Plant Cell Tissue Organ Culture 6 (1986) 41-45 © Martinus Ni]hoffPublishers Dordrecht - Printed in the Netherlands

In vitro clonal propagation of Lagerstroemia flos.reginae Retz

41

JOHN PAILY and L. D'SOUZA*

Laboratory of Applied Biology, St Aloysius College, Mangalore 575 003, India

(Received 7 December 1984; in revised form and accepted 9 December 1985)

Key words: Lagerstroemia, ornamental tree, axillary bud, multiple shoots

Abstract. Multiple shoots were obtained from nodal segments of young and mature trees of Lagerstroemia flos-reginae Retz on MS medium with 7.50-20 mg/1 of benzyl amino purine. Rooting was achieved on transfer of the excised shoots to MS medium with 1 rag/1 of indole butyric acid. The plantlets have been successfully transferred to soil.

Introduction

Lagerstroemia flos-reginae Retz is considered to be one of the most beautiful trees of India valued for its flowers, t imber and medicinal properties [ I0 ] . Since it is a dry deciduous tree adapted to withstand moisture stress and its leaves are unpalatable for cattle, it has a good potential for afforestation of degraded areas. However as the percentage of germination of seeds is low and the tree does not respond to normal methods of vegetative propagation, large scale multiplication of this species is not easy. Tissue culture techniques have been successfully used for clonal propagation of several tree species that have poor seed set or poor germination or that cannot be normally multiplied by vegetative propagation [ 1 - 8 , 1 1 - 1 3 ] . The present paper deals with a

method for rapid multiplication of Lagerstroemia flos-reginae using tissue culture techniques.

Material and methods

Trees growing on the campus of St Aloysius College, Mangalore, were used for the experiment. One was about 4 years old that had not yet started flowering YNF (Young Non Flowering) and the other was a mature tree about 30 years old that has been flowering for several years MF (Mature Flowering). The YNF tree was pruned repeatedly to induce new shoots which were used as a source of explants. In case of MF trees new shoots produced in March and April just before the flowering season were used

*To whom correspondence should be addressed.

42

for explants. The explants consisted of a nodal segment with its two axillary buds, the leaves being cut at the base of the petiole. The explants were washed in running tap water, sterilized in a solution of 0.01% HgC12 and 0.01% Sodium lauryl sulphate for 7 -10 minutes followed by 3 washings with sterile tap water. The culture medium used was MS medium of Murashige and Skoog [9] with 0.8% Agar and various concentrations of benzyl amino purine, kinetin and zeatin for induction of shoots and napthalene acetic acid and indole butyric acid for rooting. The pH of the medium was adjusted to 5.6 using NaOH and HC1 before sterilization. The explants were cultured in 25 x 150-mm tubes at 28 -+ 2°C at 16 hours photoperiod of 1500 Lux from cool day light fluorescent lamps. Ten trials were done with each experiment and the experiments were repeated four times.

Results and discussion

Shoot proliferation on explants from YNF and MF trees depended on both the type and concentration of cytokinin employed. With zeatin ranging from 0.1 to 5.0mg/1, only single shoots developed from the axillary buds of both YNF and MF trees (Table 1). In YNF trees the maximum response was with 0.5 rag/l, with 90% of the buds producing single shoots within 3 weeks, whereas the maximum response in MF trees was obtained at 1.5 mg/1 with 50% of the buds producing single shoots in 5 weeks.

With kinetin supplied at 1-17.5 mg/1, only single shoots were produced from each bud in YNF and MF explants (Table 2). In YNF explants the maximum response was obtained at 7.5 mg/1; concentrations above 15 mg/1 caused death of the explants.

YNF explants produced single shoots with 0.5-5.0mg/1 BAP while MF explants gave rise to single shoots with 1-10 mg/1 of BAP (Table 3). However BAP supplied at 7.5-15 mg/1 induced multiple shoots in both type of ex- plants. The maximum response was obtained at 12.5 mg/1 at which 95% of the explants responded producing 20-30 shoots from each axillary bud within 5 weeks (Figure 1 a). In MF explants higher level of BAP i.e. 15 mg/l was needed to induce maximum multiple shoot production. Shoot produc- tion in MF explants was lower and the time required for shoot formation longer than in YNF explants (Table 3).

Excised shoots obtained from the above experiments were transferred to MS medium containing various concentrations of NAA and IBA for rooting. The most efficient rooting was obtained in response to 1.0mg/l of IBA. After two weeks 95% of the shoots produced 4 - 8 roots (Figure 1 b). NAA was less effective as rooting was obtained after 3 weeks and only 2 - 4 roots per shoot were produced (Table 4). The rooted shoots obtained from 1.0mg/l IBA treatment were transferred to a sterilized mixture of sand, soil and leaf mold in plastic bags in the ratio 1:1:1 and covered with beakers to prevent desication. The survival rate of the plantlets was 30%.

Table 1. Effect of zeatin on shoot development in vitro from axiUary buds non flowering and mature flowering trees of Lagerstroemia flos.reginae

43

of young

Conc mg/l

Young Non Flowering Trees (YNF) Mature Flowering Trees (MF)

% of No. of Time % of No. of Time explants shoots required explants shoots required responding per bud (weeks) responding per bud (weeks)

0.01 7 ± 9 a 1 4 - - 0.25 30 ± 14 1 3 15 ± 5 a 1 0.50 90 ± 8 1 3 20 ± 14 1 1.00 8 5 ± 5 1 3 2 0 ± 8 1 1.50 45 ± 5 1 4 50 ± 14 1 2.50 12 ± 5 1 5 17 +- 5 1 3.00 - - - 20 ± 8 1 5.00 - - - 7 ± 5 1

4 4 5 5 6 8 9

a: mean ± S.D. of 10 replicates

Table 2. Effect of kinetin on shoot development in vitro from axiUary buds non flowering and mature flowering tree~ of Lagerstroemia flos-reginae

of young

Conc mg/1

Young Non Flowering Trees (YNF) Mature Flowering Trees (MF)

% of No. of Time % of No. of Time explants shoots required explants shoots required responding per bud (weeks) responing per bud (weeks)

0.50 15 ± 7 a 1 2 - - 1.00 25 ± 10 1 4 12 -+ 5 a 1 7 2.50 50 ± 11 1 4 16 ± 5 1 7 5.00 80± 8 1 4 2 2 ± 9 1 7 7.50 65 ± 12 1 5 30 ± 8 1 8

12.50 22-+ 5 1 6 1 5 ± 5 1 9 15.00 12± 5 1 8 10-+2 1 9 17.50 - - 15 ± 7 1 9

a: mean ± S.D. of 10 replicates

Table 3. Effect of benzyl amino purine on shoot development in vitro from axillary bud of young non flowering and mature flowering trees of Lagerstroemia flos-reginae

Cone mg/1

Young Non Flowering Trees (YNF) Mature Flowering Trees (MF)

% of No. of Time % of No. of Time explants shoots requireed explants shoots required responding per bud (weeks) responding per bud (weeks)

0.50 25± 10 a 1 3 15+- 7 a 1 5 1.00 80 ± 14 1 3 20 +- 10 1 5 2.50 90-+ 8 1 3 40± 11 1 5 5.00 70+- 8 1 4 60± 8 1 5 7.50 20 ± 11 5 ± 0.8a5 25 ± 12 1 7

10.00 70± 8 9 -+3.1 4 15± 5 5 . 0 ± 2 . 7 a 5 12.50 95-+ 5 25 ±2.9 4 20± 8 7.5± 1.6 7 15.00 90± 8 22 ± 2.4 5 30± 11 13.5 ± 1.3 7 17.50 IS: 5 15 ±3.8 6 20± 8 13.0+-2.0 8 20.00 - - - 10 ± 5 10.0 -+ 2.8 8

a: mean + S.D. of 10 replicates

44

Figure 1. A. Development of multiple shoots from an axillary bud of l_~gerstroemia flos.reginae. B. Excised shoot showing root formation. C. A plantlet soon after potting. D. The same transferred to soil and developed into a healthy plant ten months later.

After two weeks the plantlets were transferred to pots containing unsterilized garden soil (Figure lc). The plants were kept for 3 weeks in the green house and then transferred to the field (Figure ld).

Acknowledgements

We thank the Mangalore Jesuit Educational Society for the facilities provided and Rovin Serrao and V. Arokiadoss for their technical assistance.

45

Table 4. Effect of NAA and IBA on rooting of micropropagated shoots of Lagerstroemia flos-reginae

% of Time Nature shoots required of

Auxin Conc mg/1 responding (weeks) response

NAA 0.25 90 +- 8.1 a 3 1-2 roots 0.50 95 ± 5.7 2 2 -4 roots 1.00 80 -+ 8.1 3 Tuft of spongy roots 2.00 95 -+ 5.7 2 callus formation

IBA 0.25 80 -+ 14.4 3 4 - 8 roots 0.50 90 +- 14.1 2 4 - 8 roots 1.00 95 -+ 5.7 1 4 - 8 roots 2.50 95 -+ 5.7 4 callus and tuft of

spongy roots

a: mean -+ S.D. of 10 replicates

Refe rences

1. Brown CL, Sommer HE (1982) VegeL, J e Propagation of Dicotyledenous trees. In: Bonga JM, Durzan DJ (eds) Tissue Culture in Forestry. Martinus Nijhoff/Dr W. Junk, The Hague, pp 109-149

2. Durzan DJ, Campbell R (1974) Prospect for mass propagation, production of improved stock of forest trees by cell and tissue culture. Can J For Res 4:151-174

3. Gupta PK, Nadgir AL, Mascarenhas AF, Jagnath V (1980) Tissue culture of forest trees: Clonal multiplication of Tectona grandis (Teak) by tissue culture. Plant Sci Lett 17:259-268

4. James OP (1979) Propagation in vitro of apple trees and 3ther woody fruit trees. Methods and application. Scientific Hortic 30~.44-48

5. Koblitz H, Koblitz D, Schmander H-P, Groger D (1983) Studies on plant tissue culture of the genus Cinchona L. In vitro mass propagation through meristem derived plants. Plant Cell Report 2:95-97

6. Kotwal MM, Gupta PK, Mascarenhas AF (1983) Rapid clonal multiplication of Sapium sebiferum Roxb. by tissue culture. Plant Cell Tissue and Organ Culture 2:133-139

7. Laxmi Sita G (1979) Rapid Clonal multiplication of Eucalyptus by multiple shoot production. Curr Sci 43:350-342

8. Mehra-Palta Asha, (1982) Clonal propagation of Eucalyptus by tissue culture. Plant Sci Lett 26 :1-11

9. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497

10. Randhawa MS (1979) The queen's flower or pride of India. In: Flowering Trees. National Book Trust India, New Delhi, pp 155-159

11. Rao AN, Lee SK (1982) Importance of tissue culture in tree propagation. Proc 5 th Intl C ong Plant Tissue & Cell Culture, pp 715-718

12. Rao PS, Bapat VA (1978) Vegetative propagation of Sandalwood through tissue culture. Can J Bot 56:1153-6