CRISPR/Cas9-mediated genome editing in rice cv. IPB3S results in a semi-dwarf phenotypic mutant

Nurhayati, Ardie SW, Santoso TJ, Sudarsono. 2021. CRISPR/Cas9-mediated genome editing in rice cv. IPB3S results in a semi-dwarf phenotypic mutant. Biodiversitas 22: 3792-3800. IPB3S is Indonesian lowland rice and high-yielding cultivar. However, plant height posture makes it prone to lodging which could reduce the yield. This study aimed to edit the GA20Ox2 gene by introducing CRISPR/Cas9 GA20Ox2 construct into IPB3S and developing the semidwarf rice mutants. Immature embryo explants of IPB3S were used for the transformation process mediated by EHA105 strain of Agrobacterium tumefaciens carrying pC1300-Cas9/GA20Ox2 and by changing the regeneration medium composition. PCR analysis showed that rice cv. IPB3S genetic transformation gained putative mutant T0 lines carrying the hpt gene (growth efficiency was 47.9%, while transformation efficiency was 19.3%). Using the developed regeneration medium, we have obtained 24 putative rice cv. IPB3S T0 mutant lines carrying hpt. The best medium for regenerating IPB3S was the A medium (regeneration efficiency 73.3%). IPB 8 and IPB 14 were potential to evaluate in the next generation. The shortest plant height for the T1 generation was observed in the IPB 8-3 mutant.

Improving Adventitious Shoot Regeneration and Transient Agrobacterium-Mediated Transformation of Apricot (Prunus armeniaca L.) Hypocotyl Sections

The improvement of previously described protocols for the regeneration of shoots from ‘Canino’ mature seed hypocotyl slices has been accomplished. The effects of different factors such as the part of the hypocotyl used, vacuum-infiltration, 2,4-Dichlorophenoxyacetic acid pulse, vacuum-infiltration and sonication on regeneration and transient transformation were analyzed. When the three slices obtained from the hypocotyls were evaluated separately on regeneration medium, the highest percentages of regenerating explants were achieved in the part close to the epicotyl and in the central part. On the other hand, sonication of the explants for 30 s followed by vacuum-infiltration during Agrobacterium infection for 20 min allowed for an increase in the transformation events. The application of these modifications to the procedure increased the regeneration efficiencies, and transient transformation events and may reduce the frequency of failed experiments. An efficient regeneration/transformation protocol could facilitate its use as a biotechnological technique for apricot breeding.

Cell Suspension Culture and In Vitro Screening for Drought Tolerance in Soybean Using Poly-Ethylene Glycol

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL−1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL−1 sucrose and 7.5 gL−1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

Shoot Regeneration Is Not a Single Cell Event

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

In vitro somatic embryogenesis of superior clones of robusta coffee from Lampung, Indonesia: Effect of genotypes and callus induction media

Hapsoro D, Hamiranti R, Yusnita Y. 2020. In vitro somatic embryogenesis of superior clones of robusta coffee from Lampung, Indonesia: Effect of genotypes and callus induction media. Biodiversitas 21: 3811-3817. This study aimed to investigate the effects of genotypes and primary callus induction media on somatic embryogenesis of superior robusta coffee clones of Lampung. Leaf explants of clones Tugusari, Komari, Tugino, and Wanto were cultured on two types of primary callus induction media (PCIM). PCIM1 consisted of half-strength MS salts, 30 gL-1 sucrose, added with (mgL-1) 0.1 thiamine-HCl, 0.5 nicotinic acids, 0.5 pyridoxine-HCl, 100 Myo-inositol, 200 ascorbic acids, 150 citric acids, and 1 benzyl adenine. PCIM2 consisted of NPCM salts, 30 gL-1 sucrose, added with (mgL-1) 15 thiamine-HCl, 1 nicotinic acid, 1 pyridoxine-HCl, 2 glycines, 130 Myo-inositol, 200 ascorbic acids, 150 citric acids, 1 2,4-dichlorophenoxyacetic acid, and 2 thidiazuron. The highest percentage (100%) of primary callus formation was found in Komari and Wanto clones. PCIM2 resulted in more primary calli than PCIM1. When subcultured to embryogenic callus induction medium, primary calli of clone Komari and Wanto developed into a high percentage of embryogenic calli, while those of the other two turned brown and died. PCIM2-derived primary calli developed into more embryogenic calli. When subcultured on somatic embryo (SE) regeneration medium, these calli underwent the formation of SE of various stages. When subcultured to plant regeneration medium, these SEs developed into plantlets.

Development of an in vitro Callus Induction Protocol and Shoot Proliferation for Selected Jatropha (Jatropha curcas) Accessions

The present study was undertaken to establish a protocol for in vitro callusing of three Jatropha accessions, namely Metema, Adami Tulu and Shewa Robit from leaf explants. The medium supplemented with combination of 4.44 μM BAP and 4.52 μM 2,4-D resulted in maximum percentage of callus (100%) formed for all accessions. The maximum shoot regeneration (66.67%) from callus with 10.13 number of shoot was obtained from Shewa Robit in MS medum fortified with TDZ (2.27 μM ) and IBA (0.49 μM ). The presence of TDZ in the shoot regeneration medium has greater influence on the induction of adventitious shoot buds, whereas MS supplemented with BAP alone and combination with IBA did not induce shoot regeneration from callus culture. The results obtained in the present study would facilitate the high callus induction and regeneration responses in Jatropha for its improvement using biotechnological tools. Plant Tissue Cult. & Biotech. 30(1): 131-141, 2020 (June)

PENGARUH RETARDAN PACLOBUTRAZOL TERHADAP PERTUMBUHAN TEMU LAWAK (Curcuma xanthorrhiza) SELAMA KONSERVASI IN VITRO

ABSTRAK<br />Temu lawak (Curcuma xanthorrhiza) merupakan salah satu jenis<br />tanaman obat potensial untuk dikembangkan. Rimpangnya berguna untuk<br />mengobati penyakit hepatitis dan memperbaiki sistem kekebalan tubuh.<br />Untuk mendukung kegiatan plasma nutfah temulawak saat ini telah<br />dilakukan upaya konservasi secara in vitro di laboratorium Kultur Jaringan<br />Balai Penelitian Tanaman Obat dan Aromatik, Bogor mulai bulan Juni<br />2005 sampai April 2006. Bahan tanaman yang digunakan adalah mata<br />tunas temulawak yang telah tersedia secara in vitro. Media dasar yang<br />digunakan adalah Murashige dan Skoog (MS) yang diperkaya vitamin dari<br />group B. Sebagai sumber energi digunakan sukrosa sebanyak 30 g/l.<br />Perlakuan yang diuji adalah beberapa taraf konsentrasi paclobutrazol<br />yaitu: Paclobutrazol 1,0 mg/l; 3,0 mg/l dan 5,0 mg/l. Sebagai kontrol digu-<br />nakan media dasar MS tanpa paclobutrazol. Rancangan yang digunakan<br />adalah acak lengkap dengan tujuh ulangan. Parameter yang diamati adalah<br />jumlah tunas, panjang tunas, jumlah daun dan akar pada umur 4 dan 7<br />bulan serta penampilan kultur secara visual. Setelah dikonservasi selama<br />tujuh bulan, maka dilakukan uji regenerasi kultur setelah perlakuan<br />paclobutrazol ke dalam media MS + BA 0,1 mg/l. Hasil penelitian<br />menunjukkan bahwa penggunaan retardan paclobutrazol mampu menekan<br />pertumbuhan kultur dan dapat mengurangi periode sub kultur yang<br />biasanya setiap dua bulan menjadi tujuh bulan. Konsentrasi paclobutrazol<br />5,0 mg/l merupakan konsentrasi terbaik karena kultur masih mampu<br />beregenerasi normal setelah konservasi. Hasil aklimatisasi plantlet di<br />rumah kaca dapat tumbuh dengan baik. Plantlet tumbuh dan berkembang<br />tanpa menunjukkan adanya penyimpangan dalam penampilan visualnya.<br />Kata kunci: Temulawak, Curcuma xanthorrhiza, paclobutrazol, konser-<br />vasi regenerasi, in vitro, pertumbuhan, Jawa Barat<br />ABSTRACT<br />Effect of paclobutrazol on temulawak growth during in<br />vitro conservation<br />Temulawak (Curcuma xanthorrhiza) is one of medicinal plant<br />which is potential to be developed. The rhizome is used to heal hepatitis<br />and to improve the imune system of human body. To support the<br />medicinal germplasm conservation, in vitro conservation of temulawak<br />was conducted in Tissue Culture Laboratory of Indonesian Research<br />Institute for Medicinal and Aromatic, Bogor from June 2005 to April<br />2006. Sterile shoots in vitro were used as plant explants. The basic<br />medium used was Murashige and Skoog (MS) medium, supplemented<br />with vitamine from B group. Sucrose as carbon sources, was given 30 g/l<br />into the medium. The treatment tested were several concentrations of<br />paclobutrazol: (1) Paclobutrazol 0.0 mg/l; 1.0 mg/l; 3.0 mg/l; dan 5.0 mg/l.<br />The treatments were arranged in a completely randomized design with<br />seven replications. The parameters observed were number of shoots, shoot<br />length, number of leaves and roots during conservation. After seven<br />months conserved, shoots were regenerated into regeneration medium.<br />The result showed that paclobutrazol at 5.0 mg/l could reduce the plant<br />growth during seven months in conservatioan period and the culture could<br />regenerate normally after transferring into multiplication medium. This<br />technique can be applied to prolong the conservation culture. Plantlets of<br />temoe lawak which were acclimatisized in the glass house grew well<br />without any changes in their performance.<br />Key words : Temulawak, Curcuma xanthorrhiza, paclobutrazol , in vitro<br />conservation, regeneration, growth, West Java

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

Cryopreservation of Brazilian green dwarf coconut plumules by droplet-vitrification

ABSTRACT: This study evaluated the effect of vitrification solutions and exposure time on the cryopreservation of Brazilian green dwarf coconut plumules (BGD) using the droplet vitrification technique. Explants were excised from BGD mature fruits from the Active Germplasm Bank of Embrapa Tabuleiros Costeiros, Sergipe, Brazil. Firstly, embryos were disinfected, and after excision, plumules were pre-cultivated for 72 hours in Y3 + 0.6 M sucrose + 2.2 g L-1 Gelrite® culture medium. Plumules were exposed to PVS2 and PVS3 solutions for 15 and 30 minutes and rapidly immersed in liquid nitrogen (-196 ºC). After cryopreservation, they were thawed in culture medium solution (Y3 + 1.2 M sucrose) and cultured in regeneration medium. The experimental design was completely randomized in a 2x2 factorial scheme (vitrification solutions per exposure times), with five replicates per treatment. Data were compared by the Tukey’s test at 5% probability. Significant differences were observed in the callogenesis percentage for the solutions x exposure time interaction for non-cryopreserved cultures (-NL) and for exposure time after cryopreservation (+NL). PVS2 and PVS3 combined with 15 minutes of exposure promoted the highest callus formation (70 and 100%, respectively) in control cultures. The exposure time of 30 min, regardless of vitrification solution, resulted in 30% embryogenic callus formation after cryopreservation. These results contributed to the long-term conservation of coconut palm.

Patlıcanda In vitro Rejenerasyon Ortamında Farklı Orizalin ve Kolhisin Konsantrasyonlarının Tetraploid Bitki Üretimine Etkisi

The objective of this study is to obtain tetraploid plants in eggplant cultivars, Faselis (F1) and Karnaz (F1), by applying colchicine and oryzalin in in vitro regeneration medium (RM: MS with 10 µM BA and 1 µM IAA). In the study, leaf explants which have been incubated on the solidified RM for 5 or 7 days were cultured on the same medium with 2.5 or 3.75 mM colchicine for 8, 16 or 32 hours; or with 28.8 or 43.2 µM oryzalin for 12, 24 or 36 hours. Then the explants were transferred to RM without colchicine and oryzalin. Callus, buds or short shoots formed in the RM were transferred to MS medium with 0.5 μM BA to obtain long plants. The ploidy levels of regenerants were determined by flow cytometry. In Karnaz, higher tetraploid plant formation was achieved from 3.75 mM colchicine compared to 2.5 mM colchicine. The highest tetraploid plant rate was obtained by applying of 43.2 µM oryzalin for 24 hours to the explants after incubation on the RM for 7 days. Pollen viability of the tetraploid plants produced by application of colchicine and oryzalin were 76.99% and 81.19% and germination were 19.14% and 17.98%. In Faselis, tetraploid plants were obtained by applying 2.5 mM colchicine to the explants for 8 or 32 hours after incubation on RM for 7 days. However, in the oryzalin experiment, the higher tetraploid plants were produced when the explants were incubated for 5 days in the RM. Pollen viability of the tetraploid plants obtained from applications of colchicine and oryzalin were 86.41% and 95.68% and germination were 26.54% and 28.47%.