Of Guggul-Commiphora Wightii (Arnott.) Bhandari

Indi an Journal Experimental Biology Vol. 41 , January 2003, pp. 69-77 Establishment of embryoni-c cultures and somatic embryogenesis in callus culture of guggul-Commiphora wightii (Arnott.) Bhandari Sandeep Kumar, S S Suri , K C Sonie & K G Ramawat* Laboratory of Bio-Molecular Techn ology, Departmcnt of Botany, M.L.Sukhadia University, Uda ipur 3 13002, India Received 5 April 2002; re vised 5 August 2002 Somati c embryogenesis in ca ll us cu ltures of COllllllipilora wigiltii (Arnotl.) Bhand ari was achieved. Though the fre quency of ex plants prod ucin g embryonic culture was low, immature zygoti c embryos were the on ly suitable explants to pro duce embryoni c callus after reciprocal transfers on media cont aining 2,4,5-tri chl orophenoxy aceti c acid (0. 1 mg 1-1) and ki netin (O. lmg 1"1 ) or devoid of growth regul ators. All other medi a fail ed to produce embryoni c ca llus. Embryo ni c cells were small , densely filled with cytoplas m and isodiametri c as compared to non-embryonic cell s, \vhi ch we re large, elongated and vacuolated. Maximum growth of embryonic callus was recorded on modified MS medium (MS-2 med ium) suppl ement ed with BA (0.25 mg 1-1) and I13 A (0. 1 mg 1-1). MS -2 sa lt s supported hi gher growth of callus as compared to ti ssues grown on 135 mcd ium containing sa me concentrati ons of pl an t growth regulators. Exogenous medium nUlri cnt s had no effect on so mati c embryo developmen t whereas plant growth regulators had lillie effecl. Asynchronously growing embryos form ed plant lets regularly whi ch we re success full y tran sferred to th e fi eld conditi ons. Commiphora wighlii (A rn ott .) Bhandari is a slow calcitrant to regenerate through so mat ic embryogene grow in g woody tree of paramount medicinal impor sis. Somatic embryogenes is offers several adva ntages tance bein g over ex pl oited for med icinal purposes. over other methods of propagati on and possib ili ty fo r Plants are now ava il ab le in protected fields. It pro obtaining gum-resin by immobi li zation of differenti vides gugg ul , an oleogum-resin, used for its properti es ated ti ssues 19. against hyperlipidemi a and hyperchol esterolemia. In the present communication we report the estab Guggulsterone-Z and guggul sterone-E, the active li shment of embryo nic call us cultures from zygotic co nstituent s of resin are responsibl e for lipid lowerin g embryos, and somatic embryogenes is in such callus t s properties in human blood - . Besides, it possesses cultures of C. wightii for its rapid micropropagation. antl-ln. n· ammatory properti.es s~ . The species is under threat as an endangered spe Materials and Methods cies because of its over exploitati on for gum-resin , Immature frui ts (5-6mm) of Comniphora lViglll ii slow growth of the plant, poor seed set and excessive collected from the pl ants growing wi ld in vi ll age tapping causing threat to the plant 7 - ~. Though the Madar near Udaipur were used to initiate ca ll us cu l pl ants can be obtained from seeds to, (apogamous seed tures. Surface sterili zatio:1, dissection and culture of formation tl) seed raised natural population is very zygoti c embryos from these fru its, medium prepara limited. The plant is mainly propagated from stem ti on and culture conditions were fo ll owed as de cuttings l2. Conventi onal methods are unable to cope scribed earli erl4. In bri ef, immature green fru its (3-4 up with th e demand of large planting material s. Mi weeks old) of C. wightii were coll ected from plants cropropagati on may be useful for large-scale produc and brought to the laboratory under ice. Fruits were ti on of elite genotypes. Several attempts have been di sinfested with 70% eth anol (v/v) for 4 min followed made in the last two decades to develop methods for by 0.1 % mercuric chl oride (55min) and rin sed several its micropropagation through in vitro techniques like times with sterili zed distilled water. Young seeds (3-4 clonal propagation 13 and somatic embryogenesis mm) containing embryos (2mm, approx im ately) were through zygotic embryosl4. In spite of significant pro di ssected from th e fruits asepticall y and transferred gress made in the regeneration of several tree species onto the medium. I S 18 over past two decades - , woody pl ants are still re- Callu s was obtain ed from zygotic embryos on B5 med ium 20 contain ing various combinations of pl ant *Correspondent author growth regul ators. Callus obta ined 0 11 the medium 70 INDIAN J EXP BIOL, JANUARY 2003 containing 2,4,5-trichlorophenoxy acetic acid (2,4,5- irrespective of salt formulation as compared to media T) and kinetin (kn) was transferred and maintained on containing indole type auxin. Use of different cyto B5 hormone free (HF) medium for 3-4 passages or kinins did not influence the growth markedly. Based reciprocally transferred on medium containing 2,4,5-T on these results, phenoxy acids were used in combina and kinetin and that devoid of plant growth regulators tion with IBA and kinetin (Table 3). Trichloro (B5-HF). Embryonic callus obtained by this method phenoxy acetic acid was more effective than di was grown on B5 or Murashige and Skoog21 medium chlorophenoxy acetic acid in relation to explant re containing BA and IBA. Various salt and plant sponse and subsequently formation of embryonic cal growth regulator combinations were used for embryo lus on transfer to hormone free (HF) medium. development, maturation and germination as de Embryonic callus was obtained from light-brown, scribed under Results. watery and soft callus, produced by zygotic embryos The plantlets produced in vitro were taken out of grown on B5 medium containing 2,4,5-T and kinetin, the culture vessels after 4-8 weeks, washed and trans on transfer to B5- HF medium. No embryonic callus ferred to thermocol pots (125 ml) containing auto could be obtained directly on B5 medium containing claved garden soil. The pots were kept covered with 2,4,5-T and kinetin. Transfer on B5-HF medium was polyethylene sheets and were irrigated as and when necessary (one to a few passages) to initiate green, required with one fourth MS salts solution. After 2 granular and compact embryonic callus as sectors on months, plantlets were transferred to field. non-embryonic callus produced by the explants (Fig. Histological studies were performed by fixing the 2). All other media containing different auxin or cyto appropriate stage cultures in formalin/acetic acid/ al kinin failed to induce embryonic callus in successive cohol (5:5:90, v/v), dehydrated using a TBA (tertiary transfer on to B5-HF medium. This is a critical step butyl alcohol) series22 and embedded in paraffin wax. (concentration and combination of 2,4,5-T and ki Material was sectioned at 7-1 OJ.l.m and stained with netin) for obtaining embryonic callus. Embryonic cal various stains (safranin, methylene blue, sudan black). lus was never produced on higher concentration of any auxin. It has been concluded from several ex Results and Discussion periments of plant growth regulators (all results not Initiation and establishment of embryonic callus presented here) that the plant growth regulators were from zygotic embryos explants and somatic embryo required in low concentrations and higher concentra genesis in callus cultures was achieved in C. wightii, a tions were either inhibitory or produced non woody medicinal tree. embryonic callus. Approximately 17 % of fruits contained zygotic Isolated embryonic callus was grown on several embryos (Fig.!). Therefore, the process of explant combinations of plant growth regulators incorporated excision and transfer became slow as well as caused with either MS or B5 medium (Table 4). The cultures about 10% contamination. Approximately 20% of grew well on MS medium containing BA (0.25 mg)"l) explant response was recorded from 6000 fruits and and IBA (0.1 mgrl) and retained their colour, texture 900 zygotic embryos. Embryonic callus was obtained and growth characteristics, however on other combi in less than 1 % of explants (Table 1). nations (Table 4) of growth regulators, callus lost A large number of salt formulations with different these characteristics and became non-embryonic and combinations of growth regulators resulted in about fast growing. Optimal growth of embryonic callus 15-20 % zygotic exp!ants producing embryogenic was recorded on the medium supplemented with callus. Optimum response was recorded on B5 me BA(0.25 mgl-I) and IBA (O.! mg}-I). On B5 medium dium containing 2,4,5-T, kinetin and IBA (Table 2). embryonic callus grew slowly. Therefore, cultures Callus grew fast on phenoxy acid containing media, were maintained only on MS medium containing low Table I-Score of zygotic embryos and embryogenic callus Period Location No. of No. of fruits No. of Empty Explants contami- Response Cultures producing visits di ssected zygotic fruits nated (%) embryogcnic embryos (%) (%) (%) callus (%) I Year Different 47 6138 880 (14.3) 85.7 13.9 Only 19.2 form Less th an 1.0 20-50 km callus II Year Different 30 5600 1100 (19.64) 80.36 10.0 20 Less than 1.0 20-50 km KUMAR el at.: SOMATIC EMBRYOGENESIS IN COMMIPHORA CALLUS CULTURES 71 concentration of BA and IBA to improve the growth followed by that on MS-2 and MS medium, however, of embryonic callus, without altering the embryonic optimum growth of embryonic callus was recorded on , characteristics.

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