A comparison of conventional cloning options for annatto (Bixaorellana L.)

By N. JOSEPH, E. A. SIRIL* and G. M. NAIRDepartment of Botany, University of Kerala, Kariavattom, Thiruvananthapuram-695 581, Kerala,India (e-mail: easiril@yahoo.com) (Accepted 13 May 2011)

SUMMARYAnnatto (Bixa orellana L.) is a tree indigenous to tropical America that has been naturalised throughout the Indiansub-continent. The plant is chiefly valued for its carotenoid pigments, annatto. Methods for the clonal propagation ofhardwood and softwood cuttings, air-layering, budding, and grafting of annatto were investigated. Hardwood cuttingscollected during the wet Summer months (June-July) were treated with indole-3-acetic acid (IAA), indole-3-butyricacid (IBA), or naphthaleneacetic acid (NAA) at varying concentrations. The highest percentage rooting (63.4%),along with highest number of roots (8.33 per cutting) were achieved by hardwood cuttings after 12 h of 2.5 mM IAAtreatment. Among the various auxin treatments of softwood cuttings, 5.0 mM IBA in combination with 5.0 mM NAAfor 5 min resulted in a significant (P < 0.05) rooting percentage (56.7%). The responses of softwood cuttings to anauxin analogue (boric acid) and/or to thiamine hydrochloride (vitamin B) were inferior to synthetic auxins. Whensubjected to air-layering, hardwood branches gave 100% rooting without any application of hormone. Among thedifferent types of budding methods attempted (patch, T, or I), patch budding produced the highest efficiency (78%) ofpropagation. Splice-grafting could also be used for conventional propagation of annatto, with a 50% survival rate.Annatto can therefore be cloned by adopting these methods. Propagation based on softwood cuttings facilitatedmoderate-scale cloning of this valuable, elite germplasm.

Bixa orellana L. (family Bixaceae), or annatto, is atree native to tropical America. Annatto is the only

known source for an orange-red carotenoid pigment,bixin, which is obtained from the pulpy seed coat(Satyanarayana et al., 2003). Annatto is used extensivelyin the food, dairy, cosmetics, and textile industries.Natural colourants in foodstuffs have a higheracceptance value compared to their syntheticcounterparts (Henry, 1996). Since annatto is one of the13 basic pigments derived from natural sources that arecurrently permitted as food colourants by the US Foodand Drug Administration (US FDA), there is an ever-increasing demand for annatto pigment. Furthermore,annatto is now recognised as an ingredient in severalstandard herbal formulations (Caius, 1986).

Although B. orellana L. has considerable commercialand medicinal importance, the plant has not been fullycommercialised in India. Two major problems associatedwith the commercial cultivation of annatto are: (i)annatto is traditionally propagated by seed, as a cross-pollinated species, and so is highly heterozygous; and (ii)as a perennial woody species it has a long generationcycle, thus any strategy to increase the content of annattopigment requires a long-term breeding programme. Asan alternative, the cloning of naturally available, superiorplants could lead to more stable and/or significantlyenhanced productivity, thus making the commercialcultivation of annatto an economically viable venture.All previous attempts to micropropagate B. orellana L.used seedling-derived explants (D’ Souza and Sharon,

2001; Paiva Neto et al., 2003; Nassar et al., 2003;Parimalan et al., 2007; 2009). Unfortunately, no standardprotocol has been reported for the propagation ofmature elite plants. Therefore, conventional vegetativepropagation methods are the only way by which to cloneelite plants.

In the present work, various vegetative propagationmethods such as hardwood and softwood cuttings, layering,grafting, and budding methods were assessed for thecloning of B. orellana. Reports on vegetative propagationusing hardwood cuttings (Thirunavoukkarasu and Saxena,1997) or semi-hardwood cuttings (Navamaniraj et al., 2008)are available. Two reports (Muller et al., 1990; San-Miguelet al., 1999) on vegetative propagation used terminalcuttings, but only indole-3-butyric acid (IBA) was studied.

MATERIALS AND METHODSAll experiments were conducted at the experimental

nursery of the Department of Botany, University ofKerala (8º33'03.86" N; 76º52'38.64" E; 18 m asl).

Rooting response of hardwood cuttingsBranch cuttings (n = 300) of B. orellana L., each approx.

1.0 m in length, were collected from 4-year-old plantsduring a wet Summer (June-July 2009).The 50 cm-long tipportions of each branch were rejected. Uniform, cuttings(20 – 25 cm long; 2 – 3 cm in diameter) were then excisedfrom the basal part of each collected branch and werepooled (n = 300; then divided into ten groups, each of 30cuttings). The leaves were removed prior to indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or*Author for correspondence.

Journal of Horticultural Science & Biotechnology (2011) 86 (5) 446–451

N. JOSEPH, E. A. SIRIL and G. M. NAIR 447

naphthaleneacetic acid (NAA) treatment, each at 0.5 mM,2.5 mM, or 5.0 mM. All the auxins used were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA). Eachphytohormone was dissolved in a few drops of 1.0 MNaOH and further dilutions were made using distilledwater. One group of cuttings (n = 30) was dipped indistilled water as a control. Cuttings were arranged inbundles (n = 10) and 5 cm of the basal cut-end was dippedin each auxin solution for 24 h. The auxin-treated cuttingswere then planted in 10 m � 2.5 m nursery beds filled witha 1:1 (v/v) mix of farmyard manure (FYM) and soil. At 45d after planting (DAP), the cuttings were uprooted andthe following parameters were determined: (i) thepercentage of rooting; (ii) the number of roots per cutting;(iii) the mean root length per cutting (cm); (iv) the shootformation percentage; (v) the mean number of shoots percutting; and (vi) the mean shoot length per cutting.

Rooting response of softwood cuttingsSoftwood cuttings (n = 840) of current season growth

(October 2009), each with a shoot tip 10 cm in length,were excised from source trees and were rapidly placedin polyethylene trays containing water. Each leaf was cutin half and the cuttings were arranged in bundles, each often cuttings. Cuttings were treated for 5 min in 5 mM, 10mM, 15 mM, or 20 mM IAA, IBA, or NAA, or in variouscombinations of two auxins (i.e., IAA+IBA; IAA+NAA;or IBA+NAA) at 5 mM or 10 mM each.

In another set of experiments, softwood cuttings (n =270) were treated as above with thiamine hydrochloride(a B-vitamin) or boric acid (an auxin analogue) at eachof four concentrations (10, 20, 30, or 40 mM) for 5 min.

The phytohormone solutions for the pulse treatmentswere prepared by dissolving each auxin in 100% (v/v)ethanol and diluting this to 50% (v/v) EtOH usingdistilled water. The thiamine hydrochloride and boricacid solutions were simply prepared in distilled water.One group of cuttings (n = 30) was dipped in distilledwater for 5 min to serve as controls. The basal 2 – 3 cmportion of each cutting was dipped in each hormonesolution for 5 min. The auxin-treated softwood cuttingswere then planted in plastic cups (diameter = 7 cm;height = 8 cm) containing a 1:1:1 (v/v/v) mixture of sand,soil, and FYM and the potted cuttings were covered withpolyethylene bags to ensure a high relative humidity.Thecuttings were irrigated at 2 d intervals. Observations onthe percentage of rooting, mean root number and meanroot length per cutting, the percentage of shooting, thenumber of fully-expanded new leaves per cutting, andmean shoot growth were recorded 90 DAP. To assess thenumber of days required for rooting of stem cuttings,three randomly-chosen cuttings were uprooted in eachtreatment at 30, 45, 60, 75, and 90 DAP.

Air-layeringTo determine the potential of hardwood branches as

planting material, an air-layering experiment wasconducted. The air-layering was done during a wetSummer (June-July 2009) using six 4-year-old, healthymother trees. Air-layering was performed on the lowerbranches (2 – 3 cm in diameter and 0.5 – 1.0 m in length).An approx. 3.0 cm-wide bark ring was removed using abudding knife, without injuring the underlying wood.Thecut surface was covered with a moistened 1:1 (v/v)

mixture of FYM and soil, and then wrapped with a 220µm-thick polyethylene strip tied at both ends. Non-destructive evaluation of rooting was recorded 2, 3, and 4weeks after air-layering. After 1 month, the rooted air-layers (n = 30) were removed from the mother plants andtransplanted into pots filled with 1:1:1 (v/v/v) mix ofsoil:sand:FYM.

Budding and graftingExperiments were conducted during June-August

2009. Six-month-old seedlings, with a stem diameter of 2– 3 cm and 30 cm in height, were selected as rootstockplants and were maintained in polyethylene potscontaining a 1:1:1 (v/v/v) mix of sand:soil:FYM for boththe grafting and the budding experiments.

Three types of budding method (patch, T, or I) wereused (Hartmann et al., 1997). Single nodal buds, togetherwith a small area (1.7 cm2) of bark, were excised from themother plants and used as scions. The excised buds wereprocessed carefully so that no woody debris was retainedon the bud and they were kept in fresh water until used.The bark on each stock plant was injured approx. 15 cmabove soil level in a patch, T, or I shape, and opened, intowhich a prepared bud was inserted (Hartmann et al.,1997). The portion of the rootstock where the scion wasinserted was then protected by wrapping with apolyethylene strip (as above), starting from top. Both thegrafting and the budding experiments were conducted ina shade-house with 35 – 40% shading.

Splice grafts were prepared using a scion 2 – 3 cm indiameter from the actively-growing branches of themother trees. The scions were cut to form a whip-liketapering base.The top portion of the root stock plant wasremoved approx. 15 cm above ground level and a slit wasmade on the cut surface so that the scion was firmlyinserted into the rootstock. The inserted scion wasprotected from infection and dehydration by wrappingwith a polyethylene strip (as above). For the budding andgrafting experiments, data were collected on bud or graftsurvival rates.

Experimental design and statistical analysis of the dataExperiments using branch cuttings (both hardwood

and softwood cuttings) were performed in a randomisedcomplete block design. For each treatment, there werethree replicate blocks, each consisting of ten cuttings perblock. Data on the rooting percentage, root numbers,root lengths, the percentage of shoot formation, themean number of shoots and shoot lengths weresubjected to two-way analysis of variance (ANOVA).Prior to ANOVA, the percentage data sets were arcsine-transformed to meet the assumptions of normality. Themeans of the transformed values were re-transformedfor presentation (Snedecor and Cochran, 1962). Meansseparation was performed using Duncan’s New MultipleRange Test (P < 0.05). In both the grafting and buddingexperiments, three replications, each consisting of tengrafted or budded plants, were used to record bud orgraft survival.

RESULTS Rooting response of hardwood cuttings

The rooting responses of B. orellana L. hardwood

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cuttings showed significant (P < 0.001) effects of thevarious auxin treatments (Table I). The highest rootingpercentage (63.4%) was recorded in 2.5 mM IAA-treated cuttings, followed by 0.5 mM IAA-treatedcuttings (50%). Controls without any auxin treatmentfailed to root (Table I; Figure 1A). The type of auxin andthe concentration used had a significant (P < 0.001)effect on adventitious root formation. The highestconcentration (5.0 mM) of IAA or NAA significantlyinhibited rooting. IBA did not induce adventitious rootsirrespective of concentration. Two-way ANOVArevealed a significant (P < 0.001) interaction of auxin

type and concentration on the rooting percentage, rootlength, root number, and shoot length. Indole-3-aceticacid (at 2.5 mM) induced the maximum number of rootsper cutting (8.3) and increased mean root length (11.90cm). Indole-3-acetic acid (at 0.5 mM) resulted in thedevelopment of the maximum number of shoots (5.3)per cutting at 45 DAP. The shoots that developed on 2.5mM IAA-treated cuttings grew to 4.36 cm in size within45 d. Indole-3-butyric acid and NAA at varyingconcentrations (0.5, 2.5, or 5.0 mM) resulted in a reducedpercentage of sprouting. The emergence of shoots fromdormant axillary buds was noticed 20 DAP.

TABLE IEffect of various auxins on shoot formation and the rooting response of hardwood cuttings of B. orellana L.

Concentration Rooting Root number/ Mean root length Shooting Mean no. of Mean sproutlengthAuxin (mM) percentage rooted cutting per cutting (cm) percentage sprouts per cutting per cutting (cm)

Control (no auxin) 0.0 0.0 e† 0.0 f 0.0 f 46.6 ab 2.6 d 1.5 dIAA 0.5 50.0 b 2.8 c 6.0 b 60.0 a 5.3 a 2.6 c

2.5 63.4 a 8.3 a 11.9 a 63.4 a 4.9 b 4.3 a5.0 4.5 d 1.1 e 2.9 d 1.2 fg 0.0 g 0.0 f

IBA 0.5 0.0 e 0.0 f 0.0 f 10.0 de 1.6 e 1.2 e2.5 0.0 e 0.0 f 0.0 f 1.2 fg 1.0 f 0.9 e5.0 0.0 e 0.0 f 0.0 f 0.0 g 0.0 g 0.0 f

NAA 0.5 26.5 c 3.8 b 1.9 e 33.3 bc 4.3 c 3.7 b2.5 16.4 c 1.5 d 4.1 c 19.3 cd 1.1 f 2.9 c5.0 0.0 e 0.0 f 0.0 f 4.5 ef 0.8 f 1.1 e

Treatment Df (n-1) 9 64.32** 839.24** 2,110.75** 26.42** 368.25** 183.16**Auxin Type (T) Df (n-1) 2 20.52** 303.16** 167.99** 0.68 175.83** 94.10**Auxin conc. (C) Df (n-1) 2 7.33** 255.75** 127.90** 0.16 117.95** 129.96**T � C Df (n-1) 4 9.07** 273.85** 104.08** 0.55 18.32** 30.91**†Mean values within a column followed by the same lower-case letters are not significantly (P < 0.05) different as determined by Duncan’s NewMultiple Range Test.**Significant at P < 0.001. *Significant at P < 0.05.

FIG. 1Vegetative propagation of B. orellana L. Panel A, rooting of hardwood cuttings: 1, control; 2, IAA at 0.5 mM; 3-6, IAA at 2.5 mM; 7, NAA at 0.5mM; 8-9, NAA at 2.5 mM (scale bar = 4 cm). Panel B, 3-month-old rooted softwood cuttings (scale bar = 5 cm) Panel C, adventitious rooting ofsoftwood cuttings treated with 5 mM IBA + 5 mM NAA (scale bar = 5 cm). Panel D, rooted air-layer (1 month after layering; scale bar = 5 cm).

Panel E, patch-budding showing the emergence of a scion (scale bar = 3.5 cm). Panel F, splice-grafting (scale bar = 5.5 cm).

N. JOSEPH, E. A. SIRIL and G. M. NAIR 449

Rooting response of softwood cuttingsCuttings treated with IBA, in combination with NAA

(cumulative concentration 10 mM), showed asignificantly increased (P < 0.05) rooting response(56.7%), mean root number per cutting (18.26), and meanshoot length (2.04 cm; Table II; Figure 1B,C). Pulsetreatment (5 min) with 20 mM IBA inhibited rooting.Indole-3-butyric acid (at 15 mM) resulted in 50% rooting,which was statistically similar to 5.0 mM IAA. Thenumber of newly-formed leaves was found to be high(1.6) in the 10 mM IAA treatment.The responses to boricacid and thiamine hydrochloride were relatively poorcompared to the auxins (Table III). Among the differentconcentrations of thiamine hydrochloride and boric acidtested, 20 mM thiamine hydrochloride produced 20%rooting, with an average of 6.5 roots per cutting. Boricacid at 40 mM gave 13% rooting. Control cuttings plantedwithout any auxin treatment failed to root.

Rooting response of air-layersAir-layers raised during a wet Summer (June-July

2009) produced 100% rooting within 1 month (Figure1D). Prolific roots developed, without any auxintreatment. Two weeks after air-layering, roots werevisible. After 1 month, developed air-layers weredetached from the mother plant and transplanted,initially, in polyethylene bags filled with 1:1:1 (v/v/v)

soil:sand:FYM, then planted in the field. Callusformation at the cut surface was noticed 1 week after air-layering. Subsequently, prominent white button-likestructures were formed which later differentiated intoroots.

Budding and graftingAmong the three types of budding (patch, T, or I), the

patch-budding technique resulted in the maximum rateof survival (78%), followed by I-budding (62%), and T-budding (34%). Patch-budded rootstocks produced budswithin 4 weeks (Figure 1E). Splice grafting showed 50%survival (Figure 1F). Removal of the polyethylene stripsafter 3 weeks revealed the wound-healing activitythrough callus tissue formation at the point of scioninsertion. Graft or bud union in B. orellana L. took placewithin 2 weeks, as the wound tissue appeared and joinedthe rootstock and the scion. Successfully grafted orbudded plants were initially maintained in partial shade,then moved to field conditions. The grafted and buddedplants flowered and set fruit after 2 months.

DISCUSSION Propagation of B. orellana L. through various

vegetative means has been accomplished. The suitabilityof IAA to elicit adventitious rooting in annatto has been

TABLE IIIEffect of thiamine hydrochoride or boric acid on rooting of B. orellana L. softwood cuttings

Conc. Rooting Mean root Mean root length Shooting No. of new leaves Mean shoot lengthTreatment (mM) (%) number per cutting per cutting (cm) (%) per cutting per cutting (cm)

Control 0.0 0.0 b† 0.0 d 0.0 e 6 ab 0.0 b 0.0 dThiamine 10.0 4.0 ab 1.6 b 6.3 ab 10 ab 0.6 ab 0.4 cd

hydrochloride 20.0 20.0 a 6.5 a 4.6 abcd 20 a 0.5ab 1.3 a30.0 13.0 ab 2.5 b 5.6 abc 13 ab 0.5 ab 1.2 bc40.0 10.0 ab 2.3 b 7.3 a 10 ab 0.6 ab 0.7 abc

Boric acid 10.0 1.2 b 0.3 c 1.3 de 4 ab 0.3 ab 0.3 cd20.0 10.0 ab 1.6 b 2.1 cde 10 ab 0.3 ab 0.6 bc30.0 10.0ab 2.6 b 3.4 bcde 10 ab 0.6 ab 0.8 abc40.0 13.0 ab 6.8 a 2.1 cde 20 ab 1.1 a 0.8 abc

†Mean values within a column followed by the same lower-case letters are not significantly (P < 0.05) different as determined by Duncan’s NewMultiple Range Test.

TABLE IIEffect of various auxins on the rooting response of softwood cuttings of B. orellana L.

Conc. Rooting Mean root Mean root length Shoot No. of new leaves Mean shoot lengthAuxin (mM) (%) number per cutting per cutting (cm) formation (%) per cutting per cutting (cm)

Control (no auxin) 0.0 0.0 d† 0.0 i 0.0 j 6.7 c 0.0 f 0.0 iIAA 5.0 46.6 b 3.0 h 7.1 c 53.4 a 1.5 ab 1.5 b

10.0 16.4 c 3.1 h 7.9b 16.4 b 1.6 ab 1.2 bcde15.0 10.0 c 4.6 g 4.2 g 10.0 bc 1.3 abcd 1.2 bcd20.0 0.0 d 0.0 i 0.0 j 0.0d 0.0 f 0.0 i

IBA 5.0 10.0 c 17.0 b 2.3 i 10.0 bc 1.3 abcd 1.0 efg10.0 16.4 c 10.3 d 5.9 e 16.4 b 1.1 abcd 1.3 bc15.0 50.0 ab 13.4 c 7.0 c 50.0 a 1.3 abcd 1.3 bc20.0 0.0 d 0.0 i 0.0 j 0.0 d 0.0 f 0.0 i

NAA 5.0 16.4 c 3.0 h 3.9 gh 16.4 b 0.5 ef 0.8 g10.0 46.6 b 6.5 e 8.3 a 46.6 a 0.9 de 1.1 cdef15.0 13.0 c 4.1 g 5.8 e 13.0 bc 1.1 abcd 0.9 fg20.0 0.0 d 0.0 i 0.0j 0.0 d 0.0 f 0.0 i

IAA + NAA 5+5 10.0 c 2.6 h 3.7 h 10.0 bc 0.9 de 1.0 defg10+10 16.4 c 4.8 g 6.4 d 16.4 c 0.7 de 0.6h

IBA + NAA 5+5 56.7 a 18.2 a 4.9 f 56.7 a 1.5abc 2.0 a10+10 0.0 d 0.0 i 0.0 j 0.0 d 0.0 f 0.0 i

IAA + IBA 5+5 13.0 c 5.6 f 4.1 g 13.0 bc 1.0 bcde 1.4 bc10+10 0.0 d 0.0 i 0.0 j 0.0 d 0.0 f 0.0 i

†Mean values within a column followed by the same lower-case letters are not significantly (P < 0.05) different, as determined by Duncan’s NewMultiple Range Test.

Vegetative propagation of annatto450

reported (Thirunavoukkarasu and Saxena, 1997). Thepositive effect of IBA on root induction in softwoodcuttings was also reported earlier (San Miguel et al.,1999). In contrast, Muller et al. (1990) reported adetrimental effect of IBA on apical cuttings of annatto.In this study, softwood cuttings responded more to IBAtreatment compared to other auxins (IAA or NAA).

The rooting response of softwood cuttings variedsignificantly with the type and concentration of auxin.The enhancement of rooting using IBA indicated thatsoftwood cuttings were more responsive to this hormonethan hardwood cuttings. Similar effects have also beenreported in species such as Acacia mangium Willd.(Poupard et al., 1994) and Colutea arborescence L. (deAndres et al., 1999). The differential response ofsoftwood and hardwood cuttings to different auxinsmight be due to different ratios of endogenous root-promoting substances and/or reserve substances in eachtype of cutting. This may be explained by the greateraccumulation of reserves in hardwood cuttings (deAndrés et al., 2004).

Indole-3-butyric acid, in combination with NAA, hada synergistic effect on the induction of roots, whichbecame evident not only as a higher percentage ofrooted cuttings, but also in the number of roots percutting (Table II). Similar synergies between IBA andother auxins have been reported in woody plants such asAisandra butyraceae (Roxb.) Baehni (Tewari and Dhar,1997) and Pongamia pinnatta L. (Kesari et al., 2009).

The inferior rooting responses to boric acid orthiamine hydrochloride contrast with other reports onwoody species such as Azadirachta indica A. Juss and P.pinnata L. (Palanisamy et al., 1998), where a superioreffect of these chemicals over auxins was reported.

In the present study, using hardwood cuttings, therooting percentage, mean root number per cutting, androot length were high in 2.5 mM IAA. Supra- and sub-

optimal concentrations of IAA caused significant (P <0.05) reductions in the rooting percentage and in thenumber of roots. Reports on the dosage effects ofhormones on adventitious root formation are available;for example, in Podophyllum hexandrum Royle(Nadeem et al., 2000). The positive effect of IAA on therooting response has also been reported in Celastruspaniculatus Willd. (Raju and Prasad, 2010). The failure ofcontrol cuttings to root indicated that the endogenouslevels of auxins were insufficient for root induction, evenduring the period of active growth in this plant.

The success of air-layers and grafts in the present workagrees with previous reports (Bruckner et al., 1991;Barbosa et al., 1993). The 100% survival rate of air-layerswas achieved during the monsoon. An important factorthat promotes rooting is the ability of branches toaccumulate sugars and auxins from the leaves and theshoot tip. The practice of girdling during air-layeringstimulated the mobilisation of sugars and auxins, andconsequently their accumulation in the basal portion ofgirdled branches. The added food reserves accumulatedat the base, in turn, support root formation (Adrianceand Brison, 1955; Hartmann et al., 1997). From thepresent study, it is evident that B. orellana L. is a difficult-to-root species. However, there were differences in thesuccess of rooting between softwood and hardwoodcuttings. The vegetative propagation methods for B.orellana L. demonstrated in the present study providevarious options to multiply ‘elite’ germplasm and toincrease the productivity of annatto.

We thank Dr. Ashalatha S. Nair, Professor and Head,Department of Botany, University of Kerala for providingresearch facilities. We also thank Kerala State Council forScience, Technology and Environment (KSCSTE),Government of Kerala, Thiruvananthapuram, India forfinancial support (Project No. 028, SRSLS/ 2007/ CSTE).

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