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.     

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.     

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.     

Commercial production of Cordyline terminalis (L) Kunth. from shoot apex meristem and assessment for genetic stability of somaclones by isozyme markers

Rapid multiplication of Cordyline terminalis (L) Kunth. was achieved from shoot apex explant on Murashige and Skoog's (MS) medium supplemented with 3% (w/v) sucrose and different concentration of growth regulators at various combinations. On MS medium supplemented with BA in combination with Adenine sulphate and IAA, shoot initiation and multiplication were obtained. Best elongations of shoots were found on 1/2 MS basal medium and shoots were rooted on 1/2 MS medium supplemented with IBA. Rooted plants passed through a hardening phase prior to ex vitro transfer. Clonal propagated plants established in garden soil were uniform and identical to mother plant in respect to growth characteristics and morphology. Isozymic profiles of different micropropagated clones were assessed for their genetic stability. Ten clones were tested for six isozymes. Only a few showed variation with respect to the banding pattern in esterase and superoxide dismutase. In superoxide dismutase, the two polymorphic isoforms (Rf 0.06 and 0.45) appeared in the clone C8 of the plants transferred to the field after 15 subcultural passages. Mobility and intensity of bands were monitored in other isozymes. Isozyme markers may be used as a tool for rapid screening of genetic stability in tissue cultured clones of C. terminalis.     

In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus

This paper describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Astragalus membranaceus and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 h prior to culture induced as high as 35.3% tetraploid plants. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploids and tetraploids in A. membranaceus were compared. It was determined that leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in A. membranaceus. As well as generating tetraploids, this technique generated mixoploids in A. membranaceus. Calli derived from mixoploid leaves, were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid and tetraploid by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but no tetraploid.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Haploid plant production in Zantedeschia aethiopica ‘Hong Gan’ using anther culture

This report describes advances in the anther culture of Zantedeschia aethiopica. Important factors for improvement as compared to the earlier procedure were: (1) using flowers from inflorescences developed at relatively low temperature during winter, (2) high temperature stress treatment at 32 °C for 2 days in the beginning of the culture, (3) use of Gamborg B5 as anther culture medium, and (4) addition of sucrose at high concentration of 8% in the culture medium. Plants were obtained via a callus phase. Frequency of anthers producing calli was around 4–5%. About 87% of the calli gave regenerants, of which 52% were haploid, 36% were diploid and the rest had other ploidy levels. In addition to chromosome counting, cytological examination of the microspore development and amplified fragment length polymorphism (AFLP) analysis of the regenerants showed that haploid as well as diploid plants originated from the microspores. Finally, 12 doubled haploid (DH) plants could be produced from each inflorescence. One quarter of the DHs equaled the original cultivar in growth vigor, while more than one third showed good fertility, indicating that inbreeding depression was not so severe in this heterozygous species. The improved protocol now enables production of sufficient number of DHs for application of haploid technology in genetic improvement and breeding of Z. aethiopica.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

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.     

Regeneration and characterization of Swertia chirata Buch.-Ham. ex Wall. plants from immature seed cultures

First generation immature seeds (R₁) were collected from a field transferred micropropagated plant and seeds were induced to develop organogenic calli in Swertia chirata, a traditional revenue earning medicinal plant. Half strength MS medium with different growth regulators namely, BA, Kn (2.22–4.44 μM), NAA (2.69–5.37 μM), and 2.26 μM 2,4-D were used to induce callus and organogenesis. Isolated shoots produced roots either in the same medium or in presence of NAA (2.69–10.74 μM) or IBA (2.46–9.8 μM). Fully developed plantlets were successfully transplanted to soil and the fertile seed bearing plants developed. Occasionally plants derived from more than 56 weeks old calli showed some morphological variations. Such variations in regenerated plants is not reflected in their chromosomal constitution, with normal 2n = 26 chromosomes. Likewise, no variation was observed in DNA fingerprinting patterns among the short-term raised culture regenerants, which were morphologically similar to that of the donor plant illustrating their genetical uniformity and clonal fidelity. On the contrary, variation in DNA fingerprinting patterns was observed in long-term culture raised plants.     

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.     

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.     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.     

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.     

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.     

Micropropagation of Cryptanthus with leaf explants with attached intercalary meristems excised from greenhouse stock plants

By using the leaves with attached intercalary meristems from greenhouse grown stock plants, five cultivars of Cryptanthus were cultured on modified MS media with 4.5 μM NAA and IBA and 3 μM BA to induce adventitious shoot formation from callus tissue. Contamination was 17–21% for explants taken from stock plants which were sprayed weekly with Agribrom and 27–75% for those taken from stock plants which were not treated. More than 99% true to type plantlets were obtained from non-chimeric plants. Green and albino plantlets were obtained from chimeric plants. The chimeric C. ‘Coster's Favorite’ DeCoster also produced a few chimeric plantlets with intermarginal pink stripes in addition to the green and albino plantlets. Most of the non-chimeric plants took a shorter time to produce plantlets of transplantable size (8–12 mm) than the chimeric ones. Except for albino plantlets, survival rate of plantlets exceeded 95%. A minimum average of 500 rooted plantlets can be obtained in a year from a single well-callused leaf explant. The protocol in this report should speed up the mass production and introduction of desirable new cultivars and hybrids of non-chimeric Cryptanthus.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

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.