The use of tissue culture for fruit and nut tree species have increased substantially since the early 1970s and virtually all fruit tree species have been micropropagated with various degrees of success. Seedling rootstocks are not uniform in growth and productivity [1]. Therefore, vegetative propagation methods like cutting and stooling are used to multiply pear rootstocks. Micropropagation has shown promises for rapid and large scale clonal multiplication of disease free planting material throughout the year. In vitro propagation has been reported in several pear rootstocks viz. P betulaefolia L. [2], Wild pear [3]; OPR 157, OPR 260 and OH × F 230 [4]; P calleryana [5] and quince [6], Pyrodwarf [7] and Pyrus communis L. rootstock [8].

For forcing, dormant cuttings of Kainth of 15-20 cm in length (10-15 mm diameter) were collected and stored at 4±3°C in polythene bags. After subjecting the requisite chilling units, the cuttings were withdrawn and basal ends were re-cut by about 1 cm and placed in polythene bags containing rooting medium, covering about 5 cm of basal portion of cuttings. The cuttings were incubated in growth chamber at 23±1°C under 16 hours photoperiod with light intensity of 3000 lux. Shoots put forth by the sprouted buds served as explant source for in vitro propagation (Figure 1).

Figure 1: Forced cuttings of Kainth.

The basal media used in the study were Murashige and Skoog’s medium (MS), Murashige and Skoog’s medium with half strength of macro and micronutrients (1/2 MS) and Woody Plant Medium (WPM).

Already prepared MS medium (PT021) and WPM (PT026) purchased from Hi Media Pvt. Limited was used to prepare basal media i.e. M₁ (half strength MS), M₂ (full strength MS) and M₃ (Woody Plant Medium). Sterilized explants were inoculated in test tubes/glass jars containing autoclaved media for establishment. Different media (½ MS, MS and WPM) fortified with different combinations of 6-benzylaminopurine (BAP) {(0.5-3.0 mgl^-1) and IBA (0.01-2.0 mgl^-1)} were used. The data were recorded on necrotic cultures (%), explant establishment (%) after 3 weeks of inoculation.

Established explants were transferred to shoot proliferation media. Various shoot proliferation media i.e. ½ MS, MS and WPM were used, fortified with various concentrations of BAP i.e. 0.5-5.0 mgl-1. The optimum media and concentration of BAP for shoot proliferation was standardized. Observations on proliferated cultures (%), number of shoots/explant and average length of shoot (mm) was recorded after third subculture and the culture duration were four weeks each.

After allowing shoots to multiply on shoot proliferation medium, individual shoots were separated (20 mm long) and transferred to root regeneration medium. Different types of media i.e. ½ MS, MS and WPM containing various combinations of IBA (0.1-2.0 mgl^-1) and NAA (0.1-2.0 mgl^-1) were used. Observations on rooting (%), number of roots/ explant and average length of roots (mm) were recorded after four weeks.

The results of present investigation are described under appropriate heads supplemented with tables and plates.

Effect of medium supplemented with growth regulators on explant establishment

Data in Table 1 reveal that both media and growth regulator had significant effect on establishment (%). Highest establishment of 23.55 per cent was achieved by using M₃ medium, which was significantly higher as compared to M₂ and M₁. The data clearly shows that establishment percentage was highest using BAP (1.5 mgl^-1) and IBA (0.25 mgl^-1) and this combination differed significantly from all other combinations. Interaction studies between medium and growth regulators revealed that the highest explant establishment of 52.80 per cent was achieved from M₂ medium fortified with BAP (1.5 mgl^-1) and IBA (0.25 mgl^-1) (Figure 2).

Table 1: Effect of media type and growth regulators (mg/l) on explant establishment (%).

Figure 2: Explant establishment in MS medium fortified with BAP (1.5 mgl^-1) + IBA(0.25 mgl^-1)

M1 medium fortified with BAP (0.5 mgl^-1) and IBA (0.5 mgl^-1) resulted in the lowest establishment (%). These results are in conformity with earlier observation made by De Paoli, [9], who reported Murashige and Skoog medium as the best medium for culture initiation resulting in the highest establishment (%) with least necrotic cultures. Variations in per cent establishment of explants with different doses of auxin and cytokinin during micropropagation are in conformity with the reports given by Fan and Jiang [10] in apple; [11] in Carica papaya; [12] in pear; [13] in Citrus acida; [14] in pineapple and [15] in sweet cherry. Different media have been tried earlier for establishment of plant species by various workers and reported varied results in terms of establishment percentage. Peer et al. [16] reported better results in terms of establishment percentage in sweet cherry cv. Bigarreau Noir Grossa using Murashige and Skoog medium over Driver and Kuniyuli medium, Woody Plant Medium and Knop’s macro and MS micro-organics medium independent of growth regulators concentration. In vitro effects of growth regulator on overall establishment of explant has been reported to be influenced by growth medium composition, growing conditions and genotype [17,18].

Shoot proliferation

Established explants were transferred to shoot proliferation media i.e. M₁, M₂ and M3 containing different levels BAP. Data regarding shoot proliferation was obtained after two sub culturing as shoot growth was very slow initially irrespective of media used and growth regulator level.

From the perusal of (Table 2) it is evident that proliferated culture (%) during proliferation stage is certainly affected by type of media and BAP concentration individually as well as in interactive fashion. With regard to media, irrespective of BAP concentration, maximum proliferated culture (72.13%) was obtained in M₃, which is significantly higher than proliferated culture (%) obtained in M₂ and M₁. Maximum proliferated cultures (91.13%) resulted by using BAP at 3.0 mgl^-1, which is statistically at par with proliferated culture (%) obtained at BAP (1.5 mgl^-1). Regarding interaction between type of media and BAP concentration, maximum proliferated cultures (95.30%) were obtained by using M₃ fortified with BAP (3.0 mgl^-1) (Figure 3).

Table 2: Effect of media type and growth regulator level (mgl^-1) on proliferated cultures (%).

Figure 3: Shoot proliferation in WPM fortified with BAP (3.0 mgl^-1).

Data in (Table 3) clearly shows that with increase in level of BAP, there is increase in number of shoots produced per explant irrespective of media used. Maximum number of shoots per explant was observed in M₃ (4.39) which was followed by M₂ (3.61) and M₁ (2.63). All media differ significantly in terms of production of number of shoots per explant. The highest number of shoots per explant i.e. 4.78 was obtained with BAP (3.0 mgl^-1) and it was followed by 1.5 mgl^-1 BAP (4.43). Interaction effect revealed that there is significant interaction between media and growth regulator concentration on number of shoots produced per explant in Kainth. M₃ medium fortified with BAP (3.0 mgl^-1) resulted in maximum number of shoots per explant (6.28) followed by 5.75 in M₃ supplemented with BAP (1.5 mgl^-1).

Table 3: Effect of media type and growth regulator level (mgl^-1) on number of shoots.

Shoots of most desirable length (40.86 mm) were obtained on M₃ medium followed by 38.86 mm on M₁ medium (Table 4). From the perusal of Table 5, shoot length clearly decreases with increase in level of BAP and shoots of maximum length were obtained when no growth regulator was added to medium i.e. control. Average shoot length produced at 0.5 mgl^-1 BAP (41.79 mm) and control (42.19) were statistically at par but differ significantly from average shoot length produced at 1.5 mgl^-1 BAP (37.20 mm) and 3.0 mgl^-1 BAP (33.22 mm). There was no significant interaction between type of media and growth regulator level on average length of shoots although maximum length of shoots was obtained when basal M₃ medium i.e. control was used during shoot proliferation.

Table 4: Effect of media type and growth regulator level (mgl^-1) on av. length of shoots (mm).

These findings are in conformity with those of in P. pyrifolia cv. in Japanese pear cultivar Hosui, who reported superiority of WPM over other media with respect to proliferation rate [19,20]. BAP level was found to effect significantly per cent proliferation, shoots per explant and shoot length and these results are in conformity with studies reported by Dwivedi and Bist [19]; Sedlak and Paprstein [21]; Cosac and Frasin [22]; Karimpour et al. [18]; Hassanen and Gabr [2]; Ruzic et al. [7]; Isikalan et al. [23] and Soni et al. [24]. Hu and Wang [25] reported that cytokinins, especially BAP stimulated axillary bud development but at higher concentration shoot elongation was suppressed. Similarly, higher number of shoots per explant during proliferation stage on M2 as compared to M1 has been reported by Hassan [26] in Le Conte pear, [27] in Bartlett pear and [28] on figure due to higher nutrient concentration.

In vitro rooting

The in vitro regenerated shoots during shoot proliferation were transferred to various rooting media supplemented with different levels of IBA and NAA. Data from (Table 5) reveal that the highest rooting of 5.22 per cent was observed on M₁ medium, which is significantly different from rooting percentage observed on M₂ and M₃. Irrespective of media, IBA (0.1 mgl^-1) induced higher rooting (1.58%) as compared to IBA (1.0 mgl^-1). The interaction effect of treatment combination of M₁ fortified with IBA (0.1 mgl^-1) resulted in best rooting response (13.34%) (Figure 4), when compared to M₃ fortified with IBA (0.1 mgl^-1).

Table 5: Effect of media type and IBA level (mgl^-1) on rooting (%).

Figure 4: Rooting using ½ MS fortified with IBA (0.1 mgl^-1).

Data in (Table 6) clearly reveals that the highest number of roots per explant (1.25) was produced in M₁, which is significantly higher than those obtained on M₂ and M₃.

Table 6: Effect of media type and IBA level (mgl^-1) on number of roots per explant.

Maximum number of roots per explant (2.01) was obtained by using IBA at 0.1 mgl^-1 irrespective of media used. M₁ medium fortified with IBA (1.0 mgl^-1) resulted in significantly higher number of roots per explant (2.79) than what was obtained in M₁ medium fortified with IBA (0.1 mgl^-1).

Perusal of data in (Table 7) clearly reveals that there is significant effect of rooting media and IBA levels on average length of roots. Irrespective of IBA levels, M₁ induced average root length of 11.00 mm as compared to 7.79 mm on M3 and 7.04 mm on M₂ medium. While as IBA at 0.1 mgl^-1 resulted in maximum average root length of 27.41 mm, which is significantly higher than 7.03 mm observed at IBA (1.0 mgl^-1). Interaction effect revealed that roots of maximum length were produced in M₃ medium fortified with IBA (0.1 mgl^-1) having average length of 31.15 mm, which is significantly higher than other treatment combinations.

Table 7: Effect of media type and IBA level (mgl^-1) on average length of roots (mm).

As compared to IBA, NAA induced higher rooting irrespective of type of rooting media used (Table 8). Data from Table 9 clearly reveals that M₁ induced the highest rooting (13.49%) in Kainth. NAA at 1.0 mgl^-1 resulted in maximum rooting (24.83%), which is significantly higher than 22.35 per cent obtained at NAA (0.1 mgl^-1). A treatment combination of M₁ with NAA (1.0 mgl^-1) resulted maximum rooting of 29.61 per cent (Figure 5).

Table 8: Effect of media type and NAA level (mgl^-1) on rooting (%).

Figure 5: Rooting using ½ MS fortified with NAA (1.0 mgl^-1).

With regard to effect on number of roots per explant using different types of rooting media fortified with various levels of NAA showed that M₁ resulted in maximum number of roots per explant. From the perusal of data in (Table 9), significantly more number of roots per explant (3.07) were observed using NAA (1.0 mgl^-1), irrespective of media. M₁ fortified with NAA (1.0 mgl^-1) resulted in the highest number of roots per explant (3.40).

Table 9: Effect of media type and NAA level (mgl^-1) on number of roots per explant.

Data in (Table 10) shows that average length of roots in Kainth is significantly affected by rooting media and NAA levels. As far as media is concerned, M₃ resulted in longer roots (11.04 mm) irrespective of NAA level. NAA at 0.1 mgl^-1 resulted in significantly longer roots (20.53 mm) when compared to 16.97 obtained at NAA (1.0 mgl^-1).

Table 10: Effect of media type and NAA level (mgl^-1) on average length of roots (mm).

A treatment combination of M₃ fortified with NAA (0.1 mgl-¹) resulted in maximum average length of roots (22.97 mm) which is followed by 21.20 mm in M₃ medium fortified with NAA (1.0 mgl^-1). These two treatments were statistically at par in terms of controlling average root length.

Thakur [29] and Thakur and Kanwar [3] also reported better rooting response of Kainth as compared to scion varieties. Like the multiplication rate, rooting ability being genotype dependent [30] and rootstocks usually root with greater ability than scions [31]. The mineral concentration in the culture medium affects rooting characteristic and some researchers have proposed that reduction of salt strength to half strength improved rooting [32].

The reason behind increasing rooting rate on half strength culture medium might be due to a disorder in carbohydrate to nitrogen in nutrient medium, which lead to decreasing nitrogen level in shoot and then improving rooting rate, initiation roots, increasing root number and lengths [33]. Higher rooting response in Kainth using NAA than IBA is in conformity with [3,29]. Better rooting response of pear genotypes with NAA is in concordance to the findings of Singha [34] who preferred NAA over IBA for inducing roots in P. communis cv. Seckel to avoid the basal callus formation. Reed [35] also found that some pear genotypes rooted better on NAA than on IBA. Too high an auxin concentration in rooting media is undesirable as it leads reduction in rooting by inducing basal callus formation [36] or by inhibiting the root elongation [37]. This may be the reason for poor rooting response at higher auxin concentration in the present study.