Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange

Shoot tips obtained from in vitro grown plantlets of guava (Psidium guajava L.) were encapsulated in calcium alginate beads for short-term storage and germplasm exchange. A gelling matrix of 3% sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Maximum percent response for conversion of encapsulated shoot tips into plantlets was obtained on growth regulator free full strength liquid MS medium. The regrowth ability of encapsulated shoot tips was affected by medium strength and sucrose concentrations in the medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 30 days with a survival frequency of 25%. After 60 days of storage under minimal growth conditions (sucrose lacking medium), about 75% encapsulated shoot tips were converted into plantlets when subcultured on 3% sucrose containing medium. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

Effect of carbohydrates on in vitro low-temperature storage of shoot cultures of apricot

Procedures for cold storage of in vitro cultures can delay subculturing, reducing production costs and risks of contamination and somaclonal variation. The present work investigates the effects of media with sorbitol (116.8 mM, medium SO) or sucrose (58.4 mM) alone (medium SU), or the latter in combination with mannitol (58.4 mM, medium M) on 7-month storage at 5 °C of apricot shoots, cv San Castrese and Boreale. Shoots in SO survived in lower percentages and grew less than in the other treatments during storage, and died in large numbers after transfer to standard culture conditions. In comparison to other treatments, survival was 100% in the presence of M and both shoot weight and number of surviving proliferated axillary shoots was increased. Moreover, M improved regrowth compared to SU under standard culture conditions. The SOD and CAT activity confirmed the higher stress of shoots stored in SO than controls, and in contrast, the low stress of shoots in M.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.     

In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

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.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange

The present paper demonstrates the potential of nutrient-alginate encapsulation of axenic nodal segments of pomegranate for synthetic seed technology, which could be useful in germplasm distribution and exchange. Nodal segments from in vitro shoot cultures derived from mature nodal explants (source A) or axenic cotyledonary nodes (source B) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog's [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] medium (MS) supplemented with 4.44 μM benzyladenine (BA) and 0.54 μM naphthalene acetic acid (NAA). Of various concentrations of sodium alginate (1–6%) and the complexation solution of calcium chloride (50–125 mM), a combination of 3% sodium alginate and 100 mM calcium chloride was most suitable for formation of ideal synthetic seeds. Morphogenic response of encapsulated nodal segments to seven different planting media was evaluated. Encapsulated nodal segments of both the sources exhibited shoot development only in four selected media. Of the planting media evaluated, % sprouting (shoot development) was the highest in MS medium augmented with 4.44 μM BA and 0.54 μM NAA and lowest in (1/2) MSS medium. One step germination i.e. both shoot and root formation was possible only with encapsulated nodal segments of source B in MS, (1/2) MSS and natural soil + (1/2) MSS, with MS being most effective. Encapsulated nodal segments stored up to 30 days at 4 °C were capable of sprouting. Plants regenerated from the encapsulated nodal segments were hardened off and transferred to soil.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Conservation of Zingiber germplasm through in vitro rhizome formation

The effects of various concentrations of maleic hydrazide (MH; 2, 4, 6, 8 mg/l) and three light treatments (16-h, 24-h, 0-h) on in vitro rhizome formation and conservation of ginger (Zingiber officinale Rosc. cv. Rio de Janeiro) were studied. In vitro rhizome formation occurred in all the above treatments. Addition of MH (2–8 mg/l) to the control medium (CM) comprising Murashige and Skoog's (1962) salts, 9% sucrose, 0.8% agar-agar, 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l N⁶-benzyladenine (BA), did not show any significant positive effects on rhizome formation as well as survival of cultures. A significant effect of light treatments was observed on survival of cultures but not on rhizome formation. More than 50% cultures survived up to 14 months on CM under 16-h and 24-h light conditions as compared to 20% cultures on same medium incubated under dark. A total of 33 genotypes of cultivated and wild species of Zingiber were subsequently tested for conservation through in vitro rhizome formation on CM under 16-h light condition. All genotypes produced rhizomes of varying size with numbers ranging from 3 to 15 per culture and were conserved for at least 12 months; some genotypes could be conserved even up to 16–20 months. Viability of rhizomes was determined by in vitro regeneration of shoots upon subculture and their subsequent establishment in soil. Following the protocol described in the present paper, some 160 genotypes of cultivated and wild species of Zingiber, collected from different geographical regions of India, are being conserved at In Vitro Genebank of National Bureau of Plant Genetic Resources, New Delhi.     

Plant regeneration from stem segment-derived friable callus of “Fonio” (Digitaria exilis (L.) Stapf.)

“Fonio” (Digitaria exilis (L.) Stapf.) is a member of the grass family with excellent culinary and nutritional properties. In spite of its economic values, hardly has any improvement work been done. To enhance genetic improvement of this grain, plant regeneration protocol was developed using 8 cultivars. Stem segments of 5 mm long excised from 1 month-old seedlings germinated in vitro were cultured on 6 types of media for friable callus induction. Best result was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 g l⁻¹ casamino acid, where 91.3, 88.9 and 87.8% of the explants formed friable calli in cultivars ‘Kurelep’, ‘Churiwe’ and ‘Agyong’, respectively. Shoots appeared when friable calli were transferred to two regeneration media, i.e., MSBZ (MS medium + 0.022 mg l⁻¹ 2,4-D, 0 .22 mg l⁻¹ 6-benzylaminopurine (BA), 0.22 mg l⁻¹ zeatin) and MSBG (MS medium + 0.5 mg l⁻¹ BA, 0.1 mg l⁻¹ gibberellic acid). The highest frequency of plant regeneration was attained on MSBG, with 91.7% of the friable calli forming shoots in cultivar “Churiwe”. Regenerated plants were rooted on hormone free MS medium. Flow cytometric analysis revealed 100% of the regenerants to be diploid. The protocol developed here can be used in the transformation of “Fonio” to increase the yield potential of this crop by incorporating characteristics such as disease resistance and stress resistance.     

Multiple shoot regeneration in seed-derived immature leaflet explants of peanut (Arachis hypogaea L.)

A protocol was developed for organogenesis from immature leaflet explants derived from mature seeds of peanut. Immature leaflets pre-incubated on MS medium supplemented with 13.32 μM BAP + 4.95 μM NAA for 7 days, turned green and enlarged. The enlarged green leaflets produced multiple shoot buds after 1–2 cycles of sub-culture on MS medium supplemented with 13.32 μM BAP. Three cycles of shoot buds on the elongation medium (13.32 μM BAP) produced 6.17 ± 0.47 elongated shoots per explant. The shoot bud formation was genotype independent. All elongated shoots rooted on the medium containing 4.95 μM NAA. The complete protocol gave efficient (>81%) direct organogenesis, leading to the development of plantlets within 4 months.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

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.     

In vitro proliferation and minimum growth storage of fraser photinia: Influences of different medium,sugar combinations and culture vessels

Protocols for the in vitro proliferation and storage of fraser photinia were developed by comparing 6-benzyladenine (BA) concentrations (0.5–4 mg/L) together with different media formulations [Murashige and Skoog (MS) media and Quoirin and Lepoivre (QL) media], sugar combinations (sucrose and mannitol), culture vessels (baby food jars and vitrovents) and methods (synthetic seed technology and slow growth storage). The best responses in terms of both proliferation percentage and multiple shoot formation were obtained in QL medium containing 1 mg/L BA. Synthetic seed production was optimized by encapsulating shoot apices in 3% sodium alginate. Encapsulated shoot apices could be maintained up to 6 months at 4 °C in dark with 91.6% sprouting in MS medium. Microshoots were stored at 4 °C up to 15 months on sucrose and mannitol containing QL medium in both baby food jars and vitrovents without subculture. The stored material could be recovered and multiplied normally in 1 mg/L BA supplemented QL medium. Both in vitro propagated and conserved microshoots were rooted (∼75%) on QL medium with 1 mg/L indole butyric acid (IBA). Optimized synthetic seed and slow growth storage system can be used for short and medium-term storage of fraser photinia germplasm.     

Coconut water and BAP successfully replaced zeatin in olive (Olea europaea L.) micropropagation

The data presented report on trials conducted during 24 months using the Portuguese olive cultivar ‘Galega vulgar’. The effectiveness of coconut water, BAP, or kinetin, as possible zeatin substitutes in olive micropropagation protocols, was investigated. In all stages of the micropropagation process, the mineral and vitamin formulation of olive medium (OM) was used. Regarding culture establishment the best results were achieved when 50 ml l⁻¹ coconut water and 2.22 μM BAP were used as medium supplements. For the in vitro multiplication stage, the highest proliferation rates with an average of 3.4 new explants on each 30 days were achieved maintaining the coconut water concentration at 50 ml l⁻¹ and increasing BAP up to 8.87 μM. The effects of IBA and activated charcoal on the in vitro root induction were also studied. Rooting rates of over 85% were obtained by basal immersion of the explants in IBA solution at 3 g l⁻¹ for 10 s, followed by inoculation in the OM culture medium, added with 2 g l⁻¹ of activated charcoal and without growth regulators. All in vitro rooted plants were transferred into Jiffy-Pots filled with vermiculite–perlite 3:1 (v/v) substrate. Those were subsequently wetted with the OM mineral solution, placed into polystyrene plates each one with 100 Jiffy-Pots capacity, which were transferred to traditional rooting mist benches, on a water-cooling equipped greenhouse. Such a simple acclimatization procedure allowed for 95% of plants survival.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg L⁻¹ 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl₂ and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.