Plant Science 160 (2000) 139 – 147
Cytokinin-induced somatic embryogenesis and plant regeneration
in
Corydalis yanhusuo
(Fumariaceae) — a medicinal plant
A.P. Sagare
a, Y.L. Lee
a, T.C. Lin
b, C.C. Chen
b, H.S. Tsay
a,*
aDepartment of Agronomy,Taiwan Agricultural Research Institute,189,Chung-Cheng Road,Wufeng,Taichung41301,Taiwan bInstitute of Chinese Pharmaceutical Science,China Medical College,Taichung 40421,Taiwan
Received 30 May 2000; received in revised form 7 August 2000; accepted 4 September 2000
Abstract
An efficient method has been developed for regeneration of complete plants via somatic embryogenesis inCorydalis yanhusuo
(Fumariaceae), an important medicinal plant, using tuber-derived callus. Primary callus was induced by culturing mature tuber pieces on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg l−1 N6-benzyladenine (BA) and 0.5 mg l−1
a-naphthaleneacetic acid (NAA) in darkness. Somatic embryos were induced by subculturing the primary callus on MS medium
supplemented with 0.5 – 4.0 mg l−1 BA, kinetin, or zeatin, within 2 weeks of culture in light. Embryos with well-developed
cotyledonary leaves were transferred in half-strength liquid MS medium supplemented with 1.0 mg l−1zeatin riboside for the
development of roots. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% sucrose, and with 0.5 – 10.0 mg l−1abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5 – 5.0 mg l−1GA
3and 15 – 100 mg l−1polyethylene
glycol (PEG) 4000 for further development of plantlets and in vitro tuber formation. The development of somatic embryos over the surface of tuber and/or cotyledonary leaf base region of the converted primary somatic embryo was observed. Before ex vitro establishment of somatic embryo-derived plants, plants with well-developed tubers were cultured on half-strength MS medium with 2% sucrose and 0.1 mg l−1GA
3for 3 weeks. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:ABA;Corydalis yanhusuo; Growth retardant; Medicinal plant; Papaveraceae; PEG-4000; Somatic embryogenesis
www.elsevier.com/locate/plantsci
1. Introduction
The genus Corydalis (Fumariaceae or Papaver-aceae) of about 320 species is widely distributed in the Northern hemisphere and 70 species have been used in traditional herbal remedies in China, Japan, and Korea [1]. Corydalis yanhusuo (syn.
Corydalis turtschanino6ii f. yanhusuo), a perennial
herb up to 20 cm tall [2], is one of the medicinally
important species of Corydalis [1]. The dried and pulverized tubers of C. yanhusuo, also called as
Rhizoma Corydalis or yan-hu-suo, [3], are used in traditional Chinese medicine in the treatment of gastric and duodenal ulcer, cardiac arrhythmia disease [1], rheumatism and dysmenorrhea [3]. The tuber contains several alkaloids; some of the im-portant alkaloids are DL-tetrahydropalmatine, D -corydaline, and corydalis H, I, J, K, and L [4]. The alkaloids exhibited analgesic [5], anti-arrhythmic [6], thrombic, [7], inflammatory [8], anti-cataract [9], anti-hypertensive [10] and anti-allergic [11] activity.
C. yanhusuo is cultivated mainly in Zhejiang, Jiangxi, Anhui and Hubei provinces of China as an annual crop using tubers and the tubers are exported to other countries.C.yanhusuois suscep-tible to fungal diseases [2], especially downey Abbre6iations: ABA, abscisic acid; Ancymidol or Anc, a
-cyclo-propyl-a-(4-methoxy-phenyl)-5-pyrimidine methanol; BA, N6
-benzy-ladenine; GA3, gibberellic acid; kinetin, 6-furfuryl amino purine; MS, Murashige and Skoog (1962); NAA, a-naphthaleneacetic acid;
Pa-clobutrazol or Pac, (2RS, 3RS )-1-(4-chlorophenyl)-4,4-dimethyl-2(1,2,4-triazol-1-yl)pentan-3-ol; PEG-4000, polyethylene glycol-4000; zeatin, 6-(4-hydroxy-3-methyl-2-butenylamino) purine.
* Corresponding author. Tel.: +886-4-3302301, ext. 108; fax: +
886-4-3338162.
E-mail address:hstsay@wufeng.tari.gov.tw (H.S. Tsay).
mildew caused by Peronospora corydalis de Bary, which infect tubers and is responsible for 30 – 50% loss in yield when conditions are conducive for fungal growth [12]. To improve the produc-tivity, homogeneity and quality of the tubers, it is necessary to have pathogen-free planting mate-rial. Pathogen-free plants of C. yanhusuo could be obtained using seeds; however, the seeds have a low percentage of germination [13] and take a long time to germinate due to extremely imma-ture state of zygotic embryo at the time of dis-persal. Both warm and cold stratification treatments are required to induce germination from seeds [13]. Furthermore, the growth of the seedlings is very slow during the first year of growth forming only a small, immature tuber. Mature tubers could be obtained only in the suc-ceeding year of growth after a period of dor-mancy [13]. Plant regeneration via in vitro culture of C. yanhusuo would be useful for mass propagation of this important medicinal plant in a short time.
Somatic embryogenesis is the most common mode of regeneration for species in the Papaver-aceae [14 – 23]. The genus Corydalis has shown morphogenetic potential [24,25]; to date, how-ever, there has been no report on the complete plant regeneration of C. yanhusuo via somatic embryogenesis. We report here a method of plant production via somatic embryogenesis from tuber-derived callus of C. yanhusuo.
2. Materials and methods
2.1. Induction of somatic embryogenesis
Mature tubers of Corydalis yanhusuo W.T. Wang (syn. Corydalis turtschanino6ii Bess. f. yan
-husuo Y.H. Chou et C.C. Hsu) were obtained from Shenyang, Liaoning province, China in November 1995. Tubers were cleaned under run-ning tap water and surface-disinfected in 70% ethanol for 1 min, followed by 0.5% sodium hypochlorite (Clorox, The Clorox Co., Oakland, CA) with two drops of Tween 20® per 100 ml (Hayashi Pure Chemical Industries Ltd., Osaka, Japan) under ultrasonic vibration (Branson Ul-trasonic Cleaner, Branson Cleaning Equipment Co., Shelton, CT) for 10 min and rinsed five
times with sterile distilled water. After steriliza-tion, tubers were cut into 5×5×2 mm pieces and cultured in 22×120 mm glass test tubes, each containing 10 ml of medium. The medium consisted of Murashige and Skoog’s (MS) medium [26] (MS inorganic salts and vitamins+ 100 mg l−1 myo-inositol, referred to hereafter as MS basal medium), 3% sucrose, 0.9% Difco Bacto agar (Difco Laboratories, Detroit, MI) and 2.0 mg l−1 BA in combination with 0.5 mg l−1 NAA. The cultures were incubated at 259 1°C in darkness for 1 month. The primary callus produced from tuber pieces was subcultured ev-ery 20 days for three times on the same medium. The primary callus (200 mg each) was trans-ferred on MS basal medium with 3% sucrose, 0.9% Difco Bacto agar and supplemented with 0, 0.5, 1.0, 2.0 and 4.0 mg l−1 BA, kinetin or zeatin (Sigma Chemical Co., St. Louis, MO) (Table 1). Zeatin stock solution was filter-steril-ized (0.22 mm Millipore) and added to the
medium after autoclaving. Twenty calli were evaluated per treatment. The pH of the medium was adjusted to 5.790.1 with 1 N NaOH or HCl before autoclaving at 121°C, 105 kPa for 15 min. The culture vessels were capped with two layers of aluminum foil before autoclaving and sealed with three layers of Parafilm M after cul-ture. The cultures were incubated at 2591°C under cool white fluorescent light at 38
Table 1
Effect of cytokinins on induction of somatic embryogenesis from tuber-derived primary callus ofCorydalis yanhusuoafter 5 weeksa
Cytokinin Average number of somatic embryos
(mg l−1) produced per callus
aMeans within a column followed by the same letter are
A.P.Sagare et al./Plant Science160 (2000) 139 – 147 141
mmol·m−2·s−1 (Philips, Eindhoven, The
Nether-lands) with a 16-h photoperiod per day for 5 weeks.
2.2. Con6ersion of somatic embryos
Somatic embryos with cotyledonary leaves, dif-ferentiated on the surface of the primary calli on zeatin 1.0 mg l−1containing medium, were trans-ferred along with the undifferentiated calli in half-strength MS liquid medium (half-half-strength of MS inorganic salts+full complement of vitamins and inositol) supplemented with 3% sucrose and 1.0 mg l−1 zeatin riboside (Sigma) in 250-ml Erlen-meyer flasks with 20 ml medium. The pH of the medium was adjusted to 5.290.1 prior to auto-claving. Zeatin riboside stock solution was filter-sterilized and added to the medium after autoclaving. The flasks were placed on an orbital shaker (Model SK-302A, Sun Kuan Instruments Co., Taichung, Taiwan) with rotary motion of 100 rpm, and incubated for 2 weeks at 2591°C under
cool white fluorescent light at 38
mmol·m−2·s−1·with a 16-h photoperiod per day.
2.3. Effects of ABA, paclobutrazol, ancymidol,
GA3,and PEG on de6elopment of con6erted somatic
embryos
Converted somatic embryos with well-developed shoots and roots were transferred individually on half-strength MS medium supplemented with 6% sucrose, 0.9% Difco Bacto agar and (i) 0.5, 1, 2, 5 or 10 mg l−1 ABA, ancymidol or paclobutrazol; (ii) 0.5, 1, 2 or 5 mg l−1GA3; (iii) 15, 25, 50 or 100 mg l−1 PEG-4000 (MW 4000) (Table 2), in 22× 120 mm glass test tubes, each containing 10 ml of medium. Ancymidol (Sigma), ABA (Sigma), pa-clobutrazol (Wako Pure Chemical Industries Ltd., Osaka, Japan) and GA3 (Sigma) stock solutions were filter-sterilized and added to the medium after autoclaving. The cultures were incubated un-der the conditions described in Section 2.2. Five converted somatic embryos were evaluated per treatment.
The number of shoots and roots produced per somatic embryo derived plantlet, average length of shoots and roots, tuber diameter and number of somatic embryos developed was recorded after 1 month of culture. The experiment was repeated three times.
2.4. De6elopment of plantlets
Plantlets with well-developed tubers were trans-ferred to half-strength MS medium supplemented with 2% sucrose, 0.18% Gelrite (Sigma) and 0.1 mg l−1GA
3in 250-ml Erlenmeyer flasks with 100 ml medium, and incubated for 3 weeks under the conditions described in Section 2.2.
2.5. Ex 6itro establishment of plantlets
Sixty somatic embryo-derived plants with well-developed roots, shoots and tubers were thor-oughly washed under tap water for 2 – 3 min to remove traces of agar-gelled medium sticking to them. Plants were then dipped in 0.1% (w/v) Ben-late (Du Pont De Nemours and Co. Inc., Taoyuan, Taiwan) for 1 min and planted in 6-cm plastic pots containing a mixture of autoclaved sand and peat moss (1:1 by volume). The pots were kept in the growth chamber (Model 624 HD, Hotech Instruments Corp., Taipei, Taiwan) under a light intensity of 100 mmol·m−2·s−1 for a 16-h
photoperiod per day and day/night temperatures of 20/16°C. The plants were initially covered with polypropylene bags to maintain humidity and irri-gated once in a week with tap water. After 15 days, the polypropylene bags were removed. The percentage survival of plants was calculated after 2 months.
2.6. Statistical analysis
For statistical analysis, number of somatic em-bryos, shoot number and length, number of so-matic embryos showing tuber formation and number of somatic embryos developed over the surface of tuber and/or cotyledonary leaf base region of the converted primary somatic embryo were recorded. Least significant difference (LSD) test was used for statistical analysis.
3. Results and discussion
3.1. Callus induction
A
Effect of different treatments on somatic embryos ofCorydalis yanhusuoafter 1 month of culture on MS basal salts medium supplemented with 6% sucrose either alone or in combination with ABA, paclobutrazol, ancymidol, GA3or PEG-4000a
Number of
Treatment (mg l−1) Length of Number of Length of Tuber formation Number of somatic embryos per primary
roots roots (mm) embryoc
shoots shoots (mm)
Paclobutrazol 0.5 1.7bc 13.4bcd
0.6i 6.0f 4.6ab (100) 1.9abc (66.7)
Ancymidol 0.5 1.7bc
12.7ef
aMeans within a column followed by the same letter are not significantly different from each other at the 5% level as determined by the LSD test. bData in the parentheses within a column shows percentage of converted somatic embryos forming tuber.
cData in parentheses within a column shows percentage of the converted primary somatic embryos showing development of somatic embryos directly over the surface of
A.P.Sagare et al./Plant Science160 (2000) 139 – 147 143
mg l−1 NAA, callus initiation occurred. When the callus was separated from the parent tissue and transferred to fresh medium, it grew vigor-ously into yellow friable calli and could then be regularly subcultured at 20-day intervals.
3.2. Induction and de6elopment of somatic
embryos
Various concentrations of cytokinins (BA, kinetin or zeatin) were tested for their potential to induce either caulogenesis or somatic embryo-genesis in C. yanhusuo using tuber-derived pri-mary callus. The response is summarized in Table 1. All the three cytokinins stimulated so-matic embryo formation including the basal medium consisting of MS inorganic salts and vi-tamins, 100 mg l−1 inositol and 3% sucrose, but the efficiency varied. Somatic embryo induction usually involves the use of auxins [27], mostly 2,4-D, with or without a low level of cytokinin; however, initiation of somatic embryos on medium containing cytokinin as the sole growth regulator has also been reported in few species such as Trifolium [28], Coffea [29], Gladiolus [30],
Helianthus [31], Spinacia [32] and Medicago [33]. The development of somatic embryos occurred on the surface of the tuber-derived primary cal-lus. The highest number of somatic embryos was obtained on medium supplemented with either 4.0 mg l−1 kinetin, 0.5 – 1.0 mg l−1 BA, or 0.5 mg l−1 zeatin, however, the embryos induced on BA and kinetin reverted to callus. The emergence of somatic embryos (Fig. 1A, arrows) occurred after 15 days of culture on 1.0 mg l−1 zeatin containing medium. The embryos progressed through the globular, late-globular, heart, early cotyledonary and cotyledonary stages (Fig. 1B). Cotyledonary-stage somatic embryos were formed after 21 days of culture of primary calli on 1.0 mg l−1 zeatin containing medium (Fig. 1C). After 5 weeks of culture, somatic embryos showed development of cotyledonary leaves (Fig. 1D), however, root development was arrested.
3.3. Con6ersion of somatic embryos
Roots developed readily after transfer of em-bryos along with calli to half-strength liquid MS medium supplemented with 1.0 mg l−1 zeatin
ri-boside. In liquid medium, the embryos could eas-ily be separated without any damage, from the surrounding callus tissue. Somatic embryos (96%) converted to form well-developed roots and shoots (Fig. 1E). Over 90% of the converted so-matic embryos showed development of only one cotyledon into a cotyledonary leaf. A few species of the genus Corydalis, although being a dicot, showed a single cotyledon in the mature seeds [34]. The presence of a single cotyledon in seed-derived plantlets of C. yanhusuo has been re-ported recently [13].
3.4. Effects of ABA, paclobutrazol, ancymidol,
GA3 and PEG-4000 on de6elopment of con6erted
somatic embryos
Fig. 1.
3.5. De6elopment of somatic embryos o6er the
surface of tuber and/or cotyledonary leaf base region of the con6erted primary somatic embryo
Somatic embryos developed directly over the surface of tuber and/or cotyledonary leaf base region of the primary somatic embryo after 1 month in culture on different treatments (Table 2). The maximum number (3.7) of somatic embryos
per primary embryo was obtained on medium with 5.0 mg l−1 ABA. Somatic embryos developed on the surface of tuber showed non-synchronous de-velopment (Fig. 1J) and embryos were observed at globular (g), heart (h) and cotyledonary stage (c) of development. In contrast, somatic embryo de-velopment on the basal region of the cotyledonary leaf was more synchronous (Fig. 1K). Primary embryos cultured on either 0.5 – 5.0 mg l−1 GA
A.P.Sagare et al./Plant Science160 (2000) 139 – 147 145
2 – 10 mg l−1 ancymidol, or growth regulator-free medium did not support development of somatic embryos on either tuber or cotyledonary leaf base region. Induction of somatic embryos on the cotyledons and/or hypocotyl region of previously differentiated somatic embryos has been reported earlier in other plant species [39] (and references cited therein). The somatic embryos developed over the surface of tuber and cotyledonary leaf base region of the primary somatic embryo devel-oped normally and could be converted into plantlets (data not shown).
3.6. Ex 6itro establishment of plantlets
To prevent dormancy and promote development of new shoots, plantlets with well-developed tu-bers, formed after culture on 6% sucrose contain-ing medium without growth regulators (control treatment, Table 2), were cultured on a medium supplemented with 2% sucrose and 0.1 mg l−1 GA3. After 2 weeks of culture, the plantlets showed development of new shoots and roots. Fig. 1L shows somatic embryo derived plantlet with well-developed roots (r), tuber (t) and shoot apical meristem (sm). Plants with well-developed roots, shoots and tubers were transplanted to a sand:peat moss mixture. The acclimatization rate of plants derived from somatic embryos was 80% after 2 months of culture in a growth chamber. Fig. 1L shows a plant that survived 2 months after trans-fer to sand:peat moss.
The optimal procedure for plant production via somatic embryos inC.yanhusuois summarized as:
(i) primary callus cultures were established in 3 months by culturing mature tuber pieces on MS basal medium supplemented with 2.0 mg l−1 BA and 0.5 mg l−1 NAA in darkness; (ii) somatic embryos were induced in 5 weeks by culturing the tuber-derived primary callus on MS basal medium with 0.1 mg l−1 zeatin in light; (iii) ninety six percent somatic embryos converted in 2 weeks by culturing in half-strength MS liquid medium sup-plemented with 1.0 mg l−1 zeatin riboside; (iv) converted somatic embryos showed good shoot, root and tuber development when cultured on half-strength MS medium supplemented with 6% sucrose for 1 month; (v) before transplanting to soil:peat moss, the plants were cultured on half-strength MS medium with 2% sucrose and 0.1 mg l−1 GA3 for 3 weeks; (vi) eighty percent plants survived 2 months after transfer to sand:peat moss and incubation in a growth chamber; (vii) the entire protocol starting from establishment of the tuber-derived primary callus to well-developed plants in soil takes around 8 months. However, this period could be reduced to around 5 months once the primary callus cultures are established. Around 25 plants could be obtained from 1 g of tuber-derived primary callus after acclimatization.
4. Conclusions
Our results show that somatic embryos could be induced in tuber-derived callus of C. yanhusuo
using cytokinins. In vitro tuberization was achieved by culturing somatic embryo-derived
Fig. 1. (A – M). Plant regeneration through somatic embryogenesis in Corydalis yanhusuo. (A) Tuber-derived primary callus showing emergence of somatic embryos (arrows) after 15 days of culture on MS medium supplemented with 1.0 mg l−1zeatin.
Bar, 510mm; (B) somatic embryos at different stages of development; direction of the arrow shows globular, late-globular, heart,
early-cotyledonary, cotyledonary-stage and mature somatic embryos. Bar, 263 mm; (C) cotyledonary-stage somatic embryos
formed after 21 days of culture. Bar, 955mm; (D) somatic embryos showing the development of cotyledonary leaves after 5 weeks
of culture on 1.0 mg l−1zeatin containing medium; (E) conversion of somatic embryos after culture in half-strength liquid MS
medium supplemented with 1.0 mg l−1 zeatin riboside on a rotary shaker for 2 weeks. Bar, 5.2 mm; (F) converted somatic
embryos showing development of tubers after culture on half-strength MS medium supplemented with 6% sucrose for 1 month. Bar, 6.58 mm; (G) somatic embryo-derived tubers formed on half-strength MS medium supplemented with 6% sucrose and 0.5 mg l−1ABA. Bar, 1.77 mm; (H) somatic embryo-derived tubers formed on half-strength MS medium supplemented with 6%
sucrose and 0.5 mg l−1paclobutrazol. Bar, 1.72 mm; (I) converted somatic embryo showing emergence of numerous shoots after
1 month of culture on half-strength MS medium supplemented with 5.0 mg l−1 GA
3. Bar, 1.45 mm; (J) somatic embryos at
globular (g), heart (h) and cotyledonary (c) -stage of development, formed on the surface of the tuber after 1 month of culture of primary somatic embryo on half-strength MS medium supplemented with 5.0 mg l−1ABA. Bar, 450mm; (K) somatic embryos
differentiated directly on the surface of the basal portion of the cotyledonary-leaf of primary somatic embryo. Bar, 520mm; (L)
a somatic embryo-derived plantlet cultured on half-strength MS medium with 2% sucrose and 0.1 mg l−1 GA
3 showing a
plantlets in media supplemented with either 6% sucrose alone or in combination with ABA, an-cymidol, paclobutrazol, or PEG-4000. This regen-eration system could be used for: (i) continuous regeneration of somatic embryos for the produc-tion of pathogen-free plants and tubers of C.
yanhusuo; (ii) biochemical studies, studying the effect of precursor or elicitor feeding on accumula-tion of alkaloids in tubers of somatic embryo-derived plantlets; (iii) genetic transformation studies, especially to transfer antifungal protein gene(s) [40].
Acknowledgements
This research was supported by a grant (NSC 88-2317-B-055-001) from the National Science Council of Taiwan. We thank C.Y. Lee and S.M. Chen for technical assistance and C.T. Lu for help in statistical analysis. This paper is Taiwan Agri-cultural Research Institute Contribution no. 2030.
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