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In vitro shoot multiplication of Merton I. 793, a clonal apple rootstock, was achieved through terminal / axillary bud culture. Low concentrations of a-naphthyl acetic acid (NAA) was proved to be more effective for rooting as compared to IBA and indole-3-acetic acid.
In vitro shoot multiplication of Merton I. 793, a clonal apple rootstock, was achieved through terminal / axillary bud culture. Low concentrations of a-naphthyl acetic acid (NAA) was proved to be more effective for rooting as compared to IBA and indole-3-acetic acid.
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In vitro shoot multiplication of Merton I. 793, a clonal apple rootstock, was achieved through terminal / axillary bud culture. Low concentrations of a-naphthyl acetic acid (NAA) was proved to be more effective for rooting as compared to IBA and indole-3-acetic acid.
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In vitro multiplication of Merton I. 793An apple rootstock suitable for replantation Mohit Soni, Manisha Thakur and Manju Modgil* Department of Biotechnology, Dr Y S Parmar University of Horticulture and Forestry, Nauni 173 230 , Solan, India Received 27 April 2010 ; revised 6 September 2010; accepted 10 November 2010 In vitro shoot multiplication of Merton I. 793, a clonal apple rootstock, was achieved through terminal/axillary bud culture using Murashige and Skoog (MS) basal medium supplemented with 0.5-1.0 mg/L 6-benzyl amino purine (BA) and 0.1 mg/L indole-3-butyric acid (IBA). Time of collection of explants, duration of surface sterilents and growth regulators added in the medium influenced the survival of explants and their bud break. Although, the shoots were multiplied on different concentrations of cytokinins and auxins, improved multiplication was achieved with 0.5 mg/L BA and 0.01 mg/L IBA. Of the three types of explants evaluated for shoot multiplication, shoot tips on elongation were found suitable for rooting whereas, nodal cuttings and cluster of small shoots for further multiplication. Low concentrations of -naphthyl acetic acid (NAA) was proved to be more effective for rooting as compared to IBA and indole-3-acetic acid (IAA). To overcome profuse callus formation during rooting, activated charcoal was added. In another experiment, auxin was discontinued from the rooting medium after few days, thus, resulting in better rooting frequency (72%). Thirty plantlets of Merton I. 793 were successfully hardened after 7 wks, with around 80% survival rate. Keywords: Apple rootstocks, in vitro multiplication, Merton I. 793, in vitro rooting, micropropagation Introduction In recent years, productivity of apple orchards in Himachal Pradesh has been decreased thereby causing a serious concern for the apple growers 1 . Main causes for decline in production and productivity are (i) non-availability of high quality and healthy planting material, (ii) proper selection of varieties of rootstocks of; and (iii) inadequate adoption of advanced production technologies. Most of the commercial plantations are now more than 35- 40-year-old and have surpassed the commercial bearing life and need to be replaced 1 . Because of land limitation, growers are compelled to plant new apples on old apple sites. There has been increasing concern about poor growth, delayed fruiting and the short life of apple trees planted on sites where apple trees grew before 2 . This situation resulting in the poor growth and making plantation uneconomical is generally known as the problem of replantation. However, a clonal rootstock of Merton Immune series, Merton I. 793, is found resistant to diseases spread during replantation in orchards 2 . The Merton Immune series M.I. 788 to 793 was introduced in 1930 as a result of joint breeding programme by the John Innes Horticultural Institution and the East Malling Research Station, followed by Malling Merton (MM) series. In India, first rootstock trial was initiated in 1937 at Chaubattia (Uttarakhand) with Red Delicious, Jonathan, Rymer on M2, M13, Merton I. 779, Merton I.793. Merton I. 793 is obtained from a cross between `Northern Spy' (woolly aphid resistant variety) M.2. It was introduced in Himachal Pradesh (HP) in the year 2000-2001, from New Zealand. It is vigorous, resistant to collar rot and woolly apple aphid. In H P, the plant material is being multiplied using conventional methods but is still in short supply. It has been reported earlier that M12 rootstock should only be used where root diseases are the problem, otherwise MM115 and M.I.793 are recommended for lighter soils and M.I.793 for heavier soils with chloropicrin fumigation 3 . In vitro multiplication of plants is a commercial venture in several countries with the Netherlands, France and Italy as the world leaders. Among the fruit crops, temperate fruits share significant portion of the plants produced through tissue culture. Propagation of apple rootstocks using tissue culture methods has been developed by many scientists 4-7 . To the best of
*Author for correspondence: Tel: 91-1792-252639; Fax: 91-1792-252242 Mobile: 09459241331 E-mail: manju_modgil@yahoo.com SONI et al: IN VITRO MULTIPLICATION OF APPLE ROOTSTOCKMERTON I. 793
363 our knowledge, there are very limited reports available on M.I.793 micropropagation 8-9 . Keeping in view the recommendations of H P Directorate of Horticulture to multiply Merton I. 793 on a large- scale the present study was initiated 10 .
Materials and Methods
Collection of Plant Material The shoots of apple rootstock Merton I. 793 were collected in plastic bags throughout the year from the nursery of H P Horticulture Department, Kwagdhar, District Sirmour (HP), India. The procedure for explant collection, excision and approaches to overcome phenol exudation were similar to as described previously 5 . The terminal and axillary buds (0.5-2.0 cm) were washed under running tap water for about 1 h and treated with 70% alcohol followed by surface sterilization using sterilents 0.1% HgCl 2 for 3-4 min and 2% NaOCl for 20-25 min separately. The buds were rinsed with sterile distilled water to remove traces of the sterilent. The explants were placed on to petridish lined with filter paper, and cultured in 100 mL flasks containing basal liquid Murashige and Skoog medium (MS) 11 . The flasks were rotated on a shaker for one day to rinse the phenols, which exuded from the cut surface of explants.
Culturing of Explants For Establishment After rinsing of phenols in liquid medium, the explants were inoculated on MS medium having adsorbent polyvinyl pyrollidone (PVP) (10 g/L) and supplemented with different combinations of benzyl adenine (BA) (0.5-2.0 mg/L) and indole-3-butyric acid (IBA)/ napthyl acetic acid (NAA) (0.1 mg/L). The medium was solidified with 0.8% agar. The explants which showed vigorous bud break were selected for transfer to fresh medium for further growth and multiple shoot production.
Shoot Multiplication The shoot cultures were maintained and multiplied by the method of enhanced release of axillary buds. MS medium consisted of different combinations and concentrations of plant growth regulators like BA, kinetin (Kn), 2-isopentyl adenine (2ip), IBA, gibberellic acid (GA 3 ). Best cytokinin and auxin concentration for multiple shoot formation was determined by recording the number of shoots produced per explant and length of shoots (placed vertically or horizontally) after 5 wks of culture. Further, the effect of explant type on shoot multiplication was observed by comparing single shoot tips with other explant types such as nodal cuttings and cluster of 2-3 shoots. 6-10 cultures were examined per treatment and experiment was repeated twice.
In vitro Induction of Rooting Vigorously growing shoots from proliferating cultures were used to make cuttings of more than 1 cm long (microshoots) and cultured on 1/2 strength MS medium consisting of 25 g/L sucrose, 100 mg/L inositol, 0.5 mg/L thiamine and 6 g/L agar, which was further supplemented with different concentrations and combinations of auxins viz. IBA (0.3-0.7 mg/L), IAA (Indole-3-acetic acid) (0.5-1 mg/L) and NAA (0.1-0.5mg/L) in one-step method. Activated charcoal (0.2%) was supplemented in some of the above combinations. Two-step rooting procedure was used where microshoots were first cultured in NAA (0.1-0.5 mg/L) enriched liquid rooting medium. After one wk, these were transferred to same medium devoid of NAA but solidified with agar. Each experiment consisted of three replications with 30 cuttings. Rooting response was noted after 4 wks. All the cultures were incubated at 252C. Light consisted of white fluorescent tubes and an incandescent lamp. The intensity of light at the level of cultures was 4000 lux with photoperiod of 16 h light and 8 h dark.
Hardening Plantlets with adequate roots were removed from the culture vessels and cleaned under running tap water. After washing, plantlets were treated with 0.25% bavistin for 30 min and transferred to pots filled with coco peat which was already drenched with 5 g/L bicontrol agent (Trichoderma viridae with commercial name Defence or Ecoderma). They were maintained in shade in glass house at 202C with the help of misting, cooling pads and fans. Plants were irrigated with Knops nutrient solution and fungicide after every 15 d.
Results It has been observed that only 6-12% explants died due to phenol exudation and contamination in the buds collected during spring or summer season (Table 1). Further, the explants collected in summer survived maximum (85%) in cultures after 4 wks whereas those collected in spring showed maximum bud break (43.90%). Effect of sterilizing agents on explants revealed that treatment with 0.1% HgCl 2 for 4 min resulted in 90% uncontaminated buds but only 18% INDIAN J BIOTECHNOL, JULY 2011
364 explants survived after 4 wks, while 3 min duration resulted in 85% explants survival. On the other hand, bud explants survived after 4 wks were more (71%) by using 2% NaOCl for 25 min, as compared to explants treated for 20 min duration. Terminal/apical buds elongated into small shoots after 4-6 wks on MS medium containing 1 mg/L BA and 0.1 mg/L IBA (Fig. 1a) which increased in length upon subculturing on fresh medium (Fig. 1b) while stimulation of axillary buds led to rosette type of leaves. It is evident from Table 2 that the growth regulator composition significantly affected the establishment of cultures. Out of 14 combinations attempted, maximum bud break (87.50%) was achieved on medium containing 1 mg/L BA and 0.1 mg/L IBA followed by 0.5 mg/L BA and 0.1 mg/L IBA (70.83%). Medium supplemented with BA (0.5-2.0 mg/L) alone resulted in reduced number (12-54%) of bud breaks. On the other hand, moderate callus formation was observed in bud cultures with BA (0.5-2.0 mg/L) and NAA (0.1 mg/L) which were discarded.
In vitro Shoot Multiplication When shoots, excised from established multiple shoot cultures, were placed vertically, multiplication was seen in all the combinations tested (Table 3). Highest multiplication rate (8-fold) with 2-3.5 cm long shoots was obtained on medium having 0.5 mg /L BA and 0.01 mg/L IBA (Fig. 1c). From each explant 2-5 shoots were obtained on 1.0 mg/L BA, 0.05 mg/L IBA and 0.5 mg/L GA 3 where some of the shoots were vitrified. However, this rate of shoot multiplication was not observed in subsequent subcultures. Five-fold multiplication and longer shoots were achieved when 1.0 mg/L 2ip was substituted with BA in above combination. However, five shoots per explant were obtained in very few cultures. A 4-fold shoot multiplication was achieved when BA alone at low concentration (0.5 mg/L) was used, while at higher concentrations (1.0 & 1.5 mg/L), shoot production and length reduced. Addition of Kn (0.5-1.0 mg/L) alone or in combination with BA (0.5 mg/L) and IBA (0.05 mg/L) did not show any increase in multiplication of shoots. When the explants were placed horizontally on the medium (Table 3), 2-3 small shoots were observed to arise from nodes. While investigating the type of explant on medium supplemented with 0.5 mg/L BA Table 1Effect of time of year on browning, contamination and survival of explants in M.I. 793, on MS medium supplemented with 0.5 mg/L BA and 0.1 mg/L IBA S.No. Time of year Explants browning due to phenol exudation (%) Explants contaminated (%) Explants survived after 4 wks (%) Explants showing bud break (%) 1. Spring 10.37 (18.79) 12.26 (20.50) 77.35 (61.61) 43.90 (42.48)
S.E. 0.78 0.81 0.36 1.15 C.D.0.05 1.81 1.88 0.83 2.65 Values in parenthesis are arc sine transformed values.
Fig. 1a-iMicropropagation of clonal apple rootstock M.I. 793: a, Bud break in terminal/axillary buds on MS medium with 1.0 mg/L BA and 0.1 mg/L IBA; b, Elongation of shoot after 2 months; c, Shoot production on MS medium with 0.5 mg/L BA & 0.05 mg/L IBA; d-f, shoots with roots and callus development on 0.1 mg/L NAA, 0.7 mg/L IBA. and 0.8 mg/L IAA + 0.2 mg/l IBA, respectively; g, Rooted shoots in two-step procedure; h, Rooted shoots on activated charcoal supplemented medium; i, Hardened plants grown in paper cups. SONI et al: IN VITRO MULTIPLICATION OF APPLE ROOTSTOCKMERTON I. 793
365 & 0.01 mg/L IBA, it was found that the shoot tips grew longer (3-4 cm), with few multiple shoots (2-5) while nodal cuttings and clusters of 2-3 shoots produced smaller (2.0-2.5 cm) but comparatively more number of multiple shoots (3-8).
In vitro Root Induction When IBA, IAA or NAA were tested, rooting was observed in a few cases only (Table 4). Basal portion of the shoots formed callus and later root initials developed after about 15 d. Among the three concentrations of NAA used, highest rooting percentage (66.78) was observed with low concentration (0.1 mg/L) of NAA (Fig. 1d). Around 50% of shoots were rooted on medium supplemented with 0.7 mg/L IBA (Fig. 1e) whereas 27% rooted in case of 1 mg/L IAA. Lower levels of IBA and IAA did not initiate roots. In the presence of activated charcoal (AC), thin roots were initiated after 2 wks and 55% rooting was observed with 0.1 mg/L NAA. It is clear from Table 4 that AC did not enhance rooting in any treatment but suppressed callus formation (Fig. 1f). In two-step rooting procedure, root initials were seen one week after transferring the shoots to basal solid medium. At the end of 4 wks, 72% rooting (Fig. 1g, Table 4) was observed in shoots dipped in 0.1 mg/L NAA followed by 57 and 35.9% rooting of shoots in 0.3 and 0.5 mg/L NAA, respectively. Combined effect of IBA and IAA showed that (Table 5), 0.2 mg/L IBA and 0.8 mg/L IAA resulted in 36.36% rooting with emergence of callus (Fig. 1h), whereas low concentrations of IAA (0.2, 0.4 & 0.6 mg/L) along with 0.2 mg/L IBA gave reduced rate of rooting (22-28%) without callus. There is not much difference in rooting percentage (30-33%) on medium containing 1 mg/L IAA with 0.1 and 0.3 mg/L IBA.
Acclimatization in Potting Mixture Rooted plantlets (30) without callus were successfully acclimatized with an average of 80-85% survival rate while plantlets with callus survived less. Knops nutrient solution seemed to enhance the growth of the plantlets (Fig. 1i).
Discussion Although axillary bud cultures of Merton I. 793 could be initiated at any time of the year, the explants from actively growing shoots at the beginning of spring season generally gave best results which showed that the success in the establishment of explants was dependent on the time of year of collection and physiological state of the parent plant at the time of explant excision. In addition, surface sterilization was found to be the most critical factor for successful establishment of bud cultures. Both the sterilants (HgCl 2 and NaOCl) were effective but HgCl 2 proved better. Webster and Jones supported our findings 12 . In the present study, though HgCl 2 treatment for 4 min provided highest number of uncontaminated buds, the survival of explants in later stages was more in 3 min duration. It explains that finding the exact duration of sterilant treatment is necessary for determining the survival of explants, and higher durations may be toxic to the explants itself. Out of all the combinations, BA (0.5-1.0 mg/L) and IBA (0.1 mg/L) were found the most suitable for establishment of explants. When GA 3 was used with BA and IBA, the results were not satisfactory. In other apple rootstocks, many workers found maximum bud break on media supplemented with GA 3 along with BA and IBA 13-15 . This difference may be due to the different genotypes used in our studies. There is an earlier report on in vitro shoot proliferation of Merton I. 793 where the explants were established successfully
on MS medium supplemented with BA (0.5 mg/L) alone 8 . During shoot multiplication, BA proved the most effective cytokinin, and a relationship between BA concentration, shoot number and shoot size was Table 2Effect of different combinations of growth regulators in MS for explant initiation in M.I. 793 Concentrations of growth regulators (mg L -1 ) BA IBA NAA GA 3
Explants showing bud break after 6 wks (%) Survival of explants after 1 st
also observed in the present investigation. Higher concentration of BA alone may increase the shoot number with decreased shoot length whereas low concentrations led to longer shoots. BA (0.5 mg/L) combined with IBA (0.01 mg/L), was found to increase the rate of shoot production as well as length of shoots. After supplementing GA 3 (0.5 mg/L) with above combination, number of shoots produced were not constant in each subculture. This combination had also been found to be better for the micropropagation of several other apple rootstocks 5,14,15 . Use of 2 ip in place of BA in combination with 0.05 mg/L IBA was not found effective and on adding GA 3, the
rate of shoot production increased in few cultures only. However, in case of Java apple, shoot proliferation was stimulated by the addition of BA (1 mg/L) to MS basal medium 16 but shoot elongation and shoot number was enhanced with 2 ip (1 mg/L) and NAA (0.1 mg/L). This difference may be due to the different genotype and type of auxin used with 2ip. Kinetin with BA or IBA did not enhance rate of shoot multiplication in M.I. 793. It has been suggested that differential sensitivity to a particular cytokinin and auxin, to the concentration of each, and to the ratio between them are important factors, especially in the establishment and proliferation of cultures 17 . Removal of tip and inoculating shoots horizontally or inverted in the medium reduced the apical dominance, thus encouraging the development of Table 3Effect of different combinations of plant growth regulators on the multiplication of shoots (after 5 wks) MS medium supplemented with (mg L -1 ) BA IBA Kn 2 ip GA 3
Table 5Combined effect of different concentrations of IBA and IAA on rooting in M.I. 793 IBA Conc. (mg/L) IAA Conc. (mg/L) Rooting percentage 0.1 0.2 0.2 0.2 0.2 0.1 0.3 0.1 0.2 0.4 0.6 0.8 1.0 1.0 0.00 (0.00) 28.38 (32.19) - 22.20 (28.11) - 27.20 (31.44) + 36.36 (37.09) + 30.33 (33.49) + 32.97 (35.04) + CD(0.05) 0.438 SONI et al: IN VITRO MULTIPLICATION OF APPLE ROOTSTOCKMERTON I. 793
367 axillary buds 18 , which might have resulted in superior utilization of BA. However, there was no advantage seen in shoot production by horizontal placement of shoots in M.I. 793. This is in contrast to earlier studies on other apple rootstocks where higher multiplication rate was reported by horizontal positioning of the explants 19 . This factor may be dependent on genotype and growth regulator combinations. Three types of explants assessed for shoot multiplication in the present study resulted in multiple shoot production but, the main advantage of such type of evaluation was that shoots grew from shoot tips were used for rooting being longer whereas shoots produced by other explants were spared for further shoot multiplication. Among the three auxins, NAA was found superior to IAA and IBA, though thick and callused roots were obtained in one-step procedure. Low levels of NAA resulted in the highest rooting whereas high concentrations gave rise to more root initials, which eventually developed into callus rather than rootlets. In contrast to our studies, earlier reports on Merton I. 793 reported that IBA stimulated high rooting abilities 8,20 . This may be due to the different age of cultures, or growth regulators present in shoot multiplication medium used by them differed from ours, or these differences of in vitro rooting ability may be due to clonal variation in the rootstock used. In our experiments, after supplementing the medium with activated charcoal (AC), callus formation was stopped, but the rooting frequency decreased. These results are supported by Magyar et al
that the presence of AC in root elongation medium decreased the rooting rate and number of roots depending upon the cultivar and the concentration 21 . The favourable effect of AC on rooting was mainly due to the adsorption of NAA. In our previous studies 22 , efficacy of different concentrations of IBA in combination with activated charcoal was tested and found that the formation of roots in apple cultivar shoots growing in vitro was variable with considerable genotypic difference. During the present study, combining IAA and IBA in different concentrations did not support good rooting efficiency. On the other hand, for rooting of shoots, two-step procedure appeared to be more effective than one-step because it had the major advantage of leading to increase in rooting efficiency without callus development. It seems that both auxin type and concentration influence rooting in Merton I.793 and continuous contact with auxins led to the callus formation followed by the emergence of roots. Since too long treatment of auxin leads to root inhibition it has been suggested that the auxin treatment must be appropriately interrupted by transplanting the root induced shoots on hormone free medium 23 . In vitro rooting stage is followed by hardening, which involves the transfer of plants from in vitro conditions to soil to impact some tolerance to moisture stress, for conferring a degree of resistance against certain pathogens and conversion of the plant from heterotrophic to autotrophic stage. We have observed that rooted plantlets without callus successfully established in peat. Better root and shoot development before hardening determines the survival which is also supported by other workers 4 . Fertigation during hardening of Merton I. 793 improved the vigour of rooted plantlets, but the nutritional requirement must be adjusted to plant size and age 24 . Application of complete nutrient solution similar to that used for raising bedding plants can be applied as both liquid and foliar feed 26 . In conclusion, our results suggest that a micropropagation method has been accomplished in Merton I. 793. For the commercial scale production of plants, further efforts are in progress to increase the rooting efficiency, and thereby hardening.
Acknowledgement We acknowledge the Department of Biotechnology, New Delhi, Govt. of India for financial assistance in the form of a project, under Network Programme on Apple.
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