MATERIALS AND METHODS

Lodgepole pine (Pinus contorta Dougl.)seedlings, were grown in Beaver Plastics Ventblocks^® 160/65 (untreated controls) and Copperblocks^® (160/65) treated with ROOT TRIM^® and used once, twice or four times previously in a commercial nursery at Pelton Reforestation of British Columbia.

Seeds from 3 different seedlots (above) were sown into a peat/sawdust medium in the respective trays on January 9, 1995 (1), February 10, 1995 (2,3,4) and February 16, 1995 (5,6), and grown as per nursery schedule with nutrient solutions. Each tray had 160 cavities with a volume of 65 m . The seedlings were shipped to Thunder Bay on July 10, 1995 for critical root examination and root growth potential tests.

Plant parameters and root exam

Plant parameters were measured on five randomly selected seedlings from each tray and included seedling height, RCD, epicotyl and tap root lengths, and shoot and root dry masses. Seedling height was measured from the soil line to the terminal bud, epicotyl length was the distance from the soil line to the first lateral root, and the tap root length was the length from the soil line to the tip of the tap root. Measurements were made over a period of three weeks, from July 10 to August 2, 1995.

Root examinations were done on these same five seedlings. For comparative purposes, the root examination was based on dividing the plug root system into thirds, viz. top, middle and bottom sections, based on the portion in which the root arose from the tap root. In each third, root length was measured from top to bottom in order, for each root greater than 10 mm in length arising from the tap root in that section of the plug. Each of these is termed a main lateral. The first branch from a main lateral is termed a primary (1^o) lateral, the second a secondary (2^o) lateral, etc. Numbers of roots were counted in all sections. Lengths and degree of branching were recorded for roots in the top and middle sections. Number of laterals were counted on roots in the top section only, as numbers of laterals on lower roots were small. Means of each were calculated. In addition, total root lengths and number of laterals per mm of each main lateral root were computed.

Root growth potential

A standard test of root growth potential (RGP) was conducted on twenty randomly chosen seedlings from each tray. Five seedlings were planted into each of four 3 litre pots, in a 70:30 peat/vermiculite mix. The pots were placed in a growth chamber with 25/17^oC day/night temperatures, 70% relative humidity and 16 hour day length. After ten days, the seedlings were removed, the medium was carefully shaken off, and the roots washed so as to remove the original plug medium. As with the root exams, the root system was divided into top, middle and bottom thirds based on the origin, at the tap root, of the lateral from the top down.

White roots, which represented new root growth in the growth chamber, were counted and classified into four length categories: <10, 10 to 19, 20 to 29 and >30 mm. Average total numbers and average estimated white root lengths were calculated. White root lengths were estimated by multiplying the number of root tips in each size category by the median size of that category, eg. < 10 root numbers x 5 mm, 10 to 19 numbers x 15 mm, 20 to 29 numbers x 25 mm, >30 numbers x 40 mm. RGPs were done over two months from July 10 to September 8, 1995.

Root tubes

One randomly selected seedling from each of the trays was planted in 70:30 peat/vermiculite mix in a 90 cm long, 10 cm diameter acrylic root tube on July 19, 1995. The root tubes were placed on a stand at approximately a 60 ^o angle and the root portion encased in black plastic garbage bags to exclude light. The angle of the root tube resulted in all roots growing downward, in response to negative geotropism, and then growing along the bottom surface of the acrylic tube, where they could be measured regularly. Root tubes were fertilized with 100 ppm N 20-8-20 as needed. The medium in the root tubes reached daily minimum/maximum temperatures of 15°C and 22°C, respectively.

Weekly root growth was monitored and the length of new root growth was recorded. Seedling height was measured and bud break noted. Root tube measurements were continued until roots reached the bottom of the tube (September 14, 1995). Tubes were emptied, roots washed and photographed and measured from where they emerged from the surface of the plug, and not from where the root arose on the tap root, as in the first part of the study. This measurement did not include the root length in the plug, as was the case in the root examinations previously described.

Total root lengths were calculated for the top, middle and bottom sections.

Statistical analysis

Means were calculated for all parameters and results interpreted based on major trends.

Plant parameters

Table 1 summarizes mean seedling root collar diameter (RCD), height, epicotyl length, tap root length, shoot dry mass (SDM) and root dry mass (RDM) of five seedlings from each treatment.

Seedlings grown in untreated trays had the greatest RCD, shoot height, SDM and RDM of all treatments, with notably more roots visible, growing down, on the outside of the plug. Few large roots were visible on the outside of the plugs of seedlings grown in copper treated trays. In treated trays, in general, the more times the trays were used after the initial coating, the shorter were the seedlings produced. In seedlot #33282 second use tray seedlings were the same height as the #30818 controls, but 5th use tray seedlings were shorter. For each seedlot used, there was a trend from larger to smaller seedlings as the tray aged. Epicotyl and tap root lengths were longest in the second use trays for both seedlots.

For seedlot #30818, RCD and RDM increased with age of the tray, but none were as great as the untreated control. The difference in size between the seedlings in treated trays and the control may have been due to the one month earlier seeding in the control seedlings.

Root numbers and lengths

Table 2 presents a summary of mean numbers of roots from the three sections of the root plug, and mean lengths and total lengths of the first eight roots from the top and middle sections of five seedlings from each treatment.

Numbers of roots in the top and middle sections of the seedling plug in the different seedlings were similar, indicating that copper treatment had not influenced the pattern of main lateral root production from the tap root as it grew downwards during early seedling growth, or during later root development. In the bottom section, the most roots were on the #30818 third use seedlings, while the control seedlings had the fewest bottom roots, suggesting that copper treatment had resulted in the production of more main lateral in the lower portion of the roots, and giving rise to the `bunching' effect seen at the bottom of the plug.

The control seedlings had the longest roots of any treatment because the top lateral roots grow out to the container wall

and then down to the bottom hole. The maximum length of these main laterals was nearly 200 mm despite the fact that the container height is only127 mm, and the radius of each cell 15 mm. In control seedlings only the 5th root down was shorter (136 mm on average), but still longer than the total container depth.

Second and third use tray seedlings had dramatically shorter lateral roots (15 to 40 mm) than the controls because the lateral root apex stopped growing when it grew out horizontally and reached the side wall, presumably due to copper treatment. This reduced the total main lateral root length (Table 2) in each plug from nearly 1 metre to 140 mm in second use trays, but less severely in proportion to the number of times the tray had been used. Both fifth use trays produced seedlings with greater total root lengths, approaching the lengths of the controls, as less inhibition had taken place, presumably due to lower copper levels at the tray surface.

Degree of branching and laterals

There was no difference in degree of root branching in all treatments, which was surprising since it was believed, and published literature suggests, that apical inhibition would result in more branching (Table 3). In effect, it resulted in less total root growth.

The number of 1^o laterals and the number of 1^o laterals per mm on the first four roots of seedlings from each treatment are presented in Table 4. The control seedlings had many more laterals than any of the treatments because of their greater length, but had fewer laterals/mm in comparison with second and third use trays for seedlot #30818. The second use #30818 seedlings had the most laterals/mm, indicating that copper treatment had increased the lateral branching per unit length of lateral root, although this was not the case in seedlot #33282. Differences in response could also occur in different species.

Root growth potential

Whereas the data shows (Table 5) that the majority of the white roots on control seedlings were on roots which originated in the top and middle sections, visual examination shows that most of these emerging roots were actually situated at the bottom of the plug. This is because lateral roots, which originate at the tap root in the top third of the root system, grow out to the side wall and then down to the drainage hole, as do their laterals. These data, therefore, tell us that these main laterals resulted in the most new growth, which in untreated trays were growing most actively at the base of the plug. A better picture of where the new roots emerge on the outside of the plug is recorded in the root tube data, but it must be understood that these are mostly roots emerging originally from the top section of the plug, which have been directed down by the container configuration.

The third use #30818 seedlings had the most white roots overall (Table 5), and the second use #30818 seedlings had the most white roots in the middle and bottom section, but notably an even amount of root growth from roots arising in each section of the plug. Copper treatment, therefore, resulted in new root growth from lateral roots in each section of the plug, as compared with untreated seedlings, where the greater proportion of new root growth was from laterals arising in the top one third of the plug, although the actual root growth activity was at the bottom of the plug. As before, regardless of point of origin, most white roots were observed to emerge from the bottom of the plug for the seedlings of seedlot #30818. Seedlings of seedlot #33282 appeared to have roots emerging all over the plug.

The results for estimated lengths of white roots are similar to the above, because the numbers of roots were multiplied by the same factors (Table 6). The third use #30818 seedlings had a greater total length of white roots than any of the other treatments , and all copper treated seedlings had greater lengths of white roots than controls. There was nearly five metres of new root growth in some treated seedlings compared with half this amount in the controls.

Root tubes

Plant height

On average, there was little change in seedling height over the two month time period that seedlings were planted in root tubes. Some seedlings showed bud activity but little extension growth occurred.

Number of roots

The number of new roots growing in each treatment followed the same pattern with time (Figure 1). There was a sharp increase in root numbers visible on the tube surface in the third week, followed by a gradual decrease in the numbers of roots with active growth over the remaining six weeks. The highest peak on the curve shows the total number of actively growing roots at the acrylic tube surface. The total number did not decrease, but the actual number with active root growth did. All copper treated seedlings had a greater number of active roots in compoarison with the control seedlings. There was no difference in root numbers between all other treatments, although the fifth use #30818 and the second use #33282 seedlings both exhibited renewed growth activity with an increase in numbers in the last week. It is clear, therefore, that copper treatment stimulated root growth activity in seedlings grown in treated containers after transplanting by resulting in a greatly increased number of actively growing roots from the plug.

Average root length growth per week

All of the seedlings exhibited an initial increase in average root length from week 2 to week 3, followed by a decrease in average root length over time (Figure 2). The control seedlings had the least root growth each week on average from weeks 5 to 9. There were smaller differences in average length of root growth for the other treatments. One of the advantages of the root tubes over RGP is that it allows data to be collected over a longer period of time, and the root growth patterns can be recorded.

Total root length

The control seedling had the least total root growth on a weekly basis of all the seedlings, with an increase in the third week and then a decrease to zero by the end of the trial (Figure 3). All other treatments exhibited a large increase in total root growth initially, with up to 1 m of new root growth weekly, followed by a gradual decline over time. The fifth use #30818 seedling had the most total root growth throughout the trial, and there was no difference in the other treatments. It is obvious from these data that seedlings grown in copper treated containers had more root growth than controls.

Root position

In general, the control seedling had the fewest and shortest new roots arising from the surface of the plug in all sections (Table 7). The second use #33282 had the most new roots arising from roots originating in the top and middle sections, while the third use #30818 had the most in the bottom. Seedlings grown in second use #33282 also had the longest roots in the top section, but fifth use #30818 had longer roots in the middle and bottom sections than any of the other seedlings. All the seedlings had a majority of roots, and the longest roots, emerging from the bottom section, except for second use #33282, which had more roots emerging from the top. It cannot be said that higher copper concentrations, which were presumably present in the newer trays, inhibited growth of new roots to a lesser or greater extent after removal from the copper effect.

It must be stated that the plant parameters discussed may not necessarily be an indication of better survival and growth in the field, especially factors like height and shoot and root dry mass. The measurements made simply describe the size of a seedling which resulted from being grown in a tray which had been treated with ROOT TRIM^® and used for one or more times.

The total root length was shorter in seedlings grown in copper treated containers, as was the root dry mass and shoot dry mass. This resulted in a change in the shoot to root ratio from 1.73 in the controls to a higher 2.3 to 2.7 in the second use treatments, ie - a greater proportion of top growth to root growth. It could be stated, in fact, that second and third use trays resulted in seedlings which had half the root dry mass and 2/3 the shoot dry mass of control seedlings, but produced three times the amount of new growth in the root tubes.

Treatment	Species	Tray type	Depth (mm)	RCD (mm)	Shoot height (mm)	Epicotyl length (mm)	Tap root length (mm)	Shoot dry mass (g)	Root dry mass (g)
no copper control	Pl 1+0	VB 160/6	127	3.03	129.60	10.00	124.60	1.35	0.78
second use #30818	Pl 1+0	CB 160/6	127	2.54	123.80	14.20	130.00	1.04	0.39
third use #30818	Pl 1+0	CB 160/6	127	2.78	119.80	9.60	129.20	0.80	0.42
fifth use #30818	Pl 1+0	CB 160/6	127	2.80	81.81	8.00	123.00	0.88	0.53
second use #33282	Pl 1+0	CB 160/6	127	3.03	128.60	10.80	132.20	1.01	0.49
fifth use #33282	Pl 1+0	CB 160/6	127	2.60	120.40	7.80	126.80	1.02	0.44

*means of five seedlings

*mean of five seedlings, unless noted otherwise

#mean of four seedlings

$mean of three seedlings

^mean of two seedlings

&one seedling

Beaver Plastics containers treated with copper and used different numbers of times

Treatment	Species	Tray type	Depth (mm)	Root number
				1	2	3	4	5	6	7	8
no copper control	Pl 1+0	B 160/6	127	2.8	2.8	2.6	1.7$	3.0	2.6	2.6	2.8#
second use #30818	Pl 1+0	B 160/6	127	3.2	2.8	2.8 #	2.0&	3.4	3.0	2.8	2.0#
third use #30818	Pl 1+0	B 160/6	127	3.0	2.4	2.6	3.0$	2.6	2.6	2.8	2.4
fifth use #30818	Pl 1+0	B 160/6	127	2.8	2.6	2.4	3.0&	2.4	2.8	2.4	2.2
second use #33282	Pl 1+0	B 160/6	127	3.2	3.0	3.0 $	3.0&	2.8	3.2	2.8	2.4
fifth use #33282	Pl 1+0	B 160/6	127	2.6	2.4	2.8 #	2.7$	2.6	2.2	2.6	2.6

at Pelton Reforestation in Beaver Plastics containers treated with copper and used different numbers of times

Treatment	Species	Tray type	Depth (mm)	Root 1		Root 2		Root 3		Root 4
				# laterals	# lats/mm	# laterals	# lats/mm	# laterals	# lats/mm	# laterals	# lats/mm
no copper control	Pl 1+0	B 160/6	127	33.0	0.2	24.8	0.1	23.4	0.1	18.3 $	0.1
second use #30818	Pl 1+0	B 160/6	127	7.8	0.5	9.4	0.5	6.3 #	0.4	5.0 &	0.5
third use #30818	Pl 1+0	B 160/6	127	8.0	0.5	9.4	0.2	7.4	0.2	11.0 $	0.2
fifth use #30818	Pl 1+0	B 160/6	127	10.0	0.1	5.2	0.1	5.4	0.1	14.0 &	0.1
second use #33282	Pl 1+0	B 160/6	127	7.4	0.2	7.2	0.1	10.3 $	0.2	7.0 &	0.3
fifth use #33282	Pl 1+0	B 160/6	127	8.0	0.1	5.2	0.1	10.0 #	0.1	10.3 $	0.1

*mean of five seedlings, unless noted otherwise

#mean of four seedlings

$mean of three seedlings

^mean of two seedlings

&one seedling

Table 5  Number of white roots*, in different length categories, emerging in the RGP test, on roots originating from the top, middle, and bottom thirds of lodgepole pine (Pl) seedling plugs after 10 days in a growth chamber. Seedlings were grown at Pelton Reforestation in Beaver Plastics containers treated with copper and used different numbers of times.

Treatment	Species	Tray type	Depth mm	Top				Middle				Bottom				Total
				<10mm	10-19mm	20-29mm	>30mm	<10mm	10-19mm	20-29mm	>30mm	<10mm	10-19mm	20-29mm	>30mm
no copper control	Pli 1+0	VB 160/65	127	220.0	5.4	2.0	2.1	143.6	2.3	1.1	0.8	94.5	2.6	0.3	0.5	474.9
second use #30818	Pli 1+0	CB 160/65	127	206.7	4.5	2.6	1.6	259.4	5.8	1.8	2.0	172.9	7.0	3.2	2.1	669.4
third use #30818	Pli 1+0	CB 160/65	127	286.6	9.5	4.3	5.0	250.9	12.1	6.7	6.7	130.2	11.7	6.6	3.7	733.8
fifth use #30818	Pli 1+0	CB 160/65	127	96.5	12.4	5.3	5.3	109.7	33.2	6.9	7.2	56.2	12.3	4.2	3.5	352.4
second use #33282	Pli 1+0	CB 160/65	127	165.7	8.5	6.8	5.8	154.0	13.8	8.2	5.3	98.4	12.4	5.7	3.1	487.4
fifth use 333282	Pli 1+0	CB 160/65	127	220.3	9.0	3.7	5.3	204.0	11.3	3.7	4.3	144.6	9.2	4.0	3.8	622.8

Although control seedlings had the largest amount of roots, it does not automatically follow that they have the greatest absorbing area since a greater proportion may be suberised due to their length. Therefore, the absorptive capacity of a copper pruned system may be as great or greater, as indicated by the greater amount of white root growth which resulted after transplanting. This effect may mean that shoot:root ratios based on dry mass may be meaningless since they do not take into effect the active absorbing root area. Further research to examine when suberisation takes place in treated and untreated root systems could shed some light on this aspect. Some leads could be taken from research on corn grown in acid soils with aluminum toxicity which exhibit very similar responses, including the inhibition of mitotic division as long as the root remains in contact with the high heavy metal concentration.

In light of the above, one might question the hypothesis/theory that seedlings which have been managed so as to increase the total amount of root mass during the later part of the season, eg by drought cycles, blackout, paclobutrazol, topping, may not in fact give rise to the greatest amount of new growth roots when released from the constraints of the container. It would appear that copper treatment may do just the opposite, ie - restrict the root system and leave proportionately more dry mass in the tops (one might say have a more balanced seedling - this would create a major debate). This results in a burst of new root growth after transplanting as the seedling tries to re-establish a different/new root:shoot ratio where its roots are free to grow, and its top experiences little or no competition in comparison with the tray situation. We also recorded that copper treated seedlings had new roots appearing at the acrylic tube surface earlier than controls indicating a quicker start to new root production.

The amount of root inhibition may be related to tray management, as has been pointed out by van Steenis (1994, 1995). To overcome this, more research should be done on the coating materials such that the copper is held more `tightly' in the coating and less subject to media difusion, watering and temperature differences.

Whereas more definitive data could be obtained with more replication, the treatment differences were big enough to allow general conclusions to be made. It could be said that

ROOT TRIM^® treatment resulted in root systems with less total root length and root dry mass and fewer laterals, but with roots which were quicker to grow and had greater new growth after transplanting, and from all sections of the plug. In general, the more times a copper coated tray was used, the more like the control, untreated seedlings the root system and regrowth patterns became. As with all studies, more questions arise than answers and these should be addressed in future research.

LITERATURE CITED

Arnold, M.A. and Struve, D.K. 1989. Growing green ash and red oak in CuCO₃-treated containers increases root regeneration and shoot                           growth following transplant. J. Amer. Soc. Hort. Sci. 114(3): 402-406.

Arnold, M.A. and Young, E. 1991. CuCO₃-painted containers and root pruning affect apple and green ash root growth and cytokinin levels.                       HortScience 26(3): 242-244.

Lloyd, D. 1996. Personal communication. Pelton Reforestation, Maple Ridge, BC.

Ruehle, J.L. 1985. The effect of cupric carbonate on root morphology of containerized mycorrhizal pine seedlings. Can. J. For. Res. 15:                               586-592.

Smith, I.E. and McCubbin, P.D. 1992. Effect of copper tray treatment on Eucalyptus grandis (Hill ex Maiden) seedling growth. Acta Hort.                       319: 371- 376.

Struve, D.K. 1993. Effect of copper-treated containers on transplant survival and regrowth of four tree species. J. Environ. Hort. 11(4):                               196-199.

Struve, D.K., Arnold, M.A., Beeson, R., Jr., Ruter, J.M., Svenson, S. and Witte, W.T. 1994. The copper connection. Amer. Nurseryman                         179: 52-61.

van Steenis, E. 1994. Growing in copper treated containers requires greater awareness. Seed and Seedling Extension Topics 7(2): 4.

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