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Control of Organogenesis and Embryogenesis in Rice Calli.

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Abstract

There are two morphogenic pathvways in plant regeneration. These are adventitious shoot and root formation (organogenesis) and somatic embryo formation (embryogenesis). To establish a rice (Oryza sativa L.) culture system, in which highly-frequency of either organogenesis or embryogenesis were achieved, effective regeneration media were investigated. We tested N6 medium containing 20 g·l-1 sucrose, 35 or 50 g·l-1 sorbitol, 3 g·l-1 casein hydrolysate, lO mM L-proline, 10 g'l-1 agarose and different levels of abscisic acid (ABA), auxin, and cytokinin for regeneration. In our culture system, the type of cytokinin was the key to control the morphogenic pathways. One of the most active urea cytokinins, N-(2- chloro- 4- pyridyl)-N' -phenylurea (4PUCl), tended to induce adventitious shoots. A purine cytokinin, kinetin, in low concentration tended to induce somatic embrvos.
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... sucrose, such as albino plant frequency reduced by maltose utilization (Reinert and Bajaj 1977;Sen et al. 2011;Park et al. 2013). Besides these -sorbitol, amino acid, and AgNO 3 were found beneficial for plant regeneration from androgenesis in rice (Yoshida et al. 1994;Ogawa et al. 1995;Faruq et al. 2014). Growth regulators are also used in the in vitro culture of anther and it has a positive impact on in vitro culture (Mandal and Gupta 1995). ...
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A resurrection has started in haploid and double haploid research in the twenty-first century. The haploid and double haploid could be achieved through in vivo and in vitro anther and microspore culture techniques. Fixing the homozygosity is the most striking benefit of androgenesis. Various factors like genotypic dependency, growth condition, developmental stage of the microspore, pre-treatment, culture media, regeneration media, growth hormones, and various chemicals have a direct effect. Wheat, rice, Brassica, and tobacco are the notable crops where anther and microspore culture has been utilized. These haploidy and double haploidy through anther culture served many purposes of basic and applied research. Especially, double haploid cultivars have been cultivating around the globe. In addition, for chromosome mapping, QTL mapping, marker-assisted selection, marker-assisted backcrossing, mutation breeding, genome-wide association study, genomic engineering, and genome editing, androgenesis based haploid and double haploid plants have been exploited due to the effectiveness. Recently, researchers are trying to explain albinism that happens during anther culture from an epigenetic perspective. Further prospects of haploid and doubled haploid research through anther culture have been described in this review.
... The carbon source, usually sucrose, is also necessary due to its osmotic and nutritional properties [13]. It has also been proved that the addition of sorbitol enhances the effectiveness of the regeneration medium [14]. Other medium additives such as colchicine may increase calli induction, reduce the number of albino plantlets regenerated and increase the number of regenerated DH lines [4,15]. ...
Chapter
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Anther culture is the most used technique to produce doubled haploid lines in rice. This technique is well developed in a wide range of indica rice genotypes. However, in japonica type, and more specifically, the Mediterranean japonica, the protocols are yet to be optimized. Japonica and indica have different androgenic response, as well as different induction and regeneration rates, albinism ratios and chromosome doubling competence. The step-by-step anther culture protocol presented in this chapter allows to regenerate doubled haploid rice plantlets from anther microspores in 8 months. We also include an in vitro chromosome doubling protocol to induce doubled haploids from haploid plantlets by immersion in a colchicine solution. This chromosome doubling protocol complements the anther culture by taking advantage of the regenerated haploid plantlets.
... Also, it was the genotype that better responded to any of the three different regeneration media, being GERM the one that regenerated the most plantlets. Yoshida et al. (1994) tried different combinations of sorbitol percentage and hormone concentration in the regeneration medium of two japonica rice cultivars. It would be interesting to carry out an essay of SARM with different hormone and sorbitol concentrations to see the improvement it would have on regeneration rates. ...
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Anther culture is a fast tool to obtain double haploid plant lines for breeding purposes. In rice, this procedure is commonly performed in two steps: i) induction of calli from anthers and ii) regeneration of plantlets from calli. It has been stated that genotype highly influences the anther culture efficiency, so the media used in each step should be optimized for each variety. In this study, we tested different media modifications of an efficient protocol optimized for a medium sized grain temperate japonica NRVC980385, used as a control, in a long grain temperate japonica rice variety (NRVC20120346), and two long grain tropical japonica varieties (303012 and 303013). We found that the addition of 150 mg l⁻¹ colchicine to the induction medium worked best for all genotypes except for NRVC20120346, whose best induction was obtained with the colchicine-free medium. Referring to regeneration, increased gelling agent in the medium provided the best rates in NRVC980385, improving our former NRVC980385-optimized anther culture protocol. Sorbitol fortified regeneration medium worked the best in the case of the long grain varieties. The presence of colchicine in the induction medium was also related to a higher obtention of double haploid plantlets. This study highlights that genotype is a key factor in the performance of rice anther culture. It has set a first anther culture study on long grain japonica varieties and optimizes the anther culture protocol for temperate japonica medium grain NRVC980385 with the use of colchicine and other additives that increase osmotic stress.
... al. (1989),Emons and Kieft (1995),Wright et al. (2001),Lee and Zhang (2013),Nannas and Dawe (2015) andMushke et al. (2016) RiceOryza sativa L. +/++ +/++/+++ +Hayashimoto et al. (1990),Li et al. (1993),Hiei et al. (1994),Yoshida et al. (1994),Nhut et al. (2000) andYu et al. (2005) Sorghum Sorghum spp. +/++ +/++/+++ + Battraw and Hall (1991), Tadesse et al. (2003), Nirwan and Kothari (2004), Girijashankar et al. (2007), Paterson et al. (2009) and Do et al. (2016) Tomato Solanum lycopersicum and Solanum pimpinellifolium +/++ +/++/ + Newman et al. (1996), Ma et al. (2015), Ruma et al. (2009), The 100 tomato Genome Sequencing Consortium (2014), Ray et al. (2015), Razali et al. (2018) and Zsögön et al. (2018) Watermelon Citrullus lanatus +/++ +/++/+++ + Compton and Gray (1993), Suratman et al. (2010), Hema et al. (2004), Krug et al. (2005), Cho et al. (2008) and Guo et al. (2013) Yam Dioscorea sp. ...
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Population growth and climate change demand the constant development of new crop varieties that can produce higher yields, and better organoleptic and nutritional value under adverse biotic, and abiotic conditions. In this sense, traditional breeding and genetic transformation have been used for decades. Nevertheless, the first approach is time consuming endeavor, and is unable to keep up with increasing food demands. On the other hand, genetic transformation is often limited by consumer acceptance. Recent genome editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas9) system allows precise, specific, and low cost edition in a targeted genome region. The wide variety of applications for this technology includes increased yields and nutritional value, stress tolerance, and herbicide resistance. Crops of tropical origin have nutritional and economic importance; therefore, this review will analyze the advances and applications of CRISPR in crops of tropical origin to obtain varieties better adapted to current environmental conditions and market requirements.
... Correlation analysis between the maltose concentration in media and the frequency of albino plants revealed that the application of the maltose minimizes the frequency of albino plants from anthers (Park et al, 2013). Sorbitol has also been reported to enhance the plant regeneration of rice when applied to the regeneration medium (Yoshida et al, 1994). Besides, amino acids have also been used as nitrogen source in in vitro culture of various tissues for enhanced green plant regeneration and production (Ogawa et al, 1995). ...
Article
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In vitro androgenesis is an important component of plant biotechnology when the pollen grains are forced to switch from their normal pollen developmental pathway towards an embryogenic route. Haploid and doubled haploid produced through androgenesis have long been recognized as a valuable tool in plant breeding as it can shorten the breeding cycle, fix agronomic characters in homozygous state and enhance the selection efficiency of useful recessive agronomic traits. Recently, doubled haploids have been largely recognized as an important component of crop improvement through genome mapping, quantitative trait locus analysis, and genetic mutation, and as targets for genetic transformation programs. Thus, this review is focused mainly on various facets of doubled haploid in the chief staple food crop rice and sights its recent applications in plant breeding, genetics and genomics.
Chapter
In this chapter, we present a list of species (and few interspecific hybrids) where haploids and/or doubled haploids have been published, including the method by which they were obtained and the corresponding references. This list is an update of the compilation work of Maluszynski et al. published in 2003, including new species for which protocols were not available at that time, and also novel methodologies developed during these years. The list includes 383 different backgrounds. In this book, we present full protocols to produce DHs in 43 of the species included in this list. In addition, this book includes a chapter for one species not included in the list. This makes a total of 384 species where haploids and/or DHs have been reported up to date.
Chapter
The completely homozygous genetic background of doubled haploids (DHs) has many applications in breeding programs and research studies. Haploid induction and chromosome doubling of induced haploids are the two main steps of doubled haploid creation. Both steps have their own complexities. Chromosome doubling of induced haploids may happen spontaneously, although usually at a low rate. Therefore, artificial/induced chromosome doubling of haploid cells/plantlets is necessary to produce DHs at an acceptable level. The most common method is using some mitotic spindle poisons that target the organization of the microtubule system. Colchicine is a well-known and widely used antimitotic. However, there are substances alternative to colchicine in terms of efficiency, toxicity, safety, and genetic stability, which can be applied in in vitro and in vivo pathways. Both pathways have their own advantages and disadvantages. However, in vitro-induced chromosome doubling has been much preferred in recent years, maybe because of the dual effect of antimitotic agents (haploid induction and chromosome doubling) in just one step, and the reduced generation of chimeras. Plant genotype, the developmental stage of initial haploids, and type–concentration–duration of application of antimitotic agents, are top influential parameters on chromosome doubling efficiency. In this review, we highlight different aspects related to antimitotic agents and to plant parameters for successful chromosome doubling and high DH yield.
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الخلاصة أجريت مجموعة من التجارب في مختبر زراعة الانسجة النباتية في قسم علوم الحياة – كلية العلوم للبنات- جامعة بغداد 2013- 2014 . يهدف البحث الى إمكانية استحثاث الكالس من بادرات نبات الفلفل ودراسة تأثير ملح NaCl والتشعيع بالأشعة فوق البنفسجية (Ultra Violet) في نمو الكالس وتمايزه وانتاج نباتات متحملة للملوحة ، وكذلك التحقق من وجود جينات تحمل الملوحة في نباتات الفلفل الناتجة من اخلاف الكالس باستخدام تقنية PCR-RAPD . عقمت البذور بمادة هايبوكلورات الصوديوم (NaOCl) وزرعت على وسط(1962) Murashige and Skoog (MS) كامل القوة . زرعت الأجزاء النباتية (السيقان، الاوراق ) المفصولة من البادرات على وسط النشوء المجهز بتراكيز مختلفة من الاوكسين IAA وNAA هي (0.0، 0.5، 1،1.5، 2.0) ملغم/لتر والسايتوكاينينات BA , BAP, Kin بتراكيز (0.0 ، 1 ،2 ،3 ، 4) ملغم/لتر. قسم الكالس المستحث الى مجموعتين المجموعة الاولى فقط عرضت للتشعيع بالاشعة فوق البنفسجية لمدة عشرة دقائق. ومن ثم زرع (50) ملغم من الكالس المستحث على الوسط الغذائي MS الحاوي على التراكيز الملائمة لاستحثاث الكالس من منظمات النمو مضافاً اليه تراكيز مختلفة من ملح كلوريد الصوديوم NaCl بالتراكيز (0.0 ، 0.05 ، 0.10 ، 0.15 ، 0.20 ، 0.25)% وبعد أربعة اسابيع تم قياس الوزنين الطري والجاف للكالس ومحتوى الكالس من الحامض الأميني البرولين والكربوهيدرات . ومن ثم نقل الكالس المنمى في الاوساط الملحية الى اوساط زرعية من MS مزودة بـ 0.1 ملغم/لتر IAA + 2 ملغم/لتر BA لغرض نشوء الافرع الخضرية. وبعد شهر من الزراعة نقلت الافرع الخضرية الناتجة من الكالس الى وسط MS المزود بـ 1.0 ملغم/لتر IBA + 0.1 ملغم/لتر BA + 0.1 ملغم/لتر GA3 لغرض نشوء الجذور . كما درس تأثير توليفات مختلفة من الوسط الزراعي المكون من الرمل والبتموس في نجاح أقلمة النبيتات . كما أجريت عملية استخلاص الدنا من الاوراق الجافة للنبيتات المنتجة نسيجياً ، ومن ثم تم الكشف عن جينات تحمل الملوحة باستخدام (5) بادئات لمواقع جينية تتصف بأنها جينات تحمل الملوحة . أظهرت النتائج ان التركيز2% لمدة (15) دقيقة من NaOCl كان ذا كفاءة عالية في التعقيم. وبينت النتائج ان أفضل استجابة لاستحثاث الكالس قد تحققت عند تركيز 4 ملغم/لتر من BA + 2 ملغم/لتر من IAA كما اظهرت النتائج حدوث انخفاض معنوي في معدل الوزنين الطري والجاف للكالس بزيادة التراكيز الملحية في الوسط الغذائي. كما تفوقت المعاملة التي عرض الكالس فيها للتشعيع عن المعاملة غير المعرضة بمعدل الوزنين الطري والجاف . حصلت زيادة في كمية البرولين والكربوهيدرات في نسيج الكالس بزيادة التراكيز الملحية وتفوق الكالس الذي عرض للتشعيع على الكالس الذي لم يعرض للتشعيع في كمية البرولين والكربوهيدرات. اما فيما يخص اخلاف النباتات من الكالس المعرض للشد الملحي ، فقد اظهرت النتائج بان نسبة الاخلاف انخفضت معنوياً بزيادة تركيز الملح في الوسط الغذائي ، كما انعدم اخلاف النباتات من الكالس الذي عرض للتشعيع. كما تفوق الوسط الزراعي المكون من الرمل والبتموس بنسبة(1:3) معنوياً في اقلمة النباتات وبلغت نسبة النجاح (79%). اظهرت نتائج تفاعلات PCR-RAPD أن التركيب الوراثي لعينة الملوحة يمتلك كل تسلسلات البادئات لجينات الملوحة مقارنة مع التركيب الوراثي لعينة نباتات السيطرة .
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The huge variation in root system architecture (RSA) among different rice (Oryza sativa) cultivars is conferred by their genetic makeup and different growth or climatic conditions. Unlike model plant Arabidopsis, the molecular basis of such variation in RSA is very poorly understood in rice. Cultivars with stable variation are valuable resources for identification of genes involved in RSA and related physiological traits. We have screened for RSA and identified two such indica rice cultivars, IR-64 (OsAS83) and IET-16348 (OsAS84), with stable contrasting RSA. OsAS84 produces robust RSA with more crown roots, lateral roots and root hairs than OsAS83. Using comparative root transcriptome analysis of these cultivars, we identified genes related to root development and different physiological responses like abiotic stress responses, hormone signaling, and nutrient acquisition or transport. The two cultivars differ in their response to salinity/dehydration stresses, phosphate/nitrogen deficiency, and different phytohormones. Differential expression of genes involved in salinity or dehydration response, nitrogen (N) transport, phosphate (Pi) starvation signaling, hormone signaling and root development underlies more resistance of OsAS84 towards abiotic stresses, Pi or N deficiency and its robust RSA. Thus our study uncovers gene-network involved in root development and abiotic stress responses in rice.
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We evaluated the frequency of induction of calli and in vitro regeneration from mature seeds in four rice Venezuelan varieties, Araure-4, Centauro, Venezuela-21 (resistant), and Cimarron (susceptible) to the fungus Pyricularia grisea. Genotypic variability was identified in varieties, Cimarron was recalcitrant to all culture media, unlike the rest of the varieties where the frequency of induction of embryonic calli ranged between 3% and 81.2%, the higher frequencies were obtained in enriched media with low 2.4-D concentrations. The Venezuela-21 variety showed a 81% embryogenic calli at 1 mg.L-1 2,4-D, for Centauro variety a 67.5% was detected at 2 mg.L-1 2,4-D plus 2 mg.L-1 BA, finally Araure-4 showed 66% at 1 mg.L-1 2,4-D plus 2 mg.L-1 K. For the regeneration system, it was determined that culture media complemented with 0.5 mg L-1 IAA plus 2 mg L-1 BA, yielded a higher frequency of regeneration in both evaluated varieties, Araure-4 (44.4%) and Centauro (44%). Through morphoanatomical studies, it was confirmed that the regeneration plants occurred route indirect somatic embryogenesis.
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