Feb 24 Kim

T16 Shoot organogenesis form cotyledon explants of watermelon

Watermelon is a popular fruit originating from Africa, but cultivated in the south, southwest, and central states in the US. It is susceptible to many diseases, and thus is a good plant for genetic engineering. Somaclonal variation can produce new breeding lines, which is good for creating new cultivars. In the last assignment, we saw the negatives of somaclonal variation in organogenesis. Now we see an advantage. Seedling cotyledons are usually used for explants.

Exercises
Experiment 1 Difference in regeneration potential among distinct regions of watermelon cotyledons In watermelon, the cotyledon gives the best outcome in organogenesis. Still, not all areas of the cotyledon give uniform results. Experiment 1 will locate the best areas of the cotyledon for adventitious shoot regeneration. Procedure 16.1 1. Imbibe seeds 2. Remove seed coat and remove embryo 3. Disinfect embryo 4. Transfer embryos to vessels with germination medium and culture for 5-7 days 5. Remove germinated seedlings for dissection. Remove cotyledons. 6. Cut cotyledons into pieces and place on regeneration medium. 7. Seal with parafilm. Place dishes randomly in growth chamber. 8. Record # explants with shoots or vegetative buds. Transfer those explants to rooting medium. 9. Once roots have formed, transfer to soil. 10. Acclimatization: wash away agar from roots and transplant to soilless medium. 11. Analyze data and transfer plants to greenhouse/garden when there are favorable conditions. Anticipated results After 3-5 days explants should be 4-5x original size. After 3-4 weeks of culturing, buds, shoots, and leaves should be seen. Experiment 2 Effect of plant genotype on the ability of watermelon cotyledon explants to produce shoots Even within species, there is variation in response to a particular protocol. Thus it is important to choose those genotypes that are very regenerative for successful genetic transformation. Experiment 2 studies shoot production of 4 different cultivars. Procedure 16.2 1. Imbibe, disinfect, germinate seeds 2. Remove seedlings and follow steps 5 & 6 from above procedure. 3. Seal with parafilm. Place dishes randomly in growth chamber. At day 9-12, examine explants. Some should have bud formation. 4. At week 4, record # explants with shoots or buds. Transfer to shoot elongation medium. 5. Follow steps 9-11 above. When roots bigger than 1 cm have formed, record # that rooted.

Feb 24 Kim Anticipated results After 3-5 days explants should be 4-5x original size. After 9-12 days, some should from buds. About 75-95% shoots will root on IBA medium. About 60-90% plants will acclimate. Experiment 3 Influence of darkness In several plant species, dark treatment improves shoot regeneration. Although the role of dark treatment isnt understood, it is speculated that darkness conserves endogenous PGRs and reduces starch and oxidative polyphenols. Dark treatment shows reduced cell wall thickness and cell wall deposits, which may encourage organ regeneration. Procedure 16.3 1. Imbibe, disinfect, and culture on germination medium 2. Remove seedlings and follow steps 5 & 6 from procedure 16.1. Place plates randomly in growth chamber. 3. Examine explants at day 9-12. Some should have bud formation. 4. At week 4, record # explants with shoots or bud formation. Transfer explants with shoots to shoot elongation medium. 5. Acclimatize by following steps 9-11 in procedure 16.1. Anticipated Results Seedlings with dark treatment will be yellow, etiolated and be 2x as tall as seedlings in light. Etoilated means to develop without chlorophyll.

T17 Direct and Indirect shoot organogenesis form leaves of Torenia fournieri
Torenia fournieri is also called the wishbone flower, bluewings, or torenia. It is easy to obtain and to grow.

General considerations
Growth of plants Torenia can be grown from shot cuttings or seed. They should grow for a minimum of 8 weeks after germination. They should start flowering 12 weeks after germination. They are very vulnerable to white fly infestation. Explant preparation and basal medium Remove leaves from young and hearty plants, which will give better results than older plants. Surface disinfect and rinse. Place only 3-4 leaves per beaker with bleach solution to increase success rate. Remove leaf margins and cut into pieces. Place with abaxial side on medium. Incubate and observe weekly. Look for shoot, leaf, and callus initiation. The text talks about three types of organogenesis, which confuses me since I thought there were only two (direct and indirect). Experiment 1 Direct organogenesis from leaves of Torenia fournieri Direct organogenesis is less frequent than indirect organogenesis. However, direct organogenesis can occur if placed on media with cytokinins and auxins. The purpose of experiment 1 is to show direct organogenesis of torenia. Procedure 17.1

Feb 24 Kim 1. 2. 3. 4. Culture leaf sections on direct organogenesis medium. Place culture vessels randomly. In 4-6 weeks, count # shoots/culture vessel. If using for further experimentation, separate shoots aseptically when counting. In 3-5 weeks, remove plantlets and wash agar from roots. Transfer to soilless medium. Plants will be acclimatized in 2-3 weeks.

Anticipated results Contamination may be present 3-7 days after the culture. Discard those cultures. There may also be bleach damage, but the experiment will work if the damage isnt widespread. In 2-3 weeks, shoots should form. Each explants should have many shoots (except for damaged areas). Experiment 2 Callus formation and indirect organogenesis Indirect organogenesis has a callus formation step that is the precursor of roots/shoots. This experiment demonstrates indirect organogenesis from a callus. Given the serious problem of somaclonal variation in using indirect organogenesis, when would someone want to use indirect over direct organogenesis? Procedure 17.2 1. Culture leaf sections on callus induction medium. Place culture vessels randomly. 2. In 3-4 weeks, subculture explants on new callus induction medium. 3. After 5-6 on induction medium, remove callus and culture on indirect organogenesis medium. 4. After 4-6 weeks on organogenesis medium, count # shoots/culture vessel. Anticipated results There shouldnt be contamination because cultures will be growing in vitro. Shoots should form after 4-6 weeks on indirect organogenesis medium.

T18 Shoot organogenesis from petunia leaves

The garden petunia is a popular plant is called a cultigen because this hybrid started in domestication. It is a hybrid of 3-4 petunia species. Garden petunias are easy to tissue culture.

General considerations
Growth of plants Different petunia cultivars and genotypes will have different needs for PGRs, but almost all will produce shoots in vitro. Choose cultivars of the Grandiflora types. Avoid red flowering cultivars and mixed cultivars. Eight weeks before starting the experiment, plants seeds in a soilless medium. Do not use overhead watering, as this can cause fungal and bacterial contamination. White flies can be a problem as well. Its interesting that white flies show up in this chapter as well. They were a problem in chapter 17 as well. Seedlings should be growing rosettes when explanting. Flowering plants can be used, but may be contaminated. In each petri dish, place two leaf explants adaxial side up (upper surface up). Why is there a difference in the placement of the leaves? Chapter 17 said we should place them with the abaxial side on the medium.

Feb 24 Kim

Experiment 1 The effects of cytokinin and auxin on petunia leaf explants Experiment 1 demonstrates the effect of PGRs on organogenesis. We will look at the effects of cytokinin and auxin separately, as well as the cytokinin to auxin ratio. Procedure 18.1 1. Surface disinfect leaves 2. Remove bleach solution by swirling leaves in sterile/distilled water. Repeat once. 3. Discard edges and tip. Dissect into 1-1.5 by 0.5 cm explants. Place 2 explants per petri dish with right adaxial side up. Incubate with fluorescent lamps. Arrange petri dishes randomly. 4. Count # roots and number. Count # shoots that are at least 1 cm long. Measure callus formation with a relative scale. Anticipated Results After 1 week, roots will first start to appear on leaves growing on IBA medium. After 3 weeks, roots will mature. Shoots may be seen on leaves growing on medium with BA. After 7-10 days, callus will be seen. Best callus growth will be seen on medium with BA and IBA. Experiment 2 Regeneration of petunia from callus and monitoring somaclonal variation The purpose of this experiment is to contrast direct and indirect organogenesis. Procedure 18.2 1. Prepare explants as outlined above. 2. After 3-4 weeks, remove callus in cubes of ~4mm. Also make new leaf explants. 3. After 4 more weeks, remove shoots from callus and explants. 4. Once shoots have rooted, transplant to conditions for seedling Acclimate to greenhouse conditions. When they are large enough, move to soilless medium. 5. Arrange plants randomly 6. Each week, record length of shoots, flower corolla length, width of flowers, number stamens, plant height, number of branches, length of branches, and any abnormalities. Anticipated Results Shoots will from explants and callus. There will be greater variability in plants from callus in comparison to plants from explants or stock plants. Questions 1. Dark treatment is thought to? a. Conserve endongenous PGRs and reduce starch and oxidative polyphenol 2. What is one big problem with indirect organogenesis? a. Genetic variation 3. What is an advantage of somaclonal variation? a. Creation of new cultivars 4. What is the difference between the abaxial and adaxial sides? a. Abaxial= underside; adaxial= upper surface 5. Why is the garden petunia called a cultigen? a. It started it originated in domestication (not the wild)