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.
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
NO

). 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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