In vitro embryo germination and proliferation of pistachio (Pistacia vera L.)

In vitro development of isolated embryos and axillary bud proliferation were studied in Pistacia vera L. Different regulator-free nutrient media were compared to determine the effects of the mineral solution, agar and sucrose concentrations on seedling development from mature embryos. Nutrient-rich MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15, 473–479] and DKW [Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19, 507–509] mineral solutions were more efficient for the development of aerial parts than nutrient-poor KN [Knop, W., 1884. Bereitung einer concentrierten nährstofflosung für pflanzen. Landwersuhssat 30, 292–294] and WT [Withe, P.R., 1936. Plant tissue cultures. Bot. Rev. 2, 419–437] solutions. Reducing the agar concentration enhanced fresh matter production and balanced seedling development. When tested at different concentrations, sucrose was found to orient mature embryo development, with the best results obtained at concentrations of 2–4%, whereas high concentrations (6 and 12%) mainly inhibited elongation of the aerial parts. Plantlets obtained previously from in vitro cultured embryos were propagated by axillary budding. High bud proliferation (six shoots per explant) was achieved when using 17.8 μM benzyladenine (BA) combined with 0.5 μM indole-3-butyric acid (IBA). The addition of 2.9 μM gibberellic acid (GA₃) to the propagation medium did not improve axillary shoot yields and on average, media with GA₃ produced 2.3–2.6 elongated stems per cultured explant. Shoots were rooted in vitro in half-strength MS medium containing 12.3 μM IBA.     

Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

Micropropagation of Nolina recurvata Hemsl.: β-Cyclodextrin effects on rooting

Nolina recurvata Hemsl. is a very decorative indoor plant but difficult to micropropagate vegetatively. In vitro cuttings failed to induce adventitious rooting. Investigations for a rapid micropropagation using β-cyclodextrin added to the rooting medium has solved the problem. Rooted N. recurvata plantlets were obtained after successive stages of various culture media.Initiation and in vitro multiplication of this plant was possible with lateral buds cultured in Murashige and Skoog medium supplemented with 4.45 μmol of BA and 0.5 μmol of IBA. The number of axillary shoots by explant obtained was 6.In vitro rooting was obtained in MS medium (1/2 strength of minerals salts) supplemented with β-cyclodextrin. This substance, at 1.76 mmol associated with 5 μmol of IBA, improved quality and the rooting rate by 100%.     

In vitro response of pistachio nodal explants to silver nitrate

The effect of silver nitrate on shoot differentiation and shoot growth was examined in order to improve the regeneration efficiency of pistachio (Pistacia vera L. cv. Kirmizi) in vitro. Nodal explants of in vitro-grown seedlings were used to test various concentrations and combinations of 6-benzyladenine (BA), kinetin (KIN), gibberellic acid (GA₃) and silver nitrate (AgNO₃). Addition of AgNO₃ up to 48.0 μM to the culture medium improved the regeneration frequency and shoot growth, and reduced basal callus formation in all regenerated explants. The highest regeneration frequency (100%) was recorded on Murashige and Skoog (MS) medium containing 9.0 μM BA, 0.2 μM GA₃ and 24.0 or 48.0 μM AgNO₃ in combination. The best proliferation response in terms of both shoot formation and low callus production was obtained in the medium containing a combination of 9.0 μM BA, 0.2 μM GA₃ and 12.0 μM AgNO₃. Regenerated shoots, coming from three cycles of subculturing in proliferation media, were rooted in half-strength MS medium containing 12.0 μM indole-3-butyric acid (IBA). Well rooted plantlets were acclimatized and eventually established in peat and perlite. The development and optimization of an effective micropropagation protocol that is presented in this paper can give an important contribution to improve the quality of pistachio plants and, as a consequence, of orchard production in Middle East countries.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars

Viable shoot cultures and weaned plants were obtained from cultured apical meristems with 10 Buddleia cultivars giving viabilities of 32–72%. The number of shoots produced, the micropropagation rate and the root number produced in vitro was higher in meristem derived shoots compared to those derived from shoot-tips. The subsequent growth rate of meristem derived plants, in the greenhouse, was also greater. The number of roots produced by conventional cuttings collected from meristem derived plants was significantly higher than in cuttings which were collected from plants derived from shoot-tips or from the original stock plants.Endogenous bacteria were not detected in either shoot cultures derived from meristems or in 10-week-old weaned plants derived from meristems whereas those derived from shoot-tips showed the presence of endogenous bacteria when sterilized explants were cultured on nutrient agar or on tryptic soy broth.Factors affecting adventitious bud and shoot production in leaf and internode explants was determined for ‘Lochinch’, ‘Border Beauty’, ‘Ile de France’ and ‘Pink Delight’ using meristem derived shoot cultures. Adventitious shoots appeared after 4 weeks of culture, in both types of explant when cultured on MS supplemented with 0.5–5.0 μM TDZ. The highest percentage regeneration was achieved from bisected internode explants cultured on 0.5 μM TDZ, with 93–100% regeneration among the cultivars whereas BA was less effective. The best response was obtained using 5.0 μM TDZ which gave over 10–11 shoots per explant in all bisected explants for all cultivars.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

In vitro germination and plantlet establishment of Labisia pumila (Bl.) F. Vill.

In vitro seeds germination and plantlet establishment of Labisia pumila were studied in this report. The seeds obtained from the mature fruits of L. pumila were sterilized and cultured on Murashige and Skoog (MS) solid media supplemented with 1–3 μM of 6-benzylaminopurine (BAP) and 3% (w/v) sucrose. The presence of BAP in the medium significantly affects seeds germination. High percentage of seeds germination (up to 90%) was successfully achieved after 2 weeks of culture on medium supplemented with 2 μM BAP. Up to 70% of explants produced shoots through direct regeneration from newly emerged epicotyls after 5 weeks of culture. The average of 8.1 ± 1.0 shoots per explant obtained on media treated with 2 μM BAP. Seedlings were further transferred to growth media fortified with different types of cytokinin. Result observed after 12 weeks showed that medium supplemented with 1 μM zeatin (ZEA) promote the highest growth with an average of 2.9 ± 1.0 cm shoot length and 7.7 ± 3.2 leaves per explant after 12 weeks. In addition, medium added with 2 μM BAP and supplemented with 3–4% (w/v) of sucrose promote the best growth i.e., 3.0 ± 0.6 shoots per explant, 2.27 ± 0.2 cm length and 4.3 ± 0.5 leaves per explant.     

Comparison of shoot induction ability of different explants in herbaceous peony (Paeonia lactiflora Pall.)

Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA₃. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg L⁻¹ of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg L⁻¹ TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA₃ was beneficial for shoot and stem elongation.     

Significance of explant preparation and sizing in Aloe vera L.—A highly efficient method for in vitro multiple shoot induction

The present study was carried out to assess the effect of explant preparation and sizing for in vitro micropropagation of Aloe vera L. The stem nodal explants and shoot tips were cultured on modified Murashige and Skoog's medium (1962) supplemented with different concentrations of 6-benzylaminopurine (BA), kinetin (KIN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) either singly or in combination. The best media composition was found to be MS medium supplemented with IAA (11.42 μM), IBA (9.8 μM) and BA (8.88 μM). The explants were divided into 2 sets, with and without ensheathing leaf base. Explant sizing, pruning and retention of mother tissue was highly significant in induction of multiple shoots and roots. The stem nodal explants with leaf base performed much better than those without such covering. A very high number of shoots and roots grew from these explants. The rooted plantlets were successfully acclimatized and transferred to the green house conditions and finally to field conditions.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

An investigation on the effect of different plant growth regulating compounds in in vitro shoot tip and node culture of lemon seedlings

The possible application of some less commonly used in vitro growth regulating compounds is outlined. A number of treatments were applied to determine the best way of inducing in vitro shoot proliferation and rooting on a modified Driver–Kuniyuki [HortScience 19 (1984) 507] basal medium of lemon (Citrus limon (L.) Burm, f. cv. Interdonato) seedlings. 6-Benzyladenine (BA) alone (1, 2 and 4 mg l⁻¹) and in combination with either orange juice (10%, v/v), silver nitrate (3 mg l⁻¹), gibberellic acid (GA₃) (0.1 mg l⁻¹ at the establishment stage and 0.5 mg l⁻¹ at all combinations during the proliferation stage) or abscisic acid (ABA) (0.2 mg l⁻¹ only at the establishment stage) were used to stimulate shoot formation during the establishment and the proliferation stage. The combination of BA with ABA gave a high rate of shoot formation, while GA₃ and silver nitrate enhanced shoot elongation. When these shoots were transferred to the rooting stage, the effect of application of two different auxins (indole-3-butyric acid (IBA) and α-napthaleneacetic acid) was examined, as well as different methods of application (auxin added to the basal medium and auxin application by dipping the base of the explant in auxin solution). Dipping the base of the explants in a 50% ethanol solution of IBA at 1000 mg l⁻¹ for 5 s resulted in 80% rooting with subsequent 90% survival of these explants, during acclimatization under mist.     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

Silver nitrate and adenine sulphate induced high regeneration frequency in the recalcitrant plant Cosmos bipinnatus using cotyledon explants

Plant regeneration ability was studied in the medicinal-ornamental plant, Cosmos bipinnatus ‘Sonata white’, which is a dicotyledonous recalcitrant plant to shoot induction. Cotyledons were used as sources of explants to investigate plant regeneration. High frequency of direct shoot induction was obtained when BA (5 mg/l) and AgNO₃ (5 mg/l) were used in combination with 20 mg/l adenine sulphate (73.8%) in Murashige and Skoog (MS) medium. The highest shoot number per explant (5.7) was induced on MS medium supplemented with 5 mg/l BA, 5 mg/l AgNO₃, and 40 mg/l adenine. Eight week-old shoots were transferred to root induction media containing MS and half-strength MS medium with different concentration of IBA. The highest rate of root induction (70.8%) was obtained on half-strength MS medium with 1.5 mg/l IBA within four weeks. The plantlets were transferred to pot and kept in the greenhouse condition. Seventy percent of the plantlets successfully acclimatised.

Abbreviations: BA, 6-benzylaminopurine; IBA, Indole-3-butyric acid; MS, Murashige and Skoog; PGRs, plant growth regulators.     

Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple

Plantlets were obtained from usually dormant axillary buds, excised from the crown of pineapple (Ananas comosus L. Merr.) and grown in culture. Multiple shoots arose from single buds grown on Murashige and Skoog medium supplemented with auxins and kinetin. Shaking culture-flasks during growth increased the number of multiple shoots formed, when compared with stationary liquid cultures. Leaf explants excised from in vitro plantlets developed into a callus capable of plantlet regeneration. Subjecting developing buds to surgical segmentation also resulted in multiple shoot formation. Such shoots, when excised and grown on Murashige and Skoog medium supplemented with auxins, developed roots and grew into complete plantlets capable of being grown in soil.     

Influence of medium composition and vessel ventilation on in vitro propagation of Phillyrea latifolia L.

Micropropagation of Phillyrea latifolia L. a wild species present in Mediterranean coastal areas having drought and salt tolerance was performed using explants from adult plants. Shoots were induced from nodal explants on the Rugini’s initial medium (IM). Then these were proliferated on either Rugini olive medium (OM) or Linsmaier and Skoog (LS) medium, each supplemented with 2.22 μM 6-benzylaminopurine (BA) or 4.56 μM zeatin (Z). Rooting (66.1±11%) was induced on shoots grown in perlite soaked with half-strength Rugini olive proliferation medium (OMr) containing 2.69 μM α-naphthaleneacetic acid (NAA) and 160 mg l⁻¹ putrescine. Both shoot multiplication and rooting were performed using Magenta^® GA-7 (Sigma) vessels either non-permeable or permeable to gas exchanges. Contamination (about 40%) was observed during the first five passages notwithstanding the addition of cefotaxime to the culture medium, but a high proliferation rate (90%) of explants provided enough healthy plant material. The highest shoot proliferation was observed on LS medium and zeatin whereas the presence of the ventilated filters reduced fresh weight of explants growing on LS media and did not affect shoot growth on OM media. During rooting, the use of ventilated vessels in comparison with the closed ones enhanced development of roots, and doubled the dry weight of plantlets. The vessel ventilation combined with the artificial substrate (perlite) was beneficial for in vitro acclimatization of rooted Phillyrea plantlets.     

Aluminium-induced effects on growth,morphogenesis and oxidative stress reactions in in vitro cultures of quince

The effects of Al³⁺ [supplied as Al₂(SO₄)₃·18H₂O] addition to culture media (pH 4.0) on growth, morphogenesis (in leaf explants), and oxidative stress reactions in in vitro cultures of ‘BA 29’ quince were investigated. Aluminium (Al 0.5 mM) strongly inhibited shoot growth in the proliferation and rooting phases (Al 2.2 mM), reduced shoot proliferation (Al 1.1 mM), and induced tissue browning. Superoxide dismutase (SOD) activity was increased in shoot cultures supplemented with 2 mM Al. Malondialdehyde (MDA) content of shoots was strongly increased by Al during proliferation (starting from Al 1.7 mM) and rooting (already at Al 1.1 mM), thus serving as a good ‘marker’ for Al toxicity. Even a low concentration of Al (0.5 mM) in the shoot induction medium was found to inhibit shoot regeneration. When standard (Al 0) shoot induction medium was used, leaf explant growth was only reduced by 2.2 mM Al in the subsequent growth phases. Following a preliminary selection for their growth on Al-enriched media, 82 potentially Al-tolerant quince somaclones were selected for further trials.     

The Incorporation of Growth Hormones in Seed Dressings

Summary

The consequences of using ex vitro, single-node explants from different topophysical positions in chrysanthemum (Chrysanthemum grandiflorum /Ramat./ Kitam) were determined. In particular, how explant topophysis affected the rate of propagation, which is important for the successful micropropagation of chrysanthemum. Uniform shoots of five cultivars of chrysanthemum, cultured in vitro, were each divided into three equal zones: distal, central, and proximal. Two single-node explants were isolated from each zone and cultured on MS medium without any added growth regulators. After 10 weeks of culture, 50% of the shoots that had developed from axillary buds on each single-node explant were excised and measurements were taken in order to compare those shoots that had developed from explants from the different topophysical zones. The remaining shoots were sub-cultured on rooting medium. After 4 weeks, the numbers of roots per plantlet, and the total fresh weight (FW) of roots were recorded. The cultivars fell into two groups. ‘Lady Amber’, ‘Lady Orange’, and ‘Lady Vitroflora’ explants were topophysis-dependent, while ‘Lady Bronze’ and ‘Lady Rosy’ explants were topophysis-independent. For the three topophysis-dependent cultivars, the propagation rate, growth rate, shoot length, internode length, single leaf weight, and total plantlet FW values were highest for those shoots derived from the central and proximal zones. Topophysis failed to affect the number of leaves per shoot or the number of days between the appearance of two successive leaves. The effects of topophysis on the number of roots per plantlet and on root FW were inconsistent. The unequal growth of chrysanthemum plantlets during in vitro micropropagation can be an effect of topophysis, and this phenomenon is cultivar-specific in chrysanthemum.     

Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants,and protocorm-like bodies

In vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly priced commercial cut flower cultivars through direct organogenesis from in vitro derived foliar explants was established. Rapid clonal propagation was achieved by subsequent induction of protocorm-like bodies (PLBs) and its conversion to shoots. No significant differences were observed in the induction of direct shoots, shoot multiplication, PLBs formation and subsequent shoot development and rooting of shoots between the two cultivars. Leaf explants from flower stalk node derived shoots cultured on half-strength Murashige and Skoog (MS) medium supplemented with 44.4 μM N⁶-benzyladenine (BA) developed more than seven shoots per explant. The isolated shoots transferred onto the same medium induced more than eight PLBs from the base within 60 days, which upon transferral to fresh medium having the same level of BA facilitated rapid proliferation. More than 200 PLBs were yielded from fifth subculture. Half-strength MS medium containing 6.97 μM kinetin (Kn) facilitated conversion of more than 90% PLBs to shoots. PLBs exhibited proliferation without decline up to the 15th subculture. Half-strength MS medium with 2 g l⁻¹ activated charcoal was the best for in vitro rooting. Plantlets of the hybrids exhibited more than 80% ex vitro establishment.