Plant Cell Reports (1999) 18: 873–878
© Springer-Verlag 1999
E. Prakash · P. S. Sha Valli Khan · P. Sairam Reddy K. R. Rao
Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb
Received: 23 July 1998 / Revision received: 18 November 1998 / Accepted: 26 November 1998
Abstract A procedure was developed for plant regeneration of Hybanthus enneaspermus, a rare ethnobotanical herb from the Deccan peninsula in India, through seed-derived callus. Seeds demonstrated a high induction frequency (69.4±2.8%) and a high yield (364.4 ±2.5 mg) of light-yellow friable callus on Murashige and Skoog’s (MS) medium containing 2.6 µM NAA and 2.2 µM BA within 4 weeks of incubation. After 1 year of subculture, yellow friable and light-green compact calli types were established from initial light-yellow friable callus. Shoot differentiation was achieved from light-green compact callus, but not from yellow friable callus. Shoot differentiation resulted when light-green compact callus was transferred to MS medium supplemented with 8.8 µM BA and 2.6 µM NAA; the highest percentage of calli forming shoots (66.6 ±4.8%) and the highest number of shoots (8.9 ±0.3) were achieved in this medium. Differentiated shoot buds elongated to 4–5 cm within 4 weeks. The addition of casein hydrolysate (500 mg/l) and more potassium phosphate (1.86 mM) to the culture medium enhanced shoot differentiation. Rooting was achieved on the shoots using halfstrength MS medium containing 4.8 µM IBA. About 70% of the plants were established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. Key words Plant regeneration · Seed-derived callus · Hybanthus enneaspermus · Ethnobotanical herb · Rare Communicated by G. Phillips E. Prakash (½) · P. S. Sha Valli Khan P. Sairam Reddy · K. R. Rao Plant Tissue Culture Laboratory, Department of Botany, Sri Venkateswara University, Tirupati-517502, A. P., India Fax: 91-8574-27499 e-mail:
[email protected] P. S. Sha Valli Khan CRP-Centre Universitaire, CREBS, 162A, Av. de la Faïencerie, L-1511 Luxembourg, Grand-Duchy of Luxembourg
Abbreviations MS Murashige and Skoog’s (1962) medium · NAA 1-naphthalene acetic acid · BA N6-benzyladenine · KN kinetin · 2-ip 2-isopentenyl adenine · CH casein hydrolysate · IBA indole-3-butyric acid
Introduction
Hybanthus enneaspermus L. Muell, a member of the Violaceae family, is a rare, perennial herb found in some of the warmer parts of the Deccan peninsula in India. Popularly called ‘Ratanpurus’ by the local Yanadi and Santal tribes, villagers and herbalists, this ethnobotanical herb is known to have unique medicinal properties. The preparations made from the leaves and tender stalks of the plant are used in herbal medicine for its aphrodisiac, demulcent and tonic properties. The root is diuretic and administrated as an infusion in gonorrhea and urinary infections (The Wealth of India 1959; Nagaraju and Rao 1996). The fruits and leaves are used as antidotes for scorpion stings and cobra bites by the Yanadi tribes (Raja Reddy et al. 1989; Sudarsanam and Sivaprasad 1995). However, the natural regeneration potential of this herb is very poor due to low seed viability. Because the seeds and developing capsules are often found on the ground, loss due to rodents and inundation is considerable. Increasing human and livestock populations have already affected the status of wild plants, particularly those used in herbal medicine. In view of its ethnomedicinal importance, there is a need to conserve the wild stock of H. enneaspermus. Plant tissue culture is a useful tool for the conservation and rapid propagation of rare and endangered medicinal plants (Saxena et al. 1997; Castillo and Jordan 1997; Sanyal et al. 1998). Plant propagation through callus or suspension cultures requires the induction of an organogenic or embryogenic callus type from explants. Organogenic or embryogenic callus is generally induced from meristematic tissues such as embryos, basal meristems or shoot tips (Bajaj 1992). However, there are a few reports on callus induction and plantlet regeneration using seeds as explants (Lin and Griffin 1992; Mii et al. 1994;
874
Griffin and Dibble 1995). To our knowledge, there are only three reports on successful in vitro culture in the family Violaceae, especially on Viola patrinii DC (Han et al. 1990; Kwon et al. 1992; Sato et al. 1995). So far there have been no reports on in vitro culture and plantlet regeneration in the genus Hybanthus. This paper reports for the first time an in vitro procedure for plantlet regeneration from seedderived callus of H. enneaspermus.
Materials and methods Plant material and explant source Mature capsules were collected during November and December from plants of H. ennespermus growing at the Botanical Garden of the Department of Botany, Sri Venkateswara University, Tirupati, India. Seeds were removed from the capsules and washed with a 1% (v/v) Teepol detergent solution for 15 min. The seeds were then disinfected by immersion in 70% (v/v) ethanol for 1 min followed by immersion in 2% (v/v) sodium hypochlorite solution containing a few drops of Tween-20 for ten min. After five rinses in sterile distilled water, the seeds were transferred onto culture medium for callus formation. Callus formation Different concentrations of NAA either alone (1.3–5.3 µM) or in combination with BA (1.1 µM or 2.2 µM) were tested for their effect on callus formation from seeds. MS medium lacking growth regulators served as the control. Data on callus formation was recorded as the percentage of seeds forming callus. Seed-derived callus growth was determined as the final fresh weight after 4 weeks of incubation. Twelve seeds were used per treatment with three replications. Shoot differentiation Three sets of experiments were carried out on shoot differentiation. In the first set of experiments, two types of seed-derived calli (yellow friable callus and light-green compact callus) were transferred onto MS medium supplemented with BA either alone (4.4–16.6 µM) or in combination with NAA (2.6 µM) to determine shoot differentiation potential. MS medium lacking growth regulators served as the control. The second and third set of experiments were carried out with light-green compact callus. In these the effects of casein hydrolysate (0–1000 mg/l) and potassium phosphate (0.0–2.48 mM) were tested, respectively, on shoot differentiation during optimal growth regulator treatment. MS medium lacking casein hydrolysate and potassium phosphate served as controls for the Second and third set of experiments, respectively. For all experiments, 12 callus pieces (approximately 300 mg fresh weight) were used per treatment with three replications. Data on percentage of calli forming shoots and number and length of differentiated shoots were recorded after 4 weeks. Root formation In vitro differentiated shoots measuring 5–6 cm in length were excised from seed-derived light-green compact callus growing on optimal growth regulator treatment and cultured at four concentrations (1.2, 2.4, 4.8 and 9.8 µM) of IBA along with a control (no auxin) for rooting. Twelve shoots were used per treatment with three replications. Data were recorded on percentage of rooting, root number and root length after 4 weeks on rooting media.
Culture media and incubation conditions Murashige and Skoog’s (1962) medium supplemented with 3% (w/v) sucrose was used for callus formation and shoot differentiation. Halfstrength MS medium supplemented with 2% (w/v) sucrose was used for the rooting experiments. The media were solidified with 0.8% (w/v) agar (Bacteriological grade, Qualigens, India), and the pH was adjusted to 5.8 prior to autoclaving at 1.06 kg cm–2 for 20 min. All cultures were incubated at 25° ±2 °C and under a 16-h light/8-h dark photoperiod with a light intensity of 50 µE m–2 s–1 provided by coolwhite fluorescent lamps in combination with incandescent bulbs. Hardening Healthy plantlets with 4- to 5-cm-long roots were individually removed from the culture tubes. After washing their roots carefully with tap water, plantlets were transplanted into 10-cm-diameter plastic pots containing a mixture (1 : 1 v/v) of autoclaved soil and vermiculite. The plants were watered with half-strength MS salts solution every week and covered with a polythene bag for 2 weeks. Afterwards, the hardened plants were gradually transferred to 20-cm pots containing pure garden soil and kept in the field for developing into mature plants. Statistical analysis All experiments were repeated thrice. The effects of different treatments were quantified and the data subjected to statistical analysis using ‘standard error of the mean’.
Results and discussion
Callus formation The seeds of H. ennespermus failed to produce callus on MS medium lacking growth regulators as well as on MS medium containing only BA (1.1 or 2.2 µM). In contrast, callus initiation was achieved from the seeds within 7 days of incubation on MS basal medium supplemented with NAA either alone (1.3–5.3 µM) or in combination with BA (1.1 µM or 2.2 µM). The callus appeared light-yellow in colour and friable in texture. MS medium containing only BA (1.1 µM or 2.2 µM) stimulated seed germination and produced only seedlings. The concentration of NAA, presence or absence of BA and their interactions influenced induction frequency and the growth of seed-derived callus (Table 1). Seeds showed a lower induction frequency and decreased callus formation on media containing NAA alone (1.3–5.3 µM) than on media supplemented with both NAA (1.3–5.3 µM) and BA (1.1 µM or 2.2 µM) (Table 1). This result supports the synergistic effect of NAA and BA as reported for callus formation from Psoralea corylifolia (Saxena et al. 1997). Media containing lower concentrations of NAA (1.3–2.6 µM) and BA (1.1 µM or 2.2 µM) yielded moderate to high amounts of callus (Table 1). On the other hand, media containing higher concentration (5.3 µM) of NAA in combination with BA (1.1 µM or 2.2 µM) induced decreased amounts of callus. Seeds demonstrated a high induction frequency (69.4 ±2.8%) and a high yield (364.4 ±2.5 mg) of light-yellow friable callus
875 Table 1 Effect of growth regulators on callus formation from seeds of H. enneaspermus (after 4 weeks) (G germination of seeds) Growth regulators NAA (µ M) 0.0 0.0 0.0 1.3 1.3 1.3 2.6 2.6 2.6 5.3 5.3 5.3 a b
b
BA (µ M)
Percentage of seeds forming callus ±SE a
Mean fresh weight of callus (mg) ±SE a
0.0 1.1 2.2 0.0 1.1 2.2 0.0 1.1 2.2 0.0 1.1 2.2
0.0 G G 47.2 ±2.8 52.7 ±2.6 50.1 ±4.8 55.5 ±2.7 55.5 ±2.7 69.4 ±2.8 44.4 ±5.5 47.2 ±2.8 44.4 ±5.5
0.0 G G 255.8 ±3.6 286.8 ±3.4 297.0 ±5.8 281.4 ±3.1 332.3 ±3.4 364.4 ±2.5 209.7 ±6.1 233.6 ±0.7 215.7 ±11.9
Mean ±SE for three replications (12 seeds for each replication) MS medium lacking growth regulators served as control
Table 2 Effect of growth regulators on shoot differentiation from seed-derived light-green compact callus of H. enneaspermus (after 4 weeks culture) Growth regulators BA (µ M)
NAA (µ M)
Percentage of calli forming shoots ±SE a
0.0 b 4.4 4.4 8.8 8.8 16.6 16.6
0.0 0.0 2.6 0.0 2.6 0.0 2.6
0.0 41.6 ±4.8 55.5 ±2.7 52.7 ±2.7 66.6 ±4.8 44.4 ±2.8 47.2 ±3.8
Mean shoot number ±SE a
Mean shoot length (cm) ±SE a
0.0 3.1 ±0.2 5.8 ±0.3 2.9 ±0.4 8.9 ±0.3 3.0 ±0.2 4.5 ±0.2
0.0 2.1 ±0.1 3.1 ±0.2 3.3 ±0.2 4.7 ±0.1 1.4 ±0.1 2.9 ±0.4
a Mean ±SE for three replications (12 callus pieces for each replication) b MS medium lacking growth regulators served as control
within 4 weeks on MS medium containing 2.6 µM NAA and 2.2 µM BA. Seed-derived callus (approximately 300 mg fresh weight) was subcultured on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 2.6 µM NAA and 2.2 µM BA at 4-week intervals to maintain further growth and proliferation. The light-yellow friable callus become more yellow after the second subculture. Lightgreen compact areas appeared spontaneously on the surface of the yellow friable callus after the fourth subculture. These light-green compact areas were removed from the yellow friable callus and subcultured separately. The following two callus types were established after 1 year of subculture: light-green compact callus was associated with nodular structures, and yellow callus was associated with loosely packed friable regions (Fig. 1). Further subculture of the two types of callus on MS medium containing 2.6 µM NAA and 2.2 µM BA resulted in neither shoot nor root differentiation. Therefore, both types of calli were transferred onto MS medium containing higher concentrations of BA either alone (4.4–16.6 µM) or in combination with NAA (2.6 µM) for shoot differentiation.
Shoot differentiation MS basal medium alone did not induce any morphogenic response from either yellow friable or light-green compact calli and also did not promote further growth. Both types of calli degenerated slowly without any response within 4 weeks of incubation. However, the nodular structures of the light-green compact callus became more prominently dark green, and later developed into shoot buds in the media supplemented with BA either alone (4.4–16.6 µM) or in combination with NAA (2.6 µM) within 2 weeks of transfer (Fig. 2). These buds grew into long, green healthy shoots after another 2 weeks. The yellow friable callus failed to produce shoots. Variation in shoot differentiation ability of the light-green compact callus and yellow friable callus obtained from the petiole was also noticed in Viola patrinii (Sato et al. 1995). It was also observed that the shoot differentiation potential of light-green compact callus was influenced by the growth regulator treatment (Table 2). MS medium containing only BA (4.4–16.6 µM) induced shoots from light-green compact callus, but at a low frequency and with slow growth (Table 2). The addition of 2.6 µM NAA to MS media containing BA (4.4–16.6 µM) markedly improved overall shoot differentiation and shoot elongation. Optimum shoot differentiation resulted when light-green compact callus was transferred to medium supplemented with 8.8 µM BA and 2.6 µM NAA; in this medium the highest percentage of calli formed shoots (66.6 ±4.8%) and there was the highest number of shoots (8.9 ±0.3) per callus. Differentiated shoot buds elongated to 4–5 cm within 4 weeks (Fig. 3). In the second set of experiments, it was observed that the addition of casein hydrolysate (250 and 500 mg/l) to MS medium containing 8.8 µM BA and 2.6 µM NAA was beneficial for shoot differentiation from light-green compact callus with respect to mean shoot number (Table 3). Similar to the present finding, the stimulatory effect of CH on shoot differentiation has been reported in callus culture of Carica papaya (Hossain et al. 1993). Supplementing the MS medium with a higher concentration of CH (1000 mg/l) suppressed shoot differentiation from light-green compact callus. This clearly indicates a limit to the amount of CH which can be added to a medium to promote shoot differentiation, as reported by Anstis and Northcote (1973). Standard MS basal medium includes 1.25 mM phosphate, which may be suboptimal for shoot differentiation from callus. The shoot differentiation potential of light-green compact callus was increased by increasing the concentration of potassium phosphate (KH2PO4) in the medium from 0.0 mM to 1.86 mM KH2PO4. Further increase in the concentration of KH2PO4 (2.48 mM) suppressed shoot differentiation (Table 4). The highest percentage of callus forming shoots (74.8 ±4.9%) and the highest number (11.6 ±0.3) of shoots per callus were observed in medium supplemented with 1.86 mM KH2PO4. Differentiated shoot buds elongated to 5–6 cm within 4 weeks of incubation. It has been found to be beneficial to increase the phosphate concentration in the medium from 1.25 to 1.86 mM (Jones and Murashige 1974), 2.48 mM (Jakobek et al. 1986), 3.71 mM (Thorpe and Murashige 1970) or 5.0 mM (Barna and Wakhlu 1994) in various spe-
876 Fig. 1 A, B Two types of seeds-derived calli of Hybanthus enneaspermus. A Light-green compact callus, B Yellow friable callus. Bar: 1 cm Fig. 2 Shoot differentiation from light-green compact callus on 8.8 µM BA and 2.6 µM NAA 2 weeks after transfer. Bar: 1 cm
Fig. 3 Shoot differentiation from light-green compact callus on 8.8 µM BA and 2.6 µM NAA 4 weeks after transfer. Bar: 1 cm Fig. 4 Rooting of in vitro differentiated shoot on half-strength MS medium supplemented with 4.8 µM IBA. Bar: 1.5 cm
cific cases. In the present study, an increase in the KH2PO4 concentration of MS medium from 1.25 mM to 1.86 mM promoted shoot differentiation from light-green compact callus. In contrast, a twofold dilution of potassium phosphate in the MS basal salts mixture was more effective for shoot differentiation from the petiole callus of Viola patrinii (Sato et al. 1995). These results demonstrate the varying contributions of KH2PO4 concentration in the medium with respect to the differentiation of shoots in different plant spe-
cies. A continuous shoot harvest system was established at 4-week intervals and continued for 1 year without loss of vigour. Rooting Shoots failed to form roots on half-strength basal medium lacking auxin. Root formation was achieved, however,
877 Table 3 Effect of casein hydrolysate (CH) on shoot differentiation from seed-derived light-green compact callus (after 4 weeks culture)
Hardening
CH a (mg/l) a
Percentage of calli forming shoots ±SE b
Mean shoot number ±SE b
Mean shoot length (cm) ±SE b
0c 250 500 1000
66.6 ±4.8 69.3 ±4.8 69.3 ±2.9 63.8 ±2.2
8.9 ±0.3 9.8 ±0.4 10.1 ±0.6 7.9 ±0.4
4.7 ±0.1 4.7 ±0.1 4.8 ±0.1 3.8 ±0.1
About 70% of the plants were successfully established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. The present study demonstrates a simple and effective procedure for plantlet regeneration from seed-derived callus of the medicinally important herb species, H. enneaspermus, that could be extended to other species of the genus Hybanthus. This procedure is characterized by the use of mature seeds as novel explants for callus formation. Mature seeds are readily available and easily sterilized as explant source for the establishment of callus culture. The in vitro culture technique may help in the conservation of this species.
Plus 8.8 µM BA and 2.6 µM NAA Mean ±SE for three replications (12 callus pieces for each replication) c MS medium lacking casein hydrolysate served as control a
b
Table 4 Effect of potassium phosphate (KH2PO4) on shoot differentiation from seed-derived light-green compact callus (after 4 weeks culture) KH2PO4 a (mM)
Percentage of calli forming shoots ±SE b
Mean shoot number ±SE b
Mean shoot length (cm) ±SE b
0.0 c 0.62 1.25 1.86 2.48
36.0 ±2.7 66.6 ±4.8 66.6 ±4.8 74.8 ±4.8 60.9 ±2.6
3.9 ±0.1 5.4 ±0.2 8.9 ±0.3 11.6 ±0.3 7.6 ±0.3
2.3 ±4.8 3.1 ±0.1 4.7 ±0.1 5.1 ±0.1 3.3 ±0.3
Plus 500 mg/l CH, 8.8 µM BA and 2.6 µM NAA Mean ±SE for three replications (12 callus pieces for each replication) c MS medium lacking potassium phosphate served as control a
b
Table 5 Effect of IBA on rooting of in vitro-formed shoots of H. enneaspermus (after 4 weeks culture) IBA (µ M)
Percentage of rooting ±SE a
Mean root number ±SE a
Mean root length (cm) ±SE a
0.0 b 1.2 2.4 4.8 9.6
0.0 36.0 ±0.1 47.2 ±2.8 63.8 ±0.1 49.9±4.8
0.0 1.7 ±0.1 1.8 ±0.1 2.8 ±0.1 2.5 ±0.1
0.0 1.7 ±0.2 2.9 ±0.2 4.3 ±0.6 2.8 ±0.2
a b
Mean ±SE for three replications (12 cultures for each replication) Half-strength MS medium lacking auxin served as control
from the bases of differentiated shoots in the presence of IBA after 7 days of incubation. The best rooting response was obtained using 4.8 µM IBA (Table 5). In this medium, about 65% of the shoots produced an average of three roots, each 4–5 cm in length, within 4 weeks (Fig. 4). A simultaneous elongation of shoots was also achieved in this medium, both due to an increase in the length of internodes and the number of nodes. A higher concentration of IBA (9.6 µM) suppressed the rooting and caused extensive callusing at the basal portion of differentiated shoots.
Acknowledgements The authors are grateful to Dr. L. Hoffmann and Dr. J. F. Hausman for their valuable suggestions. EP and PSR are thankful to the University Grants Commission, New Delhi for financial assistance in the form of Junior Research Fellowship. Financial support provided to PSVK by the Ministère des Affaires Etrangères, du Commerce Extérieur et de la Coopération Luxembourgeois, Luxembourg in the form of a Post-Doctoral Fellowship is sincerely acknowledged.
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