Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

In vitro clonal propagation of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> (L.) Del

In vitro propagation technique of Balanites aegyptiaca, a multipurpose woody tree was studied. Nodal segments including axillary bud from mature tree were used as an explant and their morphogenetic potential was tested on MS media with various concentrations (2.5–15.0 μM) of 6-benzyladenine (BA), Kinetin, and Thidiazuron alone or in combination with different concentrations (0.5–2.5 μM) of α-naphthalene acetic acid (NAA). Nodal segments showed axillary bud proliferation in almost all media tried. MS medium containing 12.5 μM BA alone was effective for inducing multiple shoots (5.0 ± 0.22) with an average shoot length (3.7 ± 0.26 cm) in 67% of cultures. A better shoot differentiation and elongation was achieved in a combined treatment of BA (12.5 μM) and NAA (1.0 μM). Half strength MS medium supplemented with Indole-3-butyric acid (IBA) gave the best result for rooting. The maximum frequency of root formation (68%), number of roots (5.3 ± 0.32) and root length (4.1 ± 0.38 cm) was obtained on half strength MS medium containing 1.0 μM IBA. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the greenhouse.     

An Efficient Micropropagation Protocol for High-Frequency Plantlet Regeneration from Liquid Culture of Nodal Tissues in a Medicinal Plant,Turnera ulmifolia L.

A highly efficient, stable, and cost-effective micropropagation protocol for the conservation of a medicinal plant Turnera ulmifolia L. was established from nodal tissues via multiple axillary shoot proliferation on using Murashige and Skoog’s (MS) liquid nutrient medium. To begin with, nodal explants were placed on agar gelled medium amended with 2.0 mg L^?1 6-benzylaminopurine (BAP) and 0.1 mg L^?1 indole-3 acetic acid (IAA) for shoot induction. Subsequently, elongation of regenerated shoots could be possible on liquid MS medium supplemented with 0.5 mg L^?1 BAP and Kin (kinetin) each along with 0.1 mg L^?1 IAA where high frequency of regeneration in terms of number of shoots (47.2 shoots/explant) was achieved. Furthermore, long and healthy shoots (4?5 cm in length) were rooted on agar gelled half-strength of MS medium supplemented with 2.0 mg L^?1 indole-3 butyric acid (IBA). Finally, in vitro regenerated plantlets were gradually acclimatized in the greenhouse and transferred to the field successfully.     

Development of an efficient protocol for plant regeneration from nodal explants of recalcitrant bamboo (Bambusa arundinacea Retz. Willd) and assessment of genetic fidelity by DNA markers

The present study describes an efficient method for in vitro plant regeneration in B. arundinacea through axillary shoot bud proliferation. Nodal explants were excised, cultured on MS medium containing different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN) (0.5–5.0 mg l^?1) alone and/or in combinations with KIN/BAP (0.5 mg l^?1). The highest frequency (91.5 %) of multiple shoot bud induction with maximum number of shoots (85 shoots/explant) was noticed on MS medium + 3.0 mg l^?1 BAP + 0.5 mg l^?1 KIN. The regenerated multiple shoots were elongated on MS medium + 4.0 mg l^?1 KIN + 2.0 mg l^?1 gibberellic acid (GA₃) with maximum shoot length (4.9 cm). The elongated shoots were transferred to MS medium containing indole-3 butyric acid (IBA; 0.5–5.0 mg l^?1) alone and/or in combination with 0.5 mg l^?1 KIN and BAP. Highest frequency of rooting (75 %) was obtained on half-strength MS medium + 2.0 mg l^?1 IBA + 0.5 mg l^?1 KIN. After hardening, the plantlets were shifted to the green house and subsequently established in the field conditions with 90 % survival rate. random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the regenerants. RAPD profiles generated from the regenerated plants were found to be monomorphic, similar to the control. Results confirmed that the regenerated plants were true-to-type in nature and the developed micropropagation protocol could be used for large scale plant production of B. arundinacea.     

In vitro plantlet regeneration from Australian Red Cedar (Toona ciliata,Meliaceae)

In vitro regeneration of complete plants from nodal single-bud segments of 2-year-old Australian Cedar (Toona ciliata) trees were obtained under defined nutritional and environmental conditions. Explants were dissected from plants obtained by germination of seeds and growth in pots in a greenhouse. The best medium for shoot regeneration was that of Murashige and Skoog at 1/4 strength with 3% sucrose (1/4 MS), supplemented with 0.1 mg/l IBA and 0.5 mg/l BAP. Rooting of regenerated shoots was observed in MS medium with 0.1 mg/l IBA. Using mature tree material was more difficult. Forced flushing was used to induce shoot development on branches of a 10-year-old tree. Nodal segments of these epicormic shoots formed shoots in vitro on 1/4 MS + 0.01 mg/l IBA + 5 mg/l BAP, but rooting was never observed.     

In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP (2.0 mg·L^–1) and NAA (0.1 mg·L^–1). The optimal multiplication medium was a modified MS medium supplemented with BAP (1.0 mg·L^–1) and NAA (0.1 mg·L^–1). This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA (0.2 mg·L^–1) and acti-vated charcoal (0.5 mg·L^–1). Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproduci-ble procedure can be adopted for large-scale propagation of T. wilfordii.     

In vitro flowering of green and albino Dendrocalamus latiflorus

To propagate Dendrocalamus latiflorus, we used in vivo inflorescences to produce calli on Murashige and Skoog basal (MS) medium supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/l kinetin, 250 mg/l polyvinyl pyrrolidone (PVP), and 1% coconut milk. Multiple shoots were generated on MS medium supplemented with 0.1 mg/l thidiazuron (TDZ). The green plantlets were successfully transferred to soil. Multiple albino shoots also regenerated and were able to proliferate on medium containing cytokinins, especially TDZ. Albino multiple shoots rooted in medium containing α-naphthaleneacetic acid (NAA), and callus formation was observed in the presence of 2,4-D and picloram. Green and albino regenerates flowered after 8 months of subculture. The flowering ratio increased to 44% after three treatments in medium containing 1 mg/l TDZ. Morphological observations revealed that the in vitro green and albino flower organs were normal. However, pollen derived from the in vitro flowers of both the green and albino plants were sterile.     

Regeneration of <Emphasis Type="Italic">Melia volkensii</Emphasis> Gürke (Meliaceae) through direct somatic embryogenesis

Experiments were conducted to study plant regeneration through direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants from seeds of Melia volkensii stored for <3 and >12 months. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP, NAA and 2,4-D (0.5, 1.0 and 2.0 mg l⁻¹) alone, and BAP (0.5, 1.0, 2.0 and 4.0 mg l⁻¹) in combination with 2,4-D or NAA (0.2 and 0.5 mg l⁻¹). After 4 weeks in culture, up to 60% of cotyledonary explants from the seeds stored for <3 months produced direct somatic embryos on BAP (0.5–4.0 mg l⁻¹) in combination with 2,4-D (0.2 mg l⁻¹). The number of somatic embryos ranged from 5 to 14 per explant in BAP (0.5 mg l⁻¹) and 2,4-D (0.2 mg l⁻¹) combination. Only 20% of cotyledonary explants from seeds stored for >12 months produced somatic embryos. Mature zygotic embryos failed to produce any somatic embryos. Subcultures of somatic embryos from cotyledonary explants of seeds stored for <3 months formed clusters of shootlets on semi solid MS and 1/2 MS media. After 6 weeks of subculture on multiplication MS media augmented with BAP (0.5 mg l⁻¹) and IAA (0.2 mg l⁻¹), 70% of the shoot tips formed 4–7 shoots per explant. Up to 33% of the multiplied shoots were rooted in MS medium supplemented with 2.0 mg l⁻¹ IBA. Plantlets developed normally into seedlings in the greenhouse.     

Rapid Micropropagation of Edible Bamboo Dendrocalamus asper

Abstract

Micropropagation protocols for Dendrocalamus asper using nodal shoots and seeds culture are described. Multiple shoots were induced through forced axillary branching. Ninety-five percent of the nodal shoot explants taken from juvenile primary and lateral branches, produced multiple shoots through axillary buds activation within 2 to 8 weeks on Murashige & Skoog's (MS) medium supplemented with 0.1-15 mg/l benzyladenine (BA). The cultured seeds also produced multiple shoots (1-20) within 6 weeks on this medium. The multiple shoot differentiation was influenced by the concentration of BA in the medium. The in vitro generated shoots were excised and subculture on MS + 3.0 mg/l BAP for further shoot multiplication. Fifteen to 20 fold rate of shoot multiplication was achieved by regular subculturing. These shoots were multiplied for more than 3 years without loss of vigor. Ninety-five percent of the shoots were rooted, when propagules (each consisting of cluster of 3 shoots) were transferred on to MS medium with 3.0 mg/l NAA or 10 mg/l indole-3-butyric acid (IBA).

To date, 18,000 plants (through axillary bud initiated from nodal ex-plants) and 6,000 plants from seed culture have been hardened and acclimatized. 12,000 plants have been field transferred.     

Somatic embryogenesis and plant regeneration inAcanthopanax sciadophylloides

Somatic embryos ofAcanthopanax sciadophylloides Franch. et Sav. were differentiated from both zygotic and somatic embryos and calli, and plants were regenerated from these somatic embryos. A zygotic embryo, enclosed within a small portion of the endosperm, was incubated on Murashige and Skoog (MS) media supplemented with various combinations (range 0–10.0 mg/l) of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 months, swelling of the zygotic embryos and callus formation was observed. When the swollen embryos were transferred to MS medium supplemented with 0.5 mg/l of 2,4-D, somatic embryos were formed in one to two months. After subculture on the same medium, new embryos were differentiated from various parts of the older somatic embryos. The calli were cultured on medium supplemented with 2.0 mg/l of 2,4-D and BAP for three weeks. Proliferated calli were transferred to medium supplemented with 1.0 mg/l of 2,4-D and BAP. Somatic embryos were differentiated from the calli within one to two months. Somatic embryos were germinated on half-strength MS medium without plant growth regulators and the plantlets were grown in soil. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995) & First Asia-Pacific Symposium on Forest Tree Genetic Improvement (Beijing).     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Tissue culture ofRosa Chinensis var.Floribunda

The tissue culture of Schloss Mannheim(Rosa Chinensis var.Flaribunda) with full and unsprouting bud of stem segments as the explants was experimented. The result shows that the buds sprouted best on MS medium with the addition of 6-BA 1.0 mg/L, and differentiation was best on MS medium with addition of 6-BA 1.5 + NAA 0.05 + ZT 0.1 mg/L or KT 1.0 + NAA0.05 + ZT 0.1 mg/L. The MS medium with addition of 6-BA 0.3 + NAA 0.0 5+ ZT 0.1 mg/L or KT 0.3 + NAA0.05 + ZT 0.1 mg/L showed a good result for developing strong shoots. 1/2 MS medium with the addition of IBA 0.1 mg/L or IBA 0.1 + NAA 0.02 mg/L had best result for rooting. The plantlets should be transplanted from test-tube to soil when they grew to 2.5 ∼ 4.0 cm high and have 3 ∼ 5 strips short roots. A higher survival rate was obtained under the conditions of controlling humidity and temperature.     

Micropropagation of Anogeissus rotundifolia Blatt,and Hallb.-An Endemic and Rare Tree of the Thar Desert

Abstract

Multiple shoots and plantlets were developed in vitro from cotyledonary nodal segments of in vitro raised seedlings of Anogeissus rotundifolia (syn. A. sericea var. nummularia)-a rare and endemic tree species of the Thar Desert. About 15-20 shoots differentiated from a single cotyledonary node within four weeks on Mu-rashige and Skoog's (MS) basal medium containing 0.1 mg l^?1 indole-3-acetic acid (IAA) + 2.0 mg l^?1 6-benzylaminopurine (BAP) + additives (25 mg l^?1 each of adenine sulphate, L-arginine, and citric acid, and 50 mg l^?1 of ascorbic acid) at 26 ± 2°C temperature and 36 μmol m^?2 s^?1 photon flux density with a 12 h/day photoperiod. The shoots produced in vitro were further multiplied by subculturing on fresh medium. The original cotyledonary nodal segment was repeatedly transferred (5 to 6 times) onto fresh medium containing 1.0 mg l ^?1 BAP + 0.1 mg I ^?1 IAA + additives to yield fresh crops of multiple shoots. These shoots were rooted on half-strength MS medium supplemented with 0.1 mg l^?1 indole-3-butyric acid (IBA). Plantlets were transferred to pots containing sand-dune soil and ver-miculite it the ratio of 4:1 (v/v) and hardened in a growth chamber for two weeks and finally transferred to a greenhouse. From a single cotyledonary node about 1500 plantlets could be developed within four months. The method developed is useful for mass multiplication and for the conservation of germplasm of Anogeissus rotundifolia.     

Direct plant regeneration from nodal explants of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> L. (Del.): a valuable medicinal tree

This report describes in vitro shoot induction and plant regeneration from nodal segments of Balanites aegyptiaca on Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA), thidiazuron (TDZ) and kinetin (Kin) (0.5–20.0 μM). MS medium supplemented with BA (12.5 μM) was the most effective in inducing bud break and growth and also in initiating multiple shoot proliferation. However, the optimal level of TDZ supplementation to the culture medium was 5.0 μM. Shoot cultures were established by repeatedly subculturing the original nodal explants on the same medium. Highest number of shoots (11.5 ± 0.7) and shoot length (5.0 ± 0.2 cm) were achieved when cultures were subcultured on MS medium supplemented with 12.5 μM BA and 1.0 μM α-naphthalene acetic acid (NAA). The shoots regenerated from TDZ supplemented medium when subcultured to hormone free MS basal medium considerably increased the rate of shoot multiplication and shoot length by the end of fifth subculture. Rooting of the shoots was achieved on MS medium augmented with 1.0 μM indole-3-butyric acid (IBA) plus 0.5% activated charcoal followed by their transfer to half strength MS basal medium. The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 70% survival rate. The results of this study provide the first successful report on in vitro direct plant regeneration of B. aegyptiaca.     

Micropropagation of a mature colchicine-polyploid and irradiation-mutant of Betula pendula Roth

Tetraploid plantlets were regenerated from cultured apical and axillary buds of a 23-year-old colchicine-polyploid and irradiation-mutant Betula pendula Roth tree. Bud explants were grown on modified Murashige and Skoog medium supplemented with 2.0 mg l(-1) benzylaminopurine (BAP) and 0.01 mg l(-1) 1-naphthaleneacetic acid (NAA). The medium allowed both induction of adventitious buds and development of shoots. The cut ends of new shoots produced new buds and shoots during a 4-week culture period. The micropropagated shoots were rooted on modified Murashige and Skoog medium containing 0.1 mg l(-1) NAA as the sole growth regulator. Plantlets were transferred to a peat/soil mixture (1:1) in the greenhouse, acclimated and then transplanted to a cold frame. The regenerated plantlets had a tetraploid chromosome set (4n = 56) and an altered leaf morphology typical of colchicine-polyploid birches. The leaves were hypertrophied and asymmetrical, with curly leaf margins. The mutant nature of the parent tree was also evident in the light-green color of the leaves of the plantlets.     

Efficient in vitro regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> using immature zygotic embryos as explants

A method for efficient in vitro regeneration of Leucaena leucocephala cv. K636 was developed using immature zygotic embryos as the explant. Multiple shoot induction was achieved by culturing the explants on half-strength Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.03 to 1.5 μM). The maximum number of shoots per explants was achieved in 0.26 μM TDZ-supplemented media. TDZ concentration significantly influenced the induction of multiple shoots as well as the shoot-length. TDZ at 0.35 μM or higher concentrations resulted in abnormal stunted shoots. Full-strength MS media devoid of any plant growth regulator were used for shoot elongation. In vitro root induction was achieved in half-strength MS media supplemented either with 0.54 μM 1-naphthaleneacetic acid (NAA) or with 14.76 μM indole-3-butyric acid (IBA) in combination with 0.23 μM kinetin (Kn). Media supplemented with 14.76 μM IBA in combination with 0.23 μM Kn induced twofold–threefold more roots than media supplemented with 0.54 μM NAA. However, the average root length was lower in 14.76 μM IBA in combination with 0.23 μM Kn than in 0.54 μM NAA. Regenerated plants were maintained under normal condition after hardening. Plants, which were rooted on media supplemented with 14.76 μM IBA in combination with 0.23 μM Kn showed higher survival rate during hardening than those rooted on 0.54 μM NAA supplemented media.     

Shoot proliferation and callus regeneration from nodular buds of Drepanostachyum luodianense

This research reports on an efficient shoot proliferation and callus regeneration system for bamboo.Young, semi-lignified branches with one lateral bud from Drepanostachyum luodianense(Yi et R. S. Wang) Keng f.were used as explants. Disinfection with 0.1% HgCl_2 for 8 min was the optimum treatment and the best medium for bud initiation was Murashige and Skoog(MS) medium containing 3.0 mg L~(-1)6-benzyladenine(BA). Multiple shoots were induced from nodal shoot segments on MS medium containing 5.0 mg L~(-1) BA, 0.5 mg L~(-1) kinetin(Kin), and 1.0 mg L~(-1) naphthaleneacetic acid(NAA). The highest frequency of callus formation(65.6%) was on MS medium containing 4.0 mg L~(-1)2,4-dichlorophenoxyacetic acid(2, 4-D), 0.5 mg L~(-1) NAA, and 0.1 mg L~(-1) thidiazuron(TDZ). The optimum medium for callus proliferation was MS medium with 4 mg L~(-1)2,4-D, 0.5 mg L~(-1) TDZ and 0.5 mg L~(-1) NAA, and the optimum hormone combination was 4 mg L~(-1) BA ? 0.5 mg L~(-1) NAA for callus redifferentiation(up to 85.6%). A 100% rooting was achieved on MS medium supplemented with 2.0 mg L~(-1) NAA and 0.5 mg L~(-1)3-indole butyric acid(IBA). Rooted plantlets were acclimatized in a greenhouse in humus soil ? perlite(1:1) substrate. These micropropagated callus induction and regeneration systems for bamboo will be useful for genetic engineering and multiplication.     

In vitro regeneration of <Emphasis Type="Italic">Populus tomentosa</Emphasis> from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ‘fasta-3’. A high frequency of callus induction (>97 %) was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.     

In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes

Acacia sinuata is a valuable multipurpose tree in Southern India. The tree is over exploited, but its regeneration rate in natural habitat is low. Therefore, it is important to study if it can be regenerated through in vitro micro-propagation. Cotyledonary node and shoot-tip explants excised from 15 day-old in vitro grown seedlings were used to initiate cultures. Maximum number of shoots was induced from cotyledonary node explants on Murashige and Skoog's (MS) medium containing6.66 µM 6-benzylamino purine (BAP) and 4.65µM kinetin (Kn). Subculturing was done in the fresh medium of same composition. The number of shoots formed was comparatively greater in the first subculture. Maximum shoot elongation was achieved (5.5 cm)when subcultured on MS medium supplemented with 1.75 µMgibberellic acid (GA₃). In vitro regenerated shoots produced roots when transferred to half strength MS medium supplemented with 7.36 µM indolebutyric acid (IBA). From each cotyledonarynode 30 shoots were obtained within 90 days after two subcultures. The success rate of establishing the rooted plantlets in the field was 55%.This revised version was published online in November 2005 with corrections to the Cover Date.     

Induction of somatic embryogenesis and analysis of genetic fidelity of in vitro<Emphasis Type="Italic">-</Emphasis>derived plantlets of <Emphasis Type="Italic">Bambusa nutans</Emphasis> Wall., using AFLP markers

Bambusa nutans Wall., is an evergreen, perennial, and multipurpose bamboo having strong culms, which are largely used for construction, scaffolding, craft purposes, pulp, and paper industry. Multiple shoots from nodal segments (3–4 cm) of young branches of mature culms were established in Murashige and Skoog (1962) (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) (1.0–6.0 mg l⁻¹) or in combination with α-naphthaleneacetic acid (NAA) (0.5–1.0 mg l⁻¹) or kinetin (Kn) (1.0–2.0 mg l⁻¹). February–March and December were found to be the best seasons for culture establishments. Maximum shoots were achieved on MS medium fortified with BAP (2.0 mg l⁻¹). Embryogenic callus (slightly greenish compact, globular, and slow growing) was initiated from the base of severed sprouted buds in 2–3 subsequent subcultures on MS medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) (5.0 mg l⁻¹) under dark incubations. Maturation and germination of well-organized somatic embryos was achieved on MS medium containing BAP and 2,4-D (1.0 mg l⁻¹ each) with 20.0 mg l⁻¹ ascorbic acid. Full-strength MS medium supplemented with 2% glucose favored further development of proliferated somatic embryos into plantlets. Genetic variations of field-established B. nutans plants regenerated through tissue cultures were assessed by amplified fragment length polymorphism (AFLP) analysis using 6 primer combinations. Four hundred and seven scorable fragments were amplified, of which 402 (98.8%) have recorded conservation at various morphogenetic stages leading to plantlets regeneration, therefore, revealed a high level of genetic stability.