IntroductionNeem (Azadirachta indica) is an economically important

tropical tree species. It is esteemed for its uses in timber, medicine, pesticides and

insecticides since the ancient past not only in India but also in many countries of the Middle east, Africa, Caribbean and Latin America.

Neem seeds lose viability in a fortnight of storage.Furthermore, the seed and seedling vigour, diminishes well

before the seeds start losing viability leading to abnormalities. This has led to problems in plant genetic resource conservation, establishment of large-scale plantations, and species trials (Gamene et al.,1996).

Clonal planting offers the advantage of phenotypic selection and uniformity, and potentially rapid multiplication.

Plantlet regeneration has been reported in neem from leaf explants of a mature tree (Ramesh and Padhya, 1990; Eeswara et al., 1998) and from axillary buds of 2- to 5-year old plants (Joshi and Thengane, 1996).

Cont… Similarly , plantlet regeneration via callus was achieved from

different explants of 7-days-old seedlings (Salvi et al., 2001) and from anther tissue (Gautam et al., 1993).

We reported a bud culture method to clone the mature neem tree using basal-sprouts.

In addition, a comparison of its response and that of crown branch explants of another mature tree and of a 1-year-old plant was made.

MATERIALS AND METHODSCOLLECTION OF EXPLANTS Nodal explants of neem were collected from crown branches of a

15-years-old tree, basal-sprouts of another 15-year-old tree and a single juvenile (1-year-old) plant at the botanical garden of Pt. Ravishankar Shukla University, Raipur (India).

The three types of explants were collected from the new growth in April 2002.

STERILIZATION After removing the leaves, the shoots were thoroughly washed in

running tap water; disinfected in 0.1% (w/v) aqueous mercuric chloride for 10min and rinsed 3-4 times in sterile distilled water.

Each nodal segment with a single axillary bud was cut to 1.5cm in length and placed aseptically on MS (1962) medium with BA and PVP.

Sucrose was used as carbon source.

Cont… The media were adjusted to pH 5.6 with 1N NaOH and sterilized

by autoclaving for 20min at 15 psi pressure at 121 C. After inoculation, the cultures were incubated at 27 C under

12/12 (day/night) photoperiod with light intensity provided by cool white fluorescent tubes.

SHOOT BUD ESTABLISHMENT AND ROOTING EXPERIMENTS It were carried out in 25 150 mm glass tubes containing 15ml

medium each, and 300ml polypropylene jars containing 30ml medium each were used for shoot proliferation studies.

The bud break percentage, number and length of microshoots per explant; and number of nodes per microshoot were recorded after 30days.

After 30 days, the microshoots, which had elongated from the three types of explants, were excised and each node with a single axillary bud was excised and used for shoot proliferation.

The excised nodes were placed in 300ml polypropylene cylindrical jars containing 30ml medium.

The effect of basal media on shoot proliferation was investigated by placing the nodes on MS as well as on DKW (1984) media with BA.

For further proliferation, nodes with a single axillary bud were again excised, and placed on DKW with BA for 45 days.

Cont… Five nodes were placed in each culture jar. The number of shoots production by each nodes was recorded

after 45 days. This procedure was repeated at 45 day intervals to study the

shoot production potential of the nodes. For rooting of microshoots, half-strength DKW medium with IBA

or NAA, was used. 3 to 4 cmlong microshoots were excised and placed vertically on

the medium. Rooted microshoots were removed from the culture vessels,

washed thoroughly to remove the nutrient medium and transplanted to pots containing autoclaved fine grained black soil:sand (1:3 by volume).

These pots were placed at 30 2 C and 90% RH for 2 months.

(a) (b)

STATISTICAL ANALYSIS In all experiments, 10 explants were taken in each treatment, and

experiment was repeated 3 times. The shoot length, shoot number and node number were analyzed

by analysis of variance (ANOVA).

RESULTS Nodal explants from crown branches and basal-sprouts of mature

neem trees released phenolics into the culture medium from cut ends and eventually died.

This problem of leaching was checked by addition of PVP to the explant establishment medium.

In the case of cultures of the 1-year-old neem plant, phenolic leaching was not observed.

Axillary shoots elongation from the basal-sprout and seeding explants placed on MS medium with BA.

In tree crown explants, bud break was recorded on this medium, but the shoots did not elongate properly (data not shown).

Microshoots that elongated from the original explants were excised and their nodes were used for further shoot production.

Cont… Although MS medium was generally used in previous reports on

plantlet regeneration of neem (Gautam et al., 1993; Joshi and thengane, 1996; Eeswara et al., 1998; Salvi et al.; 2001), we have compared it with another high salt medium DKW (both containing BA), for shoot proliferation from basal-sprout explants.

DKW is more concerntrated in inorganic nutrients than MS and it contains L-glutamine.

DKW medium was significant better than MS with respect to the shoot number and node number.

The average shoot number was significant (p< 0.0001) larger in basal-sprout explants on DKW (Figure 1a) than in those on MS (2 v.s. 1).

Consequently, the number of nodes per new shoot were also higher in DKW as compared to in MS (6.6 0.4 v.s. 3.5 0.25) at p<0.0001.

Therefore, DKW with BA was used in subsequent subcultures. On this medium, shoot explants of crown branch origin did not

elongate and subsequently died after their third subculture.

Cont… However, shoots from basal-sprouts and juvenile sources proliferation

rapidly, by repeated subculturing at 45-days intervals.(table 1). The shoot number showed uniformity through five subcultures derived

from juvenile as well as basal-sprouts. However, shoot length and node numbers were significantly greater in

subcultures of juvenile origin than in those of basal-sprouts. Microshoots from crown branch explants did not elongate, hence they

were not available for rooting studies. Microshoots from basal-sprouts and juvenile explants did not root,

when placed on half DKW medium without auxin. In the presence of IBA, 90% (180/200) of the shoots from basal sprouts

(figure 1b) and (200/200) of the shoots of juvenile origin formed roots. Half strength DKW with NAA alone or in combination with IBA

induced profuse callusing at the base of microshoots.

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