91 J. Hortl. Sci. Vol. 13(1) : 91-96, 2018 Short Communication Studies on factors influencing the vegetative propagation in walnut (Juglans regia L. ) S. R. Singh*, N. Ahmed, K. K. Srivastava and P. A. Shagoo Central Institute of Temperate Horticulture, K. D. Farm, Old Air Field, P. O. Rangreth Srinagar, J&K - 190 007, India *E-mail: srajparmar@gmail.com ABSTRACT The experiment was carried out to examine the effect ofdifferent status of physiologically resting scion wood,environment and grafting time for maximum graft success in walnut. Three status of physiologically resting scion wood (apical, sub- apical and basal), were subjected to grafting on four different grafting time (15th February, 1st March, 15th March and 1st April)placed under three environmental conditions(open field,poly trench and polyhouse).Sub-apical portion of resting scion wood resulted in highest sprouting, graft success and plant growth, whereas grafting on 15th March manifested highest graft success. Poly house environmental conditions recorded maximum grafting success and plant growth. Sub apical status of scion wood with 15th March grafting under poly house conditions recorded highest sprouting, graft success and plant growth of walnut and found ideal for clonal propagation of walnut. Keywords: Juglans regia L, physiologically resting scion, environmental conditions, grafting time INTRODUCTION Wa lnu t ( J u gl a n s reg i a L . )is one of t he important temperate nut fruit praised for its high proteins, fiber, vitamin-B, minerals and anti-oxidants such as vitamin E and omega-3 fatty acids which helps in lowering the cholesterol levels in human body. Due to higher dema nd in domestic a nd international market it plays an important role in national economy with earnings of more than Rs. 300 crores annually by exporting to more than 44 countries of the world. Most of the walnut orchards of the country are seedling origin and growers are facing the problems of low price of their produce due to the large variability in quality, color, shape and size of nuts and kernels. Besides long juvenile period,low productivity and unmanageable size of these plantations are a great concern for walnut growing community of the nation. Thus varieties with high nutritive values with superior quality and yield have to be propagated, to make our walnut industry competitive in the international market. The clonal propagation of walnut is a difficult process due to low rate of callus formation in this fruit species (Kruniyuki and Ford, 1985). This is due to the p r es enc e of high c onc ent r a t ion of p henolic compounds in its tissues and their oxidation by wounding (Rangting and Pinghai, 1993, Coggeshall and Beineke,1997).Different degree of success in walnut propagation has been achieved by different propa ga tion techniques throughout wor ld with repeatable results at different places (Gandev,2007). Var ious fa ctors like, envir onmenta l condition, physiological status of scion wood and time of gr a f t ing a ff ec t gr a f t s u c c es s ( G a ndev, 2007).Optimum temperature for maximum graft success is 26-27oC (Langerstedt, 1979., Millikean, 1984) with 80-85% humidity. In walnut grafting, scion woods are prepared from resting single shoots by sectioning the different physiological positions i.e. apical, sub-apical and basal portion. Nutrient contents especially carbon nitrogen ratio plays an important role in callusing and union of scion and rootstock of a plant, which varied at different p or t ion even in sa me s c ion shoots . Op t ima l carbohydrate helps in new callus development and gr a f t su ccess , wher ea s su pr a opt ima l a mou nt restricts the activities of new callus generation. On other hand optimal threshold of nitrogen is essential for a protein and nucleic acid synthesis which is an essential constituent for cell r egener ation and callusing. Thus standardization of ideal physiological 92 resting status of scion wood, optimum environment, and time of grafting is of most importance for maximum graft success of walnut for a particular region. Keeping in view the above facts, an attempt has been made to find out the ideal physiological status of resting of scion woods, environmental conditions and grafting time for maximum graft success of walnut. T he ex p e r iment wa s c ond u c t ed a t experimental farm of Central Institute of Temperate Horticulture, Rangreth, Srinagar (J&K) in three factor factorial randomized block designs with three replications.Geographic position of the experimental site lies between latitude of 340 05 N and longitude of 74050 E at an altitude of 1640 m above the sea level. T he a ver a ge ma ximu m 19 . 6 3 æ%C a nd minimum 6.52 æ%C temperature, amount of rainfall 1 6 0 . 7 2 mm a nd r ela t ive hu midit y 58 . 3 5 % , evapora tion 2. 45 mm wa s recor ded dur ing the experimentation. There were three physiological status of resting scion wood (apical, sub- apical and basal), three environmental conditions (open field, poly trench and polyhouse),with four grafting times (15th February, 1st March, 15th March and 1st April). Two years old seedling rootstock of Juglans regia L. with uniform growth and thickness (1.5-2.0 cm in diameter at 15 cm above the ground level) were selecting for wedge grafting. 400 alkathine strips were used as tying material of graft union which has specialties of gas exchange for respiration but conserves the moisture to keep tissues live for long time. Physiologically resting scion woods (apical, sub-apical and basal), taken from one year old shoots with four to five dormant buds were used for wedge grafting. The male flower(catkins) buds were removed at the time of grafting to avoid the loss of nutrient reserves from the scion wood. 100 p la nt s wer e u s ed f or ea c h t r ea t ment . T he exper imenta l a r ea wa s pr ovided with unifor m cultural operations. The temperature under poly house and poly trench was maintained at (25+2oC) with intermittent misting and using 50% shade net during extreme temperature , which also helped in maintaining the humidity level to more than 80%. Sprouting percentage was recorded after 45 days of grafting, whereas graft success percentage, plant height and number of leaves/plant were recorded when plants started recessing their growth at the end of growing season. The pooled data of two years was analyzed as method suggested by Gomez and Gomez (1984) using R software. Physiological status of resting scion wood significantly influenced sprouting percentage, graft success a nd pla nt growth irr espective of other factors. Subapical portion of resting scion recorded highest sprouting per centage (68.56), gra fting success (56.39%) and plant height (154.86 cm) and number of leaves per plant (121.61). This may be due to better C/N ratio, which is responsible for ma x imum pa r enc hyma cell pr olifer a t ion a nd intermingling of union, which ultimately resulted in better graft union and plant growth (Hartmann et al. , 1997). Differ ent environmental conditions significantly influenced the graft sprouting, graft success and plant growth success. Environment in polyhouse recorded highest sprouting (65.50 %) graft success (54.46%) plant height ( 168.75 cm) and number of leaves per plant (120.06 ) closely followed by poly trench with 61.94% sprouting, 52.17% graft success, 145.56 cm plant height and 119.19 leaves per plant. Maximum graft success under polyhouse conditions may be due to the congenial temperature (25+2) and humidity 80-90 % which helps in new pa r enchyma tous ca llus p r olif er a t i on b et ween r oot s t oc k a nd s c ion (Hartmann et al.,1997). The active callus formation b et ween r o ot s t oc k a nd s c ion in wa lnu t is temperature specific. Grafting time significantly affected the graft sprouting success, graft success and plant growth. G r a f t ing on 1 5 th M a r c h r ec or ded ma x imu m sprouting 71.10% irrespective of other factors. This might due to rapid regeneration cambium tissues of scion and rootstock and their intermingling with a c t iva t ion of s c ion a nd r oot s t o c k on idea l temperature (25+2 oC) which occurs from second for tnight of Ma r ch in polyhouse. Results a r e corroborative with the findings of Porebsiki et al. 2002. Interaction effect of physiologically resting scion and grafting time significantly influenced the graft success and plant growth. Sub-apical scion with 15th March grafting recorded highest sprouting (80.55%), graft success (64.62%) plant height (171.17cm)and number of leaves /plant (147.67). This may be due to better C/N ratio in sub-apical Vegetative propagation in walnut J. Hortl. Sci. Vol. 13(1) : 91-96, 2018 93 scion which permits ma ximum r egenera tion of parenchymatous cells in the graft union. Interaction of environment and grafting time influenced the graft sprouting percentage graft success and plant growth significantly. Highest sprouting (76.48%) graft success (61.80%) was r ec or ded wit h 1 5 t h M a r c h u nder p ol yhou s e conditions. However, maximum plant height (188.94 cm) and maximum number of leaves per pla nt (144.33) was found under open field which was grafted on 15th March and poly trench grafted on 15th of March respectively (Table 2). This may be due to naturally active state of scion and stock tissues especially cambium during this period with the ideal temperature and humidity under polyhouse, which permits maximum regeneration of cells in cambium region and maintain their high degree of hydration level resulting the high graft success by permitting the active graft area for large period. These are inconformity with the finding of Ebrahimi et al., 2006 who obtained better success under polyhouse condition in walnut grafting. Interaction effect of environment conditions and physiological status of resting scion on graft s u c c es s a nd p la nt gr owt h wa s s t a t is t ic a lly significant. Sub-apical scion wood recorded highest graft sprouting and success under all environmental conditions. Interaction effect of physiological status of scion, environment conditions and grafting time on graft success and plant growth was significant. Sub-apical scion under polyhouse condition grafted on 15th March recorded highest sprouting 88.33%, graft success 70.85%, plant height 195.33 cm and number of leaves per plant 174.83. This may be due to better carbohydrate and nitrogen ratio and ideal bud maturity in sub-apical portion of scion, active cell regeneration stage in mid of March, c ondu c ive t emp er a t u r e a nd hu mi dit y u nder polyhouse environment which activates maximum pa r enchyma cell of c a mbiu m la yer a t higher humidity resulting in better union in scion and rootstock and higher growth (Bayazit et al. 2005). T he r esults ar e inconfor mity with findings of (Ozakan and Giimmis 2001). Sub-apical status of resting scion wood grafted on 15th of March under polyhouse condition recorded highest success and p la nt gr owt h. T he s t u dies c u lmi na t ed wit h standardization of protocol for clonal propagation of walnut. Singh et al J. Hortl. Sci. Vol. 13(1) : 91-96, 2018 Scion type x Grafting Time Sprouting (%) Graft Success ( % ) Plant height (cm ) Number of Leaves/plant Apical x 15th Feb 47.40 43.50 132.61 98.39 Apical x 1st March 60.37 51.04 144.78 110.39 Apical x 15th March 65.18 53.94 154.67 117.61 Apical x 1st April 52.59 46.52 146.06 111.56 Sub-apical x15th Feb 58.89 50.17 143.00 105.22 Sub-apical x1st March 71.85 58.14 152.00 115.61 Sub-apical x15th March 80.55 64.62 171.17 147.67 Sub-apical x1st April 62.96 52.67 153.28 117.94 Basal x15th Feb 49.26 44.58 144.44 110.11 Basal x1st March 60.00 50.84 153.22 116.39 Basal x15th March 67.59 55.43 165.50 134.44 Basal x1st April 52.59 46.53 149.33 112.17 SEM +_ 1.34 0.81 1.68 1.59 CD P= (0.05) 4.03 2.44 5.03 4.78 Table 1. Interaction effect of physiologically resting scions and grafting times on graft success and plant growth of walnut. 94 Environment x Scion status Sprouting ( %) Graft Success ( % ) Plant height ( cm ) Number of Leaves/plant Poly trench x Apical scion 57.10 49.66 123.79 111.00 Poly trench x Sub apical scion 70.41 57.47 131.92 127.71 Poly trench x Basal scion 57.50 49.39 131.88 121.46 Open field x Apical 50.97 45.57 159.29 111.21 Open field x Sub – Apical 61.94 52.03 173.63 122.50 Open field x Basal 51.66 45.98 173.33 123.88 Poly house x Apical 60.28 51.01 150.50 106.25 Poly house x Sub – Apical 73.33 59.70 159.03 114.63 Poly house x Basal 62.91 52.67 154.17 109.50 SEM +_ 1.16 0.70 1.68 1.59 CD P= (0.05) 3.49 2.11 5.03 4.78 Table 2. Interaction effect of environment and of physiologically resting scions on graft success and plant growth of walnut Vegetative propagation in walnut J. Hortl. Sci. Vol. 13(1) : 91-96, 2018 Interaction effect of Sprouting (%) Graft success (%) Plant height (cm) Number Leaves/plant environment and grafting time Poly trench x15th Feb 52.22 46.31 119.06 109.00 Poly trench x1st March 67.22 55.24 128.00 117.50 Poly trench x15th March 72.59 58.79 146.00 144.33 Poly trench x1st April 55.74 48.35 123.72 109.39 Open field x15th Feb 48.33 44.03 152.78 102.39 Open field x1st March 57.22 49.20 166.94 116.89 Open field x15th March 64.26 53.40 188.94 138.50 Open field x1st April 49.63 44.79 166.33 119.00 Poly house x15th Feb 55.00 47.91 148.22 102.33 Poly house x1st March 67.77 55.57 155.06 108.00 Poly house x15th March 76.48 61.80 156.39 116.89 Poly house x1st April 62.77 52.57 158.61 113.28 SEM +_ 1.34 0.81 1.94 1.84 CD P= (0.05) 4.03 2.44 5.81 5.52 Table- 3.Interaction effects of environmental conditions and grafting time on sprouting (%), graft success (%), plant height (cm) and number of leaves per plant. 95 Singh et al J. Hortl. Sci. Vol. 13(1) : 91-96, 2018 Table 4.Interaction effect of physiologically resting scions, environmental conditions and grafting times on sprouting,graft success and plant growth of walnut. Treatments Sprouting (%) Graft success(%) Plant height (cm) Number of Leaves/plant Apical Scion x poly trench x 15th Feb 46.11 42.76 137.83 97.50 Apical Scion x poly trench x 1st March 65.55 54.09 152.33 109.33 Apical Scion x poly trench x 15th March 68.33 55.84 152.83 124.33 Apical Scion x poly trench x 1st April 51.66 45.96 158.00 113.66 Sub-Apical Scion x poly trench x 15th Feb 61.66 51.79 150.33 101.66 Sub-Apical Scion x poly trench x 1st March 74.44 59.83 157.00 118.33 Sub-Apical Scion x poly trench x 15th March 81.66 65.08 171.16 145.83 Sub-Apical Scion x poly trench x 1st April 63.88 53.15 157.66 124.16 Basal Scion x poly trench x 15th Feb 48.88 44.37 156.50 108.00 Basal Scion x poly trench x 1st March 61.66 51.79 155.83 123.00 Basal Scion x poly trench x 15th March 67.77 55.41 144.16 145.33 Basal Scion x poly trench x 1st April 51.66 45.95 160.16 119.16 Apical Scion x Open field x 15th Feb 45.55 42.42 118.16 104.66 Apical Scion x Open field x 1st March 52.77 46.59 123.50 107.00 Apical Scion x Open field x 15th March 59.44 50.49 131.66 112.00 Apical Scion x Open field x 1st April 46.11 42.76 120.83 113.00 Sub-Apical x Open field x 15th Feb 53.33 46.91 120.66 107.83 Sub-Apical x Open field x 1st March 65.55 54.09 132.16 111.00 Sub-Apical x Open field x 15th March 71.66 57.91 147.00 122.33 Sub-Apical x Open field x 1st April 57.22 49.18 127.83 117.33 Basal Scion x Open field x 1st Feb 46.12 42.76 118.33 106.16 Basal Scion x Open field x 1st March 53.33 46.92 128.33 106.00 Basal Scion x Open field x 15th March 61.66 51.79 159.33 116.33 Basal Scion x Open field x 1st April 45.55 42.43 121.50 109.50 Apical Scion x polyhouse x 15th Feb 50.55 45.32 141.83 107.56 Apical Scion x polyhouse x 1st March 62.77 52.43 158.50 114.83 Apical Scion x polyhouse x 15th March 67.77 55.48 178.50 116.50 Apical Scion x polyhouse x 1st April 59.99 50.82 158.33 108.00 Sub-Apical x polyhouse x 15th Feb 61.66 51.79 158.00 106.16 Sub-Apical x polyhouse x 1st March 75.55 60.48 166.83 117.50 Sub-Apical x polyhouse x 15th March 88.32 70.85 195.33 174.83 Sub-Apical x polyhouse x 1st April 67.77 55.67 174.33 112.33 Basal Scion x polyhouse x 1st Feb 52.77 46.61 158.50 116.16 Basal Scion x polyhouse x 1st March 64.99 53.81 175.50 120.16 Basal Scion x polyhouse x 15th March 73.33 59.06 193.00 141.66 Basal Scion x polyhouse x 1st April 60.55 51.21 166.33 107.83 SEM +_ 2.33 1.99 1.65 1.31 CD P= (0.05) 6.99 5.97 8.22 7.82 96 Vegetative propagation in walnut J. 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