What is Polyploidy Breeding?

• Polyploidy breeding refers to the deliberate induction and utilisation of polyploid plants (those with more than two complete sets of chromosomes) to create new crop varieties or to improve existing ones. 
• It exploits a phenomenon that has been the most important mechanism of crop plant evolution over geological time — polyploidy is responsible for the origin of wheat, cotton, oat, banana, sugarcane, potato, peanut, and many other major crops.

Basic Terminology 

Classification of Polyploidy

AUTOPOLYPLOIDY : INDUCTION, FEATURES, AND APPLICATIONS

Methods of Inducing Autopolyploidy

Method 1 — Spontaneous Chromosome Doubling

• Chromosome doubling occurs occasionally in somatic tissues through endomitosis (DNA replication without cell division). 
• Unreduced (2n) gametes are also produced spontaneously in low frequencies. 
• Unreduced gamete production is promoted by certain genes — genes causing asynapsis or desynapsis, or mutant genes producing parallel spindle (ps) or omission of second division (os) — as documented in potato. 
• Spontaneous polyploidy is the natural mechanism by which polyploid crop species evolved over geological time.

Method 2 — Adventitious Bud Production

• Decapitation (cutting) of plant stem leads to callus development at the cut end. 
• Callus tissue frequently contains polyploid cells that spontaneously doubled during dedifferentiation. 
• Shoot buds regenerated from this callus may be polyploid. 
• The frequency can be increased by applying 1% IAA (auxin) at the cut ends — this promotes callus development and thus more polyploid bud opportunity.

Method 3 — Physical Agents

• Heat or cold treatment, centrifugation, and X-ray or gamma-ray irradiation may produce polyploids. 
• Heat treatment has also been successful in barley, wheat, rye, and some other crop species. 
• Cold treatment produced tetraploid branches in Datura in one cited experiment.

Method 4 — Regeneration In Vitro

• Polyploidy is a common feature of cells cultured in vitro — tissue culture itself tends to produce polyploid cells. 
• Plants of various ploidy levels have been regenerated from callus cultures of Nicotiana, Datura, rice (Oryza sativa), and several other species. 
• This is a problem in tissue culture-based propagation (producing genetically variable regenerants) but can be exploited deliberately for polyploid induction.

Method 5 — Colchicine Treatment (Most Important — Most Widely Used)

Mechanism of action:

• Colchicine binds to tubulin — the protein subunit of spindle microtubules.
• It blocks spindle formation by preventing polymerisation of tubulin into microtubules.
• Without spindle fibres, chromosomes cannot move to the poles during anaphase.
• DNA replication and chromosome duplication proceed normally — but cell division does not complete.
• Result: A RESTITUTION NUCLEUS is formed — containing all the duplicated chromosomes. The chromosome number of the cell is thereby doubled.

Important practical constraints

• Since colchicine affects only dividing cells, it must be applied to shoot-tip meristems when cells are actively dividing.
• At any given time, only a small proportion of meristematic cells are in division — repeated treatments at brief intervals are needed to double chromosomes in a large proportion of cells.
• After treatment, a chimera forms: shoot-tip contains a mixture of polyploid and diploid cells. Diploid cells often outcompete polyploid cells — careful selection and propagation needed to stabilize polyploid lines.
• The degree of competition between diploid and polyploid cells varies between species and even between varieties.

Methods of applying colchicine:

• Seed soaking — in 0.2-0.5% aqueous colchicine solution for 4-10 hours; seeds pre-soaked in water to activate metabolism
• Seedling treatment — shoot apex covered with cotton wool soaked in 0.05-0.5% colchicine
• Lanolin paste containing colchicine applied to actively growing shoot tips
• Injection of colchicine solution into developing flower buds

Alternative antimitotic chemicals:

• Oryzalin and trifluralin (dinitroaniline herbicides) — more effective than colchicine in some species; widely used in horticulture for polyploid induction in ornamentals
• Nitrous oxide gas under pressure — effective in some species

Morphological and Cytological Features of Autopolyploids

Applications of Autopolyploidy in Crop Improvement

A. Triploids — Seedlessness and Vigour

• Triploids are produced by crossing tetraploid (4x, used as female) x diploid (2x, used as male).
• They are highly sterile because three sets of chromosomes cannot pair normally at meiosis (one set remains unpaired as univalents). This sterility is agriculturally exploited:

1. Seedless Watermelon (Citrullus lanatus):

• Reciprocal cross 2x female x 4x male is NOT successful — only 4x female x 2x male works.
• Triploid plants do not produce true seeds; almost all 'seeds' are small, white rudimentary structures like cucumber seeds. 
• For good fruit setting, pollination is essential — diploid lines are planted at 1 diploid : 5 triploid ratio as pollen donors. 
• Problems: genetic instability of 4x lines, irregular fruit shape, tendency towards hollowness, production of some empty seeds, labour in triploid seed production.

2. Triploid Sugarbeet (Beta vulgaris):

• 3x is the optimum ploidy level for sugarbeet — triploids produce larger roots and more sugar per unit area than diploids; tetraploids produce SMALLER roots and LOWER yields. 
• Triploid varieties grown commercially in Europe and Japan but popularity declining 

3. Triploid Tea (Camellia assamica) — TV29:

• A triploid (3x) clone of tea has been recently released by the Tea Research Association, India for commercial cultivation in the northern parts of the country.
• The triploid cultivar TV29 produces larger shoots and thereby biomass, yields more cured leaf per unit area and is more tolerant to drought than the available diploid cultivars.' 
• Quality of TV29 is comparable to diploid CTC tea cultivars.  

B. Tetraploids — Quality and Forage Improvement

1. Pusa Giant Berseem (Trifolium alexandrinum) — First Indian Autopolyploid Variety:

• Pusa Giant Berseem is the first autopolyploid variety released for general cultivation in India. 
• It yielded 20-30 per cent more green fodder than the diploid berseem varieties.
• This is the most important Indian achievement in autopolyploidy breeding.

2. Tetraploid Red Clover (Trifolium pratense) and Ryegrass (Lolium perenne):

• Autotetraploid red clover and ryegrass are more vigorous, more digestible and palatable, and have greater resistance to nematodes as compared to the diploids.' 
• Most successful examples of autotetraploidy in forage crops.

3. Tetraploid Rye (Secale cereale):

• Varieties Double Steel and Tetra Petkus released commercially. 
• Advantages over diploid rye: larger kernel size, superior ability to emerge under adverse conditions, higher protein content. 
• Most successful application of autotetraploidy in cereal seed crops.

4. Medicinal Plants:

• Hyoscyamus niger variety HMT-1 (autotetraploid) gives 15% more biomass and 36% more crude drug yield. 
• Vetiveria zizanoides (Vetiver) variety Sugandha (autotetraploid) gives 11% more oil yield.

5. Tetraploid Maize:

• Has 43% more carotenoid pigment and vitamin A activity than diploid. 
• However, has not been commercialised as a crop due to reduced yield and fertility problems — demonstrating that nutritional improvement alone is insufficient for commercial success.

6. Overcoming self-incompatibility:

• Autotetraploidy can overcome self-incompatibility in certain cases, allowing development of self-fertile forms: in some genotypes of tobacco, white clover (Trifolium repens), Petunia. 
• This enables inbred line development in otherwise SI crops.

7. Enabling distant crosses:

• 4x Brassica oleracea x B. chinensis is successful, but when B. oleracea is diploid it is unsuccessful. 
• Similarly, autotetraploids of certain Solanum species produce hybrids with S. tuberosum, while the diploids do not.' Autotetraploidy widens the crossability range in some genera.

Generalisations on Autopolyploidy

• Autopolyploidy is more likely to succeed in species with LOWER chromosome numbers (fewer complications from multivalent formation).
• CROSS-POLLINATING species are generally more responsive to autopolyploidy than self-pollinating species.
• Crops grown for VEGETATIVE PARTS are more likely to succeed as autopolyploids than those grown for seeds (because high sterility of polyploids does not affect yield of vegetative parts).

Limitations of Autopolyploidy

• High water content, lower dry matter: Larger cells contain more water. Autopolyploid turnip and cabbage have more fresh weight but comparable or lower dry matter than diploids.
• High sterility and poor seed set: Most critical limitation for seed crops. Multivalent formation leads to irregular meiosis and unbalanced gametes. Larger seed size does not compensate for fewer seeds per plant per unit area.
• Slow progress under selection: Complex segregation in autotetraploids (Quadruplex/Triplex/Duplex/Simplex/Nulliplex system) makes selection slow — many generations needed.
• Instability of triploids and tetraploids: Triploids cannot be maintained except through clonal propagation or repeated 4x x 2x crosses. Tetraploid progeny include aneuploid plants due to irregular meiosis.
• Cannot be produced at will: Colchicine treatment success is unpredictable — not all species respond adequately. A superior diploid does NOT necessarily produce a superior autotetraploid.
• Effects unpredictable:It is impossible to predict the performance of tetraploids... the actual response has to be determined experimentally. Much trial and error is needed.

ALLOPOLYPLOIDY 

Why allopolyploidy is more important than autopolyploidy:

• Amphidiploids show REGULAR BIVALENT FORMATION at meiosis — unlike autopolyploids which form multivalents. Regular bivalents means near-normal fertility.
• Amphidiploids are GENETICALLY STABLE — progeny are uniform and predictable.
• Amphidiploids combine useful traits from both parental species — the primary objective of allopolyploid crop development.
• Most major crop species are natural amphidiploids — wheat, cotton, oat, peanut, tobacco, oilseed rape. These crops evolved naturally over geological timescales and represent refined, highly productive allopolyploid forms.

Segmental Allopolyploidy 

Significance of segmental allopolyploidy:

• Explains intermediate behaviour between diploids and allopolyploids in some crop species
• The partial homoeology allows some transfer of genes between genomes without wide hybridization — relevant to wheat improvement programmes
• Ph1 gene in wheat actively suppresses homoeologous pairing — when Ph1 is absent (ph1ph1 mutants or when Ae. variabilis is crossed to wheat), the chromosomes pair promiscuously and gene transfer between genomes becomes possible. This is used in wheat breeding to transfer alien genes onto specific wheat chromosomes

Applications of Allopolyploidy in Crop Improvement 

Utilisation as a Bridging Species

• When a wild species (donor) and a cultivated species (recipient) cannot be crossed directly (their F1 is sterile), an amphidiploid of the wild and another related species can serve as a bridge.
• Example — TMV resistance transfer to Tobacco 

• Objective: Transfer TMV resistance from N. glutinosa/N. sylvestris (wild) to N. tabacum (cultivated).
• Direct cross N. tabacum x N. glutinosa → F1 hybrid is STERILE (no chromosome pairing).
• Colchicine treatment of F1 hybrid → CHROMOSOME DOUBLING → synthetic allohexaploid N. digluta (reasonably fertile — has homologues for all chromosomes).
• N. digluta x N. tabacum (backcross to recipient) → PENTAPLOID (has full somatic complement of N. tabacum + one genome from N. glutinosa).
• Pentaploid sufficiently fertile to backcross again to N. tabacum.
• From backcross progeny: select N. tabacum-like plants resistant to TMV → cytologically verify → alien addition/substitution lines recovered.

Creation of New Crop Species

• Allopolyploidy itself has not enabled a species to become successful as a crop — many allopolyploids are weedy wild species (e.g., S. spontaneum and S. robustum are noxious weeds).
• Natural allopolyploids have evolved over a long period. Newly synthesised allopolyploids cannot be expected to become immediately successful.
• An allopolyploid that would be superior to existing diploid species would presumably already have been produced naturally.
• Partially successful examples:

• Triticale: Most successful new crop species from allopolyploidy. 
• Raphanobrassica:Cross between Raphanus sativus (radish, RR, 2n=18) x Brassica oleracea, Not commercially successful but research continues to improve it through hybridisation and selection at the polyploid level.

Widening the Genetic Base of Existing Allopolyploid Crops

• Natural allopolyploids often have a narrow genetic base because they arose from limited founder populations. Synthesising the allopolyploid afresh from current parental diploid species introduces new variation.

Limitations of Allopolyploidy

• Effects of allopolyploidy cannot be predicted — the allopolyploid may combine undesirable traits from both parents (like Raphanobrassica) or desirable ones (like Triticale).
• Newly synthesised allopolyploids ('raw polyploids') have many defects: low fertility, cytogenetic and genetic instability, and other undesirable features. Require extensive breeding.
• The synthetic allopolyploids must be improved through extensive breeding at the polyploid level — considerable time, labour, and resources required. Triticale required 50 years.
• Only a small proportion of allopolyploids are agronomically promising — a vast majority are valueless. Costly trial and error needed.

Autopolyploidy vs Allopolyploidy : Comparison

EVOLUTION OF NATURAL ALLOPOLYPLOID CROPS

Evolution of Bread Wheat (Triticum aestivum, AABBDD, 2n=42)

The evolution of bread wheat is the most important and most tested example of allopolyploidy in plant breeding.

Evolution of Upland Cotton (Gossypium hirsutum, AADD, 2n=52)

• Upland cotton is a natural amphidiploid — the most important cotton species, providing approximately 90% of world cotton production.  

Other Important Natural Allopolyploid Crops

TRITICALE — THE MOST IMPORTANT SYNTHETIC ALLOPOLYPLOID

• Triticale is the most important example of a new crop species created intentionally by allopolyploidy — the primary achievement of polyploidy breeding in the 20th century. It combines the grain quality and yield of wheat with the hardiness and disease resistance of rye.
• 1890 — Rimpau (Germany): First produced a Triticale hybrid — an octoploid. These early triticales were unstable and of little agricultural value. 
• 1930s-1960s — Early breeding: Research continued in multiple countries. Raw triticales suffered from high sterility, poor grain development, and cytogenetic instability.
• CIMMYT (1960s-present): Intensive programme at CIMMYT, Mexico produced dramatic improvements through 

• Production of large numbers of triticale strains using different wheat and rye combinations
• Hybridisation among triticale strains
• Selection against defects.

Genome Composition of Triticale

Desirable Features of Triticale

HAPLOIDS AND DOUBLED HAPLOID BREEDING

• Haploids (n chromosome number) and doubled haploids (2n, produced by chromosome doubling of haploid plants) represent a special application of polyploidy principles in plant breeding. 
• Monoploids and haploids are weaker than diploids and are of little agricultural value directly. But they are of great interest because they offer certain unique opportunities in crop improvement.

Applications of Haploids in Crop Improvement

• Rapid development of homozygous inbred lines: Chromosome doubling of haploid plants (with colchicine) produces INSTANTLY homozygous diploid lines in a single step — instead of 6-7 generations of selfing (6-8 years), near-complete homozygosity is achieved in 1-2 years. This is the most commercially important application. Used routinely in wheat, barley, rapeseed, rice, maize, and many horticultural crops.
• Mutation screening at haploid level: Recessive mutations are EXPRESSED directly in haploids — no dominant allele can mask them. After mutagenesis of haploid material, mutants can be identified and chromosome number doubled to produce homozygous mutant lines in one generation. More efficient than mutation screening in diploids.
• Selection at gametic level: Desirable gametes are more frequent (=p, that is, the frequency of desirable allele in the population) than desirable zygotes (=p²), selection based on haploids or doubled haploids may be expected to be more efficient than that based on diploid plants.
• Breeding potato at haploid (diploid) level: Autotetraploid potato (2n=48, 4x) is very difficult to breed — complex tetrasomic segregation. Breeding is much easier at the DIPLOID level (haploid potato, 2n=24, 2x) where simple Mendelian segregation applies. After selecting desirable diploid genotypes, chromosome number is doubled to obtain tetraploid varieties. This '2x-4x strategy' is a major direction in modern potato breeding.

Methods of Producing Haploids