Embryogenic Callus Induction Research Articles

Somatic embryogenesis of hypocotyl derived calli from an eggplant cultivar

Optimization of tissue culture and regeneration conditions of eggplant is necessary for achieving different goals such as gene transformation and the development of somaclonal variations. In this study, hypocotyl explants ware used to produce callus in a medium containing different concentrations of NAA and BAP. Moreover, the concentration of the elements Ca, Mn, Mg, Fe and K were measured and analysed between embryogenic and non-embryogenic calli. For shoot elongation, embryogenic calli were transferred to a new culture medium containing 3.5, 4 and 4.5 mg l-1 BAP plus 2 mg l-1 GA3. Finally, produced shoots were rooted in a culture medium containing 1, 1.5 and 2 mg l-1 NAA. Results showed that the best treatment for the embryogenic callus induction was MS medium containing 0.5 mg l-1 BAP plus 0.25 mg l-1 NAA. Two elements, Fe and K, had the highest amount in non-embryogenic calli compare to the embryogenic one. For plant regeneration, MS medium containing 4.5 mg l-1 BAP plus 2 mg l-1 GA3 and 2 mg l-1 NAA were the best treatments for shooting and rooting, respectively. In this study, the best treatments for plant regeneration produced 35 shoots from an explant with 92 % shooting. This regeneration protocol could be useful for gene transformation and micro-propagation studies.

Development of rice mutant tolerance to salinity through combination of gamma rays irradiation and in-vitro selection

Global warming caused a large rise in global sea level poses many threats, especially to a country like Indonesia. One of the threats that affected rice field located adjacent to coastal area is increase level of soil salinity. Inpari 34 and Inpari 35 varieties tolerant to salinity at seedling phase cannot perform well when planted in salinity prone area where the increase in salinity affected by the up and down of sea surface occurred during the planting season. The aims of this experiment were to obtain putative salinity-tolerant mutant (M1) at vegetative and generative phase from Inpari 34 and Inpari 35 through combination of gamma rays irradiation and in-vitro selection. The study consisted of several activities, namely embryogenic callus induction, mutation induction by gamma-ray irradiation, callus in-vitro selection by NaCl, regeneration of selected callus, and plantlet acclimatization and greenhouse grow-out. In this experiment 62 and 54 putative salinity-tolerant mutant lines obtained from Inpari 34 and Inpari 35, respectively.

Role of reduced nitrogen for induction of embryogenic callus induction and regeneration of plantlets in Abelmoschus esculentus L.

Okra (Abelmoschus esculentus L.) is a difficult species to regenerate in vitro because of its recalcitrant nature. Whitish colored ‘non-embryogenic’ callus formation is a major problem in okra tissue culture. In this study, different levels of inorganic–nitrogen (KNO3 and NH4NO3 with 1:2 ratios, 30–60 mM) in MS media with different PGRs combinations were tested to obtain friable and embryogenic callus masses. Hypocotyls produced the highest callus induction (71%) on media containing 1.0 mgL−1 BAP + 1.5 mgL−1 2, 4-D, whereas cotyledons induced (58%) calli on media containing 1.5 mgL−1 NAA + 0.5mgL−1 BAP. Regarding shooting frequency, media containing 40 mM of total nitrogen and supplemented with 1.5 mgL−1 KIN + 0.5 mgL−1 IBA produced maximum shooting (66.5%) with 3.5 shoots per calli from hypocotyl derived calli, whereas, media having 30 mM of total nitrogen supplemented with 1.5 mgL−1 BAP + 0.5 mgL−1 NAA produced (49.50%) shooting with 2.95 shoots per calli from cotyledons derived calli. In contrasts, calli cultured on media containing 60 mM total nitrogen exhibited very low shooting frequency. Activated charcoal (AC) in rooting media promoted root formation with higher survival rates. Healthy and strong roots (79%) were obtained on media containing 2.0 mgL−1 IBA and 100 mgL−1 AC. Rooted plantlets were successfully acclimatized and grew normally. Hypocotyls have more potential for callus induction than cotyledon explants. The results demonstrated that using a modified MS media with reduced nitrogen reprogrammed the compact non-friable embryogenic callus to friable embryogenic callus with high shoot induction frequency. This study provides a contribution towards the development of efficient regeneration and genetic transformation in okra.

Induction of kopyor coconut embryogenic callus using 2.4-D and TDZ

This research was conducted to obtain an effective method to produce true to type coconut kopyor seeds and determine the effect of 2,4-D with or without TDZ in inducing callus to produce somatic embryos. This research was conducted in the Lampung State Polytechnic laboratory with concentrations of 2,4-D 0, 2.5, 5, 7.5, and 10 mg.L-1 with or without the addition of 0.5 mg.L-1. Further analysis of the data used the 5% LSD test. The parameters observed in this study were: percentage of callus explants, callus diameter, callus weight. The results showed that increasing the concentration of 2,4-D and 7.5 mg. L-1 could increase the percentage of embryogenic callus, callus diameter, and callus weight and the addition of 0.5 TDZ concentration to the 2,4-D medium resulted in a decrease in parameters. The best 2,4-D concentration to induce callus embryogenic in kopyor embryos is 7.5 mg. L-1. The use of 2,4-D for embryogenic callus formation can accelerate callus growth and obtain kopyor coconut seeds true to type.

In vitro Plant Regeneration from Mature Seed Explants of Withania somnifera (L.) Dunal, an Important, Rare and Endangered Medicinal Plant

Withania somnifera (L.) Dunal a member of the Solanaceae family, is a traditional medicinal plant commonly known in India as Ashwagandha. It is used for different diseases such as hiccup, cough, rheumatism, tuberculosis, and exhibits excellent antitumor and anti-bacterial activities as well. Direct organogenesis of plants using mature seeds provides faster response and is also a time saving approach, thus the present study was conducted to investigate the optimal concentrations and combinations of plant growth regulators with MS medium for the establishment of an efficient regeneration system in W. somnifera using mature seed as an explant. Therefore, an efficient in vitro protocol for high frequency regeneration has been developed using mature seeds as explant. In the present study, the multiple shoots along with embryogenic callus induction was best seen in MS medium supplemented with BAP (1.5 mg/L) and IAA (0.5 mg/L). Furthermore, MS medium fortified with GA3 (0.3 mg/L) and IBA (3.0 mg/L) alone was suited for shoot elongation and rhizogenesis respectively. The rooted plantlets were hardened and successfully established in the soil. The establishment of a highly reproducible regeneration system would greatly influence the efforts of improvement of the hereby studied medicinal plant species through useful gene transfer technology.

Peculiarities of induction of callusogenesis and morphogenesis in fennel depending on the age of seedlings

The influence of the age of seedlings (of which hypocotyl explants were isolated) and the composition of the culture medium on the callusogenesis and morphogenesis induction of fennel cultivars ‘Mertsishor’ and ‘Oksamit Kryma’ were studied. It was established that with the use of younger seedlings (7-day of age) callus induction began in a larger number of explants for a week of cultivation earlier compared to 14 and 21-day seedlings. The frequency of somatic embryogenesis in the callus obtained from 7-day seedling hypocotyl was almost twice higher than that of the callus from more mature seedling. It was revealed that the cultivar ‘Mertsishor’ had a higher morphogenetic potential compared to ‘Oksamit Kryma’. Improved methods of embryogenic callus induction and plant regeneration can be used for obtaining the initial breeding material of fennel.

Embryogenic potential of the callus of gabirobeira, Campomanesia adamantium (Cambess) O. Berg

Campomanesia adamantium is a native plant species of Brazilian Cerrado with diverse economic potential and great medicinal importance. Its sexual propagation is impaired by the recalcitrance of its seeds, which prevents effective and profitable propagation. With the purpose of establishing commercial crops and minimizing the extractive use of vegetal resources, the aim of the present study was to induce embryogenic calli in nodal segments of gabirobeira, and to determine and characterize their embryogenic phase through the establishment of a growth curve based on cellular characteristics. Calli were induced using nodal segments inoculated in WPM culture medium without the addition of hormones (control) and with different concentrations of 2,4-D, IAA, IBA, NAA or picloram. Cytochemical and SEM analyses revealed cellular characteristics of the formation of meristematic centers that indicated 4.14 μM of picloram to be the best treatment for induction of embryogenic calli, and demonstrating their embryogenic potential. The treatment was used to establish a callus growth curve, from which it was inferred that calli should be transferred to new culture media on the 28th day to maintain cell viability

English

Coffea arabica, F1 hybrid variety, Ruiru 11 is a highly sought after crop in Kenya due to its alleviated resistance to Coffee Berry Disease and Coffee Leaf Rust coupled with high yield capacity and good cup quality. Access to the variety’s planting materials is limited due to challenges with difficulty in propagation using conventional methods of seed and vegetative propagation; and somatic embryogenesis is regarded as a suitable alternative propagation method. Therefore, the current study aimed to establish an induction protocol in F1 composite hybrid Ruiru 11. The current study investigated the effects of genotype and plant growth regulators, auxins and cytokinins, on induction of embryogenic callus in two composite genotypes of C. arabica L. F1 hybrid variety Ruiru 11. Leaf explants from the F1 hybrid were cultured on half-strength Murashige and Skoog (MS) media supplemented with varied concentrations and combinations of plant growth regulators. Callus formation was evaluated weekly until the 60th day. Genotypic effects were assessed based on the difference on callus induction rates, biomass fresh weights and callus formation. The genotypes tested showed highest callus induction 88% (Code 71) and 100% (Code 93) with respect to the formation of embryogenic calli. Highest fresh weight was obtained at 0.973 ± 0.011 g in Code 71 and 0.649 ± 0.03 g in Code 93 in MS media supplemented with 2,4-D + BAP ( 0.53 + 0.11 µM). The observed results are useful in formulating the best growth regulator concentration suitable for mass in vitro propagation genotypes of Arabica coffee hybrid Ruiru 11 through callus induction in vitro of leaf explants. Key words: Somatic embryogenesis, callus, auxins, cytokinin, in vitro.

Embryogenic Callus Induction of Aquilaira Malaccensis Lam . a nd Aquilaria Subintegra Ding Hou

Aquilaria malaccensis Lam. and Aquilaria subintegra Ding Hou belong to the family of Thymelaeaceae which is commonly known as gaharu or agarwood. It is a commercially important tree and identified as a potential aromatic plant. The overwhelming responses in the lodging sector reduce gaharu species in the forest. Mass propagation through plant tissue culture technology will substitute this problem. The present study was conducted to investigate the embryogenic callus induction between these two species. The most optimum sterilization method for both species was sodium hypochlorite 5.0% which gave the highest percentage of aseptic culture (95%) with the absence of tissue browning. The leaves of both species were cultured on Murashige and Skoog, (1962) (MS) media supplemented with combination of various concentrations of 6-benzylaminopurine (BAP) (0.5, 1.0, 2.0 and 2.5 mg/L) and 2,4-dichlorophenoxyacetic acid (2, 4-D) (0.5, 1.0, 1.5 and 2.0 mg/L) and kept under dark condition. The explants produced embryogenic, white and compact callus at the end cut of the explants after two weeks of culture in all treatments. The highest frequency of embryogenic callus formation was observed in explants cultured on 2.0 mg/L BAP and 0.5 mg/L 2,4-D for both species. From the present study, the optimum sterilization technique and embryogenic callus induction for A. malaccensis Lam. and A. subintegra were established.

The explant developmental stage profoundly impacts small RNA-mediated regulation at the dedifferentiation step of maize somatic embryogenesis

Maize somatic embryogenesis (SE) requires the induction of embryogenic callus and establishment of proliferation before plant regeneration. The molecular mechanisms underlying callus embryogenic potential are not well understood. Here we explored the role of small RNAs (sRNAs) and the accumulation of their target transcripts in maize SE at the dedifferentiation step using VS-535 zygotic embryos collected at distinct developmental stages and displaying contrasting in vitro embryogenic potential and morphology. MicroRNAs (miRNAs), trans-acting siRNAs (tasiRNAs), heterochromatic siRNAs (hc-siRNAs) populations and their RNA targets were analyzed by high-throughput sequencing. Abundances of specific miRNAs, tasiRNAs and targets were validated by qRT-PCR. Unique accumulation patterns were found for immature embryo at 15 Days After Pollination (DAP) and for the callus induction from this explant, as compared to 23 DAP and mature embryos. miR156, miR164, miR166, tasiARFs and the 24 nt hc-siRNAs displayed the most strikingly different patterns between explants and during dedifferentiation. According to their role in auxin responses and developmental cues, we conclude that sRNA-target regulation operating within the 15 DAP immature embryo explant provides key molecular hints as to why this stage is relevant for callus induction with successful proliferation and plant regeneration.

In vitro somatic embryogenesis and plantlet regeneration in anchote [Coccinia abyssinica (Lam.) Cong.

An experiment was conducted to investigate an in vitro somatic embryogenesis and subsequent plantlet regeneration in KUWE (G1) and 223098 (G2) genotypes of anchote. Concentrations and combinations of 2, 4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylamimopurine (BAP) were evaluated for embryogenic callus induction. Levels of BAP, singly and in combination with 2, 4-D were tested on full and half nutrient-strength of Murashige and Skoog medium (FMS and HMS) for somatic embryo development. Levels of BAP under FMS and HMS with and without activated charcoal (AC) were evaluated for plantlet regeneration. Highly embryogenic callus, in both genotypes, was obtained on a medium supplemented with 8 mg/L + 1 mg/L BAP. The highest (36.25) number of somatic embryos, in G2, was recorded from HMS + 2 mg/L BAP while FMS + 0.5 mg/L BAP produced 34 somatic embryos in G1. Maximum shoot number in both genotypes and the maximum number of shoots simultaneously developed with root in G2 (4) were obtained on FMS + AC + 2 mg/L BAP. While the maximum number of shoots simultaneously developed with root in G1 (3.75) was obtained on FMS + AC + 1 mg/L BAP. About 42% of plantlets in G1 and 34% in G2, survived after a month of acclimatization.

Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava

High genotype-dependent variation in friable embryogenic callus (FEC) induction and subsequent somaclonal variation constitute bottlenecks for the application and scaling of genetic transformation (GT) technology to more farmer- and industry-preferred cassava varieties. The understanding and identification of molecular factors underlying embryogenic development in cassava may help to overcome these constraints. Here, we described the Arabidopsis thaliana LEAFY COTYLEDON (LEC) LEC1 and LEC2 orthologous genes in cassava, designated as MeLEC1 and MeLEC2, respectively. Expression analyses showed that both, MeLEC1 and MeLEC2, are expressed at higher levels in somatic embryogenic (SE) tissues in contrast with differentiated mature tissues. The rapid expression increase of MeLEC genes at early SE induction times strongly suggests that they are involved in the transition from a somatic to an embryonic state, and probably, in the competence acquisition for SE development in cassava. The independent overexpression of the MeLEC genes resulted in different regenerated events with embryogenic characteristics such as MeLEC1OE plants with cotyledon-like leaves and MeLEC2OE plants with somatic-like embryos that emerged over the surface of mature leaves. Transcript increases of other embryo-specific regulating factors were also detected in MeLECOE plants, supporting their mutual interaction in the embryo development coordination. The single overexpression of MeLEC2 was enough to reprogram the vegetative cells and induce direct somatic embryogenesis, which converts this gene into a tool that could improve the recovery of transformed plants of recalcitrant genotypes. The identification of MeLEC genes contributes not only to improve our understanding of SE process in cassava, but also provides viable alternatives to optimize GT and advance in gene editing in this crop, through the development of genotype-independent protocols.

Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars

Protoplasts are useful research tools for basic and applied plant science, but the regeneration of whole plants from protoplasts is challenging in most of agronomically important crops, including grapevine (Vitis vinifera L.). Here we describe an efficient protocol for the induction of embryogenic callus, the isolation of protoplasts, and the regeneration of whole grapevine plants in two Italian grapevine cultivars. Embryogenic callus was induced successfully from stamens collected from immature flowers. Isolated protoplasts were tested to confirm their viability and then cultivated using the disc-culture method, at a density of 1 × 105 protoplasts/mL in solid Nitsch’s medium supplemented with 2 mg/L 1-naphthaleneacetic acid and 0.5 mg/L 6-benzylaminopurine. After 3–4 months, the protoplasts of both cultivars regenerated with similar efficiency into cotyledonal-stage somatic embryos. The somatic embryos were transferred to solid Nitsch’s medium supplemented with 30 g/L sucrose and 2 g/L gellan gum, and were maintained in the dark for 4 weeks. This step was necessary for the embryo to complete germination, allowing subsequent shoot elongation in response to light on a medium with 4 µM 6-benzylaminopurine. Then root elongation occurred after transferring on a medium with 0.5 µM 1-naphthaleneacetic. After ~ 6 months from the isolation of protoplasts, normal plants were regenerated, which were moved to the greenhouse. The protoplasts could also be transfected using the polyethylene glycol method, as confirmed using a plasmid carrying the yellow florescent protein marker gene. The new method is therefore compatible with biotechnological applications such as gene transfer and genome editing. This study reports an improved protocol for embryogenic callus induction, protoplast isolation and whole plant regeneration of two Vitis vinifera cultivars. Protoplasts showed high transfection efficiency.

Development-Related miRNA Expression and Target Regulation during Staggered In Vitro Plant Regeneration of Tuxpeño VS-535 Maize Cultivar.

In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpeño VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.

Molecular analysis of somatic embryogenesis through proteomic approach and optimization of protocol in recalcitrant Musa spp.

Somatic embryogenesis (SE) is a complex stress related process regulated by numerous biological factors. SE is mainly applicable to mass propagation and genetic improvement of plants through gene transfer technology and induced mutations. In banana, SE is highly genome dependent as the efficiency varies with cultivars. To understand the molecular mechanism of SE, a proteomics approach was carried out to identify proteins expressed during embryogenic calli (EC) induction, regeneration and germination of somatic embryos in the banana cultivar cv. Rasthali (AAB). In total, 70 spots were differentially expressed in various developmental stages of SE, of which 16 were uniquely expressed and 17 were highly abundant in EC compared to non-embryogenic calli and explants. Also, four spots were uniquely expressed in germinating somatic embryos. The functional annotation of identified proteins revealed that calcium signaling along with stress and endogenous hormones related proteins played a vital role in EC induction and germination of somatic embryos. Thus, based on this outcome, the callus induction media was modified and tested in five cultivars. Among them, cultivars Grand Naine (AAA), Monthan (ABB) and Ney Poovan (AB) showed a better response in tryptophan added media, whereas Red Banana (AAA) and Karpuravalli (ABB) showed maximum EC induction in kinetin and CaCl2 supplemented media respectively. Simultaneously, germination media were modified to induce proteins responsible for germination. In cv. Rasthali, media supplemented with 10 mM CaCl2 showed a maximum increase in germination (51.79%) over control plants. Thus, the present study revealed that media modification based on proteomic analysis can induce SE in recalcitrant cultivars and also enhance germination in cultivars amenable for SE.

Establishment of a regeneration system for the production of Calla plants (Zantedeschia spp.) via embryogenic callus culture

Calla lilies (Zantedeschia spp.) are monocotyledonous ornamental plants which belongs to the Araceae family. After the release of elite calla cultivar, an efficient propagation system is needed for commercial use. Despite the use of conventional propagation methods such as splitting of tubers and rhizomes of calla, rapid and efficient propagation system should be developed. In order to achieve this goal, stem segments contained apical meristems derived from calla lily cultivar (cv. Gag-si) were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of cytokinin and auxin. This was aimed at inducing embryogenic calluses, shoots and multiple shoots. As a result, about 25% of induction rates of yellow embryogenic calluses were observed with MS medium containing both 0.5 mg·L-1 NAA and 1.5 mg·L-1 BA as growth regulators. In the experiments involving the regeneration from embryogenic calluses through shoot formation, MS medium supplemented with 0.5 mg·L-1 IAA and 2.0 mg·L-1 BA showed the highest rates at approximately 85 ~ 90% with regard to the formation of shoots in calla. Moreover, multiple shoots needed for rapid propagation were generated when explants were cultured on MS medium supplemented with 0.5 mg·L-1 IAA and 2.0 mg·L-1 BA with 40% of formation rate. In this study, the combination of auxin and cytokinin showed positive effects on both the induction of embryogenic calluses, the formation of shoots as well as multiple shoots in calla. The regeneration system described here can contribute to the development of breeding programs of calla in the future.

Induction of somatic embryos of recalcitrant genotypes of <i>Theobroma cacao L.</i>

Objective: In cocoa tree ( Theobroma cacao L .), some elite genotypes have shown, in standard study conditions, an absence or a very weak response to induction of somatic embryos. This is the case of the original C8 genotype Trinidad. This study aims to improve the production of somatic embryos in this genotype Methodology and results : To do this, staminodes and petals excised from immature buds of genotypes C1, C8 and C14 were used. Genotypes C1 and C14 are embryogenic under standard conditions. These floral explants were cultured on induction media differing by the type and concentration of auxins. Callus induction obtained in the three studied genotypes ranged from 80% to 90% with the petal explants and from 70% to 80% with the staminodes regardless of the type and concentration of auxins. Transfer of callogenic explants to DKW (Driver & Kuniyuki, 1984) medium supplemented with sucrose and glucose allowed the induction of somatic embryos at mean rates varying from 5% to 20% after only 84 days with petal explants and media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2, 4, 5-trichlorophenoxyacetic acid (2,4,5-T). No induction of somatic embryos was observed on the control medium with the C1, C8 and C14 genotypes. The C1 genotype induced somatic embryos in the presence of all concentrations of auxins. The highest rate of embryogenic callus and somatic embryo induction was obtained with 18 μM of 2, 4, 5-T in all C1 genotypes (39.29 ± 0.28; 17.98 ± 0.10), C8 (36.29% ± 0.26 and 15.01 ± 0.07) and C14 (33.92% ± 0.26 and 14.50 ± 0.16). The 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T). at 18 μM is the most appropriate auxin to remove recalcitrance in cocoa genotype C8. The use of higher 18 μM concentrations of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T). and its application to other recalcitrant genotypes could confirm its beneficial effect on the removal of recalcitrance to somatic embryogenesis in cocoa. Conclusion and application of results : 18 μM concentration of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) gave the best percentage of embryogenic explants and the highest average number of embryos in the three genotypes tested. Therefore, this protocol using 18 μM of 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) could be used to overcome recalcitrance to somatic embryogenesis in cocoa. Keywords : recalcitrant, cocoa, auxins, somatic embryogenesis

High speed regeneration via somatic embryogenesis in elite Indian banana cv. Somrani monthan (ABB)

An efficient and rapid regeneration system is indispensable for germplasm conservation and valuable trait incorporation through genetic engineering. The present study was aimed to investigate regeneration potential of Musa sp. cv. Somrani monthan (ABB) via somatic embryogenesis. Meristematic shoot tips were used as explant and cultured on MS medium supplemented with 6-BAP (50 and 100 µM) and IAA (1 µM) for scalp (cauliflower like compact buds) induction. The highest percentage (~ 60%) of homogenous differentiation of shoot tip explants into scalp was observed in MS medium supplemented with 100 µM of 6-BAP and 1 µM of IAA after 6 months of culture. The scalps were sub-cultured on MS medium supplemented with different compositions of 2, 4-D (1, 2, 3, 4, 5 and 10 µM) and zeatin (1 and 2 µM) for embryogenic calli induction. The highest frequency (97%) of embryogenic calli induction was recorded in MS medium supplemented with 5 µM 2, 4-D and 1 µM zeatin after 6 weeks of culture. The histological and morphological studies suggest that, callus possessed high competence for embryogenesis and could be induced to form somatic embryos. Embryogenic cell suspension (ECS) was successfully established by culturing these embryonic calli in liquid medium. About 70% of the mature somatic embryos germinated and converted to plantlets with a regeneration capacity of 8.5 × 103. After the gradual acclimatization and hardening, the plants were transferred to nursery with 82% of survival rate. Here we report a successful regeneration system for an elite Indian Musa cv. Somrani monthan via somatic embryogenesis. The embryogenic callus and ECS are ideal explants for mass clonal propagation, germplasm conservation and genetic transformation for future research.

Micropropagation of traditional deep water rice (Oryza sativa L.) cv. TNR1 for viable seed production and germplasm conservation

Abstract Extinction of traditional crop varieties is becoming a common scenario in the world due to adoption of hybrid/improved varieties. Conservationists are very busy with protection of those verities for novel genes to develop agriculture through molecular breeding. However conservation feasibility is always questionable for traditional seeds because of recalcitrance and low viability in nature. This is the first report on the protection of traditional deep water rice (Oryza sativa L.) cultivar, TNR1 from possible extinction using callus induction and regeneration system. TNR1 is known for its traditional (antidiabetic) and genetic (salinity and submergence resistance genes) importance. The matured embryos were inoculated on LS medium prepared with 2.5 mg L−1 2, 4-D and 0.1 mg L−1 KN showed 74.5% of embryogenic callus induction. The calli was regenerated on MS medium fortified with 1.5 mg L−1 NAA and 3.0 mg L−1 BAP and obtained 83.4% of regeneration. Regenerated plants were successfully acclimatized in the field with 100% survival. The germination percentage of tissue culture derived seeds was found to be high (84.2%) compared to traditional seeds (36.4%). The outcome of this study revealed that, tissue culture enhanced the viability of recalcitrant seeds. In future, this protocol will be an invaluable tool for both transgenic plant production or conservation of threatened traditional rice germplasms.

HISTOLOGICAL ANALYSIS OF INDIRECT SOMATIC EMBRYOGENESIS INDUCED FROM ROOT EXPLATS OF OIL PALM (Elaeis guineensis Jacq)

ABSTRACT Oil palm is economically important as a crop with high oil production. Indirect somatic embryogenesis in oil palm requires a long time for callus induction and plant formation, and it is important to study the embryogenic potential of calli and the mechanisms of somatic embryogenesis. The aim of this study was to test different growth regulators and spermine in induction of embryogenic calli in root explants of oil palm (Elaeis guineensis Jacq). Apex root explants of approximately 0.5 cm were isolated from plants cultivated in vitro and inoculated in Y3 culture medium in the following treatments: A - without growth regulators; B - 1 mg.L-1 picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid); C - 1 mg.L-1 picloram and 2 mg.L-1 2ip (2-isopentenyladenine); D - 2 mg.L-1 2ip; E - 1 mg.L-1 picloram and 2 mg.L-1 BAP (6-benzylaminopurine); F - 2 mg.L-1 BAP; and G - 14.5 mg.L-1 spermine. After six months of culturing, the calli induced in the treatments were analyzed by light microscopy. The calli induced in the treatments with 1 mg.L-1picloram (B) and treatment with 14.5 mg.L-1spermine (G) exhibited embryogenic characteristics, small and isodiametric cells, forming agglomerates, besides a large amount of starch. Calli of the best treatment (Y3 com 1 mg.L-1 de picloram) were inoculated in Y3 culture medium without addition of growth regulators. After eight months, calli were once more analyzed under light microscopy. All the treatments showed callus formation, except for treatments D and A. Calli of treatment B exhibited cells with embryogenic characteristics that developed somatic embryos.