Plant tissue culture 1

Plant tissue culture 1

Which medium is specially designed for woody plants?

Woody Plant Medium WPM specially formulated by Lloyd and McCown in 1980 to meet nutritional demands of woody trees shrubs and hard to culture species which exhibit ammonium toxicity tip necrosis and vitrification on high salt MS medium. WPM lowers total nitrogen to roughly one quarter of MS, ammonium nitrate 400 mg per L versus 1650, calcium nitrate increased to improve lignification and middle lamella stability, potassium sulfate replaces potassium nitrate at lower level to reduce hyperhydricity, and microelements adjusted copper sulfate elevated for lignin biosynthesis polyphenol oxidase. Its ionic strength about 30 mM compared to 90 mM in MS prevents osmotic stress and phenol leaching inhibitory to woody explants rich in tannins. Inclusion of phloroglucinol or activated charcoal 0.1-0.5 percent adsorbs phenolic exudates preventing browning. WPM and derivatives DKW for walnut, Quoirin Lepoivre for fruit trees enable sustained shoot proliferation rooting and somatic embryogenesis of species like Rhododendron, Pinus, Eucalyptus, Quercus where MS fails. Thus WPM has become standard for forestry and woody horticulture micropropagation ensuring healthier shoots with expanded leaves and functional vascular development.

Ref: Lloyd & McCown Int Plant Prop Soc 1980 WPM; NCBI woody plant medium formulation.

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Plant tissue culture 1

Increase in secondary metabolites in cell culture is commonly achieved by:

Increasing secondary metabolite accumulation in plant cell cultures is commonly achieved by supplementation with elicitors after achieving adequate biomass. Secondary metabolism is resource costly competing with primary growth for carbon nitrogen and energy, regulated by growth defense trade off controlled by TOR kinase promoting growth versus SnRK1 promoting defense. Under normal culture high auxin low stress conditions flux stays in primary metabolism. Elicitor addition artificially activates jasmonate and salicylate mediated defense transcriptome inducing genes encoding rate limiting enzymes: phenylalanine ammonia lyase PAL for flavonoids rosmarinic acid, tryptophan decarboxylase for alkaloids, HMGR for terpenoids, and transcription factors ORCA3 in Catharanthus roseus regulating strictosidine synthase. Timing matters addition at late log phase prevents growth inhibition while maximizing product up to 10-20 fold. Combining elicitation with precursor feeding, media optimization reduced nitrogen, and permeabilizing agents DMSO enhances release. Industrial examples include methyl jasmonate raising paclitaxel in Taxus cell cultures, yeast extract raising shikonin in Lithospermum, chitosan raising berberine in Coptis, forming basis of biotechnological production of pharmaceuticals pigments and flavors in bioreactors.

Ref: Mishra & Ranjan 2008 secondary metabolite; PubMed elicitor methyl jasmonate.

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Plant tissue culture 1

Growth index of callus culture is calculated using:

Growth index measures proliferative performance of callus cultures quantitatively allowing comparison across media treatments hormone combinations and genotypes. Most accurate method utilizes change in dry weight rather than fresh weight or visual scoring because fresh weight is confounded by variable water content due to hyperhydricity, extracellular polysaccharide secretion, and vacuolar water uptake that fluctuates with osmolarity of medium. Dry weight determination involves harvesting callus, washing to remove agar, oven drying at 60 to 80 degrees Celsius to constant mass eliminating moisture, weighing analytical balance. Index calculated as final dry weight minus initial inoculum dry weight divided by initial dry weight multiplied by 100 percent, or sometimes as ratio of final to initial. This metric reflects true biomass accumulation including cell number increase and deposition of cell walls proteins and starch. Cell number counting is impractical in clumpy callus requiring enzymatic dissociation inducing error. Monitoring dry weight growth curve shows lag exponential stationary phases and correlates with metabolic activity secondary metabolite yield and requirement for timely subculture every 3-4 weeks preventing nutrient depletion and phenolic browning.

Ref: Bhojwani & Razdan 1996 growth indices; Plant Cell Culture protocols dry weight method.

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Plant tissue culture 1

Shoot tip culture is an example of:

Shoot tip culture qualifies as organ culture because explant retains organized structure of shoot apical meristem plus one to three leaf primordia and continues organized growth maintaining polarity and tissue layer arrangement tunica corpus. Unlike callus culture where organization is lost into unorganized parenchyma, or cell suspension where aggregates disperse in liquid agitation, or protoplast culture where wall removed exposing naked membrane, shoot tip preserves defined zonation central zone peripheral zone rib meristem and procambium differentiation pathway. Culture on hormone balanced medium supports leaf expansion and internode elongation converting tip into miniature shoot that can be rooted. Since meristem remains intact, organ culture ensures high genetic stability avoiding mutation accumulation common in callus phase. Practical uses range from micropropagation of horticultural species to germplasm conservation under slow growth minimal medium and virus elimination when larger meristem is needed for survival in recalcitrant species where 0.1 mm meristem excision suffers low regeneration. Thus shoot tip culture bridges meristem culture and nodal culture methods for commercial clonal production.

Ref: George et al., Plant Propagation Tissue Culture Vol1; Hartmann organ culture.

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Plant tissue culture 1

Meristematic tissues are virus free because:

Meristematic tissues remain virus free because virus multiplication and movement are inhibited in rapidly dividing apical dome cells. Most plant viruses rely on plasmodesmata intercellular connections and phloem sieve tubes for systemic translocation from source leaves to sinks; meristem dome lacks differentiated phloem and contains narrow plasmodesmata with high callose deposition limiting viral cell to cell trafficking. Additionally mitotic cycle of meristem cells is significantly shorter than time required for virus replication and assembly, so daughter cells produced outrun infection front resulting in exclusion. High metabolic rate leads to elevated endogenous auxin levels and high oxygen radical scavenging antioxidant activity creating unfavorable environment for viral polymerase function. Molecular evidence indicates strong RNA silencing response in meristem with high expression of Dicer like proteins DCL1-4 and RNA dependent RNA polymerase RDR6 generating small interfering RNAs that degrade viral genomes. Consequently even severely infected mother plant harbors distal 0.1 millimeter virus free zone exploitable for phytosanitation, providing basis for meristem culture combined with heat therapy for eradication of persistent viruses in vegetatively propagated crops.

Ref: Cassells 1991 virus free meristem; Lodish Chap. 8 viral movement.

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Plant tissue culture 1

Meristem culture is mainly used to produce:

Meristem culture technique involves aseptic excision of apical dome typically 0.1-0.5 millimeter containing tunica corpus but no differentiated leaf primordia and culture on MS based medium for regenerating plants principally to obtain virus free stocks. Foundation lies in observation that meristematic dome lacks differentiated vascular sieve elements through which many systemic viruses like Potato Virus Y, Tobacco Mosaic Virus, Cucumber Mosaic Virus travel long distance and also that rapid mitotic activity outpaces viral replication. High endogenous auxin and cytokinin concentration plus RNA silencing machinery Dicer like DCL proteins Argonaute AGO targeting viral RNAs creates antiviral niche. Protocol often combined with thermotherapy incubating donor at 35-38 degrees for weeks reducing virus titer or chemotherapy with antiviral ribavirin. Regenerants are indexed by ELISA RT PCR to confirm elimination. Because meristem cells maintain genetic stability avoiding callus mediated somaclonal variation, meristem derived plants retain true to type phenotype valuable for certified seed programs for potato garlic banana strawberry where vegetative propagation accumulates viruses causing yield decline, thus meristem culture ensures healthy foundation material.

Ref: Bhojwani & Dantu Chap. 11 virus elimination; Walkey 1978 Phytopath Z.

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Plant tissue culture 1

Elicitors enhance secondary metabolite production by:

Elicitors boost secondary metabolite production by mimicking biotic attack triggering plant innate defense signaling and activation of defence related enzymes. Biotic elicitors include fungal cell wall fragments chitin chitosan glucans yeast extract bacterial flagellin; abiotic include jasmonic acid methyl jasmonate salicylic acid heavy metals copper cadmium UV-C ozone. Perception by pattern recognition receptors triggers calcium influx cyclic nucleotide gated channels, production of reactive oxygen species via NADPH oxidase RBOH, activation of mitogen activated protein kinase cascades MPK3 MPK6 leading to jasmonate isoleucine synthesis. Jasmonate signals transcription factors MYB WRKY ORCA that bind promoters of phenylpropanoid pathway enzyme phenylalanine ammonia lyase PAL cinnamate 4 hydroxylase chalcone synthase CHS, and terpenoid pathway HMGR terpene synthase. Resulting phytoalexins alkaloids paclitaxel vincristine flavonoids accumulate up to tenfold. Commercial process employs two stage culture: biomass accumulation stage low elicitor high auxin, then production stage elicitor addition at late exponential phase to redirect carbon flux from primary to secondary metabolism without severe growth inhibition enabling industrial bioreactor harvesting.

Ref: Ramachandra & Ravishankar Plant Cell Rep 2002 elicitors; Nature Plants secondary metabolism.

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Plant tissue culture 1

Ethylene differs from other plant hormones because it is:

Ethylene stands unique among classical phytohormones being only gaseous hydrocarbon C2H4 with high diffusivity without need for active transport proteins. Biosynthesis follows methionine to S-adenosyl methionine AdoMet then ACC via ACC synthase ACS multigene family rate limiting, finally oxidation to ethylene by ACC oxidase ACO requiring iron ascorbate oxygen. As volatile it accumulates in sealed culture vessels influencing morphogenesis unless vented. Signaling occurs at endoplasmic reticulum membrane where receptors ETR1, ERS1, ETR2 function as negative regulators; in absence of ethylene they activate CTR1 Raf like kinase suppressing EIN2. Ethylene binding inactivates receptors, deactivating CTR1 allowing EIN2 C terminal cleavage translocating to nucleus stabilizing EIN3 EIL1 transcription factors which induce ERF ethylene response factors activating genes for fruit ripening polygalacturonase cellulase, abscission, triple response exaggerated apical hook shortened hypocotyl in etiolated seedlings. In vitro excess ethylene causes hyperhydricity leaf yellowing abscission, mitigated by inhibitors silver nitrate silver thiosulfate that block receptor or by using vented lids permeable closures.

Ref: Bleecker & Kende Annu Rev Cell Dev Biol 2000 ethylene; NCBI NBK21448 ethylene.

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Plant tissue culture 1

Abscisic acid is known as:

Abscisic acid designated as stress hormone and growth inhibitor originates from carotenoid pathway, cleavage of 9 cis violaxanthin and neoxanthin by NCED dioxygenase to xanthoxin then converted by ABA2 short chain dehydrogenase and AAO3 aldehyde oxidase to ABA. Under drought salinity cold endogenous levels rise sharply triggering PYR PYL RCAR receptor family that sequesters PP2C phosphatases ABI1 ABI2 releasing SnRK2 kinases OST1 to phosphorylate targets including bZIP transcription factor ABI5 inducing late embryogenesis abundant LEA genes, anion channel SLAC1 causing stomatal closure reducing transpiration, and enzymes for osmolyte synthesis proline betaine. ABA antagonizes gibberellin by stabilizing DELLA repressors enforcing dormancy and preventing precocious germination vivipary. In tissue culture ABA 1-10 micromolar improves somatic embryo quality preventing secondary embryogenesis, induces desiccation tolerance for synthetic seed storage, synchronizes maturation and enhances acclimatization by promoting cuticular wax deposition. Catabolism via CYP707A hydroxylases maintains homeostasis fine tuning stress response and developmental transitions crucial for embryo physiology.

Ref: Cutler et al., Annu Rev Plant Biol 2010 ABA signaling; NCBI NBK541119 ABA.

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Plant tissue culture 1

Gibberellins are mainly involved in:

Gibberellins are tetracyclic diterpenoid hormones with GA1 GA3 GA4 most bioactive functions centered on stem elongation, seed germination and floral transition. Biosynthesis proceeds from geranylgeranyl diphosphate via ent kaurene ent kaurenoic acid to GA12 catalyzed by terpene synthases and cytochrome P450 monooxygenases GA20 oxidase and GA3 oxidase. Signaling involves soluble receptor GID1 binding GA causing conformational change enabling interaction with DELLA repressors which are then ubiquitinated by SCF SLY1 GID2 complex and degraded via proteasome allowing transcription of growth genes. Physiologically gibberellins induce alpha-amylase and protease expression in barley aleurone via GAMYB transcription factor mobilizing starch reserves during germination antagonizing ABA. They promote internode elongation by enhancing cell division in intercalary meristem and wall extensibility through xyloglucan endotransglycosylase and expansin induction. In vitro GA3 at 0.1-1 mg per L elongates dwarf shoots, breaks bud dormancy, induces precocious flowering, though excessive level inhibits rooting and somatic embryo maturation requiring careful titration within medium composition.

Ref: Sun, Annu Rev Plant Biol 2008 GA-DELLA signaling; NCBI NBK10975 gibberellin pathway.

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Plant tissue culture 1

Cytokinins mainly promote:

Cytokinins constitute class of N6 substituted adenine derivatives including natural trans zeatin, isopentenyl adenine and synthetic benzylaminopurine BAP, kinetin, that primarily drive shoot proliferation, cell division and delay senescence. Perception occurs through membrane localized histidine kinase receptors AHK2, AHK3, AHK4 autophosphorylating upon cytokinin binding, transferring phosphate to histidine phosphotransfer proteins AHPs that shuttle to nucleus phosphorylating type-B Arabidopsis Response Regulators ARR1, ARR10, ARR12 transcription factors. Type-B ARRs directly induce expression of WUSCHEL maintaining stem cell niche, SHOOT MERISTEMLESS and KNAT1 KNOX genes preventing differentiation, and D-type cyclin CYCD3;1 accelerating G1 to S transition. Cytokinins also antagonize apical dominance by promoting axillary bud outgrowth and counteract ethylene and ABA induced senescence by stabilizing chloroplasts and photosynthetic enzymes. In tissue culture BAP 1-5 mg per L induces multiple shoots from single explant enabling exponential multiplication; high cytokinin low auxin ratio ensures caulogenesis while balanced ratio maintains callus. Thus cytokinins are essential tool for commercial clonal propagation of orchids, banana and forestry species.

Ref: Mok & Mok Annu Rev Plant Biol cytokinin metabolism; Kieber & Schaller Arabidopsis Book 2014.

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Plant tissue culture 1

Auxins mainly promote:

Auxins represent principal hormone class promoting root initiation and development, encompassing natural indole-3-acetic acid IAA synthesized via Trp dependent TAA1 YUCCA flavin monooxygenase pathway and synthetic analogues NAA, IBA, 2,4-D with higher stability and resistance to enzymatic oxidation by IAA oxidases. For rhizogenesis auxin maximum generated at basal end through PIN efflux carrier mediated polar transport triggers pericycle or phloem parenchyma cells to become root founder cells. Signaling involves TIR1/AFB auxin receptors part of SCF ubiquitin ligase complex that in presence of auxin ubiquitinates Aux/IAA repressors for degradation freeing Auxin Response Factors ARF7 ARF19 to induce downstream genes LBD16 LBD29 WOX5 defining quiescent center and columella. Cellular effect includes activation of H+ ATPase acidifying apoplast activating expansins wall loosening enabling primordia emergence. In micropropagation rooting phase exogenous IBA 0.5-2 mg per L induces adventitious roots from shoot bases within 7-14 days. High auxin represses cytokinin responses ensuring root program dominates over shoot proliferation, critical for acclimatization and field establishment.

Ref: Woodward & Bartel Ann Bot 2005 Auxin biology; Lavy & Estelle Development auxin signaling.

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