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- 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, 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 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 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 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 -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. Suitably modified B5, BTM24 and MS MS-2 medium followed by that on MS medium. medium with low nitrogen and calcium level (50mgN/I Embryonic callus transferred on MS-2 medium

of (NH4) 2 S04 and 200 mgN/I KN03, CaCI2 220mg/l; containing various combinations of IAA and BA pro- referred to as MS-2) and several combinations of IBA and BA were used (Table 5). MS-2 medium was bet­ Table 3 - Per cent explant response for production of embryonic ter in supporting growth as compared to B5 salt for­ callus in [B5 medium supplemented with different combination of mulation. Maximum growth of embryonic tissue was plant growth regulators (pGR) recorded on MS-2 medium supplemented with IBA Concentration of PGR (mgl") Explant Embryonic I (0.1 mg!"l) and BA (0.25 mgl· ). 2,4,5-T+ Kinetin + 2,4-0 + IBA producing callus The data on growth, cell size and callus texture of callus (%) (%) non-embryonic and embryonic callus are given in Table 5. It is evident from the data that embryonic 0.1 0.5 40 0.5 0.1 0.25 70 20 cells were small as compared to non-embryonic cells, 0.25 1.0 0.25 30 irrespective of the medium salts. Maximum growth of 1.0 0.25 0.25 30 non-embryonic callus was recorded on BTM medium 1.0 0.1 60 5

Table 2 - Effect of different growth regulators (PGR):. and salt strength of callus formation from zygotic embryos Medium + PGR (g I") No. of zygotic embryos % response and texture transferred B5+IBA(O .1 )+Kn (0. 1) 45 No callus B5+IBA(O. 1)+ Kn (0.25) 50 20,sofl brown callus B5+IBA(O.25)+Kn (0. 1) 44 20,soft white callus, slow growing B5+IBA(0.25)+Kn (0.25) 40 10, slow growing white callus B5+1 BA (O .I )+Picloram (0. 1) 30 20, slow growin g, brown callus B5 * +1 BA(0.50)+Picloram (0. 1) 30 B5+IBA(0. 1)+Picloram (0.25) 30 25, slow growing, brown callus B5+2,4-0(0.05)+2,4,5-T (0.05) + 25 30, brown soft callus IBA(O. I )+BA (0.20) B5+2,4-0(0.05)+2,4,5-T (0) + IBA(O.I )+BA 25 28, brown soft callus (0.1 ) B5+2,4-0(0. 1)+2,4,5-T (0.05) +IBA (0.2) +BA 25 37, white slow growing callus (0.25) B5+2,4-0(0.5)+Kn (0.1) +IBA(0.25) 40 50, white fast growing soft callus MS+2,4-0(0.5)+Kn (0.1) +IBA(0.25) 40 10, slow growing brown callus B5+2,4-T(0.5)+Kn (0. 1) +IBA(0.25) 40 70, fas t growing white callus .-..A MS+2,4-T(0.5)+Kn (0.1) +IBA(0.25) 40 10, slow growin g brown callus B5+2,4-0( 1.0)+Kn (0.25) +IBA(0.25) 30 40, fast growing white callus MS+2,4-0( 1.0)+Kn (0.25) +IBA(0.25) 30 15, slow growing brcwn callus B5+2,4-T( 1.0)+Kn (0.25) +IBA(0.25) 30 65, fa st growing soft browni sh white callus MS+ 2,4,5-T(1.0)+Kn (0.25) +IBA(0.25) 30 20, slow growing soft brown callus B5+2,4,5-T (0.5)+TOZ (0.1) 35 20, slow growing brown callus B5+2,4,5-T (0.5)+TOZ (0.0 I) 35 15, slow growing brown callus 85+2,4,5-T (0.5)+ TOZ (0.2) 35 10, slow growi ng brown callus B5+2,4,5-T (O.2)+II3A (0.2)+ TOZ (0. 1) 35 20, slow growing brownish white callus 13S+113A (O.25)+Kn(0.OS) 20 22, slow growing white callus 135 * +IBA (0.2S)+Kn(0.05) 20 20, slow growin g white callus MS+IBA (0.25)+Kn(0.05) 20 14, slow growing brown callus 135+ 2i P(O.05)+1 BA(O.2S) 20 10, slow growing compact white callus B5 3.4 +2iP(0.05)+IBA(0.25) 20 8, slow growing compact white callus MS+2iP(0.05)+IBA(0.25) 20 6, slow growing compact white callus B5+2iP(O.5)+IBA(0.25) 25 15, slow growing white callus B5 * +2iP(0.5)+IBA(0.25) 25 10, slow growing while callus MS+2iP(0.5)+IBA(0.25) 25 14, slow growing white callus B5+(NH4h S04 (-)+2,4,5-T( 1.0) +Kn(O.I) 35 18, fast growin g white callus B5 * +(NH4hS04 (-)+2,4,5-T( 1.0) +Kn(O.I) 30 22, fast growing while callus 72 INDIAN J EXP BIOL, JANUARY 2003 duced globular, torpedo and a few early cytoledonary binata somatic embryos'6. Differentiation of vascular stage embryos (Table 6; Figs 3,4). Maximum number region along with resin duct was observed only in of somatic embryos was observed on the medium torpedo shaped embryos. Torpedo stage embryos • containing IAA (0.1 mgr') and BA (0.25 mgr'). showed bilateral symmetry with two distinct cotyle­ This increase was due to high number of globular em­ dons in longitudinal sections (Fig. 5c). bryos responding this medium. Somatic embryos for­ Torpedo and cotyledonary stage somatic embryos mation from this callus was spontaneous and asyn­ obtained from experiments for development of so­ chronous. Small cotyledons were visible in torpedo matic embryos of previous observations (Fig. 4) were shaped elongated embryos having well differentiated used. Activated charcoal, ABA, and agar-agar were epidermis. With the enlargement of cotyledons during incorporated in MS-2 medium to generate stress and the development of somatic embryos, root apex was enhance maturation in somatic embryos. During this clear. A few fasciated embryos were also observed in process, it was expected to arrest the development and the cultures. growth of somatic embryos including cleavage em­ Histological studies further supported the conclu­ bryogenesis, secondary somatic embryos and globular sions derived on the basis of morphological observa­ embryos. The results obtair.cd with these experiments tions. Globular to cotyledonary stage somatic em­ showed that maximum number of cotyledonary stage bryos were produced in the cultures. Bipolar nature embryos were produced on the medium containing and separation from surrounding tissues was evident activated charcoal (0.5 gr') and sucrose (10 gl" ) in the sections (Fig. Sa-c). Outer epidermis like layer (Table 7). In this experiment embryos were placed on 25 is visible but is not markedly distinct . Similar type filter paper-bridge lIsing liquid medium (Fig. 6). Coty­ of outer epidermal layer was reported in Hardwickia ledonary stage embryos had 1-4 cotyledons. Somatic

Table 4 - Effect of BA and IBA incorporated in MS and B5 medium on growth of embryonic callus PGR (mgrl) Fresh wl. embryonic Dry wl. embryonic Medium BA IBA callus (mg±SD) callus (mg±SD) ) Control-B5 0.00 0.00 374.7±5.28 46.7±1.2 0.10 0.25 384.3±5. 13 48.2±0.60 0.25 0. 10 479.3±6.03 59.7±1.00 0.25 0.25 455±5.03 52.3±1.82

Control -MS 0.00 0.00 57 1.1 ± 10.03 70.0±0.92 0.10 0.25 68 1±11.0 84.0±1.20 0.25 0.10 979±25.94 I 280±2.10 0.25 0.25 612.5±8.66 77.4±1.46 Table 5 - Effect of different media on growth and cell size of callus cultures. Medium containing 2,4,5-T (0.1 mgrl) + Kn (0.1 mgrl) was used for non-embryogenic callus cultures and BA (0.25 mgrl) and IAA (0.1 mgrl) for embryogenic cultures rValues are mean ± SD of different replications]

Non-embr~ogenic callus Embr~ogenic callus Medium Callus texture Fresh wt Dry wl. Cell size (/!:m) Callus Fresh wl. Dry wI. Cell size (/!:m) (g) (g) Length Width texture (g) (g) Length Width B5 Light brown 1.56±0.06 0.10±0.02 I 84±85 102±26 Green I. 72±.0.15 0.11±.0.02 25.1±5.3 24.2±5.5 granular B5 J,4 Dark brownish 1.64±0.09 0.90±0.00 I 56±56 90±31 Light green 1.92±0.04 0.13±0.00 24.6±5.0 23.9±5.2 white callus MS Brown 4.23±0.32 0.24±0.02 217±90 63.8±39 Light green 3.66±0.1O 0.24±0.03 25.8±5.6 24.4±5.0 granular MS -2 Light brown 4.88±0.06 0.22±0.02 235±142 117±43 Green 5.54±0.04 O.28±O.02 24.8±5.9 24.0±S.2 granular BTM Reddish brown 6.72±0.43 0.30±0.01 2S3±129 95±40 Light green I. 87±0. 12 0.12±O.01 25.6±6.0 24.8±7.1 granular KUMAR et al.: SOMATIC EMBRYOGENESIS IN COMMIPHORA CALLUS CULTURES 73

embryos were of relatively large size on media con­ callusing. A high percentage of abnormal germination taining activated charcoal. Increased concentration of of somatic embryos was also observed in most of the gelling agent was associated with reduction in cotyle­ treatments. donary stage embryos. This reduction was markedly High frequency of conversion is a prerequisite to profound in the presence of ABA and activated char­ form an efficient micropropagation system in any so­ coal in the medium. Normally, increased stress was matic embryogenesis based protocol. Cotyledonary associated with decrease in callus formation. How­ stage somatic embryos kept on various maturation ever, except in presence of low concentration of media (Table 8) were transferred onto MS-2 HF me­ ABA, increased stress was associated with increased dium. Somatic embryos grown on media containing

Table 6-Effect of plant growth regulators incorporated in MS-2 medium on somatic embryos development PGR (mgrl ) Somatic embryos formation IAA BA Globular Torpedo Cotyledonary Precocious Total germination 0.00 0.00 28 05 02 35 0.05 0.25 32 08 04 44 0.10 0.25 48 22 06 76 0.20 0.25 26 II 04 4 1 0.50 0.25 19 07 05 3 1 1.50 0.25 16 II 04 07 38 2.0 0.25 13 09 12 34 Table 7 - Effect of ABA, AC and various concentrations of agar-agar incorporated in MS-2 mcdium on maturation of somatic embryos Medium Agar Cotyledonary No response Abnormal Callus intensity (gr') embryos (%) germination (%) (%) Control 8 17.7 52.0 30.3 C++ 8 23.3 33.3 43.4 C++ ABA(lO /L gr') + Sucrose 10 18.7 33.3 48.3 C+++ (30 gr') 12 15 .3 48.3 36.6 C+ 8 20.0 40.0 40.0 C+ ABA(20 /L gr') + Sucrose 10 16.7 41.7 41.6 C+++ (30 gr') 12 13 .3 53.3 33.3 C++ 8 30.0 43.0 26.6 C+ AC(O.5 gr') + Sucrose (10 10 16.7 43.3 41.0 C++ gr') C+++ , A 12 11.6 40.0 48.3 Tab le 8 -Germination response of cotyledonary embryos on MS-2 medium obtained from media containing various combin ation of ABA, ac ti vated charcoal and agar-agar

Maturation medium Agar-agar Embryos No. response No. embryos No. of SSE Germination MS -2 (gr') transferred producing SSE (MS-HF) (%) (%) Control 8 50 17.0 65 18 37 8 42 2 1.4 64.2 14.3 54 ABA(IO /L gr') + Sucrose (30 gr') 10 27 18.18 63.7 18. 12 88 12 33 68.6 21.4 67 8 39 15 .3 84.7 ABA(20 /Lgr ') + Sucrose (30 gr') 10 24 74.5 25.5 72 12 30 100.0 8 54 25 58.3 16.7 44 AC(0.5 gr') + Sucrose 10 36 16 75.6 (10 gr') 8.4 30 12 27 05 95.0 SSE-Secondary somati c embryos 74 INDIAN J EXP BIOL, JANUARY 2003

Figs. I-6 - ( I ) - Tcndcr grccn fru its w ith immalllrc zygoti c cmbryos uscd as an cxplant, notc th c low sccd sct; (2) - Dcvelopmen t of cmbryonic callus as sectors (arrow) on non-e mbryonic callus; (3)- Dcvclopmcnt or globul ar stagc cmbryos fro m cmbryonic ca llus (bar := 2I11 m); (4) - T orpedo and ca rl y coty lcdonary stagc embryos (bar := I mm); (5) - Histologica l scc ti ons showing globular (a), torpedo (b) and di ffercntiation of coty lcdons (c) in so mati c embryos (bar := in a, 200/1m ; b, 250/1m C, 400/1m ); and (6) - Coty lcdonary stage embryos growlI on filter pape r for maturation (bar :=5 mm). KUMAR el Cl I.: SOMATIC EM BR YOGE ES IS IN COMMIPHORA CALLUS CULTURES 75 pl ant growth regul ators, irrespecti ve of their concen­ whi ch co ul d be maintained on MS-2 HF medi um fo r trati on and co mbinati on, produced callus. Marked several month s, by usin g of static medium or fil ter increase in per ce nt germinati on was recorded in em­ paper bridge with liquid medium. Approx imately 25 % bryos obtained fro m ABA and ac ti vated charcoal sup­ torpedo shaped embryos matured into cotyledonary plemented medi a. However, increased ABA (20 IL g I-I) stage embryos and out of these approx imately 20-25 and in creased agar ( 12 g r I) were suppressi ve to em­ % were converted in pl antl ets. Thickening of hypo­ bryo co nversion on thi s medium. A hi gh percentage cotyl was observed in such pl antl ets with out elonga­ of embry os remained ungerminated, approximately tion of internodes. MS -2 med ium containing gibber­ 10-25% somati c embryos produced second ary so­ elli c acid (20!lg r l) was observed most effecti ve fo r mati c embryos. Proper selec ti on of mature embryos shoot elongati on in such pl antlets (Fi g. 8). About 200 was required for hi gh percentage of germination (Fig. 7). pl antl ets we re successfull y establi shed in ga rde n so il These second ary somati c embryos were also used in to ve ri fy the survivability of regenerants. Survivab il ­ other ex periments. Somatic embryos showed preco­ ity was 95% for the pl antl ets (Fig. 9). cious germinati on and callu sin g except those grown Somatic embryogenesis in C. wightii was possibl e on MS-2HF medium, though in low percentage, onl y with use of immature zygoti c embryos as In

Figs 7-9 - (7) - Germinati on of properly matured so mati c embryos; (8) - Germinated 3-months old so matic embryos on medium con­ ta ining gibberellin: and (9) - One month old pOlled pl ant lets covered w ith polyethylene (top) and three months old plant lets exposed to env ironment (bollom) 76 INDIAN J EXP BIOL, JANUARY 2003

~ ~~ ~~ W ~ EmIJ .. yogcllic GloIJulal' rrri'l'O'''';' "II", "". EIIlIJryos ~~q~Q,Q,~ /.:~ro~

y g ~ '5j2Clllbr o CI::;/ ~ Direct somahc ~ Callus ~CIIIIJryogcnCSiS ~ - ~ t Somatic cmlJryogensis ~A'Y'7"'Y ,mb,yo Q in C. wiglttii C Zygotic cmIJ .. yo '\(f)

Thc Plant

Pottcd plant Plantlct

Fig. 10 - Flow diagram of somatic embryogenesis in C. wighlii for cont inuous plantI et production

l R Qll erclls robur . Mature ex plants in woody are References still recalcitrant to regenerate in most of the spec ies Arora R B, Das 0 , Kappor S C & Sharma R C, Effect of 26 due to inh erent characters . Generally plant growth some frac ti ons of Co/llll1iphora IIIlIkllt on vari ous serum lip­ regu lators markedly modula.te th e growth and differ­ ids in hyperchloresterolemie chi cks and their dfectiveness in 27 I ~lyoc ardi a l infraction in rats, Indion J Exp BioI, II ( 1973) entiation , but th ese remained un affected in C. 166. wiglilii embryoni c callus cultures. The res ults ob­ 2 Tripathi S N, Sastr'i V V S & Satyavati G V . Experimenta l and ta ined in developing somatic embryogenesis in C. clinical studi es of the effect of Guggulu (c. IIIlIku/) in hyperl ip­ wiglilii are presented in Fig.lO. Cyclic somatic em­ idemia and thrombosis, J Res Indiw/ M ed, 2 ( 196B) 2. bryogenesis is an excell ent system to obtain somati c 3 Satyavati G V, Dwarkanath C & Tripathi S N, Ex perimental studies on the hypocholesterolcmi c effects of COf/lmiphora embryos continuously without loss of regenerati ve IIIlIkll/ Eng/.(Guggul), Indian J M ed Res 57 ( 1969) 1950. potenti al. These results are useful in establishin g em­ 4 Kuppu raja n K, Rajgopalan S S, Rao T K & Sitaralllan R, Ef­ bryo nic cultures of C. wighlii and possibilities of im­ fec t of guggulu (ColIIlI/ipllOra mukul Engl. ) on serum lipi ds provi ng the system for large-sca le multiplication of in obese, hypcrcholesterolemi e and hyperlipemic cases, J /\ssoc Phys icians India. 26 ( 1978) 367. this immensely important med icinal plant. Woody 5 Satyavati G V, Gum guggul (CommiphorC/ fllllklll )- th e suc­ trees are di fficult to grow and regenerate in culture as cess story of an ancient insight leadin g to a modern di scov­ it is ev ident by limited success in cell and callus cul­ cry, Illdiall J M ed Res, 1\7 ( 1988) 327. tures of tree species 's . We have yet not been success­ 6 Sharma J N & Sharm a J N, Compari son of the anti­ ful in ach ievi ng so mati c embryogenesis in liquid cul­ innammatory activity of COlllmiphora mllklll (indigenous drug) with th ose of ph.:nylbutazone and ibuprofen in experi ­ tures though we have established embryonic ce ll sus­ mental arthritis induced by mycobactrial adjuvant, Arzneimil­ pension cultures. le/forschung, 27 (1977) 1455. 7 Kumar S & Shan ka r V, Medicinal plants of tire Indian Dc­ sert: C(}IlI lIIip/lOrrt wightii (A rnott) Bhand, J /\ r id Ellviron, 5 Acknowledgement ( 1982) I. This work was supported by grants (BT/R&D/ 8 Ramawat K G, Bhardwaj L & Tewari M N, Exploitation of 08/23/95) fro m the Department of Biotechnology, India desert medicinal plants through tissue culture. Illdioll Government of Ind ia, New Delhi. Re v Lile Sci, I I ( 199 1) 3. KUM AR I't 01.: SOMATIC EMBRYOG ENESIS IN COMMIPHORII CA LLUS CULTURES 77

9 Sharma R, Suri S S, Ramawat K G & Soni e K C, Bi otcc hno­ 18 Cucnca B, San-J osc M C, Martincr M T, Ballcster A & logi cal approaches to the medi cinal plants of Aravalli Hills with Vi cit ez A M, Somati c embryogcnesis from stem and Icaf ex­ spcc ial rcfercn cc to COlIl/llip/lO ra \\'ightii, in Role 0/ Biolecil/lOl­ plants of Quercus robur L. , Plalll Cell Rep, 18 ( 1999) 538. ogl' ill lIIedicilial alld arolllatic plalll. cditcd by I A Khan and A 19 Shulcr M L, Ove r vicw of yield improvc ment stratcgics for Khanum (Ukaz Pu blicati on, Hydcrabad) 1999, 140. secondary mctabolites produ cti on in pl ant ccll cu lt urc, in 10 Prakas h J. Kase ra P & Chawan D D, A rcport on polycmbry­ Plallt cell 111/{1 tissue culture/or the productil)ll 1!//OOr/ ingre­ ony in COlllllliplw /'{/ lI'ightii from Thar dcscrt India. Curl' Sci. diell/s, cd it cdby T J Fu, G Singh and W R Cu rt is (Klu\Ve r 78 (2000) 11 85. Acadc mi c. Ncw York ) 1999.75. II Gupta P. Shi va nn a K R & MohanR am H Y, Apomi xis and 20 Phalc P, Subra mani J, Bhatt P N & Mchta A R, Viability and polycmbryony in th c guggul pl ;] i1l , COllllllipho/'{/ I\ 'ightii. 11 1111 gugg ul stcrol producti on in immobili zed ti ss uc cu ltu rcd ce ll s Bot. 78 ( 1996) 67. of CO lllllliphoJ'{[ I\ ·igillii. 'lidilill J El'p Bioi, 27 ( 1989) 338. 12 Singh P. Shanm M L & Mukhcrj cc S, Effcct or indol c bu­ 2 1 Gamborg 0 L. Mill cr R A & Ojima K, Nutri cnt rcquircment s tyri c acid on spro uting in pl ;] nt cuttings of CO llllllip/lO /'{/ of suspcnsion culturcs of soy bcan root cc ll , E l'p Cell Res. 50 ( 1968) 15 1. I\ 'ig ht ii (A rn ott ) Bhand ari , Ilidiall Dmgs. 26 ( 1989) 515 . 22 Murashi gc T & Skoog F, A rcviscd mcdium for rapid growt h l:l Bar vc D M & Mehta A R, Clonal pro pagati on of mature cli tc and bi oassays with tobacco ti ssuc cult ures. Ph.l'siol Plllilt. 15 trces or COlllllliphoJ'(l Il' ightii. Plallt Cell Tissue 0 1'.11 0 11 Cult. ( 1962) 473. ]5 ( 199]) 237. 23 Sass J E, UO/allical M icrotechll ique. (Iowa State Col legc 14 Sin gh A K, Suri S S & Ramawat K G, Somati c cmbryogcnc­ Press, Amcs, Iowa) I 95!i. sis rro m immaturc zygoti c cmbryos or CO/llllliIJhoJ'{[ I\ ·ightii. 24 Chalupa V. Clonal propagati on or broad Icvcl trce ill I'itro. Cal'leIl IHIU I\ ·isseli. a woody medi cin al pl ant . 62 ( 1997b) 44. COIiI/IIUIi Ilist Czech, 12 ( 198 1) 255. 15 Ara H, Jaiswal U & Jai swal V S, Somatic embryogcncs is and 25 Maheswaran G & Williams E G, Direct second ary somati c pl antl ct regencrati on in Am rapali and Chausa culti vars or cmbryoge nes is rrom immature scx ual cmbryos or TrijiJ/i1li 1i man go (Mallgi/era illdica L. ) Curl' Sc i. 78 (2) (2000) 1M. repellS cultured ill l'itro, 11 1111 /J ot (LOlidoll ), 57 ( 1986) 109. I (i Chand S & Singh A K. Dircct somatic cmbryogc ncsis rrom 26 Zi v M, Devclopmcntal and stru ctural pattcrn s of ill I'itro 7.ygoti c cmbryos or a tim bcr-yicld ing Icguminous trcc. plants, cditcd by WY Soh and SS Bhojwa ni MOIphogeliesis Hardl\'ic/.: ia billaw Rox b. Curl' Sci, 80 (7) (200 I) 882. ill plallt tissue cultures, (Klu we r, Dorcdrccht) 1999.235. 17 Xi c D Y & Hong Y, Rcgcncrati on of Acacia lIIaligiu/1l 27 Suri SS & Ram