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Micropropagation of mature Pongamia pinnata Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was
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  MICROPROPAGATION Micropropagation of mature  Pongamia pinnata  Pierre K. Sujatha  &  Sulekha Hazra Received: 18 October 2006 /Accepted: 6 April 2007 /Published online: 18 August 2007 / Editor: B.M. Reed # The Society for In Vitro Biology 2007 Abstract  Murashige and Skoog ’ s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), andthidiazuron (TDZ) were tested for induction of multipleshoots from mature-tree-derived axillary meristems of   Pongamia pinnata . Sprouting of buds was 64% on mediumdevoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sproutingfrequency or induction of multiple shoots. Sprouting wascompletely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was moreon explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at theaxil. Multiple shoot primordia appeared and differentiatedfrom this swelling after culturing these explants on MSmedium for six passages of 2 wk each. Shoots wereharvested and cultured on 0.45  μ  M TDZ for further  proliferation. Primary explants after harvesting of shootswere identified as  ‘ stump ’ . Reculturing of stumps on0.45  μ  M TDZ produced more shoots. This step wasfollowed for six cycles to obtain additional shoots in eachcycle. Shoots maintained on 0.45  μ  M TDZ elongated androoted (70%) on growth regulator-free medium. Rootedshoots (65%) survived transfer to a sand/soil mixture. Thisreport describes the protocol for micropropagation of   P. pinnata  using mature-tree-derived nodal meristems.Recycling of mature stock to produce a stream of useableshoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated prolifera-tion of caulogenic buds from the same srcin may also findapplication in rescue of endangered germplasm. Keywords  Biodiesel.Pongam.Pongamia.Shootdifferentiation.Thidiazuron Introduction  Pongamia pinnata  Pierre. (Syn:  Pongamia glabra  Vent.) isa multipurpose, legume tree of Indian srcin and the onlyspecies in the genus  Pongamia  and is an outbreedingspecies. Thus, seed populations exhibit high levels of heterogeneity. The seeds of this tree contain 25% oil. The potential of this oil as a substitute for diesel (Srinivasa1997; Vivek and Gupta 2004) and as a lubricant in tanning industries is recognized. Pongam oil resembles groundnut oil (  Arachis hypogaea . L) in its fatty acid composition. Theseed is a source of a number of bioactive compoundsincluding flavonoids and furanoflavonoids, which havemedicinal uses for rheumatism, skin diseases, etc. (Parmar et al. 1976; Council of Scientific and Industrial Research1988). The tree is adaptable to wide agronomic climaticconditions. There has been expansion of   Pongamia  planta-tions in India within the last few years, mainly for its oil for  biodiesel production and because of its impressive growth performance under tropical conditions.  Pongamia  can helpin restoration of fertility especially in degraded soils owingto its nitrogen fixing ability. There is a crucial need todevelop efficient methods for mass production of improvedquality planting stock. Experiments conducted by theauthors using seedling explants demonstrated the possibilityof producing shoots by reculturing the cotyledonary node inmedium containing BA (Sujatha and Hazra 2006).  In vitro In Vitro Cell.Dev.Biol.  —  Plant (2007) 43:608  –  613DOI 10.1007/s11627-007-9049-2K. Sujatha :  S. Hazra ( * )Plant Tissue Culture Division, National Chemical Laboratory,411008 Pune, Indiae-mail: s.hazra@ncl.res.in  regeneration protocols for   Pongamia  will find applicationin clonal propagation for mass propagation, germplasm preservation, and development of transgenics.Thidiazuron (TDZ) is identified as the most activecytokinin-like substance for woody plant tissue culture.Before the discovery of TDZ as a plant growth regulator (PGR), amino purine cytokinins had been used extensivelyfor micropropagation of plants via organogenesis. Compar-ative studies of amino purine cytokinins and TDZ remaincomplicated because of the requirement of extremely lowconcentrations of TDZ against the high requirement of amino purine cytokinins to stimulate axillary shoot prolif-eration (Huetteman and Preece 1993). This synthetic compound has been found to be effective at very lowconcentrations (0.0091 to 3.99  μ  M) for micropropagationof several species (Lu 1993). However, it has been used at  higher concentrations (2.27 to 145.41  μ  M) for propagationof some species including  Zanthoxylum rhetsa  (Augustineand D ’ Souza 1997), a mature forest tree species. Theactivity of TDZ varies widely depending on its concentra-tion, exposure time, the cultured explant, and species tested(Murthy et al. 1988). In the present investigation attempts were made to: (1)study the effect of both amino purine cytokinins and TDZat equimolar concentrations on axillary bud sprouting and proliferation of mature-tree-derived nodal explants of   Pongamia , and (2) develop a reliable  in vitro  protocol for micropropagation. Materials and Methods Twigs of   Pongamia  were collected during the vegetativeseason (March  –  May) from mature trees growing locally. Nodal explants of approximately 2 cm in length wereexcised and washed thoroughly. Explants were treated with1% Bavistin (Carbendazim 50%WP, India) containing afew drops of detergent for 1 h on a gyratory shaker,followed by multiple washings with sterile, distilled water and treatment with 4%( v  /  v  ) Savlon (liquid antiseptic,Johnson and Johnson Ltd., Mumbai, India) for 10 min.The explants were then surface sterilized with 0.1% HgCl 2 for 8 min followed by four times rinsing with sterile water.PGR including 6-benzylaminopurine (BA), kinetin (KN),zeatin (Z), and TDZ at concentrations of 0.45, 4.54, 9.08, or 13.62  μ  M were incorporated in Murashige and Skoog ’ s(MS) basal medium (Murashige and Skoog 1962) with 2% sucrose. Plant preservative mixture (PPM, Sameer ScienceLab, Jabalpur, India) 2 ml l − 1 and Cefotaxime (Alkem,Mumbai, India) 250 mg l − 1 were added aseptically to the primary culture media. Sprouting frequency and number of shoots produced per explant were noted after 4 wk of culture. Seven explants were used per treatment, and theexperiment was repeated five times. For TDZ cultures, thenumber of shoots produced per explant was noted after everytwo subcultures. Lengths of all the shoots were measured.In the second experiment,  Pongamia  buds were culturedfor 4 wk on MS basal media supplemented with TDZ at 0.45, 2.27, 4.54, 6.81, 9.08, or 11.35  μ  M. Eight explantswere used per treatment, and the experiment was repeatedfour times. Buds cultured on TDZ in both experiments weretransferred to PGR-free MS basal media, for six subculturesevery 2 wk. Frequency of response, number of shoots produced per explant, and their length were noted after every two subcultures. For shoot length, the number of shoots produced from the explant was considered asreplicates. Shoot primordia <0.2 cm were scored visuallyusing a stereomicroscope. The primary explant, after harvesting of shoots or meristematic buds, was identifiedas  ‘ stump ’ . Stumps were recultured on medium containing0.45  μ  M TDZ for 4 wk, followed by four subcultures every2 wk to PGR-free basal media. This process of removal of shoots and reculturing of the stumps was repeated for sixcycles. The elongated shoots (3  –  4 cm) were isolated fromthe cluster and cultured individually for two subculturesevery 4 wk for rooting and elongation on PGR-free half-strength MS medium supplemented with 0.25% activatedcharcoal (Sarabhai Chemicals, Vadodara, India). Plantletswith fully opened leaves and roots 2  –  4 cm in length weretransferred to autoclaved sand/soil mixture (1:1). Plantletswere hardened at 25±2°C for 4 wk in 70  –  80% humidityand 24-h photoperiod. The acclimatized plantlets weretransferred to the greenhouse for further growth.All media used in this study were with 2% sucrose, andthe pH was adjusted to 5.8, after supplementation withgrowth regulators. The media were gelled using 0.7% agar (Hi media, Mumbai, India) and autoclaved for 20 min.Cotton plugged boiling tubes (25×150 mm, Borosil,Mumbai, India) with 20 ml of media per tube was usedfor the cultures. Cultures were incubated at 25±2°C under a16-h photoperiod of 16  μ  E m − 2 s − 1 . Mean and standarddeviations were determined and were subjected to analysisof variance (ANOVA). The differences among the treatment means were tested using Duncan multiple range test (DMRT) at a 5% probability level (  P  <0.05). Results and Discussion Establishment of sterile cultures of   Pongamia  was limited because of microbial contamination. Use of PPM andCefotaxime were effective in controlling the growth of thecontaminants to a limited extent. Sprouting behavior of the buds and contamination frequency varied with season. In  Pongamia , each nodal bud (Fig. 1 a ) has one meristematicdome in the axil. This dome is composed of a set of three MICROPROPAGATION OF MATURE PONGAMIA PINNATA PIERRE 609  meristems, with the central meristem more developed.Sprouting of the central meristem to form a single shoot was optimum during March to May. To induce sprouting andshoot multiplication, various PGRs were tested. The centralmeristem differentiated into a single shoot in 40 to 77% of thecultures in media with or without PGRs except with TDZafter 4 wk. There was no significant variation in sproutingfrequency of the buds in media with BA, KN, and Z ascompared to the control. In media with TDZ, none of the buds differentiated in 4 wk, but the meristems were swollen.The swelling was more pronounced in higher concentrationsof TDZ.Failure of the meristems to form shoots in mediumcontaining TDZ suggests that this PGR suppresses differ-entiation of the existing meristem in the node. In tamarind, presence of TDZ in the seed germination medium sup- pressed differentiation of the apical meristem, but innumer-able meristematic domes appeared in the form of undulations circling the cotyledon node (Mehta et al.2004). These undulations differentiated into caulogenic buds and subsequently into shoots upon withdrawal of TDZ. In  Pongamia , no such undulations were noted in theaxillary meristems after 4 wk of culture on TDZ, but theswelling in the meristem was obvious. This difference in Figure 1.  Micropropagationof   Pongamia pinnata  Pierre.( a ) Nodal bud of   Pongamia  withthree meristematic domes ( m ).Central meristem ( c ) in betweenthe side buds (  s ). Cut end of stem ( n ). ( b ) Swollen meristemon removal of bud cover after 4 wk of TDZ exposure. Pair of meristematic masses ( mm )developed from centralmeristem ( c ). ( c ) Sprouting andelongation of axillarymeristem inmedium without PGR. ( d  ) Differ-entiation and elongation of shootsin medium without PGR fromexplants precultured in TDZ(0.45  μ  M). ( e ) Differentiation andlimited elongation of shoots inmedium without PGR in explants precultured in higher concentra-tion of TDZ (11.35  μ  M). Shootsare stunted and more in number.(  f   ) Elongation of shoots fromthe cluster of buds harvestedfrom the stump in PGR-free MS basal media. (  g  ) Induction of caulogenic buds ( cb ) from theside buds on reculturing thestump (  st  ) in TDZ (0.45  μ  M). Note the site of removed shoots( o ). ( h ) Rooted shoot in MSmedium. ( i ) Hardened plantletsin greenhouse.610 SUJATHA AND HAZRA  the pattern of response between tamarind and  Pongamia , inthe initial stages of meristem culture in the presence of TDZ, could be a result of variation in species or explant. Intamarind, the meristem was in the cotyledonary node of theseedling (juvenile tissue), whereas in  Pongamia , themeristem was in the axil of the mature-tree-derived nodalsegment. In tamarind (Mehta et al. 2005), histological studies revealed multiple layers of meristematic cells onand around the meristem as a result of proliferation of themeristematic cells. Caulogenic buds srcinated from thesecells upon withdrawal of TDZ. In  Pongamia , shoot  primordia appeared from the TDZ-induced swelling after two passages of 2 wk each in PGR-free medium. Keepingin mind the influence of TDZ on the proliferation of meristematic cells in tamarind (Mehta et al. 2004, 2005), it  is presumed that the swelling of the axillary bud in  Pongamia  was a result of proliferation of the meristematiccells inside the dome cover. Removal of the dome cover exposed a pair of swollen structures (Fig. 1 b ) from thecentral meristem. Caulogenic buds and shoot primordiadifferentiated from these meristematic masses upon with-drawal of TDZ for 4 wk. Thus, the frequency of sprouting,the number of shoots produced from the nodal bud, and thelength of shoots produced could be determined after 8 wk from the initiation of culture in TDZ (Table 1).The frequency of sprouting noted after 4 wk on MSmedium with TDZ pretreated explants did not increase after 12 wk. The concentrations of TDZ tested failed to inducesprouting in the remaining nonresponding buds. After 4 wk on MS medium, the sprouting noted in the TDZ pretreated buds were 59, 55, and 46% for 4.54, 9.08, or 13.62  μ  MTDZ, respectively, vs 62% for the control medium (Table 1).This repression in sprouting was not reversible uponwithdrawal of TDZ. In  Bauhinia vahlii  (Upreti and Dhar 1996), 1.0  μ  M TDZ was optimum for induction of sprouting in 96% of cultures that did not sprout in mediumwithout growth regulator.  Pongamia  buds (62%) cultured inmedium devoid of PGR sprouted and elongated (Fig. 1 c ) but did not produce multiple shoots. Multiple shoot  primordia appeared from the bud upon first transfer fromTDZ-containing medium to PGR-free medium. These primordia differentiated into shoots after repeated transfer to medium devoid of PGR. From this phenomenon, it isapparent that TDZ, at the concentrations tested, induces proliferation of meristematic cells but does not support shoot differentiation in this plant. The dome-shapedswelling appearing at the meristem after exposure to TDZwas possibly a result of development of meristematicmasses on proliferation of meristematic cells. Proliferationof meristematic cells in response to TDZ exposure wasreported in peanut (Joshi et al. 2003) and cotyledonary node meristem of tamarind (Mehta et al. 2005). Withdrawal of TDZ reduced the negative influence resulting information of shoot primordia from the dome-shapedmeristem. Larger domes in higher concentrations of TDZsuggest increased morphogenic cell proliferation leading toemergence of more number of shoots in explants precul-tured in higher concentrations (Table 1). Faster differenti-ation of shoot buds in explants pretreated in lower concentrations of TDZ was a result of reduced inhibitoryinfluence on the shoot differentiation.The second experiment with a narrower range of TDZ(0.45, 2.27, 4.54, 6.81, 9.08, and 11.35  μ  M) confirmed theinhibitory effect of TDZ on sprouting and augmentingeffect on caulogenesis. In the medium without PGR, 62.5±14.4% of the buds sprouted after 4 wk, whereas the buds onTDZ containing media did not sprout and developed thecharacteristic swelling at the node. The frequency of sprouting response was scored on appearance of caulogenic buds after 4 wk on MS medium. Compared to 62.5%sprouting on TDZ-free medium, sprouting was 53.5% on11.35  μ  M TDZ. The number of responding explants did not  Table 1.  Differentiation of shoot primordia in TDZ pretreated nodal buds of   P. pinnata  after two passages of 15 d each in PGR-free mediumConc. of TDZ ( μ  M) Elongation medium (PGR-free MS medium)Frequencyof response(%±SD)Mean number of shoots per explant  a  (±SD) Mean length of shoot (cm)  b (±SD)4 wk 4 wk 8 wk 12 wk 4 wk 8 wk 12 wk Control 61.8±5.2 1±0(28) 1±0(26) 1±0(22) 1.03±0.4(29) 1.60±0.3(42) 1.92±0.4(42)0.45 63.4±6.1 2.2±0.3(28) 2.8±0.2(28) 3.2±0.3(27) 1.65±0.3(62) 1.84±0.4(78) 2.21±0.1(86)4.54 59.2±4.4 2.6±0.4(26) 2.8±0.3(24) 3.0±0.5(23) 1.06±0.3(68) 1.65±0.3(67) 1.97±0.2(69)9.08 54.6±21.4 6.0±0.7(29) 6.4±0.8(25) 6.5±0.4(25) 0.74±0.1(154) 0.96±0.1(160) 1.06±0.1(160)13.6 46.2±13.9 7.0±0.7(24) 7.2±0.8(24) 7.3±0.3(22) 0.43±0.0(168) 0.53±0.1(173) 0.70±0.1(155)Anova NS S1% S1% S1% S1% S1% S1%Each passage is of 15 d, i.e., 4 wk=two passages, 8 wk=four passages, 12 wk=six passages. a  Figures in  parentheses  indicate the number of explants producing shoots.  b Figures in  parentheses  indicate the number of shoots measured.MICROPROPAGATION OF MATURE PONGAMIA PINNATA PIERRE 611  increase with additional passages on PGR-free medium(Table 2), confirming the nonreversible inhibition in someof the buds. In the responding explants, numerous shoot  buds appeared from the swollen node (Table 2).The total number of shoot primordia formed on eachexplant (Table 2) was higher in all concentrations of TDZcompared to the number of buds formed per explant insimilar concentration in the previous experiment (Table 1).This difference in response of the buds in two similar experiments could be a result of more accurate scoring of  buds under the microscope in the second experiment andalso a result of the physiological status of the initial explant collected at different times of the vegetative phase. In thesecond experiment, numbers of shoot primordia and shoots per explant were significantly higher in explants precul-tured in higher concentrations of TDZ (Table 2). Totalnumber of caulogenic buds per explant (Table 2) indicatesan increase in the number of multiples in higher TDZ-treated explants.Appearance of the buds after 4 wk culture on PGR-freemedium (Fig. 1 d  ) confirms differentiation of meristematiccells after withdrawal of the inhibitory influence of TDZ.Transfer of explants to fresh PGR-free medium every 15 dsupported further elongation by reduction in inhibition.Persistence of TDZ in plant tissues and suppression of shoot elongation have been described (Huetteman andPreece 1993; Lu 1993). The mean shoot height of   Pongamia  increased every 4 wk (Table 2), but the number of caulogenic buds scored initially after 4 wk on PGR-freemedium remained constant. The number of buds was morein explants precultured on higher concentrations of TDZ, but the rate of shoot elongation was less. Length of shoot did not catch up with the control or with the shoots from0.45  μ  M TDZ after six passages of 15 d each.At higher concentrations (9.08 or 11.35  μ  M), the number of shoot primordia was more and the differentiated shootswere thick and stunted (Fig. 1 e ). The shoots were close toeach other and appeared fasciated. Formation of stuntedshoots or the fasciation of shoots on TDZ-containingmedium was reported in other species (Chalupa 1988; Pradhan et al. 1998). After 12 wk on MS medium, the budsdid not attain the desired height of 2  –  3 cm for root induction. Thus, the cluster of buds was isolated from theinitial explants and was subcultured as smaller cluster tomedium for two more passages of 4 wk each to attain thedesired length (Fig. 1  f   ).The buds induced on high TDZ took longer and requiredseveral passages on PGR-free medium to elongate. Thus,the rooting step was delayed. This period was reduced bysubculturing the buds on medium with low concentration of TDZ (0.45  μ  M) for maintenance of the shoot cultures. Thestrategy of alternate culture in medium with 0.45  μ  M TDZfollowed by passages on PGR-free medium was used for maintenance of the shoots.The  ‘ stumps ’ , when recultured on medium with 0.45  μ  MTDZ for 4 wk, produced more caulogenic buds from the pair of side buds (Fig. 1  g  ). After transfer to PGR-freemedium, these buds differentiated into shoots. The cluster of buds once again isolated from the  ‘ stumps ’ , elongated to produce shoots 2  –  3 cm in length on medium, after six passages of 15 d each, and the  ‘ stump ’  produced more budsafter reculturing on 0.45  μ  M TDZ. This process of excisionof shoot buds, reculturing of the stumps on 0.45  μ  M TDZ,was repeated for six cycles to produce 25  –  30 shoots fromeach nodal bud. This strategy was used for micropropaga-tion of   B. vahlii  (Upreti and Dhar  1996) and  Dalbergia sissoo  (Pradhan et al. 1998), but the srcin of the shoots was not defined in these protocols. In  Pongamia , the first and second crop of shoots isolated from explants was fromthe central and side meristems, respectively (Fig. 1  g  ). Theorigin of the shoots obtained from the  ‘ stump ’  in thefollowing five cycles could be from the TDZ-induced Table 2.  Effect of TDZ on sprouting of meristems, multiplication, and elongation of shoots in the mature nodal bud explants of   P. pinnata Conc. of TDZ ( μ  M) Sproutingfrequency (%±SD)Mean number of shoots per explant (±SD)Mean number of caulogenic buds per explant (±SD)Mean length of shoots (cm) (±SD)4 wk 4 wk <0.2 cm >0.2 cm 4 wk 8 wk 12 wk Control 62.5±14.4 1±0 (19) 0.0±0.0 1.0±0.0 1.06 a,b ±0.24 (20) 1.78 a,b ±0.27 1.86 a,b ±0.190.45 62.8±17.4 4.5 d ±1.43 (20) 2.5±1.3 2.0±0.29 1.18 a  ±0.28 (32) 1.93 a  ±0.22 2.21 a  ±0.222.27 59.8±25.6 7.3 c,d ±2.32 (18) 4.5±1.7 2.8±0.64 0.91  b,c ±0.04 (44) 1.53  b,c ±0.22 1.76  b ±0.254.54 66.0±35.8 10.7  b,c ±2.55 (21) 8.0±1.4 2.7±1.22 0.83 c,d ±0.12 (44) 1.25 c,d ±0.21 1.55  b,c ±0.236.81 38.5±17.3 16.2 a,b ±2.95 (12) 10.8±2.8 5.4±0.29 0.67 d,e ±0.02 (81) 1.09 d,e ±0.26 1.37 c,d ±0.219.08 53.3±06.5 17.9 a,b ±2.67 (17) 11.5±2.5 6.4±0.83 0.62 d,e ±0.04 (102) 0.99 d,e ±0.25 1.28 c,d ±0.2311.35 53.5±18.6 22.0 a  ±2.97 (17) 14.8±2.5 7.2±1.18 0.55 e ±0.07 (115) 0.83 e ±0.13 0.99 d ±0.25Anova NS S 1% S 1% S 1% S 1% S 1% S 1%Figures in  parentheses  indicate number of replicates. a   –  e Duncan multiple range notation. Means followed by the same  superscripts  within a  column  do not differ significantly at   P  ≤ 0.05.612 SUJATHA AND HAZRA
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