PlantCell Reports
Plant Cell Reports (1993) 12:513-516
9 Springer-Verlag1993
Micropropagation of Madhuca Iongifolia (Koenig) MacBride var. latifolia Roxb. G.R. Rout and P. Das Plant Tissue Culture Laboratory, Regional Plant Resource Centre, Bhubaneswar
751015, Orissa, India
Received November 3, 1992/Revised version received April 26, 1993 - Communicated by G. C. Phillips
Abstract
Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca Iongifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/I N6benzyladenine (BA) singly or in combinatiobn with 1naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/I) after 18 d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil. Abbreviations: BA, N6-benzyladenine; KN, kinetin; ADS, adenine sulphate; IBA, indole-3-butyric acid; IAA, indole3-acetic acid; NAA, 1-naphthaleneacetic acid; MS, Murashigeand Skoog (1962)medium. Introduction
Micropropagation of tree species offers a rapid means of producing clonal planting stock for afforestation, woody biomass production and conservation of elite and rare germplasm (Bongaand Durzan 1982; Bajaj 1986). In general, woody taxa are difficult to regenerate under in vitro conditions. Some success, however, has been achieved in a few woody tree species (Dhawan 1989). Madhuca Iongifolia var. latifolia (Sapotaceae), a large deciduous tree, grows abundantly in India. It is a multipurpose species of high economic value. The kernel is a source of oil for the soap industry, lubricating greases and as a batching oil in the jute industry. Madhuca oil also has emollient properties and is used to treat skin diseases, rheumatism and headache. It is an edible oil and is used for cooking purposes by tribals. The flowers are a rich source of sugar and contain appreciable amounts of vitamins and calcium. The flower is used in the preparation of distilled liquors, potable spirits and Correspondence to: P. Das
vinegar. The flower also shows anti-bacterial activity against E. coil. The oil cake is used as a manure and possesses insecticidal properties. The leaves are astringent and are used as embrocations. It is also used as cattle feed. The timber is used for construction purposes (The Wealth of india 1988). Conventional methods of propagation of Madhuca, sexual as well as vegetative, are beset with many problems that restrict their multiplication on a largescale. Propagation through seed is unreliable because of disease and pest problems, short viability and heavy rains during the seeding season in the natural habitat. In vitro techniques have great potential for mass propagation of elite genotypes of this important forest species for reforestation programmes. This paper describes in vitro propagation of M. Iongifolia var. latifolia from apical and axilary meristems. Material and Methods Plant material and explant source. Seeds of Madhuca Iongifolia var. latifolia were collected from an elite tree growing in the State Botanical Garden at Bhubaneswar, India. Uniform and healthy seeds were rinsed in 70% (v/v) alcohol for 1 min, sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 15 rain, and washed 4-5 times in sterile distilled water. The seeds were aseptically germinated on Murashige and Skoog (1962) basal agar medium, kept in 16 h light/ 8 h dark photoperiod under light intensity of 50 uE m -2 s -1 provided by white cool fluorescent lamps (Philips, India) at 25 -+ 2o C with 55% relative humidity. The apical and axillary meristems (1-2 mm) derived from 10-d old in vitro grown seedlings were used as explants. Culture medium. Murashige and Skoog (MS) basal medium was variously supplemented with BA, KN, ADS, IAA, NAA or IBA. All media were adjusted to pH 5.7 using 0.1 N HCI or 0.1 N NaOH before autoclaving. The medium was gelled with 0.8% (w/v) agar (Qualigen, India). Routinely, 20 ml of molten medium was dispensed into a culture tube (25 x 150 mm) and plugged with nonabsorbant cotton wrapped in one layer of cheese cloth. The culture tubes were steam sterilized at 1.06 kg cm "2 for 15 min. Culture conditions. All cultures were maintained under the same incubation conditions used for seed germination. For each
514 treatment 20 cultures were raised and each experiment was conducted at least two times. The cultures were examined periodically and the morphological changes were recorded on the basis of visual observations. The effects of different treatments were quantified as the mean number of multiple shoots/culture. In v/tro raised shoots measuring 2-3 cm growing on medium containing 1.0 mg/I BA + 0.1 mg/I NAAwere excised and cultured on 1/2 MS or 1/2 MS supplemented with 0.5, 1.0 1.5 or 2.0 mg/I each of IAA, NAAand IBAfor induction of rooting. Plantlets with well developed roots were removed from the culture tube. After thorough washing of the roots in tap water, the plantlets were transferred to 4.5 cm earthen pots containing sterile garden soil : sand : cowdung (2:1:1) (v/v) and kept in a polythene house with high humidity to reduce desciccation. After 30 d, the plants were repotted in larger containers with garden soil and maintained in the nursery under natural light.
Results and Discussion
Bud growth and multiplication The present investigation was carried out to explore the morphogenic potential of Madhuca Iongifolia var. latifolia. Of the various cytokinins tested, BA was the most effective for inducing bud break in apical and axillary meristems (Table 1). The maximum bud proliferation was observed in nodal explants cultured on MS supplemented with 1.0 mg/I BA (Fig. A). Within 4 weeks of culture, the axillary meristems elongated up to 2-3 cm in height (Fig.B). Similar results were reported in Prunus serotina (Tricoli et aL 1985). Prolonged culture on induction medium resulted in the drying of the tip and extensive callusing at the basal end of the elongated shoots. At concentrations of BA above 1.0 mg/I, proliferation and growth of axillary shoots was inhibited. AxUlary meristems were more responsive than apical meristems. Similar results were reported in Syzygium cuminii (Yadav et aL 1990). Both for apical and axillary meristems, BA was found to be more effective for bud proliferation than were KN and ADS (0.5 mg/I to 3.0 mg/I). BA was then tested in combination with auxins (NAA, IAA or IBA at 0.1 mg/I ) to enhance the rate of shoot multiplication (Table 2). BA (1.0 mg/I) with NAA (0.1 rag/I) proved to be the most effective treatment for promoting shoot multiplication (3-4 shoots per nodal explant, each shoot having 2-3 nodes) in 4-week cultures (Fig. C). Similar observations were recorded in Eucalyptus species (LeRoux and VanStaden 1991) and in Dalbergia latifolia (RaghavaSwamy et al. 1992). Although more than 5 shoots were formed at higher concentrations of NAA supplemented with BA, growth was inhibited and only 1-2 shoots would elongate; some produced compact 9callus at the base of the explants. Similar results were obtained in Eucalyptus (Sita 1981) and Pterocarpus santalinus (Sita 1992). Initially the callus wasyellowish, but it turned dark brown within 3-4 weeks.
Table 1. Effect of cytokinins on response of apical and axillary meristems derived from 10 d old seedlings of M. Iongifolia var. latifolia. The cultures were scored 4 weeks after inoculation. The data represent the mean of two independent experiments.
MS + cytokinins (mg/I) BA
KN
Apical meristem
ADS
0.5 1.0 2.0 3.0 0,5 1.0 2.0 3.0 0.5 1.0 2.0 3,0
Axillary meristem
Mean no. of shoots/culture _+ S.E.*
1.70 2,05 1.25 1.20 1.35 1.70 1.65 1.30 1.15 1.35 1.40 i.50
-+ 0.14 -+ 0.80 -+ 0.10 _+ 0.90 + 0.10 -+ 0.14 -+ 0.15 +- 0.10 -+ 0.10 + 0.10 -+ 0.I4 -+ 0.13
2.10 3.20 1.95 1.50 1.30 2.00 2.05 1.45 1.25 1.55 1.60 1.85
-+ 0.14 -+ 0.20 -+ 0.16 -+ 0.13 +- 0.10 -+ 0.20 -+ 0.21 +- 0.16 -+ 0.10 --- 0.13 -+ 0.13 -+ 0.12
* 20 replicates/treatment ; repeated twice
Rooting of in vitro formed shoots Of the three auxins tested, only IBA induced rooting. The optimum concentration of those tested was 1.0 mg/I of IBA (Table 3). Each of the rooted shoots formed 3-4 roots in the culture containing 1.0 mg/I IBA after 18 d of incubation (Fig. D). At 1.5 mg/I IBA, rooting was reduced and basal callus formation was observed. The above results are in agreement with those of Kapoor and Gupta (1986) and Rao and Bapat (1978). A combination of NAA and IBA stimulated formation of callus at the cut end of excised shoot and inhibited root initiation. Root initiation in excised shoots was also inhibited in the medium containing NAA, IAA and IBA each at 1.0 mg/l. When the rooted shoots were transferred to 1/2 MS basal medium devoid of growth regulators, fast elongation of roots was noted within 2 weeks of culture. This phenomenon has been reported in other woody species (James and Thrubon 1981; Lane 1978 ; Yadav et aL 1990).
Figs. A & B. Proliferation of axillary buds of M. /ongifolia var. latifolia from a nodal segment cultured On MS + 1.0 mg/I BA
515 Table 2. Effect of BA in combination with auxins on response of apical meristems (axillary meristems shown within parentheses) derived form 10-d old seedlings ofM. Iongifolia var. latifolia. The cultures were scored after 4 weeks and the data represent the mean of two independent experiments.
Table 3 . Effect of auxins on in vitro rooting of isolated shoots of
M.Iongifolia var. latifolia Auxins
Concentration (mg/I)
Days of incubation
Mean percentage of rooting -+ S.E.*
IAA
0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0
30 30 30 30 28 18 30 30 30 30 30 30
a a a a 50.0 -+ 2.35 88.33 -+ 1.36 26.66 -+ 1.36a a a a a a
0.5 (each) 1.0 (each)
30 30
a a
Mean no. of shoot/explant _+ S.E. * Auxins (mg/I)
IAA 0.1 NAA 0.1 IBA 0.1 * a
BA (mg/I) IBA 0.5
1.0
1.0 -+ 0.0 (1.0 + 0.0)
1.15 +- 0.13 (1.25 ,+ 0.09)
1.37 ,+ 0.09 1.55 -+ 0.13 a (1.35 ,+ 0.10) (1.85 ,+ 0.12) a
1.0 + 0.0 (1.0 + 0.0)
1.22 -+ 0.21 (1.66 ,+ 0.11)
1.45 -+ 0.08 1.62 _+ 0.11 a (1.92 -+ 0.13) (2.05 ,+ 0.24) a
1.0 _+ 0.0 2.30 -+ 0.10 (1.15 -+ 0.08) (3.65 -+ 0.16)
1.85 ,+ 0.15 1.45 _+ 0.23 a (2.25 + 0.10) (1.75 +- 0.10) a
1.0 ,+ 0.0 (1.0 ,+ 0.0)
1.62 -+ 0.46 (2.21 ,+ 0.08)
1.5
2.0
1.92 -+ 0.13 1.42 _+ 0.07 a (2.30 ,+ 0.09) (1.87 -+ 0.15) a
20 replicates/treatment;repeated twice. Shoots with less than 0.5 cm length were not counted callusing at the basal end
NAA
IAA + IBA
]AA + 0.5 (each) 30 IBA + 1.0 (each) 30 NAA , repeated twice; 20 replicates/treatment a callusing at the basal end
Transfer of plantlets into soil Plantlets grown and rooted in vitro were washed thoroughly to remove the adhering gel, transplante to 4.5 cm earthen containers on sterile pot-compost containing garden soil : sand : cowdung (2:1:1) and kept under high humidityfor acclimatization. Only 46 out of 65 (which constitute about 70%) of the rooted plantlets were established in the soil. As the plantlets became acclimatized to the natural enviornment, they exhibited normal leaf development and no morphological variation was noticed (Fig.E).
Fig. C. Formation of multiple shoots regenerated from axillary buds of a nodal explant cultured on MS + 1.0 m g / I BA + 0.1 m g / I NAA after 4 weeks culture Fig. D. Rooting of shoots on 1/2 MS + 1.0 mg/IBA + 2% sucrose after 18 d of culture
This study illustrates a successful micropropagation system for Madhuca Iongifolia var. latifolia, a multipurpose tree species. Such methods should be helpful in the mass production of this economically important woody plant. Acknowledgements. The authors wish to thank the Department of Biotechnology, Government of India for financial assistance and Department of Forest & Environment, Govt. of Orissa for other necessary facilities for this study,
References
Fig. E. In vitro raised Madhuca/ongifo/ia var. latifolia plant, 30 d after transplanting to soil
Bajaj YPS (1986) Biotechnology in agriculture and forestry, Vol 1, Trees I. Springer, Berlin Bonga JM, Durzan DJ (1982) Tissue culture in forestry, Martinus Nijhoff, The Hague Dhawan V (1989) In : Dhawan V (ed) Application of biotechnology in forestry and horticulture. Plenum Publishing Crop. New York, pp 285-296 James DJ, Thurbon, tJ (1989) J Hortic Sci 56:15-20 Kapoor S, Gupta SC (1986) Plant Cell Tiss Org Cult 7:263-268 Lane WD (1978) Plant Sci Lett 13:285
516 LeRoux JJ, VanStaden J (1991)Hort Science 26:199-200 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 RaghavaSwamy BV, Himabindhu K, Sita GL (1992) Plant Cell Report 11 : 126-131 Rao PS Bapat VA (1978) Can J Bot 56: 1153- 1156 Sita GL (1981) In: Rao AN (ed) Proc COSTED Symp on Tissue culture of economically important plants, Singapore, pp 62 Sita GL (1992) Curr Science 62:532-535 The Wealth of India (1988) Vol VI, pp 207-216 Tricoli DM, Maynard CA, Drew AP (1985) Forest Science 31:201-208 Yadav U, MadanL, Jaiswal VS (1990)Plant Cell Tiss Org Cult21: 87-92
Plant Cell Reports (1993) 12:513-516
9 Springer-Verlag1993
Micropropagation of Madhuca Iongifolia (Koenig) MacBride var. latifolia Roxb. G.R. Rout and P. Das Plant Tissue Culture Laboratory, Regional Plant Resource Centre, Bhubaneswar
751015, Orissa, India
Received November 3, 1992/Revised version received April 26, 1993 - Communicated by G. C. Phillips
Abstract
Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca Iongifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/I N6benzyladenine (BA) singly or in combinatiobn with 1naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/I) after 18 d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil. Abbreviations: BA, N6-benzyladenine; KN, kinetin; ADS, adenine sulphate; IBA, indole-3-butyric acid; IAA, indole3-acetic acid; NAA, 1-naphthaleneacetic acid; MS, Murashigeand Skoog (1962)medium. Introduction
Micropropagation of tree species offers a rapid means of producing clonal planting stock for afforestation, woody biomass production and conservation of elite and rare germplasm (Bongaand Durzan 1982; Bajaj 1986). In general, woody taxa are difficult to regenerate under in vitro conditions. Some success, however, has been achieved in a few woody tree species (Dhawan 1989). Madhuca Iongifolia var. latifolia (Sapotaceae), a large deciduous tree, grows abundantly in India. It is a multipurpose species of high economic value. The kernel is a source of oil for the soap industry, lubricating greases and as a batching oil in the jute industry. Madhuca oil also has emollient properties and is used to treat skin diseases, rheumatism and headache. It is an edible oil and is used for cooking purposes by tribals. The flowers are a rich source of sugar and contain appreciable amounts of vitamins and calcium. The flower is used in the preparation of distilled liquors, potable spirits and Correspondence to: P. Das
vinegar. The flower also shows anti-bacterial activity against E. coil. The oil cake is used as a manure and possesses insecticidal properties. The leaves are astringent and are used as embrocations. It is also used as cattle feed. The timber is used for construction purposes (The Wealth of india 1988). Conventional methods of propagation of Madhuca, sexual as well as vegetative, are beset with many problems that restrict their multiplication on a largescale. Propagation through seed is unreliable because of disease and pest problems, short viability and heavy rains during the seeding season in the natural habitat. In vitro techniques have great potential for mass propagation of elite genotypes of this important forest species for reforestation programmes. This paper describes in vitro propagation of M. Iongifolia var. latifolia from apical and axilary meristems. Material and Methods Plant material and explant source. Seeds of Madhuca Iongifolia var. latifolia were collected from an elite tree growing in the State Botanical Garden at Bhubaneswar, India. Uniform and healthy seeds were rinsed in 70% (v/v) alcohol for 1 min, sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 15 rain, and washed 4-5 times in sterile distilled water. The seeds were aseptically germinated on Murashige and Skoog (1962) basal agar medium, kept in 16 h light/ 8 h dark photoperiod under light intensity of 50 uE m -2 s -1 provided by white cool fluorescent lamps (Philips, India) at 25 -+ 2o C with 55% relative humidity. The apical and axillary meristems (1-2 mm) derived from 10-d old in vitro grown seedlings were used as explants. Culture medium. Murashige and Skoog (MS) basal medium was variously supplemented with BA, KN, ADS, IAA, NAA or IBA. All media were adjusted to pH 5.7 using 0.1 N HCI or 0.1 N NaOH before autoclaving. The medium was gelled with 0.8% (w/v) agar (Qualigen, India). Routinely, 20 ml of molten medium was dispensed into a culture tube (25 x 150 mm) and plugged with nonabsorbant cotton wrapped in one layer of cheese cloth. The culture tubes were steam sterilized at 1.06 kg cm "2 for 15 min. Culture conditions. All cultures were maintained under the same incubation conditions used for seed germination. For each
514 treatment 20 cultures were raised and each experiment was conducted at least two times. The cultures were examined periodically and the morphological changes were recorded on the basis of visual observations. The effects of different treatments were quantified as the mean number of multiple shoots/culture. In v/tro raised shoots measuring 2-3 cm growing on medium containing 1.0 mg/I BA + 0.1 mg/I NAAwere excised and cultured on 1/2 MS or 1/2 MS supplemented with 0.5, 1.0 1.5 or 2.0 mg/I each of IAA, NAAand IBAfor induction of rooting. Plantlets with well developed roots were removed from the culture tube. After thorough washing of the roots in tap water, the plantlets were transferred to 4.5 cm earthen pots containing sterile garden soil : sand : cowdung (2:1:1) (v/v) and kept in a polythene house with high humidity to reduce desciccation. After 30 d, the plants were repotted in larger containers with garden soil and maintained in the nursery under natural light.
Results and Discussion
Bud growth and multiplication The present investigation was carried out to explore the morphogenic potential of Madhuca Iongifolia var. latifolia. Of the various cytokinins tested, BA was the most effective for inducing bud break in apical and axillary meristems (Table 1). The maximum bud proliferation was observed in nodal explants cultured on MS supplemented with 1.0 mg/I BA (Fig. A). Within 4 weeks of culture, the axillary meristems elongated up to 2-3 cm in height (Fig.B). Similar results were reported in Prunus serotina (Tricoli et aL 1985). Prolonged culture on induction medium resulted in the drying of the tip and extensive callusing at the basal end of the elongated shoots. At concentrations of BA above 1.0 mg/I, proliferation and growth of axillary shoots was inhibited. AxUlary meristems were more responsive than apical meristems. Similar results were reported in Syzygium cuminii (Yadav et aL 1990). Both for apical and axillary meristems, BA was found to be more effective for bud proliferation than were KN and ADS (0.5 mg/I to 3.0 mg/I). BA was then tested in combination with auxins (NAA, IAA or IBA at 0.1 mg/I ) to enhance the rate of shoot multiplication (Table 2). BA (1.0 mg/I) with NAA (0.1 rag/I) proved to be the most effective treatment for promoting shoot multiplication (3-4 shoots per nodal explant, each shoot having 2-3 nodes) in 4-week cultures (Fig. C). Similar observations were recorded in Eucalyptus species (LeRoux and VanStaden 1991) and in Dalbergia latifolia (RaghavaSwamy et al. 1992). Although more than 5 shoots were formed at higher concentrations of NAA supplemented with BA, growth was inhibited and only 1-2 shoots would elongate; some produced compact 9callus at the base of the explants. Similar results were obtained in Eucalyptus (Sita 1981) and Pterocarpus santalinus (Sita 1992). Initially the callus wasyellowish, but it turned dark brown within 3-4 weeks.
Table 1. Effect of cytokinins on response of apical and axillary meristems derived from 10 d old seedlings of M. Iongifolia var. latifolia. The cultures were scored 4 weeks after inoculation. The data represent the mean of two independent experiments.
MS + cytokinins (mg/I) BA
KN
Apical meristem
ADS
0.5 1.0 2.0 3.0 0,5 1.0 2.0 3.0 0.5 1.0 2.0 3,0
Axillary meristem
Mean no. of shoots/culture _+ S.E.*
1.70 2,05 1.25 1.20 1.35 1.70 1.65 1.30 1.15 1.35 1.40 i.50
-+ 0.14 -+ 0.80 -+ 0.10 _+ 0.90 + 0.10 -+ 0.14 -+ 0.15 +- 0.10 -+ 0.10 + 0.10 -+ 0.I4 -+ 0.13
2.10 3.20 1.95 1.50 1.30 2.00 2.05 1.45 1.25 1.55 1.60 1.85
-+ 0.14 -+ 0.20 -+ 0.16 -+ 0.13 +- 0.10 -+ 0.20 -+ 0.21 +- 0.16 -+ 0.10 --- 0.13 -+ 0.13 -+ 0.12
* 20 replicates/treatment ; repeated twice
Rooting of in vitro formed shoots Of the three auxins tested, only IBA induced rooting. The optimum concentration of those tested was 1.0 mg/I of IBA (Table 3). Each of the rooted shoots formed 3-4 roots in the culture containing 1.0 mg/I IBA after 18 d of incubation (Fig. D). At 1.5 mg/I IBA, rooting was reduced and basal callus formation was observed. The above results are in agreement with those of Kapoor and Gupta (1986) and Rao and Bapat (1978). A combination of NAA and IBA stimulated formation of callus at the cut end of excised shoot and inhibited root initiation. Root initiation in excised shoots was also inhibited in the medium containing NAA, IAA and IBA each at 1.0 mg/l. When the rooted shoots were transferred to 1/2 MS basal medium devoid of growth regulators, fast elongation of roots was noted within 2 weeks of culture. This phenomenon has been reported in other woody species (James and Thrubon 1981; Lane 1978 ; Yadav et aL 1990).
Figs. A & B. Proliferation of axillary buds of M. /ongifolia var. latifolia from a nodal segment cultured On MS + 1.0 mg/I BA
515 Table 2. Effect of BA in combination with auxins on response of apical meristems (axillary meristems shown within parentheses) derived form 10-d old seedlings ofM. Iongifolia var. latifolia. The cultures were scored after 4 weeks and the data represent the mean of two independent experiments.
Table 3 . Effect of auxins on in vitro rooting of isolated shoots of
M.Iongifolia var. latifolia Auxins
Concentration (mg/I)
Days of incubation
Mean percentage of rooting -+ S.E.*
IAA
0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0
30 30 30 30 28 18 30 30 30 30 30 30
a a a a 50.0 -+ 2.35 88.33 -+ 1.36 26.66 -+ 1.36a a a a a a
0.5 (each) 1.0 (each)
30 30
a a
Mean no. of shoot/explant _+ S.E. * Auxins (mg/I)
IAA 0.1 NAA 0.1 IBA 0.1 * a
BA (mg/I) IBA 0.5
1.0
1.0 -+ 0.0 (1.0 + 0.0)
1.15 +- 0.13 (1.25 ,+ 0.09)
1.37 ,+ 0.09 1.55 -+ 0.13 a (1.35 ,+ 0.10) (1.85 ,+ 0.12) a
1.0 + 0.0 (1.0 + 0.0)
1.22 -+ 0.21 (1.66 ,+ 0.11)
1.45 -+ 0.08 1.62 _+ 0.11 a (1.92 -+ 0.13) (2.05 ,+ 0.24) a
1.0 _+ 0.0 2.30 -+ 0.10 (1.15 -+ 0.08) (3.65 -+ 0.16)
1.85 ,+ 0.15 1.45 _+ 0.23 a (2.25 + 0.10) (1.75 +- 0.10) a
1.0 ,+ 0.0 (1.0 ,+ 0.0)
1.62 -+ 0.46 (2.21 ,+ 0.08)
1.5
2.0
1.92 -+ 0.13 1.42 _+ 0.07 a (2.30 ,+ 0.09) (1.87 -+ 0.15) a
20 replicates/treatment;repeated twice. Shoots with less than 0.5 cm length were not counted callusing at the basal end
NAA
IAA + IBA
]AA + 0.5 (each) 30 IBA + 1.0 (each) 30 NAA , repeated twice; 20 replicates/treatment a callusing at the basal end
Transfer of plantlets into soil Plantlets grown and rooted in vitro were washed thoroughly to remove the adhering gel, transplante to 4.5 cm earthen containers on sterile pot-compost containing garden soil : sand : cowdung (2:1:1) and kept under high humidityfor acclimatization. Only 46 out of 65 (which constitute about 70%) of the rooted plantlets were established in the soil. As the plantlets became acclimatized to the natural enviornment, they exhibited normal leaf development and no morphological variation was noticed (Fig.E).
Fig. C. Formation of multiple shoots regenerated from axillary buds of a nodal explant cultured on MS + 1.0 m g / I BA + 0.1 m g / I NAA after 4 weeks culture Fig. D. Rooting of shoots on 1/2 MS + 1.0 mg/IBA + 2% sucrose after 18 d of culture
This study illustrates a successful micropropagation system for Madhuca Iongifolia var. latifolia, a multipurpose tree species. Such methods should be helpful in the mass production of this economically important woody plant. Acknowledgements. The authors wish to thank the Department of Biotechnology, Government of India for financial assistance and Department of Forest & Environment, Govt. of Orissa for other necessary facilities for this study,
References
Fig. E. In vitro raised Madhuca/ongifo/ia var. latifolia plant, 30 d after transplanting to soil
Bajaj YPS (1986) Biotechnology in agriculture and forestry, Vol 1, Trees I. Springer, Berlin Bonga JM, Durzan DJ (1982) Tissue culture in forestry, Martinus Nijhoff, The Hague Dhawan V (1989) In : Dhawan V (ed) Application of biotechnology in forestry and horticulture. Plenum Publishing Crop. New York, pp 285-296 James DJ, Thurbon, tJ (1989) J Hortic Sci 56:15-20 Kapoor S, Gupta SC (1986) Plant Cell Tiss Org Cult 7:263-268 Lane WD (1978) Plant Sci Lett 13:285
516 LeRoux JJ, VanStaden J (1991)Hort Science 26:199-200 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 RaghavaSwamy BV, Himabindhu K, Sita GL (1992) Plant Cell Report 11 : 126-131 Rao PS Bapat VA (1978) Can J Bot 56: 1153- 1156 Sita GL (1981) In: Rao AN (ed) Proc COSTED Symp on Tissue culture of economically important plants, Singapore, pp 62 Sita GL (1992) Curr Science 62:532-535 The Wealth of India (1988) Vol VI, pp 207-216 Tricoli DM, Maynard CA, Drew AP (1985) Forest Science 31:201-208 Yadav U, MadanL, Jaiswal VS (1990)Plant Cell Tiss Org Cult21: 87-92
