Highest Callus Induction Frequency Research Articles

Determination of phosphinothricin and paromomycin selective concentrations for obtaining transgenic spelt plants

Aim. To determine the selective concentrations of phosphinothricin and paromomycin for the selection of transgenic plants of spelt wheat. Methods. Shoot apical meristem culture, mature embryo culture, Agrobacterium-mediated genetic transformation. Results. Isolation and cultivation of shoot apical meristems of seedlings from three spelt genotypes and mature embryos from three other genotypes were carried out. A high frequency (from 80 to 100 %) of callus induction from explants was observed. It was shown that the addition of 5 mg/l of phosphinothricin or 100 mg/l of paromomycin to the culture medium almost completely inhibited plant regeneration compared to the control. After Agrobacterium-mediated genetic transformation of calli with a vector containing the phosphinothricin-N-acetyltransferase gene, regeneration of spelt shoots for one genotype was observed on a selective medium with 5 mg/l phosphinothricin. Conclusions. The selective concentrations of herbicide and antibiotic for obtaining transgenic spelt wheat plants with the corresponding marker genes are 5 mg/l for phosphinothricin and 100 mg/l for paromomycin.

Optimizing Micropropagation Protocol of Rose (Rosa hybrida L.) cv. ‘Double Delight’

An experiment was conducted to develop a suitable in vitro plant regeneration technique for Rosa hybrida L. cv. ‘Double Delight’ using nodal segments and leaf tissues as explants through direct and indirect organogenesis. Aseptic explants were inoculated onto gelled MS medium contained various strengths of plant growth regulators alone and in combinations for callus and direct shoot induction. MS medium containing 2.0 mg/l BAP was found to be the most effective for direct shoot induction from nodal explants (87.5%) as it produced the maximum number of shoots per explant. The highest callus induction frequency was found to be 80% in leaf tissue, 72% in nodal segments and 96% in leaf and intermodal segments of in vitro raised plantlets in MS medium containing 2.5 mg/l 2, 4-D. The best percentage of indirect shoot organogenesis (93.33%) with maximum number and length of shoot were found when the different explant-derived callus was transferred in 3.0 mg/l BAP supplemented medium. The direct and indirectly induced shoots were multiplied on MS medium containing 3.0 mg/l BAP, where the average number and length of shoots per culture were 7.20 ± 0.80 and 5.22 ± 0.47 cm, respectively. Maximum rooting (80%) was observed in ½-strength of gelled MS medium containing 15.0 g/l sucrose with 1.0 mg/l IBA. Plantlets with the proper root system were then placed in a polybag with a 1:1:1 ratio of sand, garden soil, and compost, and they had a survival rate of about 76%. Plant Tissue Cult. & Biotech. 33(1): 25-36, 2023 (June)

Establishment of a suitable regeneration protocol for rapid propagation of Piper nigrum L. through in vitro culture

An experiment was performed to establish a suitable regeneration protocol for the rapid propagation of Piper nigrum L. using nodal segments and leaf tissues as explants through direct and indirect organogenesis. After surface sterilization, several types of explant were inoculated onto gelled MS medium containing various concentrations and combinations of growth regulators for callus and direct shoot induction. The highest callus induction frequency was 92% and 84% in the case of leaf tissues and nodal segments, respectively, in gelled MS medium containing 2.0 mg/l 2,4-D. Multiple shoots (6.43±0.35 shoots per unit callus) were obtained when the calli from both explant types were cultured on MS medium containing 1.0 mg/l BAP. Nodal segments showed the best result (85%) in terms of direct shoot induction in MS medium supplemented with 1.5 mg/l BAP, where the highest number of shoots per explant was 5.03±0.69. The directly induced shoots were multiplied and elongated on MS medium containing 1.0 mg/l BAP and 0.5 mg/l IBA, the number and length of regenerated shoots per culture being 6.07±0.39 and 5.84±0.65 cm, respectively. The best response to root induction (86.67%) was observed when shoots were transferred to ½ strength of gelled MS medium supplemented with 1.5 mg/l IBA within 16-24 days, with 13.60±1.76 roots per shoot unit. The well-rooted shoots were successfully acclimated in a mixture of soil, sand, and compost (1:1:1) with a survival rate of 88%.
 Jahangirnagar University J. Biol. Sci. 11(1 & 2): 31-40, 2022 (June & December)

In vitro regeneration through organogenesis in Costus igneus–an important herbal insulin plant

Costus igneus is an important high-valued herbal insulin plant. An efficient protocol for in vitro plant regeneration through organogenesis from leaf and nodal explants was standardized. Explants were incubated in Murashige and Skoog (MS) medium amended with diverse levels of plant growth regulators (PGRs). Optimum frequency (68.25%) of adventitious shoot regeneration was obtained from nodal segments cultured onto MS medium containing 2.0 mg/l of 6-benzylaminopurine (BAP) with an average number of 5.0 shoots per explant. Further, MS medium containing 1.5 mg/l 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 1.0 mg/l Thidiazuron (TDZ) produced a higher frequency of callus induction (55.22%) from leaf explants. Optimum shoot regeneration response (66.0%) through indirect organogenesis was observed from leaf-derived callus on MS medium fortified with BAP (2.0 mg/l) and kinetin (0.5 mg/l) with 6 shoots per callus with mean shoot length of 8.0 cm. The shootlets obtained from node and leaf cultures were rooted with a frequency of (63.52%) on MS medium with 1.0 mg/l indole-3-acetic acid (IAA). Plantlets obtained through organogenesis were successfully acclimatized in the greenhouse and field conditions. There were no significant differences among frequency of somatic plants regenerated through either direct or indirect organogenesis. The present study reports on successful plant regeneration through direct and indirect organogenesis in C. igneus. The regeneration protocols discussed here can be used effectively for large-scale multiplication, germplasm survival, pharmacological and genetic manipulation research.

In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

In Vitro Doubled Haploid Production of Bacterial Blight Resistant Plants from BC2F1 Plants (Ranbir Basmati X Pau148) Through Anther Culture

Doubled haploid plants are very important for the development of complete homozygous plants from heterozygous parents in one generation as they possess duplicate copy of haploid chromosome. Haploid production is easily obtained from in vitro anther culture. The present study was undertaken with the objective to develop doubled haploids using anthers for in vitro induction of callus on N6 medium supplemented with various combinations and concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5-2.5 mg/L), Kinetin (0.5-1.0 mg/L) and Naphthalene acetic acid (NAA) (2.0 mg/L) as callus induction medium (CIM). The highest callus induction frequency was obtained when N6 medium fortified with 2,4-D (2.5 mg/L), Kinetin (0.5 mg/L) and NAA (2 mg/L) of 10.07 per cent. The induced callus was sub cultured for shoot regeneration on Murashige and Skoog medium (MS) supplemented with growth regulators: Kinetin and NAA (0.5 mg/L each) in combination with BAP (0.0 - 2.5 mg/L). MS medium supplemented with NAA (0.5 mg/L), Kinetin (0.5 mg/L) and BAP (1.5 mg/L) was most responsive exhibiting regeneration frequency of 28.1 per cent which resulted in maximum regeneration of green plantlets and only 5.21 per cent of albinos. Individual plantlets were separated and immersed in liquid MS medium augmented with NAA (0.5-1.0 mg/L) and BAP (0.5-1.0 mg/L). Maximum rooting was observed in MS medium with NAA (0.5 mg/L) and BAP (1.0 mg/L). The survival rate of in-vitro raised plants was 51.51 per cent. Of these surviving plants, 21 plants were observed to have the sterility percentage above 50 percent and hence can be considered as the doubled haploid plants. Plant DH8 is susceptible and DH20 is heterozygous for gene Xa21. Two plants are susceptible for gene xa13

Micropropagation of Alocasia amazonica through Indirect Shoot Organogenesis

The present experiment was conducted to establish an efficient in vitro regeneration protocol for Alocasia amazonica through indirect organogenesis from young leaf segment. For callus induction, leaf explants were inoculated on MS medium containing different concentrations of 2,4-D. Highest callus induction frequency (90%) was recorded on MS medium supplemented with 4.0 mg/l 2,4-D. Remarkable results on indirect shoot organogenesis and multiplication (86.67%) with maximum shoot number per unit callus (11.64 ± 0.37) and shoot length (10.13 ± 0.24 cm) were observed when the leaf derived callus were transferred on MS medium fortified with 1.5 mg/l BAP and 0.5 mg/l NAA. Additionally, the incorporation of 10% coconut water with the medium showed satisfactory shoot growth and development. The best response towards root induction (85%) was achieved on ½ MS medium fortified with 3.0 mg/l IBA, 2.0 mg/l IAA and 2.0 mg/l NAA with an average 22.20 ± 0.87 roots per unit shoot. Well rooted plantlets were successfully acclimatized to the soil where the survival rate was 90%. Plant Tissue Cult. & Biotech. 32(1): 13-20, 2022 (June)

Standardization of efficient regeneration protocol and agrobacterium mediated genetic transformation in RPBio-226 variety of rice (Oryza sativa)

Studies related to the genome engineering in plants require an efficient protocol with high frequency callus induction and regeneration. This present study was conducted at PRR Biotech laboratory, Hyderabad in the year 2020 and aimed to develop an efficient protocol to achieve a high frequency callus induction and genetic transformation of RPBio-226, a Samba Mahsuri rice variety. In-vitro callus induction was carried out using MS media supplemented with different concentrations of 2,4-D viz., 1.0, 1.5, 2.0, 2.5 mg/L singly and in combinations with BAP viz., 0.1, 0.2, 0.4, 0.6 mg/L. Shoot induction was carried out using MS media supplemented with BAP 1.0, 1.5, 2.0, 2.5 mg/L and Kinetin 0.5, 1.0, 1.5, 2.0 mg/L and the Best response observed with 2,4-D and BAP at 2.0 and 0.1 mg/L, respectively. Agrobacterium tumefaciens strain EHA105 harboring PBI 121 plasmid (binary vector) was used in the process of rice transformation. An experiment was planned to use various parameters viz. 0.1, 0.2, 0.3, 0.4 and 0.5 ODs of Agrobacterium cells at 600nm. Embryogenic calli were co-cultivated with Agrobacterium supplemented with different concentrations of acetosyringone (AS) (0, 50, 100 and 150 μM) and 100 μM was found to give the best response for transformation. After Agrobacterium infection, infected calli were selected on the selection media with kanamycin as selection agent. The best hormone for shoot induction was BAP at 1.5 mg/L and kinetin at 0.5 mg/L and highest number of roots was achieved with 0.1 mg/L NAA in combination with 0.2 mg/L IBA. Regenerated plantlets were successfully acclimatized on soilrite mix showing the highest survival rate after 4 weeks of acclimatization. The npt II and Gus specific primers were used for PCR to identify stably transformed progeny that survived kanamycin treatment. GUS Histochemical Analysis of Callus was also carried out for the confirmation of transformation which helps us to generate transgenic plants in future.

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus caryophyllus semperflorens flore pleno) Cultivars

Abstract Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm−3 of 2,4-D and 200 mg·dm−3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm−3 of BA, 0.6 mg·dm−3 of NAA, 5 mg·dm−3 of AgNO3, and 40 mg·dm−3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm−3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Efficient callus induction and regeneration in proso millet

AbstractProso millet (Panicum miliaceum L.) is an important drought‐tolerant crop, and recently it has become a research focus because of its health‐care and nutrition significance. A study was carried out to investigate callus induction and differentiation and plantlet rooting in Shaanmi 1 and Shaanmi 2 with young leaves of the two varieties. Its result showed that in proso millet, the age of aseptic seedlings could influence on inducing calli from young leaves with the highest callus induction frequency at the seedling age of 7 d. N6 medium performed better in inducing calli from young leaves. There did appear to be no calli on MS medium without 2,4‐D, but calli were induced on N6 medium without 2,4‐D in Shaanmi 2, with its induction frequency standing 6.45% and no calli were induced on the medium without 2,4‐D in Shaanmi 1. With 2,4‐D added, there appeared the highest callus induction frequency at a 2,4‐D concentration of 3.0 mg L–1. The optimal medium to induce calli from young leaves in proso millet was N6 medium plus 2,4‐D (3.0 mg L–1) and KT (0.5 mg L–1), with its callus induction frequency at 64.33% (Shaanmi 1) and 68.67% (Shaanmi 2), respectively. The optimal callus differentiation medium was MS medium plus 1.0 mg L–1 6‐BA and 0.5 mg L–1NAA, with its differentiation rate at 87.78% (Shaanmi 1) and 96.29% (Shaanmi 2), respectively. Proline adding at 600 mg L–1 was conducive to callus differentiation. The optimal rooting medium was 1/2MS medium plus NAA (0.1 mg L–1), with its rooting rate at 100%.

Callus Induction and Phytochemical Constituents of Finger Eggplant (Solanum sp.)

This study examined the efficiency of callus induction on optimum concentrations of NAA (a-naphthaleneacetic acid) and BAP (6-benzyladenine) from culturing stem and leaf explants of finger eggplant (Solanum sp.) and investigated the phytochemical constituents of callus tissue. Seeds were sterilized by using 3 and 5 % Clorox solution, which gave the highest number of survival seeds (100 %) and were grown in vitro plantlets. The highest frequency of callus induction (100.00 ± 0.00 %) was obtained from stems and leaf explants that were excised from in vitro plantlets. The stem explants cultured on MS medium consisted of 1.0 mg/L NAA + 1.0 mg/L BAP, giving the maximum mean callus fresh weight (0.14 ± 0.05 g). Meanwhile, the leaf explants cultured on MS medium consisted of 0.5 mg/L NAA + 2.0 mg/L BAP, generating the maximum mean callus fresh weight (0.48 ± 0.10 g). The highest frequency of callus induction (88.00 ± 1.60 %) was obtained in solidified MS medium supplemented with 0.5 mg/L NAA + 2.0 mg/L BAP, producing the maximum mean fresh weight of callus (1.54 ± 0.27 g) and dry weight (0.90 ± 0.01 g). The results of the Phytochemical screenings of callus and dried leaf extracts indicated the presence of alkaloids, flavonoids, terpenoids, and saponins.

Effect of 2,4-d on embryogenic callus induction and evaluation of G418 on growth inhibition in rice calli

An efficient in vitro callus induction study in rice genotypes Kitaake and PR124 was carried out using different concentrations of 2,4-D. Effect of 2,4-D was studied using four different concentrations in Kitaake (1, 2, 3, 4 mg/l) and in PR124 (1, 1.5, 2, 2.5 mg/l). This study found that Kitaake exhibited high frequency of callus induction on MS (Murashige and Skoog) medium supplemented with 2 mg/l 2,4-D, whereas in PR124, 1.5 mg/l concentration of 2,4-D was found to be the best for calli induction. Callus induction frequencies in Kitaake and PR124 were found to be 85 % and 64.4 %, respectively. Further, the study demonstrated that the growth of rice calli was not affected at lower concentrations of G418 and necrosis of calli occurred at 100 mg/l. These optimized concentrations of 2,4-D and G418 would be helpful to obtain desirable transformants in rice through Agrobacterium-mediated genetic transformation.

High frequency callus induction and proliferation of MD2 pineapple (Ananas comosus)

Ananas comosus var. MD2 is currently the most preferred pineapple variety in the international market due to its pleasant aroma and high Brix acidity ratio. In vitro approaches such as callus culture is promising in producing disease-free plantlet. However, there are limited studies reported on callus culture of MD2 variety despite the potential of in vitro regeneration through biotechnological advances. The purpose of the study was to determine the effect of plant growth regulators (PGRs) i.e., 2,4- dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and Thidiazuron (TDZ) on callus induction from leaf explant of MD2 pineapple. Leaf base explants were cultured on Murashige and Skoog (MS) media supplemented with varying concentration of 2,4-D (0.5 to 6.0 mg/L) alone and in combination with BAP (1.0 to 3.0 mg/L). The frequency of callus induction was seen significantly highest (91.67±8.33%) with maximum callus fresh weight (0.25±0.07 g) at a combination of 2.0 mg/L 2,4-D and 2.0 mg/L BAP. The shortest duration of callus formation was seen on day 12 with the lowest concentration of 2,4-D, 0.5 mg/L. There is a moderate correlation between the earliness of callus formation and the frequency of callus induction (P<0.01). The most favourable media for callus proliferation was 6.0 mg/L 2,4-D and 2.0 mg/L TDZ as the highest fresh weight of 1.52±0.03 g was recorded. Callus culture has the potential to be a source of plant material and secondary metabolites production. In this study, 2,4-D and BAP have successfully induced callus in MD2 pineapple.

Plant regeneration from Eucalyptus bosistoana callus culture

Eucalyptus bosistoana is an important durable hardwood species selected by New Zealand Dryland Forest Initiative (NZDFI) for their adaptability to diverse environments and good quality wood. There is no report on in vitro methods for callus induction and plantlet micropropagation of this species, although these methods could assist further genetic improvement of plants. In this study, different combinations of α-naphthalene acetic acid (NAA) and benzyl adenine (BA) at concentrations ranging from 0.5 to 2 mg L−1 were investigated for callus induction, shoot regeneration, shoot elongation and root formation. High frequency of callus induction was obtained when cotyledon explants were cultured on MS (Murashige and Skoog medium 1962) with 1.0 mg L−1 NAA and 1.5 mg L−1 BA. For shoot regeneration from callus, MS medium with 0.5 mg L−1 NAA and 1.5 mg L−1 BA was an effective medium. Shoot elongation was observed on MS medium supplemented with 0.5 mg L−1 NAA, 1.0 mg L−1 BA and 1.0 mg L−1 GA3. High frequency of adventitious root formation in micro-cuttings of E. bosistoana was observed on half-strength MS medium with 0.5 to 1.0 mg L−1 NAA. Finally, pre-acclimatization of the micropropagated plantlets led to 100% survival under glasshouse conditions.

Effect of Growth Regulators on Stevia rebaudiana Bertoni Callus Genesis and Influence of Auxin and Proline to Steviol Glycosides, Phenols, Flavonoids Accumulation, and Antioxidant Activity In Vitro.

Stevia is a plant containing many active compounds, but usually propagated by stem cuttings because of low seed-yield-germination ability. The aim of this study was to investigate the impact of plant-growth regulators on stevia callus induction and growth from somatic tissue, as well as to determine the effect α-naphthalene acetic acid (NAA) and proline (PRO) on the amount of stevioside, rebaudioside A, phenols, flavonoids, and antioxidant activity. Stem and leaf segments were inoculated on a Murashige and Skoog (MS) medium supplemented with different concentrations of NAA and 6-benzylaminopurine (BAP) for callus genesis. The amount of steviol glycosides (SGs) was evaluated using high-performance liquid chromatography (HPLC), and the amounts of total phenols, flavonoids, and antioxidant activity by spectrophotometric methods. The highest callus-induction frequency and callus-mass increase were obtained from the leaf explants in MS medium supplemented with 2.0 μM NAA. The highest amount of SGs, phenols, and flavonoids, and stronger antioxidant activity were determined in the cellular compounds of callus from leaf explant. PRO reduced the amount of SGs and flavonoids. The significantly highest amount of total phenolic compounds was obtained in the callus from leaf explants in the medium supplemented with 2.0 µM NAA and 2.0 µM PRO.

High-throughput in vitro culture system targeting genetic transformation in sugarcane

Conventional recombination breeding suffers setbacks for genetic improvement of sugarcane owing to its narrow gene pool, large complex genome, rare flowering, low fertility, long breeding cycle, and complex environmental interactions. However, production of transgenic plants can be a better alternative to improve quality traits and resistance to biotic and abiotic stresses. The authors report a ready in hand efficient in vitro culture protocol for genetic transformation in a popular sugarcane cv. Sabita (CoOR 03151) of Odisha. 2, 4-D at 3.0 mg/l resulted in highest callus induction frequency (87.8%) with white, friable, and nodular embryogenic calli suitable for plantlet regeneration. 2 mg/l BAP resulted in moderately higher number of shoots/responsive callus and higher percentage of plant survival. However, addition of TDZ (0.05 m/l) with 2 mg/l BAP in R medium (modified MS) may be considered optimum for regeneration of profuse healthy plantlets. A combination of PBZ (0.05 mg/l) with BAP (2 mg/l) and TDZ (0.05 mg/l) revealed profuse multiple shoots (25 microshoots/responsive callus) and higher percentage of survival during follow-up plant establishment than above hormonal supplementation. Among various hormone recipes in MS medium with 3.0 mg/l NAA resulted in excellent rhizogenesis response (88.0%) with more or less normal rooting within 2 weeks. The above efficient and highly reproducible in vitro culture system can be amenable for genetic transformation in this crop.

Optimization of regeneration and transformation system in finger millet (Eleucine coracana L.)

A proficient in vitro regeneration protocol and transformation system is a requisite for genetic improvement of finger millet. Finger millet (Eleucine coracana L.) GN-4 was used for optimization of regeneration protocol. Using seed as a explants, highest frequency of callus induction was observed on MS supplemented with 2,4-D (1.0 mg.L-1), Proline (500 mg.L-1) and Casein enzyme hydrolysate (300 mg.L-1). Among the different combinations used for shoot initiation, MS with kinetin (1.50 mg.L-1) and BAP (0.50 mg.L-1) produced maximum number of shoots (11.00) having highest shoot length (11.2 cm). Within six days, maximum numbers of roots (12.3) were obtained on half strength MS basal media fortified with NAA (1 mg.L-1). In hardening process, maximum survival (44.51%) of plantlets with minimum days for new sprouting (4.4 days) was reported in vermicompost: sand: cocopeat (1:1:1 v.v-1) mixture. Agrobacterium tumefaciens mediated genetic transformation system was optimized and putative transgenic plants were confirmed by PCR. When the effect of the age of the callus and co-cultivation duration were evaluated, forty five days old co-cultivated callus for five days yielded 1.33% frequency of transformation.

Establishment of a maize callus regeneration system from haploid shoot tips

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize. An effective haploid maize shoot tips callus regeneration system was established which enabled homozygous transgenic plants to be obtained in a single generation.

Effect of sucrose on calus induction and green plantlet regeneration in anther culture of Indica x Indica rice

Anther culture is an important biotechnological tool. By rice anther culture homozygous pure lines in the form of doubled haploid (DH) plants can be produced within a year as compared to the long inbreeding method, which might take 8-10 years. Application of anther culture technique in breeding and genetics research is limited due to the very low regeneration frequency of anthers of rice in general, and indica cultivars in particular. Therefore, the successful use of the technique depends on the adequate production of DH plants for selection and field evaluation. Effect of various concentrations of sucrose in callus induction media was investigated on callus induction and regeneration of green plantlets from anther culture of several F1s derived from indica x indica crosses. Cold pretreated anthers of five genotype of anther donor plants (F1s) at 5 °C for 8 days were cultured on N6 callus induction medium containing sucrose at a concentration of 6.0%, 6.5%, and 7.0%. Results revealed that all genotypes showed similar response to callus induction, but a significantly different responses to plant regeneration depended on sucrose level in the callus induction media. Two genotypes, i.e. IR78788 / Inpara 5 and Dendang/Inpari 30 only regenerated albino plantlets. In this experiment, 6.5% sucrose compared to two other sucrose treatments was suitable for inducing high frequency callus induction and high green plant regeneration in all genotypes. The high concentration of sucrose (7.0%) in the culture medium not only resulted in a decrease in the number and percentage of callus formation but also decreased the regeneration of green plantlets.