The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

Background: The sequencing potato DM1-3-516-R44 played an irreplaceable role in the study of gene function. So far, no one research the transformation system about DM. Therefore, our experiment was studied from three aspects: plant regeneration system, optimization of agrobacterium infection conditions and the effect of hygromycin on DM. Results: A relatively suitable method for genetic transformation of DM was obtained: 1) The stem callus induction medium was MS + IAA 0.5 mg/L + 6-BA 2.0 mg/L, the leaf callus induction medium was MS + NAA 1.0mg/L + 6-BA 0.5mg/L and the shoot differentiation medium was MS + 6-BA 3.0 mg/L + ZT 0.5 mg/L. 2) The specific transformation condition was the agrobacterium concentration kept the OD600 = 0.3, and co-culture time consisted 3 days in the dark. The hygromycin concentration chose 8 mg/L to screen the transgenic plants. 3) Using hygromycin to screen about 100 transgenic shoots, 75 shoots were obtained and 53 strains were identified had target stripe by PCR technology. Conclusion: The efficiency of the transformation system we created was over 50%. It provided a good basis for the study of potato gene function.

Adaptation of in vitro regeneration protocol for Brazilian wheat genotypes

ABSTRACT: The availability of an efficient protocol for in vitro regeneration is imperative for genetic transformation. Using genotypes adapted to the target region as a transgenic platform accelerates the development of cultivars. Therefore, this study aimed to adapt an in vitro regeneration protocol for Brazilian wheat genotypes. For this purpose, the in vitro regeneration capacity of immature embryos from six Brazilian wheat genotypes using two protocols of regeneration of somatic embryos was analysed. Furthermore, combinations of 2,4-D and picloram in the callus induction medium were tested in order to improve regeneration efficiency. Genotypes with higher regeneration efficiency were BR18-Terena and PF020037, yielding 0.42 and 1.13 shoots per explant using the Hu and the Wu protocol, respectively. Adding 1mgL-1 2,4-D in the callus induction medium was the most favourable, producing 3.73 and 3.07 shoots per explant for PF020037 and BR18-Terena, respectively. In conclusion, a protocol for regeneration for two Brazilian wheat genotypes recommended and developed to be cultivated at the Cerrado region has been adapted.

МОРФОГЕНЕЗ ЯРОВОЙ ТВЕРДОЙ ПШЕНИЦЫ В КУЛЬТУРЕ ЗРЕЛЫХ ЗАРОДЫШЕЙ

<p>Mature wheat embryo is a convenient type of explants because of its unlimited availability at any time of the year. But the regenerative capacity of the calli derived from mature embryos is low due to the peculiarities of their hormonal status. A high-performance protocol for culturing these explants is necessary to develop to use them in various areas of applied plant biotechnology. Induction and maintenance of a high rate for unorganized growth in plant cell cultures take place on a nutrient medium with high levels of an exogenous auxin, but the presence of a cytokinin is required to induce differentiation processes. We have carried out a study of the various morphogenetic processes in mature embryo cultures of three spring durum wheat genotypes, depending on the time of their cultivation on the callus induction medium. Mature embryos were cultured in the dark at 26 ± 1 °Con Murashige &amp; Skoog (MS) medium containing MS basal salts and vitamins supplemented with 0.7% agar, 3% sucrose, as well as 2 mg L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (callus induction medium). For morphogenesis induction a part of calli was transferred every five days to a differentiating medium of the same composition of salts and vitamins supplemented with 0.5 mg L^-1 2,4-D and 0.5 mg L^-1 kinetin. Cell cultures were grown in the light at 22 – 24 °C with a 16-hour photoperiod. Six variants of time intervals for callus proliferation on the induction medium have been studied (5, 10, 15, 20, 25, 30 days). A variant of cell culturegrowing without transferring to the differentiating medium was examined too. Frequencies of callus induction, morphogenesis induction and regeneration capacity (relatively morphogenetic calli) were calculated.<strong></strong></p><p>We found active callus induction was visible on the 5th – 7th day after placing explants on the MS inducing medium. The greatest level of callusogenesis (92.3%) was discovered under incubating cultures on the original medium for 30 days. After the short-term cultivation of explants on the initiating medium (for five days) new calli on the differentiating medium were not initiated. In this variant, proliferation of the before induced cell clusters was taking place. This resulted in a low frequency of callus formation (44.3%). Development of the primary callus on the inducing medium for 20 – 30 days helped to keep the competence in somatic tissues of mature embryos and generated the largest number of morphogenetic structures of different qualities. The way of morphogenesis depended on the time interval for cell culture growing on the initial medium. Rhizogenesis decreased by 25% after increasing the incubation period to 15 days. This was followed by active nodular structure formation in calli and plant regeneration. For Oasis variety and 12S2-24 line the most effective variant for the realization of regenerative capacity of morphogenetic calli was to incubate cultures on the induction medium for 15 – 20 days and then to transfer them to the differentiating medium. For Pamyati Yanchenko variety the best variant was to grow calli on the induction medium for 25 days. We have shown the significant effects of a genotype and cultivation conditions at different developmental stages of mature embryo cultures from durum wheat. The specificity of a variety began to manifest after 5 – 10 days staying on the induction medium.</p>

Pengaruh Komposisi Media Dasar dan Jenis Eksplan terhadap Pembentukan Embrio Somatik Kakao

<em>The composition of basal medium determines the regeneration success of in vitro culture. The study aimed to evaluate the effect of basal medium in the primary callus induction medium and explant type on the formation of cacao somatic embryo. The research was conducted at the Tissue Culture Laboratory of IAARD, Bogor, from June 2014 to December 2015. Primary callus induction derived from staminoid and petal explants of ICCRI 4 clone used two types of basal medium, i.e. DKW+ 9 µM 2.4-D + 1.16 µM kinetin or WPM + 9 µM 2.4-D + 1.16 µM kinetin.</em> <em>After 14 days, callus was subcultured onto secondary callus induction medium (WPM + 2.4-D 9 </em><em>μM + kinetin 0.58 μM), and then onto DKW medium without growth regulators to induce the formation of somatic embryo. The research was designed in two-factor factorial design with five replications. The first factor was the type of basal medium on the primary callus induction medium (DKW and WPM) and the second factor was the type of explants (petal and staminoid). The results showed significant interaction effect between basal medium type and explant type on the formation of callus and somatic embryo of cacao. The highest percentage of callus formation was derived from staminoid explants on the DKW basal salt medium (92.5%). However, the highest percentage of somatic embryo formation and the number of somatic embryo per explant were obtained from DKW basal salt medium with petal explants (36.5% and 2.3).</em> <em>Therefore, t</em><em>he use of DKW basal salt medium and petal explant were recommended for the induction of somatic embryo of the ICCRI 4 clone.</em>

In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols.

Plant Regeneration from Protocorm-derived Callus of Five Paphiopedilum Hybrids

Callus was induced from protocorms of five Paphiopedilum hybrids (Paph. 03-1, Paph. 03-4, Paph. 03-5, Paph. 03-6, and Paph. 03-7) on callus induction medium [MS inorganics (412.5 mg NH4NO3 instead of 1650 mg and 475 mg KNO3 instead of 1900 mg) and vitamins plus (per liter) 0.1 g myo-inositol, 30 g sucrose, and 2.5 g Gelrite; pH 5.5] containing various concentrations and combinations of thidiazuron (TDZ; 4.5 and 45 μm) and 2,4-D (4.5 and 45 μm). Callus formation was greatest for protocorms of Paph. 03-1, Paph. 03-4, Paph. 03-6, and Paph. 03-7. Among the most competent hybrids, callus formation was greatest among protocorms induced in medium containing 4.5 μm 2,4-D and 4.5 to 45 μm TDZ. Induced calli were transferred to 100 × 15 mm petri dishes containing 25 mL of PLB and plant regeneration medium (similar to callus induction medium) containing various concentrations of either benzyladenine (BA; 0.5, 5, or 10 μm), TDZ (0.25, 2.5, or 5 μm) or no growth regulator (control). PLB and plant formation was greatest on medium containing BA.

Development of a protocol for the micropropagation of mature Eucalyptus grandis clones through somatic embryogenesis

Biotechnology techniques such as micropropagation VIa somatic embryogenesis offer potential significant advances in the improvement of forest species, which could sustain forest production in South Africa, as well as globally, without increased use of land. In order to apply such techniques to commercial breeding and clonal programmes of E. grandis species, it is necessary to develop reliable and efficient protocols applicable to explants of proven superior genotypes. Most of the research on E. grandis somatic embryogenesis has used the genetically variable embryos or seedlings as explant sources, which results in the propagation of material of unproven genetic value. In order to exploit somatic embryogenesis maximally for cloning of superior trees, somatic embryos have to be induced from highly selected and, hence, mature trees. The aim of this investigation was to develop such a protocol for E. grandis and to test its applicability to various E. grandis hybrids. Somatic embryos were induced from buds, stems, leaves and petioles, with petioles and buds giving the best results. Thus, these were selected for further studies which involved testing the effect of medium composition on embryogenic callus induction. Media used for this purpose contained MS or B5 nutrients, 1 mg.l' 2,4-D, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 or 50 g.rl sucrose. All the media tested were able to support induction of embryogenic callus, although the number of explants producing embryogenic calli was affected significantly by the media composition (10-91 %). Callus induction media with B5 nutrients seemed to have a significant effect onn the developmental stage of embryos in the callus induction medium. Presence of 50 g.r! sucrose in the callus induction medium reduced the embryo yield, but the progress of embryo development was enhanced. The callus induction medium containing B5, 1 mg.l' 2,4-D, 0.5 g.rl glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 30 g.l' sucrose was chosen for subsequent studies. Of all the media tested for embryo development, the medium with B5, 2.5 mg.l' 2iP, 0.5 g.r! glutamine, 0.5 g.r! casein hydrolysate, 4 g.r! Gelrite and 50 g.r! sucrose was found to be the most suitable for embryo development to the cotyledonary stage. Experiments involving incorporation of both ABA and 2iP aiming at maturation of E. grandis somatic embryos led to an increase in size of the cotyledonary embryos formed but not to germination.