(a) Basal media, growth regulators, sterilization and culture conditions are essential components of plant tissue culture…

(a) Essential components of plant tissue culture

1. Basal medium. The medium supplies everything a heterotrophic explant needs.

• Macronutrients: N (as nitrate and ammonium), P, K, Ca, Mg, S.
• Micronutrients: Fe (as Fe-EDTA, so that it stays available), Mn, Zn, B, Cu, Mo, Co, I.
• Vitamins and organic additives: thiamine (essential), nicotinic acid, pyridoxine, myo-inositol; sometimes glycine, casein hydrolysate or coconut water.
• Carbon source: usually 2–3% sucrose, because cultured tissues photosynthesise little.
• Gelling agent: agar (0.6–0.8%) or Gelrite for solid media; liquid media are used for suspension cultures.
• pH: adjusted to 5.6–5.8 before autoclaving.

Common formulations. MS (Murashige and Skoog, 1962) is rich in salts and ammonium nitrate and is the general-purpose medium. B5 (Gamborg, 1968) has less ammonium and is used for cell suspensions and legumes. White's medium is low in salts and was used for root cultures. WPM (Lloyd and McCown) is used for woody plants, and N6 (Chu) for cereal anther culture. Choosing and adjusting the medium for each species is often the key to success.

2. Growth regulators. They decide what the tissue does.

• Auxins (IAA, IBA, NAA, 2,4-D, picloram): cell division, callus and roots; 2,4-D is the strongest callus and somatic-embryo inducer.
• Cytokinins (BAP, kinetin, zeatin, TDZ): shoot formation and release of axillary buds.
• Skoog and Miller's rule (1957): in tobacco pith, a high auxin : cytokinin ratio gave roots, a low ratio gave shoots, and an intermediate ratio gave callus.
• Gibberellins: shoot elongation and embryo germination.
• Abscisic acid: maturation of somatic embryos.
• Ethylene inhibitors (silver nitrate): these improve regeneration in some cultures.

The same concentration can act differently in different species or tissues, so each protocol is optimised empirically.

3. Sterilisation (asepsis). Microbes grow far faster than plant cells on a sugar-rich medium, so a single contaminant can ruin a culture.

• Explant (surface sterilisation): wash in detergent, dip in 70% ethanol for 30–60 s, then treat with 0.1% HgCl₂ or 1–2% sodium hypochlorite for a few minutes. Rinse three or four times in sterile water.
• Medium and glassware: autoclave at 121 °C and 15 psi for about 15–20 min. Heat-labile substances (some vitamins, zeatin, antibiotics) are filter-sterilised through a 0.22 µm membrane. Glassware can also be dry-heat sterilised.
• Instruments and workspace: dip instruments in alcohol and flame them, or use a bead steriliser. Work in a laminar airflow cabinet with HEPA filters; a UV lamp can be used before work.

4. Culture conditions.

• Temperature: usually 25 ± 2 °C.
• Light: a photoperiod of about 16 h light and 8 h dark, with cool-white fluorescent or LED light of roughly 25–50 µmol m⁻² s⁻¹. Some cultures, such as callus induction and anther culture, start in the dark.
• Humidity: inside the closed vessel humidity is near 100%, and the growth room is kept at about 50–70%. High humidity inside the vessel can cause hyperhydricity (vitrification), so ventilated closures help.
• Gas exchange and ethylene accumulation: both affect growth.
• Physical factors: shaking at 100–120 rpm for suspension cultures, and subculture intervals of 3–4 weeks to avoid nutrient exhaustion.
• Hardening: plantlets are acclimatised gradually before transfer to soil.

All four components act together. For example, micropropagated banana (Grand Naine) and potato are produced commercially only because each step is standardised.

(b) Pollen haploids: production, diploidisation and importance

Production (androgenesis). Guha and Maheshwari (1964) first raised haploid embryos from anthers of Datura innoxia. The method has since been extended to tobacco, rice, wheat, barley, Brassica and many other crops.

1. Choice of stage: flower buds are collected when the microspores are at the late uninucleate to early binucleate stage, which is the most responsive stage. The stage is checked by acetocarmine squash. The donor plant's growth conditions also matter.
2. Pretreatment: a stress switches the microspore from the pollen pathway to the embryo pathway. Cold (4–10 °C for a few days, as in rice and Datura), heat shock (32–35 °C in Brassica), sugar starvation, or an osmotic shock can be used.
3. Culture: surface-sterilised anthers are placed on a suitable medium, such as N6 for cereals or MS for tobacco. Alternatively, microspores are isolated and cultured alone. Isolated microspore culture avoids callus from the anther wall, which is diploid, and gives more embryos, as in Brassica napus.
4. Development: the microspore divides repeatedly.

• Direct route: it forms an embryoid, which germinates into a plantlet, as in Datura and tobacco.
• Indirect route: it forms a callus, from which shoots and roots regenerate, as in rice and wheat.

5. Confirmation of ploidy: chromosome counts, flow cytometry, or stomatal size and chloroplast number in guard cells.

Problems. Albino plants are frequent in cereals, and response depends strongly on genotype. Plants can also regenerate from the diploid anther wall, or become polyploid in callus.

Diploidisation (doubling the chromosome number)

• Colchicine: it prevents spindle formation, so the chromosomes double. Common methods are dipping the roots or crowns of plantlets in about 0.1–0.5% colchicine for a few hours, applying it to axillary buds, or adding it to the culture medium. Oryzalin, trifluralin and amiprophos-methyl act the same way at lower, less toxic doses.
• Spontaneous doubling: endomitosis or nuclear fusion during the callus phase. This is frequent in rice and in maize microspore cultures.
• Regeneration from doubled cells: shoots can be regenerated from diploidised cells of haploid callus or leaf tissue.

Importance in agricultural research

• Homozygous lines in one generation: a doubled haploid (DH) is completely homozygous. Pure lines are obtained in one or two years, instead of six to eight generations of selfing.
• Efficient selection: recessive genes are expressed at once, and there are no dominance effects to mask genotypes. Selection is therefore more accurate, and fewer plants are needed for a given chance of recovering the desired combination.
• Genetic research: DH populations are "immortal" mapping populations for QTL and linkage mapping, and for genomic selection.
• Mutation breeding and in vitro selection: haploid cells reveal recessive mutations directly.
• Hybrid breeding: pure parental inbreds for hybrids are produced faster.
• Varieties released: many cultivars worldwide have come from DH methods, including numerous rice and wheat varieties from anther culture in China. In India, CRRI (now NRRI) released the rice varieties Satyakrishna (CR Dhan 10) and Phalguni (CR Dhan 801) from DH lines.
• Limits: genotype dependence and albinism are the main constraints. Maize and wheat now rely more on in vivo haploid inducers and wide crosses (wheat × maize) than on pollen culture.

(c) Polarity and symmetry in plant morphogenesis

Morphogenesis is the origin of form. Every plant organ has axes, set by polarity, and proportions, expressed as symmetry. The two are linked: an axis must exist before tissue can be arranged symmetrically or asymmetrically around it.

Polarity is the difference between the two ends, or two sides, of a cell, tissue or organ.

• Organ level: the plant body has an apical–basal axis, with the shoot at one pole and the root at the other. Vöchting's willow cuttings produced shoots at the morphological apex and roots at the morphological base, even when hung upside down. Polarity is therefore built into the tissue and is not set by gravity alone.
• Cell level: in the Fucus zygote, which starts with no fixed polarity, unilateral light (or other gradients such as a calcium current) fixes the axis. The rhizoid bulges on the shaded side, and the first division, transverse to that axis, separates a rhizoid cell from a thallus cell (Whitaker; Jaffe). In angiosperms, the zygote is already polar: its nucleus lies towards the chalazal end. Its unequal first division gives a small apical cell, which becomes the embryo proper, and a large basal cell, which becomes the suspensor. Other examples of polar cells are tip growth in pollen tubes and root hairs, and the asymmetric divisions that form stomata.
• Mechanism: the key mechanism is polar auxin transport. PIN efflux carriers sit on one face of each cell; their placement depends on GNOM, and auxin then accumulates at particular points. The embryo's root pole needs MONOPTEROS and auxin at the basal end. Mutants such as gnom and monopteros lose the apical–basal pattern.
• Tissue culture: in culture, cuttings and explants regenerate according to their polarity. A stem segment roots from its basal end, and an explant placed upside down often does poorly.

Symmetry is the regular arrangement of parts about a point, axis or plane.

• Radial symmetry: stems and roots are radially symmetrical, as are actinomorphic flowers such as Petunia and Hibiscus. Several planes through the centre divide the organ into mirror halves.
• Bilateral symmetry: zygomorphic flowers, such as Antirrhinum, pea and orchids, can be cut into mirror halves by only one plane. In Antirrhinum, the CYCLOIDEA and DICHOTOMA genes act on the dorsal side of the flower. Mutants lacking them have radial, peloric flowers. The classic peloric Linaria described by Linnaeus arises from methylation, and so silencing, of the CYCLOIDEA homologue (Cubas et al., 1999).
• Dorsiventral symmetry of leaves: the upper (adaxial) side is set by PHABULOSA, PHAVOLUTA and REVOLUTA, and the lower (abaxial) side by KANADI and YABBY. These genes repress each other, and the blade grows out where the two domains meet. If adaxial identity is lost, radial needle-like leaves form, as in phantastica mutants of Antirrhinum.
• Phyllotaxis: the regular arrangement of leaves (spiral with Fibonacci numbers, alternate, or opposite) is a higher-level symmetry. It is generated by auxin maxima that PINs create at fixed angles on the shoot apex.

How the two are linked. Polarity comes first and sets the axes; symmetry is then laid out along and around those axes. Breaking symmetry is also a polar event, as in the Fucus zygote, the leaf primordium or the zygomorphic flower. The statement holds: without polarity there would be no organised axis, and without controlled symmetry there would be no characteristic organ shape.