Lauren Mondroski

Biotechnology Senior Research Project Thesis Paper

Effects of Media on Growth of Propagates and Genetic Identities Compared to Parent Plants
 Effects of Media on Growth of Propagates and Genetic Identities Compared to Parent

Plants

The process of propagating plants involves using parts of the plant, especially cuttings

from the plant itself: its leaves, stem, root, or other area, in order to produce a genetically

identical plant from the parent plant. Many plants reproduce asexually in this manner; asexual

propagation is a practical way to maintain species, or to choose an individual plant that best

represents desirable traits of the species. For example: Bartlett pears (Pyrus communis) and the

Red Delicious apple (Malus domestica) are just two of the many plants that have been

propagated asexually for decades (Reece, J. B., Meyers, N., Urry, L. A., Cain, M. L.,

Wasserman, S. A., & Minorsky, P. V. (2014). Campbell biology (10th ed.). Pearson.). The plant

Graptoveria opalina, a species of succulent, can be propagated using the method of cutting,

where a leaf is severed from the parent plant in order to regenerate itself.

Rooting hormones can be applied to the basal ends of the leaf cuttings to help the

formation of roots and to deter the growth of disease or fungi. Root hormones supply auxin, a

naturally occurring plant hormone responsible for root development. Additionally, Graptoveria

opalina is sensitive to the type of media used. Media must have good drainage, but also hold

moisture so that exposed roots avoid drying out, and provide aeration and nutrients to the new

plant. The goal of this project is to test different types of media, separately and in established

mixtures, in order to determine the best combinations for growing Graptoveria opalina.

Common mediums used for propagation in succulent plants are cacti/succulent soil or a
combination of soils. Combinations included three parts potting soil combined with two parts

sand and one part perlite. This can serve as a guideline for propagating Graptoveria opalina.

Another goal of this project is to identify the newly propagated plants as genetic

identicals of the parent plant. To accomplish this, DNA Purification and DNA Barcoding was

used. This involves lysing the plant cell samples using chemicals and heat, and isolating nucleic

acids and precipitating it in a buffer solution. The rBCL gene will be isolated from the purified

DNA, which is present in all plants but is slightly varied between individuals (Chaveerach, A.,et

al.,2016). This gene will be compared between both the parent and propagated plant by sending

the purified DNA samples to a third party to analyze. This should prove that the plants are

genetically identical, and that the process of propagation was successful.

Materials and Methods

Using clean and dry scissors, cuttings were taken from Graptoveria opalina plants by

cutting the leaves close to the base of the plant. After this, the cuttings were left to dry and

callous on a paper towel. After approximately 2-3 days the basal ends were dipped in rooting

hormone. As the cuttings began growing roots 3-10 days later, they were transferred to pots

containing potting soil, sand, perlite, vermiculite, sphagnum moss, or varying mixtures of these

mediums in small pots. Once transferred, the cuttings were watered every other day in order to

keep the humidity levels high around the developing roots. The parent plants were kept at

north-facing window in the sunlight, while the new cuttings were kept out of direct lighting in a
warm, dry area. As the cuttings began to root, they were transferred into an area with artificial

light throughout the day.

The DNA barcoding step of the project began as cuttings grew roots and other growths.

Samples of these roots and growths were taken (about 10-20 mg worth) and then transferred to a

labeled 1.5 mL tube, where 300 uL of lysis buffer was added. The samples were then forcefully

grinded for 2-5 minutes with a plastic pestle, with a separate pestle used for each sample. The

samples were incubated for 10 minutes in a 65 celsius water bath, then centrifuged at maximum

speed for 1 minute. 150 ul of the supernatant was transferred to a new labeled 1.5 mL tube, and

the tube containing the pellet was discarded. 3uL of silica resin was added to this tube, and was

mixed by pipetting up and down before being incubated for 5 minutes in a 57C water bath. The

tubes were then centrifuged for 30 seconds and the supernatant was removed. 500 ul of ice-cold

91% isopropyl alcohol was substituted for wash buffer in the experiment, because of the lack of

wash buffer, and was added to each of the tubes. The tubes were vortexed to resuspend silica

resin and then centrifuged for another 30 seconds. The supernatant was removed again. 500 ul of

91% isopropyl alcohol were added to the tubes once more and then vortexed to resuspend silica

resin and then centrifuged for 30 seconds. The supernatant was then removed, and 100 ul of

distilled water was added to the silica resin, and the tube was incubated for 5 minutes at 57C.

After incubating, the tubes were centrifuged for 30 seconds at maximum speed. 90 ul of the

supernatant was then transferred to a new labeled 1.5 ml tube without disturbing the pellet, and

the samples were stored in the freezer until the PCR step was started.
 PCR tubes were labeled with identification numbers, and then 25 ul of Gotaq Green

master mix was added to them, along with 22.5 of rBCL primer mix. 2.5 uL of each DNA

samples were added to their corresponding labeled tubes using a new pipette tip each time. These

tubes were moved to the thermocycler and was programmed for 35 cycles, with the denaturing

step at 94 C for 30 seconds, the annealing step at 54C for 45 seconds, and the extending step at

72C for 45 seconds. After PCR, the samples were stored in the freezer until ready to be sent for

DNA Barcoding. Gel electrophoresis was performed to confirm that the DNA had been purified

from the samples. In order to make the two gels required, 1.5 grams of agarose powder was

measured and poured into an Erlenmeyer flask, as well as 100 mL of TAE buffer. The solution

was microwaved in thirty second increments until the solution was fully dissolved. 10 uL of gel

red were added to the flask, and the agarose solution was then poured into the the gel mold.

Combs were inserted into the gel as it solidified for 15-20 minutes. The gel was then placed into

the gel chamber, and TAE buffer was added until it covered the gel. 10 uL of molecular weight

marker was added to the first well of both gels, and 10uL of each sample was then added in wells

2-8 of the gels. The gel was ran at 100 V for 30 minutes. The equipment was turned off and the

gel was analyzed in UV light box.

Results

Observation Table

Date Potting Soil Washed Perlite Vermiculite Half Peat Red Group Washed Sand (Circle
(Green Sand (Blue (Blue Washed Moss (never Group)
Group) (Green Group) Group) Sand/Half Plus propagated)
group) Potting (Star
 Soil Group)
(Blue
Group)

10.1 - - - - - - 2 days
3 post-cutting
the cuttings
are
discolored
and soft

10.1 - - - - - - No evidence
7 of roots but
cuttings are
stable and
there are no
major
indicators of
bad health

10.1 - - - - - - No visible
9 changes
health is
good

10.2 - - - - - - Ends of
1 cuttings are
calloused.
No other
visible
changes.
Health is
good.

10.2 - - - - - - Slight
5 discoloratio
n. No other
visible
changes.

10.2 Took Took - - - - Cuttings are

7 cuttings cuttings slightly soft
but in good
health
overall.

11.1 Drying Drying - - - - Plant 2 is

showing
signs of
 growth near
root area.

11.3 Drying Drying - - - - No visible

changes to
report.

11.7 Plant 1 was Plant 2 is - - - - Plant 3 is in

left to dry in good failing
over health. health but
weekend others are
with no sustaining.
rooting
hormone
sprouted
roots.

11.1 The The plant - - - - Plant 3 is in

0 cuttings has no critical
root growth roots yet condition
progressed. but is and is
healthy. rotting
away. The
other two
cuttings are
healthy.

11.1 Plant 1 has Plant 2 - - - - Plant 3 has

5 been has be died. The
planted in planted in cuttings in
soil sand this group
medium medium have shown
no evidence
of growth
and will no
day to day
checks will
no longer be
maintained
on this
group, only
drastic
changes will
be
documented
.

11.1 Moved to Moved to One One cutting - One -

7 artificial artificial cutting was taken cutting
 light light. was was
Roots taken taken
have
begun to
grow
from the
end.

11.2 Plant #1 is Plant #2 No roots No roots - No -

8 growing 6+ is forming forming roots
roots and growing forming
has small 10+ roots
bulb on the
end

12.2 The bulb The roots Taken Taken Taken

has have cuttings cuttings cuttings
progressed grown
well and longer
has grown and more
bigger. plentiful
but there
is no
bulb.

12.6 Plant #1 Plant #2 Planted Planted in Beginni

continuing has no in Perlite Vermiculite ng to
to grow visible medium. medium. form
roots and changes. Small Small roots roots.
the growth Good roots are are
is becoming health. growing. growing.
larger

12.1 Two sub The roots Progress Progressed Cutting Roots

2 growths have ing well, well, has taken have
have grown begun no had no progres
from the branching problem problems sed,
main bulb out s with with root they are
root developmen about
develop t. 1.5 in
ment in
length.

1.6 Even more A decline The The roots New Small

sub growths in health roots lay have addition to branche
from the has been atop the seemed to blue s have
main bulb noticed surface dug into the group, a been
now seems and media but half sand noticed
to be 5. health the body of and half on the
 has the plant potting initial
maintain does not soil roots
ed. look mixture but
healthy. media. there is
no sign
of a
bulb.

1.10 No drastic The Health in health Roots Health

changes, decline in state has has not from this state
health has health has maintain worsened. propagate has
maintained. not gotten ed. are very maintai
visibly straight, ned.
worse. they are ~
1.5 in in
length.

1.12 Bulb has

increased in
size

1.19 More Plant Plant seems

growths seems to to have
have be in changed
formed and critical color but
plant is condition, still in good
progressing roots health
well have
died,
discolorat
ion
present
and body
is wilting

1.27 Growth has Same Cuttings Cuttings were taken

become regressio were taken
primary n in
body as leaf health, tip
is attached is
but blackenin
beginning g and
to wilt roots are
away dying
1.31 Good Sand Addition
progress, propagate al
bud has likely to branchin
many die soon-- g of
sections lack of roots
and mother change about 1
leaf is from in from
almost previous lateral
entirely condition
wilted away

2.2 Cutting was Cutting was

propagated propagated into
into potting washed sand media
soil media

2.6 Roots
progressing
promisingly
and a
possible
growth can
be seen

2.8 The new All living The possible Continuing root

growths are roots growth development
beginning have died could
to unfold. possibly be
remnants
from the
parent plant
as it has not
progressed

2.10 Changing
to a
darker
shade of
red and is
shriveling

2.14 Continuin
g as
before,
darkening
and
shriveling

2.16 All
 propagates
are as they
were last
recorded

2.21 Has
turned a
brighter
shade of
red

2.23 No visual
changes can
be observed

2.27 DNA Sand

Extraction propagate
began on is
this day declared
dead

Visual Data

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

October 19th, November 7th, November 11th, November 28, December 6th,
2016 2016 2016 2016 2016
Red Group Green group Green group Green group Snapshot of
Plants after plant 1 first root plants 1 (potting plant 1 progress of all
cutting appears, to be soil medium) continuing to plant cuttings
transferred to and 2 (sand grow roots, first
potting soil medium) sign of bud
 medium appearing

Fig. 6 Fig. 7 Fig. 8 Fig. 9

January 6th, January 12th, January 17th, February 2nd,
2017 2017 2017 Green 2017 Green
Green group Green group Group Plant 1 Group Plant 1
Plant 1 showing Plant 1 budding parent plant parent plant
significant progress beginning to withering.
growth of bud, fade.
while plant 2 is
in failing health

The results of this experiment are that the potting soil mix appears to be the most

effective at propagating the succulent cuttings. This is because the cutting in this medium was

able to grow roots, bulbs, and later a small succulent of its own. The peat moss, vermiculite,

perlite, and half sand/potting soil mixtures have shown to be able to sustain cuttings with roots,

although no growths have been noted. The sand medium was ineffective at rooting the succulent,

likely because sand is a water-permeable medium and the cutting likely did not experience

enough moisture at the roots. After gel electrophoresis, it was determined that DNA Purification

PCR had been successful in samples #6, #7, #8, #10 (Fig. 11)
Labels for DNA Barcoding

Fig. 12

Parent Plants -

Red Group #1

Green Group #2

Star Group #3

Circle Group #4

Blue Group #5

Cuttings -

Green Group/ Soil #6

Blue Group/Perlite #7

Blue Group/ Vermiculite #8

Star Group/Peat Moss #9

Blue Group/Half Soil and Sand #10

Red Group/Soil #11

Circle Group/ Sand (2) #12

 7 6 5 4 3 2 1 MWM - - 12 11 10 9 8 MWM

Fig.13 Results of gel electrophoresis for gel Fig.14 Results of gel electrophoresis for gel
#1 under ultraviolet light #2 under ultraviolet light
- - 5 4 3 2 1 MWM - - 5 4 3 2 1 MWM

Fig.15 Electrophoresis attempt #3 Fig.16 Electrophoresis attempt #4

Well Labels for Gel Electrophoresis Attempt #1

 Well # 1 2 3 4 5 6 7 8
Sample # M 1 2 3 4 5 6 7
W
M

Well Labels for Gel Electrophoresis Attempt #2

Well # 1 2 3 4 5 6 7 8
Sample # M 8 9 10 11 12 - -
W
M

Conclusion

The results of the experiment were mostly inconclusive, because enough propagates did

not grow to the point where the effectiveness of the soil media could be compared. Although a

fully-established plant developed in the soil media, it would be hasty to conclude that this is the

best media for growing Graptoveria opalina, because the second trial was concluded

prematurely because of time constraints. In the same fashion, a connection could not be drawn

between sand being the least effective at growing these plants, because of lack of confirmation

from further trials, even though it was the only media that caused the death of a propagate,. The

DNA barcoding aspect of the experiment also proved to have inconclusive results, because

although we were able to confirm that DNA was successfully extracted from sample #6, sample

#7, and sample #10 by observing results from electrophoresis, we were unable to confirm that

DNA was extracted from the parent plants, which would have been the point of comparison to

confirm that asexual reproduction had taken place. Overall, if there had been more time and
space to grow plants, the experiment could have been scaled-up to increase the chances of

propagates rooting and successfully growing new structures. There was also possibility of error

in the DNA Extraction technique, because we were unable to extract DNA from the parent

plants, so if the project could continue more trials of DNA Extraction could be run. If there were

more conclusive results, this experiment could have been applied to finding the most effective

soil media for succulents or other asexually reproducing plants to help them propagate

successfully.

Sources

Asexual Propagation. (1998). Retrieved November 15, 2016, from

http://cals.arizona.edu/pubs/garden/mg/propagation/asexual.html

Ornamental Production. (n.d.). Retrieved November 15, 2016, from

http://aggie-horticulture.tamu.edu/ornamental/a-reference-guide-to-plant-care-handling-and-merc

handising/propagating-foliage-flowering-plants/
Reece, J. B., Meyers, N., Urry, L. A., Cain, M. L., Wasserman, S. A., &

Minorsky, P. V. (2014). Campbell biology (10th ed.). Pearson.

Sorensen, D. C., & Garland, K. (n.d.). Plant Propagation. Retrieved November 15, 2016, from

https://extension.umaine.edu/gardening/master-gardeners/manual/propagation/plant-propagation/