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19 Targeted Improvement of Medicinal Plant Under Stress Condition... 347
Fig. 19.1 Major abiotic
stresses affect plant
growth
(modified from Biswas et al.
2021)
Abioc
stresses
Drought
Cold
ROS
Heat
19.2 Types of Abiotic Stress Affecting Plant Growth
Most of the plants are facing survivability in adverse environmental condition
(Chang et al. 2020). Different types of plant abiotic stresses are present in environ-
ment. In drought condition, water deficiency caused cellular osmotic stress which
affects transport and reduces the plant height and growth (Dubois and Inzé
2020).
Heat stresses causes excess water loss because of loss of membrane stability and
photosynthesis impairment (Lamaoui et al. 2018). Chilling and freezing temperature
caused facilitation of ice nucleation (i.e., formation of larger ice crystal outside the
cell by pulling out the available water molecules) and resulted in damage of
protoplast (Zhu et al. 1997). Drought and the extremes of temperature concentrate
solutes in soil as well as cellular environment through rapid dehydration, evapora-
tion, and transpiration (Rani et al. 2020). When the existing ions concentration will
reach to a toxic level, it then affects biochemical and physiological activities of
plants. Oxygen is a crucial factor for cell survival and plant growth (Geigenberger
2003). Plants require oxygen from the atmosphere through aerobic respiration. It
mainly caused in waterlogging condition during flood. Oxida tive stress is developed
in plant through the overproduction and accumulation of reactive oxygen species
(ROS) (Yousuf et al.
2012). ROS cau se cellular biopolymer damages and respective
dysfunctions (Demidchik 2015). All these stressful conditions affect the morpholog-
ical character and physiological and biochemical activities of plants (Fig. 19.1).
19.3 History of CRISPR
CRISPR-Cas9 is e asy to handle, is more efficient, can be easily targeted to almost
any genomic location of choice by a short RNA guide, and has been successfully
applied in many organisms, including both model and crop plants (Ceasar et al.
2016). The chronology of CRISPR-Cas-based gene editing has been showed in
Fig. 19.2. In 2020 E. Charpentier and J. Doudna share the Nobel Prize for develop-
ing this gen ome-editing technology.
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348 P. Shah et al.
1987
2002
2003
2007
2008
2010
2012
2013
2015
2017
2018
2019
2020
First CRISPR Mechanism published
CRISPR/Cas9 term published first me
Use of ZFN tool for genome eding
Discovery that CRISPR/Cas resistance to specific phages
Term protospacer use first me
CRISPR/Cas idenfied as bacterial and archaeal immune system
CRISPR/Cas developed as gene eding tool
CRISPR/Cas used to edit human and plant genome
CRISPR/Cas9 used to develop virus resistant tomato plant
Discovery of third generaon base editor
ABE Base editor was developed, successful applicaon of CBE in plant
Rapid applicaon of base eding tool
Prime editors in plant
Fig. 19.2 Time line of CRISPR-Cas from initial to present
19.4 Structure and Mechanism of CRISPR-Cas
CRISPR-Cas system known as molecular scissors which belong to Cas gene family
protein and CRISPR array consists of spacers, repeats, and leader sequences. Leader
sequences are presen t on upstream of the CRISPR array and activate initiation of
CRISPR transcription. Short repetitive sequences are present near about 21–48
nucleotides in length which help in the formation of loop hair. These repeat
sequences varied from species to species. Spacers have 26–72 nucleotides, and
these spacers are located between 2 repeats (Grissa et al. 2007). The coding
sequences of Cas9 represent upstream of CRISPR array and then encode a highly
conserved nucleic acid-related Cas protein which has nuclease, nickase helicase, and
other activity such as cleave d DNA sequences (Bland et al. 2007).
CRISPR-Cas has three steps of working mechanism systems: acquisition, expres-
sion, and interference (Fig.
19.3). The initial step starts when Cas1 and Cas2 protein
complexes accomplish which are known as CRISPR-Cas system. This Cas protein is
recognized by spacer protein (protospacer and protospacer adjacent motif) in foreign
nucleic acids that adjacently integrate CRISPR spacer into CRISPR array by repeat
sequences and created immunological memory of heredity materials (Pourcel et al.
2005).
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19 Targeted Improvement of Medicinal Plant Under Stress Condition... 349
Fig. 19.3 Schematic representation of CRISPR-Cas immunity (modified from Guo et al. 2022)
19.5 Application of CRISPR-Cas on Medicinal Plants
CRISPR-Cas is the most advanced genome-editing tool which has lots of utility
because of its effectiveness, being short-lived, and extensive range of uses. It is most
conveniently used in medicinal plants, such as S. miltiorrhiza, D. officinale,
C. sativa, etc., which are shown below the Fig. 19.4.
The omics technology as presented in Fig. 19.5 will also provide more efficient of
gene editing to targeted gene in medicinal plant, which promotes the synthetic
biology and biologically active materials and enhances the crop production and
refinement of medicinal plant evolution. It is shown in Fig. 19.5 as a diagrammatic
form.
There is different medicinal plants which have CRISPR-Cas application which is
elaborated below:
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350 P. Shah et al.
Assembly and transformation
CRISPR-Cas sg RNA
Biolistic
Load Agrobacterium
Mediated
Regenerate and screening Plant viruses Polyethyelene glycol
Design sgRNA
Target sequence PAM
Crispr-Cas gene editing plant
Promoter
Guide RNA
Sg RNA backbone
Next generaon
sequencing (NGS)
RRRRecombinant
plasmid
Fig. 19.4 Flow chart of CRISPR-Cas-based genetic engineering in medicinal plants
19.5.1 Camelina sativa (L.) Crantz
Camelina sativa which belongs to Brassicaceae family which is commonly known
as false flax has gained lots of attention due to its industrial and commercial benefit
and purpose. It contains high amount of oil fatty acid near about 45% which is a rich
source of saturated fatty acid and has high value in biofuel industry, human nutrition,
and pharmaceuticals especially terpenoids and cosmetics (Gugel and Falk
2006
).
False flax is a primitive plant distributed majorly in Central Asia and Europe. It has
medicinal purposes such as anti-hypercholesterolemia, antioxidant, anti-
inflammatory, anticancer, and insulin sensitivity enhancer. The CRISPR-Cas plays
an important role as it enhances the oil yield production by random mutagenesis-
mediated genome editing. In hexaploidy C. sativa, CRISPR knocks out fatty acid
desaturase 2 (FAD2) genes which enhances the oleic acid content (Jiang et al. 2017;
Morineau et al.
2017). Seed oil content is increased when enhances the oleic from
16% to 500% of total unsaturated fatty acid, decline the unwanted polyunsaturated
fatty acid such as linoleic and linoleic acid -16% to<4% from -35% to <10%,
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respectively. In allohexaploid Camelina guide RNAs targeted all three
homoeologous FAD2 genes which enhances the composition of oleic acid in
C. sativa seeds in F3 and F4 generation (Jiang et al. 2017).
19 Targeted Improvement of Medicinal Plant Under Stress Condition... 351
Bioinformatic analysis Functional Prediction
Identify functional gene Regulate metabolic Pathway
Transcriptome
Metabolome
Proteome
Genome omics
Key gene acve site
Crisper -Cas gene
eding
Genec improvement
Synthec biology
Reverse genecs
Fig. 19.5 Omics technology for gene editing in medicinal plants
19.5.2 Salvia miltiorrhiza
Salvia is belonging to the Lamiaceae family which is used as tradition medicinal
herb in Asia. It is also known as Danshen. It is used against diabetes, cardiovascular
diseases, and cerebrovascular diseases near about thousands of years. It has a rich
amount of secondary metabolite such as phenolic acids, rosmarinic acid, salvianolic
acid, and lithospermic acid (water-soluble), and some secondary metabolite is lipid-
soluble such as tanshinones which are present (Zhou et al.
2018). It is a model plant
for genetic and epigenetic research as emerging medicinal plant studies (Xu et al.
2016).
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352 P. Shah et al.
In 2017 CRISPR-Cas was used as a genome-editing tool for this plant. CRISPR-
Cas knocks out the diterpene synthase gene (SmCPS1) in tanshinone biosynthetic
pathways to obtain the 8 chimeric and 3 homozygous mutants from 26 transgenic
lines. It is Agrobacterium-mediated transformation where secondary metabolite
content was changed in mutant and wild type which is compared by LC-MS
technique. Tanshinone I and tanshinone II are completely absent in mutant type.
There is proof that SmCPS1 is key gene for tanshinone biosynthesis pathway
(Xu et al. 2016). For the phenolic acid biosynthesis pathway, hairy root mutant
transformed through the targeted knockout the rosmarinic acid (RA) synthase gene
SmRAS which is water-soluble by using CRISPR-Cas system (Zhou et al.
2018).
According to the study’s findings, Phenolic acids like RA and salvianolic acid
reduced in the wild type while RA precursor level increased in the mutant. The
conclusion is provided the study of functional gene and their targeting gene family.
19.5.3 Dendrobium officinale Kimura and Migo
Dendrobium officinale is a medicinal plant which belongs to family Orchidaceae.It
is commonly known as Tiepi Shihu. It is used as herb tonic and healthy food in
China, Southeast Asia (Niu et al. 2021 ). It is very old remedy for Yin deficiency
diseases (Tang et al. 2017). It has many more medicinal importance such as in
pharmacology, antioxidant, anti-fatigue, renoprotective, antiangiogenic,
hepatoprotective, gastroprotective, antimicrobial, antiviral, and anti-diabetes
properties (Guo et al.
2018; Kui et al. 2017). Around 190 phytochemicals including
alkaloids, phenanthrenes, polysaccharides, bibenzyls, essential oils, glycosides, etc.
have been recorded in the plant (Tang et al.
2017).
19.5.4 Cannabis sativa
C. sativa is belonging to hemp family. It is commonly known as marijuana,
cosmopolitan distribution in all over the world. It is basically cultivated in Indige-
nous as wel l as Eastern Asia.
It is used as a medicinal plant since a very long period of times. The secondary
compound which is present in Cannabis used as pharmacological is cannabinoid,
such as D9-tetrahydrocannabinol (THC) and cannabidiol (C BD) (Gao et al.
2020).
C. sativa used in the treatment of multiple human diseases, such as complex
neurological diseases and cancer. It is used as potential drug in COVID-19 period.
Cannabis is containing most efficient source of secondary metabolite cannabinoids.
It is used as socio-economic purpose and global marketing benefits (Aliekperova
et al.
2020). The CRISPR-Cas9 plays a very important role in enhancing the
secondary metabolite content in Cannabis and genetic improvement in canna binoid
synthesis gene in Cannabis. CRISPR-Cas technology is edited to phytoene
desaturase gene (CsPDS1), as a marker gene for genetic manipulation. After this
manipulation four transgenic line, albino-type phenotype is generated. They develop
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a balanced regeneration and transformation method for genetic modification of the
C. sativa plant. The transformation which is used is Agrobacterium-mediated where
T-DNA integrates in cannabis genome. The CRISPR vector which is used in GRF3-
GIF1 in plant regeneration, 1.7-fold increased gene, was edited. This research is
applied to functional and genomic studies and demonstrated potentials of CRISPR
gene editing in C. sativa.
19 Targeted Improvement of Medicinal Plant Under Stress Condition... 353
19.5.5 Nicotiana tabacum
Nicotiana tabacum belongs to family Solanaceae which is commonly known as
tobacco. It is rich source of secondary metabolites such as nicotine which is pyridine
alkaloid act as barrier in herbivores. It has lots of valuable therapeutic importance
used as secondary metabolites and transgenic proteins, including antibodies,
enzymes, hormones, growth factors, and cytokines, produced by N. tabacum; how-
ever, commercialization has not been fully achieved. The CRISPR-Cas has been
used gene inactivation in N. tabacu m (Jansing et al. 2019). It is produced
non-transgenic for nicotine-free tobacco.
19.5.6 Papaver somniferum
Papaver somniferum, commonly known as the opium poppy or bread seed poppy,
belongs to family Papaveraceae. It contains secondary metabolite biosynthesis such
as benzylisoquinoline alkaloids (BIAs) and morphine which contain clinical signifi-
cance in health and pharmaceuticals due to central nervous system (CNS) activity,
and there are additional uses of this plant in epilepsy, diarrhea, analgesic, insomnia,
sciatic pain, respiratory problem, depression, and appetite (Labanca et al.
2018).
There is type II CRISPR-Cas-based gene knockout via NHEJ (nonhomologous
end joining) repair, to target the 4OMT2 (gene which controls the BIAs biosynthe-
sis). In P. somniferum Agrobacterium-mediated transformation is used based on
synthetic binary plasmids for sgRNA transcription and Cas9-encoding synthetic
vector under AtU6 promoter to knock out the 4OMT2 gene. As a result, this kind
of genome editing method modifies the biosynthetic flux of the BIAs (morphine,
codeine, thebaine, etc.) (Alagoz et al.
2016).
19.5.7 Comfrey (Symphytum officinale)
It is commonly known as knit bone or slipper root, which belongs to family
Boraginaceae. It has lots of medicinal property used as anti-inflammatory, analgesic,
and proliferative effect (Staiger 2012). It is used since 400 B. C. for treatment of
heavy bleeding and bronchial problem. Its pharmaceutical application is inhibited
because it has high content of toxic pyrrolizidine alkaloid (PA) in the whole plant,
which may result in hepatic toxicity in humans even in low dose (Stickel and Seitz
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2000). Despite the beneficial characteristics of comfrey’s valuable metabolites, its
medicinal uses are limited (Allgaier and Franz 2015). Traditionally the secondary
metabolite isolation of extraction or purification is expensive and laborious
(Kopp et al.
2020). The CRISPR-Cas9 gene editing technology helps in the reduc-
tion of biosynthesis pathway of toxic compound from raw materials for
phytopharmaceuticals.
354 P. Shah et al.
CRISPR-
Cas9
syst
e
m
induce
d
the
mutati
on of spermidine specific enzyme (HSS)
homospermidine synthase, the initial specific biochemical reaction of the
pro-anthocyanidin biochemical pathway. The capacity of targeted gene and low
toxic improvement variety with CRISPR is introduced in this research.
19.5.8 Chrysanthemum morifolium
C. morifolium belongs to family Asteraceae, commonly known as aster, daisy. It has
lots of medicinal benefits such as anti-inflammatory, blood purifier, cold, diuretic,
swelling of feet, cosmetic purpose, etc. It is rich in folate and vitamin C. The
CRISPR-Cas9 system technology is used in medicinal and dietary plants with
possible nutraceutical and pharmacological significance (although not directly in
relation to secondary metabolite production) such as Chrysanthemum morifolium
(flower head used as dietary herbal medicine against cardiovascular and respiratory
diseases) (Kishi-Kaboshi et al. 2017; Yuan et al. 2020).
19.5.9 Grewia asiatica
It belongs to Malvaceae family, commonly known as phalsa or falsa. It is worldwide
cultivated especially in Asian countries. It is a rich source of vitamin E, carotenoid,
vitamin C, etc. It has lots of most valuable medicinal importance such as for
treatment of diarrhea and dysentery, prevention of cancer, and prevention of chronic
heart diseases and gastrointestinal disorder and so on. The CRISPR-Cas9 edits the
chalcone synthase gene (GaCHS) in order to produce commercially viable seedless
cultivars (Wani et al. 2017).
19.6 Advantage and Disadvantage of CRISPR-Cas in Abiotic
Stress
There are few disadvantages of CRISPR-Cas because it has off-target effects
and changes of germline, but CRISPR-Cas has lots of advantages as it modified
the desired gene in plants during stress condition and improved the yield and
production.
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19 Targeted Improvement of Medicinal Plant Under Stress Condition... 355
19.7 Conclusion
CRISPR-Cas9 is very important and most valuable technique for genome-targeted
engineering, because of its simplicity, robustness, and high target efficiency which
makes it powerful in medicinal plant genome editing. In medicinal plant the gene
occurs through knock in and knock out, genetic regulation, genetic replacement, and
modification of gene locus for medicinal improvement. For example, Salvia is
medicinal plant where high-quality genome sequencing, and transcriptome sequenc-
ing is done for more functional gene (Ma et al.
2016; Luo et al. 2014). CRISPR-Cas
technology enhances the medicinal secondary metabolite compound and edited
biosynthesis pathway during stress condition to enhance the yield production and
enhance the secondary metabolite content.
19.8 Future Prospective
Medicinal plant metabolic engineering plays some significant roles such as increas-
ing secondary metabolite content, being tolerant to abiotic stress, enhancing the shelf
life of plants, etc. Monogenic or polygenic traits may be used for improvement of
plants. For example, in A. annua improvement of Artemisia acid biosynthesis
pathway by using the super transformation method. Artemisinic acid is the precursor
of artemisinin, which is used to cure malaria (Fuentes et al.
2016). This whole
process is tedious, time-consuming, and laborious. These processes can be done
easily by CRISPR-Cas-mediated targeted gene insertion and can introduce several
genes via homologous recombination (Miki et al.
2018). Accessibility of mRNA and
whole genome sequences is key feature of many medicinal plants as a tool. There-
fore CRISPR- Cas helps in gathering insufficient sequences information and acts as a
limiting factor. There are some plant transformation tools used. This method is
completely diverse from transgenic technology.
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