A PHISIOLOSICAL AVALYSIS OF ROOTING IN CUTTINGS OF JUVENILE WALNUT (JUGLANS RIGIA L.) D.R, Jautam J.3.Chauban Dr.¥.S,Parmar University Department of Fruit of Forticulturs’ and Culture and Orchard Forestry, Temperate Fruit Management, Dr.Y.S.Parmar R ch Station, Kotihai University of Horticulture Distt.Shinla (H.P.) and Forestry,Solan (H.P.) INDIA INDIA jetract Juvenile phase walnut cuttings callused almost throughout the year under intermittent mist with 1A applications of 0.5 to 1.5 ver cent. Dormant hardwood cuttings rooted to some extent, whereas leafy cuttings during summer and autumn failed to root, Pre-conditioning treutments like blanching and girdling also kad no effect cn the rooting of these cuttings. These walnut shouts vere found quite rich in carbohy- drates, phenolics and amino acids and the pre-conditioning treatments further increased the C/N ratio from 12.8 to 22.7 and the total phenols from 14.0 to 23.5 mg/g. This increased bio~ chemical status also shoved no effect on the rooting of the walnut cuttings. In fresh semi-hardwood cuttings of walnut (before the Flanting), the bioactivity of roasting promoters was seen at Rf 0.2 to 0.4. After 90 days in the callused cuttings this activity was celuced and a strong inhititory activity was tested at most of the Rf values except 0.2 to 0.4. These pronounced inhibitory activities And the reduction in the level of rooting 2rosoters may vertially explain the difficult regeneration of juvenile valnut cuttings. 1, Introduction The maintenance of genetic uniforaity ty clonal propagstion through roeting of cuttings is the most convenient and cacy method of multiplication, However fruit crovs differ greatly in their ability to regenerate From cuttings. Stem cuttings of sone of fruit ereps root easily bunder ordinary field dota Horticulturae 284, 1 Walnut Sroduetion 1? 799° 33conditions while other difficult-to-root varieties cun be rooted if some factors like nutrition of stock plant, tine of taking cuttings, flowering condition end growth regulator treatments etc, are given due consideration. Still other nlant species fail-to-root even if all these factors are taken care of and probtatly walnut is one of tems Very poor results have been obtained with the rooting of softwood and hardwood cuttings of walnut under mist. Even with the layering of welnut, only callusing was found to occur in the first year of operation (Smyers, 19773 Smyers and Still, 1978; Gutenev and Bogorodiskii, 1974), The difference in ability of fruit crops to regenerate from cuttings essitated the research workers to investigator the physiological and kio-chemical factors that determine the rooting success. The possible role of co- factors was reported in rooting of plants (Hess, 1962). The rooting ability of walnut stem cuttings was tested and the probable role of the various physiological factors was also studied. Haterial and Methods 2.1 Plant Material The investigation to ascertain the seasonal changes in the rootings of walnut cuttings was carried out in a mist chamber (8x4m) situated in the Department of Fruit Culture and Orchard Management, Dr.Y.S.?armar University of Horticuiture and Forestry, Nauni, Solan during 1983-5, 20-25em long terainal cuttings were taken from 4-Syr old hedges of walnuts during spring, eumzer and autuan. ‘Wo longitudinal wounding cuts were given to cuttings tases and then dipped for 10 s2conds in I2a solutions of 0-5, 1.0 or 1.5 concentrations wade aa 50 per cent ethanol (v/v). Preconditioning treatwents like etiolation, girdling were also tried in separate coubinations. Observation on rooting was taken after 30 days. 34Cutting samples were taken during each season and chopped and dried in oven at 60 ¢ for estimation of different biochemical consitituents. Oven dried sample weighing 2.53 was boiled cn a water bath with 15-20 ml of 60 per cent ethanol for 10 minutes. after cooling supernatant was decanted ani the residue was again extracted 2~3 $imes, The alcoholic extract was filtered and final volume of 25 ml was made. This alcoholic extract vas used for estimation of phenols, For other constituents the alcoholic extract was reduced to aqueous phase and adjusted to known volume. Ortho dihydric (0.D.) phenols were estinated as per method suggested by arnow (1937), wheréas total phenols were determined with Folin ciocalteu reagent procedure given by Bray and Thorpe (1954), Total sugars were estimated as per method given by Duboi's et al. (1954) using anthrone, nt. Starch content in the samples was estimated following perchloric acid hydrolysis (Clegg 1956). Ninhydrin method developed by Moore and Stein (1948) was followed for the estimation of total amino acids. For the determination of total nitrogen, one gram of oven dried cutting sample was digested in concentrated sulphuric acid by adding digestion mixture. Total nitrogen was estimated on "Technicon Auto-analyser." 2.3. Determination of endogenous root promoters and inhibitors Twenty gram tasel two cm segments of fresh (before planting) and callused/rooted cuttings (after 90 days) of juvenile walnut ‘rere macerated in blendér with cold ethanol at 3°C and homogenates were extracted twice with eight volume of ccld aethanol. Extracted math material along with plant tissue was keot in a freezer for 24 hours and then reduced to aqueous phase by evapora- tion under reduced pressure at 40°C in dark. The aqueous extract was adjusted to pH 2.5 with 5.0 per cent hydrochloric acid and was partitioned four times with ethyl acetate. ‘The acidic ethyl acetate 2fraction was reduced to dryness and taken in 2 ml of ethanol. Huntered microlitre aliquots of these samples was strip leaded on Vhatman No.1. Chrosatography vaper., The chromatograms ware leveloped by descending aethed ty using isovropancl 1 ammonia and water (10:1:1 v/v) as solvent. The activity of rcot promoters was determined by Nung bean {P. lus aureus ) technique given by Hess (1364). The presence and activity of inhibitory substanceg was estimated by Cress seed (Lepidium sativum var. Curled) germination inhibition bioassay fasuda (1362). test procedure tiodified by 3. Results 3.1. Seasonal callusing and rooting behaviour Since poof rooting was recorded in the cuttings, hence the data on callusing was also recorded. The cuttings treated with 0.5 per cent IBA and planted in June resulted in maximum callusing (60.0 per cent) in 1383 and with 1.5 per cent in 1384, (Table 1), The dormant cuttings planted in winter only showed rooting to an extent of 14.5 per cont in 1984. Table 1. Effect of season and 134 concentrations on the = ee busin and rooting of cuttings from juvenile walnut. Season of Cuttings callused(s) Cuttings rooted planting conc. See Spas Seppe topted cuttings. (3) June, 15 0.0 5.0 30.9 0.0 0.9 (Sunaer) 0.5 60.0 30.0 0.0 0.0 1.0 37-5 5765 0.0 c.o 1.5 22.5 35.0 c.0 0.0 September, 15 0.0 0.0 0.0 0.c 0.2 Cautun) 0.5 7:5 10.0 OC 0.0 1.0 res aoe O.c 0.0 ie 0.0 540 0.0 C.0 January 15 0.0 o.0 0.0 0.0 0.0 Winter) OS 20.0 50.0 7 e.c 15 12.5 33.3 5.00.9 15 3705 23,5, 1000 14.5 363.2. Stochemical status The data on some biochemical constituents is given as below in Table 2. Table 2. Hean valuss of some biochemical constituents in juvenile walnut cutting. Year/Season Total Total c/i Total «= @.Dw =Total. of planting nitrogen carbo- ratig amino phe= _— phenol Ge hydra- acids nots _tese (mg/g) (mz/a)__ (m3/a) 383 June ,15 0.44 10.77 -24.5 1420 3.6 18.6 Sept,15 1,20 26.18 = 21.82 1414 2.6 25.4 January, 15 0.86 44652 13h 1,06 5423849 1384 Tune, 15 0.72 13.65 15491440 24 16.5 Sept, 15 0.74 11.33 16.2 0.48 3.2 22.0 January )15 0.94 11.06 14.8 0.72 3.5 25.9 From the data it 48 evident that during sumer and autuan the shoots were rich 4n C/N ratio and amino acids. ‘The trend in case of phenolics was however reverse, The rooting only occured in winter planted cuttings when phenolics were maximum. The various pre~conditioning treatments like etiolation (&) and girdling (G) had marked effect on tnese biochemical constituents (Table 3) given belowt- Zable 3. lean valurs of some blochenical constituents on oven dry weight basis as effacted ty various preconditioning treatments. Year/pre Total Total c/n Totel C.D. Total conditicn- nitro- carbo- ratio amino pheno- ghenols gen hydra. acids 1s G tes) (mg/s) (male) (m/e) 9.60 14.45 2444420035 0.72 13.00 15.2 4.40344 0.57 12.95 22.7 1.08445 4615 14.75 12.8 4478 145 3TBG Etiolated and girdled NE+NG Control. Etiolation and girdling of shbots before taking cuttings reduced the total nitrogen and amino acids. The level of C|M ratio and phenols was increased in general. This increased status also fatled-to initiate rooting in leafy cuttings. 3.4, Promoters The Bones of promotion in the basal extracts from walnut cuttings were seen at RE 0.2) 0.7 and 0.9 (Fig 1). After 20 days (in callused cuttings) the activity of the promoters was found very less at Rf 0.2 to Oud and 0.7. 3.5. Inhibitors In the extracts of fresh walnut cuttings the zones of inhibition were seen at Rf 0.4, 0.9 and 1.0 tut after 30 days, in the extracts from callused bases of cuttings very strong inhititory activities were recorded at Rf 0.4 to 0.6, 0.3 and 0.9 (Fig 2). It showed that inspite of the accumulation of inhibitory sutstances at original Rf values, some more inhibitors accumulated in walnut cuttings during callusing process at Rf 0.5 to 0.8. 4 Catlusing and rooting behaviour vimun callusing (73.5 ver cent) was obtained in cuttings planted in January}484 and ainimun (2.5 2er cent) in Septeaber,19£3. There was rooting of winter cuttings to an extent of 14,5 rer cent. dutenev and Bogoroditskit (1374) reported vary poor rcoting of softwood arid hardwood cuttings of walnut. Lot of callus production during firat year of layering of walnut shoots was also observed by schneiders (1940) und Maurer (1350). 4.2. Some hurdvood cuttings from juvenile walnut shoots which were tiolated and girdled also failed+to-root evan with 134concentration of 1.5 per cant. ‘These findings are in agrement with those of Jill et al. (1979). They also reported that ringed and mound layered shoots of Juglans regia failed to root in one season. 4.5. BL te rooting There were quite large variations in the amounts of total Ny carbohydrates, amino acids and phenolic compounds. With etiolation and girdling, there was increase in the level of carbohydrates and henols, however, there was reduction in t! amounts of N and amino acids. These findings are in opposition to those of Howard et al.(1983), who have reported increased rooting with increase in the level of phenolic cofactors, Other workers lke Tree by and Considine (1382) have reported that neither the starch nor carhoydrates were statistically related to the proportion of rooted cuttings 4.4. Promoters There appeared to be little root promoting activities at Rf 0.2, 0.7 and 0.9, After 90 days in the callused basal ends these root promoting activities were markedly reduced to strong inhititory areas except at Rf 0.2 to 0.4. Gasto et al. (1367) and Mandez et al. (1968) have observed that although toth inhibitor and promoter zones were present in easy as vell as difficult-to-root ssecies but the presence of inhibitor was much stronger dr, later case, Hess (1959) stated that in cuttings which are difficult-to-root only natural co-factors can stimulate the root lifferentiation. Since in Walnut cuttings the >romoters/ cofactors ware very less, therefore it may be due to this reason thot these cuttings failed to regenerate. 4.5. In! There was marked increase in the activity of inhibitory substances in the callused basal ends of the juvenile walnut cuttings. Besides the appearance of peak zone of inhibition at RE 0.5 and 0.7 at which A3A standards are reported to move by 39abdel Rehman et al. (1975) there was also vary strong inhibi- tory activity at &f 0.8 and 0.9sJuglone is a quinone compound which is found to occur in walnut plants (Sernthsen and Semper, 1882) and reported for its toxicity to certain plants growing near to walnut trees. Taylor and Odom (1370) nave reported that this compound was associated with one of the arces of root inhibitory activities in xecan at Rf 0.8°to 0.9. They also advocated that the factors other than rooting cofortors appear to limit the rooting of pecan cuttings. Cbviously in the present findings the inhibitory activities at taese RE (0.2 and 0.9) values scems to be due to juglone. Spiegel (1954) in grape vine, Fadl and Hartmann (1967) in pear and Fatta del Basco and De. Micheli (1963) in orange found natural inhibitors localized respectively in buds, leaves or in stem, Possibly the intact leaves of walnut continued the symthesis of the inhibitors noticed in the present studies and hence may te due to this increased inhititor level that these cuttings failed to ht initiate roots, whereas dormant cuttings rooted to an extent of 14.5 per cent. References Atdel-Rehman, M., Thomas, T.H., Doss,G.L. and Howell, L.,1975- Changes in endogenous plant hormones in cherry tomato fruits during development and maturation. Physiol. Plantarum. 34: 39-43. Arnow, L.E., 1937. CoTorinetric determination of the compone- nts of 2,4~ihyiroxy-phenylalanine in tryosine mixtures, J. Bio. Chem. 18: 531-537. Bernthsen and Semper, A., 1887. Ber. Dtsch. Chem. Ges.20: 934. Bray, H.3. and Thorpe, W.W.,1954. Estimation of phencls. Methods of 2iochem. Analysis (Glick, D3D). Interscience Publishing Inc. New York. 1:27-54. Dubois M., Gilli»s,K., Hamilton, J.K., Robers an:t Saith,F., 1951. A colornitric method for jetermination of sugars. Nature, 168: 167. Clegg, K.M31956. The application of the anthrone reagent to the estimation of starch in carzals. J. Sci. Food Agric. 7: 40-44, ill, G.3., Majica, J.P. and Avila, G.,1979. Root initiation in walnut layers, Gencia ¢ Investigation Agraria. 40 bts): 185-188,Fadl, M.S. and Hartmann.1967, Relationship between seasonal changes in endogenous promoters and inhititors in pear buds and cutting bases and the rooting of pear hardwood cuttings. Proc. Am. Soc. Hort. Sci, 91: 95-112. Fatta del Bosco, G. and Hicheli, A. Des, 1968. Indagam sco alouni as-pette fisiologici doll radicazione di talee di melangole (Citrus ahrantiua,L.). Bev. Ortofloro~ frutticoltura Ptaliana. 52. Gasto, M.D.V.,, Vanquez, A., Mendez, E., Vieltez, E.and Seoane, 55,4967. | Growth substance isolated fron woody cuttings of duercug robur L. and Juglans regia L. Phytochem., 6: 1687-1593. Gutenev, I.,and Bogoroditaskit, 1.1,1374. Vegetative propagation of walnut ty cuttings and layers in the Rostovskays oblast. Trudy Novacherkar Inzh. Mellor Instituta, 45(3)t 115-121. Hess, C.2,,1959.. A study of plant growth substance in easy and aitgicult-to-root cuttings. Proc. Fl. Propag. Sock. 2 39-95. Hese, C.£.,1962. Characterization of rooting Co-factors extracted from Hedera helix L. and Hibiscus reseasinensis L. 16th Int. Hort. Congr, 4:° 362-386. Hess, C.8.,1964. Naturally occuring substances which stimulate root initiation. pp 517-527 in J.P.Nitch (Ed) Regulators Natureie dé ta Crossane Vegetable C.N.R.S. Paris. Howard, B.H., Shephered, H.R. and Harrison Murray, R.S., 1983. Preconditioning shoots for summer cuttings. A. Rep. 8. Malling Res. Stnsy pp. 86-89. Kandez, Jey Gasto, M.D.Vs, fanquezy As and Vieitz, 3.,1363. Growth substances isolated from woody cuttings of Alnus glutinoga and Fraxinum excelsior. Phytochem. 7% 575. iiasudov, Y.,1362. £f2ct of light on grovth inhitition on wheat roots, Physiol. 2lantarum. 15: 780-730. Haurer, K.J.,1350. ‘Jegetative sropagation of walnuts, Schweiz obst. U. ieinb, 531 138-137. Moore, 8., and Stein, Y.H.,1949, Photometric method for use in chromatography of amino acids. J. Biol. Chem. 176: 367-28. Sebneiders, 2.,1940, Stool beds for walnut, propagstion Dtsch obste, Hit 11: 0.212.Shreve, L. and liles, N.¥.,1972. Propagetion black wainut clones from rooted cuttings. In 63rd A. Rep, Northern Fut Gr. Assoc. Amer. USA, 45-49. k and black ncentrations in hattan, Smyers, Don, R.,1977. Rooting study of mature red walnut stem cuttings treated with high c: of IBA. H,S. Thesis, Kansas state Uni. Kans as. Snyers, D.R., Still, S.1i.,1978, Non-rootability of mature red oak and black walnut stam cuttings. Pl. propagator 24 (4): 8-9. Spiegel, P., 1954 Bull. Auxina and inhibitors in canes of Vitis, es. Counc. Israel. 4: 176-183. faylor, G.3. and Cdom, 2..,1370, Some tiochemical associated with rooting of Carya illinoensis stem cuttings. J.Amer Soc. Hort. Sci. 95: 140-157. Tr2eby, M.T, and Congidine, J.A.,1962. Propagation of Vitis Champini. Planchon Cv. Ramsey: Relationshio between carbohydrate metablism during storage and cutting performance. Am. J. of Analogy and Viticulture 33(1):53~-56. 42PER AVG, NO, OF ROOTS CUTTING 43 JUVENILE WALNUT c FRESH ee CALLUSED FIG: 4 SR pL o4 = 10 RE 02 04 06 os 40 ACTIVITY OF ENDOGENOUS ROOTING PROMOTERS EXTRACTED FROM BASAL SEGMENTS OF FRESH AND CALLUSED/ROOTED (AFTER 90 DAYS) CUTTINGSOF JUVENILE WALNUT. EACH HISTOGRAM REPRESENTS THE ACTIVITY OF 1.09 RESH WEIGHDPERCENTAGE, PROMOTION INHIBITION JUVENILE = WALNUT CUTTINGS: = FRESH CALLUSED 6 50 «© 30 20 | 10 . =) 0 Ly a ; lo Ale 02 04 06 08 10 02° 04 06 08 10 FIG: 2 ACTIVITY OF ENDOGENOUS ROOTING INHIBITORS EXTRACTED FROM BASAL SEGMENTS OF FRESH AND CALLUSED/ROOTED (AFTER 90 DAYS) CUTTINGS OF JUVENILE WALNUT. EACH HISTOGRAM REPRESENTS THE ACTIVITY OF 10g (RESH WEIGHT.