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.     

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.     

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.     

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

Adventitious shoot regeneration and protoplast isolation and culture were examined from leaf explants of in vitro shoot cultures of several cauliflower (Brassica oleracea var. botrytis) cultivars, sourced from Europe and Australia, was investigated with the aim to develop improved nuclear and plastid transformation protocols for this vegetable crop. Eight out of 10 cultivars regenerated shoots from at least 79% of leaf explants. Mesophyll protoplasts from leaves gave high yields and division frequencies. Growth of shoot cultures in large glass vessels with vented lids was the key factor in obtaining high protoplast division frequencies of up to 71% and at least 70% of protoplast calluses regenerating shoots.     

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.     

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.     

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.     

Hybridization and in vitro culture of an orchid hybrid Ascocenda ‘Kangla’

The present study focuses on hybridization program involving two species belonging to two different vandaceous genera, viz., Ascocentrum ampullaceum (Roxb.) Schltr. var. auranticum, a narrow endemic orchid of Manipur and Vanda coerulea Griff., an endangered orchid of Appendix I of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora), to synthesize the primary hybrid genus with intermediate and improved characters in the F₁ generation. Observations on the crossability in the present bigeneric cross between V. coerulea and A. auranticum had been achieved with 60% success when V. coerulea was taken as female parent. Murashige and Skoog (MS) basal medium at half-strength was effective for the development of the hybrid seedlings of V. coerulea × A. auranticum followed by Vacin and Went (VW) and Knudson C (KC) media. The best response of seedling growth was observed on MS medium at half-strength supplemented with 2.3 μM kinetin + 0.5 μM α-naphthalene acetic acid with maximum shoot height (2.7 cm), leaf number (4.6) and root number (4.1) after 150 days of inoculation. The survival percentage and growth performance of the seedlings were found to be higher (80% survival) in potting substrate consisting of brick:charcoal in the ratio 2:1 mulched with moss (Sphagnum sp.) than in potting substrate consisting of brick:charcoal:tree fern in the ratio 2:1:1. The first flowering was observed in the hybrid seedlings of V. coerulea × A. auranticum after 2 years of transfer to the ex vitro environment. Morphologically, the flowers differed from that of the parents clearly showing the success of the hybridization experiment. Registration of the hybrid has been made with the Royal Horticultural Society with the nomenclature Ascocenda ‘Kangla’ (No. T128725).     

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.     

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.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

Phenotypic characterisation of Phaeoacremonium and Phaeomoniella strains isolated from grapevines: enzyme production and virulence of extra-cellular filtrate on grapevine calluses

Extra-cellular enzyme production of different Phaeoacremonium spp. and Phaemoniella chlamydospora isolates were used to assay the possibility of inter-specific characterisation. Isolates of Phaeoacremonium aleophilum, Phaeoacremonium angustius, P. viticola and Ph. chlamydospora were grown on solid media and the activities of extra-cellular amylases, lipases, proteases, cellulases, xylanases, laccase, polygalacturonase, pectate lyase, lignin peroxidase, manganese peroxidase, urease and chitinase were assayed. Phaeoacremonium species showed activities of a larger number of enzymes and also enzyme activity was frequently higher suggesting that Phaeoacremonium can be more virulent. To assay if the produced extra-cellular enzymes could reflect the virulence capacity of the two genera, calluses of Vitis vinifera L. (cvs. Baga and Maria Gomes) and of a rootstock (R3309) were inoculated with filtrated culture liquid medium of three isolates of Ph. chlamydospora and one of P. angustius. Filtrates from all strains decreased callus growth and membrane integrity, while soluble protein content of calluses decreased with the strains CAP 054 and 1AS. P. angustius (CAP 054) induced the more severe symptoms in all genotypes. Water content decreased together with an increase of osmolality in both cultivars but not in rootstock suggesting that osmorregulatory capacity is more affected in cultivars. Data show that: (1) Phaeoacremonium and Phaeomoniella genera have different patterns of extra- cellular enzymatic production; (2) these fungi produce extra-cellular compound(s) that induce(s) senescence symptoms in plant cells inhibiting callus proliferation; (3) among the strains tested in plant calluses the most virulent isolate (CAP 054) also produced higher amounts of some extra-cellular enzymes; (5) rootstock calluses were less sensitive to inoculation than grapevine calluses.     

Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local

A protocol for plant regeneration by somatic embryogenesis was developed in guava cv. Banarasi local by using immature zygotic embryo explants. Best induction of somatic embryogenesis was achieved from 10-week-old zygotic embryos on MS medium supplemented with 2,4-d (4.52 μM) and 5% sucrose. Maximum number of somatic embryos was produced when zygotic embryo explants were transferred to growth regulator free full strength MS basal medium after 8 days treatment with 2,4-d. Full strength MS basal medium containing 5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of conversion and normal plantlet production were recorded from elongated torpedo stages of somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted shoots survived acclimatization.     

Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter

Agrobacterium-mediated transformation for both Melastoma malabathricum and Tibouchina semidecandra were optimized using green fluorescent protein (GFP) as a reporter. The binary vector pCAMBIA1304 harboring the modified green fluorescent protein (mgfp) gene driven by the CaMV 35S promoter was used. Parameters optimized were bacterial strain, bacterial concentration, pre-culture period, co-cultivation period, immersion time, acetosyringone concentration and wounding type. Results obtained were based on the percentage of GFP expression which was observed 3 days post-transformation. Agrobacterium tumefaciens strain LBA4404 and EHA105 at concentration 1 × 10⁷ cfu ml⁻¹ (OD_600nm 0.8) showed the highest virulence on M. malabathricum and T. semidecandra, respectively. Four days of pre-culture and 2 days of co-cultivation were optimum for M. malabathricum transformation, while 3 days of pre-culture and co-cultivation for T. semidecandra. Result also showed that 60 min of immersion and addition of 200 μM acetosyringone gave the highest percentage of positive transformants for both M. malabathricum and T. semidecandra. Mild wounding also significantly increased the efficiency of M. malabathricum transformation.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

In vitro shoot regeneration from stored mature cotyledons of sweet cherry (Prunus avium L.) cultivars

Shoot regeneration was achieved from stored mature cotyledons of sweet cherry (Prunus avium L.) in vitro. The influences of different cytokinins (thidiazuron (TDZ) and benzylaminopurine (BA)) and their levels, the dark incubation for the first 10 days of the culture and TDZ pretreatments on shoot regeneration were determined. All varieties regenerated in the presence of TDZ if cotyledons were maintained in darkness for the first 10 days of the culture; however, only three varieties regenerated with low frequencies in the absence of dark incubation. Dark incubation at the early stage of culture was critical for obtaining higher regeneration efficiencies from stored cherry cotyledons. TDZ was more effective than BA in inducing shoot regeneration. The highest regeneration efficiencies were obtained with intermediate concentrations of TDZ (3.6 and 7.2 μM) in combination with dark incubation and the best regeneration frequencies for ‘Vista’, ‘Sunburst’, ‘Tehranivee’, ‘Vouge’ and ‘Heidelfingen’ cotyledons were 70.0%, 53.3%, 23.3%, 30% and 26.6%, respectively.     

A micropropagation system for cloning of Bambusa tulda Roxb.

The communication describes standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda. Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in winter and summer seasons, respectively facilitated optimum culture establishment and bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud break due to toxicity to explants. MS liquid medium enriched with 100 μM glutamine, 0.1 μM indole-3-acetic acid and 12 μM 6-benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The proliferated shoots were successfully rooted on MS liquid medium supplemented with 40 μM coumarin resulting in a maximum of 98% rooting. The procedure requires 45 days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30 days for root induction) and 80 days for acclimatized plantlet production.     

A comparison of the gelling of isubgol,agar and gelrite on in vitro shoot regeneration and rooting of variety Samsun of tobacco (Nicotiana tabacum L.)

Agar is being used to solidify media for plant tissue culture since long. Both purity and type of agar or gelling agent influence the behaviour and growth of tissues in culture. The study using leaf disc explants of variety Samsun of tobacco compares adventitous shoot regeneration on 0.5 cm × 0.5 cm leaf discs cultured on MSD4X2 medium and rooting on MSO medium gelled with different blends of agar–isubgol, gelrite–isubgol, phytagel–isubgol or isubgol singly. It was found that irrespective of some problems associated with isubgol gel, the maximum number of shoot per explant were recorded on MSD4X2 medium gelled with 7 g/l isubgol. The longest shoots were recorded on MSD4X2 medium gelled with 9 g/l isubgol. Likewise, the highest number of roots were also recorded on MSO medium gelled with 7 g/l isubgol. For a given quantity of a medium, agar, gelrite/isubgol blends are very cheap compared to agar used singly. Moreover, blends of agar/isubgol, gelrite/isubgol gelled at low temperatures indicated safe use of these gels for heat labile substances in genetic transformation or tissue culture experiments. The results emphasized the potential of the isubgol used singly or in combination with agar and gelrite for economic commercial application, replacing the costliest, though not indispensable, gelling agent agar.     

Medium-term conservation of redwood (Sequoia sempervirens (D. Don.) Endl.) in vitro shoot cultures and encapsulated buds

This study evaluated the survival and recovery of non-encapsulated and encapsulated shoots of Sequoia sempervirens after storage at 4 °C in the dark for up to 15 months on four different culture media. Survival and regrowth of encapsulated shoots declined within 3 months, regardless of the storage medium composition. By contrast, no significant decrease in survival and regrowth was noted with non-encapsulated shoots after 12 months of storage on Quoirin and Lepoivre medium supplemented, or not, with 1 mg l⁻¹ benzyladenine. Regrowth dropped to 60–61% after 15 months of storage on the same media. Medium-term conservation of S. sempervirens germplasm is therefore possible using in vitro storage of non-encapsulated shoot cultures.