Agrobacterium rhizogenes-induced Altered Morphology and Physiology in Rubber Dandelion after Genetic Transformation
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J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023. https://doi.org/10.21273/JASHS05217-22
Agrobacterium rhizogenes–induced Altered
Morphology and Physiology in Rubber Dandelion
after Genetic Transformation
David Lankitus, Yingxiao Zhang, Menaka Ariyaratne, David J. Barker, Sarah L. McNulty,
Nikita Amstutz, Lu Zhao, and Brian J. Iaffaldano
Department of Horticulture and Crop Science, Ohio Agricultural Research and Development
Center, College of Food, Agricultural and Environmental Sciences, The Ohio State University,
1680 Madison Avenue, Wooster, OH 44691, USA
Katrina Cornish
Department of Food, Agricultural and Biological Engineering, Ohio Agricultural Research and
Development Center, College of Food, Agricultural and Environmental Sciences, The Ohio State
University, 1680 Madison Avenue, Wooster, OH 44691, USA
KEYWORDS. natural rubber, Taraxacum kok-saghyz, alternative rubber crops, photosynthesis, metabolomics
ABSTRACT. Agrobacterium rhizogenes transformation is a more rapid method of obtaining transgenic and edited rubber
dandelion (Taraxacum kok-saghyz) plants than Agrobacterium tumefaciens. The hairy root rol genes are present along-
side transgenes after transformation, and they change the morphology of rubber dandelion significantly. Although
these rol genes are useful visual markers indicating successful transformation of rubber dandelion, they modify the
phenotype induced by the target transgenes and are ultimately detrimental to agronomic traits. Fortunately, the rol
genes can be removed by conventional plant breeding because they segregate in progeny separately from the targeted
transgenes. However, it is preferable to have preliminary identification of promising effects induced by transgenes
or gene edits before rol gene removal so that only the best plants are used for breeding. Therefore, the goal of this
research was to characterize rol– and rol1 plant morphology so that, in the future, rol1 transgene1 plants can be eas-
ily distinguished from rol1 transgene– plants. This requires that rol gene–induced morphological changes and simply
assayed physiological traits are first characterized thoroughly so that transgene changes may be observed. Taproot for-
mation is reduced or eliminated in rubber dandelion by rol genes, and rol-induced hairy roots are identifiable easily
because they grow shallowly in potting soil, so only partial unearthing is needed. Both leaf and flower numbers are
increased by rol genes, but leaves and flowers are smaller than in rubber dandelion wild type with longer stalks. The
rosette doming phenotype caused by the induction of a large number of leaf primordia is obvious in rooted plants as
young as 1 month old. Photosynthetic rates are reduced significantly in rol1 plants, although growth is not. An accu-
rate description of the morphology of rubber dandelion after A. rhizogenes transformation may allow for initial selec-
tion of promising transformed plants before confirmation with polymerase chain reaction, by phenotypic comparison
of plants expressing transgenes and the rol gene, with those only expressing the rol gene.
Different species of Agrobacterium are used in biotechnology research group: Agrobacterium tumefaciens and Agrobacterium
to insert genes into plants and improve traits. In nature, these rhizogenes. Although differences between these two systems
bacteria infect plants and insert their genes into a host’s nuclear will be discussed more thoroughly, greater transformation rates
chromosomes via a virulent plasmid. After this plasmid enters have been found in Taraxacum species using A. rhizogenes than
host cells, it incorporates its transfer DNA (tDNA) into the host A. tumefaciens (Bae et al. 2005; Lee et al. 2004).
genome. Researchers have modified this mechanism by replac- Agrobacterium tumefaciens causes crown gall disease in plants.
ing viral tDNA with genes of interest (Tzfira and Citovsky Crown galls are tumorous plant growths that form calluses of un-
2006). Two species of Agrobacterium have been used by our organized plant tissue (Gelvin 1990). Agrobacterium tumefaciens
infects plants by integrating tDNA from its tumor-inducing plas-
mid into host cells. Transfer DNA contains oncogenes that cause
Received for publication 16 Mar 2022. Accepted for publication 28 Sep 2022. overproduction of auxins and cytokinins, which cause galls to
Published online 22 Dec 2022.
This work was supported in part by the United States Department of Agricul- form. Oncogenes also trigger production of opines, which are low-
ture National Institute of Food and Agriculture (Hatch project no. OHO01417, molecular weight compounds that A. tumefaciens bacteria con-
accession no. 1014257). sume for carbon and nitrogen (Gelvin 1990; Tzfira and Citovsky
We thank Laura Chapin and Dr. Michelle Jones for training and use of the 2006). Researchers have removed the tDNA responsible for pro-
portable photosynthesis system. Thanks as well to Sarah Davis for help har-
vesting Taraxacum kok-saghyz plants. We also thank Dr. Shashi Kumar for ducing crown galls from A. tumefaciens while maintaining the
the plasmid used to generate plants for this study. tumor-inducing plasmid’s ability to insert a foreign DNA into
This paper was written as part of the Master’s degree requirement for David plant cells (Ream 2009).
Lankitus, Department of Horticulture and Crop Science.
K.C. is the corresponding author. E-mail: cornish.19@osu.edu.
Agrobacterium rhizogenes causes hairy root disease. Hairy root
This is an open access article distributed under the CC BY-NC-ND license disease is characterized by the abundant production of fuzzy adven-
(https://creativecommons.org/licenses/by-nc-nd/4.0/). titious roots from the site of bacterial infection (Gelvin 1990).
J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023. 21
Although A. rhizogenes produces different effects in plants than transformation. However, new methods are desired to select rol1
A. tumefaciens, both species insert virulent plasmids using similar plants solely by phenotype to reduce the number of PCRs re-
mechanisms, and both result in plants expressing bacterial tDNA quired. Almost all rubber dandelion plants are self-incompatible
(Gelvin 1990). Morphological changes are induced by the root- (i.e., cannot be self-pollinated to produce progeny) (Luo et al.
inducing virulent plasmid of A. rhizogenes. Like A. tumefaciens, 2017). Hybridization with wild-type plants allows the transgenes
plant auxin levels are increased, but the A. rhizogenes root loci and rol genes to segregate independently in the T1 and T2 gener-
(rol) genes in the tDNA increase plant cell sensitivity to auxin by ations. Effective selection criteria to remove rol1, transgene–
100 to 1000-fold. This increased sensitivity is the primary cause of plants before performing PCR or further crossing with wild-type
hairy root formation (Gelvin 1990). For clarity, in the rest of this plants would save time and resources. Thus, an efficient proto-
article, plants containing rol genes are referred to as rol1 and those col for removing hairy root phenotypes from the population,
without rol genes are referred to as rol–. without inadvertently eliminating genotypes expressing trans-
Crown galls from A. tumefaciens–infected plants rarely pro- genes, is needed.
duce new plants capable of rooting without the addition of Therefore, the goal of this research was to characterize rol–
plant growth regulators (Bae et al. 2005; Gelvin 1990). In con- and rol1 plant morphology so that, in the future, rol1 trans-
trast, A. rhizogenes–infected roots are able to regenerate new gene1 plants can be distinguished from rol1 transgene– plants.
plants that carry tDNA, whereas—most often demonstrated in In addition, we compared the wild-type morphology of plants
tobacco (Nicotiana tabacum) as a model—this has also been grown directly from seed with those selected as rol– and regen-
demonstrated in carrot (Daucus carota) and morning glory erated to assess potential changes caused by the transformation,
(Convolvulus arvensis) (Durand-Tardif et al. 1985; Gelvin selection, and regeneration protocols used.
1990; Tepfer 1984). In most species, complete plants cannot
be regenerated by A. rhizogenes–infected roots, although ap- Materials and Methods
plications are still reported. Roots of the marshmallow plant
(Althaea officinalis) were infected and propagated in liquid PRODUCTION OF PLANTS WITH SPECIFIC ROL TRAITS. T0 plants
media to produce a candidate protein for destroying human were made by transforming roots with rol1 or empty vector
immunodeficiency virus (Drake et al. 2013). Taraxacum species (rol–) plasmids using A. rhizogenes–mediated transformation
can fully regenerate and can do so without the addition of plant followed by regeneration, acclimation, and transfer to pots using
growth hormones (Gelvin 1990; Zhang et al. 2015). Thus, re- established methods (Zhang et al. 2015). For all generations,
searchers can choose between these two Agrobacterium species after 1 month of growth, rooted plants were transplanted into
based on the goals of their study. 8.9- × 22.9-cm rectangular black plastic pots (Mini-Tree Pot
Taraxacum kok-saghyz (rubber dandelion) is a plant species TP49CH; Stuewe and Sons Inc., Tangent, OR, USA) supported
that produces high-quality natural rubber within its root laticifers by 40-cm square plastic trays (Square Tray TRAY6, Stuewe and
and is being developed as a temperate climate and/or hydroponic Sons Inc.) with a combination of soilless media and field soil.
crop (Cornish et al. 2019). However, rubber dandelion currently Plants were grown in a greenhouse with a 12-/12-h (light/dark)
lacks ideal agronomic traits, impeding a profitable rubber yield, photoperiod at 22 C at The Ohio Agricultural Research and
and thus is being improved via gene insertions and gene editing Development Center, Wooster, OH (lat. 81.93 55'19.4"N; long.
(Cherian et al. 2019; Men et al. 2018; Salehi et al. 2021). Several 40 46'20.9"W). Because rubber dandelion is self-incompatible,
Taraxacum species have been transformed with A. tumefaciens flowering T0 plants were crossed with three different genetic
and/or A. rhizogenes. In Taraxacum platycarpum leaf disks, the backgrounds of nontransgenic plants to produce T1 generation
stable transformation rate (transgenic plants per number of trans- seeds as described (Zhang et al. 2015). These T1 seeds were
formed tissue pieces) using A. tumefaciens was 1% to 5% then crossed with other T1 progeny from a different nontrans-
(Bae et al. 2005). The transformation rate using root fragments genic parent to ensure seeds could be produced in the future.
via A. rhizogenes was 76.5% in the same species (Lee et al. T2 plants were grown in a greenhouse, as described earlier.
2004). Although transformation using A. tumefaciens has been Agrobacterium rhizogenes transgene segregation in a Mendelian
achieved in Taraxacum brevicorniculatum, a species closely pattern (Budar et al. 1986; Tepfer 1984) was first demonstrated
related to rubber dandelion, transformation efficiency was not in tobacco, one of several species that can produce plants from
provided (Post et al. 2012). Taraxacum brevicorniculatum trans- transformed roots. Thus, transgenic plants crossed with wild
formation using A. rhizogenes has a rate of 15.7% (Zhang et al. types produce heterozygous T1 transgenic plants for both trans-
2015). Agrobacterium tumefaciens and A. rhizogenes transfor- genes and rol genes (Budar et al. 1986). Because of the different
mation rates in rubber dandelion were 21.9% (Zhao L, unpub- genetic background of the wild-type plants used to create the T1
lished data) and 24.7% (Zhang et al. 2015), respectively. Time to generation, they were interbred to create the T2 generation and
regenerate plants fully and prepare them for soil growth has been beyond. Transgenes and rol genes segregate independently in
reported as 67 to 81 d for A. tumefaciens transformation (Collins- progeny as rol1 transgene–, rol1 transgene1, rol– transgene–,
Silva et al. 2012). However, using both these Agrobacterium spe- or rol– transgene1, the desired genotype. These differing
cies, we find that A. tumefaciens transformants require 168 d be- genotypes were confirmed by PCR. We selected rol1 trans-
fore acclimation can begin whereas A. rhizogenes transformants gene– and rol– transgene– plants for this study, and they are
are ready in only 70 d (unpublished data). Given the greater trans- called rol1 and rol– in the rest of the article.
formation rates and shorter regeneration times using A. rhizogenes, Rubber dandelion plants with rol1 and rol– phenotypes with-
we have adopted this method of genetic transformation for most out transgenes were produced by selection and PCR confirmation
of our transformation research. within a T2 population. This morphological study was performed
Currently, polymerase chain reactions (PCRs) are required to on the T3 progeny. T3 plants were used for further analysis, and
confirm the presence of both transgenes and rol genes after plants of rol1 transgene– and rol– transgene– genotypes were
22 J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023.
planted in 3.81- × 13.97-cm black plastic cones (Ray Leach 81.9308 W, respectively, according to The Ohio State Univer-
“stubby cell” cone SC7R, Stuewe and Sons Inc.) supported by sity (2022) Weather System.
98-cell plastic trays (Ray Leach “cone-tainer” RL98, Stuewe and For most plants, measurements were taken from the same leaf
Sons Inc.) containing peat-based soilless media (Pro-Mix; Pre- throughout the day. However, some similar leaves from the same
mier Tech Horticulture, Riviere-du-Loup, QC, Canada). Along- plants were used if the original leaf was torn or pulled from the
side them were wild-type rubber dandelion plants grown from plant. The external infrared gas analyzer chamber was 6 cm2,
seed of the Bravo population. Bravo is a population produced which was used as the input leaf size for all samples while meas-
by crossing a high-yield rubber dandelion with rubber dandelion urements were being taken. Because rubber dandelion leaves,
accession HR009, after which progeny were propagated clon- although long, are often too narrow to cover the entire 6-cm2
ally (Luo et al. 2018). Because Bravo is a higher rubber yielding chamber, photosynthetic rates were adjusted for actual leaf area
population, derived from U.S. Department of Agriculture acces- calculated using the ImageJ image processing program (Rasband
sion KAZ08-017 (W635172), it is useful to compare rubber 2018). Because rol1 plants have smaller leaves than rol– and wild
yield data from new rubber dandelion germplasm to Bravo. type, this issue affected rol1 plant measurements more strongly.
This will provide insight into rubber yield improvements in new RUBBER QUANTIFICATION. Rubber dandelion plants not used
rubber dandelion selections and enable progress toward estab- for photosynthesis analysis were harvested in Jul 2020, at 10
lishment of high-rubber cultivars. The number in each plant months old, whereas the plants used for photosynthesis analysis
group was rol–, n 5 8; rol1, n 5 11; and Bravo (wild type), were harvested in late Dec 2020, at 15 months old. Plants were
n 5 12. Six of each were selected randomly for diurnal photo- removed from their tree pots and excess dirt was shaken loose
synthesis measurements. from the roots. Each whole plant was weighed, then leaves
Plants were greenhouse-grown as described and were trans- were cut from the plant with a knife. The cut was made just above
planted after 1 month. During transplanting, healthy plants were the plant’s crown to prevent latex leaking from the crown or from
selected with identifiable traits; a smaller selection of plants was the roots. Roots (including crown) were weighed, then placed in
used for the rest of this study. Standard morphology and the brown paper bags and dried in a 50 C oven for at least 2 weeks
hairy “dome” morphology were used at this stage to guide plant before being ground to a powder using an analytical grinding mill
selection. These morphologies are discussed thoroughly in the (Basic analytical mill IKA A10; MilliporeSigma, Billerica, MA,
Results section. Plants were then grown to 10 months of age. USA). The rubber content in powdered roots was quantified using
an infrared spectroradiometer (FieldSpecV 3 Spectroradiometer;
R
Only rol1/transgene–, rol–/transgene–, and Bravo wild-type
plants were used for trait analysis. Analytical Spectral Devices Inc., Boulder, CO, USA) and a previ-
POLYMERASE CHAIN REACTIONS. DNA was extracted as de- ously developed computer model (r2 5 0.93, df 5 298), based on
scribed (Vilanova et al. 2020) using fresh or lyophilized rubber rubber dandelion root rubber quantification reference data gener-
dandelion leaves. For PCR, forward and reverse primer sequen- ated using accelerated solvent extraction (Ramirez-Cadavid et al.
ces were designed for rol genes and transgenes using the 2018), to predict the rubber concentration of dried root samples
Primer3 program (Koressaar and Remm 2007; Untergasser (measured in milligrams rubber per gram dry root). Rubber yield
et al. 2012). The primers used were rolC new_F: AGTCT- (measured in milligrams rubber per plant) was determined by
TAAGGTAGGCGACGT and rolC new_R: GTTGCTGGCA- multiplying root dry weight (measured in grams per plant) by pre-
TAAAGGTCGA. PCR primers and sample DNA were placed dicted rubber concentration.
in a thermal cycler (C1000 Touch™; Bio-Rad Laboratories, Her- STATISTICAL ANALYSIS. Photosynthesis data were analyzed us-
cules, CA, USA) for annealing, elongation, and denaturation. ing one-way analysis of variance (ANOVA) using statistical soft-
Times and temperatures were an initial 5-min denaturation phase ware (SAS version 9.4; SAS Institute Inc, Cary NC, USA), with
at 95 C, 35 cycles of 40 s denaturation at 95 C, 60 s annealing five replications in a completely randomized design. Time of day
at 54 C, 60 s elongation at 68 C, and a final 5-min extension was analyzed as a repeated measure using the REPEATED op-
phase at 68 C (Iaffaldano et al. 2016). The PCR products were tion within PROC GLM. Although photosynthesis was measured
then separated by gel electrophoresis using a 2% agarose gel over 2 d, there was no effect of “day,” and this was excluded
with ethidium bromide. PCR times were adjusted based on band from the ANOVA to conserve degrees of freedom. Whole-plant
clarity in gels (Iaffaldano et al. 2016). fresh weight, root fresh weight, root dry weight, and rubber con-
PHOTOSYNTHETIC RATES. Carbon dioxide assimilation rates tent were analyzed using one-way ANOVA using SAS version
(measured in micromoles per square meter per second) of rol1, 9.4, with five replications in a completely randomized design.
rol–, and rubber dandelion Bravo (seed-generated wild type) The Shapiro-Wilks test of residuals, the Levene test, plots of
plants and associated parameters collected automatically (three residuals vs. predicted values, and normal-quantile plots were
of each on the first day followed by another three on the next used to confirm that data conformed to assumptions of normality
day) were measured using a portable photosynthesis system and homogeneity of variance (data not shown). Photosynthesis
(LI-6400XT; LI-COR BioSciences, Lincoln, NE, USA) accord- and phenotypic measurements conformed to ANOVA assump-
ing to manufacturer’s instructions when plants were 10 months tions; however, stomatal conductance (gs) measurements did not
old (Jul 2020). Diurnal curves were produced by measuring the conform to the assumption of normality and were transformed
assimilation rate five times throughout the day: sunrise, mid- using x-0.5; data presented are the untransformed means whereas
morning, solar noon, midafternoon, and sunset. Times for P values are from the transformed analysis.
sunrise, solar noon, and sunset were determined using the Na-
tional Oceanic and Atmospheric Administration Solar Calculator Results
(National Oceanic and Atmospheric Administration 2022) given
latitude and longitude. Latitude and longitude of the Ohio Agri- PLANT MORPHOLOGY. Rubber dandelion leaves were thicker
cultural Research and Development Center is 40.7787 N and and more blue-green than the yellow-green leaf color of the
J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023. 23
common dandelion (Taraxacum officinale). Rubber dandelion
has a taproot or multiple roots and often branching roots. rol–
plants had the same basic morphological features as Bravo
(wild-type) plants; however, both rol– tops and roots appeared
smaller than Bravo plants (Fig. 1A and B), even though the sizes
were not significantly different (P > 0.05) because of large inter-
plant variation.
The morphology of all rol1 plants was distinctly different
from rol– and wild-type (Bravo) plants. Rosettes of rol1 plants
had many more and smaller leaves than wild-type plants, and
formed a dome, which was not seen in the wild-type or rol–
plants. rol1 plants were more variable than the wild-type or
rol– plants (Fig. 1A–C). However, root rubber concentration
was almost identical among the genotypes (Fig. 1D), and differ-
ences in rubber yield (Fig. 1E) mirrored differences in root dry
weight (Fig. 1C) because rubber yield is the product of concen-
tration and root dry weight. Plants removed from cones had a va-
riety of root lengths and shapes.
They fit into two categories: “wild type” (rol–, including
Bravo) and “hairy” (rol1). Wild-type roots had a taproot and
thick lateral roots, although the number and size of these roots
varied. Hairy roots did not have a taproot and had many tangled,
thin roots that spread through the soil. In general, hairy roots did
not penetrate as deeply as standard roots, but there was still vari-
ation in root depth within cones. Although all plants grew much
larger after their transplantation to tree pots, their general mor-
phologies remained the same.
rol1 roots grew much shallower and were much thinner than
roots of rol– plants (Fig. 2A and B). rol1 leaves were narrower
and much more abundant, causing a rosette dome phenotype of
vertical and horizontal leaves (Fig. 3A and B), a leaf trait that
can be used easily to confirm rol1 transgenesis. rol1 plants
also have smaller flowers than rol– plants. These phenotypes
were observed in all rol1 plants studied.
PHOTOSYNTHESIS. Photosynthetic rates were fit to quadratic
curves for each plant group (genotype) and showed diurnal vari-
ation throughout the day, with a general trend of a rising and
falling photosynthetic rate as the sun rose and set. On average,
rol1 plants had a lower carbon dioxide assimilation rate (Fig. 4)
than rol– or wild-type plants, which were similar to each other.
Thus, rol1 plants may sequester less carbon than rol– or wild-
type Bravo plants. The repeated measures ANOVA showed a
statistically significant time effect (df 5 4,12; P 5 0.004) and a
nonsignificant time × genotype interaction (df 5 8,24; P 5
0.839). Genotypes were significantly different at two time peri-
ods (between 1400 and 1800 HR) (df 5 2,15; P < 0.07), with
rol1 plants having lower assimilation rates than the other two
genotypes. However, gs differed over time (df 5 4,12; P 5
0.001), but not among genotypes (df 5 2,15; P 5 0.180–0.664)
or time × genotype interaction (df 5 8,24; P 5 0.675).
LEAF SENESCENCE. Although yellowed and dead leaves were
removed as a standard practice to maintain rubber dandelion
plant health, rol– and Bravo rosette leaves senesced at the base
of the rosette whereas rol1 leaves senesced throughout the Fig. 1. Whole plant fresh weight (A), root fresh weight (B), root dry weight (C),
rosette. rubber concentration (D), and rubber yield e in rol– (green), rol1 (blue), and
FLOWERS AND SEED. Seed from rol1 plants was noticeably wild-type (Bravo) (brown) rubber dandelion plants (P > 0.05). Rubber yield
was calculated by multiplying root dry weight with rubber concentration. The
smaller than rol– seed (Fig. 5A and B). Germination rates of T2 bars are means ± SD. The number in each mean varies as follows: (A) rol–,
seed grown to produce the T3 plants were inhibited by rol1. n 5 6; rol1, n 5 11; Bravo, n 5 10. (B–E) rol–, n 5 8; rol1, n 5 11; Bravo,
Although rol– was also lower than Bravo wild types, which had n 5 12.
24 J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023.
Fig. 3. Morphology of 6-month-old rol1 and rol– plants in planar (A) and
side (B) views. Plants on the left are rol1 and plants on the right are rol–.
The leaf proliferation and dome shape of the rosettes is clear after 1 month.
The most easily identified phenotype distinguishing rol1
from rol– in soil-grown plants was the dome leaf bunching ob-
served in rol1 plants (Fig. 3). This trait manifests within the
first month of rol1 rubber dandelion growth independent of
Fig. 2. Morphology of 10-month-old rol1 (A) and rol– (B) rubber dandelion other transgenes or when leaves are large enough to be sampled
plants after harvest from soil. The scale is in centimeters with the total
length shown.
for DNA extraction and PCR confirmation of transgene expres-
sion. Because this dome configuration occurred in all rol1
plants (with different underlying heterozygous genotypes), this
not passed through transformation, regeneration, and acclima- is a direct effect of rol1. This is not surprising, because auxin
tion, the 14-d germination rates were 70%, 80%, and 93% for plays a primary role in leaf primordia initiation and leaf devel-
rol1, rol–, and Bravo, respectively. The rol1 flowers and seed opment (Xiong and Jiao 2019), and rol1 plants are known to
heads with pappus were smaller in diameter than rol– plants, but
had longer flower stalks (Fig. 6). Flowers produced viable seed
when fertilized with pollen of different genotypes.
Discussion
The greatest benefits of using A. rhizogenes are increased trans-
formation efficiency during transgene introduction and rapid plant
regeneration; the greatest detriments are during breeding and selec-
tion for high-performing transgene1 plants, because the rol1
phenotype may obscure phenotypic traits conferred by the target
genes. This is opposite from A. tumefaciens–mediated transforma-
tion, because although the transformation efficiency is less, breed-
ing and selections are simpler.
This study showed that wild-type and rol– plants had similar
Fig. 4. Diurnal photosynthetic rates collected on 28 and 29 Jul 2020 for six
morphology, size, rubber yield, and carbon dioxide assimilation each of rol– (blue), rol1 (orange), and wild-type [Bravo (gray)] rubber dan-
rates, suggesting there were no lasting negative effects caused delion plants. Photosynthetic rates were fit to quadratic curves for each plant
by tissue culture and regeneration. group and the means are plotted. NS, not significant. † indicates solar noon.
J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023. 25
Fig. 6. Flowering plants of rubber dandelion in planar (A and B) and side
(C and D) vies of rol1 (A and C) and rol– (B and D) plants. Bravo plants
(not shown) are similar to rol– plants.
deliberate removal from soil, so is much less useful for sorting
rol1 from wild-type plants in greenhouses than the rosette
doming phenotype.
Taraxacum platycarpum transformed using A. rhizogenes dis-
played similar root phenotypes to rol1 rubber dandelion. High
numbers of hairy roots were reported, and no taproot formed in
rol1 plants in contrast to wild types (Lee et al. 2004). Agrobac-
Fig. 5. Seeds from rol– (A) and rol1 (B) rubber dandelion plants. Numbers terium rhizogenes–induced morphological changes in carrot and
on the ruler are centimeters. The mean weight of rol– seed was 0.546 mg/ tobacco also are similar to those observed in rubber dandelion,
seed and rol1 seed was 0.233 mg/seed.
including reduced flower size, leaf wrinkling, and reduced apical
dominance, phenotypes that persisted in the progeny of trans-
overproduce auxin as well as to cause the host plant to become formed plants (Tepfer 1984). Although rubber dandelion does
more sensitive to this plant growth regulator (Gelvin 1990). The not have a central stem, its apical meristem is located at its
greater variability of rol1 plants suggests that the susceptibility rosette, which likely relates to the leaf bunching observed in
of the underlying genotypes to elevated auxin differed even rol1 plants. Because carrot and tobacco had reduced apical
though all were more sensitive to auxin than wild-type plants dominance, the rubber dandelion equivalent may be expressed
(Fig. 1A–C). However, rubber concentration, although known as excessive leaf growth at its apical meristem rather than the
to be sensitive to environmental stimuli, such as cold tempera- apical dominance traits observed in carrot and tobacco.
tures (Salehi et al. 2021), does not appear sensitive to auxin The inhibitory effect of rol1 on photosynthetic carbon diox-
(Fig. 1D) because it was the same across the genotypes. The ide assimilation rate (Fig. 4) was not expected because elevated
more vertical aspect of the leaves in rol1 rosettes is likely an auxin levels more commonly increase plastid size and number,
indirect effect of leaf crowding, because this also happens in stomatal aperture, and photosynthetic rate, although the relation-
wild-type plants when they are planted at high density (Bates ship of auxin levels to primary metabolism is poorly understood
et al. 2019). The rol1 root phenotype is also useful in differen- (Tivendale and Millar 2022). The impact of the reduced assimi-
tiating rol1 and rol– plants because the rol1 plants usually late is unclear because although mean rol1 root and plant sizes
lack a taproot and have thinner, shallower, and more abundant were the smallest (Fig. 1A and B), these differences were not
roots. Although this root morphology can be seen readily in significant at the P < 0.05 level with the number of samples
plants grown on transparent media, it can only be observed in available. Also, although the photosynthetic carbon dioxide as-
soil-grown plants during transplanting or harvesting, or other similation rate was inhibited in the rol1 plants, gs was not. This
26 J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023.suggests that an internal inhibition is occurring. For example, Arabidopsis. Plant Cell 24:1081–1095, https://doi.org/10.1105/tpc.111.
auxin treatment of roots represses chloroplast development in 092254.
Arabidopsis thaliana (Kobayashi et al. 2012) and may reduce Koressaar, T. and M. Remm. 2007. Enhancements and modifications
growth, cause chlorosis, and induce starch accumulation inhibi- of primer design program Primer3. Bioinformatics 23:1289–1291,
tion of fixed carbon (sugars) transport (Mohajjel-Shoja et al. https://doi.org/10.1093/bioinformatics/btm091.
Lee, M.H., E.S. Yoon, J.H. Jeong, and Y.E. Choi. 2004. Agrobacterium
2010). Although we did not see significant growth inhibition by rhizogenes-mediated transformation of Taraxacum platycarpum and
rol1 apart from a reduction in seed size (Fig. 5), perhaps some- changes of morphological characters. Plant Cell Rep. 22:822–827,
thing of this nature occurred in our rol1 plants. We did not https://doi.org/10.1007/s00299-004-0763-5.
quantify the storage carbohydrate inulin, chloroplast number, Luo, Z., B.J. Iaffaldano, X. Zhuang, J. Fresnedo-Ramırez, and K. Cornish.
chlorophyll content, or leaf color in our study. 2017. Analysis of the first Taraxacum kok-saghyz transcriptome reveals
In conclusion, this description of rol1 morphology can guide potential rubber yield related SNPs. Sci. Rep. 7:1–13, https://doi.org/
researchers in differentiating between rol1 and rol– rubber dande- 10.1038/s41598-017-09034-2.
lion plants in advance of PCR tests. Plants containing transgenes Luo, Z., B.J. Iaffaldano, X. Zhuang, J. Fresnedo-Ramırez, and K. Cornish.
of interest in the T1 and T2 generation can then be compared with 2018. Variance, inter-trait correlation, heritability, and marker-trait
rol1 to detect transgene-induced changes in morphology or pho- association of rubber yield-related characteristics in Taraxacum kok-
tosynthetic rate. This will allow for a more efficient selection pro- saghyz. Plant Mol. Biol. Rpt. 36:576–587, https://doi.org/10.1007/
s11105-018-1097-8.
cess that should allow for more rapid development of transgenic
Men, X., F. Wang, G.Q. Chen, H.B. Zhang, and M. Xian. 2018. Bio-
rubber dandelion populations after A. rhizogenes transformation. synthesis of natural rubber: Current state and perspectives. Int. J.
Mol. Sci. 20:1–22, https://doi.org/10.3390/ijms20010050.
References Cited Mohajjel-Shoja, H., B. Clement, J. Perot, M. Alioua, and L. Otten.
2010. Biological activity of the Agrobacterium rhizogenes–derived trolC
Bae, T.W., H.R. Park, Y.S. Kwak, H.Y. Lee, and S.B. Ryu. 2005.
gene of Nicotiana tabacum and its functional relation to other plast
Agrobacterium tumefaciens-mediated transformation of a medicinal
genes. IS-MPMI. 24:44–53, https://doi.org/10.1094/MPMI-06-10-0139.
plant Taraxacum platycarpum. Plant Cell Tissue Organ Cult. 80:
National Oceanic and Atmospheric Administration. 2022. NOAA solar
51–57, https://doi.org/10.1007/s11240-004-8807-7.
Bates, G.M., S.K. McNulty, N.D. Amstutz, V.K. Pool, and K. Cornish. calculator. https://www.esrl.noaa.gov/gmd/grad/solcalc/. [accessed 6 Jun
2019. Planting density and harvest season effects on actual and poten- 2022].
tial latex and rubber yields in Taraxacum kok-saghyz. HortScience. Post, J., N. van Deenen, J. Fricke, N. Kowalski, D. Wurbs, H. Schaller,
54:1338–1344, https://doi.org/10.21273/HORTSCI13986-19. W. Eisenreich, C. Huber, R.M. Twyman, D. Pr€ ufer, and C.S. Gronover.
Budar, F., L. Thia-Toong, M. Van Montagu, and J.P. Hernalsteens. 2012. Laticifer-specific cis prenyltransferase silencing affects the rubber,
1986. Agrobacterium mediated gene transfer results mainly in trans- triterpene, and inulin content of Taraxacum brevicorniculatum. Plant
genic plants transmitting T-DNA as a single mendelian factor. Ge- Physiol. 158:1406–1417, https://doi.org/10.1104/pp.111.187880.
netics 114:303–313, https://doi.org/10.1093/genetics/114.1.303. Ramirez-Cadavid, D.A., S. Valles-Ramirez, K. Cornish, and F.C. Michel.
Cherian, S., S.B. Ryu, and K. Cornish. 2019. Natural rubber biosyn- 2018. Simultaneous quantification of rubber, inulin, and resins in Tarax-
thesis in plants, the rubber transferase complex, and metabolic engi- acum kok-saghyz (TK) roots by sequential solvent extraction. Ind.
neering progress and prospects. Plant Biotechnol. J. 17:2041–2061, Crops Prod. 122:647–656, https://doi.org/10.1016/j.indcrop.2018.06.008.
https://doi.org/10.1111/pbi.13181. Rasband, W.S. 2018. ImageJ. https://imagej.nih.gov/ij/. [accessed
Collins-Silva, J., A.T. Nural, A. Skaggs, D. Scott, U. Hathwaik, 6 Jun 2022].
R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish, and Ream W. 2009. Agrobacterium tumefaciens and A. rhizogenes use dif-
D. Shintani. 2012. Altered levels of the Taraxacum kok-saghyz ferent proteins to transport bacterial DNA into the plant cell nucleus.
(Russian dandelion) small rubber particle protein, TkSRPP3, result Microb. Biotechnol. 2:416–427, https://doi.org/10.1111/j.1751-7915.
in qualitative and quantitative changes in rubber metabolism. Phyto- 2009.00104.x.
chemistry. 79:46–56, https://doi.org/10.1016/j.phytochem.2012.04.015. Salehi, M., K. Cornish, M. Bahmankar, and M.R. Naghavi. 2021. Nat-
Cornish, K., S. Kopicky, and T. Madden. 2019. Hydroponic cultiva- ural rubber-producing sources, systems, and perspectives for breed-
tion has high yield potential for TKS. https://www.rubbernews.com/ ing and biotechnology studies of Taraxacum kok-saghyz. Ind. Crops
technical-notebooks/hydroponic-cultivation-has-high-yield-potential-tks. Prod. 170:1–12, https://doi.org/10.1016/j.indcrop.2021.113667.
[accessed 27 Apr 2022]. Tepfer, D. 1984. Transformation of several species of higher plants by
Drake, P.M.W., L. de Moraes Madeira, T.H. Szeto, and J.K.C. Ma. Agrobacterium rhizogenes: Sexual transmission of the transformed
2013. Transformation of Althaea officinalis L. by Agrobacterium genotype and phenotype. Cell 37:959–967, https://doi.org/10.1016/
rhizogenes for the production of transgenic roots expressing the anti- 0092-8674(84)90430-6.
HIV microbicide cyanovirin-N. Transgenic Res. 22:1225–1229, https:// The Ohio State University. 2022. CFAES Weather System. https://
doi.org/10.1007/s11248-013-9730-7. weather.cfaes.osu.edu//stationinfo.asp?id=1. [accessed 6 Jun 2022].
Durand-Tardif, M., R. Broglie, J. Slightom, and D. Tepfer. 1985. Struc- Tivendale, N.D. and A.H. Millar. 2022. How is auxin linked with cellu-
ture and expression of Ri T-DNA from Agrobacterium rhizogenes in lar energy pathways to promote growth? New Phytol. 233:2397–2404,
Nicotiana tabacum. J. Mol. Biol. 186:557–564, https://doi.org/10.1016/ https://doi.org/10.1111/nph.17946.
0022-2836(85)90130-5. Tzfira, T. and V. Citovsky. 2006. Agrobacterium-mediated ge-
Gelvin, S.B. 1990. Crown gall disease and hairy root disease: A netic transformation of plants: Biology and biotechnology. Curr.
sledgehammer and a tackhammer. Plant Physiol. 92:281–285, https:// Opin. Biotechnol. 17:147–154, https://doi.org/10.1016/j.copbio.2006.
doi.org/10.1104/pp.92.2.281. 01.009.
Iaffaldano, B.J., Y. Zhang, J. Cardina, and K. Cornish. 2016. CRISPR/ Untergasser, A., I. Cutcutache, T. Koressaar, J. Ye, B.C. Faircloth,
Cas9 genome editing of rubber producing dandelion Taraxacum kok- M. Remm, and S.G. Rozen. 2012. Primer3: New capabilities and
saghyz using Agrobacterium rhizogenes without selection. Ind. Crops interfaces. Nucl. Acids Res. 40:e115, https://doi.org/10.1093/nar/
Prod. 98:356–362, https://doi.org/10.1016/j.indcrop.2016.05.029. gks596.
Kobayashi, K., S. Baba, T. Obayashi, M. Sato, K. Toyooka, M. Ker€anen, Vilanova, S., D. Alonso, P. Gramazio, M. Plazas, E. Garcıa-Fortea,
E.M. Aro, H. Fukaki, H. Ohta, K. Sugimoto, and T. Masuda. 2012. Reg- P. Ferrante, M. Schmidt, M.J. Dıez, B. Usadel, G. Giuliano, and
ulation of root greening by light and auxin/cytokinin signaling in J. Prohens. 2020. SILEX: A fast and inexpensive high-quality
J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023. 27DNA extraction method suitable for multiple sequencing platforms Zhang, Y., B.J. Iaffaldano, W. Xie, J.J. Blakeslee, and K. Cornish. and recalcitrant plant species. Plant Methods 16:1–11, https://doi. 2015. Rapid and hormone free Agrobacterium rhizogenes-mediated org/10.1186/s13007-020-00652-y. transformation in rubber producing dandelions Taraxacum kok-saghyz Xiong, Y. and Y. Jiao. 2019. The diverse roles of auxin in regulating and T. brevicorniculatum. Ind. Crops Prod. 66:110–118, https://doi. leaf development. Plants 8:243, https://doi.org/10.3390/plants8070243. org/10.1016/j.indcrop.2014.12.013. 28 J. AMER. SOC. HORT. SCI. 148(1):21–28. 2023.
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