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INTERNATIONAL JOURNAL
OF CURRENT RESEARCH
International Journal of Current Research
Vol. 10, Issue, 10, pp.74067-74070, October, 2018
ISSN: 0975-833X DOIDOI: ht: httptps://dos://doii.or.orgg//110.20.24941/4941/ijcijcr.r.32574.10.2018
RESEARCH ARTICLE
PLANT PLANT PLANT TISTISTISSUSUSUE CUE CUE CULLLTTTURE TECURE TECURE TECHNIQHNIQHNIQUEUEUES INS INS IN CROP CROP CROP I I IMPROVMPROVMPROVEMEEMEEMENTNTNT
*Mohit and Sirohi, S.P.S.
DepartDepartmement nt of Genetof Genetics aics and nd PlantPlant Br Breedieedingng, Ki, Kisasann PG PG ColleCollege, ge, SimbhaSimbhaolioli, H, Haapur, Uttar Prapur, Uttar Pradesdesh, h,
India afIndia afIndia affififillliaiaiated tted tted to CCo CCo CCS US US Unnniiiversiversiversity, ty, ty, MMMeeruteeruteerut, Uttar Pra, Uttar Pra, Uttar Pradesh, Indesh, Indesh, Indididiaaa
ARTICLE INFO ABSTRACABSTRACTT
Article History: TTTissue issue issue cultcultcultuuure re re inininvolvevolvevolves s s thththe e e uuuse se se ooofff sssmmmall all all pppieceieceieces s s ooofff ppplantlantlant tissue tissue tissue (((exexexppplantlantlants) s) s) wwwhhhich ich ich areareare cultcultcultuuured red red ininin a a a
th nnnuuutrietrietriennnt t t mmmediuediuediummm uuunnnddder er er sterile sterile sterile cococonnnddditiitiitiooonnnsss. . . ManManManyyy ppprimarrimarrimaryyy steps steps steps inininvolvevolvevolveddd areareare thththe e e pppreparationreparationreparation ooofff nnnuuutrtrtriiient ent ent
Received 10 July, 2018 memeddiuiumm, , ththe e estaestabblishlishmment ent ooff asaseptieptic c cultcultuure,re, inoculinoculatiatioonn, , ddeveveloeloppmment ent ooff ththe e pplantlant iinn growgrowthth roroomom,,
Received in revised form
th
30 August, 2018 hhhardening ardening ardening ooofff mmmicro icro icro ppplantlantlants s s anananddd tratratrannnsfsfsfer er er tototo thththe e e gregregreenhenhenhooouuuse. se. se. PlanPlanPlant t t tististissue sue sue cultcultcultuuure re re hhhas as as a a a lllooot t t ooofff appappapplicatilicatilications ons ons
th
Accepted 05 September, 2018 ininin cropcropcrop imimimppprororovemvemvement ent ent like like like mamamaking king king succesuccesuccessssssfffuuul l l dddistant istant istant crossescrossescrosses, , , shoshoshorrrteniteniteninnng g g bbbreereereedddiiinnng g g cccyyyclesclescles, , , sososommmaaaclonalclonalclonal
th
Published online 30 October, 2018
vavariantriants, s, gegerrmmpplalassmm exexchangechange etetc., c., TThhere ere are are mmanany y ttyyppes es ooff pplantlant tissue tissue cultcultuure re techntechniques iques suchsuch asas
seeseeseeddd cultcultcultuuure, re, re, ememembbbrrryyyooo cultcultcultuuure, re, re, shoshoshoot ot ot mmmeristeeristeeristem m m cultcultcultuuure, re, re, ooovavavarrryyy ooor r r ooovule vule vule cultcultcultuuure, re, re, ppprrroootoplast toplast toplast culturculturculture,e,e,
Key Words: suspsuspension ension culcultuture re etc.,etc., TThhereereffoorre e inin tthhis is article article an an attemattemppt t hhas as bebeen en mmade ade toto givgive e a a bbriefrief idideaea aboabouutt
ththe ve variouarious s pplantlant t tissue culissue culttuure technre techniiqquues aes and tnd thheir applicationeir applications.s.
In Vitro, Meristem culture, Protoplast
culture, Somclonal variants, Totipotency.
Copyright © 2018, Mohit and Sirohi. This is an open accesaccesss article distributed under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Mohit and SPS. Sirohi, 2018. “PlPlanant t titissssuuee cucullttuurere techtechnniiqquuees s iinn crocropp impimprorovevememenntt”, IInntterernnatiatioonanal l JJoouurrnal nal oof f CCuurrerrennt t RReseeseaarcrchh, 10, (10), 74067-
74070.
Autoclave: AAn n aaututooclaclave ve iis s bbasicallasically y a a larlargege-sized but
INTRODUCTION sopsophihisticated sticated ppreresssusurere cocoooker,ker, and and isis uused sed ffoor r tthe he sterilizatisterilizatioon n
ooff ththe e mmedediiuumm, , glglasassswwarare e and and ininstrstruummentents. s. AAututooclaclaves ves ooff
Plant tissue culture is a technique of in in vitro vitro cultivation of ddififfferereentnt ssizes izes arare e avaavailabilable le ccoommmmerercialcialllyy. . HiHigghh-pressure heat
pplant lant cells cells and and oorgansrgans, , wwhhich ich ddivivideide and and reregengenererate ate ininto to callus callus is needed to sterilize media,a, wwaterater, , and and glglaassware. ssware. CertCertain ain
or particular plant organs. In In 11990022, , a a GerGermman an pphhysysioloiologigist,st, 0
GoGottlieb ttlieb HabHabererlandt landt ddeveloped eveloped ththe e coconcept ncept ooff in vitro cell sposporeres s ffroromm fungi fungi aand nd bbacteracteria ia arare e kikilledlled oonly nly at at 112211 C and
cultculture. ure. He He isolatedisolated ssingle ingle fulfulllyy ddiifffferereentntiatediated iindivndivididual ual ppllant ant 11.0.055kgkg/sq.c/sq.cmm ( (1155 p poounundds pes per r sq. sq. ininch) pch) preressssure.ure.
cells cells ffroromm ddiifferent fferent pplalantnt ssppecies ecies liklikee ppalisadealisade cellcells s ffroromm
leaves of Laminum purpureum, , glglaandulndularar hairhair ooff Pulmonaria Laminar airflow chamber: The laminar flow chambers
and pith cells from petioles of EiccEicchhoorniarnia crascrassiples siples etc and pprorovividde e clean clean filterfiltereded air air ththaat t alloallowws s ccululttures ures to to bbe e hahandndled led
wwaas s ffirirst st to to cultculture ure tthehemm inin KKnopnop’’s s salt salt solsolutution ion eenricnriched hed wwith ith under contamination-ffreree e eenvnviroironnmmeentnt.. The laminar-flow
glglucoucose. se. In In hihis s ccululttures, ures, cellscells inincrcreasedeased inin size, size, accuaccummuulatlated ed cabcabininets arets are loe locatedcated in th in the ce culultture trure transfer aansfer arerea.a.
starch but failed to divide. Thereforere, , HabHabererlandt’s landt’s pprereddiction iction
ffailedailed ththat at tthe he cultculturedured pplant lant cells cells cocoululdd grogroww, , ddivivideide and and Other equipment: TThe sterilihe sterilizatiozation of in of innstrustrummeentnts, s, susucch ah as s ththe e
ddevelopevelop ininto to eemmbbrryyoo and and tthen hen to to wwhole hole pplalantnt. . TThihis s ppootentitentialal ooff ffoorcrcepeps, s, scalpescalpel l holdeholders rs and and bblladades es is is achieachieved ved wwitith h gagas s flaflammed ed
a a cell cell iis s knknoowwnn asas totiptotipootenctency, y, a a tertermm cocoiined ned bbyy SteStewwarardd in in burner. The medium preppararatioation n rorooomm usuallusually y hahass ththee
11996688. . Despite Despite lack lack ooff susucccess, cess, HabHabererlandt landt mmadade several ffoollollowwing ing eqequiuippmmenent. t. AA rereffrigrigereratoatorr-freezer to store chemicals
pprereddictions ictions ababooutut tthehe rereqquiuireremmenents ts in in mmededia ia iin n eexperxperiimmeentntal al and and stock stock solutisolutioonsns, , wweieigghihinngg scales scales ffoor r large large aammoounts unts ooff oover ver
coconditions nditions wwhhichich cocoululdd ppoossssibliblyy inindduce uce cellcell ddiviviisisioon,n, 1100 g, g, and and analanalytytical ical bbalaalance nce wwitith1 h1 mmg g accaccuracuracyy, , a a mmaaggnenetictic
pproroliflifereratioation n and and eemmbbrryyoo iinndduction. uction. G. G. HabHabererlandt landt iis s tthus hus stirrerstirrer ffoor r ththe e agitation, agitation, aand nd a a ppH H mmetereter. An aspirator can be
regarded as father of tissue culture. attached attached to to aa wwaterater taptap ffoor r filfilter ter sterilizatiosterilization n oof f cchehemmicals icals and and
ffoor r susurrfface ace sterilizatiosterilization n ooff ththee pplant lant mmateraterial.ial. A drying oven is
Physical Components of Tissue Culture re TecTechnolohnologgyy rereqquiuireredd to to keep keep glglasassswwarare e susucch h aas s bbeaeakers, kers, fflaslasks ks and and
ccyylilindernders, s, and and is is alalso so uuseful seful ffoor r ddryry steristerilizalization of scalpels and
TThe he bbasic asic eqequiuippmmeentnt in in mmoosstt tissue tissue cculultture ure ffacilitacilities ies iinclncluuddeses glglasassswwarare, e, susucch as Ph as Petri etri ddishisheess, , ppipeipettes and ottes and oththerers.s.
the following:
Non-essential equipment: MicrMicroowwaave ve oovens vens arare e coconnveveninienent t
*Corresponding author: Mohit and Sirohi, S.P.S., ffoor r ddefrosting efrosting stock stock solsolututioionsns aand nd pprere-heating agar media.
DeDeppartmeartmennt t ofof GGeenneetitics cs and and PlPlant ant BreBreeeddining, g, KKisaisann PGPG ColCollegelege, , SimbhaolSimbhaoli, i, LLababooraratortory y gglasslasswwarare e wwasasherhers s oor r rereggulularar ddisishhwwaashsherers s cacann bbee
Hapur, Uttar Pradesh, India affiliated to to CCCS CS UUnniveiversityrsity, , MeeMeerutrut, , UUttttaarr used foor r rerepplacinlacing g mmaannual ual wowork.rk. Automatic media dispensers
Pradesh, India.
74068 Mohit and Sirohi. Plant tissue culture techniques in crop improvement
are helpful to pipette pre-set volume of media. A gyratory per second per sq. centimeter or approximately 1076 foot
shaker or a reciprocal shaker is necessary if micro-propagation candle) with some up to 5 to 10 Klux (672-1345 μmole/m2/s).
is based on liquid media or suspension cultures. Computers, The developmental stage of the plants also determines if wide
photocopiers and fax machines are helpful for easy data spectrum or cool white-fluorescent lights are to be used.
management and maintenance of records. Shelves within the growth rooms vary depending upon the
situation and the plants grown. Frames for the shelves can be
Activity specific requirements: Based on the different made from 1.25cm (half-inch) thick angle iron.
activities of a tissue culture, a facility can be divided into semi-
clean, clean and ultra-clean areas. The semi-clean areas Greenhouse facility: A critical stage in plant tissue culture is
comprise of the washing room, office and staff restrooms, the interim phase between the laboratory and field conditions.
where there is no need for maintaining sterile conditions. The In vitro derived plants need to be gradually hardened to field
clean areas encompass the media preparation and sterilization conditions. Plant hardening is usually carried out under
rooms, which have to be sufficiently clean. High sterility has to greenhouse that ensures high survival of the tissue-cultured
be maintained in the culture transfer rooms and the growth plants in the field. Appropriate light, shading and blackout
rooms, which constitute the ultra-clean areas. systems can be achieved with supplementary lighting.
Glassware washing and storage area: The glassware Process involved in Plant Tissue Culture Technique
washing area should be located near the sterilization and
medium preparation rooms. Both hot and cold water should be There are some common stages present in the different plant
available and the water still and de-ionization unit should be tissue culture methods as follows:
located nearby. Ovens or hot air-cabinets should be located
close to the glassware washing and storage area. Dust-proof 1. Preparation of nutrient medium: A semi-solid
cabinets and storage containers should be installed to allow for medium is prepared in double distilled water containing
easy access to glassware. macro elements, micro elements, amino acids, vitamins,
iron source, carbon source like sucrose and phyto-
The Store: It is advisable to have a separate area for storage of hormones.
chemicals, apparatus and equipment. 2. Establishment of aseptic culture: The starting
material for the process is normally an actively growing
Media preparation and sterilization area: The media shoot tip of auxiliary or terminal bud or shoot tip of a
preparation room should have smooth walls and floors, which plant.
enable easy cleaning to maintain a high degree of cleanliness. 3. Inoculation: Inoculation is carried out under aseptic
The media preparation and sterilization can be carried out in conditions. In this process explants or micro shoots are
the same area but preferably in different rooms, which need transferred on to the sterilized nutrient medium.
not be separated with doors. It is essential to have safety 4. Development of plant in growth room: After the
devices like fire extinguisher, fire blanket and a first aid kit in inoculation of the plant tissue, the bottles are sealed and
the media preparation room. A variety of glassware, plastic transfer red into growth room to trigger developmental
ware and stainless steel apparatus is required for measuring, process under diffused light (fluorescent light of 1000-
mixing, and media storage. 2000 lux) at 25 ± 2 o Cand 50 to 60% relative humidity.
5. Hardening of micro plants: Due to very high humidity
Sterilization room: The sterilizing room should be in inside the culture vessel and artificial conditions of
continuation with the media preparation room. The sterilization development, the plantlets re tender and are therefore
room must have walls and floors that can withstand moisture, are not ready for coping up with the field conditions.
heat and steam. For large volume media making, horizontal or 6. Transfer to the Green house: Due to very high
vertical autoclaves should be installed. environmental fluctuations in the field, young
developed plants can transfer to the green house for
Transfer room: The most important work area is the culture increasing its survival in the field. Green house have the
transfer room where the core activity takes place. The transfer less environment fluctuations as comparable to the
area needs to be as clean as possible and be a separate room field.
with minimal air disturbance. Fire extinguishers and first aid
kits should be provided in the transfer room as a safety Types of Plant Tissue Culture Techniques
measure. The personnel should leave shoes outside the room.
Special laboratory shoes and coats should be worn in this area. 1. Seed Culture: It is performed by surface sterilization
and in vitro culture for increasing efficiency of
Growth room: Growth room is an equally important area germination of seeds which that are difficult to
where plant cultures are maintained under controlled germinate in vivo.
environmental conditions to achieve optimal growth. Lights 2. Embryo Culture: Embryo culture is the sterile
directly fitted to the racks create uneven heat distribution. This isolation and growth of an immature or mature embryo
leads to high humidity within the culture containers, which in in vitro, with the goal of obtaining a viable plant. In
turn can cause hyperhydricity. Sideways illumination is an plant breeding embryo culture have been valuable tools,
alternative, which requires less number of lights, and provides especially for the transfer of biotic and abiotic
more uniform lighting. Temperature in the growth room is resistance genes from wild relatives into crop plants.
usually controlled with air conditioners. Generally, 3. In vitro pollination: When pollen is applied to stigma
0
temperatures are kept around 22 C. Some plant cultures can be of ovaries cultured in vitro or directly onto ovules
kept in complete darkness; however, most culture rooms need cultured with or without placental tissue, it is called in
to be illuminated at 1 Klux [134.5 μmole/m2/s (microeinsteins vitro pollination. Ovaries are collected from
74069 International Journal of Current Research, Vol. 10, Issue, 10, pp.74067-74070, October, 2018
emasculated flowers usually 1-2 days after anthesis and mixture of the appropriate polysaccharide-degrading
cultured intact or with the ovarian, wall removed to enzymes.
expose the placenta. Alternatively, the entire placenta or 12. In vitro mutagenesis: One of the applications of tissue
pieces of placenta bearing ovules may be cultured. culture systems is their exploitation for the induction
4. Haploid Technique: Plant tissue culture have extended and isolation of mutant cells, which can then be
the range of crop species from which haploid plants regenerated as mutant plants. While a number of
have been produced as well as the efficiency resulting mutations have been recognized in plant cells in vitro.
in large-scale haploid plant production by anther and 13. Somaclonal Variations: In plant breeding tissue
microspore culture techniques Specialized plant tissue culture in conventional micro propagation has resulted
culture methods have enabled the production of to a large extent in clonal fidelity, it has become
completely homozygous breeding lines from gametic increasingly clear that under the appropriate culture
cells in a shortened time frame compared to conditions, a great deal of genetic variability can be
conventional plant breeding. recovered in regenerated plants. In early report, most of
5. Ovary or ovule culture: In flowering plants, the ovule the variations were attributed to the readily detected
is typically located inside the ovary of the gynoecium chromosome instability of cultured plant cells.
which produces the ovules. Ovule consists of three Reorganization of this spontaneous variation inherent in
parts: the integument(s) forming its outer layer(s), the long- term culture led to the use of cell culture for
nucellus (or megasporangium), and the megaspore- mutagenesis and selection of genetic variants and for
derived female gametophyte (or megagametophyte) in direct recovery of novel genotypes from cell cultures
its center. via somaclonal variation.
6. Shoot apical meristems culture: This is a method of 14. Genetic transformation: The ability to move DNA
asexual propagation used to produce clones of a into an organism and thereby alter its genotype or
particular plant in large quantities. The shoot apex genetic makeup is central to both basic and applied
explant measures between 100 to 500μm and includes molecular biology. Genes derived from unrelated
the apical meristem with 1 to 3 leaf primordia. species and even other kingdoms, such as bacteria,
7. Nodal segment or axillary bud culture: This consists fungi, plants, animals, that would otherwise be
of a piece of stem with axillary bud culture with or inaccessible to an organism, can be combined in the lab
without a portion of shoot. When only the axillary bud using genetic transformation techniques.
is taken, it is designated as “axillary bud” culture. These
techniques are mostly applied for mass propagation. Applications of Plant Tissue Culture in Crop Improvement
8. Synthetic seeds: Synthetic seeds (somatic embryo as
substitutes for true seeds) can be produced either as Germination of seeds which that are difficult to
coated or non-coated, desiccated somatic embryos or as germinate.
embryos encapsulated in hydrated gel (usually calcium Make wide crosses with a greater number of related
alginate). Successful utilization of synthetic seeds as species of wild plants and have access to a much wider
propagules of choice requires an efficient and range of genes that can be used for genetic
reproducible production system and a high percentage improvement of crop plants.
of post-planting conversion into vigorous plants. Shortening of breeding cycle culturing immature
Artificial coats and gel capsules containing nutrients, embryos especially in marker assisted selection (MAS).
pesticides and beneficial organisms have long been Overcoming seed dormancy, self-sterility of seeds and
thought as substitutes for seed coat and endosperm. embryo abortion due to incompatibility barriers.
9. Callus cultures: Any explant i.e. any plant parts can be Embryo rescue in distant (inter-specific or inter-
cultured to initiate callus. A callus is a mass of generic) hybridization where endosperm development is
unorganized cells, which in many cases, upon transfer poor.
to suitable medium, is capable of giving rise to shoot- Production of homozygous diploid lines through
buds and somatic embryos, which then form complete chromosome doubling, thus reducing the time required
plants. In some instances it is necessary to go through a to produce inbred lines.
callus phase prior to regeneration via somatic Developing “Double Haploid (DH)” mapping
embryogenesis or organogenesis. populations for QTL analysis.
10. Suspension Culture: Tissues and cells cultured in a Unfertilized ovary and ovule culture may lead to
liquid medium produce a suspension of single cells and production of haploid plants
cells clumps of few to many cells: these are called Overcoming abortion of embryos of wide hybrids at
suspension cultures. Suspension cultures grow much very early stages of development due to incompatibility
faster than callus cultures need to be sub-cultured about barriers. Mass in vitro propagation for plantation, virus
every week, allow a more accurate determination of the free plant and desirable genotypes.
nutritional requirements of cells and are amenable to Facilitation of germplasm exchange between locations
scaling up for a large scale production of cells and even (production of clean material) and Cryopreservation
somatic embryos (SEs). The suspension cultures are (cold storage) or in vitro conservation of germplasm.
broadly grouped as batch cultures, continuous cultures One of the major pathway of regeneration and
and immobilized cell cultures. production of artificial seeds.
11. Protoplast isolation, culture and fusion: A protoplast For generation of useful somaclonal variants (genetic or
is a cell that had its cell wall completely or partially epigenetic), production of metabolites and in vitro
removed using either mechanical or enzymatic means. selection.
Cell walls are made of a variety of polysaccharides.
Protoplasts can be made by degrading cell walls with a
74070 Mohit and Sirohi. Plant tissue culture techniques in crop improvement
Combining genomes to produce somatic hybrids, For doing this there is no alternate of plant tissue culture. Plant
asymmetric hybrids or cybrids, plants that show tissue culture is now a well established technology which has
physical or chemical incompatibility in normal sexual made significant contributions to the propagation and
crosses, may be produced by the fusion of protoplasts improvement of agricultural crops.
obtained from two cultures of different species and
cybridization especially for transfer of cytoplasmic REFERENCES
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