Validating field regeneration capacity for selected accessions of Gossypium hirsutum using callus induction and regeneration capacity

Background Gossypium hirsutum undergoes rapid clonal propagation to regenerate a mature plant through tissue culture.However,the correlation between cotton leaf regeneration,callus induction,and regeneration ability was still obscure.In this research,cotton leaf regeneration level for 21 accessions in the field(new leaves)was observed after the first harvest,and a comparison between field regeneration level and callus induction with its regeneration capacity(new shoots and roots)for the same 21 accessions was carried out.Agronomic traits,including plant height,leaf area,fresh leaf weight,dry leaf weight,the number of flowers and bolls,and biochemical(proline content)and physiological(chlorophyll and carotenoid content)traits during the flowering stage of 21 upland cotton accessions,were investigated.Result A significant correlation between physiological parameters and callus induction was discovered.Callus induction and regeneration capacity of roots and shoots for hypocotyl,cotyledons,and shoot tip tissues were used to validate field leaf regeneration level after the first harvest.CCRI 24 showed significant leaf regeneration in the field and callus induction capacity through callus induction and regeneration.Conclusion We found a substantial relationship between field regeneration capability and callus induction with its regeneration capacity for the hypocotyl,cotyledons,and shoot tip.The results showed that ZS061,Lumian 378,Jimian 863,and ZS065 have the highest moisture retention capacity,while CCRI 24,Liaoyang Duomaomian,and Beizhe Gongshemian have the lowest moisture retention capacity.CCRI 24 has the highest leaf regeneration capacity in the field,while Beizhe Gongshemian has the lowest leaf regeneration capacity.All our result provides a clue for checking the regeneration capacity through leaf regeneration level in the field.

Callus Induction of Young Leaf Coconut cv. MATAG with Combination of 2,4-Dichlorophenoxyacetic Acid (2,4-D), α-Naphthalene Acetic Acid (NAA) and Benzyl Amino Purin (BAP)

This research was to study in vitro callus induction in Coconut cv MATAG from young leaf explants. Young leaf segments from mature coconut were cultured on Y3 medium supplemented with different concentrations of 2,4-D and a combination of NAA and BAP. Each of these plant growth regulators (PGR) gives different responses toward callus formation, the percentage of explants producing callus, the percentage of callus proliferation, and the morphology of callus. A series of different concentrations were used for 2,4-D (1, 5, 10, 20, 40, 60, 100 mg/L), NAA (1, 3, 5 mg/L) and BAP (1, 3, 5 mg/L) respectively. The range of days of callus formation using 2,4-D treatments is 7 - 12 months, while the 2,4-D combined with NAA is recorded at 2 - 5 months. Despite the variety of different months between these plant growth regulators for callus formation, the percentages of explants producing callus and callus proliferation are different. The highest percentage of explants producing callus (2.9%) was observed at 2,4-D (40 mg/mL), followed by 2.7% at 2,4-D (10.0 mg/mL) with NAA (1 mg/mL). At a concentration of 100 mg/mL of 2,4-D, the highest percentage of callus proliferation was found, as well.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.

Plant Regeneration through Indirect Organogenesis in Two Cultivars of Pineapple (Ananas comosus L.)

Unavailability of performant planting material of pineapple constitutes a major problem of its cultivation in Africa. For this purpose, indirect organogenesis technique is used to evaluate the in vitro responses of two cultivars of pineapple during the explant’s regeneration. Calli were induced from crown leaf and plantlets leaf of “Smooth Cayenne” and “Sugarloaf cultivars”. Murashige and Skoog medium with vitamins B5 supplemented with different growth regulators combinations were used. BAP and/or 2,4-D have been added to base medium for calli cells’ differentiation while BAP and GA3 have been added for plant elongation. The results indicated that explants from regenerated plantlets leaves cultivated on MS supplemented with copper (II) sulphate 5-hydrate concentrations incorporated had significant (p < 0.0001) influence on callus induction in pineapple cultivars. Likewise, MS medium with NAA (0.5 mg/l) + BAP (1 mg/l) had a highly significant influence with 8.8 differentiated Calli. Also, MS medium supplemented with BAP (3 mg/l) + GA3 (2 mg/l) for the “smooth Cayenne” had significantly influenced (p < 0.0001) Calli regeneration with a high rate of 55.25% plantlets. MS medium containing 0.5 mg/l of NAA + 0 mg/l IBA produced a high number of roots in Sugarloaf whereas the medium containing 1.5 mg/l NAA + 0.5 mg/l (IBA) produced high number of roots in smooth Cayenne. We have established an efficient and reproducible protocol for mass propagation and genetic transformation of pineapple though indirect organogenesis. This protocol may be used in genetics engineering studies for pineapple breeding.

In Vitro Propagation of Agave guiengola Gentry Using Semisolid Medium and Temporary Immersion Bioreactors

Agave guiengola Gentry is an endemic plant from a very small locality in Oaxaca,Mexico.Its conservation status is fragile and can rapidly worsen.Because of its scarcity,this agave has been used solely for ornamental purposes,but it could have other uses if more plants were available.In vitro propagation by enhanced axillary sprouting from stem segments was attained using Murashige and Skoog Basal Medium(MS)as well as basal medium supplemented with cytokinins 6-Benzylaminopurine(BA)or 6-(γ,γ-Dimethylallylamino)purine(2iP).The best treatment for shoot induction in semisolid medium consisted in MS supplemented with 2 mg l^(–1) BA,obtaining a mean of 3.7 shoots per explant.Other interesting responses were observed,such as nodular callus induction using combinations of BA and 2,4-Dichlorophenoxyacetic acid(2,4-D);root induction without Plant Growth Regulators(PGR);and generation of shoot clusters.These clusters constituted an excellent explant for micropropagation in temporary immersion bioreactors,obtaining a propagation rate of 43 shoots per explant with 1 min immersion and 6 h immersion frequencies.All new plants rooted and survived the transfer to soil.This study developed an in vitro propagation scheme to produce individuals that can be used either for reforestation,economical purposes,or to carry out studies in this species to assess its full potential,avoiding exploitation from wild plants.

In Vitro Screening of Cactus [Opuntia ficus-indicia (L.) Mill] Genotypes for Drought Tolerance

Drought is one of the complex environmental factors affecting growth and yield of crops in arid and semi-arid areas of the world. In this context, this investigation was carried out to select drought tolerance cactus genotypes under in vitro condition. An experiment was carried out at Laboratory of Mekelle Agricultural Research Center, Northern Ethiopia. Six cactus pear genotypes namely, Gerao, Keyih Beles, Shenkor, Limo, Lemats Beles and Suluhna were used. Areoles were used as explants in tissue culture. The non-ionic water soluble polymer polyethylene glycol (PEG) of molecular weight 6000 was used as osmoticum to simulate water stress. In the first culture, the MS medium was supplemented with (2, 4-D (4 mg/l) and BA (0.5 mg/l) for callus induction. In all cultures MS medium was supplemented with 0, 10, 20 and 40 g/l polyethylene glycol (PEG) and was solidified with 0.8% agar and 30 g of sucrose. Significant differences were observed among the genotypes, PEG levels. In the first culture highest number of explants initiated callus on medium supplemented with no PEG but had not shown significant difference with 10 g/l PEG. At 10 g/l PEG, the callus induction frequency, callus fresh weight and plantlet regeneration were recorded highest for Suluhna (83.3%, 5.5 g and 63.3%), respectively. At 40 g/l PEG, callus induction frequency, callus fresh weight and plantlet regeneration were produced highest for Suluhna (41.7%, 2.75 g and 45%), respectively but no significant difference with Gerao, Limo and Lemats Beles. However, Shenkor and Keyih Beles were induced callus but became reddish black within 35 days supplemented with 40 g/l PEG. Both shoot and root production decreased with increased PEG level in the medium. At 40 g/l PEG in MS medium, the highest shoot number was in Suluhna genotype (4.33) followed by Gerao (3.67). The highest shoot length was in Suluhna (2.11 cm) with no significant difference with Gerao (2.02 cm). Root number (5.00 and root length (1.41 cm) were in the genotype Suluhna. Survival percentage of in vitro regenerated plantlets was 100% during hardening. By taking into consideration, all the growth parameter tested revealed that Suluhna, Gerao, Limo and Lemats Beles showed better drought stress tolerance at the highest level of PEG while Keyih Beles and Shenkor appeared to be drought sensitive at the highest level of PEG.

Tissue Culture System for Different Hybrid of Indica Rice

The effect of varieties and media compositions on callus induction from rice anther and subsequent plant regeneration was studied. The results showed that the callus induction was not significantly different in different media, but mostly depended on genotypes; mean frequency of callus induction on F1 hybrid varieties showed that the medium supplemented with 100 mg · L-1 concentration of the proline was useful for callus induction; when the anther derived callus sub-cultured on MS medium supplemented with BAP (0.5 mg · L-1), KT (1.0 mg · L-1), IAA (0.25 mg · L-1) and NAA (0.5 mg · L-1) for plant regeneration, the frequency of regeneration ranged from 0 to 54.17%, and the highest differentiation index reached 30.5 after sub-cultured for three times; the plants were rooted after transferred to 1/2MS medium with NAA (0.5 mg · L-1) and IAA (0.5 mg · L-1).

In Vitro Study of Callogenesis and Regeneration Potential of Elite Wheat (Triticum aestivum L.) Cultivars

The potential for biotechnological applications in crop improvement programs requires identifying genotypes that allow cell/tissue culture with predictable plant regeneration. In the past, many genotypes of wheat (Triticum aestivum L.) have been examined for potential use in tissue culture studies. The present research work has also been designed to study in vitro callogenesis expression and regeneration potential of wheat cultivars under controlled laboratory conditions. Seeds of four elite commercial high yielding cultivars of wheat namely: NARC-2011, AAS-2011, PAK-2013 and GAL-2013, were collected from the Crop Science Institute National Agricultural Research Center (CSI-NARC) Islamabad, as the source of plant material for in vitro studies. The seeds were surface sterilized in 10% sodium hypochlorite solutions for 10 minutes with continuous shaking under laminar air flow hood. After that seeds were placed on MS (Murashige & Skoog, 1962) based callus induction and regeneration medium with various concentrations of 2, 4-D and BAP in separate test tubes. Maximum callus induction frequency of 90% for Pak-13 and AAS-11, followed by 87% and 83% for Gla-13 and NARC-11, respectively, was recorded at 4 mg/l and 6 mg/l of 2, 4-D. Similarly, maximum regeneration of 90% for AAS-11 and Pak-13, followed by 80% and 87% for NARC-11 and Gla-13 respectively, was recorded on MS basal medium containing 1.5 mg/l of BAP. An increasing trend in regeneration from 0.5 to 1.5 mg/l of BAP was observed but it gradually decreased with increasing concentration of BAP from 1.5 mg/l for all wheat cultivars. The callus formed under light was golden brown, dry nodule and smooth compact and less embryogenic while under dark conditions, it was white to yellowish white, dry nodule and compact and more embryogenic. Best results for callus induction and regeneration were obtained at temperature (24°C ± 1°C) for all wheat cultivars.

Callogenesis and Antibacterial Activity of Balanites aegyptiaca

B. aegyptiaca, it is a species of economic and cultural importance in various countries, with diverse uses that include: medicinal, charcoal, pesticides and forage and in vitro callus production is important to have many applications in both basic and industrial research on this specie. For the induction of callus, B. aegyptica seed cotyledons were surface sterilized with 90% ethanol for 1 minute and cultivated in MS media supplemented with 2,4-D, BAP and NAA. Both the callus and seed were collected and dried in an oven at 40°C - 45°C. Cotyledon’s seed and callus were grounded into the powdered form using mortar and pestle and stored at room temperature for further use. Five grams (5 g) each of the powder were mixed with 50 ml of the solvents: methanol and n-hexane (1:10) w/v, agitated vigorously and kept on an orbital shaker at 150 rpm for 24 h, then filtered. The extracts of the plant sample were evaluated in agar dilution method which was used to determine the MIC and MBC of the extracts. The auxin NAA in low concentrations (0.5 mg/L) in the presence of a dose of 0.5 mg/L of the cytokinin BAP induced 100% callus formation. The 50 and 100 mg/ml methanolic extracts were more effective than the n-hexane extracts for both the gram-positive and gram-negative bacteria. By callus extracts under 100 and 50 mg/ml reveals that methanolic extracts of callus had the highest zone of inhibition. An effective protocol for callus induction has been developed that can use for germplasm conservation or for genetic engineering. Evidence from the present study revealed both extracts possess strong broad-spectrum antibacterial effect. Therefore, methanolic extract of seed kernel callus of B. aegyptiaca can be utilized as a new source of broad spectrum antibacterial drugs for effective control of bacteria related diseases.

Plant Regeneration through Somatic Embryo in Herpetospermum pedunculosum, an Endangered Tibetan Medicinal Herb

Objective An effective reproducible protocol for complete plant regeneration via somatic embryogenesis has been developed for Herpetospermum pedunculosum, an endangered Tibetan medicinal herb. Methods The cotyledon explants used in this study were excised from seedlings germinated in vitro. Callus was induced from cotyledon explants on Murashige and Skoog's medium, supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1-1.0 mg/L) alone or in combination with 6-benzylaminopurine (BA, 0.5, 1.0, and 2.0 mg/L). Results The calli showed differentiation of globular embryos after three weeks of incubation on MS medium supplemented with various combinations of BA and NAA. Sixty-two percent of the embryogenic calli produced somatic embryos in MS basal medium supplemented with BA (1.0 mg/L) + NAA (2.0 mg/L). The addition of KN (0.5 mg/L) to MS medium containing both BA and NAA (2.0 mg/L each) significantly increased the frequency of somatic embryogenesis. The maximum percentage of embryogenic calli formation was 83%, and globular embryos formed and germinated successfully in this medium. Then, transferring the regenerated plants from this medium to hormone-free MS medium will further enhanced the development of the plants, and the healthy plantlets are formed successfully within four weeks. The plantlets were transferred to soil to acclimatize under greenhouse conditions and 75% survived. Conclusion Somatic embryogenesis protocol as reported here can play a key role in the propagation and conservation of this endangered species.