Crop Biotechnology Research Articles

PYL1- and PYL8-like ABA Receptors of Nicotiana benthamiana Play a Key Role in ABA Response in Seed and Vegetative Tissue.

To face the challenges of climate change and sustainable food production, it is essential to develop crop genome editing techniques to pinpoint key genes involved in abiotic stress signaling. The identification of those prevailing abscisic acid (ABA) receptors that mediate plant-environment interactions is quite challenging in polyploid plants because of the high number of genes in the PYR/PYL/RCAR ABA receptor family. Nicotiana benthamiana is a biotechnological crop amenable to genome editing, and given the importance of ABA signaling in coping with drought stress, we initiated the analysis of its 23-member family of ABA receptors through multiplex CRISPR/Cas9-mediated editing. We generated several high-order mutants impaired in NbPYL1-like and NbPYL8-like receptors, which showed certain insensitivity to ABA for inhibition of seedling establishment, growth, and development of shoot and lateral roots as well as reduced sensitivity to the PYL1-agonist cyanabactin (CB). However, in these high-order mutants, regulation of transpiration was not affected and was responsive to ABA treatment. This reveals a robust and redundant control of transpiration in this allotetraploid plant that probably reflects its origin from the extreme habitat of central Australia.

Regulatory policy on genetically modified breeding stack in key countries and the current status in Korea.

With an increasing interest and demand for biotechnology crops in agriculture worldwide, genetically modified (GM) breeding stacks produced by conventional breeding of previously approved GM single events remain popular for farmers in GM crop cultivation countries. However, regulations on stacks vary in each country. Currently, Korea requires approval for all breeding stacks intended for cultivation. To determine whether the stack is subject to a full safety assessment as a new GM crop, molecular characterization, protein expression, composition analysis, and agronomic characterization data are required. Korea's regulatory policy on stacks has not adopted the high-covers-low concept; therefore, subcombinations of already approved higher combination events are subject to breeding stack review if any subcombination was purposefully bred for cultivation use. This review will help promote the efficient management of GM breeding stacks in Korea in the future.

Novel and emerging biotechnological crop protection approaches.

SummaryTraditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non‐model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation.

The first GM crop in Bangladesh – Bt Eggplant

Eggplant (Solanum melongena) commonly called Brinjal is the most popular vegetable in Bangladesh. It is extensively cultivated around homesteads and in commercial fields throughout the year. It is estimated that nearly 67 percent of the eggplant crop yield in Bangladesh is lost due to the damage caused by Leucinodes orbonalis also known as eggplant fruit and shoot borer (EFSB). EFSB is reportedly the most serious pest of eggplant. To overcome this EFSB problem Bt eggplant project has been initiated in 2004. The Agricultural Biotechnology Support Project II (ABSPII) is a USAID-supported project that has funded the development of Bt eggplant in Bangladesh under the Public Private Partnership (PPP) mode. Bangladesh Agricultural Research Institute (BARI) received the Event EE-1 eggplant in Mahyco and backcrossed into nine popular eggplant varieties of Bangladesh selected based on the regional consumer preference. The efficacy of cry1Ac gene in providing effective resistance to the target pest was also assessed in terms of productively (fruit yield increase) which was studied in 9 Bt eggplant lines for a period of 7 years from 2008 to 2015. To launch the best 4 varieties, BARI applied to the National Technical Committee on Crop Biotechnology (NTCCB). For release of GM crop variety, first of all follow the recommendation of NTCCB, then submitted to the National Technical Committee on Crop Biotechnology (NTCCB) Core Committee and then to the National Committee on Biosafety (NCB). The Bangladesh government approved four varieties namely, BARI Bt Begun-1 (Uttara), BARI Bt Begun-2 (Kazla), BARI Bt Begun-3 (Nayantara) and BARI Bt Begun-4 (BARI Begun-6 or ISD006) on 30 October 2013 and these got momentum countrywide. On 22 January 2014, Bt seedlings were distributed among 20 farmers in four districts of Bangladesh as the first genetically modified (GM) crops. After the government of Bangladesh approved the cultivation of Bt eggplant in 2013, its popularity is increasing day by day and the socio-economic condition of the farmers is also improving. Bt eggplant started its journey in 2014 with only 20 farmers, in seven years it has grown to about 27012 farmers and from 8 acres to about 5,000 acres. This advancement of Bt eggplant has also faced opposition from different quarters at different times. Today, the progress of Bt eggplant continues by overcoming all obstacles. Field days, trainings, various publications, documentary making, use of mass and social media, observations, workshops, stewardship activities are acting as important regulators of this progress.

COMPARATIVE ANALYSIS OF THE STATE AND REGULATION OF AGRICULTURE IN JAPAN , SOUTH KOREA AND CHINA

The article is devoted to the study of general and special characteristics of the condition and state regulation of agriculture in three East Asian countries: China, Japan, and South Korea. It is determined that there are several historical similarities associated with the Asian way of production, low land use, high dependence on food imports, rapid urbanization and high population density. The topic of food security is relevant for all three countries. The comparative analysis made it possible to identify and specify important differences in the process and results of the modernization of agriculture in the three countries. Especially in terms of the level and speed of technical and technological development, domestic state support for agriculture, foreign trade policy, changes in the trade balance for agricultural goods, the inclusion of countries in the import of GM seeds and the production of organic food, positions in the ratings of the global food security index. The article focuses on the issues of commercialization of biotechnological crops and China's strategy aimed at future global dominance in the field of agricultural science and technology. Using the mechanism of international corporate mergers and acquisitions, the Chinese company with state participation entered the top five world leaders in the production of GM seeds and IT platforms for precision farming.

Extended investigation of field-evolved resistance of the corn earworm Helicoverpa zea (Lepidoptera: Noctuidae) to Bacillus thuringiensis Cry1A.105 and Cry2Ab2 proteins in the southeastern United States

Previous studies have reported that the corn earworm/bollworm, Helicoverpa zea (Boddie), has developed field resistance to pyramided Bacillus thuringiensis (Bt) Cry1A/Cry2A maize and cotton in certain areas of the southeastern United States. The objective of the current study was to determine the current status and distribution of the resistance to Cry1A.105 and Cry2Ab2 in H. zea. In the study, 31 H. zea populations were collected from major maize planting areas across seven southeastern states of the United States during 2018 and 2019 and assayed against the two Bt proteins. Diet over-lay bioassays showed that most of the populations collected during the two years were significantly resistant to the Cry1A.105 protein. Most of the populations collected during 2019 were also resistant to Cry2Ab2, while significant variances were observed in the susceptibility of the populations collected during 2018 to Cry2Ab2. The results showed that Cry1A.105 and Cry2Ab2 resistance in H. zea is widely distributed in the regions sampled. The resistance to Cry1A.105 appeared to have plateaued, while selection for Cry2Ab2 resistance is likely still occurring. Thus, effective measures for mitigating the Cry1A/Cry2A resistance need to be developed and implemented to ensure the sustainable use of Bt crop biotechnology.

Exploring innovation capabilities build up in the deployment of crop biotechnology innovation in Kenya

ABSTRACT Agro-biotechnology deployment has been very slow in Africa, yet it has potential for enhancing sustainable development and industrialization. This paper attempts to understand the underlying factors that characterize the new technologies’ deployment process. It takes buildup of innovation capabilities as the point of interrogation considering that very few products have received regulatory approval for large-scale commercialization in Africa. It uses Kenya’s agro-biotech innovation system as a case study. The study finds that creating and accumulating innovation capabilities is much broader than research and development (R & D) capability and entails institutional capabilities that are needed to enhance biotech innovation that may lead to a sustainable industrialization.

Who is the African Farmer? The Importance of Actor Representations in the Debate About Biotechnology Crops in Africa

The discussion about the impact of agricultural biotechnology on Africa is deeply divided and contains widely diverging claims about the impact of biotechnology on African farmers. Building upon literature on the ‘good farmer’ that highlights that farmers identities are an important factor in explaining the success or failure of agricultural change, we argue that the identity of the farmer is an undervalued yet crucial aspect for understanding the debate about the impact of agricultural biotechnology on African farmers. In this article we therefore investigate what farmers’ identities are implicated in the arguments about the impact of biotechnology on African farmers. We aim to identify the main fault lines in different accounts of the African biotechnology farmer by analysing the identities ascribed to them in two prominent cases of controversy: the debates at the 2002 World Summit on Sustainable Development in Johannesburg and the discussion about the impact of biotechnology on smallholder farmers in the Makhathini flats in KwaZulu Natal, South Africa. Our findings demonstrate that arguments about biotechnology are informed by diverging conceptions of who the African farmer is, what is important for the African farmer, and what role the African farmer has in relation to agricultural biotechnology. These findings remain relevant for current discussions on gene editing technologies like CRISPR-Cas. Openly discussing these different views on the identity of smallholder farmers is crucial for moving forward in the biotechnology controversy and can inform future attempts to elicit the farmer’s voice.

History and Outlook for Glyphosate-Resistant Crops.

Glyphosate-resistant (GR) crops, commercially referred to as glyphosate-tolerant (GT), started the revolution in crop biotechnology in 1996. Growers rapidly accepted GR crops whenever they became available and made them the most rapidly adopted technology in agriculture history. Adoption usually meant sole reliance on glyphosate [N-(phosphonomethyl)glycine, CAS No. 1071-83-6] for weed control. Not surprisingly, weeds eventually evolved resistance and are forcing growers to change their weed management practices. Today, the widespread dissemination of GR weeds that are also resistant to other herbicide modes-of-action (MoA) has greatly reduced the value of the GR crop weed management systems. However, growers continue to use the technology widely in six major crops throughout North and South America. Integrated chemistry and seed providers seek to sustain glyphosate efficacy by promoting glyphosate combinations with other herbicides and stacking the traits necessary to enable the use of partner herbicides. These include glufosinate {4-[hydroxy(methyl)phosphinoyl]-DL-homoalanine, CAS No. 51276-47-2}, dicamba (3,6-dichloro-2-methoxybenzoic acid, CAS No. 1918-00-9), 2,4-D [2-(2,4-dichlorophenoxy)acetic acid, CAS No. 94-75-7], 4-hydroxyphenyl pyruvate dioxygenase inhibitors, acetyl coenzyme A carboxylase (ACCase) inhibitors, and other herbicides. Unfortunately, herbicide companies have not commercialized a new MoA for over 30years and have nearly exhausted the useful herbicide trait possibilities. Today, glyphosate-based crop systems are still mainstays of weed management, but they cannot keep up with the capacity of weeds to evolve resistance. Growers desperately need new technologies, but no technology with the impact of glyphosate and GR crops is on the horizon. Although the expansion of GR crop traits is possible into new geographic areas and crops such as wheat and sugarcane and could have high value, the Roundup Ready® revolution is over. Its future is at a nexus and dependent on a variety of issues.

High-Throughput Profiling of Extrachromosomal Linear DNAs of Long Terminal Repeat Retrotransposons by ALE-seq.

Extrachromosomal linear DNA (eclDNA) is the reverse-transcribed cDNA intermediate derived from long terminal repeat (LTR) transposable elements (TEs) (Cho et al., Nat Plants 5:26-33, 2018). Given that the eclDNAs are the final intermediate of LTR-TE life cycle prior to integration to the host chromosomes, their presence is considered a strong indication of active LTR retrotransposons (Cho et al., Nat Plants 5:26-33, 2018; Lanciano et al., PLoS Genet 13:e1006630, 2017). Here, we describe a method of amplification of LTR extrachromosomal DNA followed by sequencing (ALE-seq) which determines the 5' LTR sequences of eclDNAs. Briefly, ALE-seq consists of two steps of amplification, in vitro transcription of adaptor-ligated eclDNAs and subsequent reverse transcription to cDNAs primed at the conserved primer binding site (PBS) (Cho et al., Nat Plants 5:26-33, 2018). ALE-seq allows the high-throughput identification of novel LTR-TEs which are active in plants that could be potentially useful for crop biotechnology.

CHALLENGES TO THE ADOPTION OF MODERN CROP BIOTECHNOLOGY: INSIGHTS FROM INDIAN AND MALAYSIAN GM REGULATORY FRAMEWORKS

The emerging use of genetic engineering technology led to the establishment of the Cartagena Protocol on Biosafety in 2001. India and Malaysia are signatories to the Protocol, having established regulatory measures governing the use of biotechnological genetic modification including regulation of genetically engineered crops from research to open cultivation and post-market surveillance. India and Malaysia have developed biosafety policies that display some similarities but also many differences, consequently impacting the practicalities of applying the technology to development and deployment of new crop varieties. The objective of this paper is to compare biosafety policies and regulatory frameworks that India and Malaysia have in place for the use of modern biotechnology. We highlight the implications of imposing rigid requirements as well as lacking harmonized policies on the approval process and trade flows, identifying these as potential barriers to the optimal use of modern crop biotechnology. We also briefly discuss how current interpretations of Living Modified Organisms and Genetically Modified Organisms in India and Malaysia will influence the pace of crops developed from new plant breeding techniques and propose options to regulate these technologies based on experience from other countries.

Genetically Modified Crops’ Technology and its Awareness among Smallholder Farmers in Nigeria

Genetically Modified (GM) crops are crops modified through genetic engineering to improve their quality. Although safety concerns about genetically modified organisms (GMOs) are still being debated, the food security benefits have led to adoption by many counties. In Nigeria, where most farmers are uneducated and likely unaware of the agricultural technology, the government approved its first biotechnology crop for commercialization in 2018. Level of farmers’ awareness is crucial to acceptance of GM crops, although; this has not been fully explored in the literature. Therefore, this study aimed to assess farmers’ awareness for GM crops and the factors that determine their awareness in Oyo state, Nigeria, using primary data collected in 2018 with the aid of well-structured questionnaires from 242 smallholder farmers. Principal component analysis and Tobit regression model were used for data analysis. Results showed that farmers were aged 43 years with farm size of 3.57 ha, farming experience of 14 years and 11 years of education. Most farmers were male (71.90%), married (67.36%), not members of farmer groups (64.46%) and sourced their seeds from non-credible sources (85.12%). Most farmers (52.07%) had either not heard of or did not know of any benefits/costs of GM crops, hence; were not aware of GM crops. Factors that influenced awareness of GM crops were being a male farmer, years of education and source of seeds. The study concluded that increasing years of education and ensuring use of credible seed sources will increase awareness of the costs and benefits of GM crops among farmers.

Overexpression of BvHb2, a Class 2 Non-Symbiotic Hemoglobin from Sugar Beet, Confers Drought-Induced Withering Resistance and Alters Iron Content in Tomato

Drought stress is one of the major threats to agriculture and concomitantly to food production. Tomato is one of the most important industrial crops, but its tolerance to water scarcity is very low. Traditional plant breeding has a limited margin to minimize this water requirement. In order to design novel biotechnological approaches to cope with this problem, we have screened a plant cDNA library from the halotolerant crop sugar beet (Beta vulgaris L.) for genes able to confer drought/osmotic stress tolerance to the yeast model system upon overexpression. We have identified the gene that encodes BvHb2, a class 2 non-symbiotic hemoglobin, which is present as a single copy in the sugar beet genome, expressed mainly in leaves and regulated by light and abiotic stress. We have evaluated its biotechnological potential in the model plant Arabidopsis thaliana and found that BvHb2 is able to confer drought and osmotic stress tolerance. We also generated transgenic lines of tomato (Solanum lycopersicum) overexpressing BvHb2 and found that the resulting plants are more resistant to drought-induce withering. In addition, transgenic lines overexpressing BvHb2 exhibit increased levels of iron content in leaves. Here, we show that class 2 non-symbiotic plant hemoglobins are targets to generate novel biotechnological crops tolerant to abiotic stress. The fact that these proteins are conserved in plants opens the possibility for using Non-GMO approaches, such as classical breeding, molecular breeding, or novel breeding techniques to increase drought tolerance using this protein as a target.

Crop Biotechnology and Product Stewardship

ABSTRACT Agricultural biotechnology is enhancing agricultural productivity, food security, and livelihoods globally. Some developing countries have established functional biosafety regulatory systems and have commercialized genetically modified (GM) crops. Release of GM crops requires enhanced capacity for regulatory compliance and product stewardship to help ensure sustainable use of biotechnology products. We conducted a survey of 66 stakeholders, mostly from Africa and Asia, in two-week international agricultural biotechnology short courses. Respondents showed knowledge of biotechnology benefits and expressed potential barriers to commercialization. They identified 16 crops in the “pipeline for commercialization.” Stakeholders also shared ideas about how to build capacity for product stewardship. Product stewardship is a concept which requires each person in the product life cycle – innovators, scientists, and technology users, to share responsibility. This paper focuses on adoption of product stewardship for post-release management of GM crops which encompasses trait performance, resistance management, integrated pest management (IPM), good agricultural practices, high-quality seeds and planting material, intellectual property management, labeling, identity preservation, consumer acceptance, and effective marketing.

Strategies of Plant Biotechnology to Meet the Increasing Demand of Food and Nutrition in India

A groundbreaking application of biotechnology research during the recent past has been improvement of crop health and production. India being one of the most rapidly developing countries with an enormous population and remarkable biodiversity, plant biotechnology promises significant potential to contribute to characterization and conservation of the biodiversity, increasing its usefulness. However, India’s green revolution was noted to be insufficient to feed the country's teeming millions. Therefore, novel approaches in crop biotechnology had to be aimed at ensuring better productivity and quality of cultivars. This paper provides a comprehensive review of research undertaken mainly in the last couple of decades along with potential strategies in plant biotechnology focusing on specific grain and seed crops of key agricultural as well as dietary importance to meet the growing demand of food and nutrition in India, while also proposing potential application of relevant global research findings in the Indian context. The analysis would help address the ever-increasing worldwide socio-economic necessity for greater food security, particularly during times of crisis such as the recent Coronavirus Infectious Disease 2019 (COVID-19) pandemic.

Environmental impacts of genetically modified (GM) crop use 1996–2018: impacts on pesticide use and carbon emissions

ABSTRACT This paper updates previous assessments of the environmental impacts associated with using crop biotechnology (specifically genetically modified crops) in global agriculture. It focuses on the environmental impacts associated with changes in pesticide use and greenhouse gas emissions arising from the use of GM crops since their first widespread commercial use 22 years ago. The adoption of GM insect resistant and herbicide tolerant technology has reduced pesticide spraying by 775.4 million kg (8.3%) and, as a result, decreased the environmental impact associated with herbicide and insecticide use on these crops (as measured by the indicator, the Environmental Impact Quotient (EIQ)) by 18.5%. The technology has also facilitated important cuts in fuel use and tillage changes, resulting in a significant reduction in the release of greenhouse gas emissions from the GM cropping area. In 2018, this was equivalent to removing 15.27 million cars from the roads.

KANDUNGAN FENOL DAN LIGNIN TANAMAN NILAM HIBRIDA (Pogostemon sp.) HASIL FUSI PROTOPLAS

<p><strong>Evaluation of phenols and lignin in the root of hybrid patchouli (Pogostemon cablin Benth.) front protoplast fusion</strong></p><p>One of the problems faced in patchouli production is nematode infection (Pratylenchus brachyurus). Improvement of the plant resistance to nematode in patchouli is dificult 10 be conducted through conventional method as the plant never llowcrs. One of the methods of improving (he plain resistance is by using protoplast fusion of Aceh patchouli (Pogostemon cablin, cv Sidikalang ) and Java patchouli (/' heyneanus. cv (iirilaya). It has been found thai Ihe plain resistance lo nematode is closely related to phenols and lignin content of the root Therefore. Ihe level of resistance of different clones of somatic hybrids lo nematode was approached by evaluating ihe content of phenols and lignin of the roots The study was conducted al the laboratory of the Research Institute for food Crop Biotechnology, Bogor in November 2000. Phenol content was determined with high performance liquid Chromatography (IIPI.C). while lignin content was determined with Klasou method. Result showed that Java patchouli (Girilaya) contained phenols and lignin respectively 76.53 and 21 900 pp. higher than those of Aceh patchouli (Sidikalang and Tapak Tuan) respectively 38 2-81 45 and 8 000-7 200 ppm. Phenol and lignin content in the somatic hybrids were dilferenl in Ihe respective pairs. In the fusion of Girilaya x Sidikalang. all clones contained phenol lower than those of Iheir parents, some clones (29%) contained lignin higher than Sidikalang but lower than (iirilaya. and the rest (71%) contained lignin lower than the two parents. Meanwhile, from Ihe fusion of Girilaya, Tapak Tuan. I clone (4%) contained phenols higher than those of (he two parents (97 ppm). 39% contained phenols higher than Tapak Tuan but lower than Girilaya (38.75-60.12 ppm) and the rests (60%) contained phenol lower than the two parents. Furthermore, the lignin content, of 78% somatic hybrids was lower than Girilaya but higher than Tapak Tuan, but the rest (22%) was lower than the two parents. Based on the distribution of phenols and lignin content. Ihe somatic hybrids can be categorized in three group The irst groups (5 clones) contained high phenols (higher than the average). Ihe second group (9 clones) contained high lignin. and Ihe third (I clone) contained high phenol and lignin.</p>

Dynamic Transcriptome Analysis Reveals Uncharacterized Complex Regulatory Pathway Underlying Genotype-Recalcitrant Somatic Embryogenesis Transdifferentiation in Cotton.

As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE—hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)—were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Crop biotechnology and the future of food

The global population continues to rise, as does the likelihood of reduced yields of major food crops due to the changing climate, thus making the development of genetically improved, stress-resilient crops a research priority. The convergence of low-cost genome sequencing with improved computational power and high-throughput molecular phenotyping technologies has accelerated the identification of genes underlying important agronomic traits relevant to food production and quality. Here, we discuss the evolution of plant improvement, and how researchers leverage genomic analyses and revolutionary new plant breeding technologies like site-directed nucleases to enhance food crop traits through agricultural biotechnology. Deployment of these products from the laboratory to the field remains hindered by biological and regulatory bottlenecks that require further development. Crop yield is stagnating in many parts of the world, and climate change threatens the worldwide agricultural system. This Review presents a comprehensive overview of current development leveraging genomic analyses and revolutionary new plant breeding technologies to enhance food crop traits through agricultural biotechnology.

Genome analysis of tissue culture-derived variations in regenerated Brassica rapa ssp. pekinensis plants using next-generation sequencing

Genetic variations have often been detected in plants regenerated from tissue culture, which is an essential step of molecular breeding and tissue culture techniques. The incidence of unintended genetic variations should be kept low to ensure the stability of biotechnological crops. In this study, regenerated Chinese cabbage plants (Brassica rapa ssp. pekinensis) were produced and sequenced to identify the genetic variation generated during tissue culture. The sequencing data of each regenerated plants were compared with reference genome sequences. A total of 6644 genetic variations, including single-nucleotide polymorphisms as well as insertions or deletions in the regenerated plants, were detected using bioinformatic analysis, and 109 mutation candidates that are expected to consistently occur in the regenerated R0 group were identified. To investigate the inheritance of the variations, the R1 and R2 lines of the two regenerated plants were also sequenced and analyzed. The results indicated that 39 of the regenerant-specific mutation candidates were expected to be maintained despite the progress of the generation. Among those, five of the SNP candidates occurred in exons, and sequence analysis was performed on these mutations. For detailed analysis, we validated the nucleotide sequences and deduced the amino acid sequences of the genes with five base substitution candidates in the exonic regions. Among the five SNP candidates, three were found to be synonymous mutations that resulted in no changes in the amino acid; however, two mutations were nonsynonymous mutations that altered the amino acid compositions. Our findings suggested a genetic diversity of the regenerated plants and revealed the mutation candidates that consistently occurred in the regenerated plants and were maintained in the progeny lines.