(a) What factors affect in vitro stages of micropropagation? Discuss the applications and limitations of micropropagation. (20…

(a) Micropropagation: factors, applications and limitations

Factors affecting the in vitro stages:

• Explant: shoot tip or axillary bud from a healthy, young, well-maintained mother plant gives low contamination and high regeneration; season and size matter.
• Medium: mineral salts (MS), sucrose, vitamins, gelling agent and pH about 5.8.
• Growth regulators: a higher cytokinin to auxin ratio favours shoot multiplication; auxin (IBA, NAA) favours rooting.
• Culture conditions: temperature about 25 ± 2 °C, 16-hour photoperiod, light intensity and humidity in the vessel.
• Sterility and phenolics: surface disinfection and control of browning by antioxidants such as ascorbic acid or activated charcoal.
• Ex vitro transfer: hardening under gradually reduced humidity.

Applications: rapid, uniform clonal multiplication of elite plants, e.g. banana, orchids, sugarcane; virus-free plants from meristem culture, e.g. potato; germplasm conservation; year-round production; and easy movement of clean material.

Limitations: high cost of labour and facilities; microbial contamination; somaclonal variation in long-term cultures; recalcitrance of woody plants; vitrification (hyperhydricity); loss in hardening; and need for skilled staff.

**(b) Male gametophyte of Gnetum; angiosperm characters**

Male gametophyte: The microsporangia form on the microsporangiate (male) strobilus. The microspore mother cells divide meiotically to give haploid microspores. In the endosporic development, the microspore nucleus divides to give a prothallial cell and an antheridial initial; the latter divides into a generative cell and a tube nucleus. The pollen grain is shed at this stage. On the nucellus the pollen tube grows, and the generative cell divides to give two non-motile male gametes, without flagella (unlike Cycas and Ginkgo), which fertilise the female nuclei.

*Angiosperm-like characters in Gnetum:*

• Vessels in the secondary wood.
• Broad leaves with reticulate venation in Gnetum.
• Flower-like strobili with a perianth-like envelope and opposite bracts.
• Non-motile male gametes and a pollen tube.
• Absence of archegonia, with a free-nuclear female gametophyte, and a second fertilisation-like event.
• Absence of resin canals.

(c) Protoplasts from leaves: yield, viability and purification

Factors affecting yield and viability:

• Source: young, fully expanded leaves from healthy, preferably in vitro grown plants, with the lower epidermis peeled or cut into strips.
• Enzymes: cellulase (1–2%) and macerozyme or pectinase (0.1–0.5%), with hemicellulase, in the correct concentration and pH (5.5–5.8).
• Osmoticum: 0.4–0.6 M mannitol or sorbitol, which prevents bursting, and calcium ions to stabilise the membrane.
• Incubation: temperature about 25–28 °C, dark or dim light, for 4–16 hours, gentle shaking.
• Genotype and physiology of the source.

Purification: the digest is filtered through nylon sieve (50–100 µm) to remove debris, and centrifuged at low speed (about 100 g); protoplasts are then floated on a 0.4–0.6 M sucrose cushion, where intact protoplasts collect at the interface. After washing with osmoticum, viability is checked with FDA (green fluorescence) or Evans blue.