297

The Korean Society of Crop Science

J. Crop Sci. Biotech. 2011 (December) 14 (4) : 297 ~ 303

RESEARCH ARTICLE

DOI No. 10.1007/s12892-011-0037-z

Comparison of Agar and Gum Karaya as Gelling Agent for invitro Regeneration of Rough Lemon (Citrus jambhiri Lush.)Plantlets from Nodal Explants

Balwinder Singh1*, Amritpal Kaur2

1Plant Tissue Culture Lab, Department of Biotechnology, Khalsa College Amritsar 143 002 (Punjab), India2Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143 005 (Punjab), India

Received: June 15, 2011 / Revised: July 28, 2011 / Accepted: September 15, 2011Ⓒ Korean Society of Crop Science and Springer 2011

Abstract

In the present study, attempt was made to compare agar with gum karaya as gelling agent in micropropagation of rough lemon(Citrus jambhiri Lush.). Initially nodal segments were cultured on agar-gel MS medium containing benzyladenine (BA), kinetin(KN), zeatin (ZN) (1.0 - 2.5 mg L-1) and malt extract (200 - 1,200 mg L-1) to standardize the medium. Maximum shoot regeneration(66.66%) was observed with KN 2 mg L-1 with an average shoot length of 0.73 cm. Gum karaya and agar was then evaluated at dif-ferent concentration and combinations in same medium. The shoot regeneration response on media gelled with 30 g L-1 gum karayawas 62.49% with an average shoot length of 0.80 cm. Regenerated shoots were rooted on MS medium gelled with agar and supple-mented with different concentrations (0.5 - 2.5 mg L-1) of indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), and indole-3-butyric acid (IBA). Maximum response (52.77 %) was observed with IBA 2.0 mg L-1 with an average number of 2.58 roots/shoot. Amaximum of 53.47% cultures showed root regeneration with an average number of 2.91 roots/shoot in 30 g L-1 gum karaya-gel medi-um. Texture measurements revealed that firmness of gum karaya-gel medium was nowhere near to that of agar. However, in theircapability of supporting growth and differentiation of explants they are equal to agar medium. Gum karaya forms less adhesive andgummy medium as compared to agar. This study indicates that gum karaya can be used as gelling agent in place of agar.

Key words: agar, Citrus, gum karaya, micropropagation, nodal segments, rough lemon

Citrus are almost universally propagated by budding ontoseedling rootstocks. Rough lemon (Citrus jambhiri Lush.) is themost commonly used rootstock for different citrus cultivars. Itforms a normal union with all scion varieties, develops a deeprooting system, produces heavy yields, and gives a long life totree when planted on a suitable soil (Altaf et al. 2008). It is animportant rootstock in North Indian states like Punjab, Haryana,Rajasthan, and Uttar Pradesh. In Punjab, there are only few C.jambhiri plants available for seed collection. Further, these seedshave a very short life span because they lose their viability

(Johnston 1968), so they cannot be stored for a longer period oftime. When seed of desired rootstock is not available in suffi-cient quantities, it can be propagated as stem cuttings. Such root-stocks have greater uniformity because of absence of the varia-tion which sometimes appears as the result of the variableseedling rootstocks of grafted plants (Hartman et al. 1997).Micropropagation offers a number of advantages over conven-tional propagation methods, such as the possibility of obtaininga large number of true-to-type, virus-free plants in relatively lesstime and in limited space (Barlass and Skene 1982). It may bealso useful in providing plant material for in vitro conservationand genetic transformation studies (Marutani-Hert et al. 2010;Perez-Tornero et al. 2010).

There are some reports about regeneration of plantlets from

Introduction

Balwinder Singh ( )E-mail: bbs171@rediffmail.com

Gum Karaya as Gelling Agent in Plant Tissue Culture Media298

nodal segments of citrus. Al Khayri and Al-Bahrany (2001)studied shoot multiplication and elongation from nodal segmentsof lime. They observed that regenerated shoots grew vigorouslyand developed into plantlets on rooting medium without anyabnormalities. Begum et al. (2004) reported shoot proliferationfrom the nodal explants of Pummelo (C. grandis (L.) Osb.).Rathore et al. (2007) developed a protocol for in vitro cloning ofmature plants of Citrus limon using nodal segments. Perez-Tornero et al. (2010) investigated the effect of basal mediumand different plant growth regulators on micropropagation ofnodal explants from mature trees of lemon cultivars. Samarina etal. (2010) reported successful regeneration of lemon plantletsfrom nodal explants. They studied the effect of sterilizationagents and the composition of the nutrient medium on nodalexplant regeneration. In spite of micropropagation of citrusgenotypes reported by several workers, very few reports on invitro propagation of rough lemon have been reported (Ali andMirza 2006; Altaf et al. 2008; Krishan et al. 2011; Savita et al.2010, 2011). Moreover, there are no reports on direct shootorganogenesis of rough lemon through nodal segments. Directshoot regeneration through nodal segments offers several advan-tages which are not possible with conventional propagation tech-niques. It provides reliable and economical method for maintain-ing pathogen-free plants that can allow international exchange ofgermplasm. The number of plants produced would not be limit-ed by their seed supply, rather more uniform disease-free andquality plant populations might be produced.

In vitro regeneration has emerged as a powerful tool for mul-tiplication and improvement of many woody plant speciesincluding Citrus. It requires the presence of highly responsiveregeneration protocol and expensive culture medium. However,establishment of large-scale tissue culture laboratories must bebased on cost effectiveness. Agar represents one of the mostexpensive media components used. The search for low costalternative gelling agents has become crucial for plant tissue cul-ture laboratories, particularly for those involved in micropropa-gation. In earlier reports, the number of substances like cornstarch (Henderson and Kinnersley 1988; Zimmerman et al.1995), tapioca (Nene et al. 1996), isubgol (Babbar and Jain1998), guar gum (Babbar et al. 2005), gum katira (Jain andBabbar 2002), and xanthan gum (Jain and Babbar 2011) havebeen tested for their gelling ability in plant tissue culture media.The development of cost effective and highly responsive culturemedium is essentially required for in vitro propagation of impor-tant plant species. Gum karaya exuded from fibrous bark ofSterculia urens, a softwood tree of family Sterculiaceae, wascompared with agar as a gelling agent in the present study. It is acomplex, branched polysaccharide with a molecular weight of 9- 16 million daltons (Le Cerf et al. 1990). The structure of gumkaraya contains a central chain of glactose, rhamnose, and glac-turonic residues with side chains of glucoronic acid (Andersonet al. 1982).

This study was conducted with the aim to explore the regen-erative ability of rough lemon plantlets on cost effective culturemedium to make their utility more effective in orchard establish-ment of citrus crop.

Materials and Methods

Source of explant and gumHealthy rough lemon plants growing at the Govt. Nursery,

Department of Horticulture (Punjab), Attari, Amritsar, Indiawere selected and used as a source of explants for raising invitro cultures. Gum karaya (Grade I) was purchased fromGirijan Cooperative Corporation Ltd. Visakhapatnam, India andintact lumps (Fig. 1A) were stored in airtight polypropylene jarsand sealed to protect from moisture. Intact lumps were pow-dered using high speed mechanical blender (Bajaj, India) andsieved through 150 size mesh before making gel.

Explant sterilization and inoculationBud sticks (10 - 12 cm) collected from the healthy rough

lemon plants were cut into small nodal segments each contain-ing a single node. Nodal segments were washed with detergentto remove any external microbes and soil particles. After this,they were washed thoroughly under running tap water so as toremove the detergent completely. The sterilization of the explantwas done under aseptic conditions in a laminar flow hood. Theexplants were disinfected with 0.1% HgCl2 for 4 min and rinsedthree times with autoclaved distilled water to remove any tracesof HgCl2.

Shoot regenerationNodal segments were cultured on MS medium (Murashige

and Skoog 1962) gelled with gum karaya and agar (HiMedia,India) either alone or in combination. Initially, the nodal seg-

Fig. 1. Micropropagation of rough lemon (Citrus jambhiri Lush) through nodal seg-ments. A: Intact lumps of gum karaya; B: In vitro shoot regeneration from nodal seg-ment on gum (30 g L-1) gelled medium containing KN (2 mg L-1); C: In vitro shootregeneration from nodal segment on agar (8 g L-1) gelled medium containing KN (2mg L-1); D: In vitro root regeneration on gum (30 g L-1) gelled medium containing IBA(2 mg L-1); E: Complete plantlet development after 45 days of culture on gum (30 g L-

1) gelled medium containing IBA (2 mg L-1); F: Plant development after transplanta-tion of regenerated plantlets into a sand/soil mixture.

JCSB 2011 (December) 14 (4) : 297 ~ 303 299

ments were inoculated on MS medium containing 3% (w/v)sucrose and 0.8% (w/v) agar. Different concentrations of benzy-ladenine (BA), kinetin (KN), zeatin (ZN) (1.0 - 2.5 mg L-1), andmalt extract (200 - 1,200 mg L-1) were tried in agar-gel mediumto find out the best suitable medium. The medium was adjustedto pH 5.6 with 1 N NaOH and autoclaved at 121ºC and 15 lb in-2

pressure for 20 min. After finding out the best suitable concen-tration of plant growth regulator in MS medium, the gum karaya(20, 25, and 30 g L-1) alone, and in combination with agar (2, 4,and 6 g L-1) was used as gelling agent. MS medium supplement-ed with suitable plant growth regulator and gelled with 8 g L-1

agar served as control in gum-gel experiments. For preparinggum karaya-gel media, overnight soaked gum were mixed withother constituents of the medium and the volume was raised tothe required level on a magnetic stirrer before adjusting the pHto 5.6. For all the experiments, 20 mL of culture medium wasdispensed in 25 x 150 mm culture tubes, which were closed withcotton plugs. All cultures were maintained at 26 ± 1ºC with aluminous intensity of 40 µmole m-2 s-1 and 16-h photoperiod. Foreach treatment, percentage of cultures showing shoot regenera-tion and shoot length per culture was recorded 30 days after ini-tial culturing.

Rooting of regenerated shootFor rooting, the regenerated shoots were separated out and

cultured on MS medium supplemented with various concentra-tions (0.5 - 2.5 mg L-1) of indole-3-acetic acid (IAA), naphtha-lene acetic acid (NAA), and indole-3-butyric acid (IBA) alongwith 3% (w/v) sucrose. Initially, 0.8% agar was used as gellingagent to find out the best suitable medium for rooting. Afterfinding out the best suitable concentration of plant growth regu-lator in MS medium, the gum karaya (20, 25, and 30 g L-1) aloneand in combination with agar (2, 4, and 6 g L-1) was used asgelling agent. Forty-eight regenerated shoots were inoculated forindividual treatment and the experiment was repeated threetimes. The percentage of cultures showing root regeneration andnumber of roots/shoots was recorded after 30 days of inocula-tion.

Hardening and acclimatizationThe well-developed plantlets were washed with water in

order to remove adhering agar and transferred to autoclavedplastic pots containing a mixture of garden soil, sand, and ver-miculite in the ratio of 3:1:1. Hardening of potted plantlets wasaccomplished in a culture room set at 26 ± 1ºC, 16-h day-length(40 µmole m-2 s-1), and covered with polyethylene bags to main-tain high humidity. After 12 - 15 days, polyethylene bags wereremoved initially for a short duration (15 - 30 min) daily forabout 1 week. Gradually, the daily exposure time was increasedby 30 min for each day. Polyethylene bags were completelyremoved after 20 days, and subsequently the plantlets weretransferred to the earthen pots containing only garden soil andkept in the polyhouse for 1 month, and thereafter transferred tothe field conditions.

Texture analysis of medium gelled with agar and gumkaraya

The agar and gum karaya-gel medium were poured into stan-dard-sized vials (25 mm diameter) and stored at 26 ± 1ºC for 24h. The gels formed in the vials were evaluated for their texturalproperties using TA/XT2 texture analyzer (Stable Micro System,England) equipped with a 1 kg load cell. Each vial was placedupright on the metal plate and the gel was subjected to compres-sion using a 5 mm diameter aluminum cylinder probe (SMS P/5)at a pre-test, test, and post-test speed of 0.5 mm s-1 to a distanceof 10 mm. The compression was carried out in two cycles togenerate a force-time curve from which hardness, springiness,cohesiveness, gumminess, and adhesiveness were calculated asdescribed by Bourne (1978). Three repeated measurements wereperformed for each gelled medium and their mean was taken.

Statistical analysisFor each treatment, 48 culture tubes were inoculated and the

experiments were repeated thrice. The data pertaining to theeffect of gelling agent and plant growth regulators on shoot androot regeneration were subjected to one-way analysis of variance(ANOVA) and the differences among means were compared byhigh-range statistical domain (HSD) using Tukey’s test.

Results

Shoot regenerationNodal segments of rough lemon cultured on agar gelled (8 g

L-1) MS medium supplemented with different concentrations ofPGRs (BA, KN, and ZN) or organic nutrient (ME) showed signsof shoot regeneration after 4 - 6 days of inoculation. The per-centage of shoot regeneration and average shoot length wasrecorded after 30 days of inoculation (Table 1). Among differentconcentrations of PGRs tested, maximum percentage of culturesshowing shoot regeneration (66.66%) was observed with KN 2mg L-1 (Fig. 1C) with an average shoot length of 0.73 cm. A fur-ther increase in concentration of KN resulted in a decrease inpercent shoot regeneration. ZN at a concentration of 2 mg L-1

was also effective for shoot formation but average shoot length(0.57 cm) and percentage of shoot regeneration (62.49 %) wasless as compared to KN. The effect of different concentrationsof gum karaya (sterculia gum) and agar, either alone or in com-bination, on shoot regeneration in MS medium supplementedwith KN 2 mg L-1 is shown in Table 2. The shoot regenerationresponse on media gelled with 25 g L-1 gum was less (41.66%)than those of 30 g L-1 gum (62.49%, Fig. 1B). Shaking of theculture tubes containing gum-karaya gel medium at a concentra-tion of 25 g L-1 often resulted in sinking of the nodal segments tosubsurface levels. Such submerged explants also responded pos-itively indicating that sufficient aeration was available in gum-gel media. Addition of agar at a concentration of 2, 4, and 6 g L-

1 along with gum (25 and 30 g L-1) increased the firmness of themedia and also improved the shooting response. However, the

overall shoot regeneration response on gum karaya-gel mediaalong with all the concentration of agar was less than those ofcontrol (agar 8 g L-1). Among all the concentration of gum andagar tested the average shoot length (0.80 cm) was maximumwith 30 g L-1 gum-gel media.

Root regenerationShoots developed from nodal segments were excised and

transferred to the rooting medium gelled with agar (8 g L-1). Theeffect of different concentrations of NAA, IBA, and IAA (0.5,1.0, 1.5, 2.0, and 2.5 mg L-1) on root regeneration after 30 daysof inoculation is shown in Table 3. The initiation of roots tookplace after 2 weeks of inoculation. In MS medium supplementedwith IBA 2 mg L-1, maximum of 52.77% cultures showed rootregeneration, with an average number of 2.58 roots/shoot. Adecrease or increase in concentration of IBA resulted in decreasein rooting response. Among IAA and NAA, the later was moreeffective with maximum of 36.10% cultures showing root regen-eration at a concentration of 2 mg L-1. The effect of differentconcentration of gum karaya and agar, either alone or in combi-nation, on root regeneration in MS medium supplemented withIBA 2 mg L-1 is shown in Table 4. Increase in concentration ofagar in gum gelled media resulted in increase in percent root for-mation and height of shoots. The quantitative response obtainedon media fortified with both gum and agar in combination wasstatistically insignificant. The rooting response (53.47%) andaverage number of roots/shoot (2.91) on gum-gel media at aconcentration of 30 g L-1 was more than those of control (agar 8

g L-1). The plantlets responded positively in 30 g L-1 gum-gelmedium after 5 - 6 days of culture (Fig. 1D) and there was nosoftening of the medium even after 45 days of culture (Fig. 1E).The plantlets, thus developed where transferred to pots contain-ing a mixture of garden soil, sand, and vermiculite for acclimati-zation (Fig. 1F), which showed 63 % survival rate after transferto field conditions

Texture measurementsIn texture analysis, firmness (height of the first peak) and

springiness (ratio between recovered height after the first com-pression and the original gel height) was determined. The nega-tive area of the curve during retraction of the probe was termedadhesiveness. Cohesiveness was calculated as the ratio betweenthe area under the second peak and the area under first peak(Bourne 1978). Gumminess was determined by multiplyingfirmness and cohesiveness. Texture measurements revealed thatagar-gel (8 g L-1) medium possessed maximum firmness (Table5). Use of agar (2 - 6 g L-1) along with gum karaya (25 and 30 gL-1) improves the firmness of the culture medium but it wasnowhere near to that of agar. Gum karaya-gel medium was lessadhesive and gummy as compared to agar-gel medium. Howev-er, addition of agar to these increased the adhesiveness and gum-miness with increase directly proportional to the concentrationof agar. Cohesiveness of gum karaya-gel medium was more ascompared to agar-gel medium and use of agar in combinationwith gum decreases the cohesiveness of culture medium. Thespringiness of gum karaya-gel (25 and 30 g L-1) medium wasmore than that of agar. Addition of agar along with gum karayadecreases the springiness of the culture medium.

Discussion

This report provides simple and cost effective protocol for themicropropagation of rough lemon. Direct shoot regenerationoffers a means for producing identical propagules from mature

Gum Karaya as Gelling Agent in Plant Tissue Culture Media300

2525252530303030-

-246-2468

Table 2. Response of nodal segments on MS medium supplemented withkinetin (2 mg L-1) and gelled with gum karaya and agar either alone or incombination

Gum karayaGelling agent (g L-1)

Agar41.66 ±1.20b

43.05 ±1.38b

61.80 ±0.69a

63.19 ±0.70a

62.49 ±1.20a

63.19 ±0.69a

65.27 ±1.40a

65.97 ±1.38a

66.65 ±1.20a

F 8 ,18 = 65.28*; HSD = 5.46

Shoot regeneration (%)*(Mean ± SE)a

0 .69 ±0.15a

0 .68 ±0.16a

0 .71 ±0.13a

0 .63 ±0.18a

0 .80 ±0.10a

0 .71 ±0.16a

0 .75 ±0.12a

0 .72 ±0.15a

0 .74 ±0.09a

F 8, 99 = 0 .44*; HSD = 0.29

Average shoot length (cm)*(Mean ± SE)a

Data shown are Mean ± SE of three experiments. Each experiment consisted of 48replicates. Observations recorded after 30 days of inoculation*Significant at P ≤ 0.05.aValues followed by the same letter with in a column are not significantly differentusing HSD multiple comparison test.

Benzyladenine

Zeatin

Malt Extract

Kinetin

1.01.52.02.5

1.01.52.02.5

20040060080010001200

1.01.52.02.5

Table 1. Response of nodal segments on agar gelled media (8 g L-1) con-taining different concentrations of plant hormones

Plant hormoneConcentration

(mg L-1)

12 .50 ± 1. 19c

16 .65 ± 1. 19b

29 .84 ± 0. 67a

26 .38 ± 0. 69a

F 3, 8 = 69 .16*; HSD = 3.9447 .21 ± 0. 69b

61 .80 ± 0. 68a

62 .49 ± 1. 19a

59 .72 ± 1. 38a

F 3, 8 = 47 .16*; HSD = 4.2141 .66 ± 1. 20b

46 .52 ± 0. 69b

54 .86 ± 1. 38a

56 .94 ± 0. 68a

56 .24 ± 1. 19a

55 .55 ± 1. 38a

F 5, 12 =31 .08*; HSD = 5.1146 .52 ± 0. 69d

62 .49 ± 1. 20b

66 .66 ± 1. 19a

54 .85 ± 0. 69c

F 3, 8 = 81 .54*; HSD = 3.97

Shoot regeneration (%)*(Mean ± SE)a

0.43 ± 0. 08a

0.48 ± 0. 07a

0.55 ± 0. 10a

0.50 ± 0. 11a

F 3, 44 = 0.97*; HSD = 0.160.44 ± 0. 08b

0.50 ± 0. 06a

0.57 ± 0. 10a

0.59 ± 0. 09a

F 3, 52 = 2.78*; HSD = 0.130.48 ± 0. 08b

0.63 ± 0. 10a

0.70 ± 0. 06a

0.80 ± 0. 07a

0.76 ± 0. 10a

0.76 ± 0. 10a

F 5, 84 = 6.70*; HSD = 0.180.58 ± 0. 13b

0.64 ± 0. 12a

0.73 ± 0. 12a

0.74 ± 0. 10a

F 3, 60 = 1.90*; HSD = 0.1

Average shoot length (cm)*(Mean ± SE)a

Data shown are Mean ± SE of three experiments. Each experiment consisted of 48replicates. Observations recorded after 30 days of inoculation*Significant at P ≤ 0.05.aValues followed by the same letter with in a column are not significantly differentusing HSD multiple comparison test.

JCSB 2011 (December) 14 (4) : 297 ~ 303 301

trees selected for desired horticultural characteristics. Shoots canbe easily derived from nodal segments of mature rough lemonplant on KN-containing medium and subsequently rooted onIBA-containing medium. In the present study, addition of KNwas found to be better than addition of ZN, BAP, and maltextract for shoot regeneration from nodal segments. Shootinduction occurred after 4 - 6 days of inoculation of nodal seg-ments in medium supplemented with BA, KN, and ZN whereasin malt extract shoots initiated occurred after 8 - 10 days of inoc-ulation. Higher concentration of KN resulted in decrease in per-centage of cultures showing shoot regeneration. These resultsare in conformity with some of the earlier studies, where KNalone or in combination with various growth regulators/supple-ments was used for organogenesis in different plant species(Havelange et al. 2000; Singh et al. 2005; Talegaokar andDangat 2010). There are some reports indicating BA (3 mg L-1)in combination with NAA (0.5 mg L-1) to be a better plantgrowth regulator for shoot regeneration (Beloualy 1991; Savitaet al. 2010) in rough lemon from callus cultures. The beneficialeffect of cytokinins in shoots proliferation has been reported innumber of citrus species (Begum et al. 2004; Gill 1992; Krishanet al. 2001; Parthasarathy et al. 2001; Saini et al. 2010; Singh etal. 1999).

The incubation of regenerated shoots on auxin-supplementedmedium induces the formation of roots. Among different plantgrowth regulators tried for rooting, IBA was found to give betterresponse (52.77%) as compared to NAA and IAA. Similarresults have been reported by Carimi and De Pasquale (2003)and Perez-Tornero et al. (2010) in Citrus species. On the con-trary, there are some reports indicating NAA to be better rootinghormone for Citrus species. (Chakravarty and Goswami 1999;Usman et al. 2005). In earlier report on rough lemon, however,

the maximum rooting response was recorded on NAA (1 mg L-1)+ IBA (1 mg L-1) supplemented MS medium (Saini et al. 2010).

In this report, effort has been made to compare gum karaya(sterculia gum) with agar in plant tissue culture media. Shootorganogenesis in gum karaya-gel medium (30 g L-1) was at parwith agar and rooting response was slightly higher than agar-gelmedium. It was observed that the initial response for shootregeneration was better on agar-gel medium as cultures establishon this medium faster compared to gum-gel medium. But oncethe cultures establish themselves on gum-gel medium theyexhibit normal growth and proliferation. Among the differentconcentrations and combinations of agar and gum karaya tested,better germination and subsequent differentiation of roots wasregistered on 30 g L-1 gum karaya-gel medium.

Texture measurements reveal firmness, cohesiveness, adhe-siveness, springiness, and gumminess of culture medium pre-pared from gum karaya and agar. This analysis shows that firm-ness of gum karaya-gel medium was much less than that of agar-gel medium. However, in their capability of supporting growthand differentiation of explants, they are equal to agar medium.In 30 g L-1 gum karaya-gel medium there was no sinking of theexplants. Gelling agent provides support and nutrients toexplants from the culture medium. It is generally accepted thatthe dynamics of the interaction between gelling agent, culturemedium, and explants play a major role in tissue growth underin vitro conditions (Scholten and Pierik, 1998). Low firmnessafforded better diffusion and uptake of ions and avoided accu-mulation of toxic metabolites around explants. It was observedthat gum karaya-gel medium dried slowly as compared to agarmedia, so it would be a better choice for long term maintenanceof cultures. Among the other parameters, adhesiveness of gumkaraya medium is comparable with agar. Less adhesivenesswould be required to wash away the culture medium from plant-lets during hardening. Possible explanations for the differencesin regeneration response include limited and lateral diffusion ofnutrients, impurities, and differences in firmness of the gellingagent (Debergh, 1983). Jain and Babbar (2011) evaluated blendsof guar gum, xanthan gum, or isubgol with agar for their rheo-

NAA

IBA

IAA

0.51.01.52.02.5

0.51.01.52.02.5

0.51.01.52.02.5

Table 3. Rooting response of shoots in agar-gel media containing differentconcentrations of plant hormones

HormoneConcentration

(mg L-1)

17 .34 ± 1. 37c

27 .33 ± 1. 41b

33 .31 ± 1. 20a

36. 10 ± 0. 69a

34 .71 ± 1. 20a

F 4 ,10 = 46 .61*; HSD = 4.8824 .99 ± 1. 19d

31 .05 ± 1. 13c

38 .88 ± 1. 83b

52 .77 ± 0. 69a

51 .38 ± 1. 38a

F 4 ,10 = 87 .80*; HSD = 5.6516 .64 ± 1. 19b

27 .77 ± 0. 69a

29 .14 ± 1. 18a

29 .84 ± 0. 67a

29 .14 ± 1. 20a

F 4 ,10 = 29 .72*; HSD = 4.42

Root regeneration (%)*(Mean ± SE)a

0.58 ± 0. 29b

0.83 ± 0. 33b

1.08 ± 0. 29a

1.57 ± 0. 28a

1.51 ± 0. 26a

F 4 ,55 = 6.62*; HSD = 0.551.08 ± 0. 16b

1.16 ± 0. 32b

1.33 ± 0. 28b

2.58 ± 0. 29a

2.59 ± 0. 16a

F 4 ,55 = 21 .27*; HSD = 0.600.83 ± 0. 15b

0.91 ± 0. 22b

1.24 ± 0. 26b

1.83 ± 0. 33a

1.91 ± 0. 29a

F 4 ,55 = 10 .91*; HSD = 0.57

Number of roots/shoot*(Mean ± SE)a

Data shown are Mean ± SE of three experiments. Each experiment consisted of 48replicates. Observations recorded after 30 days of inoculation*Significant at P ≤ 0.05.aValues followed by the same letter with in a column are not significantly differentusing HSD multiple comparison test.

2525252530303030-

-246-2468

Table 4. Rooting response of shoots on MS medium supplemented withIBA (2 mg L-1) and gelled with gum karaya and agar either alone or in com-bination

Gum karayaGelling agent (g L-1)

Agar47.91 ±1.20b

46.52 ±0.69b

52.08 ±1.19a

50.69 ±0.68a

53.47 ±1.38a

52.08 ±0.70a

52.77 ±1.39a

51.38 ±0.68a

52.77 ±0.69a

F 8 ,18 = 4.94*; HSD = 4.81

Root regeneration (%)*(Mean ± SE)a

Data shown are Mean ± SE of three experiments. Each experiment consisted of 48replicates. Observations recorded after 30 days of inoculation*Significant at P ≤ 0.05.aValues followed by the same letter with in a column are not significantly differentusing HSD multiple comparison test.

2.41 ±0.26a

2.25 ±0.38a

2.16 ±0.33a

2.08 ±0.16a

2.91 ±0.29a

2.50 ±0.38a

2.41 ±0.29a

2.33 ±0.28a

2.58 ±0.29a

F 8, 99 = 2.77*; HSD = 0.67

Number of roots/shoot*(Mean ± SE)a

logical properties and their capability of supporting a mor-phogenic response. They also reported that none of these alter-natives had rheological properties comparable to agar but theysupported good growth and differentiation of plant tissues. Gumkaraya has very low firmness but it supported regenerationresponse comparable to agar. So our results are in conformitywith them that textural properties comparable to agar may not benecessary for supporting plant growth under in vitro conditions.Culture media ingredients, especially agar, adds to the cost ofregenerated plants. Agar is the most widely used solidifyingagent for preparation of solid and semi-solid plant tissue culturemedia (Scholten and Pierik 1998). The cost of in vitro propaga-tion can be reduced by using cheap alternatives to the highlyexpensive purified agar (Ezekiel 2010). Gum karaya forms atransparent medium which was less firm but nodal segmentsremained on the surface if left undisturbed. It is a highly cost-effective gelling agent as its price in India is about one-fourth ofagar. The results of the present study offer a new possibility ofusing gum karaya as a gelling agent which may be fruitful indeveloping cost effective protocol for micropropagation of imp-ortant plants.

Acknowledgements

The authors are thankful to the University GrantsCommissions, New Delhi (India) for providing financial assis-tance. Thanks are due to Mr. K.P Rao, State Project Advisor(Mktg), Indira Kranthi Pathan, Department of RuralDevelopment, Govt. of Andhra Pardesh for procuring and sup-plying gum karaya samples from Girijan CooperativeCorporation Ltd. Visakhapatnam, India.

References

Al Khayri JM, Al Bahrany AM. 2001. In vitro micropropagation of Citrus aurantifolia (lime). Curr. Sci. 81: 1242-1246

Ali S, Mirza B. 2006. Micropropagation of rough lemon (Citrus jambhiri Lush.): Effect of explant type and hormone concen-

tration. Acta Bot. Croat. 65: 137-146Altaf N, Khan AR, Ali L, Bhatti IA. 2008. Propagation of rough

lemon (Citrus jambheri lush.) through in vitro culture and adventitious rooting in cuttings. Electronic J. Environ. Agric. Food Chem. 7: 3326-3333

Anderson DMV, McNab CGA, Anderson CG, Brown PM, Pringuer MA. 1982. Gum exudates from the genus Sterculia(gum karaya). Int. Tree Crops J. 2: 147-154

Babbar SB, Jain R. 1998. ‘Isubgol’ as an alternative gelling agent for plant tissue culture media. Plant Cell Rep. 17: 318-322

Babbar SB, Jain R, Walia N. 2005. Guar gum as a gelling agent for plant tissue culture media. In Vitro Cell Dev. Biol. Plant. 41: 258-261.

Begum F, Amin MN, Islam S, Azad MAK. 2004. A comparative study of axillary shoot proliferation from the nodal explants of three varieties of pummelo (Citrus grandis [L.] Osb.). Biotechnol. 3: 56-62

Beloualy N. 1991. Plant regeneration from callus culture of three Citrus rootstocks. Plant Cell Tiss. Org. Cult. 24:29-34

Bourne MC. 1978. Texture profile analysis. Food Technol. 32: 62-66

Carimi F, De Pasquale F. 2003. Micropropagation of citrus. In SM Jain, K Ishii, eds, Micropropagation of Woody Trees and Fruits, Kluwer, Netherlands, pp 589-619

Chakravarty B, Goswami BC. 1999. Plantlet regeneration from long term callus cultures of Citrus acida Roxb. and the uniformity of regenerated plants. Sci. Horti. 82: 159-169

Debergh P. 1983. Effects of agar brand and concentration on the tissue culture medium. Physiol. Plant. 59: 270-276

Ezekiel A. 2010. Viable options and factors in consideration for low cost vegetative propagation of tropical trees. Int. J. Bot. 6: 187-193

Gill MIS. 1992 Studies on somatic cell and protoplast culture in Mandarins, PhD Thesis, Punjab Agricultural University, Ludiana, India

Hartman H, Kester TDE, Davis Jr. FT, Geneve RL. 1997. Plant propagation: Principles and Practices, 6th Ed. Prentice-Hall, Inc. Upper Saddle River, New Jersey, USA, pp 239-385

Havelange A, Lejeune P, Bernier G. 2000. Sucrose/cytokinin interaction in Sinapis alba at floral induction: A shoot-to-root-to-shoot physiological loop. Physiol. Plant 109: 343-350

Gum Karaya as Gelling Agent in Plant Tissue Culture Media302

2525252530303030-

-246-2468

Table 5. Texture analysis of MS medium gelled with gum karaya and agar either alone or in various combinations

Gum karayaGelling agent (g L-1)

Agar0.0130.0940.2910.4670.0220.1040.3070.5340.585

Firmness (N)

0.850.820.330.240.760.480.320.240.20

Cohesiveness

0.0790.2950.6020.8760.0910.3160.4621.0070.104

Adhesiveness (N s)

0.970.840.810.770.870.930.860.810.81

Springiness (mm)

0.0110.0770.0960.1120.0160.0490.0980.1280.117

Gumminess (N)

Data shown are Mean ± SE of three experiments. Each experiment consisted of 48 replicates.*Significant at P ≤ 0.05. Observations recorded after 30 days of inoculationaValues followed by the same letter with in a column are not significantly different using HSD multiple comparison test.

JCSB 2011 (December) 14 (4) : 297 ~ 303 303

Henderson WE, Kinnersley AM. 1988. Corn starch as an alter-native gelling agent for plant tissue culture. Plant Cell Tiss. Org. Cult. 15: 17-22

Jain N, Babbar SB. 2002. Gum katira – a cheap gelling agent for plant tissue culture media. Plant Cell Tiss. Org. Cult. 71: 223-229

Jain-Raina R, Babbar SB. 2011. Evaluation of blends of alterna-tive gelling agents with agar and development of xanthagar, a gelling mix, suitable for plant tissue culture media. Asian J. Biotechnol. 3: 153-164

Johnston JC. 1968. Growing seedlings from seed. In KW Opitz, RG Plate, EF Frolich, eds, Propagation of Citrus, 1–6. Division of Agricultural Sciences University of California

Krishan K, Dhatt AS, Gill MIS, Kumar K. 2001. In vitro plant regeneration in kinnow mandarin (Citrus nobilis Lour. x C. deliciosa Tenora) Ind. J. Hort. 58: 299-302

Krishan K, Kaur H, Gill MIS, Rattanpal HS, Kanika, Gosal SS. 2011. An efficient regeneration protocol from callus culture in rough lemon (Citrus jambhiri). Ind. J. Agric. Sci. 81: 324-329

Le Cerf D, Irinei F, Muller G. 1990. Solution properties of gum exudates from Sterculia urens (karaya gum). Carbohydr. Polymers 13: 375-386

Marutani-Hert M, Evens TJ, Gregory T, McCollum GT, Niedz RP. 2010. Bud emergence and shoot growth from mature cit-rus nodal stem segments. Plant Cell Tiss. Org. Cult. 106: 81-91

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497

Nene YL, Shiela VK, Moss JP. 1996. Tapioca – a potential substitute for agar in plant tissue culture media. Curr. Sci. 70: 493-494

Parthasarathy VA, Barua A, Nagaraju V, Parthasarathy U. 2001. Quadratic response of citrus species to cytokinins and com-parative efficacy on morphogenetic characters of in vitro proliferated shoots. Ind. J. Hort. 58: 336-341

Perez-Tornero O, Tallo´n CI, Porras I. 2010. An efficient protocol for micropropagation of lemon (Citrus limon) from mature nodal segments. Plant Cell Tiss. Org. Cult. 100: 263-271

Rathore JS, Rathore MS, Singh M, Singh RP, Shekhawat NS. 2007. Micropropagation of mature tree of Citrus lemon. Ind. J. Biotechnol. 6: 239-244

Saini HK, Gill MS, Gill MIS. 2010. Direct shoot organogenesis and plant regeneration in rough lemon (Citrus jambhiri Lush.). Ind. J. Biotech. 9: 419-423

Samarina LS, Kolomiets TM, Baranova EN, Arutyunova ES. 2010. Regeneration and micropropagation of lemon cultivars in vitro from nodal explants. Russ. Agric. Sci.36: 417-420

Savita, Singh B, Virk GS, Nagpal AK. 2011. An efficient plant regeneration protocol from callus cultures of Citrus jambhiriLush. Physiol. Mol. Biol. Plants 17: 161-169

Savita, Vijay, Virk GS, Nagpal A. 2010 Effect of explant type and different plant growth regulators on callus induction and plantlet regeneration in Citrus jambhiri Lush. Environ. We

Int. J. Sci Tech. 5: 97-106Scholten HJ, Pierik RLM. 1998. Agar as a gelling agent: Che-

mical and physical analysis. Plant Cell Rep. 17: 230-235Singh B, Sharma S, Rani G, Zaidi AA, Hallan V, Nagpal A,

Virk GS. 2005. In vitro production of indian citrus ringspot virus-free plants of kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora) by ovule culture. J. Plant Biotechnol. 7: 259-265

Singh SK, Sharma HC, Goswami AM, Saxena SK. 1999. In vitro seed germination for polyembryony in Citrus spp. Proc. Intern. Symp. Citriculture NRC for citrus, India, pp 211-215

Talegaokar AP, Dangat SS. 2010. Direct shoot organogenesis of Indian cotton G. arboretum cv. PA402 from embryonic axis explants. Int. J. Integ. Biol. 9: 119-122

Usman M, Muhammad S, Fatima B. 2005. In vitro multiple shoot induction from nodal explants of Citrus cultivars. J. Central Europ. Agric. 6: 435-442

Zimmerman RH, Bhardwaj SV, Fordham IM. 1995. Use of starch-gelled medium for plant tissue culture of some fruit crops. Plant Cell Tissue Org. Cult. 43: 207-213