TIlE ROOTING OF PARAOOXWALNUTSOFTWOODCU1TINGS: THE EFFECfS OF PRIOR TISSUE CULTUREPROPAGATION E. M. BROOKS and J. A. DRIVER

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1 TIlE ROOTING OF PARAOOXWALNUTSOFTWOODCU1TINGS: THE EFFECfS OF PRIOR TISSUE CULTUREPROPAGATION E. M. BROOKS and J. A. DRIVER This rooting study was set up to compare Paradox walnut softwood cuttings from trees which had been: a) propagated through tissue culture, and b) from conventionally grown trees (seedlings). A number of studies on rooting of both hardwood and softwood cuttings of walnut trees have been made by various workers. Although it was reported by Lee, et. al. (1) and others that 1%rooting was obtained for Ju~lans hindsii, the survival of these cuttings have been a problem. Thus, this means of propagation of walnut trees is not practiced on a commercial basis by nurseries which supply the market. There is a continuing need for the clonal propagationof paradox walnut rootstock. The tissue culture propagationof walnut trees allows new selectionsof rootstock to be produced and tested for improved characteristics, yield, disease resistance, nematode resistance, virus tolerance, and trees stature and yield. The use of cutting propagation from these improved trees from tissue culture would allow more rapid utilizationof the improved selections in a cost effective manner. Therefore,this study was initiated to determine if the use of softwood cuttings was a commercial possibility and also to compare it to cuttings from conventionallygrown Paradox walnut trees. Since most studies of walnut cutting propagation have been done under greenhouse conditions, it is a further objective to determine if field propagationis possible. This would provide a significantcost advantage,save time, and labor for utilizationof improved walnut cultivars. The only known method for successful field propagation of tissue culture plantlets (7,8,9) is found in U.S. Patent No. 4,612,725 and is used in this study. Twenty different treatments were evaluated which included 3 levels of IBA hormone, 2 levels of Sulfuric acid, and use of 2 or 3 plastic cups to provide "miniature greenhouse" conditions in the field. A principal objective of this study is to determine if a commercially acceptable level of rooting of cuttings (75% to 8%, or more) is possible. A secondary objective is to determine, if possible, why rooting of conventional walnut tree cuttings does not result in survival of high percentages, and why the tissue culture derived cuttings might be superior in this vital characteristicfor commercialuse. MATERIALSANDMElHODS Plant Materials Paradox (Lazar clone) tissue culture cuttings were from trees planted September or October, 1988from micropropagated plantlets. In most cases the entire tree was cut at the base (4 to 6 inches in length) with leaves remainingat the shoot tip. Paradox conventionally grown tree cuttings were from seedlings planted in November, 1988;from the BurchellNursery. These seedlings were about 6 feet in height and cuttings were made from side limbs (to equal the diameter of the tissue culture material) of these small trees. This was deemed the most juvenile material obtainable from paradox walnut. Plant Treatment Eleven cuttings were prepared for each treatment with 2 treatments in all. The table of treatmentsis below: 54 -

2 IBARootingHonnone n 1(3. Wm) 2(5. p,pm) 3(8. p,pm) Control CO Cl C2 C3 A=IN-H2S4 - la 2A 3A B=2N-H2S4 - IB 2B 3B Two Cups CO-I Cl-l C2-1 C3-1 ThreeCups C-3 CI-3 C2-3 C3-3 Two Cups - la-l 2A-l 3A-l ThreeCups - la-3 2A-3 3A-3 Two Cups - IB-l 2B-l 3B-l ThreeCups - IB-3 2B-3 3B-3 The table above is for codes used with the tissue culture cuttings and conventionallygrown cuttings (CG). The cuttings were taken in the cool of the morning (before 9 a.m.), wrapped in wet paper towels in lots of 11 and placed in a plastic tub with a lid. These cuttings were 5 to 6 inches long and only leaves at the end were left with the last leaf cut in most cases. The cuttings in groups of 11 were then dipped in a hormone (IBA K salt) solution (about 1 inch deep) for about 15seconds. The cuttings were then dipped in a dry fungicide powder (2 or 3 inches deep) and again wrapped in wet paper towels and put into a closed plastic tub along with a dated, coded wooden stake for identification. For the cuttings with H2S4 dip, this acid solution treatment was actually first (again dipped about 1 inch) followed by water rinse, honnone dip and then the fungicide. The fungicide powder contained 5% fungicide (1/3 Tersan, 1/3 Bravo and 1/3Benlate) and 5% talc. The cuttings were taken to the field and planted. After the cutting was inserted, soil was pressed around it. Each cutting was covered with either 2 cups or 3 cups as follows: 2 cups (one styrofoam, one small clear plastic) 3 cups (2 styrofoam, 1 clear plastic). An eleventh cutting was placed with each treatment under one small clear plastic cup. Dirt was pressed around the inverted cups to keep them in ptace. The same number of cuttings were taken from conventionally grown trees as for tissue culture grown. After eleven weeks the cuttings were evaluated for rooting. The soil was removed carefully from the bottom of the cutting by firs using a shovel to loosen the soil. After the roots were cleaned, they were counted and the number per cutting recorded. The number of cuttings per treatment which had rooted was also determined to evaluate whether this was a commercially feasible process. In every case a color photograph of the rooted cutting was taken for evidence of findings in this study. RESULTS AND DISCUSSION A summary of results in presented in the two tables attached: Table I -Tissue Culture of Softwood Cuttings and Table II - Conventionally Grown Paradox Walnut Rooting of Softwood Cuttings. As indicated in Table I, a very acceptable and commercial level of rooting was found for three treatments of the tissue culture tree derived cuttings. Although a very limited number of treatments were studied, this evidence of the ability of the walnut cuttings to root in soil at very high levels provides preliminary evidence of the 55 -

3 success of this process. With the results of Table I it can be noted that covering the cuttings with 2 cups is preferable to 3 cups in those treatments where the highest percentages of rooting were found. An additional result not included in the tables was that the use of only one clear cup for covering gave unacceptableresults (all cuttings died) in all treatments. The use of a 3 cup covering evidently caused a number of the cuttings to be in a moist condition which resulted in rotting of the material. It is possible that in higher ambient temperatureconditionsthe 3 cups might have provided better results. It has been reported in a Masters Thesis by Pelletreau (2) that rooting of black walnut and paradox walnut is possible using a treatment of sulfuric acid plus indole-3- butyric acid (IDA). This study was done under greenhouse conditions (mist propagation). Many other workers have also reported high rooting percentages for walnut, but the eventual transfer of the tooted plantlet has been problematic. In the present study, rooting trials are compared in the field, as it is believed that the direct field rooting and acclimatizationis of greatcommercialvalueandimportance.. The use of acidification for preparation of cell walls in promoting growth of many plant species is reported in several studies (3) (4) (5). In our study, the sulfuric acid at both 1 Nand 2 N were effective with 5, ppm and 3, ppm respectively, in providing 8% or greater rooting of cuttings. However, it should be noted that without acidification, 5, ppm IDA provided a 9% rooting level. The requirement for the acid pretreatment should be investigated further. None of the cuttings from the conventionally grown trees reached a commercially acceptable level of 8%, in contrast to the tissue culture results. In addition, perhaps the most significant finding was the poor appearance of their roots. The conventionally grown cuttings had roots that extended out perpendicularily from the callus of the cutting. They did not provide the strong juvenile appearance of roots found with tissue culture cuttings and therefore it is doubtful that they would have produced mature trees. In many cases they were segmented and the roots grew out of only one side of the cutting which also is a negative for conventional cuttings. It has been reported in the literature that rooting of walnut is possible in high percentages, but the resultant tree percentages through transplant and acclimatization have been unacceptable. Our results appear to confirm this problem with conventionallygrown tree cuttings. As an aid to the discussion of the results of this study, an evaluation of root quality was made from photographs taken of each treatment. This summary is given below: ROOT QUALITY Geotrophism 1. Type of growth habits of roots in comparison Tissueculturederivedcuttin~sspreading to vertical downward growth type. Conventional seedlin~ cuttines flat horizontal to spreading growth type, some roots exhibiting vertical upward growth. Site of Root Origin 1. Location of Cells Involved in Root Organogenesis(appearanceonly) Tissue culture cuttin~slittle or no callus at base of treated stem with roots. Adventitiousroots appear formed in the cortex of root near vascular tissue. Conventional seedling cuttinf:sconsiderable callus at cutting stem base, roots arise from / or grow through callus. It appears Adventitious roots are 56

4 fonned a) in callus b) cortex of root. Concern over nearness to vascular tissue. 2. Sectorialorientation Tissue culture cuttin&scircular orientationof roots around stem cutting Conventional seedline cuttines non-circular orientation. usually roots clustered on one or two locations of stem cutting. Root Structure (Comparison of root morphology with seedling roots) 1. Clubbyness of roots Comparison with seedling roots Tissue culture cuttines very similar to seedling roots. size. color. diameter. Conventional seedline cuttines short, clubby roots; enlarged diameter, shorter length than seedling, TC roots. 2. Secondary roots presence / absence of secondary roots, orientation. Tissue culture cuttines branched, well developed root structure. secondary roots exhibit seedling type secondary growth habits. Conventional seedline cuttines not well developed root structure; marked reduction of secondary roots; some secondary roots growing upward initially before turning downward in growth. Table ill gives the results of a subjective analysis on the basis of the three areas of concern given in the above summary. A to 5 scale was used. with 5 being the most comparable to seedling roots, and not acceptable. CONCLUSIONS Preliminary evidence is that tissue culture produced Paradox walnut tree softwood cuttings of the Lazar clone selection can be successfully rooted in the field under cups. A commercially acceptable, high level of 9% rooting was found with the 5, ppm lea rooting honnone treatment and this was repeated in this study with the low level of sulfuric acid treatment. Conventionally grown Paradox tree cuttings did not produce rooting at commercial level with any treatment, and the roots did not appear to meet acceptable levels of quality which may influence survival and mature tree growth. New studies are now underway to optimize treatments to achieve greater success, and to test this method of propagation on a wide range of genotypes. LITERATURE CII'ED 1. Lee C.I., J.L. Paul and W.P. Hackett Root promotion on stem cuttings of several ornamental plant species by acid or base pretreatment. Proc. Inter. Plant Prop. Soc. 26: Pelletrau, K Rooting of leafy walnut cuttings and the role of mycorrhizas in the survival of transplanted seedlings and rooted cuttings. (Masters Thesis, University of California, Davis). 3. Cleland, R Auxin-induced hydrogen ion excretion from Avena coleoptile. Proc. Nat. Aca. Sci. USA 7: Hager, A. H. Menzel and A. Krauss Versehe and hypothese zur primarwiking des auxin beim steckungswachstum. Planta 1:

5 5. Rayle, D.L. and R. Cleland Auxin-induced secretion in Avena coleoptiles and its implications. Planta 114: Lynn, C. and H.T. Hartmann Rooting cuttings under mist. Calif. Agri. May, 1957 pp Driver, JA Direct field rooting and acclimatizationof tissue culture cuttings. In Vitro. 21(3.pt.2): 57 A. (Abstr.). 8. Driver, JA 1986.Method for acclimatizingand propagating plant tissue culture shoots, U.S. Patent, Number 4,612,725. (Sept. 1986) 9. Driver, JA and Suttle, GRL Nursery handling of propagules IN: Bonga,1M and Durzan, DJ (eds). Tissue Culture in Forestry, Vol. 2, Scientific Principles and Methods. Martinus JijhofflDr. W. Junk, Publishers, Dordrecht, Netherlands; pp

6 FIGURE II Tissue Culture ~ Conventional >< Q) 'C c >- 1 - fts 8 ::J , C') Ln o-m... -Ln -m C\I -t"i C\I -It) C\I -co ppm ISA / sulfuric acid, N Root Quality Based on: 1. Geotrophic growth habit 2. Sectorial orientation- root placement 3. Root structure It appears that the different treatments of tissue culture derived cuttings did not significantly affect the root quality as shown in the graph above. The similar finding is shown for the different treatments of the conventional cuttings. A very significant difference is seen in comparing tissue culture cuttings to conventional cuttings. In all cases tissue culture cuttings root quality is superior. 59

7 FIGURE 2 14-I II Tissue Culture )( 12 G) 't:i.5 1 C) c ::: 8 a:: - 6 >- - ::J 4 2 Conventional ~ ~ ~ N N N ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o M ~ ~ ~ ~ ~ ~ ~ m ppm IBA I Sulfuric acid, N When Root Quality is multiplied by Rooting Percent, the difference between tissue culture and conventional cuttings is even greater. The exception for this is for the 2N Sulfuric acid combined with 5, ppm or 8, ppm IBA. It would appear that at these high levels, the toxic effects affected negatively the tissue culture derived cuttings

8 FIGURE iii Tissue Culture fa Conventional 7 6 ::: o a: C\I C\I C\I M 1. co M 1. co M 1. co ppm ISA I Sulfuric acid, N This figure shows perhaps the most significant findings of the study. Since the commercial application of field propagation of cuttings of walnut requires a 75% to 8% survival rate, only the three tissue culture treatments meets this requirement. None of the conventionally grown cuttings achieve this level. As a preliminary analysis, the 5, ppm lea treatment either at or IN Sulfuric acid show the most promise for the tissue culture cuttings. With the higher (2N) concentration of sulfuric acid, less IBA is required (3, ppm). Thus the prior tissue culture propagation affected the rooting success more than the various treatments. 61 -

9 FIGURE OJ c: :;: 4 3 ::1 II III IIH, NMNMNMNMNMNMNMNMNMNM ~~~~~~~~~~~~~~~~~~~~ OOOOOOOO~~~~~~NNNNNN ~~~~~~~~~~~~~~~~~~~~ OOMM~~~~MM~~~~MM~~~~ m Tissue Culture 121 Conventional ppm ISA / Sulfuric acid, N / # CUP The figure above is a graph of the total treatments. provide a commercial level of rooting in any treatment. The third plastic cup did not The Tables I and II of this report provide the number of roots per cutting. These numbers compare favorably with those reported by Lynn and Hartmann (6) in a rooting study of paradox walnut under mist. Overall, it is noted that tissue culture sample 2A-l combines 9% rooting with an average of 6.8 roots per cutting. The conventional cuttings had fewer roots per cutting on average, with none as high as sample 2A

10 TABLE I TISSUE CULTURE GROWN PARADOX WALNUT: ROOTING OF SOFTWOOD CUTllNGS Code Treatment No. Rooted Average No. of Roots C -1 No IBA.No H2S4. 2 cups CO-3 No IBA. No H2S4. 3 CUDS C DDrn IBA. No H2S4. 2 CUDS 5 no data C DDrn IBA. No H2S4, 3 CUDS 2 no data C DDrn IBA, No H2S4. 2 CUDS C DDrn IBA, No H2S4. 3 CIJDS C DDrn IBA. No H2S4, 2 CUPS C DDrn IBA, No H2S4. 3 CUPS A-1 3. DDrn IBA 1N H2S4. 2 CUDS A-3 3. DDrn IBA. 1N H2S4. 3 CUDS A-1 5. DDrn IBA, 1N H2S4. 2 CUDS A-3 5. DDrn IBA, 1N H2S4, 3 CUDS A-1 8, DDrn IBA. 1N H2S4, 2 CUDS A-3 8. DDrn IBA. 1N H2S4. 3 CUDS B-1 3 DDrn IBA 2N H2S4 2 CUDS B-3 3. pprn IBA, 2N H2S4, 3 Cups B-1 5. DDrn IBA, 2N H2S4, 2 CUPS B-3 5, DDrn IBA, 2N H2S4, 3 CUPS B-1 8, DDrn IBA. 2N H2S4, 2 CUPS B-3 8. DDrn IBA, 2N H2S4, 3 CUDS 5 4 Note: Each treatment contained 1 cuttings 63

11 TABLE II CONVENTIONALLY GROWN PARADOX WALNUT (SEEDUNG): ROOTING OF SOFTWOOD CUTTINGS Code Treatment Average. No. Rooted No. of Roots CGO-1 No IBA, No H2S4, 2 cups CG-3 No IBA, No H2S4, 3 cups CG1-1 3 ppm IBA No H2S4. 2 cups CG1-3 3, ppm IBA, No H2S4, 3 cups 3 2 CG2-1 5, ppm IBA, No H2S4, 2 cups CG2-3 5, ppm IBA, No H2S4, 3 cups CG3-1 8, ppm IBA, No H2S4, 2 Cups CG3-3 8, ppm IBA, No H2S4, 3 CUPS CG1A-1 3. ppm IBA. 1N H2S4, 2 CUPS CG 1A-3 3, ppm IBA. 1N H2S4, 3 CUPS CG2A-1 5, ppm IBA, 1N H2S4, 2 CUPS CG2A-3 5, ppm IBA, 1N H2S4, 3 CUPS CG3A-1 8, ppm IBA, 1N H2S4, 2 CUPS CG3A-3 8, ppm IBA, 1N H2S4, 3 CUPS CG1 B-1 3 ppm IBA 2N H2S4. 2 CUPS 1 2. CG1 B-3 3, ppm IBA, 2N H2S4, 3 CUPS 1 1 CG2B-1 5, ppm IBA, 2N H2S4, 2 CUPS 4 4 CG2B-3 5, ppm IBA, 2N H2S4, 3 cups CG3B-1 8, ppm IBA, 2N H2S4, 2 CUPS CG3B-3 8, ppm IBA, 2N H2S4. 3 CUPS Note: Each treatment contained 1 cuttings 64

12 TABLE III r. C. CONVENTIONAL GEOTRO? SECT STRUCT GEOTROP SECT STRUCT 3/ Cl (14) (6.5) 5/ C (13) (6) 8/ C (14.5) (4) 3/ (13.5) (4) 5/ (15) (4) 8/ (14) (5) '1 U1 3/ (14) (4) 5/2 5 /. 3 (12) (3) 8/2 4?5 3.5 (11) (5)