CHAPTER1 Introduction

The Brassica juncea (Indian mustard) is also known as Laha, Banga, Sarso, Rai. Brassica
genus has many commercial importance such as vegetables and oil seed crops. It belongs to the
family Brassicaceae L.(Goswami1 et.al.,2020). Brassicaceae family comprises of about 350
genera with 3200 species. There are some important crops of this genus which are mustard
(Brassica juncea), rapeseed (Brassica campesteris and Brassica napus), cabbage and broccoli
(Brassica oleraceae) ,turnip (B. napa).It acclimatize mostly to light and colder climate
(Janick,1986). Mustard plants are branched and erect. Their leaves of brassica are green in colour
and lyrated .It is an annual herb and flowers are yellow in color. It is called rabi crop and takes 4-
5 months for complete maturation of seeds .The growing period of Brassica is approximately 40-
60 days depends on the variety and the conditions which is provided. The best environmental
temperature is 15-180 C to be considered. It is mostly grown in India mainly in Haryana and
other states like West Bengal, Asaam, Madhya pardesh,Punjab, uttar pardesh and Rajastan
because of favourable environmental condition. (Duhoon et al., 1998; Misra et al.,2010;Dutta et
al.,2008; ).

Brassica seeds contain 20–25 percent protein and 42%–45% oil. It is one of the most
significant sources of commercial oil, vegetable oil, and protein-rich foods. Mustard oil is used
for a variety of purposes, including cooking, dressing salads, and the preservation of pickles and
chutneys. The mustard oil has antifungal characteristics due to the presence of glucosinolate-like
material and is rich in vitamin E and natural antioxidants (Rahman et.al.,2011). The world's
largest occupying area for rapeseed production is India. In India, Brassica juncea L. is frequently
used to make oil and seasonings. The most significant area of scientific interest is Brassica spp.
(Goswami1 and others, 2020) The Brassica juncea plant has also been used to make pesticides
and biofuels. (Shyam et al. 2021). Because of the linolic acid and alpha linolic acid in mustard
oil, heart patients take it specifically (omega 3 and omega 6 fatty acids compositions
respectively). Other fatty acids including oleic acid, palmitic acid, eicosenic acid, erucic acid,
and linolenic acid are also present. Other fatty acids include stearic acid, arachidic acid, behenic
acid, lignoceric acid, and behenic, oleic, and eicosenic acids.

We use a traditional breeding approach to improve their key characteristics. Modern

developments may be enhancing the new traits within the species through conventional breeding.
However, because to high levels of isolation from cross-pollination and a lack of readily
accessible wild material, standard breeding strategies alone are insufficient for Brassica. Shiam
et al. (2002).

Plant biotechnology is employed in genetic engineering techniques to overcome the constraints

brought upon by conventional breeding (Narusaka et al., 2003). Various biotechnology
techniques can be used to increase crop output and nutrient levels. Plants that are resistant to
many diseases, abiotic stressors, and insect pests can be created by genetic engineering. Different
genes have been discovered and altered through the development of genetic engineering for the
study of the structure and diverse functions of genes (Kidokoro et al., 2009). In order for some
plants to develop the necessary traits, these tailored genes are transferred into them. Because of
this, several plants were transformed utilising techniques such as protoplast electroporation
(Bergman & Glimeius, 1993), Agrobacterium-mediated transformation (Narasimhulu et al.,
1992), and biolistic gene gun transformation (Chen & Beverdorf, 1994). Although
Agrobacterium-mediated gene transfer is the method most frequently utilised to change dicot
plants. The most beneficial, simple, and efficient transformation method for Brassica juncea is
one mediated by Agrobacterium tumefaciens. (Das et al., 2006; Dutta et al., 2005; Barfield &
Pua, 1991; Pental et al., 1993). This technology has been applied to Brassica juncea to increase
oil quality (Das et al., 2006; Hong et al., 2002; Sivaraman et al., 2004; Zada et al., 2013, 2013a),
fight against herbicides, against insect pests (Kanrar et al., 2002), develop cross breed seeds
(Jagannath et al., 2002), phytoremediation (Zhu et al. (2015) Ahmed et al.,2015). In Brassica
spp., the increase in genetic variety can be added to the generation of disease-resistant/tolerant
plants to overcome incompatibility barriers as well as plants with better agronomic
characteristics (Dubey and Gupta, 2014). Additionally, in vitro transformation and regeneration
may have the ability to meet breeding needs (Khan et al., 2010). The reproducible and effective
plant regeneration convention for various genotypes of Brassica must be established in order to
produce genotypes with the desired peculiarities in genetic transformation and in vitro selection
studies.  (Shyam, et al.2021)

In Brassica spp., the increase in genetic variety can be added to the generation of disease-
resistant/tolerant plants to overcome incompatibility barriers as well as plants with better
agronomic characteristics (Dubey and Gupta, 2014). Additionally, in vitro transformation and
regeneration may have the ability to meet breeding needs (Khan et al., 2010). The reproducible
and effective plant regeneration convention for various genotypes of Brassica must be
established in order to produce genotypes with the desired peculiarities in genetic transformation
and in vitro selection studies.  (Shyam, et al.2021). Additionally, patients with type 2 diabetes
mellitus are more likely than those without diabetes to experience memory loss (Mittal and
Katare, 2016; Gupta et al., 2020).
Diabetes mellitus is derived from the Latin term mellitus, which means sweet, and the Greek
word diabetets, which means siphon—to pass through. A metabolic disorder like diabetes
mellitus implies unnaturally elevated blood sugar levels. There are numerous subcategories,
primarily Type 1 and Type 2. These two are typical outcomes of impaired insulin secretion. One
such illness that has the potential to seriously harm people's health is diabetes mellitus. (Kumar
PJ, Clark M, Textbook of Clinical Medicine, Saunders, London, 2002, p. 1099–1121)
People who have diabetes are becoming more and more numerous every day. It is growing at an
alarming rate on a global scale. According to a 2011 study (Oyedemi SO, Adewusi EA,
Aiyegoro OA, Akinpelu DA, Antidiabetic and haematological effect of aqueous extract of stem
bark of Afzelia africana(Smith) on streptozotocin-induced diabetic Wistar rats), the number of
people with diabetes worldwide could reach 366 million by the year 2030. Without enough
insulin, the body's cells are unable to properly absorb glucose from the blood, leading to
hyperglycemia, or high blood sugar. If the blood glucose level remains high for an extended
period of time, it can cause long-term damage to organs like the kidneys, liver, eyes, nerves,
heart, and blood vessels, and severe issues with some of these organs can cause death (Pari L,
Saravanan R, Antidiabetic effect of diasulin, a herbal drug, on blood glucose, plasma insulin, and
hepatic enzymes of glucose metabolism in hyperglycaemic rats, Diabetes, Obe (Pradeep
Singh,et.al. ,2015). Various insulin formulations and a wide range of antidiabetic medications are
already available for the treatment of diabetes (Jacob et al., 2018). However, overcoming
diabetes without the use of medications remains a significant task. There are numerous
possibilities for herbal treatments available today that can be utilised to manage type 2 diabetes
on a long-term basis (Gupta et al., 2011; Gupta et al., 2013; Sharma et al., 2020). Sumeet Gupta,
among others, 2022)
Men have used herbal medicines as a form of traditional medicine for a long time. Herbalism or
botanical medicine were the terms used to describe this sort of treatment (Evans WC, Trease and
Evans, Pharmacognosy 15thed.,W.B Saunders, Edinburgh, 2002, 585). Herbs and diabetes have
a long history of interaction. The ethnobotanical statistics on 800 plants that may have anti-
diabetic potentiality thus show that plants are a potential source for anti-diabetic medications.
Although synthetic oral hypoglycemic medications and insulin are the most effective treatments
for diabetes and are efficient in controlling hyperglycemia, they have significant side effects and
do not significantly alter the course of diabetic issues. It becomes the main reason that more and
more individuals are learning about alternative therapies that have fewer or no adverse effects.
Annual sales of herbal medicines increase by 10% worldwide. The World Health Organization
estimates that 75–80% of people in less developed nations rely on herbal remedies for their
healthcare. Traditional medicine use has increased in industrialised nations as a result of modern
medicine's failure to effectively treat chronic diseases and the discovery of multi-drug resistance
bacteria and parasites. Allium cepa, Anacardium occidentale, Andrographic paniculata,
Momordica charantia, Azadirachtha indica, Brassica oleraccia, Cinnamomum tamala, and
Withania sominifera are a few examples of the medicinal plants that have been used to treat
diabetes in traditional medical systems.( Pradeep Singhet al. 2015)
Cruciferous vegetables, often known as brassicas, are a key component of the diabetes
treatment diet. According to one study, cruciferous vegetables operate as a good source of
organic antioxidants and have anti-diabetic properties (Li et al., 2018). Sumeet Gupta, et al., "Et
al." 2022). Brassica juncea seeds and leaves in particular are utilised to treat diabetes resistance.
Following a diet high in fructose for 30 days caused blood sugar to rise by 29.4%, insulin to rise
by 101.2%, and cholesterol to rise by 26.7%, indicating the development of insulin resistance.
However, following a fructose-based diet with 10% powdered Brassica juncea seeds
continuously for one month reduced the fasting serum glucose, insulin, and cholesterol levels but
did not return to normal. The current study's findings suggest that Brassica juncea should be
given to people who are at risk for developing diabetes because it can help reduce the pre-
diabetic condition of insulin resistance. There have been no reports of the insulin gene being
transferred to Brassica juncea to yet. In our study, Brassica juncea was transformed using
Agrobacterium-mediated genetic transformation in order to ease the functional genomics
investigation and the improvement of Brassica.

The objectives of research work are

1. Optimization of the regeneration of Brassica juncea, Variety RH 30 and PUSA

JAGANNATH
2. Optimization of transformation system and expression of insulin gene in Brassica
juncea , Variety PUSA JAGANNATH
CHAPTER 2 Review of Literature

For in vitro multiple shoot regeneration, Sanjita et al. (2021) employed MS media with varied
doses and a hormone combination. One Brassica campestris variety, Tori, and two Brassica
juncea cultivars, BARI Sarisha 11 and BARI Sarisha 16, were taken. The best responsive
explants for the regeneration of new shoots in the case of the Brassica juncea varieties were
obtained on MS with these concentrations: 2.0 mg/l BAP, 0.2 mg/l NAA, and 0.5 mg/l Kn. The
best concentrations for Tori-7 to get the most shoots to emerge from an explant are 3.0 mg/l BAP
and 0.2 mg/l NAA. BARI Sarisha 11 displays the best response out of all three kinds in terms of
shoot regeneration and the quantity of shoots per explant. Days required for induction of shoots
in BARI Sarisha-11 were also noted to be at their lowest. The best root induction was achieved
in BARI Sarisha 11 and BARI Sarisha 16 on hormone-free MS medium. The in vitro regenerated
plantlets were then successfully transplanted into soil. A few interesting in vitro flowers were
produced from plantlets that had been in vitro-regenerated on regeneration media with hormone-
free MS.
Brassica juncea is a crucial mustard species that is produced for edible oil in India, according to
C. Shyam et al. Using mature cotyledons and seeds as explants, the current study established an
effective and reliable plant regeneration convention from callus, and from embryogenic friable
calli, embryogenic cell suspension cultures were generated. The best callus induction was
achieved with MS basal media supplemented with 3.0 mgl-1, 2, 4-D. A mixture of 0.5 mg/l BAP
and 0.5–1.0 mg/l 2,4-D is added to promote organogenesis and embryogenesis and further
regeneration. It has been demonstrated that ripe seeds are superior as explants, indicating that
this explant has a larger potential for morphogenesis. However, plant growth regulator genotype,
type, relative concentrations, and mixes all had a major impact on the success of plantlet
regeneration. Future generations of Indian mustard could be selected using these techniques as
the optimum genotype for genetic modification. C. Shyam1 et al. According to Gerszberg et al.,
the ability of two explant types (cotyledons and hypocotyls) to regenerate was examined in vitro.
They were taken along with eight capitata varieties of Brassica oleracea. Both explants were
subcultured onto different callus induction media based on MS basal medium that was enhanced
with different combinations and concentrations of plant growth regulators at 10-day-old
seedlings. When examined for all cultivars, hypocotyl explants were found to be more valuable
for callus induction and organogenesis than cotyledons. The cultivar cv. "Amager" was
significantly more responsive during regeneration than the other cultivars seen and produced the
most shoots or buds per explant. The highest productive media for shoot regeneration, out of the
five types of media examined, was MS + 8.88 M 6-benzyloaminopurine (BAP) + 0.53 M -
naphthylacetic acid (NAA). All rooting media achieved rooted in 10 to 15 days, however MS
medium containing 5.37 M NAA produced the most robust and healthy roots. The majority of
plantlets (95%) successfully established themselves in the greenhouse, and the regenerated plants
showed no phenotypic alterations. Many different cabbage cultivars may be able to use this plant
regeneration convention.
According to Farooq et al., in vitro regeneration is crucial for creating transgenic plants using
genetic engineering techniques that rely on tissue culture. The identification of a set of
regeneration conditions for a single genotype of the species Brassica involved the recognition of
very large variances among different genotypes, which is likely to be useful in transformation
studies.
In this study, we used petioles, cotyledons, hypocotyls, and roots as four different explants to
explore the morphogenesis potential of one model, Westar, and four commercial cultivars (Faisal
canola, Punjab canola, Aari canola, and Nifa Gold). Three distinct protocols, BRP-I, -II, and -III,
were used to regenerate brassica. Although the regeneration respondent in cases of average
shoots per explant was observed to be 0.76-10.9, 0.2-3.2, 0-3.4, and 0-2.7 from the explants, the
regeneration efficiency was from cotyledons, petioles, hypocotyls, and roots, in the range of 6-73
percent, 4-79.3 percent, 0-50.6 percent, and 0-42.6 percent, respectively. When the commercial
cultivars were evaluated, almost all kinds showed lower regeneration than Westar, with the
exception of Aari canola. On BRP-I from cotyledon, Aari canola regeneration frequency was up
to 7.5 times higher than Westar, and it incited up to 21.9 times more shoots per explant.
According to our studies, the explant has a significant impact on the regeneration response, from
24 and 92 percent. While the regeneration circumstances provided had little to no impact on the
growth of commercial cultivars, they had twice as much of an impact on Westar. In addition to
choosing the best explant type and regeneration circumstances, we also determined the lowest
kanamycin concentration levels required for these cultivars to specifically inhibit the
proliferation of untransformed cells. The regenerated shoots of Aari canola may be successfully
grown to maturity in 16–18 weeks with normal seed yields reached and no altered phenotypic
seen. Because of this, the commercial variety Aari canola may be a promising target for future
genetic transformation investigations. In order to improve Brassica napus genetic resources,
Natalija et al. performed genetic transformation; however, an effective method of shoot
regeneration in vitro is required. In this study, shoot organogenesis from explants was used to
examine the regeneration capacity of 10 commercially important winter rapeseed cultivars
(hypocotyls and stem segments). Acc. As a result, the genotype-based pre-owned shoot
regeneration intensity. The cultivars Insider, Siska, and Kazimir H achieved the highest overall
regeneration frequency from stem segments, whereas hypocotyl explants of the cultivar Valesca
gave the highest overall capacity to induce shoots from all genotypes studied. Shoot regeneration
effectiveness may be related to the type of explant used; for example, regeneration frequencies
from stem segments were higher than those from hypocotyls by a maximum of 25.78 to 85.11
percent (Casino to Libea). Exogenous growth regulators were able to induce shoot growth from
both hypocotyls and stem segments, although their effects varied according on the genotype.
Growth hormone formulation must be tailored to each kind and genotype of Brassica napus L.
explants as a result. (Natalij et.al.). In order to regenerate the Brassica oleraceae L. variety
Capitata using a plant tissue culture technique, Daud et al. reported that tests were carried out to
establish the ideal culture conditions and to identify the higher responder explants. Growth-
regulating hormones were used to modulate the organogenesis as the explants were being
created. We employed the plant growth regulators benzyl amino purine (BAP) and naphthalane
acetic acid (NAA). Plant growth regulators were included into the MS medium in a number of
different combinations and concentrations. After two weeks of subculturing, the callus-forming
process began. The new plantlets emerged quickly when stem and root segments were cultivated
on MS medium with 1.0 mg/1 BAP and 0.5 mg/1 NAA. The explants of the petiole and leaves
showed that for the majority of the concentration, the callus growth was slower. The properly
rooted in vitro-awakened plantlets were successfully transplanted to soil, and their survival
percentage in the wild was 90%. Red cabbage (Brassica oleracea) extract's ability to protect cells
from oxidative stress was demonstrated by Kataya and Hamza. Diabetes was induced by
streptozotocin in male Wistar rats. 60 mg/kg of body weight of streptozotocin was administered.
Throughout the 60-day experiment, the diabetic rats developed a range of symptoms, including
weight loss, hyperglycemia, polyuria, polydipsia, enlargement of the kidneys, and renal failure. It
has been demonstrated that diabetic kidneys have a substantial rise in malondialdehyde, a marker
of lipid peroxidation. This was accompanied by a significant decrease in catalase activity, a
significant increase in reduced glutathione, and a significant increase in superoxide dismutase
activity. To reverse the deleterious consequences of diabetes, rats received daily oral dosages of
B. oleracea extract (1 g/kg body weight) for 60 days. A B. oleracea extract encouraged weight
loss, decreased blood sugar levels, and enhanced kidney function. B. oleracea extract also
reduced the detrimental effects of diabetes on malondialdehyde, glutathione, superoxide
dismutase, catalase activity, and the kidney's overall antioxidant capacity. As a result, the
antioxidant and antihyperglycemic properties of B. oleracea extract may offer a therapeutic
source for the treatment of diabetes. According to BK Parshad et al., effective methods for
regenerating whole plants are required for gene transfer via genetic engineering. This work was
carried out in order to develop a suitable method for plantlet regeneration from diverse explants
(namely cotyledon and plumule explants obtained from 5 day old seedlings) of five different
Indian mustard cultivars. These explants were cultivated on MS medium with supplementation
of 1.0 mg/l BAP and 0.2 mg/l IAA. Compared to other genotypes, Varuna demonstrated shoot
regeneration 85% of the time, whereas RH 30 demonstrated more shoot regenerations per
explant but with lower shoot regeneration rates than Varuna. The cotyledon and plumule from
Varuna had the highest frequency (75 and 95 percent, respectively) of shoot regeneration,
although both of the explants from RH 30 produced the most shoots per explant. It is clear that
the potential for shoot regeneration frequency and number per explant varies between genotypes
and explants.

Diabetes mellitus is one of the most common non-communicable diseases in the world. Instead
of being a single disease, it is a group of metabolic abnormalities that affect a significant section
of the global population. Despite the possibility of side effects, biguanides and sulphonylureas,
two oral hypoglycemic medications, continue to be crucial in the treatment of diabetes mellitus.
The search for alternative medicines that might have fewer or no adverse effects is growing
among people. Many different traditional medical systems all over the world have employed a
variety of medicinal plants to manage diabetes, including Allium cepa, Anacardium occidentale,
Andrographic paniculata, Momordica charantia, Azadirachtha indica, Brassica oleracea,
Cinnamomum tamala, and Withania sominifera. This review includes information on the
scientific name, plant family, and plant parts used to treat diabetes. Whether red cabbage leaf
powder or methanolic extract with or without chromium had synergistic effects on obese adults
was the subject of a study. Certain liver markers and enzymes are evaluated along with the rats'
serum lipid profiles, antioxidant levels, and dietary indications. A positive control group (basal
diet), a group with 15% cabbage powder in the diet, a group with 4.5 g/kg body weight/d
chromium, a group with 20% cabbage extract in the diet, and a group with 15% cabbage powder
+ chromium were all randomly assigned to groups of obese rats after feeding them high-fat diets
for six weeks to cause obesity (15 percent of the diet).(Pradeep et.al.). Following the
commencement of obesity, the experiment ran for 60 days. The obtained results demonstrated
that administration of chromium-fortified cabbage powder or cabbage extract may reduce body
weight gain (BWG), raise feed efficiency ratio (FER), and decrease feed intake when compared
to a positive obese control group (FI). The liver function enzymes were more noticeable in the
chromium-infused cabbage powder and cabbage extract groups as compared to the positive
control group. Both the serum and liver lipid profiles improved in all treatment groups. It is
recommended to offer red cabbage leaves or its extract to obese patients along with chromium.
(Nawal. A. Tahoon). The effects of Brassica oleracea L. var. italica Plenck flower extracts on
type 2 diabetes mellitus and associated diseases were examined in the study by Gupta et al.
Three different oral doses of each extract—petroleum ether, ethanol, and aqueous—were
administered for a total of 42 days. Histological examinations, behavioural research, and
biochemical indicators were all measured at various intervals. Up to 2,000 mg/kg, it was
discovered that there were no deaths. The groups receiving 400 mg/kg of petroleum ether,
aqueous, or ethanol extracts had a statistically significant (P0.001) improvement in serum
glucose level as compared to the negative control group. While aqueous and ethanol extracts
improved lipid profiles, aqueous extracts decreased insulin levels. Transfer delay decreased as a
result of each of the three extract treatments. Ethanol extract therapy (400 mg/kg) showed the
best percentage of inhibition in a lipid peroxidation testing. In addition, the aqueous and ethanol
extract treatments dramatically reduced the levels of tumour necrosis factor, interleukin-6, and
glycosylated haemoglobin. Histological research revealed that high doses of extracts lessened the
damage that type 2 diabetes mellitus caused to numerous bones and organs. According to the
results of this investigation, B. oleracea has the capacity to treat type 2 diabetes mellitus. Sumeet
Gupta and others. One of several is the important oilseed crop Brassica juncea (Czern & Coss.,
L.). Because B. juncea variety RAYA ANMOL is afflicted by a number of bacterial and fungi
diseases, we developed a simple and straightforward method for the regeneration and
transformation of this plant in order to produce transgenic plants that impart resistance against
several fungal diseases. The transformation was carried out using the Agrobacterium with the
chitinase gene. This gene was isolated from Streptomyces griseus HUT6037. We used
cotyledons and hypocotyls, two different kinds of explants, for the transformation. Only
hypocotyl explants demonstrated effective callus initiation. Different hormone dosages were
employed, including NAA 0.1, 0.2, and 0.3 mgL-1 and BAP 2, 4, and 6 mgL-1. However, great
transformation efficiency was observed by including the medium with a 2 mgL-1 BAP and 0.2
mgL-1 for callus initiation. Similar to this, the selection of modified calluses responded
favourably to both 200 mgL-1 of cefotaxime and 10 mgL-1 of kanamycin. Chitinase gene-
specific primers were employed in the polymerase chain reaction to see if a transgenic callus was
present. (2015) (Bashir Ahmad et al.).

According to E. Bhavya, chronic hyperglycemia caused by insulin production, insulin action, or

both is a hallmark of the group of metabolic illnesses known as diabetes mellitus. beta cells of
the pancreas produce insulin. Insufficient insulin causes blood glucose levels to rise, which
affects the metabolism of proteins, lipids, and carbohydrates. As things deteriorate, tissue or
vascular damage occurs, which causes major diabetes consequences such retinopathy,
neuropathy, nephropathy, cardiovascular problems, and ulceration. In the absence of any
effective prevention and control measures, the prevalence of diabetes will keep rising on a global
scale. Diabetes can be treated medically with insulin and oral hypoglycemic medications, or non-
medically with diet and exercise. Since insulin therapy is more effective at treating DM, it is
typically used in emergency hyperglycemic situations. Furthermore, insulin's role in glucose
homeostasis is frequently linked to both its physiological role and clinical importance. Several
insulin variants have proven successful in the treatment of DM. Insulin pen devices offer a
variety of benefits over the standard insulin bottle and syringe procedure. Insulin pumps are a
viable alternative to traditional insulin injections for the treatment of persons with type 1 diabetes
mellitus. It is therefore efficient and secure for the treatment of diabetic mellitus (DM). Kamboj
et al. developed a highly efficient and reproducible plant regeneration and transformation
technique using seedling explants in the Brassica juncea genotypes RH-406 and RH-555. Five-
day-old in vitro seedlings were used to explant the hypocotyl and cotyledon, which were then
cultured on MS medium with different concentrations and combinations of growth regulators. In
cotyledon explants cultivated on MS media enriched with 1 and 2.5 mg/L BAP, genotypes RH-
555 (60.34.19) and RH-406 (64.91.42), respectively, showed higher percent shoot development.
When one of the eight rooting media was used to plant roots in regenerated shoots, the MS
medium containing 0.2 mg/L NAA had the best rooting response. 81.8 percent of the regenerated
plants in RH-406 and 67 percent in RH-555 survived when planted in pots on a 1:1 mixture of
sand and soil. A transformation procedure was developed using the GUS reporter gene,
hypocotyl and cotyledon explants, and genotype RH-406 as a model. A histochemical GUS assay
revealed transitory GUS expressions of 75% and 80% in cotyledon and hypocotyl explants,
respectively. Kamboj et al. In order to create transgenic lines that are resistant to the herbicide
phosphinothricin, genetic modification was performed on the important oilseed crop Brassica
juncea, which is grown on more than 6 million hectares of land in North India (PPT). Hypocotyl
explants generated from seedlings were transformed using Agrobacterium tumefaciens strain
GV3101 The bar gene included in the created constructions encodes the enzyme
phosphinothricin-acetyl-transferase (PAT), which acetylates phosphinothricin (PPT). The
expression of the bar gene was controlled by the 35Sdebar CaMV35S promoter, the
35SAMVLbar CaMV35S promoter, or the 35Sbar CaMV35S promoter with or without the
leader sequence from RNA4 of the alfalfa mosaic virus. Plant viral leader sequences have been
shown to behave as translational enhancers. In vitro selections for transformed plants were made
on a medium containing PPT. Transgenic shoots were found with a frequency of 23% with the
35Sdebar gene construct and a frequency of 16% with the 35SAMVLbar gene construct. The
35Sbar design had very low transformation frequencies. Individual transgenics with the
35Sdebar and 35SAMVLbar constructs were utilised to determine the copy number on the right
and left border flanks of T-DNA using Southern hybridization. For single copy transgenic lines,
the bar gene's transcript levels were further examined by Northern blotting, and the protein levels
were examined using PAT tests. The transgenics with the 35Sdebar design displayed the widest
expression level variation. Single copy transgenics were selfed in order to produce homozygous
lines that could be used to study herbicide PPT resistance at the field level and to link this
protection with expression levels established through molecular analysis. It might be able to test
the sustainability of low-till or no-till farming in the rain-fed areas where mustard is frequently
produced by utilising herbicide-tolerant lines. (Smriti mehra et. Al.)
The BAR gene was used by Mehra et al. in 2000 to produce transgenic mustard plants in India
that were resistant to the herbicide phosphinothricin (PPT). The PAT (phosphinothricin acetyl
transferase) enzyme, which is encoded by the BAR gene, inactivates phosphonothricin. In order
to produce transgenic Indian mustard that is resistant to the herbicide 2,4-D, Bisht et al. (2004a,
b) used a gene called TFDA that codes for the enzyme 2,4-monooxygenase. The TFDA gene was
cloned under a 35S promoter with RNA4 leader sequence for enhanced expression. Transformed
shoots thrived on the selective medium containing 2.5 mg/l 2,4-D, but wild type shoots perished
even at a dosage of 0.5 mg/l. Transgenic mustard plants could withstand the chemical up to a
concentration of 500 mg/l, while non-transgenic mustard plants died at a concentration of 50
mg/l. These transgenic plants can be utilised to manage weeds, particularly Orobanche
aegyptiaca, with the help of the pesticide 2,4-D. A double-mutant acetolactate synthase gene
(ALSdm) was employed by Ray et al. (2004) to select changed tissues and cells for the genetic
transformation of B. juncea. A mutant version of the ALS gene was used as a selection agent
during the genetic transformation of B. juncea cvs Varuna and RLM198, conferring resistance to
sulfonuylureas (such as chlorosulfuron) and imidazolinones (such as imazethapyr).
Imidazolinones were shown to hold more promise as selective agents than sulfonylureas based
on the comparative transformation frequency identified in both scenarios. To create hybrid seeds,
Indian mustard Varuna and EHII transgenic lines with the barnase and barstar genes,
respectively, were created (Ray et al. 2007). They employed a mutant ALS gene as a selection
marker that exhibits resistance to the imidazolinone-based herbicide "Pursuit." Imidazolinone has
been utilised as a selection marker in the in vitro development of transgenic plants and the
production of hybrid seeds.
CHAPTER 3 Materials and Method

Plant Material

The healthy seeds of Brassica juncea (Indian mustard), variety RH 30, PUSA JAGANNATH,
PUSA MEHAK and PUSA MUSTARD 31 were obtained from two Agriculture Universities,
Haryana.

The RH 30 variety was taken from Haryana Agriculture University, Hisar. The other three
varieties were taken from Indian Agriculture Research University, Pusa, New Delhi. All these
varieties were used as the experimental materials for present investigation.

Optimization of regeneration system

Medium Preparation and Seed Sterilization

The healthiest and best seeds were taken out of the packet. 70% alcohol was used to rinse
seeds for up to one minute. Now sterilized with mercuric chloride (HgCl 2 0.1%) for 5 minutes.
They were rinsed with autoclaved distilled water upto 4-5 times.All of these steps were
processed in a good laminar air flow cabinet. Now putting all these seeds onto the autoclaved
filter whatman,s filter paper for soaking up remaining water of seeds. For the germination of
seeds, poured with the MS medium (Murashige and Skoog ,1962). The seeds were inoculated at
a density of about 20-30 seeds per petri plate. Closed petriplates with clingfilm and transferred
all of them into the culture room for the 2 days dark period and 3-4 days light period.

By mixing the ready-to-use MS medium powder with distilled water, MS (Murashige and
Skoog,1962) medium was created, containing MS major and minor salts as well as MS vitamins.
After thoroughly dissolving, 0.8 percent agar was added as a solidifying agent and 3 percent
sucrose was added as a carbon source. With the help of 1N NaOH and 1N HCl, the pH was now
brought down to 5.8. The MS medium was autoclaved for 20 minutes at a temperature of 1210 C
and a pressure of 15 psi. After this cooling the temp. of medium and autoclaved petriplates
poured with MS basal medium. Now the sterilized seeds were slightly embedded in MS medium
with the help of foreceps (sterilized) in laminar air flow cabinet. After 2 days, germination of
seeds got started in 16 hours light period and 8 hours dark period were followed in culture room.

Preparation of explants

After the foreceps and blade holder were red hot, the 4-5 days old seedlings like cotyledons,
hypocotyls were excised a no. of explants were cultured horizontally slightly embedded in MS
medium poured petriplates. In a number of petriplates covered in cling film and cultured on MS
media, both hypocotyls and cotyledons were grown under 16 hours of photoperiod at 250 C.

In vitro shoot regeneration of Brassica juncea

From seedlings that were 4 days old, the hypocotyls and cotyledonary leaves were removed and
divided into minute pieces in a laminar flow cabinet. These pieces were placed on MS media
supplemented with Benzylaminopurine in quantities of 0.5 mg/L, 1 mg/L, and 1.5 mg/L. Then,
the induced shoots and calluses were moved to the shoot regeneration medium (SIM), which
contained MS media supplemented with 1mg/L conc. of NAA. Shoot regeneration was obtained
from both, cotyledonary leaves and hypocotyls. For this purpose green and healthy portion of
seedlings was taken and slit into 0.5-1.0cm pieces and inoculated on MS medium with several
concentrations of hormones individually. Each experiment was conducted thrice by raising 10-15
calli for each analysis. To analyse shoot regeneration at various hormone concentrations, all the
petriplates were housed in a culture room at a constant temperature of 270°C, a 16-hour
photoperiod, and 60 percent relative humidity. Observations were taken after every three days
and effect of various conc. for shoot regeneration was calculated on the basis of percentage of
callus response. Root regeneration was carried out for the shoots that were raised from explants
of hypocotyls shoots and cotyledonary leaves. For this purpose healthy and green shoots were
chosen and were placed on root inducing medium (RIM) having concentration of hormone NAA
individually under the laminar air flow cabinet. Each experiment was conducted thrice by raising
1-5 shoots for each analysis. After the root induction and elongation they were transferred into
the pot .Then the pots were covered with polybags and were put in the culture room. (fig:-3.1)
Figure 3.1:- Protocol for In vitro shoot regeneration of Brassica juncea

Agrobacterium mediated gene transformation and regeneration of Brassica juncea

Inoculation of Agrobacterium tumefaciens and co-cultivation of explants
Agrobacterium strain EHA105 carrying a binary vector for gene transformation. In the study
plant expression vector used having pro- insulin gene under Glycine promoter and is encoded by
bar gene found in the produced constructs with a basta selection marker. Upcoming transgenic
lines resistant to the herbicide phosphinothricin(PPT). The enzyme phosphinothricin- acetyl-
transferase (PAT), which acetylates phosphinothricin. Through this selective agent we can easily
recognize transgenic plants visually.
 614 bp
Right NOS -Pro BAR NOS-Ter Gly-pro Pro-insulin Gly-Ter Left
order border
EcoR I Xho I

Figure 3.2:- T- DNA of the pBinGlyBar1vector containing bar gene and pro-insulin gene
under Glycine promoter.

Before experiment, bacteria was placed on solid YEP medium that contains 50 mg/l kanamycin
and 50 mg/l rifampicin having plant expression vector checked by PCR.
After confirmation, bacterial cell grown in YEP broth medium having 50 mg/l kanamycin and 50
mg/l rifamycin. They were shaken at 280 C overnight before the bacteria were centrifuged at
5000 rpm for five minutes at 250 C the following day. They were pelleted and then re-suspended
in MS liquid medium with 200 µM acetosyringone to create the resuspension medium. Then, the
separated, excised cotyledon leaves of 4-5 day-old seedlings were placed in an orbital shaker for
20 minutes while being continuously shaken at 90 rpm. The cotyledon explants were then dried
for 2–3 minutes using autoclaved Whatman filter paper. .The explants were transferred to the co-
cultivation medium MS medium supplemented with BAP (1.0mg/l) for 2 days in culture room.
The explants were washed 7-8 times with autoclaved distilled water after two days, with the last
wash containing 250 mg/l of cefotaxime autoclaved distilled water. The material has been
transferred to a callus induction medium containing MS+ 1.0 mg/l BAP+ 250 mg/l Cefotaxime
after being dried on sterile Whatman's filter paper. With 4-day-old seedlings, the aforementioned
technique was carried out, and data was gathered from repeating the procedure. Use of a control
set was made. To demonstrate that the shoots that appeared on infected explants were the result
of bacterial activity, the explants in the control group were not injected with bacteria. For shot
elongation, the shoots were moved to shoot induction media after two weeks. The SIM included
MS medium that had 1mg/l NAA added to it.(fig:-3.3)
Fig.3.3:- Agrobacterium tumefaciens mediated transformation method of Brassica juncea,
variety PUSA JAGANNATH
CHAPTER – 4 RESULTS AND DISCUSSION

4.1 Effect of type of explants excised from 4 day old seedlings in Brassica juncea var. PUSA
JAGANNATH and RH 30
Brassica in vitro-raised seedlings had hypocotyls and cotyledons, two separate types of explants,
removed. Both explants came from the Brassica juncea cultivars PUSA JAGANNATH and
RH30. From both varieties the hypocotyls formed callus and cotyledons explants formed shoots.
We observed that the best regeneration frequency in minimum days of Brassica juncea variety
was PUSA JAGANNATH. The explants were taken from 4 day old seedlings. The data were
collected after two weeks of culturing. Furthermore, we were used the cotyledon explants for
other experiments. The comparison of both explants (cotyledon and hypocotyls ) of two
varieties are seen in table 4.1 .

Table 4.1 :- Effect of type of explants excised from 4 day old seedlings in Brassica juncea
variety, PUS JAGANNATH and RH 30
Sr. Variety Type of No. of Regeneration Regeneration
No.
Explant Explants (Shoots/Callus) Frequency
co- (%)
cultivated
1. PUSA Hypocotyl 40 Callus 60
JAGANNATH Cotyledon 40 Shoots 100
2. RH 30 Hypocotyl 40 Callus 40
Cotyledon 40 Shoots 80

 Medium used Murashige and Skoog Medium (MS salts+MS vitamins+ 1mg/l BAP)
 Data were taken after 15 days
Figure 4.1:- Effect of explants cotyledon and hypocotyls in Brassica juncea , a)
Cotyledonary explants b) and c) Shoot regeneration through cotyledonary explants of
PUSA JAGANNATH and RH 30 respectively d) Hypocotyl explants e) and f) Callus
formation through hypocotyls of RH 30 and PUSA JAGANNATH respectively.
4.2 Effects of different concentrations of BAP on cotyledon explants of Brassica juncea
variety, PUSA JAGANNATH and RH 30
Brassica juncea var. PUSA JAGANNATH and RH30 cotyledon explants were used to optimise
the effects of various BAP concentrations. On different BAP concentrations, we compared the
regeneration frequency of the two types. Cotyledon explants were removed from seedlings that
were 4 days old and placed on MS medium with several concentrations of BAP probable,
including 0.5 mg/l, 1.0 mg/l, and 1.5 mg/l. Explants were taken for one control and placed on
MS basal media. After two weeks, the observations were recorded. We found that both kinds of
BAP at a concentration of 1 mg/l had the best regeneration frequency. However, when we
contrasted the two types on MS medium with conc. 1 mg/l BAP, . PUSA JAGANNATH showed
the highest rate of regeneration. The maximum average no. of shoots was arised in. PUSA
JAGANNATH, the observed data is shown in below table 4.2. By knowing the readings of
average no. of shoots regenerated, we calculated the SD±SE.

Table 4.2:- Effects of different conc. of BAP on two varieties of Brassica juncea varieties .
PUSA JAGANNATH and RH 30
Sr. Variety BAP No. of No. of Regenerati Avera Standar
No concentrati explant explants on ge no. d
. on s co- regenerat Frequency of deviation
cultiva ed (%) shoots ±
te per Standar
explan d
t Error(S
D ±SE)
1. PUSA 0 40 00 00 00 00
JAGANNAT 0.5 40 32 80 3.56 1.68±0.3
H 1.0 40 40 100 8.75 2.02±0.3
1
1.5 40 37 92.5 4.81 2.36±0.3
9
2. RH 30 0 40 00 00 00 00
0.5 40 34 85 7.58 2.14±0.3
7
1.0 40 36 90 8.30 2.96±0.4
9
1.5 40 32 80 4.21 2.34±0.4
1

 Medium used Murashige and Skoog Medium (MS salts+ MS vitamins+ diff. conc. of
BAP)
 Data were collected after 15 days
 120
Regeneration Frequency (%) Pusa Jagannath RH 30
100

80

60

40

20

0
0 mg/l 0.5 mg/l 1.0 mg/l 1.5 mg/l
BAP concentration (mg/l)

a)
Average no. of shoots regenerated

18
16 RH30
14 Pusa jagannath
12
10
8
6
4
2
0
0mg/l 0.5mg/l 1.0mg/l 1.5mg/l
BAP Concentration (mg/l)

b)
Figure a) Regeneration Frequency of Brassica juncea varieties PUSA JAGANNATH and
RH30 on various BAP hormone concentrations.

b) Average no. of shoot regeneration per explant of Brassica juncea varities PUSA
JAGANNATH and RH30 on various hormone concentrations.
Figure 4.2.1:- Effects of various concentrations of BAP on shoot regeneration of
Brassica juncea variety RH30
a) MS basal medium as a control
b) 0.5 mg/l concentration of BAP
c) 1.0 mg/l concentration of BAP
d) 1.5 mg/l concentration of BAP
Figure 4.2.2:- Effects of various concentrations of BAP on shoot regeneration of
Brassica juncea variety PUSA JAGANNATH
a) MS basal medium as a control
b) 0.5 mg/l concentration of BAP
c) mg/l concentration of BAP
d) 1.5 mg/l concentration of BAP
4.3 Effects of different cytokinins on cotyledonary explants of Brassica juncea variety,
PUSA JAGANNATH and RH 30

The effects of various cytokinins were observed in two varieties of Brassica juncea which are
PUSA JAGANNATH and RH 30. The cotyledons of both varieties were taken from 4 days old
seedlings. They were excised and put on the MS medium containing different cytokinins
concentration (BAP, Zeatin, TDz and Kinetin). The results were observed after 15 days. We
were seen the best shoot regeneration frequency in PUSA JAGANNATH on BAP cytokinin and
in RH 30 the maximum shoot regeneration frequency on Zeatin cytokinin, 100% and 75.55%
respectively. One control was also used in which medium was MS basal medium. From the
average no. of shoot regeneration frequency, the SD± SE also calculated which can be seen in
table 4.3. The maximum average no. of shoots were regenerated in PUSA JAGANNATH on
BAP and in RH 30 the max. average no. of shoot regeneration on Zeatin.

Table 4.3- Effects of different cytokinins on two varities of Brassica juncea from 4 days old
excised seedlings

Sr. Varieties Cytokinins No. of No. of Regeneration Average Standard

no. explants explants efficiency(%) no. of deviation±
co- regenerated shoots Standard
cultivated per Error(SD
explants ±SE)
1. PUSA 0 45 00 00 00 00
JAGANNATH BAP 45 45 100 8.71 3.66±0.54
Zeatin 45 42 93.33 6.14 2.224±0.35
TDz 45 39 86.66 6.38 2.04±0.33
Kinetin 45 30 66.66 6.41 2.45±0.44
2. RH 30 0 45 00 00 00 00
BAP 45 25 55.55 7.00 3.58±0.7
Zeatin 45 34 75.55 6.17 2.34±0.40
TDz 45 31 68.88 5.48 2.35±0.42
Kinetin 45 29 64.44 5.94 2.25±0.41
 Medium was used Murashige and Skoog (MS salts+ MS vitamins+ cytokinin)
 Data were collected after two weeks
 Each experiment were repeated thrice
 Pusa jagannath RH 30
180
160

Regeneration frequency ()%

140
120
100
80
60
40
20
0
0 BAP Zeatin TDz Kinetin
a) Cytokinins (mg/l)

18 Pusa jagannath RH 30
Average no. of shoot regener-

16
14
12
10
ation

8
6
4
2
0
0 BAP Zeatin TDz Kinetin

b) Cytokinins(mg/l)

Figure a) Regeneration Frequency of Brassica juncea varieties PUSA JAGANNATH and

RH30 on different cytokinins

b) Average no. of shoot regeneration per explant of Brassica juncea varities PUSA
JAGANNATH and RH30 on different cytokinins
Figure 4.3.1 Effects of different cytokinins on Brassica juncea variety PUSA JAGANNATH

a) MS basal medium as a control

b) TDz
c) Kinetin
d) BAP
e) Zeatin
Figure 4..3.2:- Effects of different cytokinins on Brassica juncea variety RH30

a) MS basal medium as a control

b) BAP
c) TDz
d) Zeatin
e) Kinetin
4.4 Effect of different varieties of Brassica juncea seedlings on different days

The effects were optimized of three varieties of Brassica juncea which were PUSA
JAGANNATH, PUSA MEHAK and PUSA MUSTARD 31. The observations were taken of
different seedlings on different days and were taken regularly. The germination and length of
seeds were observed. The maximum germination of seeds were observed in PUSA
JAGANNATH. The seeds were immersed on the MS basal medium. The readings were noted
after 3 days. All the readings can be seen in the table 4.4.

Table 4.4:- Effects of different varieties of Brassica juncea on different days

Sr. Varieties Tota 4 days old 5 days old 6 days old

no l
Germinatio Length(cm Germinatio Length(cm Germinatio Length(cm
. seeds
n ) n ) n )

1. PUSA 30 25 3.1 27 5.3 29 7

JAGANNAT
H
2. PUSA 30 13 4 15 6 17 8
MEHAK
3. PUSA 30 15 5.2 17 6.1 22 8.5
MUSTARD 31
Figure 4.4.1:- Effects of age on length of Seedlings of PUSA JAGANNATH are shown .
Figure 4.4.2 Effects of age on length of Seedlings of PUSA MEHAK are shown.
Figure 4.4.3 :- Effects of age on length of Seedlings of PUSA MUSTARD 31 are shown
In vitro shoot regeneration of Brassica juncea

The seedlings were excised after 4 days and immersed on the MS medium containing 1mg/l BAP
for shoot regeneration. After 15 days, the shoots arised and they were subcultured in MS medium
containing 1mg/l NAA for shoot elongation. When the shoots were elongates till 5-6 cm, they
were transferred on rooting media for root regeneration. The rooting was observed after some
days. When the explants were elongates till 15-20 cm with roots, they were transferred to pots in
culture room. After 2 months, they were completely ready to transfer in the field.
Figure:- Well established plant of Brassica juncea with floral twigs
Figure:- In vitro shoot regeneration of Brassica juncea :-

a) 4 day old Seedlings of Brassica juncea on MS basal medium

b) Cotyledon explants on MS medium with BAP conc.(1mg/l)
c) Shoots induction
d) Elongated shoots (After subculturing)
e) Root induction on MS medium with NAA conc. (1mg/l)
f) Explant with roots
g) Plant in pot
h) Plant with floral twig
i) A picture of floral twig
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