1. Abida Yasmeen & Bushra Mirza & Samia Inayatullah & Naila Safdar & Maryam Jamil & Shawkat Ali & M. Fayyaz Choudhry Plant Mol Biol Rep (2009) 27:20–28 DOI 10.1007/s11105-008-0044-5 In Planta Transformation of Tomato

2. Why in planta transformation? Plant transformation using tissue culture protocols is a tedious, time-consuming, and expensive. Sometimes may result somaclonal variation. Overcoming: Transformation protocol that avoids the use of tissue culture i.e. “in planta” transformation. Methods of in planta transformation:  injection of plasmid DNA or Agrobacterium tumefaciens cells into meristematic tissues or the fruit  seeds or seedlings were imbibed in DNA and grown to maturity  DNA directly dropped onto the florets (the gametophyte progenitor tissue, mature gametophyte)  DNA directly inserted into fertilized embryos In most of the in planta transformation procedures, selection is not carried out immediately after transformation; instead, the seeds of the progeny are selected.

3. Challenges in in plant transformation: In planta transformation procedures have been used successfully for various plant species, e.g. Arabidopsis thaliana, Medicago truncatula, apple, pear, tomato, peach, strawberry, and citrus However, for most plant species, except for A. thaliana and M. truncatula, only transient transformation has been successfully reported. No reports on successful stable “in planta” transformation of any cultivated plant species, including tomato.

4. Plant Material tomato (Solanum lycopersicum L. cv. Rio Grande) Bacterial Strain A. tumefaciens strain EHA 105 harboring Plasmid Constructs pROKІІAP1GUSint (cDNA sequence (1.2-kb fragment) of the Arabidopsis AP1 gene into the SmaI restriction site of vector pROKІІ ) pROKIILFYGUSint (cDNA sequence (1.2-kb fragment) of the Arabidopsis LFY gene into the KpnI and BamHI restriction site of the T-DNA region of vector pROKII) pROKIILFYGUSint (control vector carrying GUS-coding sequence and NPTII selection marker gene )

5. Transformation by In Vitro Fruit Injection Survival rate of seedlings on the selection medium was noted after 28 days. Tomato fruits on the plant by washing with 0.1% aqueous mercuric chloride (HgCl 2 ) solution for 1 min Surface sterilized fruits A. tumefaciens culture inoculated/ injected using 1 ml sterile hypodermic syringes evenly throughout the fruit. Control: Fruit injected with the culture medium Fruits were incubated at 28°C for 1, 6, 12, 24, 36, 48, 72, and 96 h Infection Co-cultivation Transformed fruits Seeds collected on ripening of the fruit, air dried on blotting paper, and soaked over night in sterile distilled water Surface sterilized seeds Seed germination Placed on selection medium i.e. 1/2 MS with 00 mg/l kanamycin.

6. For in vitro fruit infiltration experiments, a highly significant effect of the incubation period on the frequency of transformation was observed However, there was no construct nor construct×incubation effects. The seeds of the fruit incubated with A. tumefaciens for 48 h showed the highest survival rate, followed by seeds of the fruit incubated for 36 and 72 h. After 48-h incubation, seeds of the fruit injected with A. tumefaciens harboring pROKIIAP1GUSint showed 17% transformation, fruit injected with pROKIILFYGUSint showed 19% transformation, and fruit injected with the p35SGUSint construct showed 21% transformation. The incubation periods (1, 6, and 12 h) that failed to produce even a single seedling surviving on the selection medium were ignored during the analysis. Incubation period above 48 h causes decreases seedling survival regardless of the construct used. This can be explained by a reduction in activity of A. tumefaciens cells with an increasing length of inoculation period (Desfeux et al. 2000). Desfeux et al. (2000) suggested that A. tumefaciens persists for a limited period at levels high enough to achieve reasonable rates of transformation in A. thaliana.

7. Approximately 600 seeds were inoculated for each treatment. Transformation by In Vivo Fruit Injection Method The influence of mature (red color at early ripening stage) vs. Immature (green unripened) fruits was evaluated. All tomatoes were injected twice on two consecutive days with A. tumefaciens culture suspended in normal saline (sodium chloride 0.9%) was taken up into a 1-ml syringe. fruits were used for each treatment, and both experiments were carried out in triplicate Negative control: normal saline wereinjected Tomato fruits Transformed fruits Fruits were harvested at maturity Seeds were collected and air dried on blotting paper Survival rate of seedlings on the selection medium was noted after 28 days. Surface sterilized seeds Seed germination Placed on selection medium i.e. 1/2 MS with 00 mg/l kanamycin

8. In general, mature red fruit gave a higher transformation percentage (40% in case of pROKIIAP1GUSint, 35% in case of pROKIILFYGUSint, and 42% in case of p35SGUSint) as compared to immature green fruit (2% in case of pROKIIAP1GUSint construct, 5% in case of pROKIILFYGUSint, and 5% in case of p35SGUSint; Fig. 3). In mature red fruit, the higher percentages of survival on selection medium may be due to the easier penetration of the fruit by the A. tumefaciens suspension, facilitated by the loss of cell to cell contact that occurs during fruit ripening. Ripe fleshy fruit usually have large cells whose walls undergo marked changes in their structure, and the cell walls are partly dismantled by the activity of enzymes. The result of this process is the softening of the fruit, which helps in the penetration of the bacterial culture. In the in vivo fruit injection experiments, fruit maturity at the time of infiltration showed a highly significant effect on the transformation rate (p<0.05). However, the construct effect was not significant (p>0.05).

9. Transformation by the Floral Dip Method Unopened flowers before pollination and open flowers after pollination were used for transformation. Tomato flowers Transformation of flowers A suspension of bacterial cells in 30% sucrose was carefully loaded onto the stigma of flowers using a syringe and without rupturing the delicate floral organs. The treatment was repeated every 24 h for three consecutive days. After treatment, the flowers were labeled and kept under observation until fruiting. Ten flowers were used for each treatment, and both experiments were carried out in triplicate. Control flowers were treated with 30% sucrose solution. Approximately 600 seeds were inoculated for each treatment. Fruits were harvested at maturity Seeds were collected and air dried on blotting paper Survival rate of seedlings on the selection medium was noted after 28 days. Surface sterilized seeds Seed germination Placed on selection medium i.e. 1/2 MS with 00 mg/l kanamycin

10. In the floral dip transformation experiments, the floral stage as well as the construct had a highly significant effect on transformation (p<0.05). It was observed that flowers treated before pollination gave higher percentages of transformation compared to those treated after pollination. To identify the ideal stage for treatment, the stigmas of flowers were with the A. tumefaciens solution on three consecutive days. It was found that the flowers were harmed through excessive reapplication of A. tumefaciens for more than 3 days. Flowers transformed with pROKIIAP1GUSint did not survive and failed to set fruit. However, flowers transformed with pROKIILFYGUSint set fruit, and 12% of seeds derived from these fruits survived on a kanamycin medium and were deemed putative transformants. The flowers treated with the 35SGUSint construct produced the highest number of transformed seedlings, i.e., 23%. (These results indicate an adverse effect of overexpression of the flowering genes in the floral dip method.).

11.  Agroinfiltration of ripened fruit of tomato presents an excellent protocol for transformation.  A 48-h incubation period was found to be ideal in the in vitro fruit injection experiments.  In the in vivo fruit injection experiments, applications of A. tumefaciens to mature red fruits showed better results than immature fruits.  Flowers before pollination gave a higher rate of transformation.  Among the three methods (in vitro, in vivo, and floral dip transformation methods), in vivo fruit injection method gave the best transformation results.

12. Analysis of the Transformants The presence of the transgene was confirmed by GUS assays and PCR analysis of the transgenic plants. Some of the kanamycin-resistant plants failed to give GUS-positive assays in some of tissues and were probably escapes or chimeric plants. Only those plants were considered transformed, which showed GUS activity in all of the tissues tested. Among the kanamycin-resistant plants, 42.5% were GUS positive, and all GUS-positive plants were PCR positive. stem root

14. The AP1 and LFY transgenic plants were phenotypically different from the control plants. Morphological Analysis of the Transgenic Plants In vitro infiltration of fruits In vivo infiltration of fruits Floral dip treatment

15. In vitro infiltration of fruits In vivo infiltration of fruits

16. These plants not only produced flowers earlier, they were shorter in height, their stems were not as erect, and leaves were curled with different indentation patterns. The AP1 and LFY transgenic plants were phenotypically different from the control plants. AP1 LFY Although flowers of both LFY and AP1 transgenic plants appeared to be normal, they were infertile and failed to set fruit.

17. The overexpression of flowering genes had adverse effects on in planta transformation by the floral dip protocol and resulted in the failure of fruit formation in the case of the AP1 construct. Flowering time of the LFY and AP1 transgenic plants was accelerated, and the transgenic plants developed short stems with a weeping growth habit. The leaves were curled and had different sizes and shapes compared to control plants. However, the flowers did not shed normal pollens and failed to set fruit.