Immature Leaf Research Articles

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (Saccharum officinarum L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant−1) and multiple shoots (657 secondary shoots explant−1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.

Variasi Morfologi Ubi Jalar Lokal Asal Dataran Tinggi Minyambouw dan Anggi

Sweet potato is superior commodity of Papua because consumed as special food by society in the middle mountain of Papua. Therefore, sweet potato has vital value in economical, social and culture. In the highland Minyambouw and Anggi have been done exploration and characteristic morphology which is observed among other storage root, leaf and petiole. Result of research exploration and characterization are found around 18 local clons which consist of 8 clons in highland of Minyambouw and 10 clons in highland of Anggi. Clon of Simpengey has special characteristic of the primary colour of its storage root skin is brownish orange, the primary colour of its storage root flesh in crem and secondary colour its storage root is white. Clon of syeb Nntung Ninggos has characteristic among other storage root shape is elliptic, storage root skin primary colour is yellow and the most of the immature leaf colour is purple. Morphology can be proved that only 11 clone groups of sweet potato in the both area.

Effect of harmones on callus induction in Maize (Zea mays L.)

Callus induction from explants is a critical process in regeneration, micropropagation and transformation of maize (Zea mays L.) plants. Formation of callus from plant tissues on culture is affected by several factors. This study revealed to establish the effect of genotype, source of explants and auxin concentration on callus induction from five genotypes UMI 757 (G1), UMI 615 (G2), UMI 112 (G3), UMI 285 (G4) and CO 1 (G5) and one hybrid CO H (M) 5 (G6). Callus induction of the six maize varieties was investigated using immature embryos (E1), leaf bits (E2), root tips (E3), hypocotyls (E4) and seeds (E5) as explants with different concentrations of hormones. In this study, immature embryo was taken from 10 to 12 days after pollination (DAP) to get maximum response. The highest percentage of callus induction was observed (99.10) in immature embryo culture and seed culture gave the highest percentage of rhizogenic callus formation when compare to immature embryo. Among the genotypes tested, CO H (M) 5 recorded the highest callus induction percentage on (2D2K2) medium composition.

Optimization of Somatic Embryogenesis Induction of Cassava <i> (Manihot esculenta Crantz)</i>

The embryogenesis (SE) has important role for genetic engineering of cassava (Manihot esculenta Crantz). However, the success of SE induction depend on plant growth regulator s (PGR)s and treatment enriched in induction media. This experiment tried to induce callus formation of cassava from several in vitro explants: immature leaf, apical bud, and internode; and to develop somatic embryogenesis of cassava in several media enriched with tyrosine and copper sulphate (CuSO4) added into media enrich with picloram as treatment. Different response of explants source to callus induction treatment from those three varieties in callus induction as well as friable callus formation were found in this experiment. The best medium to induce varied with variety; MS media supplemented 12 mg/L picloram + 0.5 mg/L CuSO4 was the best for “Adira 4” and half MS and half GD media supplemented 12 mg/L picloram + 100 mg/L tyrosine for “Malang 6”. All treatments resulted somatic embryo which developed indirectly and in morphologically normal somatic embryos

Overexpression of PvGF14c from Phyllostachys violascens Delays Flowering Time in Transgenic Arabidopsis.

14-3-3 Proteins are a family of highly conserved regulatory molecules expressed in all eukaryotic cells and regulate a diverse set of biological responses in plants. However, their functions in flowering of Phyllostachys violascens are poorly understood. In this study, four non-𝜀 Pv14-3-3 genes from P. violascens were identified and named PvGF14b, PvGF14c, PvGF14e, and PvGF14f. qRT-PCR analyses revealed that PvGF14b and PvGF14e exhibited widely expressed in all tested bamboo tissues. PvGF14b was highest expression in root and lowest in immature leaf. Whereas PvGF14c and PvGF14f showed tissue-specific expression. PvGF14c was mainly expressed in immature and mature leaves. PvGF14f was highest expression in mature leaves. These four genes were not significantly differentially expressed in mature leaf before bamboo flowering and during flower development. PvGF14b and PvGF14c were not induced by circadian rhythm. PvGF14c displayed subcellular localization in the cytoplasm and PvFT in nucleus and cytoplasm. Yeast two-hybrid screening and bimolecular fluorescence complementation confirmed the interaction between PvGF14c and PvFT. The overexpression of PvGF14b, PvGF14c, and PvGF14e significantly delayed flowering time in transgenic Arabidopsis under long-day condition. These findings suggested that at least three PvGF14 genes are involved in flowering and may act as a negative regulator of flowering by interacting with PvFT in bamboo.

Comparative phytochemical analysis of Coffea benghalensis Roxb Ex Schult, Coffea arabica L. and Coffea liberica Hiern

Objective: To make phytochemical studies of the leaf, pericarp and seed of Coffea benghalensis (C. Benghalensis) compared with those of the widely known Coffea arabica and Coffea liberica. Methods: The sample extracts were prepared by Soxhlet-extraction. Polyphenol content was analyzed by HPLC-ESI-MS/MS, the identification was carried out based on the retention time, UV and mass spectra of standards and literature data of the detected compounds. Results: Phenolic acids like caffeoylquinic acids, dicaffeoylquinic acids, feruloylquinic acids and coumaroylquinic acid, as well as mangiferin were detected as main constituents in all extracts. Procyanidin trimers were present exclusively in the leaves. In C. benghalensis, main constituents were 5-caffeoylquinic acid and 4-caffeoylquinic acid. Flavan-3-ols were described in all immature and mature pericarp and leaf extracts. Even though 4-feruloylquinic acid was described in both immature and mature seed, dicaffeoylquinic acids were identified only in the mature seed extracts. Mangiferin was present in the leaf, mature pericarp and seed. Conclusions: These analyses provide new chemotaxonomical data for the selected coffees, especially for C. benghalensis. Due to its high polyphenol content, our results indicate its significance of providing new data as a possible source for industry.

Establishment of cell suspension cultures of Garcinia mangostana L.

A tropical medicinal fruit Garcinia mangostana L., which is highly valued for its antioxidant, anti-inflammatory and obesityrelated ailments, was evaluated for its response to callus culture. Immature leaf explant was inoculated on MS basal medium supplemented with 300 mgL−1 glutamine, 200 mgL−1 bavistin, 250 mgL−1 cefotaxime, 1.0 g L−1 activated carbon and different plant growth regulators (BAP, TDZ and 2, 4-D). About 74% of total explants responded for callus induction, with the highest callus frequency of 89% with 10 mgL−1 2, 4-D. The cultures were serially subcultured at 3–4 weeks interval. The friable callus obtained were transferred to liquid MS media supplemented with 10 mgL−1 2, 4-D or 1.0 mgL−1 TDZ and 300 mgL−1 glutamine which resulted in the production of α-and γ-mangostin, which provides a sustainable method of obtaining such xanthones.

Optimization of Somatic Embryogenesis Induction of Cassava (Manihot esculenta Crantz)

The embryogenesis (SE) has important role for genetic engineering of cassava (Manihot esculenta Crantz). However, the success of SE induction depend on plant growth regulator s (PGR)s and treatment enriched in induction media. This experiment tried to induce callus formation of cassava from several in vitro explants: immature leaf, apical bud, and internode; and to develop somatic embryogenesis of cassava in several media enriched with tyrosine and copper sulphate (CuSO4) added into media enrich with picloram as treatment. Different response of explants source to callus induction treatment from those three varieties in callus induction as well as friable callus formation were found in this experiment. The best medium to induce varied with variety; MS media supplemented 12 mg/L picloram + 0.5 mg/L CuSO4 was the best for “Adira 4” and half MS and half GD media supplemented 12 mg/L picloram + 100 mg/L tyrosine for “Malang 6”. All treatments resulted somatic embryo which developed indirectly and in morphologically normal somatic embryos

New phytoplasma subgroup identified from Arecaceae palm species in Grand-Lahou, Côte d’Ivoire

Coconut palms in the south coastal littoral of Grand-Lahou have long been affected by the Côte d’Ivoire lethal yellowing (CILY), associated with subgroup 16SrXXII-B ‘Candidatus Phytoplasma palmicola’-related strains. Recently, foliar discolouration and yellowing symptoms were observed in oil palms (Elaeis guineensis), Rônier palms (Borasus aethiopium), and raffia palms (Raphia vinifera) located in the vicinity of coconut palms (Cocos nucifera) showing CILY-like symptoms in coconut-growing villages of Grand-Lahou. Immature leaf samples were randomly collected from symptom-bearing palms, including coconut palms. Total DNA was extracted and used as a template for a nested PCR assay with universal primers that target the phytoplasma 16S rRNA gene, and the translocation protein (secA) genes. The R16F2n/R2 and SecA amplicons from representative samples of each palm species were purified, cloned, and sequenced. R16F2n/R2 sequences were over 99% identical to phytoplasmas enclosed in group 16SrXXII. Based on iPhyclassifier analyses with 17 restriction enzymes, the phytoplasmas found in C. nucifera, E. guineensis, B. aethiopium and R. vinifera were identical to each other. However, a unique Sau3AI RFLP pattern was observed, and similarity coefficients were lower than 0.97, and so a new 16SrXXII subgroup, 16SrXXII-C, was assigned. Phylogeny based on both R16F2n/R2 and SecA sequences supported this RFLP classification. The presence of the subgroup 16SrXXII-C in the same area where coconut palms are severely infected by the subgroup 16rSXXII-B could lead to different epidemiological constraints that may affect the severity of the coconut disease. Thus, the 16SrXXII-C phytoplasma poses a new threat for other Arecaceae species, which may impact CILY management and control in Grand-Lahou.

Influence of Holoparasitic Plant Cuscuta japonica on Growth and Alkaloid Content of Its Host Shrub Catharanthus roseus: A Field Experiment

Japanese dodder (Cuscuta japonica) is a flowering, parasitic weed that causes major yield losses on crop and forest production. In the present field study, the influence of Cuscuta on the growth and secondary metabolite of its host medicinal shrub Madagascar periwinkle (Catharanthus roseus) was investigated. Vegetative and reproductive growth of the host plant Catharanthus was measured at intervals from 0 to 60 days after inoculation (DAI), and content of three alkaloids, naturally produced by the host, in the host and parasite were measured by HPLC at intervals from 15 to 45 DAI. Dodder infection significantly inhibited biomass accumulation and reproductive growth of Catharanthus, and aerial parts of the host died by 60 DAI. Levels of alkaloids vinblastine, vindoline and catharanthine in the inoculated Catharanthus were lower than in uninoculated control plants, both in immature leaf and in mature leaf, except the alkaloids contents were elevated in host immature leaf compared with uninoculated control plants immature leaf at 15 days. The host plant thus actively responded to dodder infection by accumulating alkaloids acts as a defense molecule at 15 DAI. The alkaloids produced by host plants accumulated in dodder at 15–45 DAI, and the alkaloid content in dodder reached a maximum at 45 DAI. The alkaloids transported from host to parasite act as a sink, the high content of alkaloids in dodder be cytotoxic and accelerated the onset of senescence.

OsHKT1;5 mediates Na+ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice.

Salt tolerance quantitative trait loci analysis of rice has revealed that the SKC1 locus, which is involved in a higher K+ /Na+ ratio in shoots, corresponds to the OsHKT1;5 gene encoding a Na+ -selective transporter. However, physiological roles of OsHKT1;5 in rice exposed to salt stress remain elusive, and no OsHKT1;5 gene disruption mutants have been characterized to date. In this study, we dissected two independent T-DNA insertional OsHKT1;5 mutants. Measurements of ion contents in tissues and 22 Na+ tracer imaging experiments showed that loss-of-function of OsHKT1;5 in salt-stressed rice roots triggers massive Na+ accumulation in shoots. Salt stress-induced increases in the OsHKT1;5 transcript were observed in roots and basal stems, including basal nodes. Immuno-staining using an anti-OsHKT1;5 peptide antibody indicated that OsHKT1;5 is localized in cells adjacent to the xylem in roots. Additionally, direct introduction of 22 Na+ tracer to leaf sheaths also demonstrated the involvement of OsHKT1;5 in xylem Na+ unloading in leaf sheaths. Furthermore, OsHKT1;5 was indicated to be present in the plasma membrane and found to localize also in the phloem of diffuse vascular bundles in basal nodes. Together with the characteristic 22 Na+ allocation in the blade of the developing immature leaf in the mutants, these results suggest a novel function of OsHKT1;5 in mediating Na+ exclusion in the phloem to prevent Na+ transfer to young leaf blades. Our findings further demonstrate that the function of OsHKT1;5 is crucial over growth stages of rice, including the protection of the next generation seeds as well as of vital leaf blades under salt stress.

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

ABSTRACTSuccessful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.

Indirect somatic embryogenesis from transgenic immature leaf of safflower Carthamus tinctorius (Mohler, Roth, Schmidt &amp; Boudreaux, 1967) (Asterales: Asteraceae)

Highly efficient protocol is developed for indirect somatic embryogenesis from transgenic immature leaf of safflower Carthamus tinctorius (Mohler, Roth, Schmidt &amp; Boudreaux, 1967) (Asterales: Asteraceae) explants in solid culture. Here two different half strength mediums like MSG and EMM both were tested in addition to inorganic and organic nitrogen sources and also combination of the reference medium. Inorganic nitrogen source shows about 39.5 number of embryos with 37% embryo germination and organic Nitrogen source like L-glutamine (20 uM) on reference medium influenced 80.5 number of somatic embryos with 44.9% germination. However, maximum of 92.0 number of embryo induction with 52.5% germination was noticed on CH (casein hydrolysate) (1.5 mg/L) containing medium. Combination of inorganic and organic nitrogen sources like NH4NO3 (40 uM) in combinations with reduced form of organic nitrogen casein hydrolysate (CH) (1.5 mg/L) was found to be most suitable for induction of somatic embryos with a maximum of 117 number of somatic embryos with 60.9% germination in immature leaf explants. About 57% plant conversion rate showed in NARI-6 cultivar after 2 weeks of culture. However it showed maximum plant conversion rate about 68% with basal medium along with 1.5% sucrose supplemented with GA3 (1.5 uM) and spermidine (1.0 uM). Germinated embryos with shoot and root poles were isolated and subcultured on suitable root induction medium containing NAA (2.5 uM) + AgNO3 (1.5 uM) showed maximum rooting frequency of about 69.6%.

Influence of Meta-Topolin on Efficient Plant Regeneration via Micropropagation and Organogenesis of Safflower (&amp;lt;i&amp;gt;Carthamus tinctorius&amp;lt;/i&amp;gt; L.) cv. NARI-H-15

The effect of meta-Topolin (mT) was assessed to develop a reliable protocol for efficient plant regeneration of safflower (Carthamus tinctorius L.) cv. NARI-H-15. For micropropagation, 7 - 9 days old shoot-tip explants cultured on MS basal medium supplemented with 3.0 mg/L meta-Topolin (mT) + 0.5 mg/L CPPU showed 97.7% adventitious shoot formation (42.4 shootlets) than node after 45 days of culture. For organogenesis, the seedling explants of immature leaf cultured on 1.5 mg/L CPPU or 1.5 mg/L NAA fortified medium produced high amount of callus than cotyledon and stem calli after 60 days of culture. However, MS basal medium fortified with 4.0 mg/L mT + 1.5 mg/L CPPU was found beneficial to stimulate 100% organogenic response (74.7 shootlets) from immature leaf calli than cotyledon and stem derived calli after 45 days of culture. The healthy plantlets obtained from micropropagation and organogenesis process cultured on 1/4 MS basal salts, 1.5% sucrose (w/v) and 0.8% agar (w/v) medium supplemented with NAA (1.5 mg/L) and mT (0.1 mg/L) produced maximum of 96% (12.8 rootlets) and 84% (7.3 rootlets) adventitious rooting, respectively than mT and CPPU tested medium. However, maximum of 67% and 42% survival rate was noticed when in vitro raised plants from micropropagation and organogenesis were hardened in pots containing soil mix and maintained under green house condition. This optimized regeneration protocol might be helpful in regeneration of new genotypes and cultivars of safflower to improve agronomic traits through in vitro selection process and Agrobacterium-mediated genetic transformation system.

Genetic diversity of farmer-preferred cowpea (Vigna unguiculata L. Walp) landraces in Turkey and evaluation of their relationships based on agromorphological traits

Cowpea (Vigna unguiculata L. Walp) is one of the important vegetable, grain, and fodder legume species grown in the tropics and subtropics. Cowpea is grown on small farms, and locally adapted landraces or populations are cultivated, and genetic improvements are limited by the lack of knowledge of genetic diversity of the indigenous and cultivated germplasm. Characterization and classification of diversity of the germplasm is valuable for both plant breeders and germplasm curators in the development of conservation strategies and identification of plant genetic resources. In the present study, 36 qualitative and quantitative agromorphological traits were used to characterize and assess the genetic diversity of 32 farmer preferred cowpea genotypes collected from diverse cultivated locations. Genetic variation was highly significant, and phenotypic diversity was observed for agromorphological traits. Agromorphological traits were classified by principal components analysis (PCA) into 10 components, which explained 81.18% of the total variation. PCA revealed that seed properties such as weight, diameter, width, length, eye color and immature pod pigmentation, leaf and pod color properties were the primary characteristics to discriminate cowpea genotypes. The hierarchical analysis grouped the genotypes into five clusters, and significant association was not apparent between geographic origin and agromorphological traits. The promising germplasm was identified to improve the landraces for fresh pod, grain, and fodder.

Phytochemical Distribution and Bioactivity of Different Parts and Leaf Positions of Pimenta Dioica (L.) Merr (Myrtaceae)

Pimenta dioica (L.) Merr. (Myrtaceae) is an evergreen aromatic spice widely used in perfumery, food and cosmetic industry in many parts of the world. Present study compared Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC), Total Flavonoid Content (TFC), leaf area (LA), and Fresh to Dry weight ratio of Pimenta dioica leaves at different leaf positions (1st, 2nd, 3rd, 4th and 5th leaf positions) and different plant parts (immature leaf, mature leaf and bark). The TAC, TPC and TFC were determined using Ferric Reducing Antioxidant Power Assay (FRAP), modified Folin–Ciocalteu colorimetric method and calorimetric method respectively. Significantly higher TAC 562.38 ± 9.42 (mg TE/g DW), TPC 279.53±7.02 (mg GAE/g DW) were observed in leaf extract obtained from 1st leaf position. However the highest TFC 303.48 ±8.87 (mg RE/g DW) was observed in 5th leaf position. According to phytochemical distribution pattern, significantly higher TAC [619.84 ±11.98 (mg TE/g DW)], TPC [267.53 ± 5.03(mg GAE/g DW)], TFC [305.48 ±8.87 (mg RE/g DW)] were observed in extracts obtained from bud region. The potential of Pimenta dioica leaf material and bark as a fabulous raw material for food, perfumery and cosmetic industries. Further harvesting of immature leaves could be suggested for better therapeutic benefits.

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

Dioscorea rotundata, commonly known as white yam, is an important staple food crop widely cultivated in West Africa and provides food security to millions of people. Genetic improvements of this crop using the advanced biotechnology tools have been hampered hitherto by the recalcitrant nature of regeneration through somatic embryogenesis. Therefore, we have developed an efficient and reproducible system for plant regeneration via somatic embryogenesis. Explants of different types (immature leaf, node, stem internode, root segment, petiole, and axillary bud) of D. rotundata accession TDr 2436 were tested for their embryogenic potentials on Murashige and Skoog (MS) medium supplemented with various auxins (2,4-D, NAA, and picloram). Among all explants tested, axillary bud explants cultured on MS medium supplemented with picloram (0.5–12 mg/l) favored the production of calli. Maximum proliferation of calli (526 mg fresh weight/explant) was achieved on MS medium supplemented with picloram (0.5 mg/l), casein hydrolysate (600 mg/l), and proline (1 g/l). Histology analysis confirmed that the embryogenic calli produced on this medium were mixed with non-embryogenic calli. Transfer of calli on MS basal medium supplemented with activated charcoal (1 %) changed the color of calli to purple and promoted the production of somatic embryos (87 embryos/callus) as well as adventitious shoot buds. Furthermore, upon transfer to MS medium supplemented with BAP (0.4 mg/l), the embryos continued their differentiation and maturation and germinated into complete plantlets. The adventitious shoot buds produced multiple shoots on MS medium supplemented with BAP (0.4 mg/l). Well-developed germinated plantlets were acclimatized in the screen house with 90 % survivability. Histology studies confirmed that the regeneration of D. rotundata reported here followed dual regeneration pathways. The embryogenic calli regenerated through development of somatic embryos and germinated into complete plantlets, however non-embryogenic calli regenerated through organogenesis and developed multiple shoots. The developed protocol has potential for somatic hybridization, mass clonal propagation, and genetic transformation applications.

Optimisation of a somatic embryogenesis and transformation protocol for farmer-preferred cassava cultivars in Kenya

Cassava (Manihot esculenta Crantz) is a major food crop in developing countries, and holds potential for industrial use. It is, however, affected by various biotic and abiotic stresses that greatly affect its production. The existing regeneration and transformation protocols are not compatible with all cassava cultivars, thus efficient and robust transformation and regeneration protocols for farmer-preferred cultivars need to be optimised for ease of transfer of novel genes. The objective of this study was to develop an efficient transformation and regeneration protocol for a farmer-preferred Kenyan cassava cultivar. We cultured immature leaf lobe and stem explants on Murashige and Skoog (MS) medium, supplemented with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram and á-naphthalene acetic acid (NAA). Plants were recovered on media with 6- Benzylaminopurine (BAP) and GA3 under a 16 hour light/8 hour darkness photoperiod regime. Results showed high regeneration and transformation frequencies for both cultivars. High frequencies of callus induction (&gt;98%) for both cultivars, were obtained when 2,4-D and Picloram were used. Similarly, both auxins initiated somatic embryogenesis, with Picloram producing the highest frequency of somatic embryos (&gt;92%) in TMS 60444, using stem explants. Gus assays revealed high frequencies of transformation of &gt;77% (TMS 60444) and 60% (Kibanda meno mkubwa). This protocol offers promising perspectives for rapid improvement of these cultivars and, therefore, provides a platform for cleaning planting materials, as well as cassava genetic improvement programmes such as control of viral diseases.Key Words: Cytokinin, Manihot esculenta, regeneration protocol

English

Cassava production in India is drastically affected by cassava mosaic disease (CMD) caused by the Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). An attempt was done to develop transgenic cassava lines resistant to SLCMV through RNAi vector targeting a conserved 440 bp of 5’ end of SLCMV Rep (AC1) gene which also overlaps with part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of SLCMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL and finally mobilised into binary vector pART27, to construct pSCR1 which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as selection agent in cassava genetic transformation, Rep-RNAi gene cassettes of SLCMV was cloned into pCAMBIA1305.2 and the constructs was named pSCR2. Agrobacterium mediated cocultivation of cassava embryogenic calli was done with the developed RNAi constructs using two different explants namely, immature leaf lobes and somatic cotyledons. In total, 48 putative transgenic cassava shoots were regenerated on a regeneration medium containing 30 mgl/l hygromycin, of which 2 putative transgenic plants were transferred for hardening. All the putative transgenic cassava plants were PCR-positive for hph gene and Rep gene indicating integration of transgenes of interest. Key words: RNAi constructs, cocultivation, cassava mosaic disease (CMD).

Repellency of selected Psidium guajava cultivars to the Asian citrus psyllid, Diaphorina citri

Huanglongbing (HLB) is the most devastating disease of citrus worldwide. It is caused by bacteria of the genus ‘Candidatus Liberibacter’ and transmitted by two psyllid species, the Asian citrus psyllid (ACP) Diaphorina citri, and the African citrus psyllid Trioza erytreae. Considerable research has been conducted toward developing and implementing HLB and ACP management strategies. With respect to ACP control, of interest is that reports indicate guava, Psidium guajava, can be repellent to ACP. We conducted research to further evaluate repellency of guava to ACP. In one set of experiments, guava oil from five Brazilian guava cultivars (‘J3’, ‘Pedro Sato’, ‘Século XXI’, ‘Thailand’ and ‘Paluma’) was extracted from leaves (immature and mature) by hydro-distillation in a Clevenger-type apparatus and evaluated for psyllid repellency. In a second set of experiments, repellency of guava leaves to ACP was investigated using leaves (immature and mature) from two guava cultivars in Florida, ‘Pink’ and ‘Thai White’. In each set of experiments, repellency was evaluated by releasing ACP adults into a cage with two large vials, one containing a young flush shoot (= immature leaves) of Murraya exotica (a favored host plant of the psyllid, the flush of which is highly attractive to ACP) and one with M. exotica flush and the test material of interest (guava oil, immature guava leaf or mature guava leaf). The adults were free to move throughout the cage and into the vials, and the number of psyllids in each vial was counted after 24 h. The results showed that all guava materials tested had at least some repellency to ACP. Mature leaves tended to have a greater repellent effect than immature leaves. Each of the five oils exhibited repellency. A report in the literature suggested that sulfur compounds associated with guava may be responsible for ACP repellency. Interestingly, the five guava oil extracts we studied were repellent to ACP but none contained any sulfur compounds. Identification of the constituents responsible for repellency could lead to new ACP management tactics.