Journal of Environmental Research And Development

J. Environ. Res. Develop. Vol. 9 No. 3A, January-March 2015

In vitro MICRO PROPAGATION : A TOOL FOR CONSERVATION OF ENDANGERED AND MEDICIN -ALLY IMPORTANT FOREST TREES Tiwari P. Department of Botany, Plant, Tissue Culture Lab, Shivaji Science College, Nagpur (INDIA) Received September 14, 2014

Accepted February 21, 2015

ABSTRACT Ecological degradation and its corollary biodiversity loss pose a serious threat to development. Deforestation has resulted in serious damage to biodiversity and gene resources. Medicinal plants play a vital role in the modern economy and India is probably the richest, oldest and most diverse cultural tradition in utilizing medicinal plants. Oroxylum indicum, a popular medicinal tree species in Asia, belongs to family Bignoniaceae, commonly known as shyonal, sopatha is an important ingredient in Ayurvedic preparation called Dasamoola. Due to its medicinal value, indiscriminate exploitation for medicinal purpose coupled with low regeneration and habitat destruction have posed serious threat to its survival and pushed it into list of endangered species in some areas. So the conservation, rapid multiplication and rehabilitation of this species have become immediate need of the country. To overcome this threat the present study has been made to set proper protocol through Plant tissue culture technique using hypocotyl explants of Oroxylum indicum for shoot regeneration. Effect of PG’s (Plant Growth hormones) like BAP, NAA and IBA in different conc. and combinations on shoot regeneration was studied. In the present study 5 mg/l and 2.5 mg/l BAP alone and 5 mg/l BAP in combination with 2.5 and 1.0 mg/l NAA was found to be suitable combination for multiple shoot regeneration. Hypocotyl proved to be efficient for multiple shooting. 3% sucrose in MS medium was used. The observation was taken after 20-25 days of incubation. Regenerated shoots were subcultured onto the medium supplemented with different concs. of IBA for rooting. Rooted shoots were successfully grown in pots after acclimatization using vermiculite irrigated with ½ MS salt solution.

Key Words : Oroxylum indicum, Multiplication, Conservation, Hypocotyl, Plant growth hormones

INTRODUCTION Ecological degradation and its corollary biodiversity loss pose a serious threat to development. Due to deforestation serious damage has resulted to biodiversity and gene resources. Excessive collection, reclaiming or clearing of natural environment, deforestation are the main cause of extinction of economically important plant and its germplasm.1 India is endowed with rich and diverse forest resources. Medicinal plants play a vital role in the modern economy and India is probably the richest, oldest and most diverse cultural tradition in utilizing medicinal plants. The forest tree species Oroxylum indicum L. Kurz belonging to family Bignoniaceae is an economically and medicinally important tree found in deciduous forests of India popularly

known as broken bones tree, midnight horror tree , sonapatha, syonaka or shivanac by local tribes, villagers and herbalists. All the plant parts such as leaves, seeds, bark of stem and root equally possess medicinal values and hence used in folk , herbal and in ayurvedic medicine. The extracts are also used in the treatment of leprosy, snake bite, cancer, rheumatism, allergy, microbial infections, anorexia, leucoderma, bronchitis and as analgesic.2 The plant is pollinated by bats (Chirepterophily). Due to decline in Bat population that pollinate these species the survival of the species O. indicum is indirectly affected. Poor pollination leads to low seed set. Seed viability is also low (30%).3 The destruction caused by harvesting roots as a major source of drug, low seed viability, low rate of germination and slow rate

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J. Environ. Res. Develop. Journal of Environmental Research And Development Vol. 9 No. 3A, January-March 2015 of propagation has restricted the natural surface sterilised with 0.1% HgCl2 for 1-2 min populations of this tree. Due to its demand in then rinsed with sterilised distilled water. pharmaceutical companies, over exploitation for Explants were then inoculated onto the MS medicinal purpose.4 Destructive and non- medium supplemented with various conc. of sustainable collection method, habitat auxin NAA, IBA and cytokinin BAP.12 Cultures destruction, over grazing, deforestation and fire 5 were incubated at 24±2 °C under 6-8 hrs has created serious threat to the survival and photoperiods. Cultures were sub-cultured onto availability of this highly useful tree and pushed the same fresh media after 2 weeks. Plantlets this medicinally valuable O. indicum into the list with well developed shoots and roots were of Indian red data book and endangered plant transferred to plastic pots containing vermiculite, species of India.6 The existence of O. indicum in covered with transparent polythene and irrigated natural population is highly threatened and has with 1/2 MS salt solution. After acclimatization been categorized under vulnerable by the plantlets were transferred to pots containing government of India.7 Hence it is the need of normal soil. The data was collected after 2-3 hour to re establish the glory of this valuable weeks. The experiment was repeated thrice. The asset amongst all concerned and develop an results were analysed statistically and standard efficient tool for rehabilitation, mass-scale rapid error and mean was calculated. multiplication and conservation of O. indicum L. RESULTS AND DISCUSSION an endangered, economically and medicinally important tree. In vitro regeneration of this tree The organogenesis and morphogenetic response has been reported.8 Cultivation of medicinal of Hypocotyl explants to various conc. and plants is difficult due to lack of standardized combination of cytokinin (BAP) alone and in agronomic practices. To minimize the problems combination with auxins (NAA and IBA) are of their conservation and increasing interest has summarised in (Table 1 and Table 2) and been forced to use an alternative method plant (Fig. 1). Hormone free MS media was also used tissue culture technology for rapid propagation for culture but no response was reported. Out of and conservation of economically important various conc. (0.1-10 mg/l BAP) of BAP alone, plants.9 In vitro technique is a complementary moderate conc. of BAP i.e. 2.5 and 5.0 mg/l method for conservation of germplasm of various proved to be highly effective for shoot bud vegetatively propagated and recalcitrant seed formation i.e. 87% and 92% respectively. Percentage of shoot bud formation increased species.10 gradually from low to moderate conc. (0.1-5 AIMS AND OBJECTIVES mg/l) of BAP alone (i.e. 80-92%) but at higher The main purpose of the present investigation conc. of BAP (10 mg/l) shoot bud formation was is to set proper protocol for in vitro micro significantly reduced i.e. 56%. Adverse effect of propagation using hypocotyl explants of high conc. of BAP on shoot bud formation was Oroxylum indicum. Development of micro also reported by several workers in past in propagation protocol is a prerequisite for any Ophiorhiza prostrata ,13, Musa accuminata14 and successful in vitro conservation.11 Hence the Zingiber officinale.15 Various conc. of auxin present investigation is an attempt to conserve NAA alone (0.1-10 mg/l) was also tested for the the species via in vitro micro propagation shoot regeneration. Low to moderate (0.1-2.5 through plant tissue culture technique using mg/l) conc. of NAA also showed efficiency hypocotyl explants of Oroxylum indicum. towards shoot but formation i.e. 78%, 83% and 91% respectively. Low to moderate (0.1-2.5 MATERIAL AND METHODS mg/l) NAA when added to (2.5-5 mg/l) BAP Seeds of the plant were collected from local significant increase in the shoot formation i.e. medicinal plant agency and germinated 97% with 5 mg/l BAP +1 mg/l NAA and 94% aseptically on moist cotton and filter paper. 15 with 2.5 mg/l BAP + 2.5 mg/l NAA was days old seedlings were taken for explants observed. (a), (b), (c) : Multiple shoot buds and preparation. Hypocotyl explants was exercised shoot initiation in MS media with 5mg/l BAP from young seedlings. Excised explants were and1mg/L NAA (d) (e) : Shoot elongation and

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J. Environ. Res. Develop. Journal of Environmental Research And Development Vol. 9 No. 3A, January-March 2015 root initiation in MS media with 2.5 mg/L BAP shoot in MS media with 2.5 mg/l IBA alone. (h), and 5mg/lIBA. (f), (g) Rooting in regenerated (i) Hardened plant after proper acclimatization.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

Fig. 1 : Different stages of in-vitro micropropagation of Oroxylum indicum (L) vent. Increase in shoot formation by the addition of NAA i.e. the synergistic effect of auxin and cytokinin was also observed in several other plants viz. Rawvolfia tetraphyla16, Rotula aquatica.17 The present study also supports the shoot induction capacity of moderate conc. BAP with low conc. of NAA in combination as shown in Table 1. At the same time, it was also found that higher conc. of NAA when used alone declined the % frequency of shooting per explants. Subsequent sub-culture on the same medium also enhanced the shoot formation.

Cultures with good shooting were transferred to different conc. (0.5-5 mg/l) of IAA, IBA, alone. Out of the 2 auxins used IBA (5 mg/l and 2.5 mg/l) was found to be most effective for rooting response (95% and 93%). Efficiency of IBA over other auxin IAA for rooting was also observed in Accacia nangium18, Dalbergia sisso19 and Oroxylum indicum. 20-28 Plantlets thus obtained with well developed root and fully expanded leaf were introduced to growth chamber for hardening process. Plantlets were transferred to plastic pots containing vermiculite irrigated with ½

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J. Environ. Res. Develop. Journal of Environmental Research And Development Vol. 9 No. 3A, January-March 2015 strength MS salt solution for 2 weeks. shifted to natural soil condition. 85% of the Gradually the acclimatised plantlets were total micro propagated plants survived.

Table 1 : Response of growth hormones on shoot bud formation using hypocotyl explant S/N

Growth hormone

1

BAP

2

NAA

3

BAP+NAA

4

BAP+NAA

Conc. of growth hormone ( mg/l) 0.1 1.0 2.5 5.0 10 0.1 1.0 2.5 5.0 10 2.5 BAP +0.5NAA 2.5 BAP +1.0NAA 2.5 BAP +2.5NAA 2.5 BAP +5.0NAA 2.5 BAP + 10NAA 5 BAP + 0.5 NAA 5 BAP + 1.0 NAA 5 BAP + 2.5 NAA 5 BAP + 5.0 NAA 5 BAP + 10 NAA

% Explant producing shoot

Mean ± SE

80 82 87 92 56 78 83 91 74 64 89 91 94 80 61 93 97 94 84 76

7.5±0.25 8.2±0.22 8.8±0.21 9.25±0.2 6.0±0.25 8.0±0.35 8.3±0.216 8.8±0.41 7.75±0.22 6.5±0.25 9.0±0.35 9.25±0.41 9.5±0.43 7.8±0.37 6.2±0.21 9.25±0.22 9.7±0.216 9.5±0.25 8.2±0.41 7.8±0.42

Table 2 : Rooting response in shoots produced S/N 1

2

Growth regulators (mg/l) 0.5 IAA 1.0 IAA 2.5 IAA 5 IAA .5 IBA 1 IBA 2.5 IBA 5 IBA

% Shoots producing roots

Mean ± SE

14 20 22 38 61 91 93 84

2.6 ± 0.36 3.6 ± 0.35 3.6 ± 0.35 6.6 ± 0.16 6.2 ± 0.21 8.8 ± 0.41 9.25 ± 0.22 8.2 ± 0.41

CONCLUSION The present work explains efficient method for In-vitro propagation and rapid multiplication of O. indicum, a potential medicinal forest tree. MS media augmented with 5 mg/l BAP +1 mg/l NAA and 2.5 mg/l BAP + 2.5 mg/l NAA proved to be effective for shoot regeneration and IBA (5 mg/l and 2.5 mg/l) was found to bemost effective for rooting of regenerated shoot. The shoot regeneration is the synergistic

effect of different concs. and combinations of auxin and cytokinin. The protocol used in the study is reproducible and can be applied for large scale multiplication, rehabilitation and conservation of O.indicum, an endangered medicinal forest tree as the conservation of this species has become immediate need of the country. The study also opens up the way to upgrade the research for mass production and gradually the production of bioactive compounds with in-vitro culture of O.indicum.

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