Establishment and Growth of Potato Micro-Cuttings in Sand Trays

doi:10.4236/ajps.2012.39147

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American Journal of Plant Sciences, 2012, 3, 1219-1224

http://dx.doi.org/10.4236/ajps.2012.39147 Published Online September 2012 (http://www.SciRP.org/journal/ajps)

1219

Establishment and Growth of Potato Micro-Cuttings in

Sand Trays

Md. Jahangir Hossain1, Mosammat Shamsun Nahar1,2

1Research Wing, Bangladesh Agricultural Research Institute, Joydebpur, Bangladesh; 2Plant Pathology Section, Horticulture Re-

search Center, Bangladesh Agricultural Research Institute, Joydebpur, Bangladesh.

Email: [email protected]

Received February 8th, 2012; revised March 16th, 2012; accepted April 19th, 2012

ABSTRACT

Two separate experiments were conducted to evaluate the success of the establishment and growth of micro-cuttings

of potato (5 - 6 cm tall) in sand trays [38 cm (L) × 28 cm (W) × 7.5 cm (H) plastic trays] under controlled environment

(22˚C ± 2˚C, 60 - 75 μmo sm –1·s–1 light energy for 16 h daily). In the first experiment, micro-cuttings of potato cv. Dia-

mant were planted at six populations (500, 600, 700, 800, 900 and 1000 cuttings per tray) in treated sand (sun dry, 1%

formaldehyde, 0.2% Dithane M-45 and control). The mortality percentage of micro-cuttings was nil for sun dry sand

while formaldehyde and dithane M-45 treated sand had 1% - 4% against 15% in the control with the highest population

density. Mortality of micro-cuttings in formaldehyde and dithane M-45 treated sand trays were found not to be related

to pathogenic organism rather toxic effect of these two chemicals. Micro-cuttings in Sun dry and control treatments

showed better growth performance than these in chemically treated sand trays. In the second experiment, urea @ 1, 2

and 3 g per tray was applied as solid form after 15 days of planting the micro-cuttings and as liquid form @ 0.5, 1 and

2% solution sprayed in the micro-cuttings repeatedly after 15, 30, 45 and 60 days of planting. The micro-cuttings which

received urea as solid state died within 2 - 3 days and 2% urea solution was also detrimental. Urea solution @ 0.5%

found to be very effective for vegetative growth of micro-cuttings in sand trays. The control was also good for vegeta-

tive growth but at a slower rate.

Keywords: Sand Tray; Sun Dry; Fungicides; Urea Fertilizer; Plant Population; Growth; Potato Micro-Cuttings

1. Introduction

Potato is an important food crop worldwide. It is mainly

a temperate zone crop, though it is cultivated in many

tropical countries during the winter season. It is reported

that potato seed accounts for >50% of the total cost of

production [1-3]. Moreover, better seed can produce bet-

ter crop and higher yield. Availability of quality seed

tubers is the important factor for better crop and tuber

yield. Potato crop is prone to about 50 diseases [4-6], of

which about 8 - 10 are major diseases though others

caused considerable losses of this crop [7]. The fungal

and bacterial diseases could be controlled or minimize by

applying chemicals while viruses or viroids diseases are

uncontrollable. Once a plant is infected by virus or viroid,

it carried over generations through seed tubers and

caused degeneration of this crop and yield loss. Many

countries of the world are trying to use different planting

materials as seeds for producing quality seed potatoes

[8-11]. Production of nucleus seed stock with in vitro

micro-plant is prime important as this material is patho-

gen-tested (P.T.). The different planting materials like

top shoot cutting [9,12,13], nodal cuttings [9,14], sprout

cuttings [15] are used to increase the nucleus seed stock

within the shortest period. Ewing [16] used “slips” in

vermiculite to produce pathogen-tested minitubers, while

Vietnamese farmers used pathogen tested micro-plants

followed by repeated cuttings in Delat region in order to

develop the low cost planting materials to be used in

plains for seed potato production [17]. Commercial tissue

culture laboratories rather used to propagate planting

materials under ex vitro conditions under controlled en-

vironment in order to save energy, money, chemicals, etc.

The present work was aimed to standardize the methods

for better establishment and growth of micro-cuttings of

potato in sand trays under controlled environment.

2. Materials and Methods

Micro-cuttings of three Dutch potato cultivars Cardinal,

Diamant and Multa were used in two separate experi-

ments to standardize the methods for better establishment

and growth in sand trays under controlled environment

during 2006 and 2007.

Establishment and Growth of Potato Micro-Cuttings in Sand Trays

1220

In the experiments in vitro micro-plants (4 - 6 cm tall)

of 30 days old were first transplanted in plastic trays of

38 cm (L) × 28 cm (W) × 7.5 cm (H) contained 0.013

mm size sand particle. The transplanted micro-plants

were nursed under controlled environment (22˚C ± 2˚C,

60 - 75 µmosm–1·s–1 light energy for 16h daily). After 25

days of planting, top shoots (1.5 - 2.0 cm) were cut and

planted in sand trays of above.

In the first experiment, only one cv. Diamant was used.

Sand were treated with four different (sun dry, 1% for-

maldehyde solution, 0.2% dithane M-45 and untreated

control) ways. Sand at 5 cm heap were sun dried for two

days while sand were treated with formaldehyde and

dithane M-45 before 7 and 3 days respectively of plant-

ing the micro-cuttings. Micro-cuttings population (500,

600, 700, 800, 900 and 1000 per tray which was equiva-

lent to 4750, 5700, 6650, 7600, 8550 and 9500 m–2) was

maintained (Figure 1(a)). The sand trays were kept un-

der the controlled environment as stated previously. The

experiment was laid in a randomized complete block de-

sign with four replications. The experiments were set on

15 April in both the years, 2006 and 2007.

In the second experiment, three Dutch potato cultivars

Cardinal, Diamant and Multa were used. Urea @ 1, 2 and

3 g per tray in solid form and 0.5, 1 and 2% urea solution

were applied to standing crop of micro-cuttings. No urea

(a)

(b)

Figure 1. Planting of (a) mother plants in sand tray (38 cm

× 28 cm × 7.5 cm) at a plant population of 1000 per tray and

(b) luxuriant growth of microplants after four sprays of

0.5% urea solution at 60 days period.

was used in the control trays. Urea in solid form was

applied 15 days after planting the micro-cuttings in sand

trays, while spraying of urea solution started after 15

days of planting the micro-cuttings and sprayed for four

times at 15-days interval. The experiment was set in a

randomized complete block design with three replica-

tions. The experiments were set on June 20 in both the

years, 2006 and 2007.

As the recorded data on different parameters of both

the experiments conducted in two years, 2006 and 2007

did not vary statistically, thus the mean data were used to

analyse the experiment.

3. Results

Results of treated sand contained in plastic trays and po-

pulation on establishment and growth of micro-cuttings

of potato are presented in Table 1. The mortality rate of

micro-cuttings was nil for sun-dry sand, which was stati-

stically superior. About 1.38% and 1.53% micro-cuttings

were died in formaldehyde and dithane M-45 treated

sand, respectively while control trays had 4.83% which

was statistically inferior. Micro-cuttings took 12 - 15

days for developing new shoots. The chemically treated

sand required more number of days than the others. After

25 days of planting the micro-cuttings, the plantlets at-

tained the maximum height of about 10 cm for sun-dried

sand or in control treatment compared to about 6.0 cm

for chemically treated sand. Within this period, each

plantlet developed approx. 6 leaves. The longest leaf was

developed in 1% formaldehyde-treated sand which was

statistically similar to 0.2% dithane M-45, while sun-dry

and control treatment were almost equal. Rate of node

production was the best for sun dry sand and other three

were statistically similar but inferior. In case of internode

length, sun-dry and control treatment were almost equal

and the chemically-treated sand were statistically similar

but inferior. Similar trend was also shown for the number

of roots per micro-cuttings and length of the longest roots.

No disease incidence was observed in sun dry sand and

the control treatment had the maximum (Table 1).

The mortality of micro-cuttings was increased with in-

creasing plant population. Similar trend was observed for

plant height while other parameters except longest leaf

were found to be unaffected due to varied plant popula-

tion. Leaf size was higher for lower population.

Table 2 shows the results of urea management for

micro-cuttings and plant development. Solid application

of urea in sand and 2% foliar spray caused cent percent

plant died in all the three cultivars. Urea sprayed @ 0.5%

solution was found to be most effective for micro-cutt-

ings survival while 1% spray was also detrimental which

caused dead of micro-cuttings up to 40%. Micro-cuttings

sprayed with 0.5% urea solution were found to have

Establishment and Growth of Potato Micro-Cuttings in Sand Trays

1221

Table 1. Effect of different treatments of sand and plant population on establishment and growth of potato micro-cuttings of

potato cv. Diamant (mean of two years, 2006 and 2007).

Treatments

Sand

treatment

pp/

tray

Mortality

(%)

New

shooting

days

Plant

ht. at 30

day (cm)

No. of

leaves

per plant

Length of

leaf (cm)

Rate of

node prod.

(cm/wk)

Inter-

nodal length

(cm)

No. of

roots per

plant

Length of

root (cm)

Disease

status

Sundry 500 0 12 7 7 20 1.75 1.00 5 2.50 0

600 0 14 8 6 18 1.50 1.33 4 2.00 0

700 0 13 7 6 16 1.50 1.17 3 1.85 0

800 0 15 10 7 14 1.75 1.43 6 2.25 0

900 0 10 12 6 15 1.50 2.00 5 3.23 0

1000 0 12 14 7 16 1.75 2.00 4 3.00 0

Mean 0.00 12.67 9.67 6.50 16.50 1.63 1.49 4.50 2.47 0

1% FDH 500 1.5 17 6 6 24 1.5 1.00 4 3.00 0

600 1.05 15 5 7 26 1.75 0.71 3 1.62 0

700 1.13 16 6 7 22 1.75 0.86 2 2.00 0

800 1.25 16 4 6 20 1.50 0.67 5 1.50 0

900 1.48 15 7 5 23 1.25 1.40 1 1.50 0

1000

1.64 14 8 6 20 1.5 1.33 2 1.30 0

Mean 1.34 15.50 6.00 6.17 22.50 1.54 1.00 2.83 1.82 0

0.2% DM 500 1.62 13 5 6 24 1.50 1.00 2 1.52 0

600 2.02 14 5 5 23 1.25 1.00 4 1.63 0

700 1.08 15 6 6 20 1.50 1.00 3 2.50 0

800 1.55 15 7 6 20 1.50 1.17 2 2.50 +

900 1.39 16 7 7 21 1.75 1.00 5 1.20 +

1000 1.56 17 7 6 22 1.50 1.17 1 1.40 +

Mean 1.54 15.00 6.17 6.00 21.67 1.50 1.06 2.83 1.79

Control 500 3.06 12 6 5 21 1.25 1.20 3 1.50 +

600 1.09 13 7 6 20 1.30 1.17 5 2.30 +

700 2.36 11

9 7 20 1.75 1.29 2 2.00 ++

800 5.11 12 11 6 16 1.25 1.83 4 1.50 ++

900 7.22 11 12 7 14 1.75 1.71 6 1.60 +++

1000 10.16 14 12 7 15 1.75 1.71 2 2.50 +++

Mean 4.83 12.17 9.50 6.33 17.67 1.51 1.49 3.67 1.90

Mean of Plant Population (PP)

500 1.55 13.50 6.00 6.00 22.25 1.50 1.05 3.50 2.13

600 1.04 14.00 6.25 6.00 21.75 1.45 1.05 4.00 1.89

700 1.14 13.75 7.00 6.50 19.50 1.63 1.08 2.50 2.09

800 1.98 14.50 8.00 6.25 17.50 1.50 1.28 4.25 1.94

900 2.52 13.00 9.50 6.25 18.25 1.56 1.53 4.25 1.88

1000 3.34 14.25 10.25 6.50 18.25 1.63 1.55 2.25 2.05

Mean 1.93 13.83

7.83 6.25 19.58 1.54 1.26 3.46 2.00

lsd 1% for bed tr. (bt) 1.11 2.32 1.69 ns 1.56 0.09 0.14 0.87 0.26

lsd 1% for pp 1.58 ns 1.98 ns 2.06 0.16 0.24 1.06 0.34

lsd 1% for bt x pp 3.06 3.11 2.65 ns 2.85 0.29 0.36 1.56 0.52

Note: FDH = Formaldehyde, DM = Dithane M-45; pp = Plant population; +---------+++ = Degree of incidence.

Establishment and Growth of Potato Micro-Cuttings in Sand Trays

1222

Table 2. Effect of urea on micro-cuttings of three potato cultivars planted in sundried sand (mean of two years, 2006 and

2007).

Cultivars Urea dose

Plant

survival

(%)

No. of leaf

per plant

Length of

leaves (mm)

Plant

height

(cm)

Inter-nodal

length (cm)

Growth rate

(mm/day)

Node prodn

rate (no/wk)

Dry wt.

shoot (%)

Dry wt.

root (%)

Ratio

(shoot:

root)

V1 1 g /tray 0 0 0 0 0 0 0 0 0 0

2 g/tray 0 0 0 0 0 0 0 0 0 0

3 g/tray 0 0 0 0 0 0 0 0 0 0

0.5% FS 100 9 20 13 1.44 4.33 2.10 11.6 21.2 0.55

1.0% FS 40 6 15 11 1.83 3.66 1.40 10.2 22.1 0.46

2.0%FS 0 0 0 0 0 0 0 0 0 0

control 100 6 13 9 1.50 3.00 1.40 13.4 21.6 0.62

Mean 34.29 3.00 6.86 4.71 0.68 1.57 0.70 5.03 9.27 0.23

V2 1 g /tray 0 0 0 0 0 0 0 0 0 0

2 g/tray 0 0 0 0 0 0 0 0 0 0

3 g/tray 0 0 0 0 0 0 0 0 0 0

0.5% FS 100 12 16 14 1.17 4.67 2.80 10.9 23.1 0.47

1.0% FS

20 9 12 10 1.11 3.33 2.10 10.2 22.6 0.45

2.0%FS 0 0 0 0 0 0 0 0 0 0

control 100 7 14 11 1.57 3.67 1.64 12.5 23.2 0.54

Mean 31.43 4.00 6.00 5.00 0.55 1.67 0.93 4.80 9.84 0.21

V3 1 g /tray 0 0 0 0 0 0 0 0 0 0

2 g/tray 0 0 0 0 0 0 0 0 0 0

3 g/tray 0 0 0 0 0 0 0 0 0 0

0.5% FS 100 11 17 13 1.18 4.33 2.57 12 22.6 0.53

1.0% FS 32 7 14 10 1.43 3.33 1.63 10.4 21.1 0.49

2.0%FS 0 0 0 0 0 0 0 0 0 0

control 100 7 11 10 1.43 4.33 1.63 10.8 22.3 0.49

Mean 33.14 3.57 6.00 4.71 0.57 1.57 0.83 4.74 9.43 0.22

Mean of urea doses

1 g /tray 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2 g/tray 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

3 g/tray 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.5% FS 100.00 10.67 17.67 13.331.26 4.44 2.49 11.50 22.30 1.20

1.0% FS 30.67 7.33 13.67 10.331.46 3.44 1.71 10.27 21.93 1.07

2.0% FS 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

control 100.00 6.67 12.67 10.001.50 3.67 1.56 12.23 22.37 1.32

Mean 32.95 3.52 6.29 4.81 0.60 1.65 0.82 4.86 9.51 0.51

V1 = Cardinal, V2 = Diamant and V3 = Multa; Tray size: 38 cm × 28 cm, FS = Foliar spray, sundried sand.

Establishment and Growth of Potato Micro-Cuttings in Sand Trays

1223

better growth performance than 1% solution and control

treatment. The cv. Diamant with 0.5% urea foliar spray

gave better performance for most of the parameters com-

pared to the other two cultivars. Within two months pe-

riod, the micro-cuttings of the cv. Diamant attained the

maximum of 14 cm plant height to develop the maximum

of 12 leaves per plant [Figure 1(b)]. The cv. Cardinal

had the longest leaf of 20 mm. Internodal length (cm),

plant growth rate (mm/day) and node production rate

(no./week) were also the highest with 1% foliar spray in

Cardinal (1.83 cm); and 0.5% foliar spray in Diamant

(4.67 mm/day and 2.80 per week respectively). Urea

@0.5% foliar spray or the control treatment produced the

maximum dry matter in shoots and roots. These two

treatments were also produced the maximum shoot to

root ratio.

New growth of the micro-cuttings was appeared earlier

in sun dry and control treatment compared to the chemi-

cally treated sand, which probably affected adversely the

new growth. Plant height was also shown similar trend

across the treatments. Development of leaf per plant and

their size was found not to be affected due to treatment of

sand by formaldehyde or Dithane M-45. For most of the

parameters like rate of node production and internodal

length were significantly better for sun-dry and control

treatment compared to chemically treated sand. Similarly,

the number and length of roots were significantly better

for sun-dry sand. These two parameters probably, nega-

tively affected by formaldehyde or Dithane M-45 [20].

Solid application of urea at all the three levels (1, 2

and 3 g per tray) was found to be fetal to the micro-cut-

tings. Solid application of urea in sand probably reached

the root zone of the slender micro-plants before any

transformation which is necessary for making the avail-

able form (NH+/2



). And as such, it became toxic to

the micro-plants. On the other hand, urea solution above

0.5% was also detrimental. Kabir and Chowdhury

sprayed 1% - 5% urea solution at foliage of normal po-

tato crop at 30 days age and obtained 3% solution most

effective and >3% was detrimental [21]. Urea solution

(0.5%) spraying was found to be most effective for

growth and development of the micro-cuttings and the

micro-cuttings under control treatment was also good,

though some percentage of micro-plants were died. Urea

solution @1% caused dead of 40% - 60% micro-cuttings

and the growth of survived micro-plants was also ham-

pered which was inferior to control treatment. Internodal

length, plant growth rate and node production rate was

significantly higher for 0.5% foliar spray of urea while

dry matter production of shoot and root was lower com-

pared to control which indicates that urea caused lower-

ing of DM% in shoot and root of micro-cuttings [4,22];

their ratio was also higher in control treatment than 0.5%

or 1% urea foliar spray.

4. Discussion

The treatments of sand were found to be very effective in

reducing plantlet mortality. Effectiveness of sun-dry in

reducing micro-organisms from the soil was reported

earlier [18]. In the present investigation, sand was dried

under natural sun light for 48 hrs. (8 hrs daily). This

treatment seemed to be very effective in eliminating all

organisms from sand. On the other hand, 1% formalde-

hyde treatment was found to eradicate micro-organisms

from sand, though on the average 1.34% plantlet died,

may be due to toxic effect of formaldehyde. In vitro de-

veloped micro-plants are usually very soft and succulent.

Sand was treated with 1% formaldehyde 10 days before

transplanting the micro-cuttings. Formaldehyde toxicity

caused damaged of roots within 2/3 days. No disease

causing organism was detected in formaldehyde treated

sand. Dithane M-45 is a fungicide, active against late

blight of potato. This was also used to eradicate so many

fungus as reported earlier [19]. Dithane M-45 also eradi-

cates organisms from sand to some extent. Disease inci-

dence was observed with high population but the inci-

dence was not so severe, probably this chemicals che-

cked severity of the organism. The plantlets of the con-

trol treatment was seriously affected by micro-organism,

which arose with increasing plant population. With the

highest plant population, 10.16% micro-plants were died.

The attack was observed at patch. The pathological study

showed that the organisms were Fusarium oxysporum

and Fusarium solani caused damping off disease to the

micro-plants, which is indicative that normal sand are not

always safe for hardening practices of in vitro micro-

plants.

5. Conclusion

From the above discussion it may be concluded that sun-

dry sand is the most effective soil substrate for develop-

ment and growth of micro-plants under controlled envi-

ronment and spraying of 0.5% urea solution on foliage

can safely be done to enhance the growth of micro-cut-

tings.

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fusarium solani were found to cause damping off disease

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