Direct Plant Regeneration From Immature Male Inflorescence of Banana (Musa spp.)

This study describes for the first time a simple and quick direct somatic embryogenesis and plant regeneration protocol for turmeric (Curcuma longa L.) using leaf base explants. Preincubation of explants was carried out on solid Murashige and Skoog (MS) medium containing 4.49 μM 2,4-dichlorophenoxy acetic acid for 20 days under continuous dark condition. Subsequently transfer of the explants for 2 weeks to MS liquid medium containing 1.32 μM 6-benzyladenine (BA) under illuminated condition resulted in the highest percentage of somatic embryo (91.1 %) induction. Secondary somatic embryos which originated from primary somatic embryos were also observed in the same medium supplemented with 2.20 and 2.64 μM BA. Thereafter, mature somatic embryos germinated readily and the maximal plantlet development (87 %) was achieved on 1/2-strength MS medium containing gibberellic acid (GA3) under dark condition. Histological analysis revealed that the proembryogenic masses were initiated directly from the area near the vascular tissues of the leaf base explants. Presence of clear protoderm in the globular structure and procambial strands in the matured stage of somatic embryos confirmed that these structures were true somatic embryos. Somatic embryos were successfully acclimatized ex vitro at a survival rate of 72.9 % and they were normal phenotypes.

An efficient, rapid, reproducible and prolific protocol has been established for in vitro plant propagation via direct and indirect regeneration from internodal explants of Coccinia indica. The direct and indirect regeneration capability of internodal segments was studied on Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins and auxins either alone or in combinations. In direct regeneration, the highest regeneration percentage (90%) with maximum frequency of shoot length (7.2 cm) was achieved on D6: MS + 2.5 mg/L BAP (6-Benzylaminopurine) + 1.0 mg/L Kn (Kinetin). In indirect regeneration, the highest regeneration percentage (91.6%) was achieved on I5: MS + 1.0 mg/L BAP + 0.3 mg/L NAA(1-Napthaleneaceticacid). The highest frequency of shoot induction with shoot number (5) and shoot length (8.52 cm) was achieved on IS2: MS + 1.0 mg/L BAP + 0.5 mg/L Kn. Microshoots obtained best rooting response on IR3: MS + 1.0 mg/L IAA and 0.5 mg/L Kn by producing a highest frequency of root number (4.7) and root length (7.03 cm). The rooted microshoots were successfully hardened and were acclimatized in green house and subsequently established in soil with survival rate of 90%.

An efficacious, reproducible direct in vitro regeneration system has been developed from leaf base segments (LBs) of six high yielding genotypes of foxtail millet (Setaria italica (L.) Beauv.). LBs excised from 4-day-old seedling were inoculated on Murashige and Skoog (MS) medium supplemented with different types and concentrations of cytokinins. The shoots induced per explant significantly increased with the supplementation of BAP to auxin containing medium. The results showed that a maximum shoot induction, 58.8% was obtained on MS medium incorporated with 8.9µM BAP and 2.7µM NAA in 'CO5' genotype. Further, the highest frequency of multiple shoots was produced on MS(I) medium containing 8.9µM BAP, 2.7µM NAA, 700mgL-1 proline, 0.5mgL-1 cysteine, 2.0mgL-1 glycine and 150mgL-1 arginine. MS(I) medium additionally fortified with 5.0g L-1 activated charcoal (AC) was found to achieve the best precocious plant regeneration. Elongated shoots were rooted on half-strength MS medium amended with 2.9µM IAA and achieved maximum root number (8.7) within 10days. Rooted plantlets were acclimated in soil with 92% survival rate. Molecular marker analysis of in vitro regenerated and field grown plants revealed no somaclonal variations. Briefly, amino acids and activated charcoal could significantly enhance the foxtail millet direct multiple shoot proliferation and plant regeneration. Here we report, a short-term, genotype independent, direct plant regeneration protocol for future genetic transformation studies in foxtail millet genotypes.

Ramlov F., Plastino E.M. and Yokoya N.S. 2013. Growth, callus formation and plant regeneration in color morphs of Gracilaria domingensis (Gracilariales, Rhodophyta) cultured under different irradiance and plant growth regulators. Phycologia 52: 508–516. DOI: 10.2216/13-139.1The effects of plant growth regulators and irradiance on growth, callus formation and the process of direct and indirect regeneration were studied in axenic cultures of the green and red morphs of Gracilaria domingensis. Apical and intercalary segments from both color morphs were cultivated in ASP 12-NTA solid medium (0.5% agar) supplemented with auxins (indole-3-acetic acid, IAA; 2,4-dichlorophenoxyacetic acid, 2,4-D) and cytokinins (kinetin; 6-benzylaminopurine) in concentrations ranging from 0.5 to 50.0 μM and under 50 and 150 μmol photons m−2 s−1. In experiments to induce direct and indirect regeneration, explants and basal calluses isolated from apical and intercalary segments were cultivated in ASP 12-NTA liquid medium for 4 wk and then Von Stosch liquid medium for 3 wk under the same conditions as those noted for solid medium culture. The response to plant growth regulators and irradiance was dependent on the explant and color morph. In general, 2,4-D inhibited growth processes, while IAA and 6-benzylaminopurine were stimulatory. Neither plant growth regulator nor the two photon flux densities (PFD) influenced callus formation, although basal callus formation was greater than intermediate or apical callus formation. Compared to indirect plant regeneration, the percentage of direct plant regeneration was higher, and IAA (50.0 μM), 2,4-D (0.5 μM) and 6-benzylaminopurine all stimulated direct plant regeneration when color morphs were cultured under 150 μmol photons m−2 s−1. Our results indicated that plant growth regulators and PFD played a regulatory role in growth and direct regeneration. However, the color morphs of G. domingensis showed little potential for regeneration from callus, with direct regeneration being more suitable for micropropagation.

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 μM 6-benzylaminopurine (BA), 1.4 μM indole-3-acetic acid (IAA), 370 μM adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 μM indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.

High frequency direct plant regeneration from leaf and petal explants was accomplished for the first time in Streptocarpus varieties. The shoot induction frequency varied with respect to the benzylaminopurine (BAP) concentration added to the Murashige and Skoog (MS) medium. MS medium with 0.5 mg l−1 BAP exhibited the highest (69.9%) plant regeneration frequency with an average of 186 shoots per explant. A higher concentration of BAP inhibited shoot bud induction and plant regeneration along with necrosis of explants. Petal explants derived from the varieties ‘Branwen’ (pink and white) and ‘Chorus Line’ (violet and white) displayed plant regeneration frequency of 22.2–47.4% (within a total of 12 weeks) on MS medium containing 2.0 mg l−1 α-naphthaleneacetic acid and 0.5 mg l−1 BAP for 8 weeks followed by 4 weeks on MS medium with 1.0 mg l−1 BAP. Scanning electron microscopy confirmed direct plant regeneration without callus. Regenerated plants from leaf explants with well-developed leaves and roots were hardened and successfully transferred to pots in glasshouse exhibiting 86% survival at the end of 4–6 weeks. Whereas, regenerated plants from flower petal explants upon transfer to pots in glasshouse exhibited 75–82% survival at the end of 4–6 weeks.

ABSTRACTThis study aims to establish plant tissue culture and regeneration systems of five different sunflower (Helianthus annuus L.) genotypes: Trakya 259, Trakya 80, Trakya 129, Trakya 2098 and Viniimk 8931, which are commercially important for Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) media supplemented with various plant growth regulators using hypocotyl and cotyledon explants. The highest shoot regeneration was observed using hypocotyl explants with Trakya 259 genotype (40%) on MS media supplemented with 1 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (α-naphthalene acetic acid). Hypocotyl explants from other genotypes showed regeneration efficiencies as followed: Trakya 80, 33%; Trakya 129, 29%; Trakya 2098, 22% and Viniimk 8931, 19%. Shoot regeneration efficiencies with the cotyledon explants on the same medium were lower in comparation with hypocotyl explants as followed: Trakya 129, 20%; Trakya 2098, 10% and Viniimk 8931, 9%. In addition, two genotypes (Trakya 259 and Trakya 80) were non-responsive on the same media with cotyledon explants. All of the regenerated shoots were rooted on MS media supplemented with 1 mg/l IBA (indol-3-butiric acid). The results obtained in this study will be useful for the improvement of gene transfer systems to these commercially important sunflower genotypes.

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

An efficient, simple, and rapid regeneration system was established for bananas (Musa sp.) using sections of corm containing intercalary meristematic tissues as explants. Six different cultivars of banana (‘Mpologoma’, ‘Nakitembe’, ‘Mbwazirume’, ‘Pisang awak’, ‘Sukali ndiizi’, and ‘FHIA-17’) with diverse genetic constitution and ploidy levels were regenerated on Murashige and Skoog (MS) medium supplemented with 5 mgl−1 of 6-benzylaminopurine. All the cultivars showed relatively high regeneration efficiency. About 93–97% of the explants regenerated, producing, on average, 12–13 shoots from whole section and 16–19 shoots in total from quarter pieces of each section. Regeneration from intercalary meristematic tissues offers a simple, efficient method for transformation of a broad range of banana cultivars, including East African Highland bananas.

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.

In vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron (TDZ) and 6‐benzyladenine (BA) in Murashige and Skoog (MS) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA, and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L−1 TDZ and 1 mg L−1 BA. Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T0 plants and T1 progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.

Direct plant regeneration of three eggplant genotypes, viz., BL-5, BR-14 and BSR-23 was studied with hypocotyl, cotyledon and leaf explants on Murashige and Skoog (MS) medium fortified with different concentrations BAP and Kin. The interaction of genotype, medium and explant gave 80.36% regeneration from cotyledons of BL-5 on MS medium fortified with 3.0 mgl−1 BAP and 75.19% for cotyledons along with 3.0 mgl−1 BAP and 1.0 mgl−1 Kin. However, the same genotype induced 21.76 buds per cotyledon on MS medium supplemented with 2.5 mgl−1 BAP + 1.0 mgl−1 Kin. In general, maximum shoot elongation (%) was recorded in BSR-23 (58.53%) followed by BL-5 (56.16%), whereas, maximum rooting (%) was induced in BL-5 (57.50%), followed by BSR-23 (57.14%). The rooted plantlets were hardened on wet filter paper with 0.2% carbendazim solution for 20 days, transferred to polythene bags in greenhouse and thereafter, at 4–5 leaf stage to the earthen pots for growth and flowering.

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

A method has been developed for the regeneration of the banana cultivar Dwarf Brazilian (Musa spp. AAB group). Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l α-naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline. Subsequent transfer to MS supplemented with 10% coconut water produced rapidly proliferating embryogenic callus that developed into secondary somatic embryos (SE2); these were subcultured on half-strength MS supplemented with 5 mg/l 6-benzylaminopurine (BA). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l BA for development, and mature SE2 were isolated and cultured on hormone-free MS for germination and development into plantlets. Approximately 90% of the SE2 germinated and developed into plantlets, and these were subcultured onto MS medium plus 0.1% activated charcoal, 1 mg/l BA and 1 mg/l IAA where complete plantlets developed. Morphologically normal banana plants developed from all of the regenerated plantlets, the first of which were produced within 6 months of culture initiation.

Tissue culture is a rapid and effective method of propagating banana plants, especially for producing pest- and disease-free planting materials. This study aimed to: (1) optimize the Murashige and Skoog (MS) medium by supplementing it with coconut shell charcoal, (2) determine the most effective concentration of coconut shell charcoal for improving root quality and plantlet growth of Raja Bulu banana, and (3) evaluate the influence of charcoal addition during the subculture phase. The experiment was conducted at the Salaman Seed Garden for Food Crops and Horticulture, Magelang, Central Java, from February to March 2025. A Completely Randomized Design (CRD) was used with four treatments: MS only (A1), MS + 0.5 g/L charcoal (A2), MS + 1.0 g/L charcoal (A3), and MS + 1.5 g/L charcoal (A4). Parameters observed were root formation percentage, root mass, plantlet height, and stem diameter. The results showed that the addition of coconut shell charcoal significantly affected stem diameter. All treatments yielded homogeneous root formation, with 100% root formation in MS without charcoal and 83% for all other treatments. The best average root mass and stem diameter were obtained at 1.0 g/L concentration, suggesting a potential benefit of coconut shell charcoal at this level in promoting root growth and strengthening stem development.