses the requirement of H. cordata has been dedifferentiation of explants. But when explants from increasing in the last several years. However the wild phenolic-rich plants get ruptured to expose a fresh —————— tissue to the air, the cut end easily releases a large *Correspondent author amount of phenolic substances and PPO, and then Telephone: +86 835 2882108 Fax: +86 835 2882336 causes the oxidation reaction to attack the remaining 24 E-mail: [email protected]; [email protected] normal tissue rapidly . Therefore, it is hypothesized XU et al .: REGENERATION POTENTIAL OF HOUTTUYNIA CORDATA 541

that earlier dark treatment followed by wounding not sucrose and 0.6% (w/v) agar, and the pH was adjusted only can solve the problem of heavy browning but to 5.8 before sterilization by autoclaving at 121°C and also can induce dedifferentiation of the explants. 104 kPa for 20 min. In view of the above, the effects of combined dark The cultures were incubated at 24°±2°C under 12 h and wounding treatments on avoiding browning and photoperiod with light intensity of 30 µmol m -2 s-1 enhancing in vitro induction in leaf explants of H. provided by cool-white fluorescent lamps (Philips, cordata have been investigated. China). Statistical analysis —The parameters recorded were Materials and Methods the number of dead explants due to browning Plant materials and explant preparation —H. (browning rate), the number of explants induced cordata new line w01-100 with desirable traits like (induction rate) and the number of shoot buds per disease resistance, high-quality and yield, was explant. The data were subjected to statistical analysis selected out from more than one hundred H. cordata by analysis of variance and the least significant accessions present in China 14 . It belongs to chemotype difference (LSD) test at the 5% level of probability myrcene 25 and possesses 90 chromosomes 26 . It has using SAS version 9.1 (SAS Institute Inc., Cary, been planted for commercial purposes for years in NC, USA). Good Agricultural Practice (GAP) base of 999 Pharmaceutical Group (China). Results Young leaves were collected during March 2008 Effects of dark and wounding on explant and used as explants. These were first surface- browning —With corresponding wounding treatments, sterilized by immersing in 70% (v/v) ethanol for 30 s, explant browning occurred earlier and more seriously and then rinsed thrice with sterile deionized water, in L group (LI, LII and LIII) than in D group (DI, DII and finally treated with 0.1% (w/v) HgCl 2 for 8 min. and DIII). In LII the explant browning was severe These were then rinsed with autoclaved distilled and all explants died within 1 week, but not in DII deionized water 6 times to remove the sterilants. (Fig. 1a, b). In general, the number of dead explants Dark and wounding treatments —The following increased when explants were cultured directly under experiments were conducted in an attempt to examine light condition from the beginning, as compared to the effects of combined dark and wounding treatments when given a dark treatment in the early period (Fig. 2). on inhibiting browning and enhancing in vitro Wounding also had an influence on explant induction. The sterile leaves as explants were browning. In DII tissue browning began at the edge of subjected to three different wounding treatments: I. the incision and then spread to other parts (Fig. 1b), intact leaf without wounding as a control; II. wounded while in DI few small brown spots could be noticed near leaf margins with dissection blade or forceps; III. on a small number of intact leaves (Fig. 1c). Similar had one week pre-culture before uniformly wounded observations were noted in L group, the only like in II. These explants were divided into two difference being in the intensity of browning over the groups with different dark treatments: light (L) group whole explant surface. In general, a statistically as a control, directly under 12 h photoperiod with significant difference in browning between non- light intensity of 30 µmol m -2 s-1 in the tissue culture wounding (I) versus wounding (II) treatments were room, and dark (D) group, 4 week pre-culture in dark. observed both in L and D group, and the number of Accordingly, there were six combined treatments viz., dead explants in LII or DII was higher than that in LI LI, LII and LIII, DI, DII, and DIII. All the or DI, respectively (Fig. 2). One week pre-culture experiments were repeated at least three times with 50 before wounding decreased the number of dead explants each. The control explants were subcultured explants in DIII, but not in LIII (Fig. 2). Interestingly, on the fresh induction medium at 3 week interval. the explants on DIII showed a significant decrease in Culture medium and condition —The induction turning the medium brownish black compared to that medium was MS medium 27 supplemented with 4.44 in LIII. All of the explants in LII and LIII were dead µM 6-benzyl adenine (6-BA; Sigma-Aldrich, China). at last (Fig. 2). The rooting medium was MS medium supplemented In conclusion, both light and wounding can lead to with 1.14 µM indole-3-acetic acid (IAA; Sigma- explant browning rapidly and seriously. A period of Aldrich, China) 14 . Both media contained 3% (w/v) pre-culture in dark was effective in preventing 542 INDIAN J EXP BIOL, JULY 2011

Fig. 1—The effect of dark and wounding on explant browning, in vitro induction and regeneration in H. cordata [(a)-(c) explant browning on medium in LII, DII and DI after 3 d, respectively. (d)-(f) in vitro induction and regeneration in LI, DII and DIII after 5 week, respectively. Arrows show bud primordia on the surface of the leaf. (g) elongated adventitious buds or plantlets after 8 week. (h) de novo regeneration of adventitious buds. (i) rooting process on medium] browning, and 1 week pre-culture of intact leaf in dark significantly reduced the negative effect of wounding on the explant browning. Effects of dark and wounding on in vitro induction —In LI, only a small number of small bud primordia were visible on the surface of the explants after 3-5 week (Fig. 1d) while these arose earlier in DI. Pre-culture in darkness can promote dedifferentiation of explants. A mass of callus mainly originated from the incision of the explants in DII and DIII and seemed to be a direct result of the damage caused due to wounding. In later stage, callus proliferation was observed throughout the tissue of the explants in DIII but not in DII, whose Fig. 2—The percentage of dead leaf explants of H. cordata due to proliferation was additionally hampered by tissue browning after 2 week-culture [The number of dead explants due browning (Fig. 1e, f). to browning of explants inoculated initially. Values are means ± The percentage of explants induced was recorded SD of 3 replicates. Values marked with different letters are as induction rate. According to the induction rates in significantly different according to LSD test at α = 0.05] LI and DI, there was significantly different effect on with browning rates (Fig. 2). In D group, DII could the induction between L and D treatments (Fig. 3). not obtain higher induction rate than DI because of The induction rates possessed negative correlation severe browning of explants (Figs 2 and 3). However, XU et al .: REGENERATION POTENTIAL OF HOUTTUYNIA CORDATA 543

Fig. 3—The percentage of induced leaf explants (callus and buds) Fig. 4—The number of adventitious buds per explant after 8 after 4 week-culture [The number of induced leaf explants of week-culture [Values are means ± SD of 3 replicates. Values explants inoculated initially. Values are means ± SD of 3 marked with different letters are significantly different according replicates. Values marked with different letters are significantly to LSD test at α = 0.05] different according to LSD test at α = 0.05] The de novo regeneration frequency was approximately the highest induction rate was observed in DIII, 20 adventitious buds at 4 week interval. In contrast, because of low browning rate and the rapid response the number of adventitious buds per explant was (Figs 1f, 2 and 3). small in all the other treatments (Fig. 4), and Effects of dark and wounding on in vitro regeneration occurred through solitary adventitious regeneration —In LI and DI, regeneration process buds, which developed from direct bud primordium or occurred through direct shoot formations on the shoot on the surface of the leaves (Fig. 1d). surface of the intact leaves (Fig. 1d). Besides the The elongated shoots were separated and direct shoot formations, a small number of shoot transferred to rooting medium. Normal roots were formations by an intermediary callus phase near the formed at the bottom of all plantlets after 1-4 week cut site of some leaves were observed in DII (Fig. 1e). incubation (Fig. 1i). There was no significant Both somatic embryos and shoot buds, originating difference in the number and length of roots among from callus, were simultaneously observed in DIII, all of the plantlets from different groups. shoot bud regeneration being the predominant way of regeneration (Fig. 1f). The total regeneration was Discussion estimated by the number of shoots per explant. Plant propagation in vitro is a complex Comparison between DI and DII showed that phenomenon and is influenced by a number of wounding at the beginning moderately decreased composite factors. Besides phytohormones, there the number of adventitious buds per explant (Fig. 4). were several reports on environmental conditions, The absence of light moderately increased the number such as wounding 28 , chilling 21,23 and duration of initial of adventitious buds per explant in DI as compared to darkness 10 , which helped plants to regenerate. The LI (Fig. 4). It is worth noting that combination of present results on in vitro regeneration of H. cordata 4 week pre-culture in darkness and wounding after leaves showed that an average 20.64±5.94 adventitious 1 week pre-culture not only inhibited browning but buds per leaf explant can be regenerated when also significantly enhanced in vitro regeneration as a subjected to the treatment with combined 4 week result of which, the maximum average number pre-culture in dark and wounding after 1 week (20.64 ± 5.94) of regenerants was recorded in DIII pre-culture. In addition, de novo regeneration frequency (Fig. 4). Dense clusters of tight green adventitious of 20 adventitious buds in 4 week interval had been buds from the callus were induced with this treatment achieved by serial subculturing. Being an important (Fig. 1f, g). Due to the callus proliferation at the base, economical wild perennial herb with slow agamic continuous de novo production of adventitious buds propagation by stoloniferous rhizome, the highly had been achieved by serial subculturing of efficient regeneration system can meet the demand of adventitious buds on the induction medium (Fig. 1h). commercial cultivation, such as GAP base in China, 544 INDIAN J EXP BIOL, JULY 2011

and lay the foundation for the conservation of Since the genotypes differ markedly in the H. cordata germplasm resources. In addition this response to the substances and conditions which help system may be used for improving desirable traits cells to differentiate, every genotype seems to have through direct gene manipulations especially because its own specific requirements, and not all plant improvement by traditional breeding methods was species or varieties can regenerate easily under difficult due to the genetic limitations associated with in vitro 35,37-39 . For example ten Lycopersicon high heterozygosity, aneuploidy and polyploidy 26 . esculentum cultivars exhibited significant variation 35 Explants’ browning was an important factor that in regeneration response in the same conditions and impaired in vitro regeneration of H. cordata . Helianthus annuus var. RHA 266 was a poor 18 Browning rates of intact leaves declined in the present responding genotype to in vitro culture . A investigation and incubation in absence of light significant negative maternal effect for culture showed a significant decrease in tissue browning in response was noted for inbred B73 from Reid-type 40 wounded leaves. This must be due to arrest PPO maize germplasm . H. cordata germplasm resources activity, responsible for the oxidation of phenolic in geographic distribution exhibited highly complex compounds, by dark treatment 15,16 . The results should variation at chemotypes, cytological and nuclear 1,2,25,26,41 contribute to the hypothesis of the positive effects of genome level . Because of restrictions in dark treatment on avoiding browning. The identical genotype, there may be several different responses to 4 effect of dark on inhibiting browning was also the culture conditions. Chakraborti et al. reported observed in several other genera, e.g. Fragaria 23 . 11 shoot buds per explant, derived from the While there are many reports about addition of underground stoloniferous rhizome of the wild antioxidant substances or absorbents in culture H. cordata in the north-east India, on the MS medium to avoid browning in many species, such as medium containing 2.22 µmol/L 6-BA by 40 d Crocus sativus 29 , Pistacia vera 30 , Vicia faba 31 , Ocotea culture. However, only a few shoot buds could 21 be induced using the same organs as explants from bullata and Warburgia salutaris but these 14 substances were not found suitable for H. cordata H. cordata line w01-100 on the same medium . In (data not shown). the present work, effects of dark and wounding were additionally tested on the regeneration potential of Cells acquiring developmental competency leaf explants from other H. cordata lines, w01-39 (totipotency) is the first important step for in vitro 32 (54 chromosomes; Chemotype decanal) and w01-86 culture . It was a major barrier that prevented in vitro (36 chromosomes; Chemotype decanal) and only regeneration in H. cordata as the cells were difficult 14 few adventitious buds could be obtained (data to dedifferentiate . As every species seems to have its not shown). Therefore, in vitro propagation of own specific requirements 33,34 , shift of hormone H. cordata may also be genotype-dependent. balance and types in the culture medium had no significant effect on callus induction of the leaf 14 Acknowledgment explants in H. cordata . However, the present study demonstrated that wounding combined with dark This work was supported by the Scientific treatment could significantly enhance in vitro Research Foundation for the Returned Overseas induction. The role of wounding seemed as a Chinese Scholar in China. Thanks are due to triggering signal for callus in wounded leaves 35,36 . It Department of Biotechnology for laboratory facilities, has been suggested that wounding promotes transfer to Fan Liu and Wenting Yang for helpful comments, of endogenous hormones to the location of the wound, and to Dr. Sandhya Gupta (Tissue Culture & causing callus production 20 . Similar positive effect of Cryopereservation Unit, National Bureau of Plant wounding had been observed in other plant species, Genetic Resources, India) for reading the manuscript such as Populus nigra × P. Maximowiczii 20, and suggestions. Phalaenopsis 19 , Helianthus annuus 18 and Prunus avium 17 . The positive effect of wounding in the References present study was observed in DIII and the 1 Wu W, Zheng Y L, Chen L, Wei Y M, Yan Z H & Yang R W, PCR-RFLP analysis of cpDNA and mtDNA in the genus precondition here that the explants were pre-cultured Houttuynia in some areas of China, Hereditas , 142 (2005) 24. in dark, promoting cell dedifferentiation and 2 Wu W, Zheng Y L, Chen L, Wei Y M, Yang R W & Yan Z 24 inhibiting browning . H, Evaluation of genetic relationships in the genus XU et al .: REGENERATION POTENTIAL OF HOUTTUYNIA CORDATA 545

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