In vitro flowering and rapid propagation of Vitex negundo L. A medicinal plant

Transcription

1 Indian Journal of Biotechnology Vol 5, January 2006, pp In vitro flowering and rapid propagation of Vitex negundo L. A medicinal plant A V Vadawale 1*, D M Barve and 2 A M Dave 1 1 GSFC Science Foundation, Vigyan Bhavan and 2 A D & A S Department, GSFC Ltd, Fertilizernagar, Vadodara , India Received 13 July 2004; revised 23 February 2005; accepted 3 March 2005 In vitro flowering and an efficient micropropagation protocol was developed for the woody, aromatic and medicinal shrub Vitex negundo (Verbenaceae) by the in vitro culture of nodal segments of mature plant. Murashige and Skoog s (MS) medium supplemented with 4.4 μm N 6 -benzylaminopurine (BAP) and 0.53 μm α-naphthaleneacetic acid (NAA) were found to develop fully functional flowers. MS medium supplemented with 4.4 μm BAP and 0.53 μm NAA induced an average of five shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. Full strength MS solid medium with 3.69 μm indole-3-butyric acid (IBA) exhibited the best in vitro rooting. Ninety per cent of the rooted shoots survived when transferred to green house and subsequently to the field. Keywords: inflorescence, medicinal plant, micropropagation, Verbenaceae, Vitex negundo IPC Code: Int. Cl. 7 A01H4/00, 5/02 Introduction Vitex negundo L. (Family: Verbanaceae) is a woody, aromatic and medicinal shrub or a small tree. It is one of the common plants used in Indian system of medicine. Various parts of the plant are used in the treatment of bronchitis, asthama and gastric troubles. The leaves are reported to possess insecticidal properties 1. The extract of leaves showed anticancerous activities against Echrlich ascites tumour cells 2. V. negundo is also known for reclamation of degraded soil to prevent soil erosion 3. Conventional propagation of V. negundo through vegetative cuttings is very slow and a large number of cuttings do not survive during the transportation. It can also be propagated through seeds or root suckers, but these methods are also not reliable because of the poor viability of the seed and production of root sucker is strictly age dependant 4. There are very few reports on the micropropagation of V. negundo. Sahoo and Chand 4 reported callus free multiple shoot formation from axillary meristem explants in MS medium with BAP (2 mg L -1 ) and GA 3 (0.4 mg L -1 ). The shoot multiplication from nodal explant has also been achieved by Thiruvengadam and Narayansamipillai 3 in MS medium with BAP (1.5 mg l -1 ) and NAA (0.1 mg l -1 ). *Author for correspondence: Tel: ; Fax: ashutoshv@rediffmail.com The present paper reports an efficient micropropagation system for generating a large number of plants through high frequency axillary shoot multiplication and in vitro flowering in V. negundo. Materials and Methods Plant Materials Healthy nodal explants with dormant axillary buds were collected from the mature plants of V. negundo grown in the medicinal plant garden of Fertilizernagar, Vadodara. After removing leaves, the explants were washed under running tap water for 45 min, followed by a wash with a solution of detergent Extran (5%, v/v) for 10 min, treated with an antifungal agent Bavistin (1%, w/v) for 20 min and finally washed thoroughly in sterile double distilled water. They were then surface sterilized in mercuric chloride solution (0.05%, w/v) for 4 min and, following thorough washings in sterile double distilled water, the materials were cut in to appropriate sizes ( cm) and cultured on sterile medium. Culture Medium and Conditions Basal medium used for initial set of experiments for shoot proliferation consisted of Murashige & Skoog s salts 5, vitamins 30 g L -1, sucrose 8 g L -1 agar (Qualigens, India). The ph of the medium was adjusted to 5.9 before autoclaving at a pressure of

2 VADAWALE et al: RAPID PROPAGATION OF V. NEGUNDO kg cm -2. The basal medium was supplemented with various cytokinin [BAP (N 6 -benzyl aminopurine) & Kn (Kinetin)] and auxins [NAA (α-naphthalene acetic acid) & IBA (indole-3-butyric acid)] at different concentrations and combinations. The cultures were incubated at 25± o C under a 14/10 h (light/dark) photoperiod with light supplied by white fluorescent tubes at 3,500 Lux. Rooting, Acclimatization and Field Transfer The induction of roots was carried out on full strength MS solid medium supplemented with auxins (NAA & IBA) at different concentrations. Healthy shoots with well developed roots were transferred to small pots containing vermicompost and cocopit (1:1) at maintained humidity (~ 90%) through misting device inside a greenhouse. Experimental Design, Data Collection and Analysis Experiments were set up in completely randomized design. Ten cultures were raised for each treatment and all experiments were conducted thrice. Data were collected on number of shoots, shoot length, number of roots, root morphology, number of flower buds and developed flower, etc. Results and Discussion Axillary Bud Multiplication MS medium supplemented with different concentrations of BAP/Kn in combination with NAA resulted in initiation of axillary buds in explants (Table 1). The bud break was achieved on MS medium supplemented with 2.20 μm BAP within 10 d of culture. The explants cultured on MS medium without supplement of growth regulator did not grow at all. BAP was found to be better than Kn with respect to multiplication. Nodal explants induced an average of 4 shoots on MS basal medium supplemented with BAP (Table 1; Fig. 1a). All the shoots developed from single axillary bud attained a maximum length of 10 cm with 4 nodes and 8 compound leaves within 30 d. The cultures were repeatedly sub-cultured every 4 week. MS medium with BAP (> 3.3 μm) in combination with NAA showed the strongest effect with respect to the multiplication of axillary buds. Single nodal explant induced an average of 25 shoots in the presence of 4.4 μm BAP and 0.53 μm NAA within 4 subcultures. The present study exemplifies the positive modification of shoot induction efficacy by lower concentrations of an auxin in combination with cytokinin. Excision and culture of the axillary buds from the in vitro derived shoots facilitated the development of increased number of shoots (Fig. 1a). The shoot multiplication at an enhanced pace was achieved by subsequent subcultures. Shoots developed through axillary bud multiplication exhibited abscission of leaves and shoot tips after 30 d of culture. However, just below the point of abscission of the shoot tip, axillary buds got initiated and resumed the growth in the same medium. In Vitro Flowering The fully developed shoots from the nodal explant when isolated and transferred to fresh MS medium having different concentration of cytokinin (BAP/Kn) in combination with auxin (NAA) started developing inflorescence at the shoot apex but none of the flower buds developed into flower in the same medium. Effect of C/N Ratio When plantlets with inflorescence were transferred to the same media but with increased C/N ratio, the Table 1 Efficacy of MS medium fortified with different growth regulators on axillary bud multiplication of V. negundo after 4 weeks of culture. Treatment % response Mean no. of shoots/node Growth regulator free medium - - BAP (2.22 μm) ± 0.3 BAP (1.11 μm) ± 0.5 BAP (2.22 μm) ± 0.8 BAP (4.44 μm) ± 0.7 Kn (1.16 μm) ± 0.3 Kn (2.32 μm) ± 0.7 Kn (4.64 μm) ± 0.8 BAP (2.2 μm) + Kn (1.16 μm) ± 0.8 BAP (2.2 μm) + Kn (2.32 μm) ± 0.1 BAP (2.2 μm) + Kn (4.64 μm) ± 0.9 Values represents means ± standard deviation of 10 replicates per treatment in three repeated experiments

3 INDIAN J BIOTECHNOL, JANUARY 2006 Fig. I-(a-d) In vitro flowering and rapid propagation of V. negurado. (a) Axillary bud multiplication on MS pm BAP pm NAA. (b-c) In vitro flowering: (b) Explant cultured on MS pm BAP pm NAA mm sucrose, showing fully developed flower; (c) Culture showing multiplication, rooting and synchronized flowering. (d) Pollens of in vitro developed flower (14000 x). (e-f) Hardened regenerated plants maintained in greenhouse: (e) An in vitro raised plantlet acclimatized in potting mixture (1: 1, vermicompost + cocopit), (f) Hardened plants maintained in greenhouse. buds get transformed into the fully developed flower Table 2-Effect of C/N ratio on in vitro flower development of V. after 7-8 d. The increased C/N ratio was achieved by negundo in MS medium increasing the sucrose concentration and maintaining Sucrose (mm) C/N ratio % of explants Av. no. of days the concentration of NH4N03 at rnm (Table 2). in MSa producing fully required for flowers Eighty percent of cultures with inflorescence medium developed flowers to fully develop - developed fully functional flower in MS medium having C/N ratio of 7.08 and supplemented with pl4 BAP and 0.53 pm NAA (Table 2; Fig. lb). The CIN ratio plays a critical role in the transition of "Ms medium supplemented with BAP (4.4 CUM) and NAA '(0.53 vegetative parts to the flowering state637. Generally, in PJ4) Values represents means + standard deviation of 10 replicates per vitro culture with a high C/PJ ratio promotes plant,a,nt in three repeated experiments reproductive development, while a high N/C ratio promotes plant vegetative development83g. Wang et germination to root, shoot and flower formation". a1" also achieved the ir, vitr~ flowering in bitter The effects of the cytokinins (BAP and Kn) and the melon by increasing the CiN ratio. auxin (NAA) on flower induction were examined Effect of Plant Growth Regulators on Flower Induction (Table 3). MS medium containing BAP (4.4 CLM) and Plant growth regulators influence many diverse NAA (0.53 pa4) resulted in uniform and synchronized developmental processes ranging from seed flowering with 4-5 flowers per plant (Fig. lc). The

4 VADAWALE et al: RAPID PROPAGATION OF V. NEGUNDO 115 combination of BAP and NAA promoted flower bud formation has also been reported by Chang and Chang 12 in Cymbidium ensifolium. The flower buds induced on the MS medium supplemented with different concentration of BAP developed into functional flower, while those flower buds induced on the MS medium supplemented with different concentration of Kn did not developed into functional flower (Table 3). The inductive effect of BAP on flowering have also been demonstrated in vitro and described for several orchids 13. Flower, cm in length, opened up fully showing the mature male reproductive organs, i.e. anthers with long filaments, and female reproductive organs, i.e. swollen ovary at base, long style and bifid stigma. Pollens, obtained by rupturing the anthers of the in vitro developed flower, were studied microscopically. The pollens were observed to be in isolated condition (Figure 1d). Viability of Pollen The viability of pollen was examined, 6 h after flower bloom, by the germination of pollen on the Table 3 Effect of plant growth regulators on in vitro flowering of V. negundo in MS medium after 3 weeks of culture Treatment % of explants producing inflorescence Mean no. of fully developed flower per culture MS Plain 10 0 BAP (1.10 μm) ± 0.8 BAP (2.20 μm) ± 0.6 BAP (4.4 μm) ± 0.5 Kn (1.16 μm) Kn (2.32 μm) Kn (4.64 μm) Values represents means ± standard deviation of 10 replicates per treatment in three repeated experiments pollen germination medium according to procedure of Adhikari 14. About 70% of the pollen examined germinated in the pollen germination medium (data is not shown). Effect of IBA and NAA on In Vitro Rooting IBA was found to be more suitable for root induction compared to NAA (Table 4). When shoots were transferred to the rooting medium supplemented with NAA, the basal ends first developed callus within d of incubation and then few thick, stout and shorter roots were formed from the callus. The regenerated shoots, obtained from the axillary buds, rooted best on MS medium containing 3.69 μm IBA. The roots were thin, slender and with lots of root hairs in the medium with IBA. After d incubation in the rooting medium, each shoot developed an average of 7 roots (Table 4; Fig. 1c), with an average length of cm. According to Sahoo & Chand 4, IBA (1.0 mg L -1 ) alone or in combination with IAA (1.0 mg L - 1 ) was most effective for rooting in the same species. The effectiveness of IBA in rooting has also been reported in other medicinal plants, like Hemidesmus indicus 15, Withania somnifera 16 and Aloe polyphylla 17. The slow movement and slow degradation of IBA facilitates its localization near the site of application and, thus, it functions better in inducing roots 18. Rooted shoots were transferred directly to small pots filled with vermicompost and cocopit (1:1). The growth of plants revived after 15 d of transplantation (Figs 1e & f). Of the plantlets transferred to soil, 90% survived and exhibited morphological characters similar to those of the source plant. Conclusion A simple and efficient method has been developed for inducing the in vitro flowering and ion of plants in micro propagated plants of V. negundo. Plant flowering transition is a complex process, which is controlled by various factors The key factors in our experiments that increased in vitro flowering were Table 4 Effect of auxins on root induction from in vitro raised shoots of V. negundo after 4 weeks of culture Treatment Mean no. of roots/shoot Mean root length (cm) Root morphology MS + IBA (1.23 μm) 5.33± ±1.05 Thin, long and fragile MS + IBA (2.46 μm) 7.33± ±0.97 Long sturdy MS + IBA (3.69 μm) 7.44± ±1.67 Long sturdy MS + NAA (1.32 μm) 7.22± ±0.83 Thick, short with root hairs MS + NAA (2.64 μm) 4.89± ±0.87 Thick, short with root hairs MS + NAA (3.96 μm) 4.22± ±1.05 Thick, short and callus at base Values represents means ± standard deviation of 10 replicates per treatment in three repeated experiments

5 116 INDIAN J BIOTECHNOL, JANUARY 2006 C/N ratio and concentration of a particular cytokinin, BAP. The findings of the study could be helpful in understanding the detailed physiology of flowering. The protocol described here for the micropropagation of V. negundo through axillary bud multiplication would also facilitate the rapid propagation of this valuable medicinal plant. References 1 Wlth India, The wealth of India: A dictionary of raw materials and industrial products Raw material series, vol 10 (CSIR, New Delhi) 1976, Ambasta S P, The useful plants of India (Publications and Information Directorate, CSIR, New Delhi) Thiruvengadam M & Narayansamipillai J, Mass propagation of Vitex negundo L. in vitro, J Plant Biotechnol, 2 (2000) Sahoo Y & Chand P K, Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through highfrequency axillary shoot proliferation, Plant Cell Rep, 18(1998) Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) Kachonpadungkitti Y, Romchatngoen S, Hasegawa K & Hisajima S, Efficient flower induction from cultured buckwheat (Fagopyrum esculentum L.) node segments in vitro, Plant Growth Regul, 35 (2001) Tanimoto S & Harada H, Chemical factors controlling flower bud formation of Torenia stem segments cultured in vitro: Effects of mineral nutrients and sugars, Plant Cell Physiol, 22 (1981) Konar N R & Nataraja K, In vitro control of floral morphogenesis in Ranunculus sceleratus L., Phytomorphology, 14 (1964) Nitsch C & Nitsch J P, The induction flowering in vitro in stem segments of Plumbago indica II. The production of reproductive buds, Planta, 72 (1967) Wang S, Tang L & Chen F, In vitro flowering of bitter melon, Plant Cell Rep, 20 (2001) McCourt P, Genetic analysis of hormone signaling, Annu Rev Plant Physiol Plant Mol Biol, 50 (1999) Chang C & Chang W-C, Cytokinins promotion of flowering in Cymbidium ensifolium var. misericors in vitro, Plant Growth Regul, 39 (2003) Duan J X & Yazawa S, Induction of floral development Doriela tiny (Doritis pulcherrima Kingiella philippinensis), Sci Hortic (Amst), 59 (1994) Adhikari K N & Campbell C G, In vitro germination and viability of buckwheat (Fagopyrum esculentum Moench.) pollen, Euphytica, 102(1998) Sreekumar S, Seeni S & Pushpangadan P, Micropropagation of Hemidesmus indicus for cultivation and production of 2- hydroxy 4-methoxy benzaldehyde, Plant Cell Tissue Organ Cult, 62 (2000) Vdawale A V, Mehta-bhatt P and Dave A M, Rapid in vitro propagation of Ashwagandha (Withania somnifera) through axillary bud multiplication and indirect organogenesis, Phytomorphology, 54 (2004) Abri A L & van Staden J, Micropropagation of the endangered Aloe polyphylla, Plant Growth Regul, 33 (2001) Nickell G L, Kirk-Othmer encyclopaedia of chemical technology, vol 18 (John Wiley and Sons Inc., New York) 1982, Bernier G, The control of floral evocation and morphogenesis, Annu Rev Plant Physiol Plant Mol Biol, 39 (1988) Weigel D, The genetics of flower development: from floral induction to ovule morphogenesis, Annu Rev Genet, 29 (1995) Weller J T, Reid J B, Tayler S A & Murfet I C, The genetic control of flowering in pea, Trends Plant Sci, 2 (1997)