(a) Briefly explain the method of herbaria preparation. What is its role in modern taxonomic research? (20 marks) (b) Write an…

(a) Method of herbarium preparation, and its role in modern taxonomic research (20 marks)

Method (15 marks). A herbarium is a collection of dried, pressed, mounted and labelled plant specimens.

1. Collection: take a representative specimen with roots (for herbs), stem, leaves, flowers and, if possible, fruits, about 30 by 42 cm, using a digger, secateurs and a vasculum or polythene bag. Record in a field book the collection number, date, locality, habitat, altitude, plant habit, colour of parts, local name and uses, and take a GPS position.
2. Pressing: arrange the specimen flat between sheets of blotting paper or newspaper, with the leaves shown from both sides, and place it in a plant press between corrugated boards and tighten it.
3. Drying: change the driers daily in the first few days; dry in the sun, a ventilated warm place or a dryer for one to two weeks, until brittle. Fleshy parts may be sliced, and large fruits dried separately.
4. Poisoning and pest control: earlier the specimens were treated with a solution of mercuric chloride in alcohol; nowadays safer methods such as freezing at -20 °C for a few days or fumigation are preferred, along with naphthalene or paradichlorobenzene in cabinets.
5. Mounting: fix the dry specimen on a standard herbarium sheet (about 42 by 28 cm) with glue, gummed strips or stitching, with loose material in a packet.
6. Labelling: paste a label (about 11 by 7 cm) at the bottom right corner, with family, botanical name, locality, habitat, date, collector and number.
7. Storage: file the sheets in genus covers and family folders in insect-proof cabinets, arranged by a system such as Bentham and Hooker, and keep them dry with regular checks and fumigation.

Role in modern taxonomic research (5 marks):

• It is a permanent reference for identification and comparison of specimens.
• It holds type specimens that fix the application of names under the botanical code.
• It documents distribution, biodiversity and flowering time, the basis of floras, monographs and revisions.
• Old specimens yield DNA for molecular phylogeny and give data on climate change and extinct or rare taxa.
• Digitisation into virtual herbaria (for example, GBIF) makes specimens available worldwide.

(b) Types, regulation and significance of totipotency (15 marks)

Types (5 marks). Totipotency is the ability of a cell to give rise to a whole organism. In plants, most living cells retain it, but its expression differs:

• Zygote and embryonic cells are naturally totipotent.
• Differentiated somatic cells express it after dedifferentiation, forming callus and then either shoots and roots (organogenesis) or embryos (somatic embryogenesis).
• The pathway may be direct, from the explant, or indirect, through callus.

Regulation (5 marks):

• Hormones: the auxin to cytokinin ratio; a high ratio favours roots and embryogenic callus, and a low ratio favours shoots (Skoog and Miller).
• Genes: transcription factors such as WUSCHEL, BABY BOOM, LEC1, LEC2 and the SERK receptor kinases start dedifferentiation and embryogenesis.
• Epigenetic changes: chromatin remodelling, histone modification and DNA methylation.
• Stress and medium: 2,4-D, heat or osmotic stress, sugar, nitrogen source, pH and light, and the genotype and age of the explant.

Significance (5 marks):

• Micropropagation, somatic embryogenesis and synthetic seeds for clonal multiplication.
• Regeneration of transgenic plants after genetic transformation and of hybrids from protoplast fusion.
• Virus-free plants by meristem culture, and germplasm conservation by cryopreservation.
• Haploid production, in vitro selection and secondary metabolites; and the experimental proof that a plant cell has the whole genetic programme.

(c) Haploid plant production through tissue culture, and advantages of homozygous diploid lines (15 marks)

Haploid production (10 marks):

• Androgenesis: anthers or isolated microspores are cultured at the late uninucleate stage, often after cold pretreatment (about 4 °C for several days), on MS or N6 medium with sucrose (6 to 9%) and 2,4-D or other hormones. The pollen turns from its normal path and forms embryos directly or through callus, which develop into haploid plantlets. Guha and Maheshwari first obtained haploids in Datura in 1964, and the method works in tobacco, rice, wheat, barley and Brassica. Problems include low frequency and albino plants.
• Gynogenesis: unpollinated ovaries or ovules are cultured, and the haploid embryo sac forms an embryo (barley, sugar beet, onion).
• Elimination of the chromosomes of the pollen parent after wide crosses, such as the Hordeum bulbosum method in barley, followed by embryo rescue in vitro.
• Chromosome doubling with colchicine (about 0.1 to 0.2%), or spontaneously, gives a doubled haploid.

Advantages of homozygous diploid lines (5 marks):

• Complete homozygosity in a single generation, instead of six to eight generations of selfing, saving time and cost.
• Recessive traits and mutations are expressed and selected effectively.
• Stable lines fix desirable gene combinations and can be used as inbred parents of hybrids.
• Ideal populations for mapping genes and QTLs, and for transformation with homozygous transgenes.