, from the family Brassicaceae, is an economically important genus. It includes several species that are often used as oilseed crops, vegetables, fodder crops as well as condiments. Brassica
oilseed varieties producing oil low in anti-nutritive aliphatic glucosinolates and erucic acid as well as rich in unsaturated fatty acids are generally termed as ‘canola’. Conventionally, the term ‘canola’ was more often used for B. napus
but now some canola quality varieties of B. rapa
and B. juncea
are also available [1
]. Being rich in omega-6 and omega-3 fatty acids and low saturate fats, canola oil is considered as a heart-healthy oil. Due to its high quantity of proteins, its meal for poultry and livestock is considered as good as soybean [5
With the increasing world population, the demand for vegetable oil is also increasing. According to the United States Department of Agriculture, the largest importers of vegetable oil in 2018 were the European Union, the US, China and India. Pakistan also gets over 80% of its edible oil requirements from imports [7
]. Since the increase in cultivable land is not possible in the face of an increasing population, the viable option to meet the challenges of edible oil requirements, as well as industrial applications, is to develop stress-resilient high-yielding Brassica
genotypes. The development of stress tolerant Brassica
is possible by transferring genes from the plant species that are adapted to harsh environmental conditions. These species present a rich reservoir of the traits that enable them to grow under stressful conditions. However, transferring these traits to salt or drought sensitive crops is only possible by genetic transformation, as they cannot be cross bred through conventional breeding approaches. GM (genetic manipulation) tools developed in the 1980s allow the transferring of traits from a wide taxon for engineering novel traits into field crops. Herbicide tolerance, insect resistance, β-carotene synthesis (golden rice) and vitamin-enrichment (multivitamin corn) are few examples among the list of engineered traits through GM technology.
The introduction of transgenes into plants to engineer useful novel traits may seem trivial now [8
], but it has its own challenges and limitations [9
]. For example, a plant species must be responsive to in vitro regeneration protocols, and a robust regeneration system is one of the key pre-requisites for successful genetic transformation. Several indigenous Brassica
varieties developed locally have canola characteristics. Being stress-sensitive, these varieties are unable to grow on marginal lands. To develop stress-resilient transgenic versions, it is necessary to determine the morphogenesis potential of these varieties. Although transformation of Brassica
species has been reported in several studies [13
], several Brassica
genotypes remain recalcitrant to genetic transformation [2
]. Several factors including susceptibility to Agrobacterium
infection, choice of explant and tissue culture conditions mainly responsible for these variations have been identified [13
]. These factors vary from genotype to genotype, indicating a strong genetic control on in vitro regeneration and transformation of Brassica
]. Therefore, it is important to find out responsive genotypes, as well as a type of explant, which show reliable regeneration efficiency to be used in future transformation experiments.
Traditionally, model cultivars such as Westar have been used in transformation and regeneration experiments for the introduction of transgenes for desired traits [19
]. While these model cultivars are valuable for studying gene function, there is a need to work directly on elite cultivars for the development of climate-resilient crops. The undesired agronomic characters presented by the model plants require an exhaustive process of crossing and back-crossing to transfer engineered traits into field varieties [10
]. The challenge of transferring traits from model plants is greatly increased if the number of genes to introduce increases. Therefore, the transformation of commercial cultivars with desired agronomic performance adapted to the prevailing climatic conditions is highly desirable, making the process of developing transgenic plants quicker and more efficient. However, their transformation is often hampered with genotypic recalcitrance, which makes it necessary to test them for in vitro regeneration prior to genetic transformation experiments.
In this study, we evaluated the regeneration potential of different commercial varieties of Brassica grown in Pakistan using different explants and growth conditions for establishing transgenic technology in commercial varieties of Brassica. One of the commercial varieties, Aari canola, was found to be highly responsive to the given conditions. We also determined kanamycin concentrations to be used in future transformation experiments for the recovery of transformants. The in vitro Aari canola plants grown to maturity showed a normal plant morphology, with normal seed setting and without any observable phenotypic variations. The information generated in this study will be useful for developing stress-resilient Brassica varieties by directly transforming the commercial cultivars.
The present study was carried out to evaluate the regeneration potential of commercial cultivars of Brassica
belonging to Brassica juncea
and Brassica napus
. The regeneration and transformation protocols for Brassica
are mainly limited to model cultivar Westar and attempts have been made to extend these developments to transform the locally adapted Brassica
cultivars in many parts of the world [28
]. Although the success of genetic transformation depends on several factors including susceptibility of the genotype to Agrobacterium
infection, explant type as well as age, and in vitro regeneration efficiency are the key factors. Transformation of the elite varieties is manly hindered because of the high degree of recalcitrance of these varieties to regeneration protocols [23
]. Therefore, the first pre-requisite to embark on transformation experiment of elite cultivars is to evaluate their regeneration potential to available regeneration protocols.
Regeneration in Brassica
has been reported from several tissues and organs including leaves, stem sections, petioles, roots, hypocotyls, cotyledons, immature zygotic embryos, protoplasts and cell suspension cultures [16
]. Out of these, cotyledons, hypocotyls and roots have been frequently used for genetic transformation [2
]. Therefore, only the commonly used explants were tested in this study. Explant age and type also significantly influences regeneration potential. Ono, Takahata and Kaizuma [42
] compared regeneration from the explants isolated from seedlings of different ages. The study reported that explants from 4 day old seedlings gave higher regeneration response compared to the those isolated from 5 and 6 day old seedlings. In another study, when the explant age was increased from 4 to 10 days, the regeneration frequency was significantly decreased [44
]. Therefore, in this study, only 4 day old seedlings were used for explant preparation.
Cotyledonary explants showed the highest regeneration efficiency on all the tested protocols followed by petioles and hypocotyls, respectively (Figure 1
). Overall, the roots were found to be least responsive among all the types of explants used (Figure 1
d,h,l). This observation was in line with earlier studies. Zhang and Bhalla [34
] tested the regeneration potential of seven Australian commercial cultivars of Brassica napus
using cotyledons, hypocotyls, and roots. In six cultivars, the regeneration response from roots was comparatively low and slow compared to cotyledons and hypocotyls.
We observed that the regeneration protocol BRP-I was the most conducive for shoot formation from cotyledons (up to 10.9 average shoots per explant) while BRP-II was best for petioles (up to 3.2 average shoots per explant) and BRP-III promoted shoot formation both in hypocotyls (up to 3.4 average shoots per explant) and root explants (up to 2.7 shoots per explant) (Figure 2
). We applied these protocols without introducing any major modification. The regeneration of these cultivars, including that of Aari canola, could be further improved upon by optimizing the plant regeneration conditions. It has been observed that varying the media components affects the shoot formation efficiencies significantly [33
]. The fact that huge variations have been observed among different explants, a generalized recommendation cannot be made. For example, all the three protocols can be efficiently used to recover transformants of Aari canola from cotyledons, while the regeneration efficiency of other explants was greatly reduced in these protocols (Figure 1
). The poor regeneration of elite varieties except Aari canola indicates the recalcitrant nature of these cultivars to regeneration as reported in the literature. The regeneration response of Westar to BRP-III from all the explants confirms the fact that it was developed for Westar (Figure 1
and Figure 2
). Nevertheless, this study provides a snapshot of the in vitro regeneration potential of these cultivars on three different regeneration protocols.
In vitro regeneration in the genus Brassica
is highly genotype specific and huge variations have been reported in the regeneration potential of different genotypes. In the present study, all the tested genotypes exhibited variable regeneration response in a manner that appears highly genotype specific. For example, huge variations were observed in regeneration from different explants ranging from 6% to 73%, 4% to 79.3%, 0% to 50.6% and 0% to 42.6% from cotyledons, petioles, hypocotyls and roots, respectively (Figure 1
). Similar variations have been reported in Brassica
. Hachey, et al. [48
] screened six cultivars of Brassica campestris
and observed 0–70% regeneration in these genotypes. Ono, Takahata and Kaizuma [42
] investigated the regeneration potential of 100 genotypes of Brassica napus
by in vitro regeneration and reported huge variations in the regeneration response, which ranged from no regeneration at all to 97%. Zhang and Bhalla [34
] reported huge variations (0–96.7%) in seven Australian cultivars of Brassica napus
. Similar genotype-dependent variations among different Brassica
species have been reported in several studies [23
The regeneration response appeared specific to the type of cultivar and media components used. For example, Aari canola showed exceptional regeneration on all the tested regeneration protocols. The model variety, Westar, showed a good regeneration on BRP-III but lagged far behind Aari canola. Both Faisal and Punjab canola were least responsive to BRP-I and BRP-III. The results presented here show that explant type had a significant effect on the in vitro regeneration response of the tested cultivars (Table 1
and Table 2
). The effect that regeneration conditions provided was less significant compared to the explant type and often non-significant like that of the replicate. Khehra and Mathias [27
] studied the effect of genotype along with explant type on shoot regeneration frequency in four Brassica napus
varieties, one spring (Westar) and three winter (Ariana, Cobra and Libravo) by culturing cotyledons, hypocotyls and stem sections on different growth mediums. The study showed that both the genotype and explant type had significant effect on shoot regeneration frequency.
Determination of the threshold values of the selective agent, kanamycin in this study, is a preliminary requirement before starting transformation experiments that will allow the selective growth of transformants while killing the non-transformants. Different cultivars showed a complete inhibition at different kanamycin concentrations (Figure 4
) perhaps due to the genotypic variations. The findings of this study are in close agreement with [50
] who reported that the optimum kanamycin concentration for mustard (Brassica juncea
Coss.) was 30 mg/L.
A notable observation of this study is the exceptional regeneration response of an elite variety Aari canola to different regeneration conditions particularly from cotyledons. The regeneration efficiency of Aari canola from these explants was found to be up to 7.5-fold higher than the model cultivar Westar (Figure 1
a,e,i), whereas, when compared in terms of total number of shoots obtained per explant, it was up to 21.9-fold higher than Westar (Figure 2
a,e,i). Shoots of Aari canola were successfully grown to maturity using the procedure outlined in BRP-I [23
]. The complete regeneration of mature Aari canola plants took approximately 18 weeks (Figure 5
), which is 2 weeks shorter than the Brassica oleracea
for which this protocol was originally designed [23
]. The efficient regeneration of Aari canola both in terms of regeneration efficiency and as well as total number of shoots obtained, and relatively quicker recovery of mature plants make it a good candidate for genetic transformation. One of the reasons of faster recovery of mature plants grown from in vitro regenerated shoots may be since Aari canola is a short-duration variety. However, such a correlation between the cropping type, short-day or long-day, with the in vitro regeneration potential has not been observed in Brassica
]. Aari canola is a recently approved, high-yielding commercial canola cultivar with many good agronomic attributes, and is currently grown in many parts of the country [51
]. Transformation of an elite variety has several advantages over the transformation in a model genotype. Transformations are usually made in a few selected genotypes of a species, which can be easily manipulated by genetic means. Often, these genotypes have poor agronomic performance and therefore, the engineered traits must be transferred into commercial yet cross-compatible varieties through crossing. After crossing, it takes several more years breeding work, labor and cost to recover the recurrent parent genome, and to minimize the ‘linkage drag’. The associated labor and costs with the back-crossing programs increase several times if the number of traits to be transferred increases [10
]. Aari canola belongs to Brassica juncea
), which is the most widely cultivated genotype in the Southeast Asian countries like India and Pakistan [30
]. The use of Aari canola (commercial variety) in transformation experiments will help expedite the improvement of canola through genetic transformation against biotic and abiotic stresses as well as facilitate the functional genomic studies.
In conclusion, we have used simple regeneration protocols, with slight modifications, to successfully regenerate shoots from different explants of commercial Brassica varieties. Our work has identified commercial cultivar, Aari canola, which is highly responsive to regeneration conditions tested. The quicker recovery of in vitro plants and extra-ordinary regeneration potential make this variety an ideal candidate for future transformation-based studies. The regeneration conditions and the kanamycin levels identified in this work will be useful for improving canola through different genetic engineering approaches against various biotic and abiotic stresses as well as functional genomic studies.