HORT 201
Plant Propagation Laboratory
Laboratory Exercise 8
Maize Embryo Culture and Cauliflower Culture,
Reference: Embryo culture: page 658-659
Adventitious shoot formation: page 654-657
Key Words and Terms
from the CD:
Agar, auxins, aseptic procedures, anther culture, autoclave, axillary shoot culture, callus, chimera, cytokinin, ELISA, embryo culture, embryogenesis, explant, haploid, HEPA filter, in vitro, kinetin, laminar flow hood, media for tissue culture, meristem, microcutting, micropropagation, organogenesis, protoplast culture, somaclonal variation, somatic embryogenesis, subculture, suspension culture, tissue culture, virus-indexed,
Objectives:
1. To culture maize (monocot) embryos and to demonstrate that artificial nutrient media can substitute for the natural nutrition present in cotyledons and seed storage tissues.
2. To initiate cultures of floral meristems of cauliflower, with the goal of stimulating them to re-differentiate into shoot meristems.
A.
Embryo
Culture (Embryo rescue):
Embryo culture, unlike axillary shoot proliferation or adventitious shoot induction, is not a multiplication technique. Rather, it is used to encourage the development of embryos that would normally die if left within the seed. Embryo abortion is a common problem in interspecific or wide genetic crosses. Viable embryos will often develop, however, if they are dissected out and cultured at an early stage of development. For example, cultivated barley (Hordeum sativum) is sensitive to winter injury and mildew attack, while wild barley (Hordeum bulbosum) is reasonably winter hardy and mildew resistant. Unfortunately, embryos of the H. sativum X H. bulbosum hybrid fail to grow beyond a few days after pollination. When they are excised and transferred to a nutrient culture media, however, they resume normal growth and result in a viable seedling. Other examples of successful embryo rescue have been reported in cotton, tomato, alfalfa, bean, rice, lily, stone fruits and orchids.
Embryo culture techniques are also important in the commercial production of orchids. Orchid embryos often require a long time (3 months to a year) for mature seed to form after pollination. Further, once the seed is mature, it contains very little stored food; it is extremely fragile and easily killed. Embryo rescue procedures are widely used to obtain seedlings from unripe seeds as well as to capture valuable hybrids.
Procedure for Embryo
Rescue ö corn (monocot)
Each group will work
together to prepare and wash the explants; each student will establish an
aseptic culture of 2 corn embryos in one baby food jar.
1. Choose a fresh, disease-free ear of corn, and remove the silks and husks.
2. Remove several individual whole kernels, being very careful not to damage the pericarp. It is important for the pericarp (outer surface of the kernel) to be undamaged. If the pericarp is torn or punctured, the internal kernel tissues will be no longer be sterile.
3. Each student needs to practice excising the embryo of several non-sterile seeds. Hold the kernel with forceps, and gently use a razor blade to dissect out the embryo. The embryo is a pale yellow color and should be easily distinguishable from the milky-white endosperm.
4. Each lab group will have 1 corn cob, 1 baby food jar of 70% alcohol, and 1 baby food jar of 10% bleach. Each student needs to remove 5 corn kernels from the corn cob. When each member of the lab group has removed 5 kernels, place the groupâs kernels in the alcohol baby food jar for 45 seconds, then transfer the kernels into the Clorox baby food jar and cap the jar. The kernels will need to soak in the bleach for 10 minutes.
5. Take your kernels in the bleach to a hood in the tissue culture lab. Sterilize your hands with 70% alcohol and spray down the bleach baby food jar then place it in the hood.
6. Then while you are waiting for the 10 minutes to elapse go to the tissue culture incubation room and examine your baby food jar containing your African violet and mum explants. Look for any fungal or bacterial contamination. Each student will record her or his observations on the provided data sheet and turn in at end of lab.
8. Remember to disinfect your hands before you place your hands in the hood! After the kernels have soaked 10 minutes in bleach, using flame-sterilized forceps, transfer the kernels to the first baby food jar containing water. When the kernels are transferred to the first baby food jar of water, immediately begin transferring the kernels to the second baby food jar of water and leave the kernels in the second jar until it is your turn to extract out two embryos. Because the embryo inside the kernel is protected from the bleach, only two water rinses are required.
9. Transfer two kernels to a sterile Petri dish. Remove the embryos using flame-sterilized forceps and a flame-sterilized scalpel. Then place both embryos basal end down into your baby food jar containing embryo culture medium. Use the point of the forceps to make a slight depression in the media where the embryo can be inserted. Take care not to pinch or bruise the embryo with the forceps.
10.Close the
jar, remove it from the hood and write your full name, and your lab section on
the glass side (not on the lid). Do not write a lab group name, write your
individual first and last name. If you have followed aseptic techniques, the
culture will be established in 24 to 48 hours.
B. Culture of Cauliflower Floral Meristems:
The explant for this experiment is cauliflower curd, which is a 'preinflorescence' with an accumulation of many meristematic domes. These would normally develop into flowers. The cytokinin and auxin treatments described below will cause the floral meristems to de-differentiate and then re-differentiate to form shoots. Shoot growth should be evident after 7-10 days. The procedure outlined below is similar to that used commercially for the production of cauliflower seed.
Procedure:
Each group will
prepare the cauliflower explant; each student will establish a single explant
in one baby food jar.
1. Remove your baby food jar of bleach from the hood and take it to the lab classroom to obtain a piece of cauliflower curd about the size of a grape.
2. Working on a clean surface, use a razor blade to further divide your cauliflower into three pieces (one for each student).
3. Place the cauliflower pieces into 70% alcohol for 45 seconds, then transfer 10% bleach solution for 10 minutes. This will surface sterilize the tissue. Take your bleach jar to your hood in the tissue culture lab. Remember to disinfect your hands and the outside of the bleach baby food jar with the alcohol spray bottle before you place the jar in the hood. If you have not finished recording your observations of your African violet and mum tissue cultures please do so now.
5. Using flame-sterilized forceps, transfer a cauliflower piece to a sterile Petri dish. Use a flame-sterilized scalpel to shave a very thin slice (a millimeter or less) off an edge of the cauliflower piece.
6. Using flame-sterilized forceps, place your explant into your baby food jar containing cauliflower medium. Press the explant slightly into the medium, with the curd side facing up.
7. Shoots will form in about one month. Once they are 1-2 cm long, they may be transferred to an in vitro tissue culture rooting medium. Use a sterile scalpel to remove shoots and then transfer to fresh jars of rooting medium. These shoots will develop roots within 2-3 weeks, after which they can be gradually conditioned for transplantating into a soil-less growing media.
C. Additional things to do today:
1. Observe progress of the tissue cultures we initiated 2 weeks ago. Look at the mum (axillary branching) and the African violet (organogenesis). Every student needs to record their observations on the data sheet attached at the end of this handout and turn in at the end of lab today. Dispose all contaminated cultures by taking your contaminated cultures to the kettles next to the autoclaves and use a spatula to scoop out the media into a kettle. Then take your empty jars and lids to the tissue culture sink. The TAâs will show you where the autoclaves and sink are located.
2. In Zone 21, each student will need to pot up 10 apple rootstocks in 10 large black cone-tainers in preparation for the grafting/budding labs after spring break. These are bare root stocks and are in water to prevent root desiccation.
a. Fill the cone-tainer about 1/3 full with media.
b. Place the rooted end of the rootstock into the cone-tainer. The rootstock should be about 6 inches into the cone-tainer.
c. Pour media into the cone-tainer. Make sure media completely fills the cone-tainer and press the media with your fingers to pack it in so the media is not loose.
d. Place your
cone-tainers in a rack and place on bench 7.
3. Leaf and leaf-bud cuttings which had not yet rooted when scored 3 weeks ago have been moved to bench 3 in Zone 21. These cuttings need to be rescored for rooting.
a. Score by removing the cutting from the rooting media and score for rooting. Record your data on your lab groupâs leaf cutting data sheet and turn in at the end of lab.
b. Pot up what material you wish to keep and place on bench 4. Dispose the remainder.
c. Leaf and leaf-bud cuttings which you have already
moved to Zone 21 three weeks ago do not need to be scored. Pot up any cuttings
you wish to keep and place on bench 4. Any
leaf or leaf bud cuttings left on benches 1,2 or 3 will be discarded.
4. Examine your groupâs mint stem tip and mint
stolon cuttings. Both are in Zone 21.
Record your observations on your groupâs mint data sheet and turn in at end of
lab. Please pot up what mint you wish to keep and place on bench 4. Any mint left on benches 1,2,or 3 will be
discarded.
To be turned in at end of lab
1. Mum and African violet tissue culture data sheet (each student turns this in)
2. Leaf cuttings data sheet (one data sheet per group)
3. Mint data sheet (one data sheet per group)
Data Sheet for mum and African
violet tissue CULTURE -
Your name:______________________________________ Circle Lab Section # 1 2 3
If your group did not
name your jars by your individual names, please do so now so you can check the
same jars in upcoming labs. Write your name on the glass with a sharpie.
Do not pick up jars
by the lids, the lids will come off, then your jar may be contaminated.
Do not open the jars
to observe your explants because contamination will occur.
Circle what you observe.
------------------------------------------------------------------------------------------------------------------
Baby food jar #1 Explant 1 contamination shoots no change
mum present visible
Explant 2 contamination shoots no change
present visible
Baby food jar #2 Explant 1 contamination shoots no change
mum present visible
Explant 2 contamination shoots no change
present visible
-----------------------------------------------------------------------------------------------------------------
Baby food jar #1 Explant 1 contamination callus at tiny no change
African violet present edges shoots
Explant 2 contamination callus at tiny no change
present edges shoots
Baby food jar #2 Explant 1 contamination callus at tiny no change
African violet present edges shoots
Explant 2 contamination callus at tiny no change
present edges shoots
PLEASE DISCARD
CONTAMINATED CULTURES AS INSTRUCTED BY YOUR TAâs