DIKTAT

ADVANCED PLANT NUTRITION

“Genetic Assembling of Seeds of Storage Resistant Rice Plant Types (BS90)”

By:
Anggi M Marsusyi, S.P
A1L012156

AGRICULTURAL BIOTECHNOLOGY POST GRADUATE PROGRAM

UNITED NATIONS (UN)
UNIVERSITY OF NEW SOUTH WALES (UNSW)
SIDNEY, AUSTRALIA
2023

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 I. INTRODUCTION

A. Background
The Diktat on the Genetic Assembling of Seed Varieties of Storage-Resistant Rice
Plants (BS90) was prepared to fulfill the prerequisites for the assignment as a replacement
for the Advanced Plant Nutrition course, Postgraduate Program in Agricultural
Biotechnology. The preparation method refers to the Reference Research Techniques
(RRT). The citations are based on the writing conventions listed in the Culture of
Scientific Writing Techniques (CSWT) University of New South Wales, Sydney.
This Diktat contains explanations and information in accordance with the criteria
for the task of the Advanced Plant Nutrition course at the Masters level in the Department
of Agricultural Biotechnology, including the definition and classification of seeds,
handling of rice seeds, genetic assembly techniques for rice plant species (BS) resistant
to mutations, stable viability, and high genetic immunity.

B. Purpose
Presenting advanced plant nutrition coursework at the Masters level in the
Department of Agricultural Biotechnology, including definition and classification of
seeds, handling of rice seeds, genetic assembly techniques for rice plant type seeds (BS)
mutation resistant, stable viability, and high genetic immunity.

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 II. SEED DEFINITION AND CLASSIFICATION

According to Griffin et. al. (2002) seeds are plants or parts thereof that are used to
propagate and/or reproduce, both in the form of seeds and other plant materials such as
cuttings, grafts, joints, seedlings, cloves, rhizomes, and plantlets in micro propagation.
According to Hoftstein and Lunetta (2003) the classification of seeds in seed
certification includes Species Seeds, Basic Seeds, Principal Seeds, and Spread Seeds.
a. Breeders seed
According to Clarke et. al. (2012) breeder seed is seeds that are produced and
supervised by researchers and expert laboratory assistants at the national level and are a
source for basic seed propagation.

Figure 1. Breeders Seed Lable.

b. Foundation Seed
According to Alvarez and Risko (2007) foundation seeds are the first offspring of
type seeds which are produced under intensive guidance and strict supervision so that
high varietal purity can be maintained. Basic seeds are produced by agencies or bodies
determined by the Directorate of Seed Quality Development.

Figure 2. Foundation Seed Lable.

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 c. Stock Seeds
According to Lagowski (2002) stock seeds are descendants of basic seeds which
are produced and maintained in such a way that the identity and level of purity of the
variety meet the quality standards set and have been certified as stock seeds.

Figure 3. Stock Seed Lable.

d. Extension Seed
According to Novak and Gowin (1985) extension seed is the offspring of the main
seed which is produced or maintained in such a way that the identity and purity of the
variety meet the quality standards for dissemination.

Figure 4. Extension Seed Lable.

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 III. RICE SEEDS AND THEIR HANDLING

According to Griffin et. al. (2002) rice plants reproduce with seeds in the form of
seeds. The longest test of seed resistance in international seed captivity is only 60 (sixty)
days. Since 1923, genetic researchers have been trying to attach special fragments to each
variety made with the aim of extending the shelf life of rice seeds, especially for seed
types.
According to Frenkel and Wallen (2011) increasing the shelf life of rice plant
species up to 90 days (BS90) can be done by several methods with the aim of making
seed varieties that are resistant to mutations, stable viability, and have a high level of
genetic immunity during the shelf life.

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 IV. GENETIC ASSEMBLY TECHNIQUES OF RICE SEEDS
MUTATION RESISTANT

According to Alvarez and Risko (2007) mutations are changes that occur in genetic
material both at the gene and chromosomal levels. Gene-level mutations are called point
mutations, while mutations at the chromosomal level are called aberrations. Seeds of rice
plant species that have passed the shelf life of tolerance can cause mutations, thus
destroying the purity and potentially reducing the quality of the rice to be planted.
According to Robinson (2005) the resistance of rice plant species to mutation can
be carried out using the Cloning Expression Technique. This technique discusses a piece
of protein-coding DNA transplanted into a plasmid. Plasmids that contain pieces of DNA
are inserted into bacterial or viral cells. The insertion of DNA into the bacterial cell is
called transformation. The transformation is carried out by electrophoration and
microinjection methods, as follows:
a. Electrophoration method.
Electrophoration is a method of inserting DNA using an electric shock to enlarge
the pores of the cell membrane thereby increasing the permeability of the membrane. The
cell must be grown in the media until it reaches the middle of the log phase, then the
electrical signal induces enlargement of the membrane pores so that small molecules such
as DNA can enter. The electrophoration method has a higher success rate and efficiency
than other transformation methods but has a greater risk of bacterial cell death and is
relatively expensive (Silberman, 2002). The electrophoration kit used in the
electrophoration method is shown in Figure 5.

Figure 5. Electrophoration kit (Frenkel and Wallen, 2011).

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 b. Microinjection method.
According to Wendell et. al. (2007) microinjection is a DNA insertion technique
used in the forced insertion process (transgenesis). The microinjection technique,
developed from the production technique of transgenic organisms, is a technique
commonly used in gene introduction in plants. The gene to be introduced is injected into
the cell using a very small glass pipette (needle tip diameter of 0.05-0.15 mm). The
advantage of this method is the level of efficiency is higher compared to other methods.
The process of DNA transgenesis in plant cells is shown in Figure 6.

Figure 6. Microinjection method for plant DNA insertion (Carin, 1997).

After the insertion of DNA into the cell, the proteins encoded by the DNA are
expressed by the destination cell. Various techniques can be used to assist expression so
that the protein is obtained in large quantities, for example inducible promoters and
specific cell-signaling factors. The large amount of protein is then extracted in the
bacterial cells (Elisabeth et. al., 2012).

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 V. ASSEMBLING TECHNIQUES OF RICE SEED GENETICS
STABLE VIABILITY

According to Pavelich and Abraham (1977), viability is the vitality of the seed
which can show the process of seed growth. The viability parameter observed in this study
was seed germination. Viability test using tetrazolium solution. Viability tests are always
carried out by skilled researchers and laboratory assistants to determine the viability
stability of a seed.
According to Hoftstein and Lunetta (2003) the function and stability of rice plant
species is very important, because it is the first generation of seeds to produce seeds for
distribution. The shelf life of rice plant species is generally only 45 days. After passing
through the shelf life, there was a decrease in the viability of rice plant species from 98%
to 54%.
According to Daly et. al. (2004), the stability of seed viability of rice plant types
can be increased by polymerase chain reaction (PCR) techniques, so as to increase the
shelf life of these seeds.
According to Lumaret et. al. (1998) Polymerase chain reaction (PCR) is a suitable
technique for making DNA copies. PCR allows a specific number of DNA sequences to
be copied (up to millions of times) to be amplified so that it can be analyzed or modified.
PCR can be used to add restriction enzyme sites or to change certain bases in DNA. PCR
is also used to detect the presence of certain DNA sequences. So that it can increase the
stability of seed viability. The PCR Kit in the laboratory is shown in Figure 7.

Figure 7. PCR analysis kit for agricultural laboratory plants (Tasker, 1992).

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 According to Millar (2004) PCR is used to amplify DNA involving a series of
repeated temperature cycles and each cycle consists of three stages, namely:
a. Denaturation, the process of separating DNA double strands into two single
strands, the DNA template (DNA template) is separated at 94-96°C.
b. Annealing, the process of attaching or hybridizing between primers and single-
stranded DNA templates. The temperature is reduced until it reaches 45-60°C.
c. Extension or elongation, the elongation of the primer into a new DNA strand by
the DNA polymerase enzyme. One cycle of PCR will double the number of DNA
template molecules.

Figure 8. Results of running PCR in the replication process (Grupta, 2008).

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 VI. GENETIC ASSEMBLY TECHNIQUES OF RICE SEEDS
HIGH GENETIC IMMUNITY

According to May et. al. (2007) genetic immunity is the ability of a plant type seed
to maintain gene expression from changes that occur physically including light,
temperature, and humidity. Genetic immunity is only affected by physical properties, and
not by changes due to mutations. This is related to the germ cell which is the assembly
result of the DNA arrangement.
According to Clarke et. al. (2012) the shelf life of rice plant species of more than
45 days can cause physical damage to the seeds due to decreased genetic immunity of
these seeds. This damage is fatal damage because it damages (lysis) the tissue and
appearance of the seed, and it is certain that the seed does not have the ability to grow
even in suitable media or environment.
According to Lagowski (2002) genetic immunity of rice plant species seeds can be
increased by gel electrophoresis technique as follows:
a. Gel electrophoresis process.
According to Pavelich and Abraham (1977) gel electrophoresis is a technique in
molecular biology. The basic principle of gel electrophoresis is that DNA, RNA or
proteins can be separated by an electric field. The molecules are separated according to
the rate of movement by electromotive forces within the gel matrix. The rate of transfer
depends on the size of the molecule. Gel electrophoresis is performed for analytical
purposes and is used as a preparative technique to purify molecules. Electrophoresyl Gel
Kit in the laboratory is shown in Figure 9.

Figure 9. Laboratory kit gel electrophoresis (Mei et. al., 2007).

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 The gel used is a cross-linked polymer whose porosity can be adjusted as needed.
Polyacrylamide gels are used to separate small proteins or nucleic acids (DNA, RNA or
oligonucleotides). Polyacrylamide gels were prepared at different concentrations to
produce polyacrylamide networks with different cavity sizes (Afamasaga-Futa'i, 2009).
According to Kalinowski (2012) the electrophoretic process of molecular samples
is placed into wells on a gel placed in a buffer solution and electricity is applied to it. The
sample molecules will move in the gel matrix towards one of the electric poles according
to their charge. The direction of movement of nucleic acids towards the positive electrode
is caused by the natural negative charge on the sugar-phosphate framework it has. The
working principle of the gel electrophoresis technique is shown in Figure 10.

Figure 10. The working principle of gel electrophoresis (Kalinowski, 2012).

b. Post gel electrophoresis.
According to Benjamin et. al. (2009) after the electrophoresis process is complete,
a staining process is carried out so that the separated sample molecules can be seen. This
process uses Ethidium bromide, silver, or Coomassie blue dye. If the sample molecules
fluoresce in ultraviolet light after being stained then the gel is photographed under
ultraviolet light.
Bands on different lanes of the gel will appear after the staining process. The bands
equidistant from the gel wells at the end of electrophoresis, contain molecules moving at
the same speed, meaning the molecules are the same size. Markers or markers are
mixtures of molecules of different sizes that can be used to determine the size of the
molecules in the sample band. The marker strip band is compared with the sample band
to determine its size (Hansen and Lovedahl, 2004). The process of reading the gel
electrophoresis results is as shown in Figure 11.

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Figure 11. Readings of gel electrophoresis results (Chen et. al., 2011).

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