In vitro regeneration of Pakistani peanut (Arachis hypogea L.) varieties using de-embryonated coteledonary explants (original) (raw)

Evaluation of peanut genotypes for in vitro plant regeneration using thidiazuron

Journal of Biotechnology, 2007

A major limitation with the available protocols for in vitro regeneration of peanut (Arachis hypogaea L.) is their narrow application to very few select genotypes. Here, we report a protocol that can be applied across a broad spectrum of peanut market types, explant types and geographic regions using thidiazuron (TDZ). The effect of the timing of TDZ application to the culturing of both zygotic embryos and subsequent plantlet explants on MS medium is also reported. An extended use of TDZ and at a higher concentration (30 m/l) resulted in the greatest explant shoot average (∼13). However, a limited application of TDZ (10 d) was sufficient to induce shoot formation in peanut. Hypocotyl was the best explant type that induced the greatest shoot average (15) across market types followed by lamina (7.4). Spanish and Valencia were the most efficient market groups that induced shoots across explant types, consistently.

Multiple shoot regeneration in seed-derived immature leaflet explants of peanut ( Arachis hypogaea L

Scientia Horticulturae, 2009

A protocol was developed for organogenesis from immature leaflet explants derived from mature seeds of peanut. Immature leaflets pre-incubated on MS medium supplemented with 13.32 mM BAP + 4.95 mM NAA for 7 days, turned green and enlarged. The enlarged green leaflets produced multiple shoot buds after 1-2 cycles of sub-culture on MS medium supplemented with 13.32 mM BAP. Three cycles of shoot buds on the elongation medium (13.32 mM BAP) produced 6.17 AE 0.47 elongated shoots per explant. The shoot bud formation was genotype independent. All elongated shoots rooted on the medium containing 4.95 mM NAA. The complete protocol gave efficient (>81%) direct organogenesis, leading to the development of plantlets within 4 months. ß

Factors promoting efficient in vitro regeneration from de-embryonated cotyledon explants of Arachis hypogaea L

Plant Cell Tissue and Organ Culture, 2008

Highly efficient (>90%) protocols were developed for in vitro regeneration from de-embryonated cotyledon explants of peanut (Arachis hypogaea L.). Phytohormone combinations and concentrations, explant source and orientation, period of incubation and the response of genotypes were examined for optimization of the regeneration efficiency. Adventitious shoot primordia could be induced from de-embryonated cotyledon explants when (1) the proximal end of the explant was kept in contact with the shoot induction medium-I supplemented with 5 mg l−1 BAP + 2 mg l−1 2,4-D for 4 weeks, or (2) the distal end was kept in contact with shoot induction medium-II supplemented with only BAP at 20 mg l−1 for the first 2 weeks, followed by subculture in the same medium containing 15 mg l−1 BAP for the next 2 weeks. Orientation of placing the explant on the above media was critical for in vitro regeneration. The factors affecting the morphogenic responses like, repetitive organogenesis, shoot elongation, in vitro flowering and rhizogenesis were examined. Shoot bud formation was genotype independent. Histological studies showed multicellular origin of adventitious shoot primordia. The protocols gave healthy and fertile plants within 4 months.

Optimization of factors for efficient recovery of transgenic peanut (Arachis hypogaea L.)

Plant Cell, Tissue and Organ Culture (PCTOC), 2012

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing b-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L L-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glasshouse conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study. Keywords Agrobacterium tumefaciens Á De-embryonated cotyledon Á Genetic transformation Á Grafting Á Peanut Abbreviations AS Acetosyringone BAP 6-Benzylaminopurine DEC De-embryonated cotyledon GUS b-Glucuronidase enzyme hpt Hygromycin phosphotransferase gene nptII Neomycin phosphotransferase gene PCR Polymerase chain reaction SIM Shoot induction medium uidA b-glucuronidase gene Electronic supplementary material The online version of this article (

In Vitro Regeneration Protocol of Kenyan Adapted Groundnut (Arachis hypogaea L.) Genotypes using Cotyledonary Node Explants

Journal of Plant Biochemistry & Physiology, 2019

A reproducible regeneration protocol for ICGV 12991, CG 7 and Red Valencia groundnut genotypes using Cotyledonary Node explants has been optimized. The effect of different BAP concentrations combined with either 2,4-D or TDZ was tested to determine optimum conditions for high shoot induction. Different BAP concentrations were tested to determine an optimum concentration for shoot elongation. Different NAA concentrations were similarly evaluated to determine the best concentration for rooting. Media containing combination of 5 mg/L BAP and 1 mg/L TDZ was the best concentration for shoot induction while media containing BAP at 5 mg/L was the best for elongation of shoots. NAA concentration of 1 mg/L gave the highest number of plants with roots. This works provides a very good protocol which will be beneficial during groundnut tissue culture as well as genetic transformation of groundnuts.

Response of Groundnut Varieties to Plant Growth Regulator (BAP) to Induce Direct Organogenesis

An efficient protocol for plant regeneration has been developed for crop improvement programs. The present study was designed to assess the regeneration response of cotyledonary node explant taken from the germinating seeds of peanut (Arachis hypogaea L.). Complete plants were regenerated from in vitro cultured sectioned cotyledonary nodes. Multiple shoots arose on 6-benzylaminopurine (BAP) supplemented Murashige and Skoog medium (1-50 mg L ), with maximum production occurring at 15 mg L in most varieties. 1 1 1

Regeneration of transgenic peanut plants from stably transformed embryogenic callus

Plant Science, 1993

Embryogenic tissue cultures of Arachis hypogaea L. (peanut or groundnut), have been transformed via microprojectile bombardment. We introduced a gene (hph) conferring resistance to the antibiotic hygromycin under the control of the CaMV 35S promoter. Selection for resistant callus was initiated 4-5 weeks post-bombardment on medium containing 10-20 mg/1 hygromycin. Twelve percent of the bombardments resulted in recovery of a transgenic cell line. An average of two transgenic embryogenic cell lines was isolated per bombardment experiment over 4-6 months of continuous selection. Each bombardment experiment consisted of 11-19 plates, and each plate contained approximately fourteen 25 mm 2 embryogenic callus pieces. Thus, nearly 1% of the bombarded callus pieces produced a stably transformed cell line. Over 100 plants have been regenerated collectively from all of the transformed cell lines. The presence and integration of foreign DNA in hygromycin-resistant callus lines and regenerated plants has been confirmed by polymerase chain reaction amplification of a defined portion of the chimeric gene and by Southern hybridization analysis. Hygromycin resistance was expressed in leaflets from transformed plants which remained green when cultured on basal medium containing hygromycin. Leaflets from control, non-transformed plants turned brown within 3 weeks on the hygromycin-containing medium.

Protocol for regeneration in vitro of Arachis hypogaea L

Electronic Journal of Biotechnology, 2000

The regeneration of peanut Runner varieties grown in the South of the province of Cordoba, Argentina, was studied to count on a regeneration protocol, which is essential to perform genetic transformation of the plant. The induction of somatic embryos was evaluated in different peanut explants cultivated in Cordoba: cotyledons with and without embryos, epycotils and leaflets, of the Tegua, Nahuel and Florman INTA varieties. The induction medium consisted of MS salts with 30 g.l-1 sucrose, supplemented with the following hormones and herbicides: BAP, Picloram, DMA and 2,4-D. The highest percentage of embryogenic callus was produced cultivating epicotyls with 50 mg.l-1 2,4-D. The embryos obtained produced plantlets which, once acclimatized, reached the reproductive stage.

Somatic embryogenesis in peanut: Influence of growth regulators and sugars

Plant Cell, Tissue and Organ Culture, 1993

Somatic embryogenesis and plant regeneration were induced from immature embryonal axes and immature cotyledons of peanut (Arachis hypogaea L. fastigata type cv JLM-1). Influence of different auxins, cytokinins and sugars on somatic embryogenesis from immature cotyledon explants was also investigated. Among the different auxins tested, 2,4-dichlorophenoxyacetic acid (2,4-D) was most effective, producing the highest frequency of responding cultures and highest average number of somatic embryos per responding culture, while dicamba, picloram, indolepropionic acid, a-naphthaleneacetic acid, 2,4,5-trichlorophenoxypropionic acid and a-naphthoxyacetic acid were also effective for embryogenesis. Indolebutyric acid, indoleacetic acid, p-chlorophenoxyacetic acid and trichlorophenoxyacetic acid were not beneficial. Among the four cytokinins tested, zeatin slightly enhanced the frequency of somatic embryogenesis, while kinetin, 6-y-y-dimethylallylaminopurine and benzyladenine were relatively inhibitory. Among the different carbon sources tested, sucrose was the best for embryo induction and at 6% sucrose the highest frequency of responding cultures and average number of somatic embryos per explant were obtained. For inducing embryogenesis from embryonal axes, 2,4-0 was more effective than picloram. Highest plant conversion frequency from somatic embryos was obtained in presence of dicamba or NAA and using cotyledon explants.