Factors influencing high callusing proliferation in cotton ( Gossypium hirsutum L . )

In vitro regeneration of cotton (Gossypium spp.) has been a subject of intense research for the last two decades because of the commercial value of the crop. A study was conducted to assess the callusing potential of two local cotton genotype varieties viz., MCU-5 and SVPR-2 and two exotic genotypes, Coker 310 and 312. High significant difference was observed between media composition, genotype and explant types used for callus induction. Among the 24 media composition screened for callusing, the medium CIM3 (MS + 0.1 mg/l 2,4-D + 0.5 mg/l kinetin) exhibited successful and faster induction of calli. Callus initiation was found to be quicker from hypocotyls than cotyledons. Calli developed from hypocotyl explants were large, rough and friable, while cotyledon-derived calli were friable and medium sized. Significantly higher callus induction frequencies were observed in hypocotyl (97.3%) than cotyledon (95.8%) explants which were collected from 7 day-old seedlings irrespective of genotype. Explants collected from younger (4 day-old) and older (14 day-old) plants showed poor and low callus induction frequencies.


INTRODUCTION
Cotton (Gossypium hirsutum) which belongs to the family Malvaceae is an important fibre crop of global importance.It is also a source of oil and high quality protein meal and plays a significant role in the Indian economy.Globally, the cotton industry contributes millions of dollars annually in exports and imports.Among the cotton producing countries, India has the distinction of having the largest cotton growing area in the world with all four cultivated species under cultivation (Basu et al., 1995).
Genetic improvement of cotton through conventional breeding is limited by several factors including incompatibility barriers and time period for improved variety development (Munro, 1987).Although plant biotechnology seems to be an attractive way to improve cotton plant, its use requires an effective in vitro culture system using somatic tissues of plant.In vitro culture allows circumventing these difficulties: for example, callus obtained from which explant will be an ideal material for genetic transformations (Finer and McMullen, 1990).Cotton somatic embryogenesis was first observed by Price and Smith (1979), in Gossypium klotzschianum, but complete plantlets could not be regenerated from somatic embryos.Plant regeneration in cotton through somatic embryogenesis was first reported by Davidonis and Hamilton (1983) in two years old calli derived from cotyledons.Since then, numerous reports on somatic embryogenesis and regeneration (Leelavathi et al., 2004;Chaudhary et al., 2003;Shoemaker et al., 1986;Trolinder andGoodin, 1987, 1988a, b;Cousins et al., 1991;Kumeria et al., 2003;Rajasekaran et al., 1996) have been published.With regard to Indian cotton varieties *Corresponding author.E-mail: pushpa.saravanan@gmail.com.success was reported so far only in two varieties viz., MCU 5 (Kumar and Pental, 1998) and SVPR 2 (Ganesan and Jayabalan, 2004).Factors involved in the initiation and maintenance of Gossypium species callus have been investigated by number of laboratories (Rao et al., 2006;Sun et al., 2006;Xie et al., 2007).The main factors determining the tissue culture response in cotton and other recalcitrant crops include genotype (Seabrook and Douglas, 2001), donor plant (Lu et al., 1984), type of growth regulators (Trolinder and Goodin, 1988;Sun et al., 2006) and sugar type (Ishii et al., 2004).An in-depth study of such factors would enable the development of genotype-specific culture methods to enhance the tissue culture response of the recalcitrant crops.The purpose of this study was to screen different cotton genotypes, media composition, explant age and type of explant (cotyledon, hypocotyls) for cotton callus induction.

Genotype
In the present investigation, acid-delinted cotton seeds of elite cotton varieties, MCU 5, SVPR 2 and two Coker genotypes viz., Coker 310 and Coker 312 in which genetic transformation work was reported earlier (Kumar et al., 1998;Sakhanokho et al., 2001) were obtained from the germplasm collections of the Department of Cotton, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore.

Surface sterilization
Delinted seeds were surface sterilized with 70% ethanol for 2 min and then washed three times with sterile distilled water.They were again surface-sterilized with 0.1% mercuric chloride for 10 min followed by three washes with sterile distilled water.

Seed germination
The surface sterilized seeds were germinated on half strength MS (Murashige and Skoog, 1962) medium supplemented with one percent (w/v) sucrose and 10 g/l agar.The pH of the medium was adjusted to 5.7 to 5.8 (by using 0.1 N KOH or 0.1N HCl) prior to autoclaving.

Explant type
Hypocotyl and cotyledonary explants obtained from seedlings (5 to 7 day-old) were used for callus induction.Both hypocotyl (4 to 6 mm) and cotyledonary leaf (16 mm 2 ) sections were plated onto callus induction medium (Figure 1).The explants of each genotype were plated on the following MS salts based compositions in three replications (Table 1) and subcultured on the same medium once in two weeks.The observations were recorded in each replication for all the genotypes and percentage of callusing was worked out.

Effect of plant growth regulators on callus induction
Both hypocotyl and cotyledon explants were cultured on MS basal medium supplemented with different concentrations and combinations of plant growth regulators to identify a medium that would produce, proliferate and maintain the callus cultures.The different media compositions and explants were tested for their effect on callus induction frequency as listed in Table 1.After autoclaving the media, the filter sterilized plant growth regulators were added onto the media at 42°C.The pH of the media was adjusted to 5.8 prior to autoclaving the media for 15 minutes at 121°C and 15 psi pressure.
Callus initiation was evaluated one month after culture on the callus induction medium.The frequency of callus induction was expressed as percentage of the number of explants cultured and was calculated as given below: Number of explants produced calli Frequency of callus induction (%) = x 100 Total number of explants cultured

Effect of explant age
To test the effect of age of the explants on callusing response, hypocotyls and cotyledon explants were excised from 5, 7, 10 and 12 day old seedlings.The callus induction frequency of hypocotyls and cotyledons was assessed on CIM3 medium.

Experimental design and statistical analysis
All tissue culture media were carried out in a completely randomized block design with three replications.Statistical analyses of the experiments were performed using AGRES-AGDATA software.

RESULTS AND DISCUSSION
The frequency of callus induction and plant regeneration influenced was by several factors, including composition of the culture medium, explant sources and genotypes (Shengwei and Jingsan, 2000).The analysis of variance (Table 2) revealed significant differences in callus induction and callus characteristics due to media compositions, genotypes and explant types.The interaction effects were also significant.
Callus induction was observed in all media compositions in both explants.Among the various media compositions tested, CIM3 (MS + 0.1 mg/l 2,4-D + 0.5 mg/l kinetin) recorded maximum callus induction frequency with hypocotyl and cotyledon explants irrespective of genotypes studied (Table 3).Among the four genotypes, highest callus induction frequency of 97.7% was observed in Coker 310.Callus maturation was achieved on MS basal medium in duration of 2 months.Most of the published works had also reported MS based medium containing 2,4-D and kinetin as the best for callus induction (Trolinder andGoodin, 1987 and1988a, b;Kumria et al., 2003;Choudhary et al., 2003;Haq and Zafar, 2004;Tohidfar et al., 2005;Zhao et al., 2006).
In the present study, it was observed that 2,4-D was more effective than NAA in producing embryogenic calli.Moreover, low levels of 2,4-D induced callus quickly and readily, whereas NAA required a longer time to produce significant amount of callus.This was in accordance with the results obtained by Trolinder andGoodin (1987, 1988a, b).Finer (1988) indicated that the callus induction was better at lower concentrations of auxins and cytokinins.Among the two explant types used in callus induction, hypocotyl explants (97.7 %) recorded higher callusing percentage than cotyledons (95.8 %).Such high callusing in hypocotyls was also reported by Trolinder and Goodin (1988a) and Sakhanokho et al. (1998Sakhanokho et al. ( , 2001Sakhanokho et al. ( and 2004)).Rapid callus development in hypocotyl tissue may shorten the culture duration, thus reducing the occurrence of somoclonal

Medium
Media composition* 2,4-D (mg/l) Kinetin (mg/l) NAA (mg/l) 2-ip (mg/l) Picloram (mg/l) * All media were supplemented with MS salts and 30 g/l maltose and solidified with 8 g/l agar (CIM: callus induction medium).variation, a major problem in cotton tissue culture (Sakhanokho et al., 2004).Among the cotton genotypes studied, Coker 310 exhibited the highest callus induction frequency than MCU 5, SVPR 2 and Coker 312.Callus induction frequency was more in hypocotyl explants than cotyledon explants isolated from 7 day-old seedlings.Significantly higher callus induction frequencies were observed for hypocotyl (97.3%) than cotyledon (94.6%) explants collected from 7 days-old seedlings irrespective of genotypes studied (Figures 2 Values represent the mean ± standard error of three replications.In a column, means followed by same letters are not significant at 5% level by LSD. and 3).Explants collected from younger and older seedlings of 4 and 12 days old exhibited only low callus induction frequencies.It was also observed that 7 days-old seedlings provided explants (cotyledon and hypocotyls) which were superior in callusing response.Such variable responses in callus induction for different age have been reported in cotton (Sakhanokho et al., 1998;Nobre et al., 2001) and also in other species (Pereira et al., 2000;Dhar and Joshi, 2005).Such variations can be attributed to the physiological condition of the explant, which is determined by genetic factors (Nagarathna et al., 1991).Younger explants exhibit greater morphogenic potential than older explants in view of their higher metabolically active cells.From the study, it is clear that the effectiveness of various callus initiation media for each of the cotton genotypes  tested suggested that the optimal media combinations and genotypes are dependent on each other.Hormonal and nutritional conditions could also contribute to the differential callusing response.Callus selection is an important step in cotton tissue culture which plays a major role in the successful regeneration of cotton somatic embryogenesis.

Figure 2 .
Figure 2. Effect of age of explants on callus induction frequency on Indian genotype.

Figure 3 .
Figure 3.Effect of age of explants on callus induction frequency on Coker genotype.Cotyledon; hypocotyl.

Table 1 .
Different media compositions used for callus induction from seedling explants.

Table 2 .
Analysis of variance for callus induction in cotton genotypes with different explants and media compositions.

Table 3 .
Effects of plant growth regulators on callus induction from cotyledon and hypocotyls explants of cotton variety.