Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
AGRICULTURAL PRODUCTION
BY ACHIEVING TWO CROPS
SIMULTANEOUSLY
WITH ONE SEED
CONCEPT
(https://mrsmatheson.wordpress.com/2012/11/30/higher-genetic-engineering-and-somatic-fusion/)
ABSTRACT
Todays Scenario is more or less like this.
1. Agricultural lands are used for industrial or mining or
for land filling of Solid Wastes or for dwelling thus
causing diminishing agricultural lands available.
2. Labour is becoming scarce and expensive
discouraging farmers to grow.
3. Wild Animals, Rats and untimely rains, hurricanes,
cyclones etc are damaging agricultural fields and thus
reducing total available yields.
4. Pests and insects are becoming more and more
pesticide resistant and reducing the yields.
5. Seasonal fluctuations in market prices sometimes
lead to heavy losses to the farmers leading to suicides
in many developing and under-delveloped countries
where agriculture is the main occupation.
MISSION:
I am looking for associates who can involve with us in technology,
production and marketing on contract basis.
PROLOGUE
of the food price crisis, the financial and economic crisis, and climate
change.
Rajul Pandya-Lorch, Chief of Staff and Head of the 2020 Vision for Food,
Agriculture, and the Environment Initiative, International Food Policy
and Research Institute (IFPRI), said that, despite efforts to improve
food security by the international community, the number of people
going hungry has remained relatively constant over the last half
century, which forced the question: What do we need to do
differently?
On an inquiry about the tension between supporting smallholder
farmers and the need for substantial, larger-scale agricultural
investment to improve food security, he said it was an issue of
maximum efficiency, and that one way to encourage that could be
through support of cooperatives. Our main job now is to show that,
with the available resources, we can get good results during the next
few years, he said.
(http://www.un.org/press/en/2009/gaef3242.doc.htm)
HISTORY
The concept of grafting related potatoes and tomatoes so that
both are produced on the same plant was originally developed in
1977 at the Max Planck Institute for Developmental
Biology in Tbingen, Germany, and although healthy, the plant
produced neither potatoes nor tomatoes.[2] The Max Planck
Institute for Plant Breeding Research in Kln produced a plant
with fruit in 1994.[2]
As with all grafts, this plant will not occur in nature and cannot be
grown from seed, because the two parts of the plant remain
genetically separate, and only rely on each other for nourishment
and growth. Like most standard types of plant grafting, a small
incision is made in the stem of both plants and they are strapped
together. Once the cuts have healed and the plants are joined,
the leafy top of the potato plant can be cut away and the roots of
the tomato can be removed, leaving the leaves of the tomato
plant to nourish the roots of the potato plant.[3] The rootstock
(potato) acts as a stable and healthy root system and the scions
(tomato) are chosen for their fruit, flowers or leaves. The
tomatoes should be ready to harvest after about 12 weeks during
the summer months, the potatoes should be ready after the
tomato leaves begin to die back, normally in early autumn.
[4]
Grafting in this way can be used to produce many different
related crops from the same plant, for example the growing
popularity of 'Fruit salad' trees, which is a single tree that
produces multiple types of citrus fruits, or a tree with a variety of
fruits with stones (peach, plum etc.).
((http://en.wikipedia.org/wiki/Pomato)
In 1972, Carlson and his associates produced the first inter-specific
somatic hybrid between Nicotiana glauca and N. langsdorffii. In 1978,
Melchers and his co-workers developed the first inter-genetic somatic
hybrids between Solanum tuberosum (potato) and Lycopersicon
esculentum (tomato). The hybrids are known as Pomatoes or Topatoes
(http://www.biotechnology4u.com/plant_biotechnology_applications_cell_tissue_c
ulture.html)
Grafted pomato plants have been launched in the United Kingdom in
September 2013 by horticultural mail order company Thompson &
Morgan, who sold pre-grafted plants branded as the "TomTato".
The Incredible Ediblenursery in New Zealand announced a "DoubleUP
Potato Tom" in the same month
Grafting is a difficult process because the tomato and the potato stems
have to be the same thickness, and Thompson & Morgan trialled the
hybrid for several years before selling it. Production and grafting of
tomtatos begins in a specialist laboratory in the Netherlands, before
being shipped back to the UK and grown in greenhouses until they are
ready to be sold.
(http://en.wikipedia.org/wiki/Pomato)
In Florida, most orange trees have lemon roots. In California, nearly all
lemon trees are grown on orange roots. This sort of thing is not unique
with citrus. With the stone fruits, there is a certain latitude. Plums can
be grown on cherry trees and apricots on peach trees, but a one-to-one
relationship like that is only the beginning with citrus. A single citrus
tree can be turned into a carnival, with lemons, limes, grapefruit,
tangerines, kumquats and oranges all ripening on its branches at the
same timeMost of the trees on the Ridge [a mountain range in Florida
renowned for its orange tree orchards] are growing on Rough Lemon
As a rootstock, it forages with exceptional vigor and, in comparison
with others, puts more fruit on the tree.
Although grafting woody plants, like fruit trees, is an ancient
horticultural technique, grafting soft-stemmed vegetables is a much
more recent agricultural practice. Perhaps nurseries will soon start
selling mixed vegetable shrubs alongside fruit salad trees. Brassica
oleracea seems like a particularly good candidate for such an
experiment. This one species includes cabbage, broccoli, cauliflower,
Brussels sprouts and kale. Yes, all these plants are cultivars of the
exact same speciestheir appearances and characteristics have been
altered through artificial selection over the generations, in the same
way people have created so many different dog breeds. A
broccauliflower sprouts plant sounds particularly delicious. Maybe its
time for a family reunion.
(http://blogs.scientificamerican.com/brainwaves/2012/09/10/thescience-of-pomato-plants-and-fruit-salad-trees/)
Previously NONGWOO BIO developed a new CMS line (NWB-CMS) for
radish and the NWB-CMS line indeed provided lots of sub-lines of
Radish. And the Biotechnology Institute has developed the new
Cruciferae plants by fusing the NWB-CMS cells of Radish to the ordinary
cells of Chinese Cabbage and Cabbage.
(http://www.nongwoobio.com/nongwoo/html/tech_02.html)
SCIENCE
The seed is the mature, fertilized ovule. After fertilization, the haploid
cells of the embryo sac disintegrate. The maternally derived diploid
cells of the ovule develop into the hard, water-resistant outer covering
of the seed, called the testa, or seed coat. The diploid zygote develops
into the embryo, and the triploid endosperm cells multiply and provide
nutrition. The testa usually shows a scar called the hilum where the
ovule was originally attached to the funicle. In some seeds a ridge
along the testa called the raphe shows where the funicle originally was
pressed against the ovule. The micropyle of the ovule usually survives
as a small pore in the seed coat that allows passage of water during
germination of the seed.
In some species, the funicle develops into a larger structure on the
seed called an aril, which is often brightly colored, juicy, and contains
sugars that are consumed by animals that may also disperse the seed
(as in nutmeg, arrowroot, oxalis, and castor bean). This is distinct from
the fruit, which forms from the ovary itself.
The embryo consists of the cotyledon(s) , epicotyl, and hypocotyl. The
cotyledons resemble small leaves, and are usually the first
photosynthetic organs of the plant. The portion of the embryo above
the cotyledons is the epicotyl, and the portion below is the hypocotyl.
The epicotyl is an apical meristem that produces the shoot of the
growing plant and the first true leaves after germination. The hypocotyl
develops into the root. Often the tip of the hypocotyl, the radicle, is the
first indication of germination as it breaks out of the seed. Flowering
plants are classified as monocotyledons or dicotyledons (most are now
called eudicots ) based on the number of cotyledons produced in the
embryo. Common monocotyledons include grasses, sedges, lilies, irises,
and orchids; common dicotyledons include sunflowers, roses, legumes,
snapdragons, and all nonconiferous trees.
The endosperm may be consumed by the embryo, as in many legumes,
which use the cotyledons as a food source during germination. In other
species the endosperm persists until germination, when it is used as a
food reserve. In grains such as corn and wheat, the outer layer of the
endosperm consists of thick-walled cells called aleurone, which are high
in protein.
SEARCH OF LITERATURE
APPLICATIONS OF SOMATIC HYBRIDIZATION
a) Creation of hybrids with disease resistance - Many disease resistance
genes (e.g. tobacco mosaic virus, potato virus X, club rot disease) could be
successfully transferred from one species to another. E.g resistance has been
introduced in tomato against diseases such as TMV, spotted wilt virus and insect
pests.
b) Environmental tolerance - using somatic hybridization the genes conferring
tolerance for cold, frost and salt were introduced in e.g. in tomato.
c) Cytoplasmic male sterility - using cybridization method, it was possible to
transfer cytoplasmic male sterility.
d) Quality characters - somatic hybrids with selective characteristics have
been developed e.g. the production of high nicotine content.
TRADITIONAL METHODS
(https://www.google.co.in/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0CFUQFjA
J&url=http%3A%2F%2Fwww.scirp.org%2Fjournal%2FPaperDownload.aspx
%3FpaperID%3D35851&ei=t5PxVI-GI4yjugSi4LoBg&usg=AFQjCNEOVeQ2SGm5UNLSRVqlQBxp_Tkecw&bvm=bv.87269000,d.
c2E)
ii. High pH and Ca++ treatment: This technique lead to the development
of intra- and interspecific hybrids. It was demonstrated by Keller and
Melcher in 1973. The isolated protoplasts from two plant species are
incubated in 0.4 M mannitol solution containing high Ca ++(50 mM
CaCl2.2H2O) with highly alkaline pH of 10.5 at 37C for about 30 min.
Aggregation of protoplasts takes place at once and fusion occurs within
10 min.
diploids
leads
to
formation
of
for two weeks and then shifted to alternating light (of intensity 200
m2 s 1) and dark regimes of 14 and 10 h respectively. Samples
were tested periodically for viability and wall regeneration Further
culturing of protoplasts to obtain cell colonies and plant regeneration
was followed for N. tabacum cv Thompson applying the procedure
described earlier by Sankara Rao and Gunasekari (1991). All media
were autoclaved for 15 min at 15 p.s.i. Growth regulators used were
filter sterilized. All operations were carried out under aseptic
conditions. 3. Results The results obtained from the various protoplast
isolation experiments performed are summarized in table 1. With the
method described now, tissues of species explanted were most readily
acted upon by the enzymes and microscopic examination carried out
following digestion showed that the protoplast release was complete.
All species investigated yielded protoplasts (figures 1, 2, 5 and 6).
These species include herbaceous dicotyledonous plants, woody
sandalwood and the mucilage-containing herbaceous vine, Basella. On
an average, the incubation time required for complete release of
protoplasts varied between 4 and 10 h depending on the species
examined. Protoplast release from the explants of N. tabacum,
Lycopersicon esculentum cultivars and Capsicum frutescens cultivars,
particularly, needed only 4-6 h. Digestion of these explants carried out
by another method which was adopted earlier by Sankara Rao and
Gunasekari (1991) required a longer time (8-12 h) for protoplast release
(table 1). The yield of protoplasts was also comparatively low. The Novo
enzymes used in the present method maintained protoplast releasing
ability at temperatures beyond 25C. Increase of temperature during
tissue incubation using these enzymes, in fact, aided tissue digestion
further. The yield of protoplasts also increased slightly in N. tabacum cv
Thompson (table 1). More than its beneficial effect on protoplast yield,
increased incubation temperature had reduced the time needed for
complete release of protoplasts (table 1). The optimal conditions
identified with the protocol were, an incubation temperature range
between 28-30C and a pH between 58 and 6. These were also
conditions observed to be ideal for protoplast preparation from the
plant species used in the experiments as the protoplast viability did not
suffer the least. Using the present protocol, the yield of protoplasts
derived from chopped explants as also from leaves with epidermal
layers peeled off were comparable (table 1). Filtration of incubation
mixture through nylon mesh reduced the amount of contaminating
debris but only incompletely. That isolated protoplasts were free of cell
walls was determined by microscopic observation. Virtually a pure
population of intact protoplasts was readily isolated by flotation
separation on sucrose. The yield data for each of the plant species used
is shown in table 1. Washes and sedimentation helped further
purification of the protoplast preparation. Protoplasts were most stable
if allowed to equilibrate in the wash medium for about 30 min before
plating. In aliquots of purified protoplasts tested for viability, a fairly
good number were found positive. Protoplasts plated in modified 8E
medium formed cell wall readily (figures 3, 4, 6 and 8). The growth
regulators and osmolarity of the culture medium were different for each
species/cultivar investigated
Protoplasts of niger, tobacco cultivars, C. frutescens cv Punjab Jwala
and L. esculentum cv Arkavikas divided within six to seven days of
Somatic Embryo
Somatic
Embryo Chart
(https://www.google.co.in/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0CFUQFjAJ&url=ht
tp%3A%2F%2Fwww.scirp.org%2Fjournal%2FPaperDownload.aspx%3FpaperID
%3D35851&ei=t5PxVI-GI4yjugSi4LoBg&usg=AFQjCNEOVeQ2SGm5UNLSRVqlQBxp_Tkecw&bvm=bv.87269000,d.c2E)
Protoplast Isolation:
PROTOCOLS
When epidermis cannot be peeled, leaf may be cut into Ca. 1 mm 2 pieces and
treated with the enzyme mixture; vacuum infiltration may be used to facilitate
the entry of enzymes into the tissues. After the period of incubation, protoplasts
are washed with a suitable washing medium in order to remove the enzymes and
the debris.
The protoplasts may be cultured in a suitable medium in a variety of ways: (i)
Bergmanns plating technique (in agar medium), (ii) in a thin layer of liquid
medium or (iii) in small microdrops of 50-100 l. Protoplasts readily regenerate
cell wall (within 2-4 days) and undergo mitosis to form macroscopic colonies,
which can be induced to regenerate whole plants. The conditions for isolation
and culture of protoplasts and regeneration of complete plants have been
standardized for a large number of plant species, but cereals still present some
problems.
Generally, MS and B5 media, and their modifications are used for protoplast
culture. The media are supplemented with a suitable osmoticum and, almost
always, with an auxin and a cytokinin, their types and concentrations depending
mainly on the plant species.
After 7-10 days of culture, protoplasts regenerate cell wall, and the osmolarity of
medium is gradually reduced to that of normal medium. The macroscopic
colonies are transferred onto normal tissue culture media. Protoplasts are very
sensitive to light; therefore, they are cultured in diffuse light or dark for the first
4-7 days.
2. Protoplast Fusion:
The techniques for protoplast fusion are pretty well refined and highly effective
for almost all the systems. A number of strategies have been used to induce
fusion between protoplasts of different strains/species; of these the following
three (Fig. 8.10) have been relatively more successful.
Protoplasts of desired strains/species are mixed in almost equal proportion;
generally, they are mixed while still suspended in the enzyme mixture. The
protoplast mixture is then subjected to high pH (10.5) and high
Ca2+ concentration (50 m mol l-1) at 37C for about 30 min (high pH-high
Ca2+ treatment). This technique is quite suitable for some species, while for some
others it may be toxic.
Polyethylene glycol (PEG) induced protoplast fusion is the most commonly used
as it induces reproducible high frequency fusion accompanied with low toxicity to
most cell types. The protoplast mixture is treated with 28-50% PEG (MW 1,5006,000) for 15-30 min, followed by gradual washing of the protoplasts to remove
PEG; protoplast fusion occurs during the washing.
The washing medium may be alkaline (pH 9-10) and contain a high Ca 2+ ion
concentration (50 m mol l-1); this approach is a combination of PEG and high pHhigh Ca2+ treatments, and is usually more effective than either treatment alone.
PEG is negatively charged and may bind to cation like Ca 2+, which, in turn, may
bind to the negatively charged molecules present in plasma lemma; they can
also bind to cationic molecules of plasma membrane.
During the washing process, PEG molecules may pull out the plasma lemma
components bound to them. This would disturb plasma lemma organisation and
may lead to the fusion of protoplasts located close to each other (Fig. 8.11).
The above fusion techniques are nonselective in that they induce fusion between
any two or more protoplasts. A more selective and less drastic approach is the
electrofusion technique, which utilizes low voltage nonuniform alternating
electric current pulses to bring the protoplasts in close contact (Fig. 8.12). Fusion
of protoplasts is brought about by a short pulse of high voltage.
The duration of high voltage is a few microseconds, and the voltages ranges
from 500 to 1,000 V/cm. The high voltage creates transient disturbances in the
organisation of plasma lemma, which leads to the fusion of neighbouring
protoplasts. The entire operation is carried out manually in specially designed
equipment, called electroporator.
Electrofusion has been mostly used with the members of Solanaceae often with
very high rate (over 50%) of fusion. This approach induces general fusion among
protoplats and there is no control on the type of protoplasts entering fusion. In a
modification of electrofusion, protoplast pairs are individually transferred into
microfusion chambers with the help of a micromanipulator set up.
Thus each microfusion chamber has one pair of protoplasts, which are induced to
fuse by a single or multiple, negative DC-pulse of 800 to 1,800 V /cm for 50
This would allow the microscopic identification of hybrid protoplasts from the
parental ones. This approach can even be adapted to automatic cell sorting
permitting the recovery of large numbers of heterokaryons in a very short time.
This approach is, however, time consuming, and requires considerable skill and
effort.
Several workers have attempted to devise systems, which specifically select for
hybrid cells. In simple words, (2) these systems exploit some properties (usually,
deficiencies) of the parental species, which are not expressed in the hybrid cells
due to complementation between their genetic systems.
These properties may be sensitivity to culture medium constituents,
antimetabolites, temperature, etc., inability to produce an essential biochemical
(auxotrophic mutants), chlorophyll or some other pigmentation, etc. These
properties may be naturally present in the parental species or may be artificially
induced through mutagenesis.
The following example should be enough to clearly bring out the essential
features of the complementation approach. Protoplasts of Petunia hybrida form
calli on the MS medium, while those of P. parodii produce only small colonies.
This selection strategy exploits those natural properties of the two parental
species, which show complementation in the hybrid cells and, at the same time,
permit their selection. These strategies are simple, highly effective and the least
demanding, but their application is drastically limited by the nonavailability of
suitable properties (both natural and induced) in most of the parental species of
interest to the experimenters.
A more general and widely applicable strategy, but demanding more work than
the previous approach, is (4) to culture the entire protoplast population without
applying any selection for the hybrid cells. All the types of protoplasts form calli;
the hybrid calli are later identified on the basis of callus morphology,
chromosome constitution, protein and enzyme banding patterns, etc.
In some cases, the identification may be delayed till plants are regenerated. In
such an approach, it will be desirable to culture the protoplasts in very low
But even today, it has not been possible to recover hybrid plants and/or calli
from a number of somatic combinations; this phenomenon is called somatic
incompatibility. The reasons for somatic incompatibility are not clearly
understood. The somatic hybrids are of the following two types: (1) symmetric
and (2) asymmetric hybrids.
Symmetric Hybrids:
Some somatic hybrid plants retain the full or nearly full somatic complements of
the two parental species; these are called symmetric hybrids. Such hybrids
provide unique opportunities for synthesizing novel species, which may be of
theoretical and/or practical interest. Frequently, somatic hybrids between
distantly related sexually incompatible species are sterile, precluding their
incorporation into a breeding programme.
plants may be expected to be partially fertile. These somatic hybrids can now be
used in breeding programmes for limited gene/chromosome introgression from
the species contributing the haploid protoplast.
The available evidence suggests that such hybrids are likely to show a limited
introgression of chromosome segments from the eliminated genome(s) due to
drastically enhanced chromosomal aberrations and/or mitotic crossing over in
vitro.
This is reflected in the plants regenerated from these cells. The same applies to
mitochondria as well. In addition, the distribution of chloroplasts is independent
from that of mitochondria.
Therefore, a somatic hybrid plant may contain chloroplasts from one parental
species and mitochondria from the other fusion parent. There is considerable
evidence that the genomes of both chloroplasts and mitochondria, particularly
the latter, undergo recombination in the hybrid cells; this produces recombinant
organelles in the progeny.
5. Cybrids:
Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one
species but cytoplasm from both the parental species. They are produced in
variable frequencies in normal protoplast fusion experiments due to one of the
following: (i) fusion of a normal protoplast of one species with an enucleate
protoplast or a protoplast having an inactivated nucleus of the other species, (ii)
elimination of the nucleus of one species from a normal heterokaryon, or (iii)
gradual elimination of the chromosomes of one species from a hybrid cell during
the subsequent mitotic divisions. Cybrids may be produced in relatively high
frequency by (i ) irradiating (with X-rays or gamma-rays) the protoplasts of one
species prior to fusion in order to inactivate their nuclei, or (ii) by preparing
enucleate protoplasts (cytoplasts) of one species and fusing them with normal
protoplasts of the other species.
The objective of cybrid production is to combine the cytoplasmic genes of one
species with the nuclear and cytoplasmic genes of another species. But the
mitotic segregation of plasmagenes, as evidenced by the distribution of
chloroplasts, leads to the recovery of plants having plasmagenes of one or the
other species only; only a small proportion of the plants remain cybrid, which
would further segregate into the two parental types.
RESULTS
DISCUSSION
IP
EXPLORING MARKETEERS
STATUTORY REGULATIONS TO BE TAKEN
CARE
Regulation of GMOs
WEB REFERENCES
REFERENCES
S 1992 Rapid isolation of mesophyll and guard cell protoplasts from pea
and maize leaves; Indian J. Exp. Biol. 30 424-428