African Journal of Biotechnology Vol. 8 (21), pp. 5718-5726, 2 November, 2009
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB09.822
ISSN 1684–5315 © 2009 Academic Journals
Full Length Research Paper
Micropropagation of superior eucalyptus hybrids FRI-5
(Eucalyptus camaldulensis Dehn x E. tereticornis Sm)
and FRI-14(Eucalyptus torelliana F.V. Muell x E.
citriodora Hook): A commercial multiplication and field
evaluation
I. D. Arya2, Sudhir Sharma1* and Sarita Arya2
1
Tissue Culture Discipline, Botany Division, Forest Research Institute, Dehradun (Uttarakhand)-248006-India.
Forest Genetics and Tree Breeding Division, Arid Forest Research Institute, Jodhpur (Rajasthan)-342005-India.
2
Accepted 31 August, 2009
Micropropagation protocols for multiplication of highly important Eucalyptus hybrids have been
achieved. Two Eucalyptus hybrids namely as FRI-5 (Eucalyptus camaldulensis Dehn x Eucalytpus
tereticornis Sm) and FRI-14 (Eucalyptus torelliana F. V. Muell x Eucalyptus citriodora Hook) selected for
micropropagation using nodal segments as explants from the mature trees (30 - 32 years old) for the
two hybrids. These Eucalyptus hybrids were produced by Forest Research Institute, Dehradun, in the
early 1960 - 70 through controlled crossing. These are available in limited number and need
multiplication true to type for field - testing to ascertain its superiority under varied agro - climatic
conditions. These hybrids showed 3 - 5 superior in terms of total biomass and wood volume when
compared to their parents. Clonal cultures were established and multiplied on MS medium
supplemented different concentration of BAP either alone or in with combination of auxins i.e. IBA/NAA.
MS medium was found to be effective and suitable for all the experiments during plant production. 85 92% rooting was achieved on half strength MS medium supplemented with IBA. 90 - 98% of the plantlets
survived hardening and acclimatization prior to field transfer. Parameters like height, DBH, clear bole
length and self pruning capability of both the hybrids were recorded up to three years to identify the
suitability of these hybrids in different varied climatic zones of Uttarakhand.
Key words: Eucalyptus hybrids FRI-5 and FRI-14, field testing, diameter at breast height, micropropagation.
INTRODUCTION
Eucalypts are credited with high growth rate and multiple
uses and are planted extensively. Eucalyptus produces
some of the heaviest, hardest and most durable wood,
thus making this genus the most valuable source of
hardwood in the world. To meet the increasing demand
*Corresponding author. E-mail: sudhirfri2007@gmail.com. Tel:
+919927832700.
Abbreviations: BAP, 6-Benzylaminopurine; NAA, αnaphthalene acetic acid; IBA, indole-3- butyric acid; MS,
Murashige and Skoog’s (1962) medium; DBH, diameter at
breast height.
for timber in the future, fast growing Eucalyptus plantations are preferred. Eucalyptus hybrids have been a
widely planted species in India owing to its adaptability in
different eco-climatic zones. Hybridization is a known
process by which the desirable traits of the two parents
may be combined in F1 offspring. It also offers a means to
capture the benefits of hybrid vigour (heterosis), which is
often manifested in certain specific parental species combinations in hybrids. Development of hybrids, their clonal
multiplication and post juvenile character studies still
remains a serious problem due to various reasons.
Forest research Institute, Dehradun, has 13 mature F1
Eucalyptus hybrid combinations. At Forest Research
Institute, Dehradun, work on hybridization in Eucalyptus
Arya et al.
5719
Table 1. Effect of cytokinin (BAP) in MS medium on axillary bud induction using nodal segments
of FRI-5 (data recorded after 5 weeks).
BAP (mg/l)
Control
0.1
0.5
1.0
1.5
2.0
2.5
Significance
CD at 5%
Response (%)
12.50± 0.29
20.83 ± 0.48
45.83± 0.48
83.33 ± 0.19
66.66 ± 0.38
41.66± 0.38
33.33 ± 0.19
***
1.09
Mean shoot number
0.50 ± 0.22
0.83 ± 0.31
1.83 ± 0.31
3.33 ± 0.33
2.67 ± 0.33
1.67 ± 0.21
1.33 ± 0.21
***
0.81
was initiated during 1970s (Venkatesh and Sharma,
1977c). Based on cross - ability pattern, studies were
initiated to produce controlled hybrids as well as natural
hybrids from half-sib progenies raised from seeds
collected from stands of two intercrossable species
growing in vicinity to each other. The hybrids are artificially produced or spontaneous selected from New Forest
area at Forest Research Institute campus, Dehradun. Out
of these different interspecific hybrids developed, two
hybrids were selected for study i.e. FRI-5 and FRI-14.
Both the Eucalyptus hybrids FRI-5 and FRI-14 were
selected for present study has shown superior character
over their parental combination. They have also displayed a very high degree of hybrid vigour and proved 35 times superior in growth parameters than the parent
combinations. However, when F2 population raised
through seeds a lot of segregation was observed which
had reduced the average yield per unit area per unit time.
These hybrids produced higher biomass than the parental progenies and the Mysore Gum (Venkatesh and
Sharma, 1977a,b). We developed micropropagation
protocol for cloning and large scale production as nonconventional methods of clonal propagation for commercial cultivation of these valuable hybrids of Eucalyptus.
MATERIALS AND METHODS
Nodal shoots segments containing axillary buds were harvested
from the two natural F1 hybrid of Eucalyptus, that is FRI-5 (E.
camaldulensis Dehn x E. tereticornis Sm) and FRI-14 (E. torelliana
F. V. Muell x E. citriodora Hook). The explants were harvested from
newly developed fresh shoots during early in the morning and
proved to be the best time for explant collection. It was found that
the explants collected during January to February and August to
September were the best for in vitro studies as they showed least
phenolic exudation and gave 65 - 70% bud break response as
compared to other months.
RESULTS OF IN VITRO STUDIES
Collection and Preparation of Explant
The nodal shoot segments containing axillary buds were
Mean shoot length (cm)
0.13 ± 0.06
0.23 ± 0.09
0.77 ± 0.04
0.89 ±0.03
0.92±0.04
0.55 ± 0.03
0.38 ± 0.02
***
0.14
collected from 30-32 years old mature trees of Eucalyptus hybrids FRI-5 and FRI-14 growing at New Forest
experimental field of Forest Research Institute, Dehradun. Axillary bud/nodal segments measuring 2-3 cm were
cut. Explant treated with bavistin (1%) and antibiotics
(Streptomycin and Chloramphenicol) for 3-5 minutes
followed by surface sterilization with (0.15%) HgCl2 (12
min for FRI-5 and 10 min for FRI-14) was found to be
very effective in controlling 78 - 80% contamination with
good survival rate of 65 - 75%.
Axillary bud/Shoot Initiation
Four nutrient media viz. MS medium (Murashige and
Skoog, 1962), Woody Plant Medium (Llyod and McCown,
1980), B5 medium (Gamborg et al., 1968) and SH
medium (Schenk and Hildebrandt, 1972) were tested for
the establishment of aseptic cultures from axillary buds
and for shoot multiplication. All the media containing 2%
sucrose solidified (in case of FRI-5) and 3% (in case of
FRI-14) with 0.6% bacteriological agar and supplemented
with different concentrations of BAP individually and
along with NAA was used for the establishment of bud
induction. Initiation of buds started without an intervening
callus phase within 3 weeks from the date of inoculation
of the nodal segments. Among the four media, MS
medium was found to be very effective for axillary bud
induction and shoot initiation for both the hybrids. Nodal
segments of FRI-5 when cultured on MS medium supplemented with 1.0 mg/l BAP, gave 83.33% bud break. In
FRI-14, 70.83% bud break response was obtained on
(MS+1.5 mg/l BAP + 0.5 mg/l NAA (Tables 1 and 2).
Shoot multiplication
Axillary buds were inoculated onto the MS media supplemented with different concentrations of BAP alone and in
combination with NAA. Out of the different media combinations tested, best shoot multiplication occurred on MS
+ 1.0 mg/l BAP + 0.1 mg/l IBA in FRI-5 and on MS + 1.0
5720
Afr. J. Biotechnol.
Table 2. Effect of hormonal interaction (BAP+NAA) in MS medium on axillary bud induction using
nodal segments of FRI-14 (data recorded after 5 weeks).
BAP + NAA (mg/l)
Control
Response (%)
4.16 ± 0.09
Mean shoot number
0.17 ± 0.17
Mean shoot length (cm)
0.07 ± 0.07
0.1 + 0.1
0.1 + 0.5
0.1 + 1.0
0.1 + 1.5
0.5 + 0.1
0.5 + 0.5
0.5 + 1.0
0.5 + 1.5
1.0 + 0.1
1.0 + 0.5
1.0 + 1.0
8.33 ± 0.19
12.50 ± 0.29
8.33 ± 0.19
16.67 ± 0.39
12.50 ± 0.29
20.83 ± 0.48
25.00 ± 0.58
20.83 ± 0.48
29.16 ± 0.09
41.66 ± 0.38
37.50 ± 0.29
0.33 ± 0.21
0.67 ± 0.21
0.67 ± 0.21
1.00 ± 0.00
1.67 ± 0.21
2.17 ± 0.17
2.83 ± 0.31
3.33 ± 0.21
3.83 ± 0.31
4.83 ± 0.31
4.00 ± 0.37
0.13 ± 0.08
0.40 ± 0.13
0.50 ± 0.16
0.72 ± 0.03
0.77 ± 0.01
0.82 ± 0.02
0.84 ± 0.00
0.81 ± 0.02
1.01 ± 0.04
1.50 ± 0.04
0.94 ± 0.00
1.0 + 1.5
1.5 + 0.1
29.16 ± 0.09
45.83 ± 0.48
3.33 ± 0.21
3.00 ± 0.26
0.99 ± 0.02
1.05 ± 0.02
1.5 + 0.5
1.5 + 1.0
70.83 ± 0.48
58.33 ± 0.19
5.67 ± 0.33
4.50 ± 0.22
1.86 ± 0.01
1.68 ± 0.07
1.5 + 1.5
2.0 + 0.1
54.16 ± 0.09
50.00 ± 0.58
3.67 ± 0.21
2.67 ± 0.33
1.58 ± 0.01
1.26 ± 0.01
2.0 + 0.5
2.0 + 1.0
41.34 ± 0.19
37.50 ± 0.29
2.00 ± 0.26
1.67 ± 0.21
1.16 ± 0.03
0.92 ± 0.02
2.0 + 1.5
2.5 + 0.1
2.5 + 0.5
2.5 + 1.0
2.5 + 1.5
Significance
CD at 5%
33.33 ± 0.19
29.16 ± 0.09
20.83 ± 0.48
12.50 ± 0.29
8.33 ± 0.19
***
0.95
1.50 ± 0.22
1.33 ± 0.21
1.33 ± 0.21
0.83 ± 0.17
0.67 ± 0.21
***
0.67
0.73 ± 0.02
0.50 ± 0.03
0.35 ± 0.03
0.17 ± 0.03
0.13 ± 0.04
***
0.15
***Significance at 0.1%, ± values represent the standard error.
Table 3. Effect of hormonal interaction cytokinin and auxin (BAP + IBA) in MS medium on in vitro shoot
multiplication of FRI-5 (data recorded after 5 weeks).
BAP + IBA (mg/l)
Mean shoot number
Mean shoot length (cm)
Multiplication rate
Control
1.0 + 0.1
10.33 ± 0.42
57.17 ± 1.38
0.50 ± 0.03
1.51 ± 0.04
2.07 ± 0.08
11.43 ± 0.28
1.0 + 0.5
***
45.83 ± 0.75
37.17 ± 0.83
1.33 ± 0.08
0.61 ± 0.06
9.17 ± 0.15
7.43 ± 0.17
1.0 + 1.0
1.0 + 1.5
24.33 ± 1.20
23.80 ± 0.96
0.44 ± 0.01
1.12 ± 0.07
4.87 ± 0.24
4.70 ± 0.19
***
2.81
***
0.16
***
0.56
Significance
CD at 5%
mg/l BAP in case of FRI-14. Multiple shoots were obtained after 5 weeks in both the hybrids (Tables 3 and 4).
A regular sub-culturing was carried out every 5 weeks on
fresh medium. Cultures were incubated at 25 ± 2°C 16
hours in light (illuminated by 40 watt fluorescent tubes,
1200 lux) and for 8 hours in dark cycle irradiance by cool
fluorescent tubes. The cultures were regularly transferred
into fresh medium to check the browning of cultures.
Arya et al.
5721
Table 4. Effect of cytokinin (BAP) in MS medium on in vitro shoot multiplication of FRI-14 (data
recorded after 5 weeks).
BAP(mg/l)
Mean shoot number
Mean shoot length (cm)
Multiplication rate
Control
22.33 ± 1.12
1.07 ± 0.02
5.58 ± 0.28
0.5
33.50 ± 1.26
2.01 ± 0.12
8.38 ± 0.31
1.0
51.33 ± 2.35
3.03 ± 0.16
11.83 ± 0.16
1.5
40.80 ± 1.40
2.63 ± 0.05
6.80 ± 0.15
2.0
30.17 ± 0.91
1.97 ± 0.06
5.20 ± 0.12
2.5
Significance
20.50 ± 0.62
***
1.46 ± 0.05
***
3.53 ± 0.18
***
4.00
0.26
0.42
CD at 5%
***Significance at 0.1%; ± values represent the standard error.
Table 5. Effect of strength of different basal media supplemented with 1.0 mg/l BAP + 0.1mg/l IBA in MS
medium on in vitro shoot multiplication of FRI-5 (data recorded after 5 weeks).
Mean shoot number
Mean shoot length (cm)
Multiplication rate
MS
Media
1x
52.17 ± 0.60
1.14 ± 0.04
10.43 ± 0.12
B5
1x
11.83 ± 0.79
1.10 ± 0.01
2.37 ± 0.16
WPM
1x
29.17 ± 0.95
0.75 ± 0.05
5.83 ± 0.19
SH
1x
11.50 ± 0.76
0.60 ± 0.04
2.30 ± 0.15
Significance
CD at 5 %
***
***
***
2.06
0.10
0.41
Table 6. Effect of strength of basal medium supplemented with 1.0 mg/l BAP in MS medium on in vitro
shoot multiplication of FRI-14 (data recorded after 5 weeks).
Media
MS
1x
B5
1x
WPM
1x
SH
1x
Significance
CD at 5 %
Mean shoot number
Mean shoot length (cm)
Multiplication rate
53.67 ± 1.05
14.83 ± 0.79
30.00 ± 0.58
11.17 ± 0.48
***
1.96
3.14 ± 0.02
3.56 ± 0.00
0.76 ± 0.00
1.59 ± 0.03
***
0.03
10.73 ± 0.21
2.97 ± 0.16
6.00 ± 0.12
2.23 ± 0.10
***
0.39
***Significance at 0.1%; ± values represent the standard error.
Effect of different basal media and their strength
Maximum shoot multiplication rate (9 - 12 folds) was
obtained on MS medium supplemented with 1.0 mg/l
BAP + 0.1 mg/l IBA mg/l in case of FRI-5 and in case of
FRI-14 maximum shoot multiplication rate (10 - 12 folds)
was obtained on MS medium supplemented with 1.0 mg/l
BAP. Amongst all media, shoot multiplication was highest
(found optimum) on MS medium with a shoot multiplication rate of 10 - 12 folds (Tables 5 and 6). It was found
that maximum shoot multiplication rate was obtained on
full strength MS medium. In vitro shoot elongation was
obtained on both ½ and ¼ strength MS medium without
PGR.
Regeneration of roots
After elongation individual shoots measuring 2.5 to 3.0
cm were inoculated on half strength MS medium supplemented with IBA (0.1-2.0 mg/l) alone and in the
combination with NAA (0.1-2.0 mg/l) for rooting. 87.50%
rooting was achieved in FRI-5 on ½ MS supplemented
with IBA (1.0 mg/l) and 91.66% rooting was achieved in
5722
Afr. J. Biotechnol.
Table 7. Effect of IBA on rooting of in vitro shoot on half strength MS medium of FRI-5 (data
recorded after 5 weeks).
IBA (mg/l)
Control
0.1
0.5
1.0
1.5
2.0
Significance
CD at 5%
Rooting (%)
12.5 ± 0.29
20.83 ± 0.48
66.66 ± 0.38
87.50 ± 0.29
70.83 ± 0.48
54.16 ± 0.09
***
1.11
Mean root number
0.50 ± 0.22
8.67 ± 0.56
17.17 ± 0.48
20.17 ± 0.60
18.83 ± 0.70
11.33 ± 0.67
***
1.62
Mean root length (cm)
0.35 ± 0.16
0.73 ± 0.05
1.02 ± 0.03
2.34 ± 0.04
1.47 ± 0.05
1.30 ± 0.03
***
0.22
Table 8. Effect of IBA on rooting of in vitro shoot on half strength MS medium of FRI-14
(Data recorded after 5 weeks).
IBA (mg/l)
Control
0.1
0.5
1.0
1.5
2.0
Significance
CD at 5%
Rooting (%)
12.5 ± 0.29
45.83 ± 0.68
91.66 ± 0.86
79.16 ± 0.24
62.50 ± 0.46
41.66 ± 0.54
***
1.11
Mean root number
0.50 ± 0.22
8.67 ± 0.56
17.17 ± 0.48
20.17 ± 0.60
18.83 ± 0.70
11.33 ± 0.67
***
1.62
Mean root length (cm)
0.35 ± 0.16
0.73 ± 0.05
1.02 ± 0.03
2.34 ± 0.04
1.47 ± 0.05
1.30 ± 0.03
***
0.22
***Significance at 0.1%; ± values represent the standard error.
FRI-14 on ½ MS supplemented with IBA (0.5 mg/l)
without intervening callus phase (Tables 7 and 8).
Acclimatization and hardening and field transfer
Micropropagated and rooted plantlets were hardened in
vitro in liquid ¼ MS medium having 2% sucrose. Absorbent cotton soaked in this liquid medium was used for
supporting root system of in vitro raised plantlets.
Plantlets were maintained in this step for 2 weeks, transferred to mist chamber in polythene bags containing a
mixture of soil, sand and manure (1:1:1) and covered with
perforated polythene bags and then transferred to the net
house. Holes were made in the polythene bags, which
were withdrawn periodically, and the plantlets were finally
transferred to the field. 85 - 95% success in field survival
rate was observed in both the hybrids.
lyptus hybrids FRI-5 and FRI-14 was done on following
aspects viz survival rate and growth parameters like
height, diameter and clear bole length. So far 200 tissue
culture raised plants of each hybrid were planted in field
during monsoon season of 2005 in different eco-climatic
zones of Uttarakhand (Table 9).
Tissue culture raised plants of FRI-5 and FRI-14 were
planted in three meter apart rows and the plant to plant
distance was also 3 m (monsoon year of 2005). Each
treatment had 20 plants in a row. The total rows were 10.
The control plants raised by locally available seeds were
also maintained at the same site. Casualty replacement
was done during the second year. Other cultural operations such as weeding, fertilizing and mulching were done
up to 3 years. The height, collar diameter and clear bole
length were taken twice a year (June and December).
The survival count was also noted during this period.
RESULT OF FIELD TRIALS
Field plantation
All the experimental sites were leveled and at places
where irrigation facility was available for irrigation of the
plantation was done every fourth night. The trial was laid
out in block plantation along with the control local hybrid.
Field study of tissue culture raised plants of both Euca-
In the present study tissue culture raised cloned plants
were planted at three sites of different climatic conditions
of Uttarakhand. An average height of 7.5-9.5 m were
observed with an average diameter of 6.5-8.5 cm and a
survival rate of 90-92% at the sites of Dehradun, while at
Pantnagar and Haldwani fields a survival rate of 86-90%
Arya et al.
5723
Table 9. Geographical distributions of different agro-climatic sites of Uttarakhand State for field plantation.
Plantation Site
Forest Research Institute, Dehradun
G.B.
Pant
Agriculture
and
Technology University, Pant Nagar
Tanda Range, Haldwani
Soil
type
Sandy
Loam
Loamy
Sand
Loamy
Sand
pH
range
6.4 - 7.5
Altitude
Latitude
Longitude
661 - 665 m
30°N
78°E
6.6 - 6.9
243.84 m
29°3’N
79°30’E
1500 - 1600
6.5 - 7.1
680 - 685 m
29°13’N
79° 31’E
878
,
Annual
rainfall (mm)
2180
Major climatic
condition
Humid sub-tropical
Mean
temperature
1 - 41°C
Sub-tropical, water
logged, high rainfall
Sub-tropical,
Tarai
area
3 - 43.2°C
1.5 - 39°C
Table 10. Field performance after three years of tissue cultured clones of Eucalyptus hybrids (FRI-5 and FRI-14) under different varied climatic conditions of Uttarakhand State.
Plantation
sites
Dehradun
Pant Nagar
Haldwani
Significance
CD at 1%
Avg. height (cm)
Avg. collar dia. (cm)
Volume (m 3)
Avg. clear bole length
FRI-5
FRI-14
Control
FRI-5
FRI-14
Control
FRI-5
FRI-14
Control
FRI-5
FRI-14
Control
695.71
705.76
704.19
**
765.96
579.02
594.02
**
129.04
546.35
493.62
511.67
**
6.93
7.04
7.35
***
7.89
7.16
7.33
***
0.617
4.00
3.86
4.05
***
174.0
213.2
216.6
NS
283.5
183.4
194.5
NS
-
133.4
123.8
129.5
NS
0.011
0.011
0.011
0.016
0.009
0.009
0.003
0.002
0.003
-
*** Significance at 0.1%; ** Significance at 1.0 %; * Significance at 5.0%; NS- non significant.
Note: Average of 50 plants.
was recorded after three years of plantation. Total
height and collar diameter taken on breast height
(~137.0 cm) were recorded after three years and
variation was observed in height and collar diameter at different plantation sites (Table 10).
The growth performance and survival percentage of both Eucalyptus hybrids FRI-5 and FRI-14
showed their suitability in particular environmental
conditions. The analysis of survival percentage
showed that the hybrids as well as the localities
also when compared do not differ significantly. It
indicates that localities or hybrids have no effect
on survivalabilty of plants. Hence, it was considered to the best for commercial plantation at
these three plantation sites.
Both Eucalyptus hybrid FRI-5 and FRI-14
performed better for all these traits when compared with control. However, statistical analysis
indicated non-significant differences among the
hybrids and control for all the traits studied. The
analysis of average height showed that the
hybrids differ significantly but localities show no
significant difference among them. The average
height of the hybrids is significantly higher than
the control. Like wise, the analysis of average
collar diameter shows that the hybrids differ
significantly but localities show no significant difference among them. The average collar diameter
of the hybrids is significantly higher than the
control. While the analysis of average clear bole
length shows that the hybrids as well as the
localities do not differ significantly. The uniformity
obtained in the plantation with micropropagated
plants confirms the feasibility of using this
technique for commercial scale multiplication of
both Eucalyptus hybrids (FRI-5 and FRI-14). The
overall data collected after three years shows that
both FRI-5 and FRI-14 performed well and are
suited to the varied climatic conditions of
Dehradun, Pantnagar, Haldwani (Figures 1H-J) and
also showed good self pruning up to three years
of age. Now the plants are in fourth year of age.
5724
Afr. J. Biotechnol.
Figure 1. Commercial multiplication of Eucalyptus hybrids FRI-5 and FRI-14 from mature tree (30 - 32 years old). (A)
Axillary bud induction using nodal explant collected from mature mother plant (30 - 32 years old); (B) In vitro shoot
multiplication in FRI-5 (after 5 weeks); (C) In vitro rooted plantlets; (D) Hardened and acclimatized plants in poly bag
ready to field transfer; (E) 6th month old plant in field; (F and G) 3 years old field plantation at different plantation
sites.
DISCUSSION
The present investigation demonstrates the successful
multiplication by axillary meristem and field plantation at
different eco-climatic zones of Uttarakhand state. Systematic field evaluation data for tissue culture raised plants
Arya et al.
is not available. Although number of tissue culture raised
plants have gone to the field but large-scale field trials is
limited. Clonal plantations of Eucalyptus are being raised
by conventional vegetative propagation method by
private organizations having captive consumption (Lal,
1994). Plantation has displayed a very high degree of
vigour (positive heterosis) both in height, diameter and
wood quality. Data showed vigorous growth when compared with local Eucalyptus species in terms of total
height, collar diameter and clear bole length. This may be
attributed due to genetic constitution of the hybrid as well
as environmental interaction constituting various physical
factors like moisture content, soil pH , mean annual rainfall, mean temperature, etc. Preliminary investigation
showed that hybrids vigoursity of the hybrids was
transferred in clonal material fully and expressed even
after the age of 36 months. The present trials had also
shown heterotic superiority of hybrids but with variation
due to variable interaction between genotype and
environments. The same is reflected in these hybrids
grown in the different climatic conditions as data reflected
variations in the entire traits studied. Sreedhar and Rao,
(1998) developed commercial scale micropropagation
technique for E. citriodora and E. camaldulensis and
introduced their field plantation at various locations with
selected clones of E. citriodora. They have recorded
performance after 13 months of plantation and found that
micropropagated clones were distinctly superior and
highly uniform. Kaur and Saxena (2002) evaluated
multilocational trials of E. tereticornis in Haryana. They
also collected parameters like height, DBH, clear bole
height and self pruning capability of different clones for
the identification of suitable clones for different agro
climatic zones of Haryana. Biswas et al., 1999 also reported field trial of tissue cultured raised E. tereticornis and
establishment environment factor as a key to the
developmental stages in Eucalyptus. Johnson (1955)
also reported that hybrids can express true to type vigour
only under similar site of environmental conditions.
Venkatesh and Sharma (1977a) reported that F1 hybrids
may display varied hybrid vigour at one stage or other
during their development period, thus it is necessary that
the present study under different climatic condition may
be carried out till maturity of these cloned hybrids. The
heterotic effects of hybrids are mainly due to the specific
gene combination in the hybrids. pH and moisture content from all the three sites were also recorded to see
their effect on growth performance. Average pH as
recorded from all the three plantation sites was in the
range of 6.5-7.5 which does not affect the survival and
growth performance of Eucalyptus (Table 9). It was found
that Eucalyptus hybrid made satisfactory growth in soils
with pH less than 9.0 and TSS less than 0.3 but failed to
establish in soils of higher pH and salt content and in
soils with a kankar pan. Kaushik et al. (1969) showed that
Eucalyptus failed to grow in saline-alkali soils which have
a pH above 10 and soluble salt content above 0.7% and
5725
posses compact indurate sub soil due to kankar. Its
growth was found to be arrested on the soils which have
pH below 8.5 but salt content above 1.0%. Moisture
content was recorded maximum as 40.21 in December
and minimum as 13.34 in June. It was generally observed
that under conditions of restricted soil moisture, water
conserving mechanisms are in operation and the plants
tend to restrict the water loss so that the growth is not
seriously affected. These results showed that high rate of
transpiration by Eucalyptus are adaptability operative
under the conditions of adequate soil moisture (Rawat et
al., 1984).
Conclusion
In the present study effective and commercially viable
micropropagation techniques were successfully developed for mass multiplication of two superior Eucalyptus
hybrids FRI-5 and FRI-14. Preliminary investigation after
three years of field plantation at different eco-climatic
sites having varied environmental conditions showed that
Eucalyptus hybrids FRI-5 and FRI-14 are promising in
terms of height, diameter, clear bole length and selfpruning capability. However, suitability of hybrids in a
particular eco-climatic zone can be determined by making
continuous observations over a period of about 8-10
years. Present finding is based on a period of three
years.
In the light of above findings it may be concluded that
both the Eucalyptus hybrids FRI-5 and FRI-14 produced
on large scale through tissue culture technology performed well in terms of growth parameters up to three years
of age when compared to control and now can be considered as site specific plants species for all the three
respective plantation sites in Uttarakhand state viz.
Dehradun, Pantnagar and Haldwani.
Summary
Eucalyptus hybrids are an important forest tree species
under the social and commercial forestry programme with
a view to get more yield per unit area. The wide spread
application of in vitro propagation on its cost-competitiveness and is profitable only when there is an associated advantages over conventional propagation
methods. The field testing of these hybrids revealed their
suitability in a particular climatic zone. Also these results
had shown their performance with respect to the total biomass production. The hybrid if found suitable even after
7-8 years (rotation cycle) with improved timber and oil
production will be of immense value for commercial
growers of Eucalyptus. The trial of in vitro raised plantlets
which had completed after three years will provide
requisite material for testing the hybrids for various quailtative traits for pulp and paper based industries. Beside
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Afr. J. Biotechnol.
this clonal propagation is the only answer to tap the
productive potential of these hybrids which have been
achieved successfully.
REFERENCES
Biswas RN, Karunakaran M, Moon AR (1999). Field trials on tissue
culture in Eucalyptus. Indian For. 125(3): 264-271.
Gamborg OL, Miller RA, Ojima L (1968). Nutrient requirements of
suspension cultures of Soyabean root cells. Exp. Cell Res. 50: 151158.
Johnson AG (1955). Southern pine hybrids, natural and artificial, Proc.
rd
3 Southern Conference on Forest tree improvement, New Orleans,
USA, pp. 63-67.
Kaur A, Saxena V (2002). Evaluation of multilocational clonal trials of
Eucalyptus tereticornis in Haryana. In: Bagchi SK, Varghese M and
Siddapa M (Eds.). Proc National Workshop on Eucalyptus held at
Coimbatore and Bangalore (2001) IWST, Coimbatore Indian, pp. 1-8.
Kaushik RC, Qureshi IM, Yadav JSP, Prakash J (1969). Suitability
of soils for Eucalyptus hybrids (Mysore gum syn. E. tereticornis) in
Haryana and Punjab. Indian For. 95: 377-388.
Llyod G, McCown BH (1980). Commercially feasible micropropagation
of mountain laurel, Kalmia latifolia, by use of shoot-tip culture.
International Plant Propagator’s Society. Combined Proceedings, 30:
421-427.
Murashige T, Skoog F (1962). A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant. 5: 473-497.
Lal P (1994). Economics of mass multiplication of forest trees. Indian
For. 120(2): 85-96.
Rawat PS, Gupta BB, Rawat JS (1984). Transpiration as affected by
soil moisture in E. tereticornis seedlings, Indian For. 110: 35-39.
Schenk RU, Hildebrandt AC (1972). Medium and techniques for
induction and Growth of Monocotyledonous and Dicotyledonous
Plant Cell Cultures. Can. J. Bot. 50: 199-204.
Sreedhar D, Rao MM (1998). Commercial scale micropropagation of
Eucalyptus camaldulensis. Indian For. 124(4): 217-224.
Venkatesh CS, Sharma VK (1977a). Hybrids vigour in controlled
interspecific crosses of Eucalyptus tereticornis and E. camaldulensis.
Silvae Genetica, 26(4): 121-124.
Venkatesh CS, Sharma VK (1977b). Differential heterosis in reciprocal
interspecific crosses of Eucalyptus camaldulensis Dehn. and
Eucalyptus tereticornis Sm. FAO III World Consultation on Forest
Breeding, Canberra, Australia. FO- FTB-77-3/18.
Venkatesh CS, Sharma VK (1977c). Rapid growth rate and higher yield
potential of heterotic Eucalyptus hybrids FRI-4 and FRI-5. Indian For.
103: 795-802.