Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l^-1 N⁶-benzylaminopurine (BAP) and 0.5 mg l^-1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l^-1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO₃ or CoCl₂, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l^-1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

Etiolation as a pretreatment for in vitro establishment and multiplication of mature chestnut

Localized etiolation of branches in the crown of a 30-year-old chestnut tree produced plant material that responded much better to establishment and multiplication in vitro than unetiolated material, whose cultures were very difficult to maintain (response being measured in terms of the percentage of cultures established, the mean number of shoots formed per explant, the number of 8-mm segments per new shoot, the length of longest shoot in each culture, and the coefficient of multiplication). Only 22% of the initial explants from unetiolated material were successfully established, as against 79% for etiolated material, with similar differences between the coefficients of multiplication of the two lines in successive cultures. Accordingly, partial etiolation of branches is proposed as a suitable pretreatment for in vitro propagation of selected mature trees, when physiologically juvenile materials such as stump sprouts, epicormic shoots or root suckers are not available.     

Shoot regeneration in response to carbon source on internodal explants of Annona muricata L.

Adventitious shoot regeneration from internodal explants of mature plants of Annona muricata L. was obtained on Nitsch media. Meristems were induced with sorbitol as the sole carbon source supplemented with 2 mg l^–1 of benzylaminopurine and 0.5 mg l^–1 naphthaleneacetic acid. Adventitious shoots were developed only when the explants were transferred onto growth regulator-free media containing sucrose, galactose, or glucose. A hypothesis is proposed for the involvement of sorbitol in the induction and development of de novo shoots from internodal explants of mature trees of A. muricata.     

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 M of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.Abbreviations BA 6-benzyladenine - GD Gresshoff and Doy Medium - IBA indole-3-butyric acid     

Micropropagation of Elaeocarpus robustus Roxb.

Multiple shoots were obtained from shoot tips and nodal explants of 20-year-old trees of Elaeocarpus robustus on Murashige and Skoog's medium supplemented with 0.5 mg l^–1 each of BA and Kn. Explants taken from in vitro-proliferated shoots subsequently produced multiple shoots when cultured on the same basal medium containing 0.5 mg l^–1 each of BA and Kn. Repeated subculture resulted in rapid shoot multiplication at an average rate of 10 new shoots per subculture. The addition of CM (10%) and CH (100 mg l^–1) to the medium enhanced the number of shoots up to 20 per subculture and increased the length of shoots. In vitro-raised shoots were rooted on half-strength MS medium containing 1.0 mg l^–1 IBA and 0.5 mg l^–1 IAA. Following transplantation in the field 85% of the plantlets survived and grew uniformly. Received: 17 March 1995 / Revision received: 30 December 1997 / Accepted: 9 January 1998     

The Effect of Explant Type on the Establishment of Sitka Spruce [Picea sitchensis (Bong.) Carr.] in Culture

Five different types of shoot tip explant from three-year-oldand 13-year-old Sitka spruce trees were established on a rangeof media and shoot growth was recorded. While Schenk and Hildebrandtmedium gave the greatest shoot elongation, Webb and Street mediumproduced the healthiest-looking shoots. Poorest growth was recordedfor the explants cultured without the crown or any vasculartissues from the previous year's growth. [¹⁴C]sucrose tracerstudies indicated that poor uptake was not responsible for thereduced growth and it was suggested that damage due to inadequatewater uptake may be responsible. Results demonstrated that peelingthe protective scales from resting buds and trimming the shoottissues below the crown to a wedge shape and excluding the barkwas a reliable method of explant preparation for establishingmature and juvenile Sitka spruce in culture. Picea sitchensis, Sitka spruce, mature, juvenile, crown, [¹⁴C]sucrose, micropropagation     

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

Summary A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

Genotype- and age-dependent patterns of lignin and cellulose in regenerants derived from 80-year-old trees of black mulberry (<Emphasis Type="Italic">Morus nigra</Emphasis> L.)

Proliferating shoot cultures of black mulberry (Morus nigra), derived from axillary buds of two donor trees designated as Mn1 and Mn2, more than 80 years of age, were established in vitro. Subsequently, shoot-tips were used to induce both axillary and adventitious shoot regeneration following incubation on Murashige and Skoog medium containing 14 different treatments of various concentrations of plant growth regulators, including 6-benzyladenine (BA), thidiazuron (TDZ), and combinations of BA with indole-3-butyric acid (IBA) and TDZ with BA. The highest shoot proliferation of 5.3 shoots per explant for the Mn1 tree and 6.9 shoots per explant for the Mn2 tree were obtained when explants were incubated on a medium containing 0.5 mg l⁻¹ BA and 0.1 mg l⁻¹ IBA. The maximum frequency of adventitious rooting was similar for both genotypes. Changes in lignin and cellulose content, macromolecular properties of dioxane and Klason lignins, lignin monomer composition, and macromolecular properties of cellulose were determined in 1-year-old and 3 year-old micropropagated plants, as well as in the donor trees. Lignin and cellulose properties were significantly dependent on the genotype, the age and the mutual interaction of both these factors. The syringyl to guaiacyl weight ratio in lignin rose with the age of the micropropagated plants. Moreover, the tensile strength of wood in 1-year-old plants was supported by a high cellulose degree of polymerization. The highest polydispersity index of cellulose was detected in 3-year-old plants.     

Regeneration of `juvenile' plants of black plum,Syzygium cuminii Skeels,from nodal exp of mature trees

Nodal explants, excised from young shoots from mature trees of Syzygium cuminii, were cultured on MS medium supplemented with different concentrations of BA or kinetin. Among these, BA (0.5 or 1.0 mg l^–1) induced greening and opening of the incipient shoot buds, which however did not elongate. Elongation of the shoot buds was facilitated on MS medium with 1.0 mg l^–1 BA supplemented with casein hydrolysate (1.5 g l^–1) or glutamine (200 mg l^–1). Nodal explants (microcuttings), taken from shoots developed in vitro, also developed multiple shoots when cultured on MS with1 mg l^–1 BA. These explants did not require an additional supply of reduced nitrogen, for further normal development. Shoots developed from explants from mature trees and microcuttings were rooted by sub-culturing them on Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The plants that developed in vitro were planted in soil and were transferred to the field after an acclimatization period of 7–8 months. These plants have been thriving well for more than three years and have no apparent phenotypic aberrations.     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Improved organogenic capacity of shoot cultures from mature pedunculate oak trees through somatic embryogenesis as rejuvenation technique

Theoretically, complete rejuvenation of mature trees should occur through somatic embryogenesis, however, this has not been extensively studied. The main objective of the present study was to increase the efficiency of in vitro clonal propagation for mature Quercus robur (100–300 years old), by induction of somatic embryogenesis as rejuvenation step prior to establishment of shoot culture through micropropagation of somatic embryo-derived plantlets. Shoot culture lines of “mature” origin were established from epicormic shoots of two centenarian oak genotypes (Sainza and CR-0) and maintained by axillary shoot proliferation. Embryogenic lines were also initiated from epicormic leaf explants of the same genotypes and maintained by secondary somatic embryogenesis. Although the frequency of somatic embryo conversion into plantlets was low in pedunculate oak, shoot culture lines could be established and maintained by axillary branching from several germinated somatic embryos. For each genotype and shoot culture line of the two origins (mature tree and somatic plantlets), shoot multiplication rate and elongation as well as rooting ability parameters were compared. Compared with “mature-origin” shoot cultures and after more than one year propagation in vitro, shoot lines established from somatic plantlets produced a significantly higher proportion of elongated, rootable shoots (from 26.0–31.6 to 36.8–40.5%) with increased rooting ability (from 3.3–45.6% to 23.2–89.8%). In the case of 300-year-old Sainza genotype such a high organogenic capacity was similar to shoot cultures initiated from basal sprouts. Basal sprouts are considered as “mature” material that retains juvenile characteristics compared with epicormic shoots forced from crown branches. Somatic embryogenesis only slightly improved plant regeneration of shoot cultures from basal sprouts, thus validating their use as “juvenile control”. The present results provide evidence that some rejuvenation occurred during the process of somatic embryogenesis and resulted in improved shoot growth and rooting of somatic embryo-derived culture compared with “mature” shoot culture. The results reported in this study might be useful in embryogenic systems with low plant conversion rates. The proposed experimental model might also be useful in finding molecular markers of plant ontogeny.     

In vitro response of apical bud explants from mature trees of jackfruit (Artocarpus heterophyllus)

A tissue culture technique for rapid vegetative propagation of mature jackfruit trees using apical bud cultures has been developed. Shoot-tip cultures were established on MS medium with 5–10 mm explants dissected from terminal buds of new growth from trunk. After initial culture of bud explants, one- to two-node pieces were taken from the microshoots formed and used to proliferate further axillary shoots for multiplying and maintaining shoot cultures. Benzyladenine and kinetin (4.5–9.0 µM), either separately or together, supported shoot proliferation; higher concentrations of the cytokinins inhibited bud breaking and favoured callus formation at the explant bases. Bud explants taken from emerging trunk sprouts invariably produced clumps of multiple shoots, whereas buds obtained from actively growing top branches generally elongated to form a solitary shoot. November to January was the best season for initiation of cultures from field-grown trees. Shoots proliferated at the initial subcultures had mature morphology and were difficult-to-root. Shoots assumed to be juvenile-like developed at the later passages and could be rooted with 60–80% success using 1/2-MS salts and 10 µM of indolebutyric acid or naphthaleneacetic acid. Regenerated plantlets were transferred to the soil and about 50% survived.     

Establishment of juvenile-like shoot cultures and plantlets from 4–16 year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees

A method was developed for the establishment of shoot cultures from Douglas-fir trees selected for outstanding growth and form in a 12-year-old genetic test. Vegetative buds from the lower crown were sterilized and grafted in vitro onto juvenile clonal rootstock. The rootstocks were produced from adventitious buds induced on cotyledons, and were maintained through micropropagation. Buds that established grafts slowly elongated into shoots, which were harvested and multiplied through micropropagation. Grafts often grew several new shoots which in turn could be harvested. In 1987, 2830 buds were grafted from 18 superior trees. Twenty nine grafts (1%) produced shoots which established 11 of the 18 trees in culture. Their appearance and behavior in vitro became more juvenile over 1–3 years, as indicated by shoot and needle morphology, disappearance of episodic growth pattern, increase in multiplication rates, and ability of needles to produce adventitious buds.The five most prolific of the 11 clones were given a pre-rooting treatment and planted in soil under fog. The success of rooting and subsequent establishment in soil varied from 5 to 17% depending on clone. In contrast, trees multiplied in vitro for 1–2 years longer showed soil establishment rates from 8–60%. This technique allows establishment, multiplication, and maintenance in vitro of cultures from high value Douglas-fir genotypes. Such cultures may serve as a starting point for further research on rejuvenation and cloning.     

Rapid Micropropagation of Five Cultivars of Mulberry

Multiple shoots were initiated from nodal and shoot tip explants collected from mature trees of Morus alba L. cultivars Chinese White, Kokuso-27 and Ichinose, and M. multicaulis Perr. cultivars Goshoerami and Rokokuyaso after 2 weeks of culture. Nodal explants were more responsive than shoot tip explants. Murashige and Skoog basal medium was found to be most suitable medium and 6-benzylaminopurine was the most effective cytokinin for shoot induction. Explants collected between April and September evoked better response than the explants collected between October and March. Shoots were multiplied by transferring nodal explants excised from in vitro raised shoots onto a medium containing cytokinins. Sucrose was the most suitable carbon source examined for shoot multiplication. An increase in shoot multiplication rate was noticed upto 4 – 5 subcultures. Nodal explants rooted on an auxin-supplemented medium. The acclimatized plants were successfully transplanted in the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro multiplication and field establishment of Adhatoda beddomei C. B. Clarke,a rare medicinal plant

Clonal propagation of Adhatoda beddomei C.B. Clarke (Acanthaceae), a rare medicinal shrub, was achieved through callus-free axillary meristem proliferation from stem node explants of field-grown plants cultured in SH medium. Shoot multiplication was a function of cytokinin activity but sustained growth of the shoots was dependent on the synergistic effect with the auxin, IAA. An optimum number of 5–10 shoots per explant were obtained in 6 weeks using 3.0 mg.l^–1 BAP, 0.5 mg.l^–1 2-ip and 1.0 mg.l^–1 IAA, Upon subculture, vertical halves of the precultured node with the differentiated shoots yielded a larger aggregate number of shoots (23–27) than the uncut precultured node left intact (15–17). Shoot multiplication was rapid and consistent over prolonged periods when the hormonal concentrations were reduced to 1.0 mg.l^–1 BAP and 0.2 mg.l^–1 IAA during subculture, and reculture of the nodal explants derived from shoot cultures. Rooting of 3–5 cm shoots thus obtained was greatly accelerated in stationary liquid medium containing 0.2 mg.l^–1 IBA or IAA. Hardening of the rooted plantlets in the humidity chamber was essential for high frequency (95%) survival. Micropropagated plants established in the field flowered after fifteen months and were free from apparent defects in cytological, growth and flowering characteristics.Abbreviations SH Schenk and Hildebrandt (1972) basal medium - BAP 6-benzylaminopurine - 2-ip 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid     

Micropropagation of Eucalyptus tereticornis Smith.

Axillary shoot bud multiplication has been achieved in Eucalyptus tereticornis Smith. using explants from different regions of 8–10 years old elite trees, growing in the field. Results showed that addition of NAA at 0.1 mgl^-1 and BAP at 1.0 mgl^-1 to modified MS medium induced maximum number of shoot buds. For inducing axial growth in regenerated bud promordia, the hormone concentration of the medium was lowered. The addition of charcoal and gibberellic acid to the medium were beneficial. Rooting was best in Knop's medium containing 1.0 mgl^-1 IBA. The key factor in root induction was primarily a dark incubation for a short period. The percentage of both rooting of shoots and survival of the rooted shoots was 60–80.Continuous trials using explants from the elite trees throughout the year showed that the period between July–September was the best season for the explant source for rapid and increased multiplication of axillary buds. Phenolic exudation was also minimum at this period. The experiments were repeated using 50 populations from different plantations. It was observed that during culture, genotypically different populations responded differently in spite of optimal growth conditions.