Plant Biology

Somatic Embryogenesis Of Cotton Protocol

Cotton is one of the most important fiber crops in the world. Genetic improvement of cotton through conventional breeding is limited by several factors such as lack of useful variation and long time periods that are required. Although plant biotechnology is an attractive means for improving cotton, its use requires an effective regeneration system from somatic tissues of cotton plants. Compared with many other crops, it is more difficult to obtain somatic embryogenesis and plant regeneration from cotton.

Cotton somatic embryogenesis was first observed by Price and Smith (1979) in??Gossypium koltzchianum.

??Materials and Methods

Seed Germination and Cultivation of Sterile Seedlings

Seeds of cotton (Gossypium hirsutum??L.) cv Simian-3 were delinted with sulphuric acid. Plump, mature seeds were chosen and sterilized by the following procedure: seeds were initially sterilized for 20 min using 70% alcohol, scorched in the flame of an alcohol burner for a moment, then dipped and kept in sterile water for 30 min to soften the seed coats and allow their complete removal. The sterilized seeds without seed coats were then sown in test tubes containing MSB [MS (Murashige and Skoog, 1962) salts with B5??(Gamborg et al., 1968) vitamins] medium supplemented with 6 g/L agar for germination at 28 ?? 2??C under 24 h photoperiod conditions with the light intensity of approximately 2000 lx.

Induction and Proliferation of Callus

Hypocotyl sections (3-5 mm length), cotyledon pieces (10~16 mm2??surface area), and root segments (3-5 mm length) of 7-day-old sterile seedlings were placed on MSB??medium supplemented with various concentrations of ZT for the induction of callus.

Selection of High Frequency Embryogenic Cell Lines

After 80 days of culture, embryogenic callus was chosen and transferred onto MSB medium supplemented with different hormones or MSB medium without any hormones for the proliferation of embryogenic callus. After 28 days of subculture, embryogenic callus with high frequency of embryogenesis was chosen for the next subculture. Then continual subcultures were carried out in the same way. Subsequently, embryogenic callus was subcultured every 28 days on MSB medium supplemented with 0.1 mg/L ZT (zeatin) and 2 g/L activated charcoal.

Differentiation of Somatic Embryos and Plant Regeneration

High frequency embryogenic cell lines were chosen and transferred onto embryo differentiation medium (MSB medium supplemented with 0.1 mg/L ZT) for the induction and development of somatic embryos. Mature embryos were chosen and transferred onto embryo germination medium [MSB or ZH (Zhang et al., 1996) medium supplemented with 0.1 mg/L ZT and 2 g/L activated charcoal] after 30 days.

All media were supplemented with 30 g/L sucrose, and were solidified with 7 g/L agar. The pH of medium was adjusted to 5.8 before autoclaving at 121??C for 15 min. All cultures were incubated at 28 ?? 2??C under a light intensity of approximately 2000 lx provided by cool white fluorescent lamps with 16 h photoperiod.

Protocol For Protein Extraction

Plant cells are rich in compounds that interfere with the 2DE separation method such as salts, organic acids, phenolics, pigments, terpenes, among others. A common protocol used in our lab for extraction proteins from plant tissues consists in the homogenization of mortar-grounded material in liquid nitrogen with an extraction buffer (20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 50 M leupeptin, 1 M pepstatin A, 10 M 3, 4-dichloroisocumarine, 1 mM phenylmethylsulfonyl fluoride and 0.05 % SDS). Although, proteins extracted following this protocol produce good SDS-PAGE separations they are not suitable for 2DE. Therefore, we adopted the widly applied (see references) protocol developed by Damerval and introduced some modifications. We were able to generate 2DE separations with superior resolution and recovery from various plant parts. Moreover, we tested this method to prepare and separte proteins from mycelium of the filamentous fungus Aspergillus nidulans with great success.

Protocol

  • Ground tissue in a mortar with liquid nitrogen. Collect grounded material in a eppendorff tube (tube weight 1.0 g).
  • Weight the material
  • Add 10 % w/v trichloroacetic acid and 0.07 % v/v 2-mercaptoethanol in COLD (-20 C) acetone (approx. 1ml for 0.3 g of tissue
  • Incubate for 2 hs at -20C (Other protocols leave it over night at -20 C)
  • Centrifuge the precipitated proteins in microfuge for 15-20 min. at 14,000 rpm
  • Wash pellet with COLD acetone containing 0.07 % v/v 2-mercaptoethanol
  • (approx. 1 ml) to remove pigments and lipids until the pellet is colorless.
  • Dry proteins under vacuum (5- 10 min.)
  • Resuspend proteins in the appropriate rehydration buffer
  • Sonicate to extract proteins in a water-bath sonicator, 15-30 min.
  • Centrifuge and collect the supernatant containing predominantly soluble
    proteins.
For a liquid sample (such a plasma, lysides or a grounded tissue??resuspended in a buffer) add 4 vol. of the 10 % w/v trichloroacetic acid??and 0.07 % v/v 2-mercaptoethanol in acetone.

Protocol For Lyophilization Of Cotton Leaves

  1. Freeze cotton leaves sample at -70 C in a deep freezer.
  2. Transport the sample in an ice chest box.
  3. Check to see that drain plug is closed (left side of condenser) and be sure that <break> switch is off and ballast open.
  4. Turn on bottom <cool> switch of the condenser.
  5. Turn on top <pump><cool><control> switches of chamber and set “shelf temperature control” to -400 or -50 C. Then, wait for chamber temperature to drop to set level (it usually takes 1 to 3 hr).
  6. Turn on bottom <pump> switch.
  7. Place the frozen samples on trays. Arrange in even layers. Placing second layer of tubes in bottom 2 trays is OK). It may be fun to place the probes into samples to watch the changing temperature.
  8. Close the chamber and wait for 1 to 2 minutes till the samples and chamber temperature equalize. During this time, ice crystals will coat metal parts of the chamber.
  9. Turn on <gauge> switch and close ballast, and wait for vacuum to reach <100 mT (about 5 to 10 min). In the mean time, condenser temperature should be at least -40 C.
  10. Set “shelf temperature control” to -20 C and let run for 3 hr at least or overnight.
  11. Check the condenser periodically to be sure that the coil has not collected enough ice to lug it up (rarely a problem on a fresh run).
  12. After the run is complete, turn off <gauge> switch and open ballast.
  13. Turn on <break> and turn off vacuum <pump> switches simultaneously.
  14. Remove the samples and turn off top <pump><cool><control> switches.
  15. Turn off <break> switch.
  16. Close the cap of samples tightly immediately.
  17. Place samples in dry, air tight environment as soon as possible and store out of direct sunlight.

Protocol For Accumulation Fixation Of Plant Metaphase Chromosomes

REAGENTS

  1. Metaphase arresting agents:
  2. Choose one of the following (Note 2) and shake vigorously to aerate before putting in living plant material; except for ice water, the solution should be the same temperature as that where the plants grow to avoid shock
    (i)??ice water??(for cereals and temperate grasses): put distilled or deionized water in a clean plastic bottle, shake to aerate and keep at ???20??C until the water starts to freeze, shake again.??
    (ii)??0.05% (w/v) colchicine??(for most plant tissues). Can be stored in the dark at 4??C for several days to weeks??
    (iii)??2 mM 8-hydroxyquinoline??(for dicotyledonous plants, particularly those with small chromosomes such asArabidopsis??thaliana??). Can be stored in the dark at 4??C for several days to weeks
    (iv) a -??bromonaphthalene??: store water above liquid a -bromonaphthalene in a bottle (typically 100 ml over 50 ml). Shake, allow to separate, and then pipette out aliquots of the a -bromonaphthalene-saturated water-phase into small vials for treatment of material. Can be stored in a dark bottle indefinitely
  3. Alcohol:acetic acid fixative: 3 parts 96% ethanol (or 100% methanol) to 1 part glacial acetic acid (prepare immediately before use; do not store for more than 30 min as the compounds degrade)
MATERIALS
For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used

  1. For germination of many seeds, put onto filter paper saturated with bottled drinking water at 20-25??C in the dark and leave until roots are about 10-20??mm long (Note 1). Seeds of trees that grow long single roots are best germinated in a pot of vermiculite, while small seeds are best germinated under sterile conditions on agar minimal medium, e.g. Murashige and Skoog without sugar (Notes 3 and 4). Seed suppliers will give advice about germination of difficult species; moving between 4 ??C and 25 ??C every 3-14 days often assists germination
  2. For hydroponic growth, suspend plantlets or bulbs cleaned from soil above an aerated plant nutrient solution (commercial ‘complete’ plant fertilizers, used at 1/10 the strength recommended as a plant feed are suitable; e.g. Phostrogen); existing roots should be immersed in the solution, but not the plant itself. Root growth is normally initiated within a few days.
  3. Plants established in soil (e.g. trees such as oil palm) may produce actively growing roots within a few weeks when compost is applied on the surface around the stem
METHODS

The following steps are carried out in small glass or plastic containers (5-10??ml) or 1.5 ml microcentrifuge tubes. Use generous amounts of solutions: typically 1 ml per specimen. Material is transferred carefully by clean forceps or a pipette

  1. To accumulate metaphases, treat excised root tips (5-20mm long) or other material with one of the metaphase arresting agents as follows??

    (i) ice water for 24 h
    (ii)colchicine for 3-6 h at room temperature or 10-24 h at 4??C??
    (iii) 8-hydroxyquinoline for 1-2 h at room temperature, then 1-2 h at 4??C
    (iv) a -bromonaphthalene saturated water for 2-6 h at room temperature

  2. Quickly blot material and transfer to fixative
  3. Fix for at least 16 h at room temperature. If fixed material is to be kept (up to several months), leave for 2??h at room temperature and then transfer to new fixative (or 70% or 96% ethanol) and store at -20??C??
NOTES

  1. The response to the metaphase accumulation reagents is different from species to species and has to be established by trial and error. Some guidelines for choosing are given; Dolezel??et al??. (1992) discusses alternative reagents, including spindle poisons used a herbicides.
  2. It is very important not to expose seedlings, roots and plants during germination and metaphase-arrest to chemicals and fumes, particularly fixatives (e.g. in a cold room also used for chemical storage) and to use clean labware with tight lids (disposable plastic is ideal), clean forceps, and distilled water.
  3. Root tips from germinating seeds, and plants grown in controlled conditions, often show waves of cell division that may follow internal or environmental rhythms (e.g. light) or correlate with root length. At certain times, there may be no divisions at all, so it may be helpful to make several fixations.
  4. Representative times are given. For best results fix material after different times of treatment, experiment with different reagents and check the mitotic index by making chromosome preparations. Treating material for too long in arresting agents, particularly colchicine, results in over-condensation of the metaphase chromosomes which might be desirable for counting chromosomes, but not for??in situ??hybridization where spatial resolution along chromosomes is wanted.
  5. Fixative should note be contaminated with water, so careful blotting or an extra rinse in fixative is advised.

Fluorometerluminometer Wallac Victor Ii

Samples for both Glucuronidase (MUG) and Luciferase assays should be prepared according to the “GUS & LUC bombardment assay” protocol.

1. Switch the Victor II and the computer on. Windows 95 launches automatically the “Wallac 1420 manager” program.

2. In the “Tools” menu, check that the “User level” is set on “Advanced”. Otherwise, the “Routine” menu would disable any system operation which means that you could not change any setting, even in your own protocols.

3. Select a protocol available in the list in the “Instrument control” menu (then go to step 5) or create your own protocol (see step 4).

4. Creation of a new protocol menu

4.1 Click with the left button (LB) on “Explorer” in the “Tools” menu in order to launch the “Wallac 1420 Explorer” program. By clicking with the right button (RB) on the “Users” folder, you will be able to create a new protol and then name it.

4.2 Open your new protocol (double-LB). This operation starts the “Protocol Editor” window which should be left open all the time you will work with the Victor II. This program enables you to define all the operations to perform (see step 4.4), to select which wells will be measured (step 4.3) and other things such as the format of the saved files, etc.

4.3 Select the wells which should be measured in the “Sample” menu (LB), the default setting is 96 wells. NB: we use the standard 96 wells white microtiter plates.

4.4 Define the operations to perform in the “Measurement” menu. Select measurement “by plate”. To add an operation, you can either click (LB) on the small icons displayed on the left or click in the white operation window (RB). The following operations can be chosen : dispense (if injectors installed), delay (to wait inbetween the measurement of two wells), shake, or label (to select the kind of detection : fluorometry, luminescence, …). To perform a MUG assay, select “label” (LB) then, in the “fluorometry” menu, select the icon represented with a locker “Umbelliferone (1.0s)” (LB). You have now the possibility to select this protocol (double-LB) and use the default Umbelliferone measurement or make a “copy” of this protocol with an other name. Important : you can not change the settings of any default protocol represented as an icon with a locker. If you want to change some parameters (such as the counting time), you have to create a new protocol (by copying a default one). The default “Umbelliferone (1.0s)” protocol is well adapted to our MUG assays.

4.5 Save your protocol which should now appear in the listing of the “Wallac 1420 Manager” window in the “Instrument control” menu. Select it (LB)

5. Run your protocol by pressing “Start”. You can have a “Live display” of the measurement. I would recommend to try your protocol once with an empty microtiter plate to check if everything works fine (good connexion with the Victor II, no mistake when you selected which wells should be measured,…).

6. How to run a MUG assay : mix both the extract and the substrate (MUG buffer + methanol) according to the related protocol (GUS & LUC bombardment assay) but do not stop the reaction with CaCO3. Indeed, you can measure the activity at various time (t0, t1h, t2h,…) from the same wells. To do so, incubate the microtiter plate at 37C in an incubator (remember to cover the plate to avoid evaporation). It is recommended, especially when the GUS activity of the sample is totaly unknown, to make a series of dilution (dilute the extract in the extraction buffer) to check wether activities will be proportional.

7. The data will be automatically saved in the same folder than your protocol (classified upon the date). You can open them (double-LB) then export them as an Excel file or other formats.

8. Leave both Fluorometer and computer on during the week. Switch them off during the weekend.

Gus Histochemical Staining

1) determine number of slides needed, multiply by 0.75ml

2) make up required vol of stain:

for 10ml(4C) 5mg X-Gus

50ul nn dimethyl formamide, dissolve

+ 10ml 50mM NaPO4 pH 7

3) sections best cut with vibrating knife

for sections w/ chlorophyll, put in cell-wells w/ 500ul stain

for sections w/out chlorophyll, put directly on slides w/ stain

4) inc o/n 37C in humidity chamber

5) asp, inc 10’in FAA:

for 200ml (4C) 10ml formaldehyde

10ml HAc

75ml EtOH

H2O > vol

6) inc 2′ 50% EtOH

7) inc 2′ 100% EtOH

8) inc 1′ H2O

Protocol For Crossfertilization Of Arabidopsis

<table border="0" width="866">
<tbody>
<tr bgcolor="#ebebeb">
<td colspan="2"><strong>PREPARE SOLUTIONS</strong></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><div align="left">Two iNOX #1 forceps (or equivalent forceps with a fine grasp)</div></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><div align="left">95% EtOH (in a vial to hold the forceps)</div></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><div align="left">dH2O (in a vial to hold the forceps)</div></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><div align="left">Kimwipe surface (to remove plant parts from forceps)</div></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><div align="left">Scissors</div></td>
</tr>
<tr>
<td>??</td>
<td>??</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><strong>PROCEDURE</strong></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">1. Grow Arabidopsis until the flowers are at a stage where they are not open, yet not closed (right before the white petals become visible)</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">2. Cut all flowers that are too old or too young from the inflorescence, leaving 2-6 flowers por pollination</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">NOTE: if the stamens are already mature with pollen, do not use that flower</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><p>3. Remove all flower parts except the ovary</p></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">Do this by first placing a piece of kim-wipe on the bottom surface of a discecting scope and the place the flowers there before starting to remove the flower parts. This helps in keeping the moist flower parts from sticking to the forcep and instead stick to the kim-wipe surface</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">4. Obtain pollen from a donnor plant and place it on the tip of the exposed ovary. Repeat this at least twice to ensure proper pollination</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">5. Stick the plant and cover the pollinated ovary with Saran-wrap and let it develop to maturity</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">6. Label each pollinated inflorescence accordingly</td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2"><p>7. Siliques should develop in about 5 days. Saran-wrap can be removed at this point</p></td>
</tr>
<tr bgcolor="#ebebeb">
<td colspan="2">8. Harvest the seed once the siliques are dry (don’t let it dry too much or the seed will be lost)</td>
</tr>
<tr>
<td>??</td>
<td>??</td>
</tr>
</tbody>
</table>

Rna Extraction Protocol

Protocol

??

1)????????Use 100uL of Plant RNA isolation mix with 800uL of lysis binding mix.

2)????????Collect 100mg of tissue and quickly Freeze tissue in liquid nitrogen.

a.??????????Grind with DEPC treated mortar and pestle

b.??????????Add 450uL buffer prepared in 1) to half of the ground tissue in a 1.5mL tube

c.??????????Repeat with the other half

d.??????????Mash tissue further with small pestle

3)????????Spin for 5 min at 10,000 x g, while spinning get Elution solution and preheat to 70oC

4)????????Transfer supernatant to a new 1.5mL tube

5)????????Add an equal volume of 64% ethanol (~400uL)

6)????????Assemble filter cartridge and collection tube, add lysate from 5) to top of filter cartridge

7)????????Spin for 1 min at 10,000 x g, discard flow through.????(The RNA is now on the filter)

8)????????Add 700uL of wash solution #1, spin for 1 min at 10,000 x g, discard flow through

9)????????Add 500uL of wash solution #2/3, spin 1 min at 10,000 x g, discard flow through

10)??Repeat 9)

11)??Spin 10-30 sec to remove the last traces of Wash solution from the column

12)??Get a fresh collection tube

a.??????????Add 40uL elution solution (preheated in step 3)) to centre of filter in column.

13)??Spin for 30 sec at 10,000 x g.????(The RNA is now in the collection tube)

14)??Add a second aliquot (20uL) of elution solution to the centre of the filter in the column.????Spin for 30 sec at 10,000 x g.????Use the same collection tube as used in 12)

15)??Mix RNA with 30 uL of LiCl precipitation solution

a.??????????Incubate at -20oC for at least 30 min

16)??Spin for 15 min at top speed

17)??Carefully remove supernatant with a pipette and wash in 70% cold ethanol

18)??Air dry pellet (~10min)

19)??Re dissolve pellet in the desired amount of DEPC water (I use 60uL)

Quick Test For Restriction Enzymes

QUICK TEST FOR RESTRICTION ENZYMES

Preparation:

  1. Grow bacterial strain in desired growth medium to late logarithmic growth stage.
  2. Pellet 1.5 ml cells in microfuge tube (7.5X for 5 min in Beckman Microfuge 11). Decant supernatant.
  3. Wash cells in 10 mM Tris (pH 8), 1mM EDTA. Pellet cells. Decant supernatant.
  4. Add 1 ml 1X low salt restriction enzyme buffer.
  5. Place tube in ice for 5 min.
  6. Sonicate sample with microprobe:??
    30 sec. 50% pulsed #2 setting??
    30 sec. 50% pulsed #3 setting??
    30 sec. 50% pulsed #4 setting
  7. Pellet sonicated lysate (13.5X for 2 min.)
  8. Transfer 500 ul of supernatant from top to new tube.
  9. Assay:??
    10 ul Lambda DNA (50 ng/ul in 1X low salt restriction enzyme buffer)??
    10 ul sonicated lysate
  10. Incubate 2 hr at 37 C. Heat inactivate with 5 min at 68 C. Place on ice.
  11. Run sample on gel.

Lambda DNA stock: 55 ul DNA (0.03 ug/ul sol’n), 40 ul 10X low salt, 305 water. should be about 0.004 ug/ul.

Bacteroid Isolation

BACTEROID ISOLATION

  1. Crush 7 g nodules with mortar and pestle in 5 ml Grinding Buffer (0.15 M NaCl, 50 mM KH2PO4 (pH 7.6)) at 4 C.
  2. Filter through 4 layers of cheesecloth and wash the cloth with grinding buffer.
  3. Fill a 50 ml PC centrifuge tube with 70% Percoll: 24.5 ml Percoll concentrate; 3.5 ml 0.5 M KH2PO4, 1.5 M NaCl (pH 7.6); 7.0 ml water.
  4. Layer 1 ml of crude extract on top of gradient.
  5. Centrifuge for 45 min at 48400 g in a JA20 rotor 4 C.
  6. Collect bacteroids by suction (peristaltic pump):
     Cytosolic fraction  -  Plant debris        -                      -  Bacteroids          -  High density pellet - 
  7. Dilute fraction 1:5 with grinding buffer; centrifuge at 12000 g for 10 min.
  8. Resuspend bacteroids (pellet) in 0.15 M NaCl, 50 mM KH2PO4 (pH 7.6).