Crop Improvement in Fruit Crops

Crop Improvement in Fruit Crops

Fruit Breeding

Desirable changing and improving the heredity of fruit plants is known as fruit breeding.

Centre of Origin

• The concept of centre of origin was given by N.I. Vavilov.

• He gives the 8 centres of origin.

Primary Centre of Origin

The cultivated plant species are supposed to have originated.

Secondary Centre of Origin

The cultivated plant species show much greater variation but did not originated.

1. China Centre of Origin

Largest and oldest centre of origin.

Examples: Pear, peach, apricot, plum, orange

2. Hindustan Centre of Origin

It is divided into two groups: Indo-Burma & Siam–Malaya–Java.

Examples: Mango, sour lime, mandarin, coconut, banana.

3. Central Asia Centre of Origin

Also called as Afghanistan centre of origin.

Examples: Grape, almond, apple, pistachio nut.

4. Asia Minor Centre of Origin

Also known as Near East or Persian centre of origin.

Primary centre of origin: Fig, pomegranate

Secondary centre of origin: Chestnut, pistachio nut

5. Mediterranean Centre of Origin

Example: Pipper mint

6. Abyssinian Centre of Origin (Ethiopia)

Example: Coffee

7. Central American Centre of Origin

Also known as Mexican centre of origin.

Examples: Papaya, guava, avocado

8. South American Centre of Origin

Example: Pineapple

Breeding Methods

A. Traditional (Conventional) Methods

Introduction, selection, hybridization, mutation, polyploidy etc. methods included.

B. Non-traditional (Modern) Methods

Biotechnology (plant tissue culture) & genetic engineering methods are used.

1. Introduction

Direct utilization of germplasm as cultivars after their performance has been tested and evaluated for their adaptation under local conditions.

• The systematic introduction started in 1946 by Division of Botany, IARI, New Delhi.

• It is quick and economic method for crop improvement.

• Introduction must be routed through the NBPGR, New Delhi.

• Introduced material directly used as improvement variety or used as a rootstock.

Some Important Introduced Varieties

2. Selection

Most of the fruit cultivars are open pollinated seedling selection from the mixed population.

• Selection does not create the genetic variability but merely acts on the genetic variability already available.

• For selection variation must be present in the population which are heritable.

• It is very effective because in fruit crops high degree of variability due to heterozygosity.

Examples

Clonal Selection

Vegetative progenies of a single plant are known as clone.

• Clones are stable, homozygous but individual clone is heterozygous in nature.

• Genetic variation in clones is due to mutation, mechanical mixture and sexual reproduction.

• Clonal selection is only method of breeding in vegetative propagated fruit plants.

Mango – Tomy Atkins from Haden, Pusa Surya from Elden, Clone No. 52 from Dashaheri

Grape – Pusa Seedless from Thompson Seedless

3. Mutation

Sudden heritable changes in characteristics of the plants are known as mutation.

• The occurrence of a large number of natural bud-sports and chance seedling in apples and pears made the fruit breeding interested to breed through application of mutagens.

• Mutations are generally recessive, harmful, random and recurrent but dominant mutation also occurs.

• Induced mutations commonly shows pleiotrophy due to mutations in closely linked gene.

Kinds of Mutation

Macro Mutation

Large mutation recognised on single plant basis.

Example: Change in colour and shape.

Micro Mutation

Quantitatively small effects are recognized. More important for direct use in plant breeding.

Example: Mutants with large or smaller or higher yield.

Point Mutation

Also known as gene mutation, involves changes at individual loci (point).

Somatic Mutation

Mutation appearing in vegetative parts. Also referred to as ‘bud sport’.

a. Spontaneous Mutation

Naturally occurring mutants which arise somatically. Frequency is extremely low i.e. 1 in a million.

b. Induced Mutation

Induced by using various mutagens.

Types of Mutagens

a. Physical Mutagens

Ionising: X-ray, α ray, β particles, fast neutrons

Non-ionising: UV rays, γ rays

b. Chemical Mutagens

i. Base analogues: 5-bromouracil and 5-bromodeoxyuradine (analogues of thiamine) pairs with adenine and 2-aminopurine (analogue of adenine) pairs with thiamine and cytosine.

ii. Antibiotics: Azasorine, Streptonegrin, mytomycine. Limited practical use in plant breeding.

iii. Alkalating agents: Most important group.
Examples: Methyl Methane Sulphonate (MMS), Ethyl Methane Sulphonate (EMS), Sulphur, Mustard gas.

Achievements

• Mango: Rosica from Peruvian variety Rosadodelca

• Papaya: Pusa Nanha from local type

• Grape: Marvel Seedless from Delight.

• Banana: Highgate from Gros Michel, Moota Poovan from Poovan

• Orange: Wahington Navel

• Grapefruit: Marsh and Thompson

4. Hybridization

Combination of desirable characters by mating of dissimilar genotypes.

• Intervarietal/Intraspecific hybridization – Parents involved in hybridization belongs to same species or varieties.

• Interspecific hybridization – Cross between two species of the same genera.

• Intergeneric hybridization – Cross between two different genera.

Achievements

• Mango: Mallika, Amrapalli, Pusa Arunima, Arka Puneet, Arka Aruna, Ratna, Sindhu, PKM-1, PKM-2.

• Guava: Arka Amulya, Safed Jam, Kohir Safed

• Litchi: Sabohar Madhu, Sabohar Priya

• Sapota: CO-1, DHS-1, DHS-2

Non-Traditional (Modern) Methods

• 21st century is the ‘Century of Biotechnology’.

• Agriculture biotechnology is known as Green biotechnology.

• Biotechnology term was coined by Karl Ereky in 1919.

• Gottleib Haberland (1902) is the father of tissue culture and gave the concept of totipotency.

• Van Overbreek (1941) used coconut milk for embryo development & callus formation of Datura.

• Miller (1955) – discovered Kinetin.

• Steward (1958) – discovered Embryogenesis.

• Morel – Rapid propagation of orchids by micropropagation.

• Skoog & Miller (1957) – Auxin-Cytokinin balance theory.

• Mahesgwari & Rangaswamy (1950) – Regeneration of somaclonal embryos from nucleus of citrus ovules.

• Guha & Maheshwari – first produced haploid plants by culturing pollens of D. Innoxia.

• Larkin & Scowcraft (1981) – coined the term somaclonal variation.

• Term genetic engineering by A. Just (1941).

• Paul Berg is the father of genetic engineering.

• Friable callus is used for making cell suspension culture.

• High concentration of auxin results in induction of roots and high concentration of cytokinin results in induction of shoots and buds.

• In tissue culture generally shoot formation followed by roots.

• White’s medium – Earliest medium for plant tissue culture developed for root culture.

• Murashige & Skoog medium (MS) – widely used.

• B5 medium – developed for cell suspension/callus culture but with modifications used for protoplast culture.

• For tissue culture most critical vitamin is thiamine.

• Activated charcoal – it is used when phenolic compounds are a problem for in vitro culture.

• Zeatin is a natural cytokinin.

• ABA (Abscissic acid) – used to induce embryogenesis.

• Agar – it is solidifying/gelling agent obtained from sea weed. Difco Bracto agar is commonly used.

• Embryo culture is used for wide hybridization, productions of haploids, overcome seed dormancy and overcome seed sterility.

• Callus cells are parenchymatous and non-homogenous in nature.

• Callus culture needs sub-culturing every 3–5 weeks to cell growth, nutrient depletion and redus to medium drying.

• Callus shows ‘S’ sigmoid growth curve.

• Callus culture is used for induce somaclonal variation, development of cell suspension & protoplast culture and production of secondary metabolites.

• Cell suspension culture is used for production of secondary metabolites and development of resistant to salt, drought and toxic substances.

• Direct somatic embryogenesis – somatic cells will directly develop into somatic embryos without intervening formation of callus.

• Indirect somatic embryos – the explants cultured will first give rise to callus from which the somatic embryos formed.

• 2,4-D is used at high concentration for embryo initiation, while no or low concentration is used for embryo formation.

• Artificial seeds/Synthetic seeds/SYN seeds – somatic embryoids are encapsulated with a hydrogel. Encapsulation is done by various hydrogel likes sodium alginate which are water soluble.

• Micropropagation used creating genetic variability, germplasm conservation, virus elimination, development of somatic hybrids and gene transfer.

• In India commercial exploitation of micro-propagation is limited to oil palm, strawberry and banana.

• Meristem culture – meristem tips of 0.2–0.5 mm produce virus-free plants because of strong growth potential and low virus concentration.

• Meristem culture and micrografting are used to obtain virus-free planting material of both stock and scion.

• Embryo culture technique in coconut saves bulk transportation of nuts from distant areas.

• Micro cloning has been achieved in embryo culture of banana involving a cross between M. acuminata and M. balbisiana.

Somaclonal Variation

Somaclonal variation – genetic variability generated through tissue culture.

• Term: Larkin & Scowcroft (1981)

• For somaclonal variations various agents like toxins, drought, cold, heat etc. are added to culture medium.

• Example: Thornless blackberries ‘Linoln Longan’ (Rubus).

Haploid

• Generally the term haploid is referred to any plant originated from a sporophyte (2n) and containing ‘n’ number of chromosomes.

• Androgensis – production of haploid plants from male gametophyte.

• Gynogensis – production of haploid plants from ovary & ovule culture (unfertilized).

• Endromitosis – chromosomal duplication without nuclear. Generally haploid cells are unstable in culture.

• Haploids are used for development of homogenous lines, hybrid development, induction of mutations and induce of genetic variability.

• Production of inbred (isogenic lines) through anther or pollen culture for use in heterosis breeding and linkage analysis.

• Anther culture has shown great promise in custard apple, sapota, papaya and guava.

Protoplast Culture

Protoplast culture – a plant cell without cell wall.

• Hanstein (1880) – gave the term protoplast.

• Cocking (1960) – first time used enzymes to release protoplast from the fungus Myrothecium verrucaria to degrade cell wall.

• For protoplast isolation cellulose & Rho, Rhizome HP 150, Macerase enzymes are used.

Types

• Cytoplast – protoplast lack nucleus and each cell contains entire cytoplasm.

• Karyoplast – cells contain nucleus surrounded by some cytoplasm.

• Microplast – cells contain only a fraction of cytoplasm and outer membrane.

• Pollen viability is decreased in hybrids.

• Cybrids – somatic hybrids which have nucleus from one parent and cytoplasm from the both parents.

• The first cybrid plants using donor recipient was reported in citrus and micro citrus genotype.

• Somatic hybridization are used for production of interspecific and intergeneric cross, gene transfer, hybridization of plants which are in juvenile phase and fusion of protoplast of sexually sterile plants.

• Protoplast fusion and somatic hybridization technique provide the opportunity to facilitate mixing of both nuclear and cytoplasmic genetic traits between species through the by passing of reproductive isolation barriers.

Germplasm Storage

• In situ conservation – germplasm is conserved in its original habitats.
  Example: Gene sanctuaries.

• Ex situ conservation – germplasm is conserved away from its original habitats.
  Examples: Gene banks/seed gene banks, field gene banks.

• Orthodox seeds are stored in seed gene banks.

• Vegetatively propagated species and recalcitrant species are stored as field gene banks.

• Cryopreservation – Greek word ‘Cryo’ means frost.

• Plant material is frozen and maintained at liquid N₂ (-196°C).

• Deep freezers – -80°C

• Dry ice/solid CO₂ – -79°C

• DMSO (Dimethyl Sulfoxide) is an excellent cryoprotectant.

• Plant material is suspended in culture medium and treated with suitable cryoprotectants and transferred to sterile propylene cryovials or ampoules to prevent plasmolysis and protect the cells against osmotic shocks.

Genetic Engineering

Gene cloning is the isolation of individual gene sequence by insertion of that sequence into a bacterium through a vector where it can be replicated.

Restriction Endonucleases (RE’s)/Molecular Scissors

• Enzymes used for cutting DNA fragments.

• Nomenclature – Smith & Nathans (1973).

• First RE’s was isolated in Haemophilus influenzae.

• SAM is the precursor for methylation.

DNA Ligases

• The enzymes used for joining DNA fragments/molecules.

• Examples: E. coli DNA Ligases and Bacteriophage T4 DNA Ligase are two important DNA ligases.

• Kinases which extracted from bacteriophage is DNA modifying enzymes.

Linkers

There are two synthetic single stranded oligonucleotides, which self-associate to form symmetrical double stranded molecules containing the recognition sequence for a restriction enzymes.

Adaptor

These molecules are chemically synthesized DNA molecules within the DNA sequence to be cloned.

Vectors/Cloning Vehicles

• It is an extra chromosomal DNA molecule which has the ability to replicate autonomously in an appropriate host cell.

• Any extra chromosomal genome like plasmids, phages or viruses may be used as vectors.

Types of Vectors

1. Cloning Vector

Used for propagating of DNA insert in a suitable host to get multiple copies of the DNA insert.

2. Expression Vectors

Used for expression of DNA insert for which regulatory sequences like promoter, operator and ribosome binding sites are essential.

• E. coli is most commonly used as a host.

• E. coli K12 strains is the standard host used in gene cloning.

• E. coli supports several vector systems like plasmids, bacteriophage, cosmids, phagemids and shuttle vectors.

• R-Plasmid – carries the genes for antibiotic resistance.

• F-Plasmid – carries the genes for conjugation i.e. tra & mob genes.

• Col Plasmid – carries the genes which provide immunity to colic (antibacterial proteins).

• Size of plasmid ranges from 1–200 kb (1 kb = 1000 bp).

• ColE1 is the first plasmid.

• pBR322 – first artificial cloning vector constructed and most widely used cloning vector up to now.

• Cosmids are the plasmids vectors that contain a bacteriophage λ cos site which directs insertion of DNA into phage particles.

Gene Transfer in Plants

Two methods:

• Transformation – the process of uptake of foreign DNA/transgene by plant cell is known as transformation.

• Agrobacterium tumefaciens – It is a biological/natural genetic engineer which can be used for gene transfer in plants which contains Ti plasmid (tumour inducing plasmid).

• A. rhizogenes – contains Ri plasmid (root inducing plasmid).

1. Vector-Mediated Gene Transfer

Ti plasmid, viruses etc. are used.

• Agrobacterium mediated and direct gene transfer.
  Examples: Passion fruit, papaya, apple, strawberry, peach, plum, kiwi fruit, pecan nut and walnut.

• Mango has been successfully transformed by co-cultivation within embryonic cultures with A. tumefaciens.

2. Direct Gene Transfer

It is spontaneous uptake of DNA. Frequency of transformation is low. These are below:

i. Particle Bombardment/Microprojectile/Biolistic/Gene Gun/Particle Gun Method

1 to 2 µm tungsten or gold particles coated with the DNA to be used for transformation. DNA can be delivered into cells of shoot meristem, embryo which makes gene transfer independent of regeneration ability of the species.

Bombardment of leaf and apical meristem followed by A. tumefaciens co-cultivation in grape and banana.

ii. Electroportion

iii. Microinjection

DNA solution is directly injected inside the cell by using capillary glass micropipettes. Most successful with densely cytoplasmic non-vacuolated embryonic cells.

Examples: Banana, papaya and cranberry.

iv. Lipofection

Introduction of DNA into plant cells via liposomes.

• First method DNA delivery into plant protoplast.

v. PEG (Polyethylene Glycol)

PEG-mediated DNA delivery.

Role of Important Genes in Fruit Crops

• Most common bioinsecticides is crystal protein from Bacillus thuringiensis are more potent that which shows activity against caterpillars of the order Lepidoptera.

• Transgenic papaya plants constitutively expressing the coat protein gene of papaya ring spot virus (PRSV) have been obtained by microprojectile bombardment.

• Genes for resistance to commercial herbicides are bar, aro A and als.
  Example: ‘Royal Gala’ apple (plants expressing als).

• Nematode resistant genes are collagenase, Bt exotoxin and cp Ti.

• Transgenic plants of ‘Hayward’ kiwifruit expressing rol/ABC genes showed an increasing rooting ability.

• M 26 rootstocks of apple expressing RiT-DNA exhibited higher rooting.

• rol genes to improve rooting in hardwood cutting in apple, pear, peach, plums, mango etc.

• Transgenic plants of apple transformed with rol C gene were dwarf.

• co gene controlling columnar shape has been incorporated in apple and known to be dominant when single inherited.

• S-adenosyl methionine hydrolase (SAMase) has been introduced in ‘Meeker’, ‘Chiliwaek’ and ‘Canby’ red raspberry.

• SAMase lowers the concentration of substrate for ACC synthase and therefore reduces the ability of the plants to produce ethylene thus delaying ripening.

• Viral coat protein gene – apricoat, citrus, grape, papaya and plum.

• Bt endotoxin gene – apple, walnut and cranberry.

• E gene – bacterial blight resistance in apple.

• als gene for herbicide tolerance in apple and bar gene in papaya.

• rol genes enhancing rooting ability in kiwifruit.

• ipt gene modification of tree structure in apple.

Important Gene and Their Role

Micropropagation Technology Developed for Important Fruit Crops