Effect of daminozide on survival, growth and yield of micropropagated potatoes

1988) 353

EFFECT OF DAMINOZIDE ON SURVIVAL, GROWTH AND YIELD OF MICROPROPAGATED POTATOES

j. Sipos l, j. Nowak 2, and G. Hicks I

Abstract

The effect of daminozide [Butanedioic acid mono (2,2-dimethyl hydrazide)] on growth, field establishment and productivity of micropropagated Kennebec and Russet Burbank potato was studied. Addition of daminozide to the medium significantly reduced stem length of in vitro propagated plantlets. Optimal dose was 0.6 mg active ingredient per plant. This dose did not significantly alter the number of nodes and leaf area. Three transplanting methods were examined: direct from tissue culture to the field without protection; the same but with a plastic covering for protection; and after 7 days acclimation in a greenhouse but without protection in the field. Stand establishment ranged from 83.3 to 100%. The second transplanting method had the highest percentage establishment for both cultivars. The application of daminozide improved survival for all transplants of Kennebec and the directly planted plantlets of Russet Burbank. Independent of the daminozide treatment, plants of both cultivars hardened in the greenhouse prior to transplanting produced the highest number and yield of tubers. For Russet Burbank, daminozide caused a delay in tuberization but did not affect tuber bulking and haulm growth.

Compendio Se estudi6 el efecto de la daminozida [Acido Butanodioico mono

(2,2-dimetil hidrazida)] sobre el crecimiento, establecimiento en el campo y productividad de papas Kennebec y Russet Burbank micropropagadas. La adici6n de daminozida al medio de cultivo redujo significativamente la longitud del tallo de las plantitas propagadas in vitro. La dosis 6ptima rue de 0,6 mg de principio activo por planta. Esta dosis no alter6 significativamente el mimero de nudos ni el ~irea de hoja. Se examinaron tres m6todos de trasplante: directamente del cultivo de tejidos al campo, sin protecci6n; lo mismo, pero con una cubierta de pl~istico para protecci6n, y despu6s de siete dias de aclimataci6n en un invernadero, pero sin protecci6n en el campo. El establecimiento de las pl~ntulas vari6 de 83,3 a 100%. El segundo m6todo de

I Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3 H 3J5. 2Department of Plant Science, Nova Scotia Agricultural College, P.O. Box 550, Truro, Nova Scotia, Canada B2N 5E3. Address correspondence to J. Nowak. Accepted for publication February 26, 1988. ADDITIONAL KEY WORDS: Solanum tuberosura, in vitro, transplanting, growth retardant, butanedioic acid mono (2,2-dimethyl hydrazide).

354 AMERICAN POTATO JOURNAL (Vol. 65

trasplante tuvo el mhs alto porcentaje de establecimiento para ambos cultivares. La aplicaci6n de daminozida mejor6 la supervivencia para todos los trasplantes de Kennebec y las plfintulas directamente trasplantadas de Russet Burbank. Independientemente del tratamiento con daminozida las plantas de ambos cultivares, fortalecidas en el invernadero antes del trasplante, produjeron el mils alto ntimero y rendimiento de tub6rculos. Para Russet Burbank, la daminozida provoc6 un retardo en la tuberizaci6n, pero no afect6 el erecimiento de los tub6rculos ni el del follaje.

I n t r o d u c t i o n

Propagation of potato by tissue culture is now an important step in clonal multiplication of disease-free material. In the elite seed production system, in vitro grown plantlets are transplanted into containers and are either grown to maturity in greenhouses or, after a short hardening in a greenhouse, are planted in the field (4, 17, 18, 19).

Micropropagated plants yield from 30 to 97% compared to those propagated by tubers (4, 19) but often produce more tubers but of a smaller size. Goodwin and Brown (4) found that eight-week-old transplants gave higher yields than six-week-old plants, but had a lower rate of survival in the field. Field establishment is one of the major factors limiting yield of tubers grown from in vitro produced plantlets. A survival rate of 95% was recorded by Thornton and Knutson (17) and Wiersema, et aL (22). According to Levy (9), 17 to 75% of plantlets survived when directly transplanted to the field. Hardening in speedling trays prior to planting increased their survival rate to 98%.

Transplant vigour is one of the major factors determining plantlet survival. Elongated transplants usually have poor transplant vigour. Spindly potato plantlets are often the result of insufficient light intensity or overgrowth in culture or trays before transplanting. Goodwin and Brown (4) reported that micropropagated plantlets, when transplanted late, had only 17% field establishment.

Growth retardant daminozide (2) is used in the greenhouse production of cut flowers to shorten stem length (16). It also reduces stem elongation in potato when applied as a foliar spray in a field ( 1,5, 6, 7, 12, 14, 21 ). Kunkel and Holstad (7) found that foliar application of daminozide on potato produced more rigid, upright plants, which were less likely to contract soil borne pathogens. Riddell, etal. (12) found that foliar spraying with daminozide reduced top growth by 48%, without reduction of tuber yield. In other experiments either no increase (21 ) or increases in tuber number (3, 5) were reported.

The compound also reduces stem length of potatoes grown in vitro ( 10, 11, 20). The plantlets appear more sturdy than controls (10). Such plantlets can remain in culture longer prior to planting out without reduction in

1988) SIPOS, et al." DAMINOZIDE EFFECT 355

their vigour. This allows a more flexible transplanting schedule and may influence yield. Shorter plantlets have also less tendency to lodge when planted in the field.

In a preliminary study performed on Kennebec (Ratmi, M. and J. Nowak, 1985, Nova Scotia Agricultural College, Potato Technology Project) it was found that daminozide not only shortens internodes but also changes other morphological characteristics of micropropagated plantlets. Roots of the plantlets treated with daminozide were shorter and more uniformly distributed than those of the control. Leaves and stems were darker and plants appeared more vigorous. The objectives of this study were: 1) to examine field establishment, growth pattern and productivity of daminozide treated plantlets, and 2) to determine whether treatment with daminozide would allow direct planting of in vitro material to the field and eliminate the costly greenhouse step presently in use.

Materials and Methods

Micropropagation Cultivars Kennebec and Russet Burbank (Solanum tuberosum L.) were

micropropagated by nodal cuttings using virus-free stock plantlets cultured on Potato Nodal Cutting Medium (PNCM) containing MS salts supplemented with 1.02 g/l NaH2PO4• 100 mg/l myo-inositol, 0.4 mg/1 thiamine- HC1, 30 g/l sucrose and 6 g/1 agar, pH 5.7. The medium protocol and virus free plantlets were obtained from Plant Propagation Center, Fredericton, New Brunswick, Canada.

Approximately 8 nodal cuttings were taken from each plantlet discarding the top and bottom nodes and all leaves. The cuttings were cultured in 25• mm test tubes on 12 ml medium, one node per tube. Plantlets were grown under 80/.tmols m-2s -1, 400-700 nm, fluorescent light (Photosyn- thetic Photon Flux Density, PPFD), 16 h photoperiod and 23/19 C day/night temperature.

Daminozide Ef~ct on Plant Growth 3 Butanedioic acid mono (2,2-dimethyl hydrazide), daminozide was

added to the P N C M medium at concentrations of 0, 25, 50 and 75 ppm of pure preparation (dosage per plant, respectively: 0, 0.3, 0.6 and 0.9 mg). Stem length, leaf number and dry shoot weight were determined on four plantlets of each concentration every week, for six weeks. Leaf area was measured on five-week-old plantlets. The analyses were done on Russet Burbank.

3Daminozide--B-NINE, Plant Products Company Ltd., Bramelea, Ontario, Canada, 5% active ingredient.


Field Trials Field trials were performed on 21 (Russet Burbank) and 24 (Kennebec)

day old plantlets grown in GA7 Magenta vessels (Magenta Corp.) on 72 ml of P N C M per vessel. Each vessel contained 12 nodal cuttings with leaves attached. Daminozide was added to the medium at concentrations of 0 (control) and 100 ppm (per plant dose of 0.6 mg). The environmental conditions were as specified above. Stem length, leaf number and weight of shoot and roots were determined on plantlets of each treatment prior to planting to the field. Four Magenta vessels with 12 plantlets per jar were used for this analysis.

The plantlets were transplanted to the field on June 11 (Kennebec) and June 19, 1986 (Russet Burbank). A completely randomized block factorial design (8) with two levels of daminozide and three transplanting methods was used. The transplanting methods were as follows: (A) direct from sterile culture to the field, (B) direct from sterile culture to the field and covered with a plastic cap for 10 days (14 oz. inverted, transparent cup), and (C) 14 day old plantlets transplanted into cell paks, 40 plants per covered tray, containing 1:1 Nova Mix:Perlite (Mapplebeck, L., 1985, Nova Scotia Agricultural College, Unpublished Report) and grown for 7 days in a greenhouse prior to field planting without a cover. The plantlets were transplanted with the top 5-6 leaves above ground. Each field plot consisted of a row of 24 plants. Spacing within the row was 25 cm and between rows 91 cm. A guard row of cut seed tubers was planted between plots. The end plants of each plot were also used as guards.

Seed bed preparation consisted of fall plowing, a broadcast application of 700 kg/ha 15-15-15 in mid-May followed by disc and spring tooth cultivation. The soil was a sandy loam, organic matter of 5.5% and pH of 5.9 located at Plumdale Field Station, Nova Scotia Agricultural College, Truro, Nova Scotia. Each transplant was given 200 ml water containing 0.2 g of 10-52-10 fertilizer at the time of planting. No further irrigation was applied.

Crop management included weeding (June 23, July 1 and 17), application of insecticides for control of Potato flea beetle and Colorado potato beetle and fungicides for late blight. The rows were hilled once in late July.

Field establishment was recorded after 30 days in the field. Kennebec plants were harvested after 100 days of field growth (four replicates of 20 plantlets) and analyzed for the number of tubers greater than 20 g and total weight of tubers greater than 20 g. Russet Burbank was used for growth analysis. One replication consisting of 10 plants per plot was harvested 48, 62, 76 and 90 days after planting. The harvested plants were individually analyzed for total tuber weight, number of tubers greater than 1 g, number of tubers greater than 40 g and dry shoot weight.

Statistical Analysis ANOVA and Duncan Multiple Range Test (13, 15), were performed

using 8AS computer program (SAS Institute Inc., Cary, NC).

1988) SIPOS, eta[." DAMINOZIDE EFFECT 357

Results and Di scuss ion

Daminozide reduced internode growth of in vitro propagated potato (Figure 1). Stems were significantly shorter but leaf number remained similar to the control (Table 1). Root mass in daminozide treated plantlets was similar to the control. The roots, however, were shorter making transplanting of the in vitro material easier. Optimal dose of daminozide per plant was 0.6 mg judging from the morphological characteristics (number of nodes, leaf area and root system). The analysis of daminozide effect on in vitro performance of Russet Burbank confirmed the results of earlier experiments done in our laboratory with Kennebec (Ratmi, M. and J. Nowak, 1985, Nova Scotia Agricultural College, Potato Technology Project). In other studies in which similar concentrations of daminozide were used, reduction in longitudinal plant growth with no ill effects on subsequent growth of the plantlets was also reported (10, 11).

Treatment with daminozide significantly improved field establishment of plantlets of Kennebec. 100% of the treated plantlets survived transplanting. Field establishment of the controls varied from 82% for plantlets planted out without protection to 98% for the plantlets transplanted directly to the field with protection (Figure 2).

One of the factors which likely contributes to transplant survival is stem length. Daminozide treated plantlets were significantly shorter, darker and more vigorous. Field establishment of Russet Burbank plantlets was 90-97% (Figure 2). Neither the treatment with daminozide nor the transplanting method significantly influenced field establishment for this cultivar. The different stem length of the controls at transplanting, 6.4 and 4.1 cm for Kennebec and Russet Burbank respectively (Table 1 ), may have contributed to the difference in their field establishment.

The plantlets covered with plastic cups after transplanting had the highest rate of survival (Figure 2). The cover protected them from desiccation, lodging or washing out by heavy rain, wind damage, frost damage (night temperatures below 0 C were recorded between June 12 and 14, right after transplanting of Kennebec) and injuries caused by flea beetle. Under the environmental conditions of this experiment, treatment with daminozide resulted in the same effect as plantlets protected with the plastic cap. Under more severe conditions, a combination of daminozide and plantlet protection might be required to achieve a good field establishment of the transplants planted directly to the field. The results of this experiment also indicate that the costly greenhouse step in the elite seed production system could be eliminated by following these procedures.

Compared to direct transplanting, plantlets hardened in the greenhouse gave significantly higher tuber yield in both cultivars and produced more tubers in Kennebec (Figure 3). In the case of Russet Burbank, which is a long season cultivar (approx. 140 days) an extended period of growth could reduce yield differences among transplanting methods. Analysis of variance


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FIG. 1. Daminozide effect on growth of t'n vitro propagated potatoes Russet Burbank. Mean and standard error was based on four plantlets for each harvest date. Plantlets were grown on 12 ml medium containing a per plant dose of 0 (O), 0.3 (�9 0.6 ( l l L or 0.9 (A) mg daminozide.

1988) SIPOS, et al: DAMINOZIDE EFFECT 359

TABLE 1. -- Effect of daminozide (0. 0 mg per plant) on the growth of micropropagated potato.

Plant Russet Burbank Kennebec character Control Daminozide Control Daminozide

Stem Length (cm) 4.1 2.4 ** 6.4 4.0 ** Leaf Number 8.4 8.0 ns 8.8 9.2 ns Dry shoot weight (mg) 20.8 15.6 ns 25.5 20.4 * Dry root weight (mg) 6.8 5.0 ns 12.6 11.1 ns Shoot/root ratio 3.6 3.4 ns 2.1 1.9 ns

Means for 4 jars with 12 plantlets per jar after 21 (Russet Burbank) and 24 (Kennebec) days growth. P<0.01, P<0.05 and not significant are denoted by **, *, and ns respectively.

determined an interaction between transplanting method and daminozide treatment in Kennebec only (Figure 3). T h e growth retardant increased tuber number and tuber yield for plantlets transplanted directly to the field, with or without protection. It caused a decrease in tuber number and tuber yield and an increase in mean tuber weight in greenhouse hardened plantlets.

Daminozide also caused a significant delay of tuberization in Russet Burbank (Table 2). However, it did not influence tuber yield and tuber number after 90 days of growth. Daminozide treated plantlets had similar haulm growth to that of the control.

Humphr ies and Dyson (5) found a higher incidence of secondary growth on the tubers which had received a foliar spray of daminozide. When daminozide at 0.6 mg per plant was added to the media, there was no evidence of abnormal secondary growth.

T h e cut seed produced more tubers greater than 40 g and gave higher tuber yield than the micropropagated plantlets (Table 3). Stem density for the cut seed was 2.4 times higher than the micropropagated transplants. In order to obtain the same stem density as that of cut seed and spacing within plant rows for the micropropagated transplants should be 10 cm instead of 25 cm. Another option for increasing stem density could be the propagation of transplants as clusters of two or three plantlets (18).

T h e experiments indicate that the application of the growth retardant daminozide in combination with certain methods of plant protection might allow the direct transplanting of in vitro material to the field. In recent trials good protection was provided by replacing plastic cups with inverted biodegradable jiffy pots (George W. Ball Co.) with the bot tom removed. An alternative method of adding protection might be to use a floating plastic mesh cover, e.g., Agronet, or to plant a large foliage cover crop.

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FIG. 2. Effect of daminozide and transplanting method on the field establishment of micropropagated potatoes 30 days after transplanting. Transplanting methods:/"N--direct, no protection; �9 --direct, protected with a plastic cap for 10 days; m--after 7 days acclimation in a greenhouse. Convergent or divergent lines indicate a statistical interaction (8).

1988) siPos, eta/." DAMINOZIDE EFFECT 361 ,,o[ x 100 ifl m

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FIG. 3. Yield characteristics of Kennebec after I00 days of growth in the field. Total tuber weight and number represent a plot of 20 plants. Transplanting methods:/N,--direct, no protection; �9 --direct, protected with a plastic cap for i 0 days; [ ] - after 7 days acclimation in a greenhouse. Convergent or divergent lines indicate a statistical interaction (8).

3 6 2 A M E R I C A N P O T A T O J O U R N A L ( V o l . 65

T A B L E 2. - - Ft'eld growth performance of the plantlets Russet Burbank, micropropagated with and without damt'nozide.

Harvest

Plant Daminoz ide (days after p lan t ing to field)

character (mg) 48 62 76 90

Total tuber 0 12.5 * 144.5 ns 341.1 ns 546.2 ns weight (g) 0.6 5.0 163.9 363.2 522.9

Tuber n u m b e r 0 2.5 ** 9.6 ns 10.6 ns 10.4 ns >1 g 0.6 1.0 9.6 10.3 9.4

Tuber n u m b e r 0 0.0 ns 0.6 ns 3.7 ns 5.8 ns >40 g 0.6 0.0 0,8 3.4 5.4

Dry shoot 0 31.0 ns 53.3 * 79.2 ns - - weight (g) 0.6 35.8 63.8 82.3 - -

Means for 30 plants per daminozide t r ea tment level for each harvest date. P<0 ,01 , P<0 .05 and not significant are denoted by **, * and ns respectively for differences between means.

- - D r y shoot weight was not de termined.

T A B L E 3. - - Yield characteristics of mt'cropropagated potatoes Russet Burbank. Three transplanting methods (A, B, C) and a seed

tuber control (19) were analyzed.

Harvest

Yield Transplant ing (days after p lant ing to field)

character me thod 62 76 90

Total tuber A 132.9 a 335.3 ab 426.5 a weight (gj B 161.8 a 312.9 a 474.7 b

C 168.0 a 408.0 b 602.5 b D 387.8 b 566.0 c 820.0 c

Tuber n u m b e r A 9.9 a 10.6 a 10.0 a >1 g B 10.0a l l . 0 a 9 . 8 a

C 8.8 a 9.8 a 10.0 a D 8.5 a 11.1 a l t . 2 a

Tuber n u m b e r A 0.5 a 3.4 ab 4.6 a >40 g B 0.4 a 3.0 a 6.0 ab

C 1.2 b 4.4 b 6.2 bc D 4.8 c 6.1 c 7.5 c

Means for pooled data consist ing of 20 plants per t ransplant ing me thod and 10 plants for the cut seed control for each harvest date were analyzed. Means not followed by the same letter were significantly different at P = 0 . 0 5 (Duncan Mul t ip le Range Test).

P lant ing method: A- -d i rec t , no protection; B- -d i rec t , protected with a plastic cap for 10 days; C - - a f t e r 7 days acclimation in a greenhouse; D - - p r o p a g a t e d from cut seed tubers.

1988) SIPOS, eta]." DAMINOZIDE EFFECT 363

Acknowledgments

The authors thank Mrs. Shirlyn Coleman from the Plant Propagation Center, Fredricton, New Brunswick for the virus indexed plantlets. The project was funded by the Plant Science Department, Nova Scotia Agri- cultural College and the Canadian International Development Agency (Awards for Canadians Program).

Literature Cited

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Effect of daminozide on survival, growth and yield of micropropagated potatoes

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