Indian 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

Central Research Institute for Dry land Agriculture, Santoshnagar, Saidabad P.O,Hyderabad 500059, Indi a Fax : 040-4531802; E-mail : vbandi @crida.ap.nic.in

Received 22 June 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 clonal plantation in Andhra Pradesh. A medium containing half-strength MS salts + BAP ( I mg/L) + sucrose (2%) produced optimum bud break in nodal explants. The same basal medium with reduced hormone level (0.5 mg/L) supported maximum multiplication of secondary cultures of P.fortuneii ( I :6 in 6 weeks). Specific treatments were tested to enhance thi s rate of multiplication. In one approach, five to six week o ld in vitro grown shoots were ratooned (cutting the main shoot at the bottom leav ing one node). The stumps (ratooned basal node) produced 2 to 3 axillary shoots, which grew into 4 to 5 nodes by 3 weeks; thus, prov id ing additional shoots from the sa me ex plant. T hi s prov ided 30% additional shoots in 4 cyc les. Secondly, reducing the light intensity to 1200 lux resulted in higher shoot e longation, i.e, formation of 8 nodes in 5 weeks with hea lthier shoots than the normal intensity of 3000 lux under which o nly 6 nodes were produced in 6 wee ks. /11 vitro­grown shoots could be successfully 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 house for 2 weeks and shifted to shade net for fu rther hardening. After one month, the plants could be success fully transplanted to field wi th 95% survival. Micropropagated plants showed an excellent growth in the fi eld attaining a height of 1.5 m and a collar diameter of 2 .8 em in 3 months.

Paulownia, popularly known as empress tree is native of Eastern Asia 1

• 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 of United States2

. Paulownia has many recognized species, but four species are important com mercially u (P. tom en rosa, P.fortunei i, P. kawakami and P. taiwaniana) . It is widely grown in temperate areas of Taiwan, China and Australia, but P. fortuneii has also been planted successfully in many tropical and subtropical areas of the world3

. P. fortuneii has been recogni zed as hav ing good potenti al for agroforestry in tropical zones of India as well4

• P.fortuneii has been introduced in India by the Forest Research Institute, Dehradun- in 1992, but not much attention has been paid on this species till recently5

. 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 one year it grows as a pole (8 feet) and useful timber can be obtained in 6 years under good management4

• The wood is strong yet light weight (16 pounds per cubic foot) making it ideal for manufacturing of crates, musical instruments and toys 1• Paulownia can be propagated through seeds, stem cuttings root suckers and ti ssue culture2

. 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 from root and shoot cuttings or rooted shoots from ti ssue culture6

. Root suckers offer a cost effect ive alternative to produce planting material under Indi an conditions. However, currently the planting stock in the country is inadequate to produce sufficient material by this method. Micropropagation 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 juvenile and mature expl ants2

·7

·8

. Even nodule cultures of P. taiwania11.a have been tried for automated micropropagation in China with high multiplication ratios9

. Micropropagation of P.fortuneii has been reported from shoot tips, nodal explants and leaves5

·7

·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 micropropaga ti on protocol has been reported for P. fortun eii with a significant enhancement of in vitro multiplication rate through a novel technique of in vitro ratooning.

VENKATESWARLU e/ a/.: MICROPROPAGATION OF PAULOWNIA 595

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 Paulownia 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 rinsing in mercuric chloride (0.1 %) for 4 min followed by repeated rinsi ng with sterile distilled water. Full strength and half strength MS 11 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/cm2 for 15 min . For each treatment, 12 explants were included. The cultures were incubated in a growth room at 28°C under 14 hr photoperiod with a light intensity of 3000 lux . The data on frequency and nature of response 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 lower than the explants from mature tree2

. Two explants 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 .

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 during July,1999 with a plant to plant spacing of 3 m. Saplings transpl anted during May, l999 were given protective irrigation 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 response was

596 INDIAN J EXP BIOL, JUN E 200 1

fo und from those collected from September till February . Among different media and hormone combinations tried, half-strength MS with BAP(J mg/L) + sucrose (2%) showed the best response (80%). Addition of auxins in the medium resulted in callus formati 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 .

with varying concentrations 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 multiplication have been reported from leaf and nodal explants of P. elongata, P. to/1/entosa, P. fo rtuneii, particularly when auxi ns have been used in the medium, but in all such cases the shoots ori ginated adventitiously through a callus Further ex periments on secondary multiplication

Medi a combinations

Table I- Response of primary cultures of P. j ortu11eii on di fferent medi a and hormone combin ations

Observations (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 unhealthy (yellowish) Ca llusi ng of total explant Ax ill ary buds sprouted; small shoot fo rmed but no further growth

MS(F) + BAP ( I mg/L) MS(F) + BAP (2 mg/L)

2 nodal length lateral shoot formed but stopped growth Small lateral shoot emerged, but fa iled to grow.

MS(H)+ BAP (0.1 mg/L) + NAA (0. 1 mg/L) MS(H) + BAP ( 1.0 mg/L)

Green callus formed Healthy shoot formed with 3-4 nodes.

MS(H) + BAP ( 1.0 mg/L) + Sucrose (20g/L) Healthy and excellent shoot developed which grew up to 4-5 nodes .

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

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

Treatment Length of the main shoot (em) after 6 weeks

Average no. of cultu rable nodes after 6 weeks

Light intensity (3000 lux) 6-7 Li ght in tensity ( 1200 lux) 8- 10 Shifting fro m low to high intensity after 8-9 2 weeks of inoculation Shifting from high to low intensity after 10-l l 2 weeks of inoculation

6 8 6

8

Table}- Effect of i11 l'itro culture of basal stalk remained after excision of shoot on the shoot multiplica ti on of P. j ortu11eii

[Culturable nodes obtained are from two ex plants placed in one culture vesse l]

Normal multipli cation In vitro ratooning

Mult ip li cation cyc le (6week)

0

2 3 4

No of culturable nodes

2 12 72

432 2592"

Multiplication cycle

(6 week)

0 I 2 3 4

No of culturable nodes from

normal cycle

2 12 72

432 2592"

"obt::tined in 24 weeks from 4 cyc les through normal multiplica ti on. bobtained in 12 weeks from 4 cycles th rough culturing of regenerated shoots.

No. of multiplication

cyc les for regenerted

shoots (3week)

0 I 2 3 4

o of culturable nodes (additional)

obtained from regenerated shoots

0 16 96

576 3456b

VENKATESWARLU e/ a/.: MICROPROPAGATION OF PAULOWNIA 597

Fig. 1- Stages in micropropagat ion of P fo rtuneii : A - Establishment of primary explant. B - in vitro multiplication, C - ex l'itro rooting. D - rooted plant let. E - Field transferred plant (2 month old)

phase6·12 However, Rao et a /8 have obtained 40 shoots

fro m each leaf explant over a fo ur month peri od through direct regeneration. The multiplication rate of 36 shoots per explant in 12 weeks obtained in the present study is considerably higher than reported earli er fo r other species of Paulownia.

Light in tens ity had a signi ficant impact on the growth of secondary cultures. Under 3000 lux light in tensity, the shoot e longati on was slow with short internodes and fo rmation of rosette type shoots. When the light intensity was reduced to 1200 lux, the internodes elongated w ith significantly more leaf area ex pansion and shoot thickness . This led to higher shoot length and more number of culturable nodes. As against 6 nodes produced per explant in 6 weeks, reduced light intensity resulted in 7-8 culturable nodes

in 5 weeks (Table 2) . However, initi al normal intensity fo r 2 weeks fo llowed by shi fti ng to low (1200 lux) intensity was more effective as compared to a shift fro m low to normal or continuous low. Continuous normal, however, was the least effecti ve. In other words, P. fo rtuneii cultures need reduced light intens ity fo r achieving higher multipli cation rate either continuously or at least 2-3 weeks in the multiplication cycle as compared to the light intensities required fo r other tree species .

Another approach fo llowed to achieve enhanced shoot production in secondary multiplicati on was in vitro shoot pro liferation. When the main shoot was excised leaving the lower most 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 the normal 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 earlier2

·5

·8 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 thickness (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 plants 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 expanded leaves. The ex vitro transplant success was 90%. Fully hardened plants when transferred to field showed 100% survival and rapid growth during the rainy season. On an average, plants transplanted during May , 1999 and prov ided tmgation have attained a height of 2.45m and collar diameter of 4.5cm in five months (Fig. 2). The growth ceased thereafter as the plants entered into dormancy. The other set of plants transplanted in the field during July, 1999 and raised under rainfed conditions have put up much less growth (height of 0 .68m and collar diam of 2cm). These plants also entered into dormancy within 2 months after planting.

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

2.5 ... . ... R.rr

.-. 2 _§, li 1.5 .SP .. = 1

0.5

----..-CD.Irr

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

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

•. ,.··''*:·.:::·_·:::: .................. ... .. . .. 0 +-------.-------.-------.--------+

0 10 20 30 40 Weeks after planting

6

5

4 e ~

3:6 loo

2 :g u

0

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

Nevertheless, they survived throughout the rainless period from November to April. There are reports on micropropagation of different species of Paulownia including P.fortuneii1

, but the protocol standardi zed in the present study enabled high rates of multiplication 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 evaluation under farmers' conditions .

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Biotechnology in Agriculture and Forestry.Trees II , Vol. 5 ed ited by Y P S Bajaj (Springer Verlag Berlin, Heidelberg, New York) 1989,359.

2 Burger D W, Lin L & Wu L, Rapid micropropagation of Paulownia tomentosa, Hart Sci 20-4( 1985) 760.

3 Yang J C, Ho C K, Chen J J & Chang S H, Paulownia X taiwaniana in Biotechnology in Agriculture and Forestry,Trees IV, Vol 35 edi ted by Y P S Bajaj (Springer­Verlag Berlin,Heidelberg,New York) 1996,269 .

4 Singh C & Arora Y K, Paulownia- the tree of the future , Indian Farm ( 1999) 15.

5 Chauhan J M S & Emmannuel C J S K, In vitro c lonal propagation of Paulownia fortun eii, Indian J For, 2 1-4( 1998) 327.

6 Bergmann B A & Moon H K, In vitro adventitious shoot production in Paulownia, Plant Cell Rep, 16(1997) 3 15.

7 Song S L, Sato T, Saito A.& Kihachiro 0, Meristematic cu lture of seven Paulownia species, J Jpn For Soc, 71-11(1989) 456.

8 Rao C D, Goh C-J & Kumar P P, High frequency adventitious shoot regeneration from excised leaves of Paulownia spp. cultured in vitro, Plallf Cell Rep 16( 1996) 204.

9 HoC K, Chen Z Z, Tsai J Y &Yang J C, Nodule cultures of Paulownia X taiwaniana, Taiwan J.For Sci, 12-1 ( 1997) 39.

10 Ho C K, Jacobs G & Donald D G M, Organo genetic

YENKATESWARLU el a/.: MICROPROPAGATION OF PAULOWNIA 599

capacities of different explants of four Paulownia species, Taiwan For Res lnst New Series. 9-4(1994) 397.

11 Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15

(1962) 473. 12 Marcotrigiano M & Stimart D P, In vitro organogenesis and

shoot proliferation of Paulownia tomentosa Steud. (Empress tree), Plant Sci Lett, 31 (1983) 303.