1. Introduction

Citrus spp. is linked to their enormous economic and nutritional values ^[1]. However, citrus cultivation has been confronting many challenges, including control of diseases ^[2][3][4][5]. Solutions to the problems will rely mostly on breakthroughs in breeding, which is also hindered by problems, such as sterility, self- and cross-incompatibility ^[6], widespread nucellar embryony, and long juvenile periods that are associated with traditional breeding practices ^[7].

Genetic engineering by transformation has been widely adopted for crop improvement ^[8][9][10], including citrus ^[11]. The main advantage of the technique is that it allows modification of interestingtrait(s) without altering the overall genetic makeup, which is useful in making desirable changes in elite cultivar(s) ^[12][13]. The common practice of citrus genetic transformation studies is 

Agrobacterium tumefaciens ^[14][15]-mediated transformation of epicotyl segments of in vitro-germinated seedlings ^[14][16][17]. However, seed availability is seasonal and genotype-dependent. For example, many citrus cultivars are seedless or few-seeded. In addition, genotypes showed a strong impact on citrus organogenesis and genetic transformation ^[18][19][20]. Notably, juvenile tissues from mandarins hybrids are more difficult to be transformed by 

A. tumefaciens ^[21][22], reducing seriously genetic transformation efficiency ^[15]. On the other hand, the use of mature materials for 

A. tumefaciens-mediated transformation could result in earlier fruit production, bypassing or reducing the juvenile phase ^[23][24][25]. However, mature tissues show recalcitrance for de novo organogenesis induction in tissue culture and have a high occurrence of chimeric transformation and losing transformed cell lines in transgenic plants ^[26].

Genetic transformation using embryogenic cell suspension cultures could be a better alternative for having higher organogenetic potential ^[27][28]. Regeneration of putatively transformed cells and subsequent grafting of transgenic micro-shoots on rootstocks may shorten the juvenile period for flowering and fruiting ^[29]. The classical conception of somatic embryogenesis (SE) is based on the unicellular origin of somatic embryos ^[30], and this mode of somatic embryo development was the most frequently noticed in embryogenic cell suspensions of 

D. carota

Musa

Cocos nucifera

Santalum album

S. spicatum

H. vulgare

Citrussinensis

Citrus

Citrus reticulata) ^[37][38] is a popular local mandarin and “W. Murcott”’ (

C. reticulata

C. sinensis

Agrobacterium tumefaciens

Citrus

Citrus

2. Embryogenic Callus Induction

The EME, DOG and H+H have commonly used media for somatic embryogenesis ^[28][40]. In this experiment, embryonic calli were successfully induced from ovules of all three cultivars (“Sweet orange”-Egyptian cultivar, “Shatangju” and “W. Murcott”) on all three different solid media (EME, DOG and H+H). As shown in 

2. Embryogenic Callus Induction

The EME, DOG and H+H have commonly used media for somatic embryogenesis [28,40]. In this experiment, embryonic calli were successfully induced from ovules of all three cultivars (“Sweet orange”-Egyptian cultivar, “Shatangju” and “W. Murcott”) on all three different solid media (EME, DOG and H+H). As shown in 

Figure 1A, the highest callus induction occurred on EME medium, while the lowest was on H+H, although no statistically significant difference was found among the three media used in the study. However, in some other cases and other plant species, media have shown to have significant effects on callus induction ^[41].

A, the highest callus induction occurred on EME medium, while the lowest was on H+H, although no statistically significant difference was found among the three media used in the study. However, in some other cases and other plant species, media have shown to have significant effects on callus induction [41].

Figure 1.

 Somatic embryo induction.Data were recorded after 6–8 weeks of incubation. The statistical difference (

p

 ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using Statistical Package for the Social Sciences (SPSS-version 23), and results were expressed as mean ± standard error of three independent experiments. (

A

) Culture media effect, all explants on the individual medium were considered as one group irrespective of genotypes (

B

) Genotypes effect, all explants of the individual genotype on all three media were considered as one group.

Figure 1B is the callus induction rates of the 3 cultivars in the case of 8 to 10 weeks old ovules. The highest induction rate, around 74%, was from “Sweet orange”*, whereas the lowest, around 71%, was from “W. Murcott”. Previous studies used excised nucelli ^[42], abortive ovules ^[43], unfertilized ovules ^[44], undeveloped ovules ^[45][46], isolated nucellar embryos ^[47], juice vesicles ^[48], anthers ^[49], styles and stigmas ^[50], leaves, epicotyls, cotyledons and root segments of in vitro grown nucellar seedling ^[51] for somatic embryogenesis in 

B is the callus induction rates of the 3 cultivars in the case of 8 to 10 weeks old ovules. The highest induction rate, around 74%, was from “Sweet orange”*, whereas the lowest, around 71%, was from “W. Murcott”. Previous studies used excised nucelli [42], abortive ovules [43], unfertilized ovules [44], undeveloped ovules [45,46], isolated nucellar embryos [47], juice vesicles [48], anthers [49], styles and stigmas [50], leaves, epicotyls, cotyledons and root segments of in vitro grown nucellar seedling [51] for somatic embryogenesis in 

Citrus. We chose undeveloped ovules as callus induction material because previous studies showed that undeveloped ovule is a preferable material for somatic embryogenesis not only for having higher regeneration capacity but also for being mostly virus-free ^[52]. Gmitter and Moore reported the explants regeneration percentage from undeveloped ovule was between 0% and 70%, depending on genotypes ^[45], but all the 3 genotypes used in the study showed a higher than 70% induction rate.

Embryonic callus induction is closely associated with the differentiation status of the material (ovule) used ^[40][53]. In our experiment, the age of ovules was indeed showed a significant influence on the embryonic callus induction. As shown in 

 We chose undeveloped ovules as callus induction material because previous studies showed that undeveloped ovule is a preferable material for somatic embryogenesis not only for having higher regeneration capacity but also for being mostly virus-free [52]. Gmitter and Moore reported the explants regeneration percentage from undeveloped ovule was between 0% and 70%, depending on genotypes [45], but all the 3 genotypes used in the study showed a higher than 70% induction rate.

Embryonic callus induction is closely associated with the differentiation status of the material (ovule) used [40,53]. In our experiment, the age of ovules was indeed showed a significant influence on the embryonic callus induction. As shown in 

Figure 2, the callus induction percentage varied from around 41% to 74% across the whole age group used in the study. However, it was neither the younger nor, the older age groups, but the middle age group (8 to 10 weeks) was the best in terms of callus induction rate.

, the callus induction percentage varied from around 41% to 74% across the whole age group used in the study. However, it was neither the younger nor, the older age groups, but the middle age group (8 to 10 weeks) was the best in terms of callus induction rate.

Figure 2.

 Effect of ovule age on embryonic callus induction. Data were recorded after 6–8 weeks of incubation. All explants of the individual age group (i.e., 4 to 6) from all three cultivars on all three media were considered as one group. The statistical difference (

p

 ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

3. Suspension Cell Culture and Plant Regeneration

In this experiment, suspension cell culture for all three genotypes was established in liquid H+H medium, as previous studies demonstrated that the medium (H+H) was suitable for citrus cell suspension culture ^[28][54]. Maintenance of suspension culture involves regular subculture (every two to three weeks), which is laborious but important for subsequent experiments and plant regeneration ^[28][55]. In this experiment, we investigated factors affecting intervals of suspension cell subculture and subsequent embryo development. Our results showed that adding a smaller amount (1ml) of suspension cells to fresh media (~50 mL) could extend subculture intervals to 8 weeks without affecting the following embryo production rate (15~16 per plate) (

3. Suspension Cell Culture and Plant Regeneration

In this experiment, suspension cell culture for all three genotypes was established in liquid H+H medium, as previous studies demonstrated that the medium (H+H) was suitable for citrus cell suspension culture [28,54]. Maintenance of suspension culture involves regular subculture (every two to three weeks), which is laborious but important for subsequent experiments and plant regeneration [28,55]. In this experiment, we investigated factors affecting intervals of suspension cell subculture and subsequent embryo development. Our results showed that adding a smaller amount (1ml) of suspension cells to fresh media (~50 mL) could extend subculture intervals to 8 weeks without affecting the following embryo production rate (15~16 per plate) (

Figure 3

). However, there was a significant reduction in the number of embryos (~8) produced per plate when the same 1 mL of suspension cells was used from 2 weeks subculture intervals, perhaps from an insufficient founder population. When larger volumes of suspension cells were used in the subculture, the subculture intervals were proportionally shortened. For example, using 5 mL of inoculation volume reduced subculture intervals to 2 weeks since longer intervals significantly reduced embryogenic capacity, possibly a result of nutrient exhaustion. Three milliliters inocula in 50 mL fresh medium was good for regular experiments (

Figure 4B). This allowed the cells to grow and ensured sufficient cells for the experiments. However, for the maintenance of suspension cells, 1 mL inocula in 50 mL fresh medium was suitable for its significantly extended subculture intervals.

B). This allowed the cells to grow and ensured sufficient cells for the experiments. However, for the maintenance of suspension cells, 1 mL inocula in 50 mL fresh medium was suitable for its significantly extended subculture intervals.

Figure 3.

 Effects of inoculation volume of suspension cells and subculture intervals on embryo production. The suspension cells were subcultured in H+H medium, and then the embryos were produced on EME-malt medium. The embryos were counted after 10 to 12 weeks of incubation on an EME-malt medium. All embryos produced from all genotypes of an individual subculture interval (i.e., 2 W) of the same inoculation volume (i.e., 1 mL) were considered as one group. The statistical difference (

p

 ≤ 0.05) among the means was analyzed by Duncan’s multiplerange test using SPSS (version 23), and results were expressed as mean ± standard error of three independent experiments.

Figure 4.

 Embryonic callus induction, somatic embryo production, suspension cell culture establishment and plant regeneration of the “Sweet orange” (

Citrus sinensis

) cultivar. (

Aa

) Embryonic callus induction from 8 weeks old ovule on EME medium (

Ab

) and somatic embryo development, (

B

) Suspension cell culture establishment in an H+H medium, (

C

,

D

) Callus formation and embryo germination from suspension cell culture on an EME malt medium (the bars represent 0.5 mm), (

E

) Axis elongation of germinated embryos on an B+ medium, (

F

) Plants on RMAN rooting medium for root induction, (

G

) In vitro shoot grafted rootstock plant and (

H

) In vitro rooted plant transplanted on the soil. The bars represent 1 cm, except C and D.

In this study, 

In this study, 

Agrobacterium

-mediated transformation and regeneration of suspension cellsderived from “Sweet orange”, “Shatangju” and “W. Murcott” were successfully accomplished (

Figure 4

). BASTA (20 mg/L) was added to the media to suppress the growth of nontransgenic cells. The transformation percentage was 32 to 35 and not significantly different among the cultivars. Genetic transformation with desirable genes is an effective alternative for

Citrus improvement ^{[56][57][58][59]}. Apparently, higher transformation efficiency is preferable since more transformants mean the chance of selecting an ideal transgenic line is high. In this regard, cell suspension culture is better than other materials, such as commonly used epicotyl segments prepared from in vitro germinated seedlings ^[15][21][22] and mature stem pieces ^[22] that normally showed a very low transformation rate (less than 10%).

 improvement [56,57,58,59]. Apparently, higher transformation efficiency is preferable since more transformants mean the chance of selecting an ideal transgenic line is high. In this regard, cell suspension culture is better than other materials, such as commonly used epicotyl segments prepared from in vitro germinated seedlings [15,21,22] and mature stem pieces [22] that normally showed a very low transformation rate (less than 10%).

4. Transgenic Plant Recovery and Molecular Analysis of Transgenic Plants

The BASTA-survived in vitro micro-shoots were propagated in two ways: grafted on rootstocks (

4. Transgenic Plant Recovery and Molecular Analysis of Transgenic Plants

The BASTA-survived in vitro micro-shoots were propagated in two ways: grafted on rootstocks (

Figure 4

G) or rooted on RMAN medium and then planted on soil (

Figure 4

F,H). Both methods gave a survival rate of higher than 90%. However, the growth of the in vitro rooted shoots was poorer than the grafted (

Figure 4

H,G). All plants regenerated from selection pressure of 20 mg/L BASTA contained the transgenes, as shown by PCR analysis of leaf genomic DNA (

Figure 5

), indicating that a concentration of 20 mg/L BASTA was high enough to screen out the transformants in our case. Tissues from different organs, including the apical and the basal leaves and even roots from invitro rooted plants, were examined by PCR. It seemed that no chimeric plant was detected, demonstrating that the chances were very slim for single-cells and/or a very small group of cells to produce chimeras (

Figure 4C,D). Additionally, no visual phenotypic changes were observed on all transgenic lines so far.

C,D). Additionally, no visual phenotypic changes were observed on all transgenic lines so far.

Figure 5. Gel electrophoresis of PCR amplified DNA from transgenic plants. 2000 kb DNA ladder (M), transgenic plants samples (1–5), positive control (+), negative control (−). (A) PCR amplicon (1139 bp) from CaMV35S forward and CsDMR6 reverse primers.(B) PCR amplicon (429 bp) from Bar (bacterial bialaphos resistance gene) forward and reverse primers. (C) T-DNA constructs showing corresponding primer sites.

RT–PCR results showed that the 

RT–PCR results showed that the 

DMR6

 in all three tested transgenic lines had a higher expression level than the control, and particularly, transgenic line 2 showed the highest expression level (10-fold) (

Figure 6). This may be because of the insertion of different numbers of gene copies in different transgene lines. Transgene expression level depends on transgene copy number and/or site of gene integration ^{[60][61][62][63]}. Different copy numbers in different transgenic lines could lead to variable gene expression levels in independent transformants ^[64]. Gene silencing could be induced by transgene ^[65], but no silencing was observed in PCR-tested transgenic lines in this study.

). This may be because of the insertion of different numbers of gene copies in different transgene lines. Transgene expression level depends on transgene copy number and/or site of gene integration [60,61,62,63]. Different copy numbers in different transgenic lines could lead to variable gene expression levels in independent transformants [64]. Gene silencing could be induced by transgene [65], but no silencing was observed in PCR-tested transgenic lines in this study.

Figure 6.

 RT–PCR-mediated expression level analysis of 

CsDMR6

 in transgenic plants. Results were expressed as mean ± standard error of three independent experiments.