Indi an Journal of Experimenta l Biology

Vol. 39, June 200 I, pp. 594-599

Micropropagation of Paulownia fortuneii through in vitro axillary

shoot proliferation
B Yenkateswarlu , J Mukhopadhyay, E Sreenivasan & V Moses Kumar
Ce ntral Researc h Institute for Dry land Agriculture, Santoshnagar, Saidabad P.O ,Hyderabad 500059, Indi a
Fax : 040-4531802; E-mail : vba ndi @crida.ap.nic.in

Received 22 Jun e 2000; revised 23 Januwy 2001

Primary cultures were es tabli shed with nodal segments from juvenile shoots o f two- year-old Paulownia fort uneii trees
from a clo nal plantation in Andhra Pradesh. A med ium containin g half-strength MS salts + BAP ( I mg/L) + sucrose (2 %)
produced o ptimum bud break in nodal explant s. The same basal medium w ith reduced hormone level (0.5 mg/L) suppo rted
maximum multiplicatio n of seco nd ary cultures of P.fortun eii ( I :6 in 6 wee ks). Specific treatments were tested to en hance
thi s rate of multiplicatio n. In o ne approach, five to six week o ld in vitro g row n shoots were ratooned (c utting the main shoot
at the bottom leav ing o ne node). The stumps (ratooned basal node) produced 2 to 3 axillary shoots, whi ch grew into 4 to 5
nodes by 3 weeks; thu s, prov id in g additi o nal shoots from the sa me ex plant. T hi s prov ided 30% additi o nal shoots in 4 cyc les.
Secondl y, reducing the li g ht intensity to 1200 lux res ulted in hi gher shoo t e lo ngati o n, i.e, formation of 8 nodes in 5 weeks
w ith hea lthier shoots than the no rmal intensity of 3000 lu x un der whi ch o nly 6 nodes were produced in 6 wee ks. /11 vitrogrow n shoots could be successfull y rooted ex vitro in verm iculite + cocopeat mixture ( I: I v/ v) under 90% humidity.
transferred to soil in polybags for hardening in the green ho use for 2 weeks and shifted to shade net fo r fu rther hardening.
After o ne mo nth, the plants co uld be success fully transplanted to field wi th 95 % survival. Micropro pagated plants showed
an excellent g rowth in the fi eld att ainin g a heig ht of 1.5 m and a collar diameter of 2 .8 em in 3 mo nths.

Paulownia, popularly kno wn as empress tree is native

1
of Eastern Asia It has revoluti onized the agroforestry
in China and has been introduced successfully in
North and South America. Paulownia has become
naturalized in Southern and Central hardwood forests
2
of United States . Paulownia has many recognized
are
important
spec ies,
but
four
spec ies
(P. tom en rosa,
P.fortunei i,
P.
com mercially u
kawakami and P. taiwaniana) . It is widely grown in
temperate areas of Taiwan, China and Australia, but
P. fortun eii has also been planted successfully in
3
many tropical and subtropical areas of the world . P.
fortun eii has been recogni zed as hav ing good
potenti al for agroforestry in tropical zones of India as
well 4 P.fortun eii has been introduced in India by the
Forest Research Institute, Dehradun- in 1992, but not
much attention has been paid on thi s species till
rece ntly 5 . From 1997 onwards many companies and
farmers have shown interest in different states of
lndi a. Fast growth is an outstanding character of
Paulownia. In o ne year it grows as a po le (8 feet) and
useful timber can be obtained in 6 years under good
management 4 The wood is strong yet light weight (16
pounds per cubi c foot) making it ideal for
manufacturing of crates, mu sical instruments and
1
toys Paulownia can be propagated through seeds,
stem cuttings root suckers and ti ssue culture 2 . The

seeds
have
a
low
germinati on
percentage
(P.fortun eii:29%; P.kawakami: 13 %; P.tomentosa :59% ;
P.taiwaHiana :23 %) and need special treatments. Early
seed ling growth is slower than vegetative ly
propagated plants produced fro m root and shoot
6
cuttings or rooted shoots from ti ssue culture . Root
suckers offer a cost effect ive alternative to produce
planting material under Indi an conditi ons. However,
currently the planting stock in the country is
inadequate to produce suffici ent material by thi s
method. Mi cropropagation is being developed as an
alternative approach to sapling production in China,
Australia and USA. Various spec ies of Paulownia
have been propagated through ti ssue cultures using
27
juvenile and mature ex pl ants 8 . Even nodul e cultures
of P. taiwania11.a have been tri ed for automated
mi cropropagation in China with high multipli cation
ratios 9 . Mi cropropagati on of P.fortuneii has been
reported from shoot tips, nodal ex plants and
leaves 57 10 with
varying
multiplication rates.
However, most of these protoco ls are based on in
vitro rooting followed by potting of indi vidual plants
and little data on growth of field transferred plan ts. In
the present paper, an effi cient mi cropropaga ti on
protocol has been reported for P. fortun eii with a
significant enhancement of in vitro multiplication rate
through a novel techniqu e of in vitro ratooning.

VENKATESWARLU e/ a/.: MICROPROPAGATION OF PAULOWNIA

Successful ex vitro rooting and field performance of

transplanted plants has also been reported.

Materials and Methods

Collection and establishment of primary
explants-Actively growing juvenile shoots from
axillary branches of field grown mature tree of
Paulo wnia fortuneii (Seem.) Hemsley . were collected
locally during the months of April, 1999 to
February,2000 at monthly intervals up to August, 1999
and bimonthly intervals thereafter as the source of
primary explant. The mother plant was selected from
a plantation established in a farmer's field in the
Karimnagar district of Andhra Pradesh using clonal
planting material of Paulownia fortuneii (clone I)
imported in the form of plugs (small hardened green
plantlets in cell-trays). The shoot segments were
thoroughly washed with tween-80 solution and cut
into 0.5 to !em single nodal segments, surface
sterilized by rin sing in mercuric chloride (0.1 %) for 4
min followed by repeated rinsi ng with sterile distilled
11
water. Full strength and half strength MS media
containing 2% sucrose solidified with 0.8 % agar (pH
5.8) and supplemented with different combinations of
auxins and cytokinins were used for the experiments.
The media were sterilized by autoclaving at 1.1
kg/cm 2 for 15 min . For each treatment, 12 explants
were included. The cultures were incubated in a
growth room at 28C under 14 hr photoperiod with a
light intensity of 3000 lux . The data on frequency and
nature of respon se was recorded after 4 and 6 weeks
of culture.
In vitro multiplication- Based on the response of
primary cultures, axillary shoots of 3-5cm in length
were used for further multiplication . Single nodes
were cut and placed in half strength MS medium with
reduced hormone levels (0.1, 0.5 , 0.75 and 1.0 mg/L)
as hormone requirement for secondary multiplication
of Paulownia has been reported to be significantly
2
lower than the explants from mature tree . Two
exp lants were placed in each culture vessel. From
each explant 2 to 3 axillary shoots emerged initially
but only one or two could grow eventually into
healthy shoots of 6-8cm length in six weeks.
Reduced Light intensity -To enhance the rate of
multiplication, the cultures were incubated at two
light intensities of 3000 and 1200 lux by regulating
the number of fluorescent tubes. Twenty replications
(culture bottles) were included for each treatment and
two explants were inoculated in each vessel. The
shoot length and total number of culturable nodes
were determined at the end of 6 weeks of incubation .

595

In vitro shoot proliferation-In this approach ,

fully grown shoots comprising 6 nodes from
secondary cultures were cut (with a specially designed
surgical scissors having a 2 em bent tip) and used for
subculture leaving the 2 basal nodes culture for
further incubation. Within 3 weeks, 2 -3 axillary
branches grew from these stumps. Such regrown
shoots were healthy and had the same leaf size and
stem thickness as the normally cultured nodal
explants within 3 weeks as against 6 weeks required
for a normal subcultured explant . These stalks of
shoots were either subcultured again or rooted just as
the normal ones.
Rooting and hardening - In vitro- grown micro
shoots of 3-4 inches length with 4-5 nodes were used
for rooting ex vitro. The cultures were gently removed
from the bottles, basal portions were cut and dipped in
IBA (800 mg/L) solution for 10 min . These were
transferred to a rooting medium (soilrite; an equal
volume mixture of vermiculite and cocopeat) filled in
plastic trays at the rate of 60 plants per tray. The trays
were placed in a poly tunnel to maintain 90-95 % RH .
All the shoots produced excellent root system by 7-8
days. The rooted shoots were then transfened to
polybags (15 x 10 em) filled with garden soil. The
bags were placed in the mist chamber under a
gradually reducing humidity regime from 90 to 55 %
RH for 2 weeks. Thereafter, the plants were shifted to
shade house for secondary hardening and kept for 4
weeks under ambient temperature and humidity
conditions.
Field transfer and evaluation-Fully hardened
plants of about 10 em of height were transplanted in
the field for studying their survival and growth. About
120 plants were transplanted in pits (45 X 45 X 45
em) dug in a loamy sand soil (pH 7.5 , OC: 0.45 %,
total N: 0.047 %) on two dates viz. 20 plants during
May, l999 and 100 plants durin g July,1999 with a
plant to plant spacing of 3 m. Saplings transpl anted
during May, l999 were given protective irri gation
twice a week (7Liplant) till the beginning of the
monsoon, where as the plants in the field were grown
solely on the rainfall (290 mm received in 23 rainy
days) . The height and collar diameter of the plants
were recorded at 8 week intervals.
Results and Discussion
Explants from field grown trees could be
established successfully on MS medium containing I
mg/L of BAP (Table 1). Buds collected during June
showed maximum sprouting followed by those
collected in May, April and July. No respon se was

INDIAN J EXP BIOL, JUN E 200 1

596

fo und from those collected from September till

February . Among different medi a and hormone
combinations
tried,
half-strength
MS
with
BAP(J mg/L) + sucrose (2 %) showed the best
res ponse (80%). Additi on of auxins in th e medium
resu lted in callus form ati on. Burger et aP have also
reported better response of primary explants of P.
to111entosa on MS with reduced salts. Subcultured
shoots produced 6 nodal microshoots (from each
explant) in 6 weeks which could be either subcultured
or rooted success fully .
Further ex periments on secondary multiplicati on

with varying concentrati ons of BAP (0.1 , 0.5, 0.75 ,

1.0 mg/L) showed optimum response at 0.5 mg/L.
Burger et at? have also reported a lower hormone
requirement for secondary multiplication of P.
to111entosa when compared to primary cultures. A
multiplication ratio of 1:6 was obtained in 6 weeks
where the shoots were healthy and roored well.
Hi gher rates of multiplicati on have been reported
from leaf and nodal expl ants of P. elonga ta, P.
to/1/entosa, P. fo rtun eii, parti cularly when auxi ns have
been used in the medium, but in all such cases the
shoots ori gin ated adventitiously through a callus

Table I - Response of primary cultures of P. j ortu11 eii on di fferent medi a and

hormone combin ati ons
Medi a combinati ons

Observati ons (after 6 weeks)

MS(F) + BA P (0. 1 mg/L)

MS(F) + BA P (0. 1 mg/L) + NAA (0. 1 mg/L)
MS (F) + BAP (0. 1 mg/L) + KIN (0.1 mg/L)

Shoot with 2-3 nodes developed, but unh ealth y (yellow ish)
Ca llusi ng of total ex pl ant
Ax ill ary buds sprouted; small shoot fo rmed but no further
growth
2 nodal length lateral shoot formed but stopped growth
Small lateral shoot emerged, but fa iled to grow.
Green callus formed
Health y shoot formed with 3-4 nodes.
Health y and excellent shoot developed which grew up to 4-5
nodes .

MS (F) + BAP ( I mg/L )

MS(F) + BA P (2 mg/L)
MS( H)+ BAP (0.1 mg/L) + NAA (0. 1 mg/L)
MS(H) + BAP ( 1.0 mg/L)
MS( H) + BAP ( 1.0 mg/L) + Sucrose (20g/L)

Mediu m contain ing : F-- Full strength salts; H- Half strength salts

--~------------------------------------------

Table 2- Effect of light intensity on i11 vitro multiplicati on of P. jortu11eii

Treatment

Length of th e main shoot (e m) after 6

wee ks

Average no. of cultu rable nodes

after 6 weeks

6-7
8- 10
8-9

6
8
6

10-l l

Light intensity (3000 lu x)

Li ght in te nsity ( 1200 lu x)
Shifting fro m low to hi gh intensit y after
2 wee ks of inoculati on
Shifting from hi gh to low intensit y after
2 weeks of inoculati on

Table}- Effect of i11 l'itro culture of basal stalk remained after excision of shoot on the shoot
multiplicati on of P. j ortu11eii
[Culturable nodes obtained are from two ex plants placed in one culture vessel]

In vitro ratoonin g

Normal multipli cati on

Mult ipli cation
cyc le (6week)

0
2
3
4

No of culturab le
nodes

Multiplication
cycle
(6 week)

No of culturab le
nodes from
normal cycle

No. of
multiplicati on
cyc les for
regenerted
shoots (3week)

o of culturable
nodes (additional)
obtained from
regenerated shoots

2
12

2
12

72

2
3
4

72

432
2592"

2
3
4

0
16
96
576
3456b

432
2592"

"obt::tined in 24 weeks from 4 cyc les through norm al multiplicati on.

bobtained in 12 wee ks from 4 cycles th ro ugh culturing of regenerat ed shoots.

VE NKATESWARLU e/ a/.: MICROPROPAGATION OF PAULOWNIA

Fig. 1- Stages in micropropagation of P fo rtuneii : A - Establishment of primary explant. B rooting. D - rooted plant let. E - Field transferred plant (2 month old)

phase 6 12 However, Rao et a /8 have obtained 40 shoots

fro m each leaf ex pl a nt over a fo ur mo nth pe ri od
through direct regenerati o n. Th e multiplicati on rate of
36 shoots pe r ex pl ant in 12 weeks obtained in th e
present study is considerabl y higher th an reported
earli er fo r othe r spec ies of Paulownia.
Light in tens ity had a signi ficant impact o n the
growth of secondary c ultures. Under 3000 lux li ght
in tensity, th e shoot e lo ngati on was slow w ith short
internodes and fo rmati on of rosette type shoots. Whe n
the li ght inte nsity was reduced to 1200 lu x, the
internodes elongated w ith signifi cantly more leaf area
ex pansion and shoot thi ckness . This led to hi ghe r
shoot length and more number of culturable nodes. As
against 6 nodes produced per expl ant in 6 weeks,
reduced li ght intensity res ulted in 7-8 culturabl e nodes

in vitro multiplication, C -

597

ex l'itro

in 5 weeks (Tabl e 2) . However, initi al normal

intensity fo r 2 weeks fo ll owed by shi fti ng to low
(1200 lux) inte nsity was more effective as co mpared
to a shift fro m low to normal or co ntinuous low.
Continuous normal, however, was the least effecti ve.
In other word s, P. fo rtuneii cultures need reduced
li ght intens ity fo r achi ev ing hi gher multipli cation rate
either co ntinuously or at least 2-3 weeks in the
multiplication cycl e as compared to the light
inte nsities required fo r other tree spec ies .
An othe r approach fo ll owed to achi eve en hanced
shoot producti on in seco ndary multiplicati on was in
vitro shoot pro liferati on. Whe n the main shoot was
exc ised leaving the lower mos t node, it led to the
growth of 2-3 lateral shoots from each stump
producing nearly 8 nodes in 3 weeks at the rate of 4

598

INDIAN J EXP BIOL. JUNE 200 1

nodal segments from each lateral shoot. In other

words, by axillary shoot regeneration 30% more
shoots could be obtained in half the time as compared
to th e norm al multiplication cycle (Table 3). Shoot
regeneration was also tried for second time (on the
same explant), but the regrowth was poor and lateral
shoots were not healthy enough either for subculture
or rooting. The medium also started drying after first
cycle of shoot regeneration. The results therefore
indicated that in vitro shoot regeneration could be a
useful method for obtaining enhanced multiplication
Qf Paulownia, but it can be done only once.
The shoots could be rooted successfully both in
vitro and ex vitro with equal effeciency. However, the
survival during primary hardening was 80% with in
vitro rooting while it was more than 95% with ex vitro
method. We followed only ex vitro rooting due to
better root formation and short protocol. This protocol
is more simple and efficient as compared to in vitro
root ing, potting and field transfer method reported
2
earli er 58 With IBA (800 mg/L) treatment for 10 min,
98% of the shoots produced excellent roots in 8-10
days. Application of hormone through chalk coating
did not have any advantage over dipping. The
plantlets continued to grow while rooting, with
considerable leaf expansion, increase in height and
stem thi ckness (Fig. I A-E). Rooted plants when
transferred to polybags and continued in hoods with
95% humidity for 48hr, fo llowed by gradual reduction
showed better survival (98%) while hardening, as
compared to those transferred out side within the mist
chamber on open benches. Based on repeated
observations by us Paulownia pl ants were found
highly susceptible to excess wetness on leaves at the
time of primary hardening, therefore an optimum
humidity may be maintained.
At the end of 30 days of secondary hardening in the
shade (50%), the plants grew up to 6 inches with 6-8
fully ex panded leaves. The ex vitro transplant success
was 90%. Fully hardened plants when transferred to
field showed 100% survival and rapid growth during
the rainy seaso n. On an average, plants transplanted
during May , 1999 and prov ided tm gation have
attained a hei ght of 2.45m and collar diameter of
4.5cm in fiv e months (Fig. 2). The growth ceased
thereafter as the plants entered into dormancy. The
other set of plants tran splanted in the field during
Jul y, 1999 and rai sed under rainfed conditions have
put up much less growth (height of 0 .68m and coll ar
di am of 2cm). These plants also entered into
dormancy
within 2
months after planting.

3Tr==~==~--------------~ 6
.---lkJrr

2.5

.... ... R.rr

.. ..... .. O>.rr

----..-CD.Irr

.-.

_,

4 ~

3:6

li 1.5
.SP

=..

loo

...,.''*:.:::_:::: ....................... .

0.5

............

:g

+-------.-------.-------.--------+ 0
0

10

20

30

40

Weeks after planting

Fig. 2 - Height and coll ar dimeter of micropropagated plant of
P fortuneii under irrigated (irr) and rain fed (rt) conditi ons

Nevertheless, they survived throughout the rainless

period from November to April. There are reports on
micropropagation of different species of Paulownia
1
including P.fortuneii , but the protocol standardi zed in
the present study enabled high rates of multipli cation
without an intervening callus stage, while the rooting
and hardening time was also reduced and provides
excellent quality saplings for field planting in a short
time.

Acknowledgement
This work was supported by AP-NL Project on
Micropropagation of multipurpose tree species and
their field evalu ation under farmers' conditions .

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YENKATESWARLU el a/.: MICROPROPAGATION OF PAULOWNIA

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