This document outlines the process of meristem and shoot tip culture in horticultural crops. It discusses (1) the establishment of explants in culture media, (2) the multiplication of propagules through axillary shoot proliferation using cytokinins, and (3) the regeneration of adventitious roots using auxins to complete the tissue culture process. Meristem and shoot tip culture is an effective method for cloning plant material and producing disease-free plants for agriculture and industry.
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
https://www.youtube.com/watch?v=IZwrkgADM3I
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
OVARY CULTURE:-
"the in-vitro culturing of ovaries in an aseptic condition from the pollinated or un-pollinated flowers, in an appropriate nutrient medium and under optimal conditions." And
OVULE CULTURE:-
"Ovule culture is an experimental system by which ovules are aseptically isolated from the ovary and are grown aseptically on chemically defined nutrient medium under controlled conditions."
Somaclonal Variation in Plant tissue culture - Variation in somaclones (somatic cells of plants)
Somaclonal variation # Basis of somaclonal variation # General feature of Somaclonal variations # Types and causes of somaclonal variation # Isolation procedure of somaclones via without in-vitro method and with in-vitro method with their limitations and advantages # Detection of isolated somaclonal variation # Application (with examples respectively related to crop improvement) # Advantages and disadvantages of somaclonal variations.
https://www.youtube.com/watch?v=IZwrkgADM3I
Also watch, Gametoclonal variation slides to understand, how to changes occur in gametoclones of plants.
https://www.slideshare.net/SharmasClasses/gametoclonal-variation
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
Meristem tip culture for the production of the virus free plantsArjun Rayamajhi
This presentation gives general idea on the meristem tip culture for the production of the virus free plants. The principles, methods and procedures of the meristem tip culture included. General idea on different in vitro culture techniques for virus elimination meristem tip culture viz. thermotherapy, cryotherapy,chemotherapy and electrotherapy are provided.
Micropropagation and commercial exploitation in horticulture cropsDheeraj Sharma
Micro-propagation – principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.
Micropropagation (tissue culture or invitro culture) refers to the multiplication of plants, in an aseptic condition and in artificial growth medium from plant parts like meristem tip, callus, embryos anthers, axillary buds etc. It is a method by which a true to type and disease free entire plant can be regenerated from a miniature piece of plant in aseptic condition in artificial growing medium rapidly throughout the year.
A presentation covering the process of protoplast culture including protoplast isolation, protoplast fusion, culture of protoplast, its application, factors affecting protoplast culture and the future of protoplasts.
Invitro culture of unpollinated ovaries and ovules represents an alternative for the production of haploid plant
First successful report on the induction of gynogenic haploid was in barley by San Noeum in 1976
Haploid plants are obtained from ovary and ovule culture of rice, wheat, maize, sunflower, tobacco, poplar, mulberry etc
Whites or MS or N6 inorganic salt medium supplement with growth substances are used
Organogenesis, in plant tissue cultureKAUSHAL SAHU
Introduction
Definition
Types of organogenesis
Organogenesis through callus formation (indirect organogenesis)
Growth regulators for indirect organogenesis
Organogenesis through adventitious organ (direct organogenesis)
Growth regulators for direct organogenesis
Factor affecting the soot bud differentiation
Organogenic differentiation
Application of organogenesis
Conclusion
References
A process where an embryo is derived from a single somatic cell or group of somatic cells. Somatic embryos (SEs) are formed from plant cells that are not normally involved in embryo formation.
Embryos formed by somatic embryogenesis are called Embryoids.
The process was discovered for the first time in Daucas carota L. (carrot) by Steward (1958), Reinert (1959).
Embryo culture is a laboratory method for producing plant lets from a fertilized or unfertilized embryo in invitro condition. there are several advantages are associated with the embryo culture like production of haploid plants, making distant crosses successful, sometimes aborted embryos can be rescued from a unsuccessful hybridization.
Meristem tip culture for the production of the virus free plantsArjun Rayamajhi
This presentation gives general idea on the meristem tip culture for the production of the virus free plants. The principles, methods and procedures of the meristem tip culture included. General idea on different in vitro culture techniques for virus elimination meristem tip culture viz. thermotherapy, cryotherapy,chemotherapy and electrotherapy are provided.
Micropropagation and commercial exploitation in horticulture cropsDheeraj Sharma
Micro-propagation – principles and concepts, commercial exploitation in horticultural crops. Techniques - in vitro clonal propagation, direct organogenesis, embryogenesis, micrografting, meristem culture. Hardening, packing and transport of micro-propagules.
Micropropagation (tissue culture or invitro culture) refers to the multiplication of plants, in an aseptic condition and in artificial growth medium from plant parts like meristem tip, callus, embryos anthers, axillary buds etc. It is a method by which a true to type and disease free entire plant can be regenerated from a miniature piece of plant in aseptic condition in artificial growing medium rapidly throughout the year.
Definition of hairy root culture ,multiple shoot culture ,Production of hairy root and multiple shoot , advantages an disadvantages of hairy root and multiple shoot culture, Sterilization and sterilizing agents wit concentration and exposure time
Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
Multiplication of Apical or Axillary bud, Shoot tip or meristem culture
Production of Disease free plants by Micropropagation techniques: their Advantages and Disadvantages
Explore meristem culture—a technique for virus-free plant propagation. Learn how apical meristems drive growth and enable cloning. Discover its applications in agriculture and research.”
INVITRO CULTURE: TECHNIQUES, APPLICATIOSNS & ACHIEVEMENTS.
INVITRO TECHNIQUES AND BIOTECHNOLOGY USE IN AGRICULTURE AND CROP IMPROVEMENT. APPLICATIONS OF VARIOUS BIOTECHNOLOGICAL TECHNIQUES AND METHODS. TISSUE CULTURE, MICROPROPAGATION, EMBRYO CULTURE, ANTHER CULTURE, POLLEN CULTURE, ENDOSPERM CULTURE, OVULE CULTURE, OVARY CULTURE, ETC.
Plant tissue culture has been widely employed in area of agriculture, horticulture, forestry and plant breeding. It is an applied biotechnology used for mass propagation, virus elimination, secondary metabolite production and in vitro cloning of plants. Recently, plant tissue culture has been used for the conservation of endangered plant species through short and medium term conservation also known as slow growth and cryopreservation also known as long term conservation. These methods had been effectively used to conserve plant species with recalcitrant seeds or dormant seeds and showed greater advantage over the conventional methods of conservation. At present plant cell culture has made great advances. Possibly the most significant role that plant cell culture has to play in the future will be in its association with transgenic plants. The ability to accelerate the conventional multiplication rate can be of great benefit to many crops countries where a disease or some climatic disaster wipes out crops. Mr. Rohan R. Vakhariya | Rutuja R. Shah "Over Review on Plant Tissue Culture" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29619.pdfPaper URL: https://www.ijtsrd.com/pharmacy/other/29619/over-review-on-plant-tissue-culture/mr-rohan-r-vakhariya
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
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Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Meristem and shoot tip culture in horticultural crops
1. 1. Media preparation
2. Explant selection
3. Establishment of explant in media
4. Callus development
5. Plantlet development
6. Hardening or acclimatization
7. Open field planting
Lecture 9: Meristem and shoot tip culture in horticultural crops
Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPs
2. Introduction to Tissue Culture
⚫Tissue Culture (also known as Micropropagation or In vitro culture) is:
⚫The growing of plant cells, tissues, organs, seeds or other plant parts in
a sterile environment on a nutrient medium.
4. 2) Explant:-
Totipotent somatic cell are used.
Immature inflorescence and Scutellar tissue of
immature seeds are used for the research. Ex:-
Triticum aestivum .
Epidermis, Procambial tissue are also produced
somatic embryo.
5. EXPLANT PREPARATION
EXPLANT : It is defined as a portion of plant body, which has been
taken from the plant to establish a culture
•Explant may be taken from any part of the plant like
root,stem,leaf,or meristematic tissue like cambium, floral parts like
anthers, stamens etc..
•Age of the explant.
• Homozygous plants are preferred.
5
13. STAGES OF MERISTEM
CULTURE
Murashige reported that there are three stages of culture:
Stage 1 is the culture establishment stage when explant may develop into
single shoot or multiple shoots.
At this stage explant are supplements with cytokinin like BA, kinetin and
2iP.
14. In stage 2 the objective is to multiply the propagule and for this axillary
shoot proliferation is followed as it maintains higher genetic stability.
In axillary shoot proliferation, high levels of cytokinin are utilized to
overcome the apical dominance.
15. The stage 3 purpose is regeneration of adventitious roots from the shoots
obtain in stage 2
Numerous studies have indicated that NAA is followed by IBA,IAA, 2,4-D
and other auxins are used for induction of root generation.
16. Components of medium
⚫ Inorganic nutrients (N2,P,Ca,Mg,S)
⚫ Carbon source (sugar)
⚫ Organic supplements including
Vitamins (Thiamine, nicotinic acid, panthonic acid, pyridoxine)
Amino acids (L-glutamine, L-asparagine, L-cysteine, L-glycine)
Complex organics (casein hydrolysate, coconut milk, yeast extract,
orange juice, tomato juice)
Plant growth hormones
Auxins (root)
Cytokinins (shoot)
Gibbrellins (internode elongation, meristem growth)
Abscissic acid (for culturing woody species)
Solidifying agent (agarose)
pH (optimum is 5.8) lower than 4.5 or higher than 7.5 greatly inhibit the
growth
17. PROTOC
OL
Remove the young twigs from a healthy plant. Cut the tip (1 cm)
portion of the twig
Surface sterilize the shoot apices by incubation in a sodium hypochlorite
solution (1% available chlorine) for 10 minutes. The explants are
thoroughly rinsed 4 times in distilled water
Transfer each explants to a sterilized petri dish.
18. Remove the outer leaves from each shoot
After the removal of all outer leaves, the apex is exposed.
Cut off the ultimate apex with the help of scalpel and transfer only those less
than 1 mm in length
Incubate the culture under 16hrs light at 25°C
As soon as the growing single leafy shoot or multiple shoots obtained from single
shoot tip or meristem, transfer them to hormone free medium to develop roots.
The plants are later transferred to pots containing compost and kept under
green house condition for hardening.
19. Application of Shoot-tip or Meristem
Culture
1. Virus Elimination
• Plants are often infected with more
than one type of virus, including
some even not known.
• A general term virus- free is used
by commercial horticulturist by this
method.
20. 2. Micro Propagation
• Asexual or vegetative propagation
(vegetative part) of whole plants using
tissue culture techniques referred to as
micro propagation.
3. Storage of Genetic Resources
• Many plants produce seeds that are
highly heterozygous in nature or that is
recalcitrant. Such seeds are not
accepted for storing genetic resources.
So , the meristem from such plants can
be stored in vitro.
21. 4. Use in Plant Breeding:
•In many plant breeding experiments the hybrid plants produce abortive
seeds or non viable seeds. As a result, it makes a barrier to crossibility in
plants where non-viable seeds are unable to develop into mature plants.
Shoot-tip or meristem from such hybrid plant can be cultured to speed up
breeding programme.
5. Quarantine
• Plantlets derived from shoot-tip or meristem
cultures are easily accepted by the quarantine
authority for international exchange without any
checking.
• Therefore, using this technique , crop plants can
be easily exchanged in crop improvement
programmes that are based on materials from
different parts of the world.
22.
23. List of the plants from which viruses have been eliminated by
meristem cultures
24. ADVANTAGES:
Lack of vascular tissue.
High auxin concentration.
Production of virus free plants
Facilitation of exchange between locations
Cryopreservation or in-vitro conservation of germplasm
• DISADVANTAGES:
Isolation is difficult
Low survival rate & regeneration time for explants may be long(abo
months for potato explant)
25. CONCLUSION
It is very effective method of cloning of plant material and to
develop disease free clean plant stock. Shoot Tip Culture is a
part of plant tissue culture which is a sun-rise technology and
working as a catalyst of agricultural and industrial
development
26. What is meristem?
A meristem is the tissue in all plants consisting of undifferentiated cells
(meristematic cells), found in zones of the plant where growth can take place.
2
6
PTC course for Horticulture students. By: Dr. Rafail S. Toma
27. What is meristem?
Groups of cells that are the source of new cells form
tissue called meristem.
Meristem cells aren't specialized, but when they
divide, some of the new cells specialize into tisues.
Areas of growth that lengthen the tips of roots and
stems are called apical meristems.
Lateral meristems, found all along woody roots and
stems, increase the thickness of these plant parts.
2
7
PTC course for Horticulture students. By: Dr. Rafail S. Toma
28. Meristem Culture History
2
8
PTC course for Horticulture students. By: Dr. Rafail S. Toma
• Though meristem culture technique is known since
1933 it was made successful only in 1965 by Morel.
• In the early period (1949) of this adventure,
Wetmore and Morel regenerated plantlets from the
meristem of ferns on simple defined medium.
• But this did not work well with the angiosperms
since they required complex medium for their in
vitro development.
29. Meristem Culture History
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
1. In 1946, Ball found the initiation of root primordium formation and the
plantlets regeneration from the young meristems of Trpaeolummajus and
Lupinus albus.
2. Following this, Morel established the technique of meristem culture with orchids.
3. Of late meristem culture technique is being increasingly applied in
micropropagation as an alternative means for sexual propagation of economically
important crop plants.
30. Explants
The explant of meristem culture may either be
the apical dome (apical meristem) or more
frequently, the apical dome plus a few
subjacent leaf primordia (the sub apical
meristematic region).
The apical meristem is located at the extreme
tip of a shoot and measure 0.1 mm in
diameter and
0.25 to 0.30 in length.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
31. The culture of meristem involves three stages:
1. culture establishment,
2. multiplication of the propagules and
3. ro t regeneration
Meristem Culture Technique
3
1
PTC course for Horticulture students. By: Dr. Rafail S. Toma
32. • Culture can be established from meristem, shoot tips or axillary buds.
• For shoot regeneration from meristem, young development stage of meristem has been
found to be optimum.
• Therefore, it is desirable to excise terminal explants for culture.
• Axillary buds are preferred since there would be only one terminal bud per shoot.
• Further, the explants should be larger enough for getting successful results.
• So larger explants like shoot tips and buds have to be chosen instead of minute meristems.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Culture Establishment
33. After explant excision, they are inoculated into culture medium.
Generally there is no necessity for the addition of exogenous hormones in
the medium since sufficient quantity of endogenous hormone is present in
the shoot apices.
However, there are cases in which exogenous auxin is applied to get better
results.
Among the auxins, NAA is the auxin routinely used for meristem tip and bud
cultures.
3
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Culture Establishment
34. The propagules multiplication in meristem/
shoot
tip/axillary bud culture can be accomplished by the three
methods as given below:
Explant - callus - meristemoids -shoot/roots plantlets
Explant - callus - embryoids/embryos- plantlets
Explant –axillary buds - multiple shoots-roots plantlets
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Multiplication of propagules
35. Among the three methods, axil ary shoot proliferation is
considered as the best because of the lower risk of genetic
instability than the other two systems of multiplication and
is easily achievable in most plant species.
In this system, the concentration of cytokinin used is
comparatively higherand is done to overcome the apical
dominance.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Multiplication of propagules
36. The incorporation of cytokinin enhances the branching of
lateral buds from leaf axils.
Too high a concentration of auxin may not only inhibit axillary
bud branching but also induces callus formation, especially
when 2,4-Dis used.
Multiplication of propagules
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
37. The purpose of this stage, in
the meristem culture is to
induce regeneration of roots
from the shoot multiplied in
the previous stage.
Adventitious root formation
can be induced quite readily in
many species, but it can be
very much recalcitrant in most
woody species.
Roots Regeneration
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
38. The induction of rooting does not
always have to be carried out in
vitro.
Good rooting can be obtained in
greenhouse by placing shoots into
pasteurized sand underintermittent
mist.
For better rooting, the proliferated
shoots may be dipped in auxin
solution or commercial rooting
powder before planting into rooting
medium.
Roots Regeneration
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
39. Size of the explant
The size of the explant determines the survival of the
culture.
In general, the larger the explant, the better the chance of
survival.
Meristem of the smal est size within the regenerable range
should be used for virus elimination.
When very smal explants are used, the presence of leaf
primordia appears to determine the capability of an
explant to develop.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Factors influencing meristem culture
40. Physiological state of the explant
Explants taken from the tip of a shoot are in a younger
stage of development than explants taken from the base.
Young developmental stage has often been found to be
optimumfor better shoot regeneration.
Factors influencing meristem culture
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
41. Culture media
White's medium(1943) was the most widely used medium
during the early days of meristem culture.
There is no general purpose medium yet available for
meristem, shoot tip and bud culture.
Murashige and Skoog (1962) medium with some
modification is the one used more frequently and with
great success.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Factors influencing meristem culture
42. Growth regulators
The requirement of growth regulators varies form species
to species, from one stage of culture development to
another.
Presence of cytokinin at higher level during proliferation
stage is felt to overcome the apical dominance.
Similarly, presence of auxin is headed for good rooting.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Factors influencing meristem culture
43. A. In vitro micropropagation,
B. Production of pathogen free plants, and
C. Cryopreservation of germplasm.
Applications of meristem culture
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
44. A. In vitro micropropagation
The micropropagation technique through meristem
or shoot tip culture favors production of thousand
and thousands of plants from a single explant
within a short period.
Moreover, once a stock of multiple shoot culture is
established, it can continuously serve as the source
material instead of having to restart from fresh
explant cultures periodical y.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
45. A. In vitro micropropagation
The greatest success using this technique has been
achieved in most of the herbaceous horticultural
species.
Compared to herbaceous plants, the
micropropagation of woody species has lagged far
behind.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
46. A. In vitro micropropagation
The major problems encountered with the propagation of
woody species are:
1.Most of the forest species are recalcitrant to culture condition
because of the presence of large quantity of polyphenolic compounds
in the tissues and
2.The other difficulty experienced is rooting of in vitro cultures.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
47. B. Production of pathogen free plants
The most important application of micropropagation
technique via meristem culture is the production of
pathogen free plants, especially viruses as they are
absent in apical meristem.
Applications of meristem culture
48. B. Production of pathogen free plants
General y, viruses infect plant species systemical y
making the plants to die.
But the evidences for decrease in virus particles
toward apical meristem made Morel and Martin
(1952) to postulate the concept of culturing apical
meristem of systemical y infected plant in vitro in
order to obtain virus free plants, genetical y
identical to the "mother plant”
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
49. B. Production of pathogen free plants
For example potato virus X infection could not be total y
eradicated since these viruses maintaintheir replication in
actively growing meristem.
In some cases of meristem tip culture the heat therapy has
necessarily to be fol owed to eliminate the viruses.
For example, in the carnation, heat therapy of plants at
38°C for two months fol owed by meristem culture
eradicated al the viruses.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
50. B. Production of pathogen free plants
The proposed reasons for meristem’s virus freeness:
1. The absence of vascular connections
2.The high metabolic activity of the meristematic cel s
which prevent virus multiplication
3.The high activity of the affective virus abolishing group
in meristems
4.The high auxin levels in apical meristems inhibits virus
multiplication
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Applications of meristem culture
51. C. Cryopreservation of germplasm
The conventional system of seed storage has the fol owing
disadvantages:
1) the loss of viability of seeds,
2) destruction by pathogen and pest attacks,
3) problems in clonal y propagated crops,
4) high cost of maintenance and transport and
5) material loss due to environmental hazards.
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PTC course for Horticulture students. By: Dr. Rafail S. Toma
Applications of meristem culture
52. C. Cryopreservation of germplasm Considering the above
disadvantages, the feasibility of in vitro storage was extensively
studied.
The potential advantages of this method are:
1) relatively little space is needed,
2)the plants are maintained free from pest and pathogens,
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Applications of meristem culture
53. C. Cryopreservation of germplasm
3)maintenance of vegetatively propagated species is easier,
4)the materials can be multiplied as and when needed
and
5)the pest pathogen free nature favors easy and quick
international germplasm exchanges.
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Applications of meristem culture