Enhanced micropropagation protocol of ex vitro rooting of a commercially important crop plant Simmondsia chinensis (Link) Schneider

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1 JOURNAL OF FOREST SCIENCE, 62, 2016 (3): doi: /80/2015-JFS Enhnced micropropgtion protocol of ex vitro rooting of commercilly importnt crop plnt Simmondsi chinensis (Link) Schneider A. Singh, P.K. Agrwl Centrl Slt nd Mrine Chemicls Reserch Institute, Council of Scientific nd Industril Reserch, Bhvngr, Gujrt, Indi ABSTRACT: A micropropgtion protocol ws developed by further improvement of previling methods using proven germplsm for ex vitro rooting nd ddressed the effect of the number of subcultures on the rooting bility of shoots. A comprtive study ws done between in vitro rooting method nd ex vitro rooting method. Using the ex vitro rooting method plntlet could be produced in 135 dys, which ws in shorter time compred to the in vitro rooting method 180 dys. The best xillry shoot bud induction ws observed on Murshige nd Skoog (MS) medium supplemented with 4.6 µm thidizuron (TDZ) with 5 shoot buds per explnt. In the shoot cluster, which ws subcultured on MS medium supplemented with 2.3 µm TDZ, the rte of shoot multipliction incresed in the 3 rd subculture. The mximum men number of shoots per explnt (20) ws obtined t the 3 rd subculture on the sme medium. Shoots were hrvested t the 1 st, 3 rd nd 5 th subculture nd pulse treted for root induction. The highest rooting (95%) ws chieved from the 3 rd subculture onwrds with pulse treted shoots for fifteen dys. The rooted plnts could be estblished in greenhouse with 99% survivl. Ex vitro rooting is promising method to reduce the time for plnt genertion. The resultnt plntlets well estblished in pots nd fruiting ws observed within yer. Keywords: fruiting; genotype; Jojob Jojob (Simmondsi chinensis) is shrub ntive to the rid regions of Northern Mexico nd South Western United Sttes. Jojob is in very high demnd, mking it profitble crop due to its potentil ppliction in cosmetic, petroleum, nd phrmceuticl industries (Agrwl et l. 2002; Tbres et l. 2004). Jojob oil is liquid gold from the desert nd one of nture s gifts to the humn rce (Bhrdwj et l. 2010), nd it is desert whle s Jojob oil is recognized s n lterntive to sperm whle oil nd hs lmost the sme properties s the oil obtined from the whle sperm (Low, Hckett 1981), which is now listed s n endngered species. Jojob cn be propgted through seeds but due to low nd inconsistent seed yield the cultivtions were not tken up s expected (Chturvedi, Shrm 1989; Cotes et l. 2006). Vegettive propgtion could be used for the genertion of selected clones with limited success (Loreto et l. 1998). In order to overcome the difficulties in vegettive propgtion methods for micropropgtion hve been developed using n in vitro rooting method (Chturvedi, Shrm 1989; Mills et l. 1997; Llorente, Apóstolo 1998; Roussos et l. 1999; Hmm et l. 2001; Tygi, Prksh 2004; Llorente et l. 2007; Singh et l. 2008). Ex vitro rooting is promising method, skipping the phse of in vitro rooting, shortens the micropropgtion cycle by 2 3 weeks or more nd so minimizes the time period of the plntlet genertion (Debergh, Mene 1981; Mrtin 2003). However, very few ttempts hve been mde in different plnts (Bh- Supported by the Centrl Slt nd Mrine Chemicls Reserch Institute (ProCSIR-CSMCRI), Communiction No. 196, nd by the Council of Scientific nd Industril Reserch in New Delhi, Project No. MLP0014. J. FOR. SCI., 62, 2016 (3):

2 ti et l. 2002; Mrtin 2003; Xu et l. 2008; Singh et l. 2014) using this technique nd no such ttempt hs been mde in Jojob. Mny reserchers hve reported the effect of the number of subcultures on the rooting cpcity of microshoots. In mny tree species such observtions were mde e.g. in pple (Mlus pumil Miller cv. Fuji, Strkrimson), shoots cquired high rooting bility with incresing the number of subcultures (Chng et l. 1991; Noiton et l. 1992); wlnut (Juglns regi Linneus) shoots hve high rooting rte fter they hve been subcultured for 4 yers (Pei et l. 2002; Wng, Guo 2007) when high rooting rte ws found until the 8 th subculture. During dventitious root formtion endogenous hormones re involved. Indolecetic cid oxidse (IAAO), peroxidse oxidse (POD) nd polyphenol oxidse (PPO) re found to ply role in dventitious root formtion (Hung et l. 2002; Xio et l. 2002; Kochhr et l. 2005). The successful rooting depends upon optiml levels of endogenous phytohormones required for the rooting of tht prticulr plnt (Pn, Tin 1999; Wng et l. 2005). As stted bove, the number of subcultures ffects the formtion of dventitious roots in mny species of woody plnts. However, no such studies hve been done erlier to revel the effect of successive subcultures of Jojob microshoots on rooting. Recently, we hve developed the lrge-scle micropropgtion protocol from nodl (Singh et l. 2008) nd lef (Singh et l. 2011) explnts. In this study, we report the clonl multipliction of Jojob using ex vitro rooting nd effect of the number of subcultures on multiple shoot buds nd their rooting bility upon subsequent subcultures. MATERIAL AND METHODS Plnt mteril nd culture conditions. Highyielding four femle (CSMCRI 1-1, CSMCRI 12-8, CSMCRI 10-4 nd CSMCRI 20-3) nd two mle genotypes (Mle-1 nd Mle-2) with n bundnce of flowering were selected from the Centrl Slt nd Mrine Chemicls Reserch (CSMCRI) experimentl sttion Znjmer (21.50 N, 17.53'E, N, 44.96'E), Bhvngr, Gujrt. Actively growing young shoots (5 10 inter nodes) were collected erly in the morning nd processed for surfce steriliztion s described by Singh et l. (2008). Uniform culture conditions were pplied in ll experiments. Murshige nd Skoog (1962) medium (MS medium) ws supplemented with plnt growth regultors (PGRs) in different concentrtions nd combintions. The ph of the medium ws djusted to 5.8 using 1 N KOH or HCl, prior to utoclving t pressure of 105 kp t 121 C for 20 min. Explnts were inoculted into mm culture tubes contining 40 ml of culture medium which ws eqully distributed using n utomtic dispenser (ASE chenit PM05). The cultures were mintined t 25 ± 2 C under 16 h photoperiod with µmol m 2 s 1 irrdince (cool white fluorescent tubes). Axillry bud induction nd multipliction. The nodl segments of pproximtely 2 3 cm length of ll genotypes were cultured on MS medium supplemented with TDZ (thidizuron, µm) nd 0.62% gr (w/v, Quligens, Glxo Fine Chemicls, Mumbi, Indi). After 4 weeks, explnts were subcultured on MS medium supplemented with different concentrtions of TDZ ( µm) for shoot multipliction nd elongtion. The bsl mss remining fter hrvesting the shoots ws subcultured onto fresh shoot multipliction medium for further multipliction. The multipliction rte, shoot elongtion nd number of shoots per explnt were recorded fter 5 weeks of culture. In vitro rooting. Shoots were hrvested t different subcultures, 1 st, 3 rd nd 5 th, nd were pulse treted in LM (½ strength liquid MS medium) supplemented with 49.0 µm indole-3-butyric cid (IBA) nd 5.40 µm 1-nphthlenecetic cid (NAA) for 5 dys nd subsequently trnsferred to ½ strength hormone-free MS medium supplemented with chrcol 0.02% (CM) for rooting. Ex vitro rooting nd cclimtiztion. Grownup shoots obtined from the 3 rd subculture onwrds from the medium supplemented with 2.3 µm TDZ were used in ll the rooting experiments. To determine the rooting efficiency, shoots were pulse treted by plcing in test tube contining filter pper bot filled with 20 ml of ½ strength MS liquid medium (LM) supplemented with μm IBA, μm NAA, µm 2,4-dichlorophenoxycetic cid (2,4-D) for 10 nd 15 dys nd trnsferred to polythene bgs (five shoots per bg) contining sterilized snd irrigted with sterile distilled wter contining 500 mg l 1 Bvistin (Singh et l. 2010) nd covered with trnsprent plstic bgs to mintin humidity. The rooting percentge ws recorded fter 3 weeks. The rooted plnts were trnsferred to greenhouse for further hrdening nd the survivl of plnts ws recorded fter 3 4 weeks. The tretment effects were ssessed by SPSS sttisticl softwre (IBM, New York, USA). The 0.05 level of probbility ws used for sttisticl significnce in the nlyses. 108 J. FOR. SCI., 62, 2016 (3):

3 120 CSMCRI 1-1 CSMCRI 12-8 CSMCRI 10-4 CSMCRI 20-3 Mle-1 Mle-2 Shoot bud induction (%) b b b b b b c c c c c c d d d d d d TDZ (µm) Fig. 1. Effect of thidizuron (TDZ) on shoot bud induction of different genotypes of Simmondsi chinensis (Link) Schneider. The vlues represent mens ± SE of the tretment of 20 explnts in three replicte experiments, different letters for TDZ tretments re significntly different t 0.05 probbility level (CSMCRI 1-1, CSMCRI 12-8, CSMCRI 10-4, CSMCRI 20-3 femle genotype, Mle-1, Mle-2 mle genotype) RESULTS Effect of TDZ on xillry bud induction, shoot multipliction nd elongtion The nodl segments of mle nd femle genotypes cultured on MS medium without PGRs did not show ny xillry bud growth, however, the ppliction of 0.46 to 6.90 µm TDZ into the medium induced shoot buds. The best xillry bud induction ws recorded t 4.60 µm TDZ (Fig. 1). The induction of shoot buds ws more thn 90% in ll the genotypes. Four weeks fter inocultion, positively responding explnts (the medium supplemented with 4.60 µm TDZ) were subcultured on MS medium supplemented with µm TDZ for shoot prolifertion nd elongtion. All the concentrtions of TDZ fcilitted multiple shoot bud induction with very smll mount of cllus t the proximl end irrespective of the genotypes studied. The best shoot prolifertion nd elongtion were observed on MS medium supplemented with 2.3 µm TDZ. After the first culture explnts of ll the genotypes cultured on 2.3 µm TDZ developed 8 10 multiple shoot buds in the first subculture which reched the pek of shoots per culture on the 3 rd subculture (Figs 2 nd 3). The rte of shoot multipliction incresed on the third subculture. These shoots ttined the growth of 10 to 12 cm nd were used for rooting. Axillry buds t the nodes of the in vitro developed shoots lso showed 6 8 multiple shoots (Fig. 3c). In vitro rooting nd cclimtiztion Shoots from ll the genotypes were hrvested from the 1 st, 3 rd nd 5 th subculture nd were pulse treted Tble 1. Effect of subcultures on in vitro rooting percentge of different genotypes of Simmondsi chinensis (Link) Schneider over 4 weeks Genotype Number of subculture 1 st 3 rd 5 th Femle CSMCRI ± ± ± 2.26 CSMCRI ± ± ± 2.56 CSMCRI ± ± ± 2.29 CSMCRI ± ± ± 1.35 Mle Mle ± ± ± 2.45 Mle ± ± ± 2.41 vlues represent mens ± SE of the tretment of 20 explnts in three replicte experiments J. FOR. SCI., 62, 2016 (3):

4 () Number of shoot buds/explnt CSMCRI 1-1 CSMCRI 12-8 CSMCRI 10-4 CSMCRI 20-3 Mle-1 Mle-2 (b) Number of shoot buds/explnt TDZ (µm) Fig. 2. Effect of the number of subcultures on the number of shoot buds, first subculture (), third subculture (b) on different genotypes of Simmondsi chinensis (Link) Schneider (CSMCRI 1-1, CSMCRI 12-8, CSMCRI 10-4, CSMCRI 20-3 femle genotype, Mle-1, Mle-2 mle genotype, TDZ thidizuron) in LM for 5 dys nd then trnsferred to CM for rooting. The rooting percentge of shoots in ll the genotypes ws very close to ech other nd did not vry very much. The highest percent rooting ws chieved in the rnge of 92 to 95% from the 3 rd nd 5 th subculture shoots in ll the genotypes (Fig. 3, Tble 1). From the third subculture onwrds, the rooting percentge ws high s compred to the 1 st subculture (39 41%). The results revel tht the rooting bility of shoots incresed from the 3 rd subculture onwrds. The shoots in the control filed to root in CM. In vitro hrdened plnts were trnsferred to greenhouse for further hrdening nd 91% survivl ws chieved (Fig. 4). Ex vitro rooting nd cclimtiztion During in vitro rooting experiments, we obtined better rooting using shoots from the 3 rd culture onwrds in ll the genotypes. Hence, for ex vitro rooting shoots were rbitrrily selected from ll the genotypes from the 3 rd subculture onwrds. In shoots subjected to pulse tretment in LM for 15 dys with IBA (24.5 µm) lone 68% rooting with 1.5 ± 0.1 cm root length ws chieved. The ddition of 2,4-D (4.5 µm) into the medium enhnced rooting up to 95% with the root length of 6.1 ± 0.2 cm (Tble 2, Fig. 3). 2,4-D nd IBA hve synergistic effects not only on rooting but lso on root length. The root length of pulse treted shoots for 10 dys ws 4.2 cm nd in 15 dys it ws 6.1 cm; hence 15-dy pulse tretment ws optimum for ex vitro rooting nd estblishment of plnts. Shoots in the control filed to root. The high rooting bility of the 3 rd subculture shoots ws conferred by ex vitro rooting results s rooting percentges obtined by both the methods were quite similr (> 90%). Ex vitro rooted plnts were further hrdened in green- 110 J. FOR. SCI., 62, 2016 (3):

5 () (b) (c) (d) (e) (f) (g) (h) (i) Fig. 3. Micropropgtion of Simmondsi chinensis (Link) Schneider using nodl segments: multiple shoot bud induction upon 1st subculture (br = 1.0 cm) (), genertion nd elongtion of shoot buds on Murshige nd Skoog (MS) medium supplemented with 2.3 µm thidizuron (TDZ) (br = 1.0 cm) (b), multiple shoot bud induction from xillry buds of in vitro shoots on MS medium supplemented with 2.3 µm TDZ (br = 1.5 cm) (c), in vitro rooted shoots in ½ strength hormone-free MS medium supplemented with chrcol 0.02% (br = 1.5 cm) (d), pulse treted shoots (4 5) plced in single poly bg contining sterile snd for ex vitro rooting covered with poly bg, ex vitro rooted plnts in bgs fter 3 weeks (br = 2.0 cm) (e), ex vitro rooted plnt removed from the bg fter 4 weeks (f), flowering resulted into successful fruiting (br = 5.0 cm) (g, h), nd ex vitro rooted plnts flowered within yer (i) house with 99% survivl rte. No visul morphologicl bnormlities were observed in generted plnts. Ex vitro rooted plnts flowered within yer (Fig. 3) nd flowering resulted in successful fruiting (Fig. 3). In the comprtive study of these rooting methods, the number of roots developed through in vitro rooting ws higher (1 15) s compred to ex vitro (4 5) rooted shoots. Roots developed ex vitro were long nd with the secondry root system without ny cllus t the bse. The totl time tken for plntlet genertion through ex vitro rooting ws 135 dys nd ws very much shorter s compred to in vitro rooting 180 dys (Fig. 4). J. FOR. SCI., 62, 2016 (3):

6 Tble 2. Effect of hormones on ex vitro rooting of Simmondsi chinensis (Link) Schneider Hormones (μm) Pulse tretment (dys) IBA NAA 2,4-D rooting (%) root length (cm) rooting (%) root length (cm) ± 2.1 c 0.5 ± ± ± ± 1.2 f 1.0 ± 0.1 b 68 ± 1.2 d 1.5 ± 0.1 b ± 1.2 d 0.6 ± ± 1.2 d 0.8 ± ± ± ± ± ± 1.8 c 0.8 ± ± ± 0.1 b ± 1.1 b 1.7 ± ± ± ± 1.9 c 1.2 ± 0.2 b 43 ± 1.9 b 1.2 ± 0.2 b ± 1.8 d 1.5 ± 0.2 b 49 ± 2.8 c 1.6 ± 0.1 b ± 1.7 c 1.4 ± 0.1 b 45 ± 1.6 b 1.5 ± 0.1 b ± 1.5 f 3.8 ± 0.1 b 90 ± 2.3 d 5.2 ± 0.1 b ± 1.6 f 4.2 ± 0.1 c 95 ± 2.2 f 6.1 ± 0.2 c ± 1.4 f 4.1 ± 0.2 c 91 ± 2.1 d 6.0 ± 0.1 c vlues represent mens ± SE of the tretment of 20 explnts in three replicte experiments, mens followed by different letters re significntly different t 0.05 probbility level; IBA indole-3-butyric cid, NAA 1-nphthlenecetic cid, 2,4-D 2,4-dichlorophenoxycetic cid DISCUSSION A non-purine phenylure derivtive TDZ is widely used for shoot development (Mlik, Sxen 1992; Bhgwt, Lne 2004). It is found to mimic cytokinin-like ctivity, nd to promote relese of lterl buds from dormncy (Wng et l. 1986). It hs lso been reported s potentil plnt growth regultor to induce high frequency of shoot regenertion, prticulrly in woody plnt species (Huettemn, Preece 1993) but no ttempts hve been mde in Jojob, typicl hrdwood plnt. In the present investigtion, xillry bud induction ws observed in the medium supplemented with 4.60 µm TDZ, however, induced shoot buds filed to elongte t the sme TDZ concentrtion. Similr results were reported in chili pepper by Hyde nd Phillips (1996). Upon subsequent subcultures, 20 shoots per explnt with cm length were obtined on MS medium contining 2.30 µm TDZ fter 5 weeks of culture. Similr results were reported in Cmelli sinensis Linneus nd Mus cumint Coll (Mondl et l. 1998; Frhni et l. 2008). The effect of lone 6-benzylminopurine (BAP) nd in combintion with uxin on micropropgtion of Jojob hs been documented by different rtes of shoot multipliction (Driver, Kuniyuki 1984; Chturvedi, Shrm 1989; Mills et l. 1997; Llorente, Apóstolo 1998; Tygi, Prksh 2004; Singh et l. 2008). In the present study, TDZ lone could give rise to 20 shoots. Such response my be due to n increse in the level of endogenous cytokinins. Aprt from its cytokinin-like ctivity, it hs been suggested to be modultor of the endogenous uxin levels (Murthy et l. 1995). This my be the reson why during the present study TDZ might 1 st culture Totl time tken for plnt genertion by in vitro rooting method (180 dys) In vitro hrdening Greenhouse 91% survivl in nurser y In vitro rooting 2 nd culture 3 rd culture Ex vitro rooting nd hrdening Greenhouse 15 dys 99% survivl in nurser y Totl time tken for plnt genertion by ex vitro rooting method (135 dys) Fig. 4. Schemtic representtion of time tken for plntlet genertion using in vitro nd ex vitro rooting methods of Simmondsi chinensis (Link) Schneider 112 J. FOR. SCI., 62, 2016 (3):

7 hve scored higher numbers of shoots. BAP is reported to produce higher number of shoots per explnt, however, it induces cllus nd hyperhydrted shoots (Llorente, Apóstolo 1998, 2000; Mills et l. 2004; Tygi, Prksh 2004). We could chieve more thn 90% in vitro rooting, which ws higher thn erlier (Chturvedi, Shrm 1989; Mills et l. 1997; Tygi, Prksh 2004). The rooting percentge of shoots incresed from round 39 to 92% from the first to the third subculture, hence from the 3 rd culture onwrds shoots re more suitble for rooting, which my be due to the better physiologicl condition of shoots for better rooting. Similr results were observed in Euclyptus L'Heritier (Gonclves 1980; Gupt et l. 1981; Bdi 1982; Mccomb, Bennett 1982). Also in perennil plnts/tree species the growth of the culture my be slow s compred to nnul plnts, nd restore their juvenile trits essentil for rooting upon subsequent subcultures, hence this my be one of the resons for high rooting percentge in older cultures/3 rd subculture onwrds. A progressive increse in the rooting bility of chestnut microshoots occurred t the 8 th subculture (Feijó, Pis 1990; Wng, Guo 2007). In Chinese chestnut (Cstne mollissim Blume) it ws observed tht upon subculture the levels of endogenous indolecetic cid (IAA) in microshoots grdully incresed, nd endogenous levels of bscisic cid, cytokinin nd gibbrelic cid in microshoots decresed. The level of 3-IAA ws directly correlted with subculture numbers nd rooting rtes in the chestnut plnt (Hou et l. 2010). In ll the genotypes rooting bility ws very close to ech other, which my be due to similr growth regultor uptke, trnsloction rte nd metbolic processes mong genotypes (Blkesley 1991). Similrly to in vitro rooting, high ex vitro rooting (> 90%) ws chieved from the shoots of the 3 rd subculture onwrds. However, shoots generted ccording to Singh et l. (2008) resulted in 82.5% ex vitro rooting (dt not shown). Ex vitro rooting using IBA in combintion with 2,4-D ws found to be best s compred to lone IBA; similr results were observed in Stchousi tryonii (Bhti et l. 2002). Rooting in snd or in uxin-free medium corrobortes other reports tht lthough uxins re essentil for root induction, they my not be required for root growth nd their continued presence my even inhibit the root growth (Singh et l. 2008). Ex vitro rooted plntlets re usully more suited for trnsplnttion s compred to in vitro rooted plntlets (Andersen 1986). The root system developed ex vitro ws different from the root system developed during in vitro rooting prctice. The ex vitro rooting method ws lso found to be better for the survivl nd estblishment of the plnt s compred to the in vitro rooting (Singh et l. 2011, 2014). We report here rooting in one step through ex vitro rooting to minimize time nd lbour for plntlet genertion. Numerous studies hve ddressed in vitro rooting in this plnt to the best of our knowledge till now but there is no report on ex vitro rooting of micropropgted shoots of S. chinensis mle nd femle plnts. The study clerly figured out the effect of subculture on rooting efficiency nd lso found tht the rooting pttern of ex vitro rooting is preferble for the better estblishment nd survivl of the plnt. This protocol will be useful for mss propgtion in short time. References Agrwl V., Prksh S., Gupt S.C. (2002): Effective protocol for in vitro shoot production through nodl explnts of Simmondsi chinensis. Biologi Plntrum, 45: Andersen A.S. (1986): Environmentl influences on dventitious rooting in cuttings of non-woody species. In: Jckson M.B. (ed.): New Root Formtion in Plnts nd Cuttings. Dordrecht, Mrtinus Nijhoff: Apóstolo N.M., Llorente B.E. (2000): Antomy of norml nd hyperhidric leves nd shoots of in vitro grown Simmondsi chinensis (Link) Schneider. In Vitro Cellulr nd Developmentl Biology Plnt, 36: Bdi N.K. (1982): Euclyptus rudis Endl.: Techniques de micropropgtion pr l culture de noeuds in vitro. In: Colloque Interntionl sur l Culture in vitro des Essences Forestières, IUFRO, AFOCEL, Nngis, Frnce: Bhgwt B., Lne W.D. (2004): In vitro shoot regenertion from leves of sweet cherry (Prunus vium) Lpins nd Sweethert. Plnt Cell nd Tissue Culture, 78: Bhrdwj M., Uppl S., Jin S., Khrb P., Dhillon R., Jin R.K. (2010): Comprtive ssessment of ISSR nd RAPD mrker ssys for genetic diversity nlysis in Jojob [Simmondsi chinensis (Link) Schneider]. Journl of Plnt Biochemistry nd Biotechnology, 19: Bhti N.P., Bhti P., Ashwth N. (2002): Ex vitro rooting of micropropgted shoots of Stckhousi tryonii. Biologi Plntrum, 45: Blkesley A. (1991): Uptke nd metbolism of 6-benzyldenine in shoot prolifertion of Mus nd Rhododendron. Plnt Cell nd Tissue Culture, 25: Chng Y., Chen S., M B., Lu Z. (1991): Studies of the reltionship between some hormones nd dventitious root formtion in shoot-tip culture of pple (Mlus pumil mill cvs). Journl of Agriculturl University of Hebei, 14: 1 5. J. FOR. SCI., 62, 2016 (3):

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