Arabidopsis Methods

Seed Sterilization Methods
Conduct the following steps using standard aseptic technique.
1. Soak the seeds in sterile water for 30 minutes.
2. Decant the water, add 95% EtOH and soak for 5 minutes exactly.
3. Decant the EtOH, add 10% Chlorox bleach and soak for 5 minutes exactly.
4. Rinse seeds 5 times with sterile water.
5. Add ampicillin (Sigma) at 100 ug per mL of final medium to control bacteria.
An easy way to sow large quantity seeds to soil is to mix seeds with fine sand grains and spread the mixture evenly over the soil.
The method we usually use to sow large numbers of seed is to suspend the seed in 0.1% agar. The agar solution is liquid and about the same density as Arabidopsis seed so the seed will be suspended. This solution can then be pipetted onto the surface of the soil such that the seeds are distributed evenly.
Try using a 1 ml disposable Gilson tip for sowing lots of seeds evenly. Half fill with seed and by tapping gently with finger you can get a just a few seeds dispensed at a time. If your sowing lots of different lines a new tip each time gets over seed contamination problems.
Sterilization by Microwave
I have a very useful hint to sterilize Arabidopsis and tobacco seeds I want share with the network. It was suggested to me by Mark Holland, Biochemistry Department, Columbia, Missouri (by the way, thanks, Mark). At the beginning I could not believe it. Now, it had become in my method of choice, because it works for me thousands of times better than the combined method of imbibing seeds into an ethanol and clorox solutions. I use a microwave oven. I take some seeds (not many) and put them into a small paper envelope. Try to avoid those having metal parts. Do not seal them, let the air to escape. Once the seeds are inside, I shake the envelope a little bit to let the seeds spread all over the envelope (trying to separate them). Then, I turn the microwave on to maximum power for 8-15 min (depends upon the microwave power), and repeat the treatment after 5-8 min for another 8-15 min. You can use several envelopes at the same time, but do not pile them. Hot can make them into fire!. Oil and water content of these seeds are low enough to avoid an excessive warming, and they survive the treatment. In my hands, fungi and bacterial contaminations were gone without significantly loosing the percentage of seeds that germinate (in fact I could say that I do not kill any seed!). You should try several times before getting the optimal conditions for your ovens. I do not guarantee that you can get rid of all kind of "hard" contaminants, but I think is worthy to try it. This method, however, is not useful for big seeds, like soybeans, pea, etc. I presume the water and oil content of these seeds are too high. I would be pleased to answer any questions, and to receive your own experiences on this respect.
Good luck to everybody

Antonio R. Franco
Dpto Bioquimica, Biologia Molecular y Fisiologia
Facultad de Ciencias
E-14071 CORDOBA
SPAIN

Alternative E-mail address: BF1RODRI@CC.UCO.ES.BITNET
EMS mutagenesis
In our EMS mutagenesis (seed was shaken with EMS for 17 hours, rinsed several time in H20, then shaken in H20 for 24 hours) 10 mM EMS resulted in 50% of the M1 plants segregating for embryo lethals. However, both germination of the treated seed and growth rate of the seedlings were not significantly affected up to 20 mM EMS (germination was tested on soil and not in a petri dish, so we could have missed some dead seed). Delayed germination occurred with seed treated with 30 mM EMS. Clear losses in germination rate together with severe stunting were obvious at 60 mM. Most seed was killed at 80 mM. Although M1 plants from seed treated with 20 mM EMS appeared pretty healthy, their pods were mostly empty. I have only looked at M1 seed kill on petri plates once. Based on that experience, I think that this is a very poor indicator of mutagenesis. The kill rate for 0.3% EMS overnight treatment was very low (13%), so the signal to random noise ratio is likely to be rather low. Embryo lethal rates might be a better (albeit more painful) indicator of mutagenesis rate. Let me know if anybody has had a different experience with M1 seed kill as an indicator, or if you come up with a clever way to titrate mutagenesis without going to M2.
Mutagenesis suggestions
1. With regard to mutagen dose vs. seed survival: 37% survival
Simple theoretical models of mutagenesis predict that the maximum yield of mutants, when considering the combination of mutagenicity and lethality, will occur at 37% survival (see appendix). This of course ignores the problems of multiple mutants per individual, but everyone backcrosses mutants 5 or 6 or 7 times anyway. In practice I usually use try various mutagen doses until I get 40 to 50 % survival.
2. Lot size of M1 pools a suggestion some may find useful
A possible balance point between pooling all the M2 seeds into one large pool, versus collecting seeds from each M1 plant individually: Project the size of your M1, then take the square root. Pool the seeds from this many plants together in a single M1 pool.
Appendix for the theoretically inclined:
M = total number of mutants
N = total number of individuals
l = fraction alive after mutagenesis
m = fraction mutant among those alive
d = dose of mutagen
assume l = e ** (k1 d) which is exponential survival
assume m = k2 d which is linear dose response
M = mlN
M = k2 d e** (k1 d)
if you now take dM/dd = 0, and solve for d
the maximum mutant yield will occur at d = e**1 = 37%
This message is in response to the question about the number of hits sustained per genome during conventional EMS mutagenesis. Several years ago I made such calculations based on observations of mutation frequencies at several loci following EMS mutagenesis. I estimated that a M1 population of 125,000 plants should carry every possible EMS induced mutation and that any given M2 plant would be either heterozygous or homozygous for approximately 215 mutations.
If you would like to check the logic behind these calculations they were published : Haughn and Somerville 1987. Herbicide resistance at the whole plant level. In: Biotechnology in Agricultural Chemistry, LeBaron, Mumma, Honeycutt, Duesing eds, ACS Symposium series #334 pp 98107.
FAST NEUTRON IRRADIATED ARABIDOPSIS SEED

As interest appears high, I thought it appropriate to mention that LEHLE
SEEDS expects to have fast neutron irradiated M2 seed available
sometime in January 1992. M2 seed will be available in three
backgrounds, Columbia, Landsberg erecta, and RLD and will be bulked
in parental groups of about 1000 M1 parents.

The irradiation dosage of the seed to be offered was 60 Gy and was
performed by the IAEA in Vienna Austria. Seeds were irradiated dry in
paper envelopes. I have grown out a small sample of these seeds
(Landsberg erecta background only) and have made the following
preliminary observations.

Establishment is about 15% of untreated controls. I define
establishment as the percent of seeds which produce a fertile M1 parent.
I estimated this by germinating the seeds on nutrient agar plates and
then transferring all viable seedlings to potting mix.

I also selected 4 siliques each from fertile M1 parents and counted the
number which showed segregation for albino embryos. It is my
understanding that albino embryos are rarely seen in unmutagenized
material. The work is still in progress, but 16 out of 78 (20%) of the
fertile M1 parents exhibit segregation of albino embryos among green
embryos.

If you wish to be notified as soon as these fast neutron irradiated seeds
are offered, kindly send a card, letter, or FAX with your name and
address to the following location.

LEHLE SEEDS
6531 North Camino Katrina
Tucson AZ 85718 USA
Phone 602-544-0733
FAX 602-797-9009 (THIS IS OUR NEW FAX NUMBER)
Arabidopsis thaliana transformation protocols
Dirk Valvekens
Mieke Van Lisjebettens
Marc Van Montagu
Laboratorium voor Genetica
Rijksuniversiteit Gent
Belgium
1. Arabidopsis thaliana root transformation media
(1) germination medium (GM)
1 X Mursahige and Skoog salt mixture (Flow Laboratories)
1% sucrose
vitamins (1000 X stocks)
100 mg/l thiamine
0.5 mg/l pyridoxine
0.5 mg/l nicotinate
(2) 0.5/0.05 callus inducing medium (CIM)
1 X Gamborg's B5 medium (without 2,4-D), kinetin, and sucrose (Flow Labs)
2% glucose
0.5 g/l MES (pH 5.7 adjusted with 1 N KOH)
0.8 g/l agar (for solid medium)
0.5 mg/l 2,4-D, stock: 10 mg/ml in DMSO
0.05 mg/l kinetin, stock: 5 mg/ml in DMSO
(3) 0.15/5 Shoot-inducing medium (SIM)
Gamborg's B5 medium
2% glucose
0.5 g/l MES (pH 5.7)
0.8% agar
5 mg/l N6-(2-isopentenyl)adenine (2ip), stock: 20 mg/ml in DMSO
0.15 mg/l indole-3-acetic acid (IAA), stock: 1.5 mg/ml in DMSO

(4) 0.15/5 V750 K50
As 0.15/5, but supplemented with
750 mg/l vancoymcin (Vancomycin HCl, Eli Lilly), stock: 200 mg/ml water
50 mg/l kanamycin, stock: 50 mg/ml in water
(5) 0.15/5 V500 K50
As 0.15/5 but supplemented with
500 mg/l vancomycin
50/mg/ml kanamycin
(6) GM K50
As GM, but supplemented with
50 mg/l kanamycin
Nine cm diameter Petri plates (Falcon Optilux 1005) were used in all cases, except for GM, which was normally poured in 15 cm Falcon 1013 plates. All Petri plates were sealed with Urgopore (France) gas permeable tape during tissue culture. (Nescofilm was found to lower regeneration frequencies at all stages.)
note: from recent experiments, kinetin and IAA can both be left out of the 0.5/0.05 CIM and the 0.15/5 SIM, without affecting regeneration efficiencies.
Growth of Arabidopsis plants.
(1) Seed sterilization
(i) place seed in 70% EtOH for 2 minutes;
remove EtOH with pipette
(ii) put seeds in 5% bleach, 0.05% Tween 20 for 15 minutes; shake
(iii) Wash seeds in sterile water 5 X
(iv) After first wash, keep seedsin 0.5-1.0 ml water;
take them up with a 2 ml pipette
inoculate 9 cm Petri plates spread with GM agar
disperse seeds with pipette tip
(2) Seedling growth.
(i) incubate seeds on GM for 7 days (22C, 16 hr light)
(ii) transfer cotyledons to fresh GM in 15 cm Petri plates (15-30 seedlings per dish)
note: sterilized seeds can also be immediately transferred individually to 15 cm Petri plates; however, in our experience. germinated seedlings are easier to manipulate.
Regeneration from roots
(for both generation and transformation, white roots of any age can be used. After 8 weeks growth, however, the roots will begin to turn green.)
(1) pull plantlets gently out of GM agar using a forceps (whole roots will easily extricate). Alternatively, the aerial parts of plants can be removed with a scalpel and forceps so that only roots remain in agar. Because only roots grow in agar, it can be overturned and roots then easily isolated.
(2) In a sterile petri plate, cut all green tissue away from roots.
(3) incubate root on solid 0.5/0.05 medium; take care that roots completely contact medium.
(4) incubate for 4 d in growth chamber.
(5) transfer roots to 0.15/5 medium.
within 4 weeks, the explants produce shoots over their entire length. Shoots can be cut off to set seed after transfer to GM in 15 cm Petri plates. Shoots should easily produce > 100 transformed seeds, even without rooting. However, only 2 to 4 shoots should be put in any one petri plate; dish lids must be absolutely condensation free. High humidity inhibits anthers to dehisce. Rooted regenerants are obtained on GM with 50% efficiency and can eventually be transferred to soil to set seed.
Transformation of root explants.
(1) Gently pull plantlets out of GM agar using a forceps.
(2) put plantlet in a sterile Petri plate; remove all green tissue.
(3) incubate roots on solid 0.5/0.05 medium; ensure that entire root contacts media
(4) Incubate 3 days in growth chamber.
(5) stack 3 to 5 callus-induced roots in a sterile petri dish; cut roots into 0.5 cm slices
note: one root explant is composed of multiple root segments and can be considered as a cutting through one branch of a root system. When performing infections or washings, the explants sometimes fall apart. In this case, 5 to 10 root explants are restacked for further incubation.
(6). transfer the root explant to a Petri plate containing 10 to 20 ml liquid 0.5/0.05 medium.
note: for ease of Agrobacterium infection and root explant washings, little autoclaved plastic baskets, 3 to 4 cm high by 6 cm diameter) with 100 um mesh bottoms are used. roots explants are put in these sieves to be inoculated or washed. In this way, explants can be removed very efficiently rather than "fishing" them from liquid medium. This reduces time for infections and washings.
(7) add 0.5 - 1.0 ml induced, overnight grown Agrobacterium culture. shake gently 2 minutes.
(8) blot root explants on a sterile filter paper to remove most liquid. Transfer to 0.5/0.05 solid medium.
(9) incubate up to 50 explants per 9 cm petri plate in growth chamber 2 days to allow Agrobacterium transformation. After 2 days, explants should be completely overgrown with agrobacteria.
(10) Transfer explants to 10 to 20 ml liquid 0.5/0.05 medium; shake vigorously to remove agrobacteria; blot root explants several seconds on sterile filter paper; transfer explants to solid 0.15/5 medium containing 750 mg/l vancomycin and 50 mg/l kanamycin. Ensure that root explants contact medium well.
(11) Incubate explants in growth room 2 to 3 weeks. After 3 weeks, tiny green calli appear.
(12) Transfer explants to solid 0.15/5 medium containing 500 mg/l vancomycin and 50 mg/l kanamycin. Two weeks later, green calli form shoots that are often vitreous (watery), due to vancomycin. Lower vancomycin concentrations to 250 or even zero after 5 to 6 weeks tissue culture.
(13) Transfer normal shoots to GM in 15 cm petri plates to allow further development. Among several types of containers, Optilux petri dishes were found to yield most efficient shoot regeneration and seed production.
To obtain seeds in vitro, the transformants should be cultured at very low densities (2 to 4 shoots per petri dish) and lids must be kept free of condensation. Rooting occurs with 50% efficiency on GM medium and rooted transformants can eventually also be transferred to soil to set seed.
Notes: This transformation procedure has been developed with Arabidopsis C24 or "European Columbia". C24 plants have (except on very young leaves) no trichomes (glabrous) whereas true Columbia has a very hairy (pubescent) stem and leaves.
Besides C24, the transformation and regeneration system also works with Columbia and Landsberg erecta. These three are the only ones tested. For Columbia, the regeneration time is longer and the overall efficiency is lower. however, at least 20% of the transformed root explants give rise to seed producing regenerates. Landsberg is similar to C24; transformation at the callus level (roots yielding transformed calli) is usually 100%. However, regeneration is both slow and less efficient than C24. Also, in vitro seed yield and rooting efficiency is lower. Overall transformation efficiency (root explants producing transformed seeds) is usually more than 20% per 4 month experiment.
Using this root transformation procedure, "escapes" (which yield exclusively bleached seedlings when T1 seed is germinated on kanamycin) typically occur between 5 to 10%. Hastening regeneration (weekly media refreshment) yields more escapes, presumably due to shorter kanamycin selection time and higher medium feeding capacities. Possibly, this can be overcome by increasing kanamycin to 60 or even 70 mg/l (this has never been tested). With C24, transformed seeds could be harvested after 7 weeks with bi-weekly medium changes.
Germination of F1 progeny.
(1). F1 seeds are germinated on 9 cm petri plates spread with GM plus 50 mg/l kanamycin. Plates are sealed with Urgopore tape.
(2) Place dishes in the dark at 4C (reefer) for one week (vernalization). If seeds have been previously vernalized, this step is unnecessary.
(3) incubate petri plates in growth room for 2 weeks. Sensitive seedlings don't form roots nor leaves, and show white cotyledons; transformed seedlings are phenotypically normal.
(9)
Transformation of A. thaliana - root cultures
We have had good luck with roots grown in liquid culture: 1020 seeds in 50mls of Gamborgs B5 grown in a 250 ml flask for 23 weeks.
We obtain 510 gms/flask and most of it is roots. You should be able to make RNA or DNA from this material by any method that you prefer.
Transformation of A. thaliana - seed from transgenics
We've had some improvements in working with primary transgenics. Several steps are important.
1. First, isolate a SINGLE shoot with a small ball of callus on the bottom. (Cut this from a kan resistant shoot forming mass).
2. Let it grow on a plate until the SINGLE shoot is about 1 cm. Typically 35 days.
3. Next, dip the callus end in Rootone (F) so that the ball is cover with the white powder (I got my rootone from a store like Kmart).
4. Place this shoot/callus structure in thoroughly wet soil. Be sure the callus ball is buried and that the soil goes about halfway up the shoot. Put one shoot per 2 &1/2 inch pot. Put TWO 2.5 inch pots in a bag (THE size and thickness of the plastic bag is critical). We use 6X12 inch by 2mm bags from Consolidated Plastics (#90392). If the bag is too big the shoots get fungus, too small they rot. Fold the bag over and staple it close.
5. Give low (80 uE) light at 22 C for at least 45 days or until VISIBLE growth starts. DO not open the bag til the little weed starts to grow. After 45 daysremove the staple in the evening. Next dayopen the bag (don't touch the plant).
6. Twothree FURTHER days later cut 34 inches off the bag. Another day, cut a few more inches. All changes should be done in the evening. If the flowers are showing hyperstyllic developmentpollinate with a wildtype plant.
This method has worked well for us. (Forgot one thing before you put the plant in the soil, make a hole in the soil and put a little fertilizer in it. We use Peters. Be sure the soil is entirely in contact with the plant when you place it there).
The critical point is to let the plant tell you when it's ready to come out of culture or a bag.
From dish to soil, another recipe...
We do our transformation work with ecotype No0. We usually obtain some pods from KanR shoots growing on AGM plates.
Here is the recipe: AGM plates 0.5X Murashige and Skoog salt mix (Gibco 1117) 1% sucrose 0.6% Gibco Phytagar 1X Gamborg B5 vitamins (add after autoclaving) pour in 10x2.5 cm plates These plates are sealed with porous surgical tape (3M 15301). While most shoots seem to produce seed, we prefer to transfer them to soil where the seed yield is much higher and seeds are easier to collect. According to our experience, failure to establish a rooted shoot in soil is due to damage or loss of root system during removal of the plant from the agar medium.
The following protocol solves this problem and gives over 90% survival of transplants. Applying care, one can actually get 100% survival. By the way, this method also works for seedlings selected by whatever screen on petri dishes.
>From Dish to Soil
1. Root shoots from transformation on AGM plates. As soon as rooting is evident, transfer the rooted shoot to a new AGM plate in which the medium was solidified with 1.5% Phytagar. During transfer try not to break the new roots. If necessary, cut out the agar plug containing the roots
2. Incubate the AGM plate with the rooted shoots vertically. All new roots will grow on the surface of the agar medium
3. Once the root system reaches 5 cm in length the plant can be moved to soil
4. Fill a small pot with potting mix (I think that most formulations should work, send me an Email message if you want ours). Drench the mix with water, drain excess, open a crack into the mix with a spatula or a knife
5. Open the petri dish with the plant, using benttips forceps carefully lift the plant off the agar. Do not close the forceps, think of it as lifting a tiny baby out of the cradle: put your forceps tips under the leaf rosette (the arms of the baby) and lift without squeezing
6. Drop the root system into the crack allowing the roots to reach as far down as possible. Close the crack by applying gentle pressure on the side of the plant.
7. Put the plant in growth room or in a shaded area of the greenhouse where temperature can be kept below 25 C, preferably at 18 to 22 C. Set relative humidity to 80% or cover the potted plant with clear plastic. Remove the cover after two days and grow as usual Note that Arabidopsis does not like extreme humidity, so do not leave the plastic cover on for longer than two days and do not waterlog the soil.
In response to a question from Rob Last (Boyce Thompson Inst) on seed collection from transgenic A.t. plants: We have reasonable good success when Kanr shoots of ca 5 mm are directly transferred to Magenta boxes containing GM medium as described by Valvekens. When plants develop fully, we use a Magenta vessel coupler (Sigma Plant Cell Culture Catalogue C0667) to place another vessel on top. The vessels should then be placed away from heat sources as lights to prevent condensation. PLant can reach considerable height and make a large number of flowers. Please summarize all improvements you get on this subject and put it on the BB.
Common denominator: high BAP : ANA allows shoot regeneration, but high BAP conc. (> 2.5 mg/l) inhibits shoot development. Sterilize seeds i30 min. in conc. sulfuric acid (!), then wash (> 1 hr) in water. Use halfstrength MS medium plus 0.1 mg/l BAP and 0.01 mg/l ANA, 1000 lux, 16 hr light.
Transformation of A. thaliana - root transformation
We modified the root transformation procedure from Valvekens et al.(1988) and have had good success with WS.
ARABIDOPSIS TRANSFORMATION
1. Seed Sterilization. Sterilize WS (Wassilewskija) seed for 10 min in a solution of 50% Chlorox with 0.1% SDS in ependorf tubes. Rinse 3 to 5 times with dH20. Dry thoroughly on sterile filter paper.
2. Root cultures. Place 35 seed into each of 35 250 ml Belco flasks with 50 ml B5 medium. Place on shaker (7080 rpm) at 23!C, 24 hr light for about 3 weeks. Roots will fill flask and provide plenty of tissue for transformation. Use roots while still white and healthy.
3. Preculture on CIM. Removing entire root mass from flask and cut off shoots and any green roots. Using forceps, pull off small bundles of roots and lay them on MSKig medium. Place several root bundles on each 25x100 plate. Seal dishes with filter tape (Carolina Biologicals) and incubate at 23!C, 24 hr light for 2 to 4 days.
4. Inoculation. Pour 4050 ml MSKig liquid medium into a tripour 100 5m filter sitting in a 25x100 petri dish. (We make these filter baskets by cutting off the bottom of tripour beakers and melting a piece of 100 5m mesh to the bottom.) Place Arabidopsis root bundles from MSKig into a petri dish and cut them into 0.5 cm segments. Transfer root pieces into filter units with medium. Pipet 1.5 to 2 ml of an Agrobacterium overnight culture into each filter with roots. Mix gently and let sit a few minutes. Blot the roots on sterile filter paper. Place bundles of roots on MSKig medium containing 100 5M Acetosyringone Our root bundles cover about 1 cm2. Seal with filter tape and incubate at 23!C, 24 hr light for 23 days.
5. Rinse and Transfer to SIM with Selection. Place roots bundles into a tripour filter which is sitting in a 25x100 petri dish. Pour 3050 ml liquid MSKig medium over the roots and shake the filter unit vigorously in the solution. Repeat rinse with a clean petri dish until liquid is clear. Blot the roots on filter paper. Transfer root bundles to MSg v500 containing 50 mg/l kanamycin, 58 bundles per 25 x 100 petri dish. Make sure the roots are in contact with the medium. Seal with filter tape and place at 23!C, 24 hr light for 1020 days.
6. Transfer to No Hormone medium. Green nodules and the first indications of shoots should appear within a few weeks. When shoots are first visible (they can look like dark spots to the naked eye) transfer entire explant, or break up explant into smaller pieces, to GM v500 k50. Secure lid to dish with two pieces of tape. Incubate 7 to 14 days at 23!C, 24 hr light. As the shoots develop, it is helpful to carefully break up the explants to allow the shoots to expand.
7. Root Induction: When shoots are large enough, excise them from the callus and place them on MSRg v500 k50 medium in a 25x100 petri dish. Secure lid to dish with two pieces of tape and incubate for 24 days.
8. Plantlet Growth: Transfer shoots to GM v500 k50 medium in 25x100 petris. Secure lid to dish with two pieces of tape. This ensures good air exchange and helps to harden off the seedlings. Roots should form in 1020 days.
9A. Transfer to Soil: When shoots have produced a good root system, transfer them to potting mix as follows: Place MetroMix in 2" pots and moisten with Hoaglands solution. Carefully pull robust plants from the agar. Rinse well with water from squirt bottle and use fingers and/or forceps to gently remove agar and vitreous tissue from base of shoot. Place root in a hole made in the MetroMix and gently press the soil around the root. Water the plantlet into the soil using the squirt bottle. Immediately place the potted plant into a Magenta box with lid. Return the plants to the culture room (23!C, 24 hr light). Within a day or two, the stems should straighten up and start to grow. As quickly as possible, without allowing the plants to wilt, remove the lid from the Magenta box in stages, starting one to two days after transplanting and removing the lid within a week. Water the plants as necessary to keep them moist. We have had up to 80% success rate with this procedure, however, the plants must not be vitreous. When well established, the plants can be placed in a growth chamber.
9B. Grow to Seed in Magenta boxes: Transfer rooted plants to GM v500 k50 medium in Magenta boxes (about 100 ml medium per box). To facilitate air exchange and to allow for room to grow, the lid is replaced with a modified Magenta box using the Coupler for Magenta Vessel (Sigma C 0667). The Magenta box is modified by drilling a 6 mm hole in the bottom of the box (which will become the top); this hole is covered by a Suncap Closure (Sigma C6920), held on by a rubber band during autoclaving. If condensation occurs, replace the Magenta box lid with a dry one. About half of the plants will set seed but plants in soil will set seed much faster.
ARABIDOPSIS MEDIA
Basic Medium
1 pkg. Murashige and Skoog Minimal Organics Medium without Sucrose (Gibco #5103118 or Sigma # M6899)
10 ml DM Vitamin Supplement
0.05% MES (05 g/l)
0.8% agar (8 g/l)
pH 5.8
DM Vitamin Supplement 100 X Stock
10 mg/l thiamine
50 mg/l pyridoxine
50 mg/l nicotinic acid
GM = Germination Medium
Basic Medium 1% sucrose (10 g/l)
MSKig Callus Inducing Medium
Basic Medium
2% glucose (20 g/l)
0.5 mg/l 2,4D 2.3 5M
0.3 mg/l Kinetin
1.4 5M 5 mg/l IAA 28.5 5M
MSg Shoot Inducing Medium
Basic Medium
2% glucose (20 g/l)
0.15 mg/l IAA 0.86 5M
5.0 mg/l 2iP 24.6 5M
MSRg Root Inducing Medium
Basic medium
2% glucose (20 g/l) 12 mg/l
IBA 58.8 5M
0.1 mg/l Kinetin 0.46 5M
Notes v500 = 500 mg/l
vancomycin B5 medium = Gamborg's B5 medium (Gibco # 5001153)
Use 50 mg/l kanamycin for selection.
Background

Agrobacterium genetically colonizes wounded plant cells by transferring a defined segment(TDNA) of its Ti plasmid into the plant nuclear genome.Although this natural gene transfer mechanism has been widely adopted in plant transformation,the possibility of generating transformed progeny by Agrobacterium infection in planta has not been successfuly exploited.Here we show stably transformed progenies of a crucifer plant,Arabidopsis ,are efficiently obtained by simple incision of primary shoots at their bases and by subsequent Agrobacterium inoculation at the wound site.Adventitous shoots arising from the infected wound site were transformed at a high frequency and produced transformed progenies later.

The Transformation Procedure

1. Seed germination and plant growth
(1) Soak seeds on the fillters wetted in 10 mM KNO3,10 mM NaPO4 pH 7.0 and place at 4 degree C. in the dark for 48 hours for vernalization.1)
(2) Sow coldtreated seeds on compounded soil or vermiculite soaked with Hogland's solution and cover the pots with saran wrap.2)
(3) Transfer germinated seedlings to a ligh controlled(16hr light/8hr dark) growth chamber.
(4) For healthy growth,water and fertilize sufficiently,and protect from fungal or insects attack.3),4)

2. Wounding and Agrobacteria infection
(1) When the primary inflorescences grow to 23 cm in hight,remove them by severing the basal part with a scalpel or scissors and remove auxillary buds as completely as possible with a fine forceps.This step is most critical.What you want to do is to remove as many preformed shoot meristema as possible,and to make reorganization of shoot meristems from infected and transformed cells.5)
(2) Sometimes it helps to make deep wound into the remaining stem with a needle or toothpick.
(3) Inoculate wounds with Agrobacteria from overnight culture grown in YEP media.6)
(4) Place inoculated plants in the shade or low ligh intensity for 23 days at 28 degree C.
(5) Transfer plants (POTS) to standard growth conditions,until secondary inflorescences emerge (about 1014 days).
(6) One or more inflorescences should develop from the wound site.Remove the early emerging inflorescences and reinoculate Agrobacteria onto the wound sites by repeating the procedure described above.
(7) Efficiency of transformation may be tested by assay of leaves on the newly formed shoots.7)
(8) The later emerging inflorescences are maintained through maturity for seed generation.

Notes

1) Seeds may be directly sown on soil without imbibition and/or cold treatment,but the germination will not be uniform.
2) Metromix 200 or any proper soil.
3) For efficient transformation,plant materials must be healthy.
4) Hogland's solution or commercial mixture ( such as Hyponex).
5) The timing of infection is important.
6) Overnight culture agrobacteria are concentrated to 1/5 volume by centrifugation at 600 rpm for 5 min,followed by resuspension in the remaining media.
7) We used pBI121,a binary vector containing the NPT II and GUS genes within the TDNA region.
My fax # is 82526792199,and my address is
Dr.HongGil Nam
Dept. of Life Science
POSTECH,P.O.Box 125
Pohang, Kyungbuk,790600
South Korea
Transformation of A. thaliana - IN PLANTA TRANSFORMATION
An overnight culture of an appropriate Agrobacterium strain (see last Email message) was prepared by inoculating 25 ml of LB + antibiotics in a 250 ml flask with a single colony in the previous Email message and growing for 16 to 20 hrs at 28oC (225 r.p.m.).
Plants were grown at 22oC under continuous light. 25 seeds were planted (evenly spaced) in each of 4 (4"). Plants germinating late were rogued such that all remaining plants (80) were at the same stage of maturity. At the time of bolting, when the primary shoot was 2 to 5 cm in height, the shoot and 1 to 3 of the upper rosette leaves were removed with fine scissors.
Using a pasteur pipette, one drop of culture (35 ul) was applied to the wound. After all the plants in the pot were innoculated once, a second drop of inoculum was applied. Plants were returned to growth chamber for approx. 7 days. When the largest lateral shoots were 5 cm high, all visible lateral shoots were removed regardless of size and the wounds inoculated with a drop of overnight culture as before. Plants were then allowed to set seed. Putative transformed seedlings were selected by sowing sterilized seed on minimal agar medium containing 50 mg/L Kanamycin.
First message: Dear networkers: This is in response to the requests of Tom Moran and Bob Whittier (Aug 9) for information concerning the in 'planta' transformation method of Chang et al.
We have preliminary results which suggest that the method works. In our experiments we used the Columbia ecotype grown in continuous light at 21 oC and Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz and Schell,1986, MGG 204:383396) transformed with a binary plasmid constructed by Dr. Ragu Datla: binsyn backbone with a GUSNPT fusion gene behind a CaMV 35S promoter. Otherwise, we followed the protocol as described at the Vienna meeting last summer.
Our first attempt was last summer immediately following the Vienna meeting. We treated 1520 plants and got nothing. However, encouraged by the Email report last Fall that a group at Dupont had had success, we tried again with more plants (80 treated). All of the seed obtained from those plants (I estimate over 100, 000 separated into four batches) were tested for resistance to Kanamycin. Seven plants were clearly resistant and all of these tested positive for GUS activity. We know that the seven resistant plants represent at least four independent events since at least one resistant plant was obtained from each of the four batches. Both resistance to kanamycin and the GUS activity cosegregated 3:1 (KanR/+GUS: KanS/GUS) among the progeny of one of those lines. The other lines have not been analyzed. We have not yet tested for the presence of the TDNA in any of the lines. We have initiated a third and fourth transformation experiment but have no results as yet so it is too early to tell how reliable the method will be but we feel that the preliminary results are quite promising.
Zygotic Embryo Transformation in Arabidopsis thaliana1
1. Collect green siliques from A. thaliana containing immature and mature embryos.
2. Surface sterilize siliques for 10 minutes in Clorox, and rinse 3 times in sterile distilled water.
3. Incubate removed embryos on BM2 (or BM3) callus inducing medium for 3-5 days.
4. Cut embryos longitudinally.
5. Incubate cut embryos with 107 cells Agrobacterium tumefaciens in BM (no agar) with gentle shaking, then blot explants dry on sterile filter paper.
6. Co-cultivate cut embryos on BM2 (or BM3) for 2 days (24C, 16 hour days, 20 E/m2/s [low light]).
7. Wash embryos with BM1 medium (no agar) and blot dry.
8. Transfer to BM5 (or BM4) for 3-4 weeks to select kanamycin resistant calli.
9. Subculture calli on BM5 (or BM4) for shoot induction.
10. Elongate shoots on BM6.
11. Shoots longer than 4 cm can be transferred to BM7 for root induction.
12. Plants can then be transferred to tubes, or soil, for seed setting.
BM (per liter): 4.3 g Murashige & Skoog basal salts (Gibco), 1.0 g myo-inositol, 30 g sucrose, 0.5 g MES (pH to 5.7 with 1 M KOH), 7 g Phytagar. Add 1 ml 1000X Nitsch's modified vitamins
BM1: BM plus 750 mg/L vancomycin (or cefatoxime)
BM2: BM plus 1 mg/L BAP, 1 mg/L NAA
BM3: BM plus 1 mg/L 2,4D, 1 mg/L kinetin
BM4: BM plus 1 mg/L BAP, 0.1 mg/L NAA, 15 mg/L phloridzin, 50 mg/L kanamycin, 500 mg/L vancomycin (or cefatoxime)
BM5: BM plus 5 mg/L 2iP, 0.1 mg/L IAA, 15 mg/L phloridzin, 50 mg/L kanamycin, 500 mg/L vancomycin (or cefatoxime)
BM6: BM plus 1 mg/L BAP, 0.4 mg/L NAA, 0.1 mg/L GA3, 50 mg/L kanamycin, 100 mg/L vancomycin (or cefatoxime)
BM7: ½BM plus 0.1 mg/L IAA, 50 mg/L kanamycin, 100 mg/L vancomycin (or cefatoxime)
1RS Sangwan, Y Bourgeois, & BS Sangwan-Norreel (1991) MGG 230:475-485
Plant DNA Isolation
Adapted from Focus 12(1): 13-15 (1990) by GH

1. Preheat isolation buffer to 60 C.
2. Grind fresh tissue in clean mortar:pestle w/ liq N2 (tissue:buffer = 0.5 g/ 5 ml).
3. Immediately add preheated isolation buffer and place in 60 C water bath.
4. Incubate at 60 C for 30 min.
5. Extract once w/ an equal volume of chloroform:isoamyl-OH (24:1).
6. Spin in GLC-1 for 5 min. on RmaxS.
7. Transfer supernant to a separate tube.
8. Add E 2/3 volume -20 C isopropanol to supernant (7).
9. Gently rock tube back and forth until DNA precipitates.
10. Pellet or spool DNA - spin E 2 min GLC-1.
11. Transfer pellet to 1.5 ml eppendorf tube filled w/ 0.75 ml 60 C isolation buffer.
12. Incubate at 60 C for 30 min.
13. Extract once w/ an equal volume of chloroform:isoamyl-OH (24:1).
14. Spin in Microfuge for 5 min. on RmaxS.
15. Transfer supernant to a separate tube.
16. Add E 2/3 volume -20 C isopropanol to supernant (15).
17. Gently rock tube back and forth until DNA precipitates.
18. Pellet or spool DNA - spin E 2 min in Microfuge 40% power.
19. Pour liquid off and rinse with r.t. 70% EtOH (twice).

Dry thoroughly in Speed-vac (15-20 min.).
Resuspend in TE - store at 4 C.

Isolation buffer: 100 ml

CTAB (2% solution w/v) 2.00 g
NaCl (1.4 M) 7.68 g
b-mercaptoethanol (0.2% v/v) 200 ul
EDTA (20 mM) 4 ml of a 0.5 M
Tris-HCl, pH 8.0 (100 mM) 10 ml of a 1.0 M

Add ddH20 for a final volume of 100 ml
Store at room temp

Our lab has used this protocol for various different species and have had great success with it. Good Luck.
Greg Harlow, David Mount's Lab.
We posted this protocol a few months back - it is a modification of Tai & Tanksley's procedure for DNA from dried material. Not necessarily the "best" prep, but it works fine. It was developed by Tom Tai and Doug Dahlbeck.
Plant DNA Mini-Prep (modified from T. Tai and S. Tanksley, 1991, Plant Mol. Biol. Rep. 8:297-303)

Solutions:

EXTRACTION BUFFER for 10 ml for 600 ml
100 mM Tris-HCI pH 8 1 ml 1M 60 ml 1 M
50 mM EDTA pH 8 1 ml 0.5 M 60 ml 0.5M
500 mM NaCI 1 ml 5 M 60 ml 5 M
1.25% SDS (w/v) 1.25 ml 10% 75 ml 10 %
8.3 mN NaOH 83 ul 1N (8.3 of 10 N) 0.5 ml 10 N
0.38% Na bisulfite 38 mg 2.28 g

T5E
50 mM Tris pH 8
10 mM EDTA pH 8

5 M Potassium acetate

PROTOCOL:

1. Collect leaf tissue (up to 0.15 g) in eppendorf tube. Freeze by dipping in liquid nitrogen to fill tube. Process in sets of about six tubes: fill all tubes with liquid nitrogen, grind them all and proceed to step 2 before starting another set. Alternatively, can allow liquid nitrogen to evaporate and then store samples at -70o for later use.

Note: Leaf tissue can also be dried overnight in a 45oC forced air oven, stored dry at room temperature, and then powdered prior to step 2.

2. Add 0.7 mls of extraction buffer that is preheated to 65o C and mix thoroughly using a toothpick or pipet tip. Put in 65o C water bath for 10 min.

3. Add 0.22 mls 5 M Potassium Acetate, mix well and put on ice for 20 to 40 min.

4. Pellet at 4o C for 3 min.

5. Filter supernatant. Pour into blue tip that has a small kimwipe "filter", force liquid into another eppendorf tube using pipetman. Add 0.7 vol. (0.4 ml?) of IPA and mix well.

6 Pellet at 4o C for 3 min., pour out supernatant, and rinse pellet twice with 70% ETOH. Drain (on test tube rack or paper towel) for 1 min.

7. Add 300 ul ml T5E, vortex 2 sec., into 65oC 5 min., vortex 2 sec (be sure pellet resuspends).

8. Add 150 ul 7.4 M NH4OAc, vortex 2 sec. and pellet 3 min.

9. Transfer into a new 1.5 ml tube, add 330 ul IPA and mix well.

10. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, and drain inverted for 2 min.

11. Add 100 ul T5E, vortex 2 sec., heat 65o C for 5 min., and vortex 2 sec.

12. Add 10 ul 3 M NaOAc, mix, add 75 ul IPA, and mix well.

13. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, and drain inverted for 2 min.

14. Dry pellet in speedvac for 15 min., add 25 ul of TE and let pellet resuspend overnight at 4o C.

15. Vortex 2 sec., heat 65o C 5 min and vortex 2 sec. Store at 4o.

Storing at -20o is better idea for longer periods. Scale-ups of this (for 50 ml tubes, etc.) also work fine.
Review:
Methods in Enzymology Volume XXXI Biomembranes Part A, Sidney Fleischer and Lester Packer, eds. 1974
*has a number of different protocols for different organelles and a discussion of various factors affecting the yield and intactness of the prep
Isolation of nuclei:
Luthe, D.S. and Quatrano, R.S., Transcription in Isolated Wheat Nuclei, Plant Physiol. (1980) 65, 305308.
*three methods given: a crude prep, one utilizing a Percoll gradient, and one utilizing a sucrose gradient

Slater, R.J., Venis, M.A., and Grierson, D., Characterization of Ribonucleic Acid Synthesis by Nuclei Isolated from Zea Mays, Planta 144, 8993. (1978)
*gives intact nuclei but probably contaminated with chloroplasts as well?

Wilson, P.S. and Bennett, J., 1976, Transcription in Nuclei Isolated from a Higher Plant, Biochemical Society Transactions 4:812813.
*references Hamilton et al., 1972 below. Notes that the initial nuclear pellet was heavily contaminated with chloroplasts but these were removed by repeated washing in hyperosmotic sucrose solution containing Triton X100 and Mg2+ ions.

Hamilton, R.H., Kunsch, U. and Temperli, A., (1972) Anal. Biochem. 49:4857.
Chloroplast References:

Mulligan, B.G., Purification of Chloroplast DNA using Hexadecyltrimethylammonium Bromide (CTAB), (1989), Plant Molecular Biology Reporter 7(2):144149.
Dally, A.D. and Second, G., Chloroplast DNA isolation from Higher Plants: An improved nonaqueous method, (1989), Plant Molecular Biology Reporter 7(2):135143.

Mourad, G. and Polacco, M.L., Minipreparation of Highly Purified Chloroplast DNA from Maize, (1989), Plant Molecular Biology Reporter 7(1):7884.
Chloroplast DNA

Ref: Palmer et. al. in Hillis and Moritz's "Molecular systematics" (1991) is probably pretty standard.

My material is incredibly tough and fibrous, and so I have to up the initial quantity of material to get a reasonable amount of DNA out (I use restriction enzymes and vizualize with P32 dCTP end labelling). I have had difficulty in getting the DNA sufficiently pure, although the source of the impurity has not been finally identified; nuclear contamination or sheared cpDNA. I tried everything I could think of to purify the extract; two sucrose gradients, two CsCl gradients, purification by running the extract through an agarose gel to get the smaller fragments of imputies out and then extracting the "purified" cpDNA out of the gel. Nothing seemed to really work, and I put it down to the toughness of the tissues, as a spinach extract I did as a trial worked OK.

I therefore reverted to the following method; total DNA extraction. I will (when time permits) blot and probe with suitable cpDNA probes.

Ref: I am using a slightly modified method of the Doyle and Doyle (1987) procedure; Phytochemical Bulletin 19: 1115.

I am not exactly sure what modifications have been made (haven't looked), but I use it because it has been used on the grasses before (Dr. E.A Kellogg, Harvard Univ.). You can also try Doyle and Doyle (1990) in Focus 12:1315 (I haven't got this If you find it I would appreciate a copy).

Other odd tips: I have tried getting cpDNA out of fig leaves (Ficus species) and the Palmer method using fress leaves DOESN'T WORK. The latex glues everything up and it pellets out at the sucrose gradient stage. This was a "smash and grab" experiment, and so wasn't persued too hard, but we did discover that freeze / vacuum drying the material before hand and then following the normal steps did provide some cpDNA, but the yeild was rather low. The purity was also below average (definately unpublishable as a figure or plate in a paper!)
Mitochondria Isolation and DNA Extraction
Sparks, R.B., Jr. and Dale, R.M.K., Characterization of 3HLabeled Supercoiled Mitochondrial DNA from Tobacco Suspension Culture Cells, Molec. Gen. Genet. (1980) 180:351355.

Mitochondrial Isolation from Suspension cells
1. Begin with 300600 g of cells (can be scaled down to 100 g)
2. Filter cells through miracloth (this step optional if cells not clumpy)
3. Weigh cells
4. Dilute cells 1:1 with RDMP buffer #1
5. Break cells by passing through french press at 3,000 psi
6. Collect cells in chilled flask in ice
7. Filter solution through two layers miracloth
8. Centrifuge 1,500 g at 4 C
9. Discard pellet
10. Centrifuge 1,500 g at 4 C for 15 minutes
11. Discard supernatant and resuspend in RDMP buffer #1 w/o PVP, adjust to 10 mM MgCl2, and then treat with DNase (50 ug/ml) for 1 hour at 20 C.
12. Adjust sample to 20 mM EDTA and centrifuge again at 15,000 g for 15 min
13. Resuspend pellet by homogenization in a glass homogenizer in 36 ml of RDMP buffer #2.
14. Layer on discontinuous sucrose gradient made in the same buffer.
Gradient: 6 ml 1.6 M sucrose
8 ml 1.4 M sucrose
8 ml 1.2 M sucrose
4 ml 0.9 M sucrose
4 ml 0.6 M sucrose
6 ml sample
15. Centrifuge 1 hr in a SW27 rotor at 25,000 rpm at 4 C.
16. The mitochondria will band at the 1.4/1.2 interface. Remove them with a syringe through the side of the tube.
17. Dilute 1:3 with RDMP Buffer #2
To obtain DNA:
18. Centrifuge mit. solution for 20 min. at 15,000 g.
19. Resuspend in 7.5 ml of RDMP Buffer III made 0.5% in Sarkosyl.
20. Immediately layer on a CsClEtBr gradient and centrifuge for 40 hours in a Ti50 rotor at 38,000 rpm.
21. Opencircular/linear and supercoil bands can be seen by long wave illumination and removed with a syringe through the side of the tube.
22. Extract EtBr with CsCl saturated isopropanol.
23. Dialyze the DNA sample against two changes of TEN made 0.3 M in sodium acetate and then EtOH ppt.
24. Resuspend the ppt'd DNA in water, TEN, or TE and store at 4 C.
Buffers:
RDMP Buffer #1
0.3 M mannitol
0.05 M TrisHCL
3 mM EDTA
before use add:
1 mM 2mercaptoethanol
0.1% BSA
1% Polyvinylpyrrolidone, PVP40
pH to 8.0

RDMP Buffer #2
0.3 M sucrose
0.05 M TrisHCL
0.02 M EDTA
before use add:
0.1% BSA
pH to 8.0

RDMP Buffer #3
0.05 M TrisHCL
0.02 M EDTA
pH to 8.0

TEN
10 mM Tris
50 mM NaCl
5 mM EDTA
pH to 8.0
Arabidopsis DNA MiniPrep for single plants
3/91 N.Walker: Adapted from Dellaporta et al Plant Mol. Bio. Reporter 1(4):1921 1983
EB = Extraction Buffer BTE

100mM Tris pH8 50mM Tris pH8
50mM EDTA pH8 1mM EDTA pH8
500mM NaCl
10mM BME NaOAc = 3M NaOAc
0) Water bath to 70 C, precool Eppies on Dry Ice.
1) Weigh tissue, ~0.1g (the whole plant!) Freeze in liquid nitrogen, grind to fine powder in eppendorff.
2) Add 1ml EB, pipette up & down (cut end off blue tip).
3) Add 70ul 20% SDS, mix well (vortex). Incubate @ 6570 C, 10 mins.
4) Add 260ul 5M Potassium Acetate, shake tubes vigorously. => ice for 20 min.
5) Spin @ 4 C, 14K rpm for 20 min. Split supe into two tubes containing 700ul Isopropanol/NaOAc (10:1).
6) Mix gently, incubate 30 min @ 20 C
7) Pellet DNA, 14K rpm, 15 min @ 4 C, dry gently by inverting tube on paper towel for 1520 min.
8) Dissolve pellets in 100ul BTE, combine. Spin for 10 min to pellet "goop"
9) Transfer supe. to new eppendorf tube containing 200ul isopropanol/NaOAc (10:1) mix well, 20 min on ice, spin @ 14K for 30 min.
10) Wash with cold 75% ETOH,
11) Dry in speed vac, redissolve in 20ul TE with RNAse, let rehydrate for 1h 4 or 5ul should be enough for a southern.
Notes: Steve Dellaporta claims that this DNA may be hard to cut, he recommends using ~20units of enzyme/5g & digesting for at least 3hrs, if not O/N. I would recommend adding 12 mM spermidine to the digest and running the digest in a volume of 50100ul, you can then dry it down to a more manageable volume by putting it in a speedvac for 510 mins. This prep works best on young plants, around 34 weeks old.
A. thaliana Plant DNA MiniPrep
Modified By Doug Dahlbeck from T. Tai and S. Tanksley, 1991, Plant Mol. Biol. Rep. 8:297303.
This protocol works very well for Arabidopsis, and can be easily scaled up for larger amounts of tissue.
EXTRACTION BUFFER
100 mM TrisHCI pH 8
50 mM EDTA pH 8
500 mM NaCI
1.25% SDS (w/v)
8.3 mN NaOH
0.38% Na bisulfite
T5E
50 mM Tris pH 8
10 mM EDTA pH 8
5 M Potassium acetate
PROTOCOL:
1. Collect leaf tissue (up to 0.15 g) in eppendorf tube. Freeze by dipping in liquid nitrogen to fill tube. Process in sets of about six tubes: fill all tubes with liquid nitrogen, grind them all and proceed to step 2 before starting another set. Alternatively, can allow liquid nitrogen to evaporate and then store samples at 70o for later use.
Note: Leaf tissue can also be dried overnight in a 45oC forced air oven, stored dry at room temperature, and then powdered prior to step 2. To grind the tissue we use disposable Kontes pellet pestles which are available from VWR (we reuse the pestles). This works best if the tissue is harvested and frozen in 2.0 ml microcentrifuge tubes.
2. Add 0.7 mls of extraction buffer that is preheated to 65o C and mix thoroughly using a toothpick or pipet tip. Put in 65o C water bath for 10 min.
3. Add 0.22 mls 5 M Potassium Acetate, mix well and put on ice for 20 to 40 min.
4. Pellet at 4o C for 3 min.
5. Filter supernatant. Pour into blue tip that has a small kimwipe "filter", force liquid into another eppendorf tube using pipetman. Add 0.7 vol. (0.4 ml?) of IPA and mix well.
6. Pellet at 4o C for 3 min., pour out supernatant, and rinse pellet twice with 70% ETOH. Drain (on test tube rack or paper towel) for 1 min.
7. Add 300 microl T5E, vortex 2 sec., into 65oC 5 min., vortex 2 sec (be sure pellet resuspends).
8. Add 150 microl 7.4 M NH4OAc, vortex 2 sec. and pellet 3 min.
9. Transfer into a new 1.5 ml tube, add 330 microl IPA and mix well.
10. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, and drain inverted for 2 min.
11. Add 100 microl T5E, vortex 2 sec., heat 65o C for 5 min., and vortex 2 sec.
12. Add 10 microl 3 M NaOAc, mix, add 75 microl IPA, and mix well.
13. Pellet 3 min., pour out supnt., rinse pellet twice with 70 % ETOH, and drain inverted for 2 min.
14. Dry pellet in speedvac for 15 min., add 25 microl of TE and let pellet resuspend overnight at 4o C.
15. Vortex 2 sec., heat 65o C 5 min and vortex 2 sec. Store at 4o.
We have had great success using this procedure to prepare DNA from large leaves of older Arabidopsis plants. To grind the tissue we use disposable Kontes pellet pestles which are available from VWR (we reuse the pestles). This works best if the tissue
is harvested and frozen in 2.0 ml microcentrifuge tubes.
I would add to the list of DNA extraction methods the CTAB extraction, which uses cetyltrimethylammonium bromide as a detergent. This method reduces the problem of contaminating polysaccharides which can render the miniprep DNA undigestible by restriction enzymes. The method is quick, easy and inexpensive.
Provided here are references which have detailed protocols:
1) Rogers, S. and Bendich, A. (1988) Extraction of DNA from plant tissues. IN
"Plant Molecular Biology Manual", S.B. Gelvin, R.A. Schilperoort, D.P.S. Verma,
eds. Kluwer Academic Publishers.
2) Murray, M.G. and W.F. Thompson (1980) Rapid isolation of high molecular
weight plant DNA. Nucleic Acids Research 8: 43214325.
Dear Network,
the following is the description of a cheap and functional seed harvester that also prevents cross pollination. If you want to see a real drawing, send a self-addressed (stamped?) envelope.
Regards,
Luca Comai

Pods-a-plenty, an Arabidopsis seed harvester

Mike Neff, Doug Ewing, and Luca Comai.
Department of Botany KB-15, University of Washington, Seattle, WA 98195
E-Mail: Comai@milton.u.washington.edu

1. Collect 1.5 liter PET (the clear plastic) soda bottles. In the US these bottles are used for all sodas and bubbly water. This is a good chance to recycle, but if you need a lot of seed collectors, contact a local bottle maker (look in the yellow pages). They have new "reject" bottles by the hundreds.

2. Cut the bottle as described. Remove the bottom of the bottle, then make a longitudinal cut from the bottom end to the shoulder. The result is a cilinder open at one end, sliced along one side and "closed" at the other end by the funnel constriction leading to the neck of the bottle. Cut a circular opening 5 cm in diameter in the shoulder of the bottle where the longitudinal cut ends. Punch four small holes with a paper hole puncher on the side of the long longitudinal cut . The pattern of the longitudinal cut, the large hole, and the four small holes is represented (hopefully) below.

neck of the bottle

x
x x
x x
x x
x x < 5 cm diameter hole
x x
x x
x x
x
x
x x x < small side hole
x
x
x
x
x x x
x
x
x <longitudinal cut
x
x

open end

(the above looked like a crude drawing at the time of mailing..)

3. Tie a small paper bag around the neck of the bottle. Any paper will do and it is preferable to plastic because it allows drying of the pods and seed even if some water is splashed around.

4. Thread two 30 cm wooden sticks (applicators) through the small holes.The sticks will be parallel to the longitudinal cut.

5. Apply the collector to the pot after Arabidopsis has bolted and the first pods are beginning to mature. The sticks are driven into the soil mix of the pot and support the modified, upside down bottle. You can set the height of the collector by sliding it up or down the wooden sticks. Open the collector by spreading the longitudinal cut and gently push the inflorescence into the collector. New inflorescences can be added as they form and the collector can be easily removed to harvest early-shed seed. Multiple collectors can be added to a large pot. The tube can be extended in length by taping to it an additional cylinder cut from a bottle. However, we usually coil the inflorescence inside the tube rather than increasing the tube length.

6. Once the pods are dry thresh them with a stick (beat it around inside the pod collector) and push seeds stuck to the side of the collector into the pouch. Used collectors are easily stored in nested stacks.

They are probably tetraploids. A lot of cells in Arabidopsis are apparently tetraploid, and one often obtains 4N regenerants which are simplex for the introduced genes. There are many ways to check: seed size is larger for tetraploids (one easy way to see this is to drop 20 seeds into a capillary that lines them up the long way, and a population of 2N and 4N plants will give a bimodal distribution of length of capillary filled with seeds). Chromosome counts are simple: a lactoacetic orcein squash of roots (and probably any other stain, as well) shows centromeric heterochromatin even in interphase. One sees 610 spots in diploids, 1420 in tetraploids. If you catch any in metaphase, the extra chromosomes are really apparent.
Segregation of tetraploids is funky, as well, but is not usually an appropriate way to see if the plants are tetraploid, because it takes a couple of generations. One simple genetic way to check, if you have time to wait 2 generations, is to cross the suspected tetraploids to known diploids, then self the F1. The F1 will be mostly sterile, and have lousy seed set, if one of the parents was tetraploid, because the F1s are triploid, and necessarily have a lot of nondisjunction. This is how trisomics are usually made.
There are other ways to check, also. In many plants, stomata are bigger in tetraploids. I have never looked in Arabidopsis, but I'm certain that someone has. Et cetera. If you wish, I will dig for references on tetraploid Arabidopsis: 4N Arabidopsis plants have been described several times in the literature, but I don't recall offhand by whom.
Best regards (and happy new year to all). Elliot Meyerowitz
reference for tetraploids
Two methods to check for tetrapoidy (nr of chloroplasts per epidermal guard-
cell and metaphase chromosomes in root tip squaches) are included in my paper
on "Insertional mutagenesis in A. thaliana : isolation of a T-DNA linked
mutation that alters leaf morphology (1991) Theor. Appl. Genet. 81:277-284
by M Van Lijsebettens, R. Vanderhaegen and M. Van Montagu." Lab protocols are
available. Typical Morphological features of tetraploid lines : much bigger
plants, flowers and seeds, delay of about 4 weeks in flowering time.
Mieke Van Lijsebettens.
My laboratory follows the Arabidopsis transformation protocol of
Valvekens et al. (1988, PNAS 85,55360) as adapted by Roger Innes (for
RI address contact "liam1@violet.berkeley.edu". The protocol is very
efficient. However, harvesting of root explants is time consuming. We
have found a rapid way to harvest roots. We grow Arabidopsis ecotype
No-0 on large plates (15 cm in diameter, 2.5 cm high -- Falcon 3025) in
the following medium:
0.5X Murashige and Skoog salt base pH5.7, 1X Gamborg's B5 vitamins, 1%
sucrose, 1% Phytagar. Using an L-shaped glass rod, we form a line of
about 300 to 400 seeds dividing the plate asymmetrically in one third
and two third sections (two parallel lines can also be used). We
incubate the plate vertically with the seed lines parallel to the shelf
on which the plate rests. The root system of the seedling will grow on
the surface of the agar and in about two to three weeks the root tips
will approach the edge of the plate. We harvest the roots by cutting
them below the crown with a sharp blade. Since the roots are growing on
the surface of the agar they can all be removed in a few seconds. We
incubate them on CIM as in Valvekens et al and cut them into sections
just before incubating with Agrobacterium.
Good Luck,
Luca Comai
University of Washington
Russell Malberg suggested that the community assemble a collection of
"kanamycin resistant transformants with constitutive reporter alleles"
(e.g. 35S-GUS fusions). This collection would consist of strains having
such markers distributes across the genome. I agree that this would prove
most useful to genetic manipulations. We have been using such a tool in
our chromosome walking experiments aimed at the det1 (de-etiolated 1) locus.
I will summarize the strategy below.
---IN PRINCIPLE---
In any walking experiment, one needs to establish orientation toward the
gene sought, and later determine an interval within which the gene resides.
Both of these objectives may be achieved through the mapping of RFLP markers
that have been identified in the course of the walk, and then determining
the genotype of mutant plants in the F2 of a cross between your mutant of
interest and another Arabidopsis ecotype. As one walks toward the gene,
the RFLP profiles will always display the RFLP pattern of the mutant parent,
as the gene is approached. Walks away from the gene can show the profile of
either parent. At higher resolution, one can map the RFLP pattern of the
rare plants in such an F2, that have undergone a crossover between the gene
of interest and the start point of your walk. These plants are valuable since
they contain a junction between the DNAs of both the mutant parent and the
other parent in the cross. RFLP analysis across this interval can show the
genotype at each of the RFLP sites examined, and thus help to delineate a
region in the walk that can contain the gene sought. The resolution of
this approach is a function of two factors:

-having sufficient plants that have undergone recombination between
the start point of the walk, and the gene of interest, and
-having the RFLP markers in this interval that permit genotypic
analysis.
Limitation of either of these resources will hinder delimiting the region
of the gene, using this method. Obtaining the RFLP markers is not discussed
here. However identifying the rare recombinants that have undergone
recombination within a centimorgan of the gene is fascilitated through using
plant lines carrying insertions such as those mentioned by Russell Malberg.
---IN PRACTICE---
We are using this method to identify plants recombinant between one of our
genes of interest (de-etiolated-1) and a closely linked T-DNA insertion line
(agamous). The F2 from a cross of these lines will segregate det1/kan-res
progeny, which necessarily contain at least one recombinant chromosome
between each of these lines. The value of this approach is that we can plate
the F2 out on selective medium, killing 25% of the progeny, and then screen
out det1 segregants. It is necessary to identify polymorphisms between the
det1 parent (Col-O) and the insertion line (Ws-O). We have had some success
at this, but it is early to generalize. The bottom line is that we have a
means for identifying dozens (if not more) crossovers between the start
point of our walk and DET1. In comparison with screening random det1
individuals from an F2, I estimate that this approach increases the
yield of desired recombinants about fourfold (i.e. hundreds of fewer DNA
preps). This approach relies on the dominant selectable kan-res marker
carried in many transformation vectors as well as on the T-DNA insertions.
In this example, the selectable marker was about 24 map units from the gene
of interest. Closer markers would of course be even more useful.
As people seem to be walking all over the map, or at least plan to,
a resource of Arabidopsis lines carrying single insertions of this kind
would prove extremely useful. Ideally such a collection would contain
the popular mapping ecotypes (Col-O, La-O, Nd-O), obviating the need
to identify new RFLPs, at least for the standard RFLP probes from
E. Meyerowitz' and H. Goodman's laboratories.
Terry Delaney
(DELANEY@SALK)
(Alan Pepper has contributed to the work discussed here. Both myself
(TD) and AP are postdoc's in Dr. Joanne Chory's laboratory at the
Salk Institute.
If you're isolating polyA+ RNA, I would like to bring to your attention a
useful but largely unknown paper:
Bantle, Maxwell and Hahn (1976) Specificity of oligo (dT)-cellulose chrom-
atography in the isolation of polyadenylated RNA. ANAL. BIOCHEM.
72:413-427.
This paper makes several useful observations:
1) RNA can be lost due to non-specific binding to oligo-dT columns. This
non-specific binding can be eliminated by pre-empting the column with
E.coli DNA.
2) Much of the RNA in "poly-A+" RNA preparations is not really polyA+
RNA at all, but rather rRNA that is complexed with mRNA. The authors
demonstrate that this rRNA contamination can be removed by the following
steps:
a) adsorb RNA onto oligo-dT column
b) elute RNA from column
c) Heat at 55C in the presence of 80% DMSO 0.1 M LiCL
to break up aggregates.
d) Dilute and re-run on oligo-dT column
This technique works reasonably well. Also, make sure you use reagents
and glassware treated with DEPC.

Subject: RE:nuclear runon transcription
For a start, check paper by Feinbaum and Ausubel (1988) Molecular and
Cellular Biology, 8(5), 19851992. Good luck! (and Happy New Year, all!)
My group is chromosome walking towards a locus, fg, which causes late flowering in Arabidopsis. We have now narrowed the gene down to a 600kb region and hope in the next few weeks to know its position precisely enough to be able to start complementation. At present we have a lysate of the cosmid library made by Neil Olszewski in pOCA18. However, we were interested by Gerard Lazo's abstract for the Tucson meeting describing a library that is already gridded into microtitre dishes. Is this library available? If so in what form do you send it out?
We are also considering the possibility of subcloning from our YACs into a cosmid vector. Would your vector pLZ03 and the Agrobacterium strain AGL1 be available for this purpose?
Thanks for your message inquiring about our Arabidopsis genomic library in Agrobacterium. Indeed, we are allowing the library to be copied by other laboratories and for research purposes only.
However, you have a logistical problem. As the library is stored on 225 96well microtiter dishes, it occupies a volume of some 50 liters. As we do not have a robot to pour microtiter dishes (although we hope to procure, or access, one soon), there is considerable labor involved in replicating the library.
You have two options. You may either make a copy of the 30 matrix organized pools, requiring 30, sterile, 96well microtiter dishes, or of the entire library. If you opt for the former, we will be happy to send you explicit clones from the complete library identified by your (PCR, hybridization, etc) analyses. In either case, while we will provide you with media ingredients, you must supply the labor to both pour and replicate any requisite microtiter dishes. We have a 96well replication device; the replication step is trivial.
In agreeing to accept a complete or partial copy of the library, you must also, in turn, agree to allow your copy to be further copied by up to four others designated by us. The library is being distributed as a pyramid, with all distributees condoned by us.
It is possible to transport a complete copy of the library on a direct, longdistance airplane flight. This has been accomplished several times. Again, there are considerable logistical problems that need be overcome.
There is one further possibility. A Nottingham group (contact: Zoe Wilson) also wishes to obtain a copy of the library. If you could arrange to do so together, two people could more easily move one copy to the UK, and then further replicate it in situ.
Thanks for your interest in our library. We hope it will prove useful in your experiments.
I am interested in different crosses of Col0 to either the Ms0 or Fr2 ecotype of Arabidopsis. I am interested in these two ecotypes because they exhibit differences in the distribution pattern of cauliflower mosaic virus. Information on other ecotypes crossed to Col0 would be greatly appreciated.
Subject: tetraploids
I have done root tip squashes using colchicine, and have been able
to determine tetraploidy quite easily. I used a combination of several
protocols, and would be happy to send you my method. Judy Brusslan,
Department of Biology, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-
1606.