Plant Biotechnology Research Articles

Antibacterial, Phytochemical and Antioxidant Activity of Gliricidia sepium Crude Extract against Human Pathogenic Bacteria

Aim: The present study is aimed at examine the antibacterial effect of crude extract of G. sepium to human pathogenic bacteria and its contents of its phytochemical and antioxidant properties.
 Place and Duration of the Study: The study was conducted in the Department of Science Laboratory Technology. Ekiti State University Ado Ekiti Nigeria under ambient temperature and humidity between May and September 2023.
 Methodology: The collected plant sample was identified by Mr. Omotayo F.O. of the Department of Plant Science and Biotechnology at Ekiti State University Ado Ekiti, Ekiti State. The plant was oven dried at 61.9°C for two hours and thirty minutes, then ground into a fine powder using an electric blender after being airdried for four weeks in a cold environment. 100 g of powdered leaves were steeped in 500 ml of distilled water, N-hexane, dichloromethane, and methanol, respectively, to prepare the extracts. Following the mixes' filtration via muslin fabric. Salmonella typhi, Staphylococcus aureus, Escherichia coli, and Proteus vulgaris were obtained from Federal Teaching Hospital Ido Ekitti in Ekiti State, Nigeria. The antibacterial activity of was determined using the agar diffusion method.
 Results: The results of the crude extract's antibacterial activity showed that the water extract had the largest zone of inhibition (12.00 ± 1:00 mm) against E. coli, while N-hexane had the lowest zone of inhibition (3.33 ± 5.77 mm) against S. typhi and P. vulgaris. Phenol content ranged from the lowest (6.19±0.01) in water extract to the highest (47.19 ± 0.02) in DCME. The plant demonstrated good amount in the Ferric Reducing Antioxidant Power (FRAP) and DPPH scavenging assays. Methanol extract had the highest DPPH (23.53 ± 0.03) at the lowest concentration of 100 µg/ml, and it also produced the highest DPPH (62.63 ± 5.77) at the highest concentration of sample, 500 µg/ml. 
 Conclusion: The present work highlights the possible use of G. sepium leaf extracts as a source of antibacterial agents that can be used to prevent human diseases.

Big Data in Plant Biotechnology: Leveraging Bioinformatics to Discover Novel Anticancer Agents from Flora

Big Data in Plant Biotechnology: Leveraging Bioinformatics to Discover Novel Anticancer Agents from Flora

Efficient selection of a biallelic and nonchimeric gene-edited tree using Oxford Nanopore Technologies sequencing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease system is a versatile and essential biotechnological tool in the life sciences that allows efficient genome editing. When generating gene-edited trees, T0-generation plants are often used for subsequent analysis because of the time that is required to obtain the desired mutants via crossing. However, T0-generation plants exhibit various unexpected mutations, which emphasizes the need to identify mutants with expected mutation patterns. The two critical checkpoints in this process are to confirm the expected mutation patterns in both alleles and to exclude somatic chimeric plants. In this study, we generated gene-edited Cryptomeria japonica plants and established a method to determine chimerism and mutation patterns using fragment analysis and Oxford Nanopore Technologies (ONT)-based amplicon sequencing. In the first screening, fragment analysis, i.e., indel detection via amplicon analysis, was used to predict indel mutation patterns in both alleles and to discriminate somatic chimeric plants in 188 candidate mutants. In the second screening, we precisely determined the mutation patterns and chimerism in the mutants using ONT-based amplicon sequencing, where confirmation of both alleles can be achieved using allele-specific markers flanking the single guide RNA target site. In the present study, a bioinformatic analysis procedure was developed and provided for the rapid and accurate determination of DNA mutation patterns using ONT-based amplicon sequencing. As ONT amplicon sequencing has a low running cost compared with other long-read analysis methods, such as PacBio, it is a powerful tool in plant genetics and biotechnology to select gene-edited plants with expected indel patterns in the T0-generation.

Stem cutting: A novel introduction site for transporting water-insoluble particles into tomato (Solanum lycopersicum) seedlings

The introduction of exogenous particles into plants has promising applications in agriculture and biotechnology. Nanoparticles can be transported into plants through foliar application or root uptake. However, both methods have limitations in terms of the size of the particles (<40 nm) that can be transported due to the barriers of the cell wall and cuticle. In the present study, we proposed a novel method to deliver particles of up to 110 nm into plants by cutting the stem of tomato seedlings. We demonstrated for the first time, using water-insoluble silica colloids, that not only nanoparticles but also submicron particles can be transported toward the leaves when the plant stem is used as the entry point of particles. Thirty-five-day-old tomato seedlings were used as the target plants. When the cut stem seedlings were immersed in the colloidal particle suspension for up to 24 h, significant particle accumulation was observed in the nodes and leaves. The relatively low particle concentrations (10 mg/L) allowed effective transport throughout the plants. Silica particles with average diameters of 10 nm and 110 nm were both well transported and moved through the stem. Even after the particles entered the plant, adventitious roots were formed, resulting in the formation of whole plants with roots, stems, and leaves. This method can be applied not only to tomatoes but also to other food crops for various applications in plant biotechnology.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

The "Duckweed Dip": Aquatic Spirodela polyrhiza Plants Can Efficiently Uptake Dissolved, DNA-Wrapped Carbon Nanotubes from Their Environment for Transient Gene Expression.

Duckweeds (Lemnaceae) are aquatic nongrass monocots that are the smallest and fastest-growing flowering plants in the world. While having simplified morphologies, relatively small genomes, and many other ideal traits for emerging applications in plant biotechnology, duckweeds have been largely overlooked in this era of synthetic biology. Here, we report that Greater Duckweed (Spirodela polyrhiza), when simply incubated in a solution containing plasmid-wrapped carbon nanotubes (DNA-CNTs), can directly uptake the DNA-CNTs from their growth media with high efficiency and that transgenes encoded within the plasmids are expressed by the plants─without the usual need for large doses of nanomaterials or agrobacterium to be directly infiltrated into plant tissue. This process, called the "duckweed dip", represents a streamlined, "hands-off" tool for transgene delivery to a higher plant that we expect will enhance the throughput of duckweed engineering and help to realize duckweed's potential as a powerhouse for plant synthetic biology.

First Report on Mesophyll Protoplast Isolation and Regeneration System for the Duboisia Species.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 105 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L-1 TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species.

The advent of plant cells in bioreactors.

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols.

In vitro plants: How to open a jar containing bioactive phytochemicals

Due to the increasing demand for phytochemicals, plant in vitro cultures are a noteworthy, environmentally sustainable method, enabling the biotechnological production of bioactive compounds. Medical writers can find writing scientific articles in this field quite tricky due to the knowledge required at the intersection of botany, chemistry, pharmacy, and biotechnology. This article aims to provide helpful tips for writing about plant biotechnology to minimise the risk of article rejection by scientific journals due to inaccurate description of methods or inadequate data analysis.

Exploring the Sustainable Exploitation of Bioactive Compounds in Pelargonium sp.: Beyond a Fragrant Plant.

This review article aims to present an overview regarding the volatile compounds in different scented species of Pelargonium and their biological activities, immunomodulatory activity, cytotoxic activity, high larvicidal activity and ethnopharmacological uses. Although the Pelargonium genus includes many species, we focused only on the scented ones, with the potential to be used in different domains. Pelargonium essential oil showed great properties as antioxidant activity, antibacterial activity (against K. pneumonie, S. aureus or E. coli strains) and antifungal activity (against many fungi including Candida sp.), the responsible compounds for these properties being tannins, flavones, flavonols, flavonoids, phenolic acids and coumarins. Due to the existence of bioactive constituents in the chemical composition of fresh leaves, roots, or flowers of Pelargonium sp. (such as monoterpenoid compounds-citronellol, geraniol, linalool, and flavonoids-myricetin, quercetin and kaempferol), this species is still valuable, the bio-compounds representing the base of innovative substitutes in food processing industry, nutraceuticals, or preventive human or veterinary medicine (substitute of antibiotics). Highlighting the volatile chemical composition and properties of this scented plant aims to rediscover it and to emphasize the vast spectrum of health-promoting constituents for a sustainable approach. Future research directions should point to the application of plant biotechnology with a significant role in conservation strategy and to stimulate commercial interest.

Relevances of Ethnobotanical, Chemical and Biotechnological Aspects of Andiroba (Carapa spp.) As a Source of Applications in Strategic Sectors. Review

Objective: This study sought to synthesize in the form of a literature review three aspects widely studied in relation to andiroba and its impacts on the science and oncology sectors. Theoretical framework: Because andiroba (Carapa guianensis Aubl) is widely used in popular medicine, it has led to the emergence of studies investigating its uses, followed by chemical characterization, and more recently with a biotechnological product, which gives the plant an important role. Method: A survey of the literature was applied, observing three points, which were ethnobotanical, chemical and biotechnological aspects that together comprise the strategic sectors of andiroba studies. Results and conclusion: The diversity of molecules already discovered, such as fatty acids and limonoids, which show that this plant is a powerful source of new substances with chemical and biological potential, and also their use for the development of plant biotechnology, which indicates new potential uses for andiroba to generate new materials and products. Implications of the research: It can be considered that the application of more advanced studies towards the generation of products from andiroba inputs still occurs as a result of the little appreciation of the potential of this species within the industry, especially the pharmaceutical industry. Originality/value: This study analyzes not only aspects that are commonly discussed about andiroba, which are the botanical aspects of the species, but also the characteristics of the plant.

Engineering Crassulacean Acid Metabolism in C3 and C4 Plants.

Carbon dioxide (CO2) is a major greenhouse gas contributing to changing climatic conditions, which is a grand challenge affecting the security of food, energy, and environment. Photosynthesis plays the central role in plant-based CO2 reduction. Plants performing CAM (crassulacean acid metabolism) photosynthesis have a much higher water use efficiency than those performing C3 or C4 photosynthesis. Therefore, there is a great potential for engineering CAM in C3 or C4 crops to enhance food/biomass production and carbon sequestration on arid, semiarid, abandoned, or marginal lands. Recent progresses in CAM plant genomics and evolution research, along with new advances in plant biotechnology, have provided a solid foundation for bioengineering to convert C3/C4 plants into CAM plants. Here, we first discuss the potential strategies for CAM engineering based on our current understanding of CAM evolution. Then we describe the technical approaches for engineering CAM in C3 and C4 plants, with a focus on an iterative four-step pipeline: (1) designing gene modules, (2) building the gene modules and transforming them into target plants, (3) testing the engineered plants through an integration of molecular biology, biochemistry, metabolism, and physiological approaches, and (4) learning to inform the next round of CAM engineering. Finally, we discuss the challenges and future opportunities for fully realizing the potential of CAM engineering.

Prospects of urban biotechnology for ornamental plants

Plant biotechnology methods can be used in urban environments. Cell selection, genetic engineering, and micropropagation hold great promise among biotechnological methods. Two main applications for the use of cell selection for urban plants are possible in future. The first is the use of this technology in urban greening to improve plant resistance. The second is phytoremediation of contaminated urban soils. Micropropagation can be used for urban plant reproduction. The use of genetic engineering is promising for obtaining new varieties of urban plants with high ornamental qualities. When using genetic engineering techniques, environmental risks should be taken into account. The use of biotechnology for the urban environment will contribute to the development of applied biotechnology, urban biology, urban natural sciences and urban technical sciences. Future ornamental urban plants will change, but they will always play an important role in people's lives.

The MtWOX genes in the regulation of Medicago truncatula somatic embryogenesis

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form embryos in a process called somatic embryogenesis (SE). SE can occur naturally in some plant species and it is widely used for the plant’s genetic transformation and regeneration.&#x0D; This process is regulated by hormone treatment and many proteins, among which WUSCHEL-related homeobox (WOX) transcription factors are believed to play crucial roles. Our previous studies have shown thatMtWOX9-1stimulates SE inMedicago truncatula. The aim of the present research was to search for newMtWOXgenes regulating SE. In this study, using transcriptomic data and literature data, we had selected several genes with an increased expression level during SE or in the generative organs and examined the overexpression effect of these genes on the SE ability. It was found that explants of theM.truncatulaembryogenic line, transformed with the construct forMtWOX6-likeoverexpression, develop more somatic embryos compared to the control.&#x0D; Our findings could be a helpful point for searching and studying new morphogenic regulators controlling SE and could have a positive impact on plant biotechnology in improving the transformation and regeneration capacity for other legumes.&#x0D; This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”.

Transplastomic plants — new approaches to solving “old” problems

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system.&#x0D; This work was supported by the Russian Science Foundation grant No. 23-24-00545.

Transgene-free genome editing of plants

The presence of foreign DNA is the main obstacle to the application of biotechnological plant varieties. However, transgene-free technologies in the field of genome editing make it possible to overcome this problem. In most countries that already have legislation in this area, plants without foreign DNA do not require field trials and safety tests.&#x0D; The easiest way to avoid integration of transgenes is the delivery of RNP complexes directly into the cell without the use of plasmids. However subsequent selection of edited cells in the absence of a selective marker and plant regeneration are quite difficult. Therefore, traditional genetic constructs are used more often, despite that the elements of the CRISPR system are integrated into the genome. Backcrossing and cross-pollination are used to get rid of unwanted inserts. There are opportunities to accelerate the selection process, such as the Transgene Killer CRISPR system, which ensures the death of plants carrying transgenes in the early embryonic stages [1].&#x0D; Constructs based on viral replicons integrated into T-DNA are an alternative option. They provide a high level of transient expression of CRISPR elements which are not integrated into the genome. Such vectors were created on the basis of geminiviruses, rhabdoviruses, potexviruses, potyviruses, bunyaviruses [2]. The ability of viruses to move between cells can be both preserved and lost due to the removal of the corresponding proteins.&#x0D; One of the newest approaches is grafting of shoots onto roots expressing Cas and guide RNA [3]. The addition of tRNA-like motifs to the transcripts ensured their mobility and dispersal along the shoot. Heritable edits were observed in the progeny of grafted plants.&#x0D; Thus, for transgene-free editing technologies of plant genomes are rapidly developing, which will accelerate the commercialization of new varieties with economically valuable traits.

Role of Plant Biotechnology in Agricultural Sustainability and Improvement of Crops

Role of Plant Biotechnology in Agricultural Sustainability and Improvement of Crops

In Vitro Propagation of Ruta Chalepensis Through and Callus Culture

Plant tissue culture is a field that that enables culturing of various plants and parts of plants usually treated under a nutrient medium and in highly sterile conditions. Out of them callus culture is one of the very interesting arenas of plant biotechnology that encompasses many pivotal benefits. The study focuses on such callus enrichment using different hormones that there by enhance its biological activities. The plant namely Ruta chalepensiswas chosen upon wherein the callus growth was noticed. Ruta chalepensis has multiple medicinal activities like anti-cancer, anti-ulcer, anti-diabetic and many more pharmacological properties that yields in treating and curing of illness. Ruta chalepensis the leaf and internode were taken for the study and to analyse which of those at what concentration of plant growth regulators showed a better callus induction. The MS medium as well as Various hormone concentrations was used for the study like auxin and cytokinin from 0.5mg to 2 mg (2,4 – D, NAA, IAA, IBA). Increased concentration of 2,4-D (1.0 mg/L) alone in the MS medium showed profused callus growth. Both the explants used such as leaf and Internode were also tested in the MS medium which was devoid of hormones/plant growth regulators which was treated as control for comparison. From the data obtained, MS medium supplemented with hormones showed better growth rate and callus induction when compared with that of MS medium without hormones/plant growth regulators. Among the plant growth regulators 2,4-D (1.0 mg/L) showed maximum callus initiation from both leaf and internode explants. Further work was carried out in single and combination of the plant growth regulators for callus proliferation and accumulation. Further analysis is being done to study the growth pattern on combination of hormones and fix the hormone concentrations for the mass propagation of callus from the explants.

Evaluation for Parental Polymorphism and Identification of Microsatellites Linked to Drought Tolerance in Rice (Oryza sativa L.)

Aims: To identify the ready-to-use polymorphic microsatellite markers associated with drought tolerance for marker-assisted backcross breeding through a polymorphism survey between Jyothi and Chuvannamodan rice varieties.&#x0D; Place and Duration of Study: Centre for plant biotechnology and molecular biology, Thrissur, Kerala, India, during January to May 2023.&#x0D; Methodology: The genomic DNA of Jyothi and Chuvannamodan was isolated by following CTAB (Cetyltrimethyl Ammonium Bromide) method with modification. Isolated DNA from both varieties was subjected to PCR amplification using 208 Simple Sequence Repeats (SSRs) primer pairs distributed in 12 chromosomes. The amplified PCR products were electrophoresed in 3% agarose gel and separated fragments were visualized and documented in the Gel Documentation System. Different allele sizes produced by the same SSRs primers between two varieties are identified as polymorphic markers. Polymorphism per-cent was calculated, and frequency distribution and chromosome distribution of polymorphic markers were analyzed.&#x0D; Results: A total of 208 SSR primers were surveyed for the parental polymorphism. Out of which, 85 SSR primers exhibited clear polymorphism between Jyothi and Chuvannamodan and the remaining 123 were monomorphic primers. The amplicon size ranged from 83bp (RM430) to 495bp (RM18919) among the different primers. The survey revealed maximum parental polymorphism on chromosome 4 (69.23%), followed by chromosome 5 (64.28%), and minimum polymorphism on chromosome 8 (21.73%). The average per cent of polymorphism between the parents was 40.86%. Among the 85 polymorphic markers, 66 had dinucleotide repeats, 17 had trinucleotide repeats, and 1 had tetranucleotide repeats.&#x0D; Markers with dinucleotide repeats showed higher level of polymorphism. A group of 28 polymorphic markers were identified to be linked with traits including root-related traits, grain yield, leaf rolling and leaf drying under drought conditions.&#x0D; Conclusion: The polymorphic markers identified in the present study form the basis for tagging drought-tolerant QTLs/genes, fine mapping of those genes, and subsequently in marker-assisted breeding programs. The polymorphic markers linked with the QTLs for grain yield under drought can be pyramided in Jyothi through marker-assisted backcross breeding.

Plant chromosome engineering - past, present and future.

Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.