Genetic Engineering of Male Sterility for Hybrid Seed Production # Methods of Hybrid Seed Production - Hybridization techniques # Examples of Male Sterile Hybrid Seed

1. D r . D i v y a S h a r m a
A s s i s t a n t P r o f e s s o r
GENETIC ENGINEERING OF
MALE STERILITY FOR HYBRID
SEED PRODUCTION
A Biodiction (A Unit of Krishna Academy)

2. HYBRID SEED TECHNOLOGY
Hybrid seed technology is based upon the cross
pollination of diversely related two pure lines showing
desire traits, produces the hybrid seed that show the
inferior traits then parents.

3. REQUIRED FOR HYBRID SEED
PRODUCTION
Breeders responsibilities:
➢ Develop inbred lines
➢ Identification of specific parental lines
➢ Develop system for pollen control
Major problems for breeders & producers:
➢ Maintenance of parental lines
➢ Separation of male and female reproductive organs
➢ Pollination

4. CHARACTERISTICS OF PARENTAL LINES
➢ Female parent, Male parent
➢ High seed yield --→ Good pollen production
➢ Good seed characteristics --→ Long shedding period
➢ Male sterility --→ Plant height

5. BASIC PROCEDURE FOR HYBRID SEED
PRODUCTION
➢ Development and identification for parental lines
➢ Multiplication of parental lines
➢ Crossing between parental lines and production of F1
➢ Commercial hybrid seed production demands crossing technique which is
easy and also economic to maintain parental lines.
Only few crossing mechanisms have been adopted for commercial hybrid seed
production they are;
❑ Hand emasculation and pollination
❑ Self-incompatibility
❑ Dicliny: Monoecious and Dioecious
❑ Male sterility

6. Why we prefer hybrid seed technology
on open pollinated?
Distinct yield advantage compared
to open pollinated varieties
Ability to produce hybrid seed on
commercial scale at economic
level

7. HYBRID SEED PRODUCTION IN
ALLOGAMOUS PLANTS
➢ In nature to create genetic variability and for its wider
adaptation in different environmental conditions, flowering
plants has adopted many mechanisms for cross pollination.
➢ Cross-pollination results in genetic heterogeneity and show
wider adaptations. In cross pollinated plants it is easy to
produce hybrids as there are many mechanism that promote
cross pollination in nature i.e.,
▪ Dicliny(Unisexuality),
▪ Dichogamy,
▪ Herkogamy,
▪ Self Incompatibility,
▪ Male Sterility

8. HYBRID SEED PRODUCTION IN
AUTOGAMOUS PLANTS
➢ For self-pollinating plants, the plant must be male sterilized
during the crossing process in order to avoid self-fertilization.
➢ There are various mechanism / contrivances that promote /
facilitate self-pollination.
➢ New tools and technology were discovered, which proved the
impact of hybrid breeding, and utilized for self-pollinating crops
including Cytoplasmic male sterility (cms).
➢ Cytoplasmic male sterility is a condition where the plant is
unable to produce functional pollen and would rely on other
pollen source to produce seeds. This greatly facilitates large
scale hybrid seed production, by-passing hand pollination

9. How male sterility manifest in plants?
➢ The absence or mal formation of male organs (stamens) in
bisexual plants or no male flowers in dioecious plants.
➢ Failure to develop normal anther.
➢ Abnormal micro sporogenesis leading to inviable pollen.
➢ Abnormal pollen maturation inability to germinate on
compatible stigma.
➢ No dehiscent anthers but viable pollen sporophytic control.
➢ Barriers preventing pollen from reaching ovule.

10. CLASSIFICATION OF MALE STERLITY
Male sterility classified into two types:
Phenotypic
classification
Genotypic
classification

11. PHENOTYPIC CLASSIFICATION
Phenotypic classification includes:
❑ Structural male sterility (anomalies in male sex organs)
❑ Sporogenous male sterility (stamens form, but pollen absent)
❑ Functional male sterility (viable pollen form, but barrier prevents
fertilization, inability of pollen to migrate to stigma or other
factors that affect fertilization)

12. GENOTYPIC CLASSIFICATION
Genotypic classification includes:
❑ Cytoplasmic Male Sterility (CMS):
The pollen sterility which is controlled by cytoplasmic genes is
known as Cytoplasmic Male Sterility (CMS).
Usually the cytoplasm of zygote comes primarily from the eggs cell
and due to this progeny of such male sterile plants would always
be male sterile.
❑ Utilization in Plant Breeding:
CMS has limited application. It cannot be used for development of
hybrid, where seed is the economic product.
Can be used for producing hybrid seed in certain ornamental
species or asexually propagated species like sugarcane, potato,
and forage crops.

13. GENETIC MALE STERILITY
➢ Male sterility is controlled by mutations in nuclear genes in the
single recessive genes affect stamen and pollen development,
but it can be regulated also by dominant genes.
E.g: Safflower
➢ The maintenance of the male sterile line. Normally, a GMS line
(A-line) is maintained by backcrossing with the heterozygote B-
lines (Maintainer lines), but the progeny produced are 50%
fertile and 50% male sterile.

14. UTILIZATION IN PLANT BREEDING
➢ Male fertile plant from the female is costly operation and due to
this cost of hybrid seed is higher.
➢ Therefore, GMS has been exploited commercially only in few
crops by few countries.
E.g.: In USA used in castor while in India used for hybrid seed
production of Arhar (cajanus cajan).

15. THREE LINES:
HYBRID SEED TECHNOLOGY
➢ Three lines system involves from three lines (A, B and R line)
➢ The hybrids developed by utilizing this system are called as
Three line hybrids.
Requirements for 3 Lines in CMS System:
A-line
Stable sterility
Well developed floral
traits for outcrossing
Easily, wide spectrum &
strongly to be restored
B-line
Well developed floral
traits with large pollen
load
Good combining
ability
R-line
Strong restore ability
Good combining ability
Large pollen load,
normal flowering traits
and timing

16. Steps:
➢ CMS multiplication (A x B)
➢ Commercial hybrid seed production (A x R)

17. TWO LINES:
HYBRID SEED TECHNOLOGY
➢ Hybrids produced by chemically induced male sterility are also
called Two line hybrids.
Multiplication of EGMS lines
Hybrid rice seed production

18. Steps:

19. ADVANTAGES OF TWO-LINE AND THREE-LINE
HYBRID SYSTEM

20. THREE-LINE AND TWO -LINE HYBRID SYSTEM OF
UTILIZING MALE STERILITY FOR HYBRID RICE
PRODUCTION

21. EXAMPLES: PRODUCTION OF
HYBRID SEED STERILITY

22. SOLANACEOUS CROPS: TOMATO
Techniques of Hybrid Seed Production -→ Hybridization techniques:
➢ Indeterminate tomato varieties staked and trained with either
single stem or double stem, while determinate varieties are
trained with 3 stems
➢ Usually 1st to 4th cluster on each branch selected
➢ The emasculation involves in holding the corolla at the base
and with a single upward pull pick off the corolla along with all
the stamens
➢ Usually the anthers are picked off a day before anthesis with the
help of forceps leaving the petals intact

23. ➢ Fresh pollen collection on the day of anthesis by a vibrator are
shed by vibrating the flowers
➢ Pollens are collected in a glass tube or on a glass plate from the
male line and are transferred to the stigma by finger or by
inserting the stigma into a glass tube containing the pollen
grains. Left over pollen grains in the glass tube are not used on
the next day because its viability is reduced considerably

24. SOLANACEOUS CROPS: TOMATO
Physiological parameters affecting Tomato seed yield and vigor:
➢ Prolonged high temperature affect pollen fertility and
physiology of fertilization leading to poor seed set
➢ Age of pollen and stigma, abundant or scarce pollination,
height of inflorescence and fertilizer application exert an
influence on vigor of plant

25. SOLANACEOUS CROPS: TOMATO
Seed extraction and Dying:
➢ By fermentation method
➢ By acid or alkali treatment: 10cc or 36% NaOH added in 4kg
tomato pulp for 15 minutes.
Seed Yield:
➢ 1kg tomato produce 3-4g of seed yield. Av.
➢ Seed yield: 60-70kg/ha

26. SOLANACEOUS CROPS:
EGGPLANT AND PEPPER
Manifestation of Heterosis:
➢ Heterosis in sweet pepper ranged 35-40%
➢ Eggplant 50-150 %
➢ In eggplant, it is manifested by earliness, fruit number per plant
& fruit weight
Sweet Pepper:
➢ Plant height, days to flower, fruit weight, early and total yield

27. SOLANACEOUS CROPS:
EGGPLANT AND PEPPER
Techniques of Hybrid Seed Production -→ Hybridization techniques:
➢ Stigma is receptive a day prior to anthesis in eggplant
➢ Bud pollination possible giving good fruit set and seed yield
➢ In sweet pepper, emasculation done a day prior to anthesis,
whereas, pollination done in the morning on the day of anthesis
➢ Natural cross pollination ranged from 0.2-46.8% in eggplant
flowers
➢ Pepper flowers are visited by honey bees occasionally. Fresh
pollen grains are collected on the day of anthesis by a vibrator
and can be stored for a period of 1 to 2 months at O°C using
silica gel for proper drying

28. SOLANACEOUS CROPS:
EGGPLANT AND PEPPER
Use of Male Sterility in Hybrid Seed production in Eggplant and
Sweet Pepper:
➢ Male sterile lines available in eggplant (genic male sterility) &
sweet pepper (genic and ems) but not successful at
commercial seed production
➢ Seed set on male sterile lines ranged from 46-67 % in capsicum
➢ Cytoplasmic male sterility used now in chilli pepper to produce
F1 hybrid commercially by several seed companies

29. SOLANACEOUS CROPS:
EGGPLANT AND PEPPER
Seed Extraction:
➢ Eggplant harvested 50-55 days after anthesis and stored for 10
days for post harvest ripening
➢ Sweet pepper: 60-65 days after anthesis
➢ Ripe fruits crushed and seeds separated by washing. Dry air at
28-30ºC
Seed Yield:
➢ Eggplant: 150-200 kg/ha
➢ Peppers: 100-200 kg/ha. 1kg yields 5-7g of seed.

30. CURCURBITS CROPS:
CURCURBITACEOUS VEGETABLES
Techniques of Hybrid Seed Production -→ Hybridization techniques:
➢ Production of inbred lines by inbreeding for 3 to 5 generations.
➢ Selection of inbred parents through combining ability tests and
potential hybrid production ability.
➢ Production of hybrid seeds (preferably single cross hybrids and
pistilate parent preparation is relatively easy, and single fruit
produces quite a large number of seeds).
➢ Maintenance of inbred parents

31. CURCURBITS CROPS: WATERMELON
Techniques of Hybrid Seed Production -→ Hybridization techniques:
Hybrid seeds of watermelon can be produced by two ways:
➢ Through artificial pollination.
➢ Removal of male-flower and use of insect pollination.

32. CURCURBITS CROPS: WATERMELON
Artificial Pollination:
➢ Field lay-out
➢ Selection of female flowers
➢ Bagging of female & male flowers
➢ Collection of male flowers
➢ Period of pollination
➢ Seed fruit management
➢ Harvesting
➢ Extraction, washing and drying of seeds
➢ Seed yield 150-300kg/ha based on varieties, extent of
pollination & field condition

33. CURCURBITS CROPS: WATERMELON
Removal of Male flowers and use of Insect pollination:
➢ In this technique, male flowers of female plant completely
removed before opening
➢ Fruits from female parent harvested as crossed fruits and other
variety as self fruit of male variety
➢ Used in commercial hybrid seed production
➢ Also known as crossing block method

34. CURCURBITS CROPS: PUMPKIN
Production of Inbred lines:
➢ For winter ecotypes, Seed sowing of diversified genotypes in
Nov-Jan.
➢ For summer ecotypes, seed sowing after the1st monsoon
➢ Bagging of male and female flowers one day before anthesis
➢ Select the vigorous inbreds after 4-5 generations of inbreeding

35. CURCURBITS CROPS: PUMPKIN
Production of Hybrid seeds:
➢ Planting of female and male inbred parents in 4:1 ratio
➢ Spray 50-100 ml of ethephon per litre of water on pistillate
parent to increase female flowers
➢ Bagging of flowers before anthesis. Rebag the female flowers
after pollination for another two days
➢ Harvest fruits after 60 days of pollination
➢ Remove seeds from the fruits, wash and dry for 3-4 days and sun
dry for another 3-4 days.
➢ Preserve the seeds in sealed polyethylene bags at low
temperature (4-5°C)

36. CURCURBITS CROPS: BOTTLE GOURD
Production of Inbred lines of diverse genetic background:
➢ Bagging of male & female flowers
➢ Inbreeding at anthesis through following morning and
rebagging of the female flowers for another 2 to 3 days.
➢ Inbreeding done for 4-5 generations and select better types by
discarding the poor performers.

37. CURCURBITS CROPS: BOTTLE GOURD
Production of Hybrid seeds:
➢ Planting of female and pollinator inbreds in 4:1 ratio.
➢ Bagging of female and male flowers before anthesis
➢ Harvesting of mature fruits after senescence of the plant,
remove seeds, wash and dry. Store in sealed polyethylene bags
at low temperature
Maintenance of Inbred Parents:
➢ Inbreeding of parents to produce seeds should be done when
the parental stock is depleted. Produce large quantity of seeds
to maintain genotypic & phenotypic integrity of the hybrid.

38. ROOT AND BULB CROPS: ONION
Heterosis in Onion:
➢ Ranged from 14-67 %. It is manifested in uniform bulb size, bulb
weight & efficient source sink ratio
Male Sterility in Onion:
➢ Controlled by combination of cytoplasmic factor ‘5’ together
with recessive nucleus gene in its homozygous form
Hybrid Seed Production Techniques:
➢ For the production of hybrid seed in onion, male and
cytoplasmic female lines planted in the ratio of 2:8
➢ Success of hybrid seed production depends upon the pollen
distribution pattern from fertile to sterile plants in the crossing
block

39. ROOT AND BULB CROPS: ONION
Factors affecting Hybrid Seed in Onion:
➢ Weak inbred lines
➢ Abnormal florets where ovary started to develop but failed to
produce seed
➢ Aborted ovule
➢ Excessive heat damaging the flowers
Seed Yield:
➢ Hybrid seed yield in onion ranges from 300-350 kg/ha

40. COLE CROPS
Manifestation of Heterosis:
➢ Heterosis in cabbage: 25-61%
➢ Cauliflower: 20-60 %
➢ Broccoli: 26-58 %
➢ Manifested by head/curd size, early maturity, head/curd weight
and plant weight

41. COLE CROPS
Manifestation of Heterosis:
➢ Male Sterility and Hybrid Seed Production
➢ Cytoplasmic male sterile lines and their maintainers have been
developed in cauliflower, cabbage and broccoli using radish
cytoplasm for male sterility
➢ Cytosterile plants of broccoli, cabbage and cauliflower are
petaloid with large nectarines responsible for bee attraction
and good female fertility
Seed Yield:
➢ Varies from 500 - 800 kg/ha

42. THANK YOU
A Biodiction (A Unit of Krishna Academy)