Indian Journal of Biotechnology Vol 11, October 2012, pp 481-485

In vitro propagation of humilis L. through proliferation of axillary shoots and shoot tips of mature

P S C Sri Harsha, Mohammad Imtiyaj Khan, P Giridhar* and G A Ravishankar Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India

Received 6 September 2012; revised 17 January 2012; accepted 8 March 2012

In vitro propagation of Rivina humilis L. was attempted through proliferation of axillary shoots and shoot tips obtained from mature plants. The shoot tips and nodal shoot segments exhibited 70 and 80% shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with 4.44 µM 6-benzylaminopurine (BAP)+2.85 µM indole-3-acetic acid (IAA) and 8.88 µM BAP+2.68 µM naphthalene acetic acid (NAA), respectively. Shoots (2 to 3) were obtained from both shoot tip and nodal explants on MS medium supplemented with BAP (2.22 µM), IAA (2.85 µM) and silver nitrate (4 µM AgNO3). Multiplication was maximum on MS medium supplemented with BAP (8.88 µM), IAA (2.85 µM) and AgNO3 (4 µM) wherein 3-4 shoots per nodal explant were obtained. Microshoot elongation was achieved on MS medium containing BAP (2.22 µM) and gibberellic acid (2.89 µM GA3). Shoots (3-4 cm) exhibited 100% rooting within 4 wk in medium containing half MS with IAA (2.85-5.71 µM) or IBA (2.45-4.90 µM) alone or in combination with BAP (2.22 µM), resulting in simultaneous rooting and shoot growth. About 70% of micropropagated plants were established successfully in micropots and transferred to the field after 3 months. Keywords: In vitro rooting, Rivina humilis, , multiple shoots, nodal explants Introduction has been reported8 and was found to be one of the Rivina humilis L. (Family: Phytolaccaceae), most promising ways for multiplying a selected popularly known as pigeon , grows well in the variety true to its type, showing the same agronomic , tropical America and Africa1, and now characteristics with genetic similarity. Similarly, widely naturalized in Indo-Malaysia and Pacific shoot tip culture has been a powerful technique to islands. It is also found as a neo-tropical plant in the eliminate virus infection and thereby resulting in central parts of Taiwan2. Seeds are propagated production of healthy and vigorously growing through dispersal by birds. R. humilis is widely used planting material9. So far, there is no report on in vitro as an , while red berries are used for multiplication of R. humilis through shoot tip or cosmetics. Antibacterial activity of the plant has axillary shoot proliferation. Hence, we report a been reported3. The red berries, rich in betalains method for successful and rapid micropropagation of (0.3% fresh wt; 1.7% dry wt), were used by native R. humilis through shoot tip and axillary shoot Americans as a red dye. Betalains rich Rivina berry proliferation from mature (6-month-old) plants. juice was also found to be safe in rats4. Betalains are Materials and Methods accumulated in plants of only 11 families under 5 Ripened of R. humilis were collected from Order: . Betalains are functional 6-month-old plants available on the campus of the hydrophilic colourant suitable for food stuffs in pH 6 institute. The botanical confirmation of the plant was 4-6 range . In view of the rich betalains content, done7. Seeds were collected from the pulp, recently authors have characterized the pigments, nutritional components and antioxidant activity of the washed 3-4 times with distilled water and then sown 7 in pots containing garden after soaking overnight berries . In view of these developments, mass in 2, 3, 4 and 6% urea to increase permeability in seed multiplication of R. humilis is very much required for coat. Within 4-5 d of sowing, the percentage of seed sustainable production of betalains from its berries. germination obtained was recorded. The germinated Plant regeneration through axillary shoot seedlings were then transplanted into individual pots proliferation using nodal explants from mature plants for their further growth, and this could reduce the ——————— *Author for correspondence: microbial load while preparing the explants, because E-mail: [email protected]) R. humilis plants in general grow under tree shades 482 INDIAN J BIOTECHNOL, OCTOBER 2012

where high litter exists. For explants preparation, Bavistin and transferred to micropots containing sand, 6-month old healthy plant was selected and nodal and soil and compost (1:1:1) for hardening under shoot tip explants (each ~1 cm in length) were greenhouse conditions for 2 months (28±5°C & 80% prepared. The explants were washed, treated with RH) and then to the field. Bavastin WP (1%, w/v) for 15 min, mercuric chloride To initiate shoots from both shoot tip and nodal (0.15%, w/v) for 3 min and later rinsed 4-5 times with segment for each combination of PGRs, 10 explants sterile distilled water10,11. were used and the experiment was repeated twice. For shoot induction, the medium containing Similarly, for elongation of microshoots and in vitro Murashige and Skoog (MS) basal salts12, vitamins, rooting, 10 explants were used and repeated twice. 3% sucrose and supplemented with plant growth All the values obtained were expressed as mean±SE. regulators (PGRs), such as, 6-benzylaminopurine The growth parameters were recorded and the data (BAP: 2.22-8.88 µM), α-naphthalene acetic acid within each experiment was subjected to one way (NAA: 2.68-10.74 µM), kinetin (Kn: 4.65 µM) and ANOVA. The comparisons between the mean values of indole-3-acetic acid (IAA: 2.85-11.42 µM) either treatments were made by Duncan’s multiple range test. alone or in combination, was used. The medium pH was adjusted to 5.8±0.2 before gelling with gelrite Results and Discussion (0.2%, w/v). All the cultures were incubated at an Ex vitro seed germination of R. humilis was found temperature 25±2°C and 16 h photoperiod (45 µmol to be the best when the seeds were soaked overnight m–2 s–1) using fluorescent tubes (Philips India Ltd, in 4% urea and then sown in pots; wherein Mumbai, India). Similarly, in another set of 95% germination was observed in 4-5 d. The experiment, silver nitrate (AgNO3, 1-15 µM) was seedlings reached a height of 5-6 cm within 15 d. The incorporated into the MS basal medium containing seeds treated with 2, 3 and 6% urea only recorded sucrose (2%), BAP (8.88 µM) and IAA (2.85 µM), 30, 60 and 50% germination, respectively. However, and the nodal and shoot tip explants were inoculated seeds without urea treatment did not germinate onto it. In order to see the influence of different till day 5. All the germinated seedlings grew into explants, explants having 1-4 nodes were taken. healthy plants on being transplanted into individual The microshoots obtained on shoot initiation medium pots. Among these, the best plant (2-month-old) was were transferred to shoot elongation medium, wherein selected and its nodal and shoot tips were used as the combination of BAP (2.22 µM) with indole-3- explants for further studies. butyric acid (IBA: 2.45 µM), IAA (2.85 µM) or The morphogenic induction potential of different

gibberelic acid (GA3, 2.89 µM) was tried. Similarly, growth regulators in R. humilis was evident from the the microshoots were also transferred to AgNO3 results presented in Table 1. The nodal and shoot tip (1-4 µM) containing medium supplemented with IAA explants showed visible growth and most of them (2.85 µM) and BAP (8.88 µM) at 2% sucrose. grew into microshoots of 0.8-3.0 cm and 0.6-1.0 cm The number of shoots, and shoot length was length, respectively within 30 d. Shoot formation was recorded after 8 wk. The newly formed shoots from affected by the concentration of growth regulators these primary shoots were further transferred into used in the medium. Proliferation of shoots was media containing GA3 (0.72 µM) and BAP (2.22 µM) noticed with protuberant appearance from the nodal for further elongation. explants. In 3 wk, microshoots appeared prominently In vitro rooting was initiated by sub-culturing the on medium containing BAP along with IAA/NAA at in vitro shoots of ~4 cm length into ½ strength MS different concentrations (Table 1). Apart from this, medium supplemented with IAA (2.85-5.71 µM), IBA granular greenish callus mass was noticed from the (2.45-4.90 µM) or NAA (2.65-5.37 µM) alone or in base of nodal explants, particularly in combination of combination with BAP (2.22 µM). Number of roots, BAP (4.44 µM) and NAA (5.37 µM). Similar root length as well as number and size of the leaves developmental changes were also noticed in shoot tip were documented after 8 wk of culture under 16 h explants. Appearance of 1 cm long shoot with photoperiod. The rooted plants were taken out from 2-4 leaves was observed in 6-8 wk of culture in the the culture medium and washed gently with tap water medium supplemented with BAP+IAA or BAP+NAA to remove the traces of agar and nutrients. After combinations (Table 1). Among different auxin washing, the plantlets were dipped in 0.05% (w/v) (NAA, IAA) to cytokinin (BAP) ratios used for shoot HARSHA et al: IN VITRO PROPAGATION OF R. HUMILIS L. THROUGH AXILLARY SHOOTS 483

Table 1—Effect of auxin and cytokinin combination on in vitro shoot proliferation from nodal and shoot tip explants of R. humilis PGRs % response No. of shoots/explant Shoot length (µ M) (cm) BAP IAA NAA A B A B A B 2.22 2.85 0 30 0 1.0 ±0 de 0 0.85 ±0.08 d 0 4.44 2.85 0 80 0 2.6 ±0.26 a 0 2.04 ±0.08 b 0 8.88 2.85 0 60 70 1.8 ±0.24 bc 1.0 1.51 ±0.05 c 0.75 ±0.04 c 2.22 0 2.63 30 30 1.8 ±0.24 bc 1.0 0.76 ±0.06 d 0.66 ±0.06 cd 4.44 0 2.63 50 60 1.2 ±0.13 c 1.0 2.98 ±0.09 a 1.03 ±0.06 ab 8.88 0 2.63 70 70 1.4 ±0.16 c 1.0 0.86 ±0.08 d 0.59 ±0.06 d A= nodal explants; B= shoot tip explants. Values represent the mean±SE. Means followed by the same letter were not significantly different at the 0.05 level. initiation from the nodal explants, BAP (4.44 µM) separated and transferred into the same media +IAA (2.85 µM) and BAP (4.44 µM)+NAA (5.37 containing AgNO3 (4 µM), BAP (8.88 µM) and IAA µM) showed good response, in which 80 and 70% (2.85 µM) at 2% sucrose. New shoots (2.7±0.27) with nodal explants responded for shoot initiation, 3.41± 0.07 cm length were produced on either side of respectively (Table 1; Figs 1a & b). A maximum of the node with 14.1±0.67 leaves (Fig. 1g). 2.6 shoots per node with a shoot length of 2 cm and Among the different auxins tried for in vitro at least 10 leaves were recorded when medium rooting, IAA (5.71 µM) stimulated the best response, containing BAP (4.44 µM)+IAA (2.85 µM) (Fig. 1a) wherein 2.6±0.26 roots with a root length of 3.13± was employed for shoot initiation. In Kn (2.35-9.30 0.22 cm were produced. IBA (2.45 µM) also µM)+IAA (2.85-5.71 µM) combination, the response supported in vitro rooting of microshoots, wherein was poor. 2.5±0.26 roots with a root length of 1.52±0.22 cm Maximum number of shoots per explants from were obtained. Medium devoid of hormones did not shoot tip (2.6±0.2) and nodal (2.8±0.28) explants support in vitro rooting. The combination of IAA were evident after 1 wk in the presence of AgNO3 (2.85 µM)+ BAP (2.22 µM) produced 2.2±0.06 roots (4 µM) in medium containing BAP (8.88 µM), IAA with 1.58±0.11 cm root length and the number of (2.85 µM) and 2% sucrose. Medium devoid of AgNO3 leaves were found to be 7-10 with a area of ~50 could only induce single shoot in both nodal and mm2 (Fig. 1h). NAA (2.65 µM)+BAP (2.22 µM) was shoot tip explants. In the presence AgNO3, more than also able to induce rosette like appearance along with one shoot was evident irrespective of the type of in vitro roots of length (~1-1.6 cm) and 30-40 small explant and mode of inoculation. This indicated that leaves. Callus was formed in most of the treatments. presence of AgNO3 in medium triggered multiple On addition of hormones like BAP (2.22 µM) and shoot proliferation in nodal and shoot tip explants. IAA (2.85 µM) into the medium, thick and robust Shoots of 2.5 cm long were obtained (Fig. 1c) on rooting was observed within 10 d of sub-culture. medium containing BAP (2.22 µM) and IAA Upon hardening, 70% of plants survived. The plants (2.85 µM). Similarly, dwarf shoots with long petioles developed from in vitro cultures did not show any were observed on BAP (2.22 µM)+IBA (2.45 µM) variation in morphological or growth characters when supplemented medium (Fig. 1d). The microshoots compared to the mother plant. Blooming and fruit attained 2-2.5 cm length in the presence of BAP setting was also observed to be normal (Fig. 1i). (2.22 µM) and IBA (2.45 µM) in 6 wk. But, on In the present study, the response from nodal and medium containing BAP (2.22 µM)+NAA (2.65 µM), shoot tip explants of R. humilis for multiple shoot dwarf shoots with many rosette leaves were produced initiation on MS media fortified with BAP and auxins (Fig. 1e). Upon transferring into shoot elongation (IAA or NAA) was presented. BAP was considered to medium comprising GA3 (2.89 µM) and BA be one of the most useful cytokinins and when used in (2.22 µM), an increase in the shoot length up to 3-4 combination with IAA or NAA induced cm with 3-4 shoots was observed (Fig. 1f). The multiplication and micropropagation of plants11. shoots, produced from the nodal explants cultured Though Kn was also tried, the response was poor for initially on AgNO3 supplemented medium, were both nodal and shoot tip explants (data not shown) in 484 INDIAN J BIOTECHNOL, OCTOBER 2012

cultures13. It was found to be beneficial in regeneration and clonal propagation of many other economically important plants8 as well as in the somatic embryogenesis of coffee14. Earlier reports showed that silver nitrate promotes shoot formation from nodal explants in Vanilla planifolia8, Coffea sp.15 and in vitro rooting in Decalepis 16 hamiltonii , wherein upto 40 µM AgN03 supported shoot formation and in vitro rooting but, in the present study, even at 4 µM AgNO3 significant response was noticed. In conclusion, BAP (8.88 µM) and IAA

(2.85 µM) along with AgNO3 (4 µM) favoured multiple shoot induction and the combination of BAP and GA3 was found to be the best for shoot elongation. This is the first report on micropropagation of R. humilis.

Acknowledgement The authors acknowledge with thanks Department of Biotechnology, Government of India, New Delhi for providing financial assistance. MIK thanks Council of Scientific and Industrial Research, New Delhi for providing senior research fellowship.

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