Brassica species include many oil seed crops, vegetable crops and weeds. B. napus, with its 19 chromosomes, originated about 1,000 years ago from a hybridization between B. oleracea and B. rapa 1. The same is true for B. juncea, which originated from a hybridization between B. nigra and B. rapa. Many Brassica species such as Chinese cabbage and oilseed rape have been cultivated since prehistoric times for their seeds, edible roots, stems, leaves, buds, and flowers. Most of these crops require a prolonged winter cold for flowering through a process called vernalization2. For breeding of these crops, earliness of bolting and flowering is one of most important traits.
The Brassica genus is closely related to the model species Arabidopsis thaliana since both of them belong to Brassicaceae family. Comparative analysis between Brassica species and Arabidopsis thaliana suggested that diploid Brassica genomes descended from a hexaploid ancestor3,4. This species contain either three or six copies of orthologous genomic regions of A. thaliana5,6. The current genome structures were shaped by whole-genome triplication followed by extensive diploidization7.The Brassica species displays extreme morphological diversity8. The varieties of Brassica crops are different in shape, size, curvature, and color of leaves. Leaf variation in B. rapa has been studied extensively, and many regulatory genes have been isolated9,10. On the other hand, production of Chinese cabbage, cabbage and oilseed rape is seriously affected by a number of biotic and abiotic constraints. Genetic transformation is required for reverse genetic experiments, positional cloning, and the insertional mutagenesis, fostering diverse forms of scientific inquiry and technology development for study of genetic basis and molecular breeding of Brassica crops. Genetic improvement of Brassica crops for high yield and quality is largely dependent on our understanding of molecular mechanism of leaf variance and of stress response. Efficient genetic transformation system would provide a valuable tool for functional genomics studies and genetic improvements of Brassica crops.
Agrobacterium-mediated transformation of Brassica crops
Agrobacterium-mediated transformation has become the most common method for Brassica transformation. Inoculation of hypocotyl segments with Agrobacteriun rhizogenesis generates many hairy roots, from which the transformed plants are regenerated11-13. A. tumefaciens-mediated transformation depends on the susceptibility of plants to Agrobacterium infection and delivery of T-DNA from the binary plasmid into plant cells. The ability to regenerate transgenic plants from these transformed cells is also vital for successful transformation. In B. rapa, Agrobacterium-mediated transformation of cotyledonary explants has led to the generation of stable transgenic plants with over-expression of N-acyl-homoserine lactonase (AHL-lactonase)14, rice leucine-rich repeat protein15, pineapple fruit bromelain gene (BAA)16, and GUS reporter gene17; hypocotyl segments were inoculated with Agrobacterium suspension in MS liquid medium for T-DNA insertional mutagenesis18 and metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants19, and a mannose selection system20; and peduncular segments from flower stalk were used to transform polygalacturonase-inhibiting protein 2 (PGIP2)21. In B. napus, an efficient protocol for Agrobacterium-mediated transformation has been described using seedling explants that is applicable to various Brassica varieties22. Using the explants, site-specific marker gene excision is accomplished23, and glyphosate-tolerant plants are selected24 from the transgenic plants B. napus.
In planta_ transformation by Agrobacterium mediation
In planta transformation is first described in Arabidopsis thaliana25. The plant transformation procedures involve vacuum infiltration, floral dip, and spraying. Physically, vacuum generates a negative atmospheric pressure that causes the air spaces between the cells in the plant tissue to decrease. An increase in the pressure allows the infiltration medium, including the infective transformation vector, to relocate into the plant tissue. Agrobacterium containing a vector with a gene of interest contacts the aerial portions of a plant at flowering stage under vacuum conditions. The vacuum applied is of sufficient strength to force the Agrobacterium cells into intimate contact with the plant such that the T-DNA transfer to the plant takes place. Vacuum infiltration method makes it possible to transform plants without tissue culture or regeneration. Floral dip method further simplifies the genetic transformation by simple dipping of flowering plants into a solution containing the engineered Agrobacterium26.
The successful infiltration is achieved first in Arabidopsis27. Since then, a number of laboratories have tried to use Agrobacterium vacuum infiltration through independent approaches in Arabidopsis28,29 (Ye et al. 1999; Bechtold et al. 2000). All the results in Arabidopsis show that ovule, the female reproductive tissue, is the target of transformation in planta30.
Vacuum infiltration method for in planta transformation
A breakthrough in Arabidopsis research is the invention of the vacuum-infiltration procedure, a simple and reliable method of obtaining transformants at high efficiency while avoiding the use of tissue culture. Vacuum infiltration methods have been reported to enhance the efficiency of Agrobacterium-mediated transformation of recalcitrant pakchoi (B. rapa ssp. chinenesis)31,32. Two transformants were reported in the progeny of the infiltrated plants with Basta resistant assay. However, there is no report on successful transformation of the other Brassica crops.
Brassica crops are different from Arabidopsis, in that its plants, flowers and seeds are much big, cold period of vernalization is required for flowering, cross pollination is necessary for seed set, and most of the cultivars are self-incompatible. To apply vacuum infiltration to Chinese cabbage and oilseed rape, we have tried the new treatments such as vernalization, bud opening, hand pollination, and use of non-infected pollen, and obtained a number of the transgenic plants by in planta transformation (Figure 1).
Limitation of Arabidopsis vacuum infiltration method for Brassica crops
Inflorescences of Brassica crops are much big than that of Arabidopsis. Fully-bolted inflorescences of Chinese cabbage and oilseed rape are too big for normal vacuum chambers. Most of Brassica crops need go through a period of vernalization for reproductive growth. For induction of inflorescences, the germinated seeds or seedling must be exposed to cold condition for a period. The thick sepals and petals in floral buds of Brassica crops prevent the penetration of the Agrobacterium harboring T-DNA onto stigmas, and cause low or non-transformation. Brassica crops are cross-pollinated. In the absence of honeybee and the other pollinator, hand pollination must be applied to pollen collection and pollination. Most inbred lines of Brassica crops are self-incompatible, and emasculation or bud opening by hand should be performed for fertilization.
Advantages of vernalization-infiltration method
Vacuum infiltration method for Arabbidopsis is not applicable to Brassica species. Unlike Arabidopsis, the genus Brassica that comprises many important vegetable, oil seed and weed crops are vernalization-required, cross-pollinated, and self-incompatible. Vernalization-infiltration method makes it possible to transform Brassica crops in planta. This method is a simple and reliable, at high efficiency of transformation, while avoiding the use of tissue culture and eliminating the need for in vitro regeneration of plants. Brassica crops are reciprocal to in vitro regeneration. There is a reduction in somaclonal variation because there are no tissue culture steps. This method facilitates high throughput testing using the germinated seeds because the process is fast. Therefore, vernalization-infiltration method is especially useful for transformation of Brassica crops and could be extended to the other important crops such as wheat that is recalcitrant to plant tissue culture and regeneration.
Key factors for successful transformation
Cold-treatment of germinated seeds for vernalization
A period of cold condition (vernerlization) during seed germination and/or vegetative growth is necessary for bolting and flowering of Brassica crops. When the germinated seeds of Chinese cabbage (Bre) and oilseed rape (Hus) are used for vernalization, the seed set is much earlier (one month at least) than that of the seedlings of 2-4 leaves. Meantime, the inflorescences derived from the vernalized germinated seeds are much small, compared with that from the vernalized seedlings, and thus are more suitable for vacuum infiltration. Vernalization of the germinated seeds gives rise to the adaptable size of plants for vacuum infiltration and shortens the period of transformation experiments.
To obtain the bolting plants of Chinese cabbage for vacuum infiltration, we examined the time and duration of vernalization using the germinated seeds of Bre, an inbred line of Chinese cabbage. Compared to the seedlings, the germinated seeds are more suitable for purpose of vernalization and in planta transformation. To define cold duration thresholds for vernalization, we germinated the seeds of Bre at 22°C for 1 day and placed the germinated seeds at 4°C for various periods. Then, the germinated seeds after cold exposure were transferred into normal growth temperature (22 °C and 16 h light) in growth room of SIPPE Phytotron. When the germinated seeds were exposed to cold condition for 5, 10, and 15 days, the plants were not bolted and continued vegetative growth at normal temperature. 20 days of cold exposure were enough to saturate the vernalization requirement of Bre plants. After cold treatment, the germinated seeds were transplanted to the soil and grown in growth room for 35 days, meaning that bolting time and flowering time were dependent on duration of cold exposure for vernalization. Further adjusting of cold treatment indicated the shortest cold duration during which Bre plants were able to saturate vernalization was 18 days. The longer the duration of the cold exposure, the shorter the period of normal growth temperature for bolting. We chose a combination of the vernalizaton (4 °C for 18 days) and normal temperature (22 °C and 16 h light for 36 days) to perform vacuum infiltration. Vernalization treatments of the germinated seeds caused early bolting of the plants. This size of the plants was suitable for manipulation of vacuum infiltration, since it shortened the cycle of transformation procedures.
We tested Bre seedlings for cold treatment too. The 3-week-old seedlings took much more days to go through vernalization than the germinated seeds, especially when big plants were used for cold treatment. On the other hand, the resultant big plants were not suitable for vacuum infiltration.
Vacuum infiltration of the primary inflorescences
Inflorescences of Brassica crops are much big than that of Arabidopsis. Fully-bolted inflorescences of Chinese cabbage and oilseed rape are too big for normal vacuum chambers. Meanwhile, the growth and flowering of secondary inflorescences are much later than that of the primary inflorescences. Therefore, the primary inflorescences are major sources of transformants in the whole plants. Comparison of primary with secondary inflorescences indicated that the seed set rate and transformation frequency of the primary inflorescences are much higher than that of the secondary inflorescences.
In the vacuum chamber, the primary inflorescence of bolting plants were immersed in Agrobacterium suspension medium. The inflorescences at different stages were used separately for vacuum infiltration. The primary inflorescences with a few of open flower gave rise to high transformation frequency, compared with the elongated inflorescences with many open flowers, meaning that bolting time for vacuum infiltration was critical for successful transformation.
In Arabidopsis, the primary inflorescences are usually cut off for growth of more branches and floral buds. However, this was not applicable to Chinese cabbage. The primary inflorescences generated more transformants than the branches. For the early maturity of seeds in the infiltrated plants, the branches were sometimes clipped at their early stages.
Opening up floral buds by hand before Agrobacterium infection
As in Arabidopsis, the Agrobacterium cells containing the vector were suspended in vacuum infiltration medium, and Bre seedlings to be transformed were immersed in the suspension and subjected to vacuum infiltration (See Material and Methods).
Chinese cabbage is not a self-crossed plant. In growth room, hand pollination was used for fertilization. After vacuum infiltration, we opened the infiltrated floral buds by hand and pollinated the stigmas with the pollens themselves. The seeds produced from the infiltrated plants were germinated on the mediums containing 50 mg/L phosphinothricin (PPT). Unfortunately, no PPT-resistant plants were selected. We saw that the stigmas and pollens of the infiltrated plants, when opened by hand, were very wet and thought that the wetness prevented the stigmas from fertilization. To solve this problem, the wet stigmas and pollens of the infiltrated plants were pollinated after natural drying for 10, 30 and 60 minutes. Still, no PPT-resistant plant was obtained.
A 6-fold higher rate of transformation was obtained with a mutant that maintains an open gynoecium30. We suspected that thick sepals of Chinese cabbage were the obstacle for the Agrobacterium and transformation vector to relocate onto the stigmas, and otherwise the wet stigmas were not adaptable for hand pollination. We opened the floral buds before Agrobacterium infection, and pollinated the stigma after vacuum infiltration. The naked stigmas were quickly dry when ventilation was applied. Importantly, a few of seedlings resistant to PPT were selected. We explained that opening floral buds just before vacuum infiltration facilitated the direct contact of the Agrobacterium with the stigmas and was helpful for delivery of T-DNA to developing ovules.
Hand pollination of Agrobacterium-infected stigmas with the non-infected pollens of siblings
Bre is a highly self-incompatible line. Normally, pollination of open flowers with its own pollens in the same plants seldom produces seeds unless the floral buds were opened by hand at bud stage. Nevertheless, pollination of the naked stigmas in Bre plants at bud stage generates seeds even though seed set is not high. On the basis of venalization and bud opening before Agrobacterium infection, we tried two types of the pollen: one from its own infiltrated plant and the other from non-infected siblings. Non-infected pollens resulted in much more transformed plants and higher seed set ratio than its own pollens. This confirmed that ovules the target of productive transformation in planta30. In this case, the pollens of siblings is not only useful for seed set, but also helpful for integration of extrogenous DNA in the genome of the maternal plants.
Selection of transformants using hygromycin
Four types of binary vectors were used in this study. pCAMBIA1300, pCAMBIA2301, and pCAMBIA3301 contains a hygromycin phosphotransferase gene HPT, Neomycin phosphotransferase gene NPT II, and phosphinothricin acetyl transferase gene PAT , respectively. pSKI015-h is a modified enhancer trapping vector that contains the enhanced CaMV 35S promoter. In total, more than 20 regulatory genes isolated from Chinese cabbage were constructed in these vectors for functional study. The binary constructs were delivered into Agrobacterium tumefaciens strain GV3101 (pMP90RK) using a freeze–thaw method (Weigel and Glazebrook, 2006).
Among the transgenic lines we obtained, only one line was kanamycin-resistant. This transformant was ever selected from about 50000 seeds derived from the infiltrated plants in the many transformation experiments. The germinated seeds or seedlings of Chinese cabbage were insensitive to kanamycin. When the germinated seeds or seedlings were transferred on the medium containing kanamycin, the concentrations of knanmycin from 10-100 mg/L did not make the difference repeatedly in plant phenotype between Agrobacterium-infected and non-infected seedlings. The only transformant showed stronger growth vigor than the wild-type. Actually, growth vigor was not a good morphological marker for the transgenic plant.
12 transgenic lines were PPT-resistant. The germinated seeds were selected on the solid medium containing 50 mg/L PPT or spaying PPT on seedlings with 2 primary leaves. This concentration of PPT killed or wilted the non-transformed seedlings, but less affected growth of the transformants. Southern hybridization of one transgenic line with empty indicated that the GUS gene in pCAMBIA3301 was integrated in the genome of three plants of this transgenic line.
39 transgenic lines were HPT-resistant. The germinated seeds of Chinese cabbage were much more sensitive to hygromycin than to kanamycin and phosphinothricin. On the solid medium containing 25 mg/L hygromycin, the hypocotyls of the transformants were much high than that of the wild type, and the roots looked hairy. PCR experiments indicated that all the transformants were transgenic. However, about 6% of the transformants grew with only two cotyledons but without growth of primary leaves. We considered that the high concentration of hygromycin inhibited growth of shoot apex. To solve this problem, we reduced hygromycin concentration to 17 mg/L in the medium. The transformants selected in this way were normal and produced seeds as the wild-type. According to the phenotype and identification, we suggested that hygromycin resistance was the best selectable marker for the transformants of Chinese cabbage.
Use of AA6 promoter
We tried four binary vectors. pCAMBIA3301 contains phosphinothricin acetyl transferase gene PAT and the intron-containing GUS gene that are under the control of CaMV 35S promoter in the T-DNA region; pCAMBIA1300 contains a hygromycin phosphotransferase gene HPT (Cambia, Canberra, Australia); and pSKI015-h is a modified enhancer trapping vector that contains the enhanced CaMV 35S promoter with the replacement of PAT with HPT. In total, more than 20 regulatory genes isolated from Chinese cabbage were constructed in these vectors for functional study. The binary constructs were delivered into Agrobacterium tumefaciens strain GV3101 (pMP90RK) using a freeze–thaw method33.
The expression of the regulatory genes induced by CaMV 35S promoter is not stable, and usually decreases in T3 generation and thereafter. Then we tried AA6 promoter isolated from tomato to replace 35S promoter. The expression of extrogenous genes under AA6 promoter is genetically stable in T3 and T4 generation, compared to 35S promoter. For the genetic stability of the trangenes, AA6 is much better than CaMV 35S.
BRASSICA SEEDS. Bre (B. rapa) and Hus (B. napus) are recommended for initial experiments as the positive controls.
AGROBACTERIUM STRAIN GV3101 (PMP90RK). It carries a binary vector harboring the gene of interest and a selectable marker such as hygromycin.