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VENKATESWARLU e/ a/.: MICROPROPAGATION OF PAULOWNIA 595

Successful ex vitro rooting and field performance oftransplanted plants has also been reported.

Materials and MethodsCollection a nd establishment of primar y

exp lants Actively growing juvenile shoots fromaxillary branches of field grown mature tree ofPaulo wn ia fortuneii (Seem.) Hemsley . were collectedlocally during the months of April, 1999 toFebruary,2000 at monthly intervals u p to August, 1999and bimonthly intervals thereafter as the source ofprimary explant. The mother plant was selected froma plantation established in a farmer 's field in theKarimnagar district of Andhra Pradesh using clonalpl anting material of Paulownia fortuneii (clone I)

imported in the form of plug s (small hardened greenplantlets in cell-trays). The shoot segments werethoroughly washed with tween-80 solution and cutinto 0.5 to em single nodal segments, surfacesterilized by rin sing in mercuric chloride (0.1 ) for 4min followed by repeated rin si ng with sterile distilledwater. Full strength and half strength MS 11 mediacontaining 2% sucrose solidified with 0.8 agar (pH5 .8) and supplemented with different combinations ofauxins and cytokinins were used for the experiments.The media were sterilized by autoclaving at 1 1kg/cm 2 for 15 min . For each treatment, 12 explantswere included. The cultures were incubated in agrowth room at 28°C under 14 hr photoperiod with alight intensity of 3000 lux . The data on frequency andnature of respon se was recorded after 4 and 6 weeksof culture.

n vitro multipli ca t i o n Based on the response ofprimary cultures, axillary shoots of 3-5cm in lengthwere use d for further multiplication . Single nodeswere cut and placed in half strength MS medium withreduced hormone levels (0.1, 0.5 , 0.75 and 1.0 mg/L)as hormone requirement for secondary multiplicationof Paulownia has been reported to be significantlylower than th e explants from mature tree

2 Two

exp lant s were placed in each culture vessel. Fromeach explant 2 to 3 axillary shoots emerged initiallybut only one or two could grow eventually intohealthy shoots of 6-8cm length in six weeks.

Red uced Light int ensit y T o enhance the rate ofmultiplication, the cultures were incubated at twolight intensitie s of 3000 and 1200 lux by regulatingthe number of fluorescent tubes. Twenty replications(c ulture bottles ) were includ ed for each treatment andtwo explants were inoculated in each vessel. Theshoo t length and total number of culturable nodeswere determined at the end of 6 weeks of incubation .

n vitro shoot proliferation In this approach ,fully grown shoots comprising 6 nodes fromsecondary cultures were cut (with a specially designed

surgical scissors having a 2 em bent tip) and used forsubculture leaving the 2 basal nodes culture forfurther incubation. Within 3 weeks, 2 -3 axillarybranches grew from these stumps. Such regrownshoots were healthy and had the same leaf size andstem thickness as the normally cultured nod alexplants within 3 weeks as against 6 weeks requir edfor a normal subcultured explant . These stalks ofshoots were either subcultured again or rooted just asthe normal ones.

Rooting and hardening - n vitro grown microshoots of 3-4 inches length with 4-5 nodes were usedfor rooting ex vitro. The cultures were gently remov edfrom the bottles, basal portions were cut and dipped inIBA (800 mg/L) solution for 10 min . These weretransferred to a rooting medium (soilrite; an equalvolume mixture of vermiculite and cocopeat) filled inplastic trays at the rate of 60 plants per tray. The tr ayswere placed in a poly tunnel to maintain 90-95 RH .All the shoots produced excellent root system by 7-8days. The rooted shoots were then transfen ed topolybags (15 x 10 em) filled with garden soil. Th ebags were placed in the mist chamber under agradually reducing humidity regime from 90 to 55 RH for 2 weeks. Thereafter, the plants were shifted t o

shade house for secondary hardening and kept for 4weeks under ambient temperature and humidit yconditions.

Field transf er and eva luat ion Fully harden edplants of about 10 em of height were transplanted inthe field for studying their survival and growth. About120 plants were transplanted in pits (45 X 45 X 45em) dug in a loamy sand soil (pH 7.5 , OC: 0.45 ,total N: 0.047 ) on two dates viz. 20 plants duringMay , l999 and 100 plants durin g July,1999 with aplant to plant spacing of 3 m Saplings transpl ant edduring May , l999 were given protective irri gation

twice a week (7Liplant) till the beginning of themonsoon, where as the plants in the field were grownsolely on the rainfall (290 mm received in 23 rainydays) . The height and collar diameter of the plant swere recorded at 8 week intervals.

Results and iscussion

Explants from field grown trees could beestablished successfully on MS medium containing Img/L of BAP (Table . Buds collected during Jun eshowe d maximum sprouting followed by thosecollected in May, April and July. No respon se was

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VE NKATES WARL U e / a / . : MI CROPROPAGATION OF PAULOWN IA 597

Fig. 1- Stag es in micr opropa gat ion o f rtun e ii : A - Establishm ent o f pr imary exp lant. B - i n v t r o multiplic ation, C - ex l i t roroo ting. D - root ed p lant le t. E - Fie ld tran sfe rred plant 2 month o ld)

phase 6·12 However, Rao e t a /8 have ob tain ed 40 shoo tsfro m eac h leaf ex pl a nt over a fo ur m o nth p e ri odthr o ug h dir ec t r ege ne ra tio n . Th e multipli ca tio n r a te of36 s hoo ts pe r ex pl a nt in 12 w ee ks o bt a in ed in th eprese nt s tud y is con s idera bl y h ig he r th a n re por tedea rli er fo r o the r s pec ies of P a ul w n ia.

Light in te ns ity had a s ig ni fica nt imp ac t o n th eg row th of seco nd ary c ultur es. U nd er 3000 lu x lig h tin te ns ity , th e s hoo t e lo nga ti o n was s low w ith sho rtint e rn odes a nd fo rm a tio n of ros e tte ty pe s hoo ts . Wh e nthe li ght int e ns ity wa s red uced to 1200 lu x , th eint e rn o des e lo nga ted w ith s ig nifi cantly m o re lea f areaex pansio n and shoo t thi ckn ess . Thi s led to hi g he rshoo t l e ng th a nd m o re numb e r o f cultur abl e nodes. A saga in st 6 n odes produ ce d p e r e xpl a nt in 6 w ee ks,redu ce d light int ens ity res ult ed in 7- 8 c ultur a bl e nodes

in 5 wee ks Ta bl e 2) . Howeve r , initi al normalint e ns ity fo r 2 wee ks fo llow e d by s hi fti ng to l o w12 00 lux ) int e ns ity was m o re ef fec t ive as co m pared

to a s hift fro m l o w t o no rm al or co ntinu o us lo w .Co ntinuou s nor m a l, howeve r, was th e least effecti ve .In o th e r wo rd s, P . rtun e ii c ultur es nee d r edu cedlig ht int e ns ity fo r ac hi ev in g hi g her multipli catio n ratee ith e r co ntinu o us ly o r a t least 2-3 wee ks in themultipli ca tio n cy cl e as co mp ared to th e lig htint e ns itie s requir ed fo r o th er tree spec ies .

An o th e r a pp roac h fo llow e d to ac hi eve en han cedshoot pr oducti o n in seco nda ry multiplic a ti o n wa s nvitr o sho o t pr o li f e ra tio n. Wh e n th e ma in shoo t wasexc ise d lea vin g th e lo we r m os t n o de, it l ed to theg ro wth of 2-3 la te ra l sho ot s f ro m ea ch s tum ppr odu c in g nea rl y 8 nodes in 3 wee ks a t th e rate of 4

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598 INDIAN J EXP BIOL. JUNE 20 0 1

nod al segments from each lateral shoot. In otherwords, by ax illary shoot regeneration 30 moreshoots could be obtained in half the time as comparedto th e norm al multiplication cycle (Table 3). Shootregeneration was also tried for second time (on thesa me explant), but the regrowth was poor and lateralshoo ts were not healthy enough either for subcultureor rooting. The medium also started drying after fir stcyc le of shoot regeneration. The res ults thereforeindicated that in vi tro shoot regeneration could be auseful m ethod for obtaining enhanced multiplicationQf Paulownia, but it can be done only once.

The shoo ts could be rooted succe ss f ully both invitr o and ex vitr o with equal effeciency. However, thesur vival during primary hardening was 80 with invitro rooting w hile it was more than 95 with ex vitromethod. W e followed only ex vitr o rooting due tobetter root formation and short protocol. This protocolis more s imple and efficie nt as compared to in vitroroot ing, potting and field tran sfe r method reportedea rli er 2·5·8 With IBA (800 mg/L) treatment for 10 min,98 of the shoot s produced excellent roots in 8-10days . App lication of hormone through chalk coatingdid not have any advantage over dipping . Theplant lets co ntinued to grow while rooting, withco nside rable leaf expansion, increa se in height andste m thi ckn ess ( Fig. I A-E). Rooted plants when

transferred to polyb ags and continued in hoods with95 humidity for 48hr, fo llow ed by gradual reductionshowed better surviv a l (98 ) while hardening, ascompare d to tho se tran sfe rred o ut s id e within the mi s tcha mb er on open benche s. Bas ed on repe a tedobse rvat ions by u s Paul ow ni a pl a nts were foundhighly susce ptibl e to excess wetness o n leaves at thetime of primary ha rde nin g, th e refo re a n optimumhumidity may be m aint a ined .

A t the end of 30 d ays of seco nd ary hard e ning in theshade (50 ), the plant s grew up to 6 inches with 6 -8full y ex panded leaves. The ex vi tro tra nsplant succe ss

was 90 . Fully harden ed plant s when transferred tofield show ed 100 survival and rapid g rowth duringthe rainy seaso n. On an average, pl a nt s tra nsplant eddurin g May , 1999 and pr ov ided tm ga tion h aveattained a hei g ht of 2.45m and collar diameter of4.5cm in fiv e month s (Fig. 2). Th e grow th ceasedth ereaf ter as the pl ants entered into dor mancy. Th eothe r se t of pl a nts tran splanted in the field duringJul y, 1999 and rai se d under rainfed conditions haveput up much l ess growth (height of 0 .68 m and co llardi a m of 2cm). The se plant s also entered intodorm ancy within 2 month s a fter planting.

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6

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Fig. Height and co llar dim e te r of mic ropro pagated plant ofP fortuneii und e r irri ga ted (irr) and rain fed (rt) co nditi ons

Nevertheless, they survived through o ut th e rainless

period from No ve mber to April. Th ere a re r e port s onmi cropropagation of different specie s of Paul owniaincluding P.fortun ei i 1 but th e protoc ol s tand ardi zed inthe present study enab led high rates of multipli cationwithout an intervening callus s tage, while th e rootingand hardening time was also reduc ed and provid esexce llent quality sa plings for field pl anting in a shorttime .

AcknowledgementThis work was s upport ed by AP-NL Pr oject on

Micropropag ation of multipurpose tree species and

their field ev a lu ation under farmers' conditions .

ReferencesI B urge r D W , Empress tree P aulownia toment osa) in

Biotechnology in Agriculture and Fore stry .Trees II , Vol. 5ed ited by Y P S Baja j (Springer Ve r lag Berlin, Heidelb e rg,New York) 19 89,359.

2 Burger D W , Lin L & Wu L, Rapid micropropagation ofPa ul ow ni a tomentosa , Har t Sci 20-4( 1985) 760.

3 Yang J C, Ho C K, Che n J J & C ha ng S H, Paul ow nia Xta iw aniana i n Biote chn ology in g ri cultur e andFo res try,Trees IV , Vol 35 edi ted by Y P S Bajaj (SpringerVe rla g Berlin,Heidelberg,New York) 1996 ,269 .

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

5 Chauhan J M S & Emma nnu el C J S K , n vitr o c lonalpro pa ga tio n of P a ul ow nia fortun eii , Indi an J For , 2 1-4( 1998)32 7 .

6 Bergmann B A & M oo n H K, n vit ro adve ntiti o us shoo tprod uc tio n in Paulo wnia, Plant Cell Rep, 16(1997) 3 15.

7 So ng S L , Sato T , Saito A.& Kih ac hir o 0 , M er is tematiccu lture of seve n Paulownia species, J Jpn For Soc, 71-11(1989 ) 456.

8 Rao C D, Goh C -J & Kumar P P, Hi g h freque ncy adventiti o usshoo t reg e nera tion from exc ise d leaves of Pa ul ownia spp.cultured in vitr o, Plallf Cell Rep 16( 199 6) 204.

9 H oC K, Chen Z Z , Tsai J Y &Yang J C, Nodule cu ltur es ofPaulownia X tai wan ia na , Tai wa n J.For Sci, 12-1 ( 1997 ) 39.

10 Ho C K, Jacobs G & Do na ld D G M, Organo ge neti c

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ca pa c itie s of diff ere nt ex plant s of four Paulownia species ,Taiwan F r Res ln st New Series. 9-4 1994) 397.

11 Murashige T koog F A revised medium for rapid growthand bioassays with tob acco tis sue cultures, Physiol Plant 15

1962) 473.12 Marcotrigiano M Stimart D P, n vitro organogenesis and

shoot proliferation of Paulownia tomentosa Steud. E mpr esstree), Plant Sci Lett 31 1983) 303.