Abstract
Interspecific hybridization creates new genetic variants. Embryo formation and subsequently fertile seed development are the indicators of successful interspecific hybridization. Often interspecific hybridization is limited due to variations in genome and ploidy levels. The technique of embryo rescue is commonly used in interspecific hybridization to facilitate the survival of embryos from abortion. The effectiveness of an embryo rescue program in the Brassica species depends on embryo age, embryo development stage and media composition. Embryo rescue protocol could be effectively utilized to solve some major problems of the oilseed Brassica growers including blackleg, clubroot and Alternaria disease resistance, salinity, and drought tolerance etc. This review article discusses the prospects of developing novel Brassica hybrid genotypes with desirable traits through embryo rescue.
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Key words: Oilseed, Brassica, Mustard, Interspecific hybridization, Tissue culture, Embryo rescue
INTRODUCTION
Oilseed Brassicas are the second most popular source of vegetable oil and one of the third most widely traded commodities worldwide (
Anupriya et al. 2020). Improve-ment of
Brassica genotype is required to integrate stress resistance, quality traits and yield performance. One of the most successful methods of transmitting characteris-tics of commercial interest since the historical backdrop of
Brassica breeding is interspecific hybridization (
Seyis and Aydin 2014). Interspecific hybridization may result allo- polypoid hybrids between two different species, infertile haploids and resynthesized parental type polyploids (
Fig. 1). In Brassicaceae, interspecific hybridization is often carried out for the modification of phenotypic traits with disease and insect resistance and/or to modify nutritional quality in terms of the restricted availability of variations within the species level (
Kaneko and Bang 2014). Nevertheless, development of interspecific hybrid is challenging due to abortion of hybrid embryos associated with pre- and post-fertilization barriers and also due to the existence of a U-triangle relationship between six
Brassica species (
Zhang et al. 2001). Utilizing embryo rescue protocols is expected to achieve success from the interspecific hybridization by overcoming the associated natural barriers (
Momotaz et al. 1998; Weerakoo
et al. 2009;
Niemann et al. 2012;
Sharma et al. 2017). Distant crosses between crop plants and their wild relatives most often require embryo rescue technique to save developing embryos. Since its first used by Nishi in
Brassica (1959) several studies have been conducted to strengthen the techniques for higher seed set acquisition (
Inomata 2002;
Zhang et al. 2003;
Zhang et al. 2004). The technique of embryo rescue is a possible means of regenerating haploid plants to ensure a shortened breeding period (
Zhang et al. 2004). For commercial exploitation of oilseed
Brassica, self-incompatibility trait was transferred through embryo rescue to facilitate hybrid seed production (
Ripley and Beversdorf 2003). On several occasions environmental stress tolerance and quality traits have been transferred to oilseed
Brassica cultivars through embryo rescue (Sharma
et al. 2017). However, the successful embryo culture depends on the optimization of several factors. In case of
Brassica, embryo rescue is suggested at 10 to 30 days after pollination (
Chen et al. 1988;
Quazi 1988). The effectiveness of embryo rescue in
Brassica varieties depends on the embryo maturation stage, the composition of the medium, and, to some extent, the genotype (
Rahman 2004). Hence, research should focus on developing an improved embryo rescue protocol. Besides, identification of desirable variation sources and combining them through interspecific hybridi-zation and embryo rescue is necessary for enriching
Brassica gene pool. This paper describes the suitability, applicability and role of embryo rescue in oilseed
Brassica improvement. This review also discussed various factors that affect the success of embryo rescue process and also explains potential strategies to follow in oilseed
Brassica varietal develop-ment.
GENOME RELATIONSHIP AMONG THE BRASSICA SPECIES
Brassica and its close relatives have common plant forms and more than one genus or species has identical morpholo-gical characteristics, so it is very hard to distinguish genera and species. A number of challenges are faced by the early taxonomists in the separation and classification of the different species and types within the family of Brassi-caceae (
Prakash 1980). That’s why numerous changes and modifications have been occurred from original species names and arrangement (
Schulz 1919). The cytological studies by Morinaga (
Morinaga 1929;
Morinaga 1931;
Morinaga 1933) and his student, Nagaharu U clarified the broad relationships among the economically important
Brassica species in which chromosome pairing clearly showed difference among the species with the distant chromosome number (
Nagaharu 1935).
Around 40 different species of
Brassica plants have been used in the world for the commercial processing of fatty oils (
Weiss 1983). The genetic relationships among the six most economically important
Brassica species were described by the Triangle of Nagaharu U (
Fig. 2). Three of these
Brassica species are diploid,
B. rapa (AA genome, 2n = 20),
B. nigra (BB genome, 2n = 16) and
B. oleracea (CC genome, 2n = 18), and the other three are allotetraploids species
B. juncea (AABB genome, 2n = 36),
B. napus (AACC genome, 2n = 38) and
B. carinata (BBCC genome, 2n = 34) considered as hybrids of AB, AC and BC, respectively. The diploid species are thought to have originated from a common ancestor with the basic chromosome number x = 6 (
Prakash 1980) and the allotetraploid species derived from each pair of the three diploid species by spontaneous hybridization (
Nagaharu 1935).
Microscopic inspection at the zygotene stage of meiosis of the interspecific crosses was effective in identifying and verifying genome relationships between the
Brassica interspecies crosses between tetraploid and diploid plants (
Nagaharu 1935). For instance, F
1 plants were produced with a chromosome number of n = 19 when crosses between
B. napus (n = 19) and
B. rapa (n = 10) were performed. Microscopic inspection detected two sets of chromosomes in the
B. rapa genome, but only ten pairs of bivalent chromosome pairs were observed at zygotene stage in meiotic cells of F
1 plants (
Morinaga 1929). There are the other nine
B. napus chromosomes that can never develop normal bivalent pairs due to the non-availability of homologous chromosome pairs and were affected by the chromosomes of the A genome of B. rapa (
Cheng et al. 2014). Theoretically, 10 chromosomes of
B. napus are similar to the 10 chromosomes in B. rapa (
Morinaga 1929). Related studies using
B. napus and
B. oleracea crosses revealed that homologous condition of nine chromosomes between those species. Taken together, findings of those studies lead to a conclusion that
B. napus is a tetraploid between
B. rapa and
B. oleracea (
Morinaga 1929;
Cheng et al. 2014). This U’s triangle has been utilized effectively to better explain the genetic relationships between these species of
Brassica (
Fig. 2).
EMBRYO RESCUE AND VARIETAL RE-SYNTHESIS IN BRASSICA VARIETAL DEVELOPMENT
Embryo rescue is a potent method for developing novel
Brassica genotypes. Improvement of the
Brassica gene pool is hard to achieve through traditional breeding approaches. Sexual incompatibility is the major cause that averts the gene transfer process among
Brassica species (
Kumar and Srivastava 2016). In addition, self-incompatibility (sporop-hytic) and biennial lifecycle also curbs the efficiency of conventional breeding approaches (
Pavlović et al. 2010). One of the notable ways to create novel plant species and breed new varieties is wide hybridization (
Chen et al. 2018). One way to increase the range of genetic variation accessible to breeders is by interspecific crossing between
B. oleracea and
B. rapa for resynthetic oilseed rape (
Rahman 2013). In many characteristics the resynthesized
B. napus show abundant variability including the flower size, flowering time, waxy layer features and leaf shape and size (
Gaeta et al. 2007;
He et al. 2017). The stabilization of target characteristics in the case of artificial heteropolyploid species takes a long time, making it difficult to develop new cultivars for field application (
Mestiri et al. 2010;
Tian et al. 2010;
Wei et al. 2018). Previous studies hybridized Chinese cabbage and broccoli, red cabbage and Chinese cabbage to validate the stability of target characteristics (
Wei et al. 2018;
Wei et al. 2019).
B. juncea resynthesis by using the broader gene pool of available diploids provide an important method for the discovery of novel genetic variants. In an earlier study, the interspecific hybrids of
B. juncea were achieved at a greater frequency by using direct ovule culture (
Bhat and Sarla 2004). Embryo rescue technique can also be used in the commercial exploitation of oilseed
Brassica. In order to generate a commercially viable hybrid production system, there are three requirements: the existence of substantial heterosis, an efficient pollination control system and sufficient transfer of pollen across the parental lines (
Grant and Beversdorf 1985). In addition to that self-incom-patibility has been used to promote the development of hybrid seeds in
B. oleracea for many years (
Ockendon 1982). Self-incompatibility was transported to
B. napus from
B. oleracea var. italica by interspecific hybridization, followed by embryo rescue (
Ripley and Beversdorf 2003). Ogu-CMS (Ogura Cytoplasmic Male Sterility) and its associated restorer genes (
Rfo) were relocated from radish to rapeseed using interspecific hybridization (
Heyn 1976;
Pelletier et al. 1988). These CMS lines were characterized by poor agronomic performance, high glucosinolate content and weak female fertility (
Delourme et al. 1991). Resear-chers were able to develop a low glucosinolate - restored line (R2000) using gamma-ray irradiation with desirable agronomic performances (
Primard-Brisset et al. 2005). In Chinese kale, a fertile Ogu-CMS line was resynthesized by incorporating the fertility-restored Ogu-CMS gene (
Rfo) after an interspecific hybridization with the rapeseed restorer lines followed by a successful embryo rescue (
Yu et al. 2016). Interspecific hybridization was also used to transfer other agronomic characteristics like seed coat colour. From interspecific reciprocal crosses between yellow-seeded
B. campestris (AA) and
B. carinata (BBCC), hexaploid Brassica (AABBCC) was developed to transmit the yellow seed coat genes from both genomes A and C to
B. napus (AACC) (
Meng et al. 1998).
INTERSPECIFIC HYBRIDIZATION IN BRASSICA THROUGH EMBRYO RESCUE
There is widespread interspecific hybridization process in nature, where it may contribute to the development of new species or the useful introgression of inter-species adaptive characteristics. The presence of closely related wild relatives that easily hybridize with other
Brassica species made interspecific hybridization an excellent choice for
Brassica species improvement (
Katche et al. 2019). Previous studies corroborated the superiority of using embryo rescue techniques for the development of interspecific hybrids in
Brassica compared to other techniques (
Bennett et al. 2008;
Wang et al. 2009).
Brassica cultivars can be improved through attaining resistance to various stresses and improving certain quality characteristics.
EMBRYO RESCUE FOR TRANSFERRING BIOTIC AND ABIOTIC STRESS RESISTANCE
Over a hundred wild species, weedy relatives and specific genetic tools are available to establish resistance to environmental stresses in
Brassica crops (
Zhang et al. 2014;
Kumar et al. 2015;
Šamec and Salopek-Sondi 2019). Among the biotic stresses, oilseeds
Brassica are suffered from the attack of insect pests and diseases. Three of the large and commercially recognized oilseed
Brassica namely
B. napus,
B. rapa and
B. oleracea, suffer from the significant yield loss due to pests such as mustard aphid, whitefly aphids, cabbage thrips, root flies, caterpillars, etc. (
Mafakheri and Kordrostami 2020;
Zheng et al. 2020). It is always hard to apply effective control against insect pest, therefore resistance cultivars can be extremely advanta-geous (
Gulidov and Poehling 2013;
Springate 2016;
Springate 2017). Often resistance sources of a cultivated species that carry insect-resistance are not the same genus but their wild relatives or landraces. After identifying the source, it is crucial to detect the putative genes in order to effectively transform the crop of interest (
Broekgaarden et al. 2011). Researchers under the “PGR Secure Project” program in UK conducted a detailed series of studies on the production of resistant varieties to cabbage whitefly and aphid in
B. oleracea and Chinese cabbage (
Vosman et al. 2016). Resistance was found in less-known species of
Brassica including
B. villosa,
B. incana, and
B. montana; aphid resistance was found in some crop wild relatives (CWR) (
Pelgrom et al. 2015). Interspecific hybridization with these resistant cultivars followed by embryo rescue could be a valuable tool to incorporate insect resistance in oilseed
Brassica. Among the biotic stresses Alternaria blight, blackleg, black rot, clubroot, powdery mildew diseases are notable.
Alternaria blight is a recalcitrant disease caused mainly by
Alternaria brassicae and
Alternaria brassicicola. Previous studies incorporated Alternaria resistance into oilseed Brassicas through embryo rescue. For instance,
Alternaria resistance was transferred into
B. juncea from
B. alba (
Yadav et al. 2018) and to
B. juncea cv. RH 30 from B. tournefortii (
Yadav et al. 1991).
Gupta et al. (2010) also used interspecific hybridization in combination with in vitro ovule culture to incorporate high tolerance to
Alternaria blight and white rust from
B. carinata cv. Kiran to low erucic acid TERl (OE) M21 lines. Clubroot resistance breeding greatly benefits from embryo rescue techniques that facilitate interspecific transfer (Diederi-chsen
et al. 2009). In 1987, the first interspecific crosses were made between an inbred broccoli line and Chinese cabbage cv. Parkin, which contained a single dominant resistance gene (
Harberd 1969). Later, club root resistance was transferred to
B. napus by interspecific hybridization between
B. rapa and
B. napus (
Liu et al. 2018). Blackleg is another deadly disease of oilseed
Brassica. Blackleg resistance was transferred to
B. napus by interspecific hybridization with
B. carinata (
Rahman et al. 2007;
Rahman 2012). Powdery mildew resistance was incorpo-rated to
B. oleracea from
B. carinata by interspecific hybridization (
Tonguç and Griffiths 2004). Black rot resistance was transferred from
B. carinata to
B. oleracea (
Sharma et al. 2017). Among the abiotic stress, drought and salinity are the most notable. In contrast with diploid species,
B. campestris,
B. nigra, and
B. oleracea, amphidi-ploid species including
B. carinata,
B. juncea, and
B. napus showed a superior ability to tolerate salt (
Ashraf et al. 2001). Interspecific hybridization between these species in various combinations can be a way for salt tolerance breeding in oilseed
Brassica.
B. fruticulosa,
B. tourneforti and
B. carinata were described as drought tolerant in previous studies (
Rashidi et al. 2017;
Prakash and Bhat 2007). They can be used as an important tool for inter-specific gene transfer for drought tolerance in oilseed
Brassica.
EMBRYO RESCUE FOR QUALITY IMPROVEMENT OF BRASSICA GENOTYPES
Interspecific hybridization has a great promise to modify the ecotypes of the member of Brassicaceae to improve disease resistance, insect resistance and also to alter nutritional quality and oil content (
Kaneko and Bang 2014). Embryo rescue accompanied by interspecific hybridization is utilized to incorporate beneficial quality characteristics to create desirable genetic variation (
Mei et al. 2010;
Niemann et al. 2015;
Niemann et al. 2012). But pre- and post-fertilization barriers related to compatibility are the major hindrances of successful embryo maturation process and hybrid development. Therefore, rescuing of developing embryo isolated from the immature zygotic embryos under an aseptic condition and culturing them in an aseptic growing medium may result in viable regenerated plants (
Torres 1989;
Inomata 1993). Oil quality is described as far as saturated, monounsaturated and polyunsaturated fatty acids. Mustard oil contains the most minimal measures of saturated fatty acids as compared to other vegetable oils and also has a very good proportion of n3 and n6 polyunsaturated fatty acids, in this way viewed as valuable for food utilization. Mustard oil is a special oil which contains oleic acid (8–15%), linoleic acid (13–20%) and linolenic acid (6–14%), erucic acid (41–50%), palmitic and stearic acids in small amounts (
Banga 1996;
Prakash et al. 2000). In mustard oil, erucic acid alone contributes nearly half of the total fatty acids but it is least desirable for human nutrition and health (
Sauer and Kramer 1983). There has been limited feeding value of rapeseed-mustard meals because of the glucosinolates present in the vegetative tissues and seeds of cruciferous plants (
Fenwick et al. 1983). These compounds help in flavor and odor to
Brassica vegetables and condiments but it may reduce palatability and adversely affect iodine uptake by the thyroid glands. Thus they reduce food quality and feed efficiency in terms of development and weight gain (
Fenwick et al. 1983;
Bille et al. 1983). In view of these evidences, the production of low erucic acid and glucosinolate varieties is a vital breeding goal for
Brassica quality breeders (
Agnihotri et al. 2004).
The doubled haploid homozygous lines of
Brassica carinata after UV treatment followed embryo rescue measured the reduced content of glucosinolates from an average of 80.6 µmol g
−1 seed to 37.5 µmol g
−1 seed in eight lines (
Barro et al. 2003). Interspecific crosses and embryo rescue are the viable methods to alter the fatty acid composition of mustard. Canola quality
B. napus cultivars from Western Australia were crossed with
B. juncea accessions for the improvement of the agronomic characteristics (
Iqbal et al. 2006). In another research, some traits, such as pollen characteristics, seed growth and proliferation, nuclear DNA content and genomic com-position were analyzed throughout the vegetative and generative stages of interspecific hybrids. Those hybrids obtained from interspecific crosses are valuable as new germplasm that can strengthen
Brassica-breeding programs (
Kamiński et al. 2020).
The changing climatic conditions have the adverse effect on oilseeds productivity due to unpredictable temperature fluctuations, and evolution of new biotypes/pathotypes/ races of insects and disease as a result of narrow genetic base. The utilization of tissue culture, molecular and bio- analytical strategies was implied in breeding for improved quality requirements and fungal disease resistance traits in Indian rapeseed-mustard (
Agnihotri et al. 2009). A wide number of interspecific hybrids have been developed through embryo rescue with a view to fulfill specific objectives such as content of anthocyanin in Chinese cabbage, colour in cabbage etc. These results favour the assumption that development of
Brassica genotypes with desired quality traits is possible (
Pen et al. 2018).
FACTORS AFFECTING THE EFFICACY OF EMBRYO RESCUE IN BRASSICA GENOTYPES
Effect of genotypic variability
The genome structures of
Brassica were formed by entire genome triplication followed by broad diploidiza-tion, which assumes a significant function in the speciation and morphotype enhancement of
Brassica plants (
Cheng et al. 2013). Including the allotetraploid species
B. juncea (AABB),
B. napus (AACC), and
B. carinata (BBCC), the diploid
B. rapa (AA genome),
B. nigra (BB), and
B. oleracea (CC) are commercially valuable and grown as vegetables, condiments, and oilseed sources (
Cheng et al. 2013). Those A, B and C genomes have different level of potentials in fertilization. For instance, the rate of ovule fertilization declined when female parent contained the CC genome. In crosses where the female parent possessed CC genome, the growth and development of embryos was slower than those in crosses where the female was BBCC genome species (
Rahman 2004).
On the other hand, when foreign pollen was allowed to germinate and infiltrate into the stigmas, there were significant variations between maternal genotypes, contributing to a wide variety of interspecific fertilization. That’s why embryos degenerated as early as 10–15 days after pollination and embryo rescue was needed (
Meng and Lu 1993). The genetic variations that occur in nature are the tools of researchers to transfer the traits of interest through interspecific hybridization (
Pen et al. 2018).
The great influence of genotypic variability was observed on in vitro growth and differentiation patterns in a number of crop plants including
Brassica (
Etedali and Khandan 2012). Plant regeneration capacity may vary significantly between species, as well as between genotypes within species (
Ockendon 1985). The frequency of callus growth and the appearance of the tissues of six species of
Brassica showed variation in chromosome number and ploidy level. Eventually, they concluded that the variability in the growth rate of callus within B. oleracea was as high as that of among the
Brassica species (
Dietert et al. 1982). A remarkably significant genotypic variation was also observed between parents and interspecific hybrid genotypes where embryos obtained from the cross between BARI Sharisha-8 and BARI Sharisha-12 exhibited higher regeneration ability probably because one of the parental genotypes BARI Sharisha-12 had the highest regeneration potentiality (81.45%) (
Ripa et al. 2020). Genetic manipulation either by introducing genes or cytoplasms from a suitable cultivar increased the number of regenerated plants in embryo culture (
Özgen et al. 1998).
Considering the genetic variation present in different species and subspecies in Brassicaceae it is generally suggested to collect embryo from 10 to 30 d after pollination to rescue them from abortion (
Chen et al. 1988;
Fig. 3). Moreover, considering the composition of the medium, Murashige and Skoog (MS) (
Murashige and Skoog 1962) and Gamborg’s (B5) media are the most regularly used as basal media supplemented with plant hormones to establish a new protocol for embryo rescue (
Bridgen 1994). Full salt concentration was good for several genotypes but the reduction of salt levels from full to 1/2 or 1/4 gave better results for some genotypes in vitro (
Saad and Elshahed 2012). If there should be an occurrence of organogenesis, embryo rescued in 1/2 MS media represented upto 51% higher shoot length, up to 61% higher root length, up to 21% higher leaves and up to 43% shorter days to flowering in various cross combinations compared with full-strength MS media (
Ripa et al. 2020). It is agreed that half-salt concentration resulted in better plant growth dynamics most likely because of the lower nutritional requirements for initial regeneration (
Saad and Elshahed 2012;
Sato et al. 1989;
Munshi et al. 2007;
Siong et al. 2011).
The young proembryo of
Brassica that depends on the endosperm for nutrition requires a complex medium with amino acids, various vitamins, and natural extract for embryo initiation. Since young embryo needs a high osmotic concentration in the medium, the osmotic concen-trations need to be reduced with the embryo growth (
Raghavan and Srivastava 1982). Usually the first step of indirect plant tissue culture is callus induction that is depended upon the growth medium (George
et al. 1972). The callus growth and plant regeneration efficiency of 17 cultivars of
B. oleracea was less vigorous in a medium containing coconut milk (CM) with a high level of 2,4-D compared to a medium containing CM and with a low concentration of 2,4-D. Rate of root and shoot organo-genesis was enhanced from the callus that was grown in a medium lacking 2,4-D for one week (
Dietert et al. 1982).
The age of the embryo or embryo maturity is a crucial factor in the success of embryo rescue studies. The germination of excised embryos from ovule is mainly influenced by two factors such as the maturity or age of the embryo excision and culture media which is used as nutrient media (
Uma et al. 2011;
Burbulis and Kupriene 2005). When the embryos are more developed
in vivo the probability of successful regeneration is increased (
Quazi 1988;
Uma et al. 2011). Thus the embryos isolated at their early age are more prone to degeneration (
Raghavan and Srivastava 1982). In
Brassica, the developmental stages of embryos after pollination are globular, heart, torpedo, and cotyledonary shapes (
Pen et al. 2018;
Ilić-Grubor et al. 1998;
Fig. 3). It is highly recommended to imply embryo rescue techniques before seed maturation at 10 to 30 d after pollination (
Chen et al. 1988;
Quazi 1988).
The immature zygotic embryos are more responsive compared to mature zygotic embryos (
Burbulis and Kupriene 2005). Embryos isolated at the torpedo stage took 11-15 days for callus induction in a medium supplemented with 1.0 ppm BAP, 0.5 ppm NAA and 1.0 ppm 2, 4-D (
Ilić-Grubor et al. 1998) but the embryos isolated at the earlier stages took 3–6 days additional time for callus induction (
Maheswaran and Williams 1986). However, success in callus induction was also noted from the very young embryos at their globular stages in
B. juncea (
Liu et al. 1993). Mature embryos (> 700 μm) were cultivated from five genotypes of
B. rapa species that produced plantlets directly within 7 to 12 days (relatively less time) after inoculation through direct organogenesis (
Zisan et al. 2015). In an another experiment, the cotyledonary-shaped embryos reached the maximum plant regeneration rates (overall 83.3 percent) and it was around 20 percent higher compared to torpedo-shaped embryos (63.1 percent) (
Ripa et al. 2020). The embryos at their full maturity generally regenerate plantlets directly (
Uma et al. 2011). Similarly, in winter wheat, the earlier phases of embryonic development induced calli but the mature embryos exhibited direct regeneration of plantlets (
Uma et al. 2011). Varietal variation also interacted to both success and response to embryonic development in both mature and immature embryo culture. For example, some Bangladeshi varieties of rapeseed (e.g., Agrani, BINA Sarisha-6 and BARI Sarisha-6) produced a greater percentage of calli compared to other varieties in similar culture media (
Zisan et al. 2015). The cotyledonary-shaped embryos (15 DAP) from an interspecific cross of
B. rapa and
B. oleracea revealed considerably higher rates of plant regeneration. In addition, the regeneration from the cotyledonary-shaped embryos at 20 days post pollination from interspecific crosses between
B. napus and
B. rapa was significantly higher compared to the earlier dates (
Pen et al. 2018).
PROTOCOLS OF EMBRYO RESCUE IN INTERSPECIFIC HYBRIDIZATION
According to
Ripa et al. (2020), 14 days old siliqua was selected for immature embryo rescue. The obtained siliquae were first sterilized in 0.1 mercuric chloride for 20 seconds followed by sterilization by 70% ethanol for 30 seconds. In addition, the sterilized siliquae were properly rinsed three times in sterile distilled water. In a sterile cabinet with laminar flow, the growing embryos were dissected carefully using a stereoscopic microscope. After that, separated embryos were transferred directly in 1/2 MS medium. The cultivated embryos were kept at a regulated temperature of around 22°C, 250 mol m
−2 s
−1 PPFD, and 75% relative humidity while being exposed to fluorescent light for 16 hours per day and 8 hours for dark. Regenerated plants were sub-cultured on freshly produced and sterilized media 10 days following inoculation. Lastly, the plantlets from these vials were moved into bigger vials at the 20-day mark after they had grown to an appropriate size. The established plantlets were then transferred to a plant culture room for flowering and fruit-setting.
PROSPECTS OF EMBRYO RESCUE
The changing climate and rising population are two major challenges for crop production in the upcoming year. Oilseed Brassica improvement is crucial to mitigate the vast demand for vegetable oil across the globe. The rise of temperature may create many obstacles to complete the lifecycle of Oilseed Brassicas grown in the winter season. Besides this, the increase of drought prone areas became a major concern for oilseed Brassica cultivation. That is why it is hard time to breed for the climate-smart oilseed crops. Wide hybridization can be used to exploit the wild relatives of the Brassicaceae family for abiotic stress tolerance making embryo rescue a potential tool for the future breeding program. Apart from stress tolerance, quality traits should be introgressed to save the vast population from malnutrition. Developing cultivar with a healthy fatty acid profile has become a prime concern for the researcher over the years. Embryo rescue can integrate quality traits from other species into oilseed Brassica cultivars. In addition, varietal re-synthesis may be required due to the rapid change of public dietary habits as well as climate change. We already discussed the role of embryo rescue in the varietal re-synthesis of oilseed Brassica. Taken together all of the above facts, Brassica breeder should focus on identifying novel genetic variation across Brassica gene pool and other related species for wide hybridization followed by embryo rescue. At the same time, the embryo rescue protocol should be modified frequently to accelerate the success rate.
CONCLUSION
Oilseed Brassica is one of the largest suppliers of vegetable oil worldwide. Their improvement largely depends on wide hybridization and embryo rescue because of the complex genome relationship between the species. The embryo rescue technique offers great scope to incorporate desirable traits from other related species to oilseed Brassicas. Optimization of some factors such as maturation stage, culture media, and genotype determine the efficiency of embryo rescue. Careful manipulation of these factors is required for developing a more effective embryo rescue protocol. Brassica breeders can practice embryo rescue more frequently to combat future challenges in oilseed production.
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AUTHOR CONTRIBUTIONS
AHKR planned the study. RSR and SDJ collected reviews and wrote the manuscript. AHKR organized the figures and edited the manuscript. All authors approved the final version of the manuscript.
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FUNDING
The primary authors received monthly grants from Bangladesh Agricultural University Research Systems Grant no. 2021/5/BAU.The first author gratefully acknowledges her NST fellowship.
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CONFLICT OF INTEREST
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. It is to specifically state that “No Competing interests are at stake and there is No Conflict of Interest” with other people or organizations that could inappropria-tely influence or bias the content of the paper.
Fig. 1Possible pathways and protocols of embryo rescue from interspecific hybridization between two (A) homologous species under same genus and (B) distant species from two different genera.
Fig. 2“Triangle of U” diagram, showing the genetic relationships among six species of the genus Brassica (modified from Moringa 1933;
Nagaharu 1935;
Ripa et al. 2020).
Fig. 3Developmental stages of embryo in the siliqua from 7 to 20 days of age of Brassica rapa at 40× magnification.
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