DOUBLING WORLD DOUBLING WORLD AGRICULTURAL PRODUCTION AGRICULTURAL PRODUCTION BY ACHIEVING TWO CROPS BY ACHIEVING TWO CROPS SIMULTANEOUSLY SIMULTANEOUSLY WITH ONE SEED WITH ONE SEED CONCEPT
Today’s 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.6. Younger generations are attracted to more remunerative occupations.7. Over generations inherited lands got divided and became smaller with lesser cultivable land after volumes of the boundaries are increased. In this context a need arises to solve all these problems. A better proposition is to enable a farmer to use one seed which can yield one crop above the ground and simultaneously another crop under the ground like POMATO which was designed to produce Tomatoes above the ground and Potatoes under the ground; thereby farmer will be having a chance to have a better price atleast to one of them.In this article efforts are made 1. to compile the available technologies2. to better understand the protocols involved in simpler terms3. to use improvised versions of the methodologies to augment sustained world food production in the coming Years.
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SEARCH O LITERATURESEARCH O LITERATURE
APPLICATIONS O SOMATIC HYBRIDIATION
a) Crat+!" !. h17r+%s $+th %+sas rs+sta"& - Many disease
resistance genes (e.g. tobacco mosaic virus, potato virus , club
rot disease) could be success!ully trans!erred !rom one species to
another. ".g resistance has been introduced in tomato against
diseases such as #M$, spotted wilt virus and insect pests. b)
E"6+r!"m"ta3 t!3ra"& - using somatic hybridi%ation the
genes con!erring tolerance !or cold, !rost and salt were introduced
in e.g. in tomato. c) C1t!p3asm+& ma3 str+3+t1 - using
cybridi%ation method, it was possible to trans!er cytoplasmic male
sterility. d) Za3+t1 &hara&trs - somatic hybrids with
selective characteristics have been developed e.g. the production
o! high nicotine content.
CHROMOSOME NUMBER IN SOMATIC HYBRIDS #he chromosome number in
the somatic hybrids is generally more than the total number o! both
o! the parental protoplasts. &! the chromosome number in the
hybrid is the sum o! the chromosomes o! the two parental
protoplasts, the hybrid is said to be symmetric hybrid. 'symmetric
hybrids have abnormal or wide variations in the chromosome number
than the eact total o! two species.
(http://www.biotechnology4u.com/plant_biotechnology_applications_cell_tissue_c
ulture.html)
pectinase or macero%yme (+.7-7.38) and cellulose (7-*8) is
appropriate !or most plant materials. emicellulase may be necessary
!or some tissues. 0enerally crude commercial preparations o!
en%ymes arc used.
#he p o! en%yme solution is ad9usted between 4. and .+ and
the temperature is ept at *;-<+=>. #he osmotic concentration
o! en%yme miture and o! subse?uent media is elevated (usually, by
adding ;++-++ m mol l-7 sorbitol or mannitol) to stabili%e the
protoplasts and to prevent them !rom bursting. 6sually, ;+-7++ m
mol l-7 >a>l* is added to the osmoticum as it improves
plasma membrane stability. #he cells and tissues are incubated in
the en%yme miture !or !ew to several (generally, 7@-7) hours1 naed
protoplasts devoid o! cell wall are gradually released in the
en%yme miture.
2rotoplasts have been isolated !rom virtually all plant parts, but
lea! mesophyll is the most pre!erred tissue, at least in case o!
dicots, !or this purpose. &n general, !ully epanded leaves are
sur!ace-sterili%ed, their lower epidermis is peeled oA with a pair
o! !orceps and the peeled areas are cut into small (>a. 7 cm*)
pieces with a scalpel and suspended in the en%yme miture.
Bhen epidermis cannot be peeled, lea! may be cut into >a. 7
mm* pieces and treated with the en%yme miture1 vacuum
inCltration may be used to !acilitate the entry o! en%ymes into the
tissues. '!ter the period o! incubation, protoplasts are washed
with a suitable washing medium in order to remove the en%ymes and
the debris.
0enerally, MF and ; media, and their modiCcations are used !or
protoplast culture. #he media are supplemented with a suitable
osmoticum and, almost always, with an auin and a cytoinin, their
types and concentrations depending mainly on the plant
species.
'!ter -7+ days o! culture, protoplasts regenerate cell wall, and
the osmolarity o! medium is gradually reduced to that o! normal
medium. #he macroscopic colonies are trans!erred onto normal tissue
culture media. 2rotoplasts are very sensitive to light1 there!ore,
they are cultured in diAuse light or dar !or the Crst 4-
days.
'# Pr!t!p3ast s+!":
#he techni?ues !or protoplast !usion are pretty well reCned
and highly eAective !or almost all the systems. ' number o!
strategies have been used to induce !usion between protoplasts o!
diAerent strains/species1 o! these the !ollowing three (5ig. .7+)
have been relatively more success!ul.
2rotoplasts o! desired strains/species are mied in almost e?ual
proportion1 generally, they are mied while still suspended in the
en%yme miture. #he protoplast miture is then sub9ected to high p
(7+.;) and high >a*G concentration (;+ m mol l-7) at
<=> !or about <+ min (high p-high >a*G treatment). #his
techni?ue is ?uite suitable !or some species, while !or some others
it may be toic.
#he washing medium may be alaline (p 3-7+) and contain a high
>a*G ion concentration (;+ m mol l-7)1 this approach is a
combination o! 2"0 and high p- high >a*G treatments, and is
usually more eAective than either treatment alone.
2"0 is negatively charged and may bind to cation lie >a*G,
which, in turn, may bind to the negatively charged molecules
present in plasma lemma1 they can also bind to cationic molecules
o! plasma membrane.
Huring the washing process, 2"0 molecules may pull out the plasma
lemma components bound to them. #his would disturb plasma lemma
organisation and may lead to the !usion o! protoplasts located
close to each other (5ig. .77).
#he above !usion techni?ues are nonselective in that they
induce !usion between any two or more protoplasts. ' more selective
and less drastic approach is the electro!usion techni?ue, which
utili%es low voltage nonuni!orm alternating electric current pulses
to bring the protoplasts in close contact (5ig. .7*). 5usion o!
protoplasts is brought about by a short pulse o! high
voltage.
#he duration o! high voltage is a !ew microseconds, and the
voltages ranges !rom ;++ to 7,+++ $/cm. #he high voltage creates
transient disturbances in the organisation o! plasma lemma, which
leads to the !usion o! neighbouring protoplasts. #he entire
operation is carried out manually in specially designed e?uipment,
called electroporator.
"lectro!usion has been mostly used with the members o! Folanaceae
o!ten with very high rate (over ;+8) o! !usion. #his approach
induces general !usion among protoplats and there is no control on
the type o! protoplasts entering !usion. &n a modiCcation o!
electro!usion, protoplast pairs are individually trans!erred into
micro!usion chambers with the help o! a micromanipulator set
up.
microseconds I a!ter mutual dielectrophoresis (7 M%1 @;-+ $/cm).
#his techni?ue is called microelectro!usion1 it leads to highly
speciCc and nearly 7++8 pairwise !usion. Many worers !eel that this
!usion techni?ue is more desirable than the others !or a number o!
important reasons.
*# S3&t+!" !. H17r+% C33s:
&n somatic hybridi%ation eperiments, only the heteroaryotic or
JhybridD protoplasts, particularly those resulting !rom !usion
between one protoplast o! each o! the two !usion partners, are o!
interest.
owever, they !orm only a small proportion o! the population
(usually +.;-7+8). #here!ore, an eAective strategy has to be
employed !or their identiCcation and isolation. #his step is called
the selection o! hybrid cells, is the most critical, and is still
an active area o! investigation.
' number o! strategies have been used !or the selection o! hybrid
protoplasts. (7) Fome visual marers, e.g., pigmentation, o! the
parental protoplasts may be used !or the identiCcation o! hybrid
cells under a microscope1 these are then mechanically isolated and
cultured.
5or eample, the protoplasts o! one species may be green and
vacuolated (derived !rom mesophyll cells), while those o! the other
may be non-vacuolated and non-green (obtained !rom cell cultures).
Bhere such !eatures are not available, the protoplasts o! two
parental species may be separately labelled with diAerent
Kuorescent agents.
#his would allow the microscopic identiCcation o! hybrid
protoplasts !rom the parental ones. #his approach can even be
adapted to automatic cell sorting permitting the recovery o! large
numbers o! heteroaryons in a very short time. #his approach
is, however, time consuming, and re?uires considerable sill and
eAort.
deCciencies) o! the parental species, which are not epressed in the
hybrid cells due to complementation between their genetic
systems.
#hese properties may be sensitivity to culture medium
constituents, antimetabolites, temperature, etc., inability to
produce an essential biochemical (auotrophic mutants), chlorophyll
or some other pigmentation, etc. #hese properties may be naturally
present in the parental species or may be artiCcially induced
through mutagenesis.
#he !ollowing eample should be enough to clearly bring out
the essential !eatures o! the complementation approach. 2rotoplasts
o! 2etunia hybrida !orm calli on the MF medium, while those o! 2.
parodii produce only small colonies.
5urther, actinomycin H (7 Eg ml-7) inhibits cell division o! 2.
hybrida protoplasts, but it has no eAect on those o! 2. parodii.
#hus protoplasts o! both these 2etunia species !ail to produce
macroscopic colonies (calli) on MF medium supplemented with 7 Eg
ml-7 actinomycin H.
owever, their hybrid cells (2. hybrida G 2. parodii1 it may be
noted that somatic hybrids are denoted by a G sign as against the
seual hybrids being designated by a symbol) divide normally on this
medium to produce macroscopic colonies.
#his selection strategy eploits those natural properties o!
the two parental species, which show complementation in the hybrid
cells and, at the same time, permit their selection. #hese
strategies are simple, highly eAective and the least demanding, but
their application is drastically limited by the nonavailability o!
suitable properties (both natural and induced) in most o! the
parental species o! interest to the eperimenters.
' variation o! the complementation approach uses selectable marers.
&n this approach, (<) diAerent selectable marers lie
antibiotic and herbicide resistance are engineered into the two
!usion partners. #he hybrid cells, in such cases, are resistant to
both the concerned selection agents, while the parental cells will
be sensitive to one and the other selection agent.
' more general and widely applicable strategy, but demanding more
wor than the previous approach, is (4) to culture the entire
protoplast population without applying any selection !or the hybrid
cells. 'll the types o! protoplasts !orm calli1 the hybrid calli
are later identiCed on the basis o! callus morphology, chromosome
constitution, protein and en%yme banding patterns, etc.
densities since neighbouring colonies are liely to !use at higher
densities1 ideally, they should be cultured in microdrops, each
drop containing but a single cell. Many worers tend to !avour this
approach since it does not depend the presence o! appropriate but
diLcult to Cnd marers in the parental species.
# R,"rat+!" !. H17r+% P3a"ts:
nce hybrid calli are obtained, plants are induced to regenerate
!rom them since this is a prere?uisite !or their eploitation in
crop improvement. 5urther, the hybrid plants must be at least
partially !ertile, in addition to having some use!ul property, to
be o! any use in breeding schemes. #he culture techni?ues have been
reCned to a state where plant regeneration has been obtained in a
number o! somatic hybrids (#able .;).
Ta73 #;# A 3+st !. s!m %+sta"t s!mat+& h17r+% p3a"ts:
ut even today, it has not been possible to recover hybrid plants
and/or calli !rom a number o! somatic combinations1 this phenomenon
is called Jsomatic incompatibilityD. #he reasons !or somatic
incompatibility are not clearly understood. #he somatic hybrids are
o! the !ollowing two types: (7) symmetric and (*) asymmetric
hybrids.
Symmetric Hybrids:
Fome somatic hybrid plants retain the !ull or nearly !ull somatic
complements o! the two parental species1 these are called symmetric
hybrids. Fuch hybrids provide uni?ue opportunities !or synthesi%ing
novel species, which may be o! theoretical and/or practical
interest. 5re?uently, somatic hybrids between distantly related
seually incompatible species are sterile, precluding their
incorporation into a breeding programme.
plants may be epected to be partially !ertile. #hese somatic
hybrids can now be used in breeding programmes !or limited
gene/chromosome introgression !rom the species contributing the
haploid protoplast.
' possible approach to the improvement o! apparently useless
somatic hybrids, e.g., nonKowering somatic hybrid Haucus carota G
'egopodium podagraria, is to !use protoplasts !rom the hybrid with
those o! one o! the parental species. #he !usion o! a somatic
hybrid protoplast with that !rom one o! its parents is called
somatic bac hybridi%ation.
Bhen protoplasts !rom the above somatic hybrid were !used with
carrot protoplasts, the resulting somatic hybrid produced Kowers.
Fuch hybrids can now be ordered into breeding programmes with the
aim o! gene/chromosome introgression.
Asymmetric Hybrids:
Many somatic hybrids ehibit the !ull somatic complement o! one
parental species, while all or nearly all chromosomes o! the other
parental species are lost during the preceding mitotic divisions1
such hybrids are re!erred to as asymmetric hybrids.
#he available evidence suggests that such hybrids are liely
to show a limited introgression o! chromosome segments !rom the
eliminated genome(s) due to drastically enhanced chromosomal
aberrations and/or mitotic crossing over in vitro.
'symmetric hybrids can be obtained even !rom those combinations,
which normally produce symmetric hybrids by the !ollowing approach:
protoplasts o! one o! the parental species are irradiated with a
suitable dose o! -rays or gamma-rays to induce etensive chromosome
breaage.
&n such cases, chromosome segment introgression may be maredly
enhanced. &t may be pointed out that asymmetric hybrids are
essentially cytoplasmic hybrids or cybrids, ecept !or the
introgressed genes.
$ate o% &lasma'genes:
#he cytoplasmic genes (generally studied in terms o!
chloroplast types or chloroplast HN', cp-HN' mitochondrial HN',
mtHN') appear to be distributed randomly during the mitotic cell
divisions. 's a result, some cells receive chloroplasts o! one
parental species, some others o! the other species and a small
proportion retains the chloroplasts o! both the species.
#his is reKected in the plants regenerated !rom these cells.
#he same applies to mitochondria as well. &n addition, the
distribution o! chloroplasts is independent !rom that o!
mitochondria.
#here!ore, a somatic hybrid plant may contain chloroplasts
!rom one parental species and mitochondria !rom the other !usion
parent. #here is considerable evidence that the genomes o! both
chloroplasts and mitochondria, particularly the latter, undergo
recombination in the hybrid cells1 this produces recombinant
organelles in the progeny.
;# C17r+%s:
>ybrids or cytoplasmic hybrids are cells or plants containing
nucleus o! one species but cytoplasm !rom both the parental
species. #hey are produced in variable !re?uencies in normal
protoplast !usion eperiments due to one o! the !ollowing: (i)
!usion o! a normal protoplast o! one species with an enucleate
protoplast or a protoplast having an inactivated nucleus o! the
other species, (ii) elimination o! the nucleus o! one species !rom
a normal heteroaryon, or (iii) gradual elimination o! the
chromosomes o! one species !rom a hybrid cell during the subse?uent
mitotic divisions. >ybrids may be produced in relatively high
!re?uency by (i ) irradiating (with -rays or gamma-rays) the
protoplasts o! one species prior to !usion in order to inactivate
their nuclei, or (ii) by preparing enucleate protoplasts
(cytoplasts) o! one species and !using them with normal protoplasts
o! the other species.
#he ob9ective o! cybrid production is to combine the
cytoplasmic genes o! one species with the nuclear and cytoplasmic
genes o! another species. ut the mitotic segregation o!
plasmagenes, as evidenced by the distribution o! chloroplasts,
leads to the recovery o! plants having plasmagenes o! one or the
other species only1 only a small proportion o! the plants remain
JcybridD, which would !urther segregate into the two parental
types.
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R,3at+!" !. GMOs #he overseeing o! testing, use and
commercialisation o! 0Ms I whether plants, animals or
microorganisms I re?uires a special regulation system. #his system
establishes the legal and institutional !ramewor !or the control o!
potential negative eAects o! 0Ms on the environment or human and
animal health (Obeanu, *++<). &n the 6F, transgenic plants
are only introduced into the environment or on the maret !ollowing
approval !rom the !ollowing governmental agencies responsible !or
environmental, and human and animal health protection: 7. 6F
Hepartment !or 'griculture (6FH'), *. "nvironmental 2rotection
'gency ("2'), <. 5ood and Hrug 'dministration (5H'). &n the
6F and >anada, transgenic plants are grown and used !or human
and animal !ood, and separate storage and labelling are not
mandatory. Fince 733+, in the "uropean 6nion, special legislation
has been drawn up, enhanced and etended, with the purpose o!
providing environmental and human health protection, and creating a
common maret in the Celd o! biotechnology. #hus: - "6 Hirective No.
*73/733+ (amended by Hirective No. 7/733) regulates the contained
use o! genetically modiCed microorganisms (!or research and
commercialisation), - "6 Hirective No. **+/733+, concerning the
deliberate release o! genetically modiCed organisms into the
environment, was the main initiative taen by the "6, and was
subse?uently supplemented by several >ommission Hecisions
(@*<, 77, 7*, 7</*++<), - "6 Hirective No. 7/*++7
regulates the deliberate release o! genetically modiCed organisms
into the environment. #his Hirective repealed Hirective No.
**+/733+. aving come into !orce on 7 ctober *++*, Hirective 7/*++7
both updates and consolidates 7; eisting regulations. #his
Hirective also deals with mandatory in!ormation to the public, the
long-term monitoring o! eAects, labelling and traceability, in all
stages o! 0M introduction on the maret. #wo other acts have been
adopted and published in the Lcial ournal o! the "uropean
>ommunities, with respect to the >ommunity system o! 0M
traceability, the labelling o! genetically modiCed !ood and !odder,
and the continuous procedure o! authorisation or introduction o!
0Ms in the environment as !ood or !odder: - "> Pegulation No.
7*3/*++< (** Feptember *++<) on genetically modiCed !ood and
!odder, and - "> Pegulation No. 7<+/*++< (** Feptember
*++<) on the traceability and labelling o! genetically modiCed
organisms, and 0M-based !ood and !odder. #hese regulations
amended "6 Hirective No. 7/*++7.
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