RAPD analysis of long term micropropagated rootstock plants of Apple Malling 7.

Apple is the most dominating fruit crop of Himachal Pradesh, constituting about 40 per cent of total area under fruit crops and about 90 per cent of the total fruit production. Plant tissue culture has encouraged production of quality planting material which is disease free and true to type. Due to current trends in favour of high density apple plantations and to fulfill the requirement of planting material from the farmers, commercially viable in vitro propagation method of semidwarf clonal apple rootstock Malling 7 (M7) was developed by axillary branching, initially developed from shoot tip explants. A few thousand plants were transferred to field conditions. In order to check any tissue culture induced variation in these long term micropropagated plants, the genetic fidelity was assessed using Random Amplified Polymorphic DNA (RAPD) markers. Out of the 20 primers screened, 12 generated a total of 44 scorable and distinct bands with an average of 3.7 bands per primer. All the banding patterns for each primer were highly uniform and monomorphic across in vitro multiple shoot clusters and field transplanted micropropagated plants as well as comparable to the field grown mother clone from which the cultures had been established. Our results have shown that the micropropagation protocol developed by this study is appropriate and applicable for clonal propagation of apple rootstock M7 on large scale over a long period without any risk of genetic instability.

Assessment of genetic fidelity of micropropagated apple rootstock plants, EMLA 111, using RAPD markers.

EMLA 111(East Malling Long Ashton), a clonal rootstock of apple (Malus pumila Mill.) was micropropagated using axillary bud and shoot apices. The present work was carried out to assess the genetic fidelity of micropropagated plants using random amplified polymorphic DNA (RAPD). Ten arbitrary decamer primers have been used to amplify genomic DNA from in vitro raised field material and mother plant. A total of 57 amplified products were obtained, out of which 53 were monomorphic across the mother tree and its tissue culture raised progenies. Of the ten primers used, 8 showed RAPD profiles which were identical to the mother plant. Similarity matrix based on Jaccard's coefficient revealed that pairwise value between the mother plant and its tissue cultured plants ranged from 0.93 to 1.00 and among tissue cultured plants, it was 0.92 to 1.00, thus indicating a high degree of genetic fidelity.

Factors affecting induction and development of in vitro rooting in apple rootstocks.

Shoots of apple rootstocks raised in vitro were transferred to various rooting media to study the effect of different factors on root initiation and development. Various concentrations of indole-3-butyric acid (IBA) initiated rooting but maximum rooting percentage was found with 2.0 and 2.5 mg l(-1) of IBA in M7 and with 1.0 mg l(-1) of IBA in MM106. The drawback was that the roots were thick, short and with profuse callus. The presence of activated charcoal (AC) in the rooting medium improved the rooting quality but reduced the rooting percentage in both the rootstocks. In high auxin dip of 70, 80 and 90 mg l(-1) IBA for 2, 2 and 1 hr showed 75-85 per cent rooting in M7, but lacked reproducibility of the results. Whereas in MM106, 66 - 70 % rooting was achieved with 70 mg l(-1) of IBA dip for 3 h. Root induction in shoots in IBA containing liquid medium (LM) in dark for few days and root elongation in IBA--free medium in light proved most effective. On the other hand, continuous light treatment showed reduced rooting. Reduction of MS salts and sucrose in root elongation medium showed decreased rooting. Plantlets from two--stage rooting procedure showed more rapid growth and satisfactory survival during hardening of plants and on transfer to field.

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds.

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.

Distinguishing clonal apple rootstocks by isozymes banding patterns.

Molecular characterisation of clonal apple rootstocks using isozymes was carried out to identify isozyme polymorphism in seven clonal apple rootstocks and to identify the most characteristic and stable enzyme markers for each individual rootstock. Five enzyme systems were studied out of which polyphenol oxidase, malate dehydrogenase, acid phosphatase and peroxidase were useful in discriminating among the rootstocks. The peroxidase enzyme system showed maximum variation and esterase showed the least variation among the rootstocks. Out of seven rootstocks, three were distinguished on the basis of one enzyme system only (M.3 with MDH or PER, M.7 with PPO or PER and MM. 111 with MDH). Out of the sixteen loci studied seven were found to be polymorphic. Genetic variation among the rootstocks was explained on the basis of various parameters. The percentage of polymorphic loci varied from 13.33 to 35.71 per cent.

Successful propagation in vitro of apple rootstock MM106 and influence of phloroglucinol.

Successful in vitro propagation of clonal apple rootstock MM106 was achieved by culturing axillary buds on MS basal medium with BAP (1 mg/L), GA3 (0.5 mg/L) and IBA (0.1 mg/L). Use of liquid medium (LM) in initial cultures reduced phenol exudation to a greater extent and gave maximum sprouting percentage when transferred to solid MS medium. Phloroglucinol (PG) did not enhance sprouting of buds but increased the rate of multiplication when added in the medium. Maximum number of shoots were obtained when MS medium was supplemented with BAP (0.5 mg/L), GA3 (1 mg/L), IBA (0.1 mg/L) and PG (100 mg/L). For rooting, in vitro regenerated shoots were placed in IBA (30 mg/L) for 3 hr and transferred to solidified auxin free medium. Rooting was recorded in about 80% of shoots. Inclusion of PG in rooting medium was not beneficial but shoot cultures grown in its presence gave higher rooting percentage. Rooted plantlets showed about 70% survival rate in potting mixture of sand:soil:perlite (1:1:1).