Plant growth regulators (PGRs) are the variables that determine what a piece of plant tissue in culture actually does — whether it forms a callus, multiplies as shoots, or initiates roots. Of all the PGRs used in tissue culture, BAP and NAA are the most commonly encountered. Understanding how they work and how they differ gives you real control over your protocols.
The Auxin–Cytokinin Ratio: The Fundamental Control
Skoog and Miller established in 1957 that the ratio between auxins and cytokinins in culture medium determines the morphogenic response of plant tissue:
- High auxin : low cytokinin → root formation
- Low auxin : high cytokinin → shoot formation and multiplication
- Balanced auxin : cytokinin → callus (undifferentiated cell mass)
This ratio principle, not absolute concentrations, is what you’re working with when you design a TC protocol. BAP and NAA sit on opposite sides of this balance.
BAP — 6-Benzylaminopurine (Cytokinin)
BAP is a synthetic cytokinin — one of the most widely used in TC work. Its role is to promote cell division and, at appropriate ratios with auxin, shoot organogenesis (the formation of shoot buds from callus or explants).
What BAP does:
- Promotes axillary bud break and shoot multiplication
- Suppresses apical dominance (encourages branching in culture)
- Stimulates callus proliferation in combination with auxin
- Can inhibit root formation at high concentrations
Typical concentrations:
- Shoot multiplication: 0.5–5.0 mg/L, depending on species
- Callus induction: 0.1–2.0 mg/L in combination with auxin
Caution:
High BAP concentrations or extended exposure can cause hyperhydricity (vitrification) — cultures become watery, translucent, and non-viable. If you see this, reduce BAP concentration or reduce subculture intervals.
NAA — Naphthaleneacetic Acid (Auxin)
NAA is a synthetic auxin. It mimics the action of the natural plant hormone IAA (indole-3-acetic acid) but is more stable in autoclaved medium and more persistent in culture.
What NAA does:
- Promotes root initiation and elongation (primary use in rooting stage)
- Induces callus formation at high concentrations
- Suppresses shoot formation when dominant
Typical concentrations:
- Root induction: 0.1–2.0 mg/L on ½ MS
- Callus induction: 1.0–5.0 mg/L in combination with cytokinin
NAA vs IBA for rooting:
IBA (indole-3-butyric acid) is often preferred over NAA for rooting because it produces more natural root morphology in many species. NAA can produce thick, stubby roots that transition poorly to ex vitro conditions. IBA is metabolised in the plant into IAA, making it gentler. In practice, try both: some species root better on NAA, others on IBA.
A Practical Protocol Example
Shoot multiplication stage (generic):
MS + 1.0 mg/L BAP + 0.1 mg/L NAA, pH 5.7
Rooting stage:
½ MS + 0.5 mg/L IBA (or NAA), no cytokinin, pH 5.7
The shift to cytokinin-free medium for rooting is important — residual cytokinin from multiplication medium inhibits root formation. Some protocols include an auxin pulse (high concentration for 3–5 days) followed by transfer to hormone-free ½ MS for root development.
Stock Solutions
BAP and NAA are both poorly soluble in water. Dissolve in a small volume of 1N NaOH (BAP) or 70% ethanol (NAA) before diluting to working concentration. Prepare 1 mg/mL stock solutions, filter-sterilise (0.22 µm), and store at 4°C. Add to medium after autoclaving when the medium has cooled to around 50°C.