HYBRID SEED PRODUCTION TECHNOLOGY IN VEGETABLES

Brinjal
Manifestation of Heterosis Adaptability, Number of long &medium styled flowers Fruit set, size , weight and number of fruits plant Early yield. consideration size, colour shape, quality & resistance factors

DEVELOPMENT OF HYBRIDS
A wider gene base as purelines and varieties.

Diallel or partial diallel or line x tester method.

TECHNIQUE OF HYBRID SEED PRODUCTION

150 to 200 g of hybrid seed is enough to cover one hectare of land.
On an average, each crossed fruit yields 3 g seed. 70 crossed fruits enough.

F1 hybrid seed - small scale the female and male parents of a cross are grown side by side but away from other genotypes.

 The mature long-styled flower buds of female emasculated on previous day afternoon - covered with butter paper bags. Long-styled mature buds are also covered with butter paper bags for collection of pollen.

Hand pollination - next day between 9hr to 13 hr. in winter and 7 hr to 12 hr in summer. Fertilized flowers (ovaries) get prominence about 10 days of pollination. Fruits matured in 30 to 55 days White, green- yellow, dark purple - become yellowish brown in colour Seeds extracted - after harvesting fruits or after a few days of storage under shade.

 Male and female parents of a cross are grown 200 meters in isolation
For pollinating the emasculated flowers, anthers from male parent - mature flowers buds collected in large Petri dishes -anthers are kept under light during night to dehisce the pollen grains - pollen grains sieved through muslin cloth - collected into a container - used for hand-pollination. A part of the petals of pollinated flowers are cut with scissor to indicate the pollination - in 10 days the fertilized flowers (ovaries) develop into prominence.

MAINTENANCE OF PARENTAL LINES Plant type and other morphological characters are also given due consideration in maintaining the purity of parents. Strict rouging of undesirable plants is observed to keep the purity of parental lines. The purity of parental lines can also be maintained vegetatively.

TOMATO (Lycopersicon esculentum)

Commercial production -cross combination have shown a high value on evaluation. The largest seed producer - which has profuse pollen

 Female flowers are emasculated and covered with pollen as soon as they are exposing after emasculation. Mechanical aids are powered electric bee which shakes the anthers and pollen is collected in a glass vial of thumb nail. Pollens is applied to stigma by brush or inverting the emasculated flower over the thumb nail.

 For temperate regions earliness is the main attribute to be incorporated in hybrids. Genic male sterility is also used in developing hybrids. Exerted stigma condition used with a marker gene (potato leaf) K1-4 (Pusa Divya)

SWEET PEPPER (Capsicum annuum)

Production the F1 hybrids of sweet peppers are commercially exploited in India due to the followin reasons :
Sufficiently large flowers facilitates easy emasculation and pollination Large number of seeds obtained per pollination. Manifestation of heterosis for yield and its components. The highly remunerative prices obtained from the produce.

 Emasculation should be done in the bud stages. Stigmas are normally receptive at the late bud stage and pollen from the male parent can be applied successfully at the time of emasculation. Cytoplasmic male sterility 200 seeds per fruit - matures when fruit colour changes from green to brown and red.

POLLINATION BEHAVIOR
In sweet peppers, anthesis starts at 7.15 am and continues upto 11.15 am with peak at 7.15 am. Anthers dehisce after 30 minutes of anthesis, stigma becomes receptive from the day of anthesis and remains receptive upto 2 days. Fruit set was maximum on the day of anthesis with around 80% set and pollen fertility around 96%.

Selecting a bud - day prior to anthesis may be desirable since dehiscence of anthers takes place before anthesis in some of the genotypes.

 200 seeds per fruit - matures - fruit colour changes from green to reddish brown. On a single plant 3-5 crossed fruit can give good hybrid seed yield - crossing attempted in 10-12 flowers

The other flower bud should be pinched to avoid setting of selfed fruit.

CHILLIES
Floral biology and pollination : Flowers usually born single and are terminal, corolla deeply 5 lobbed, while greenish in colour. Stamens 5-6 inserted near the base of corolla, anther dehiscing longitudinally, ovary 2 celled, style simple, white or purple. Bees, are the possible agents of pollination. Both self and cross pollination occur, the latter to about 16 per cent. Flowers remain open for 2-3 days. The percentage of fruit setting is 40-50.

METHODS FOR DEVELOPMENT OF F1 HYBRID IN CHILLIES

Production of inbred lines : The inbreds used for production of F1 hybrids should be genetically pure.

Sibbing or selfing helps in production of genetically pure inbred lines.

Chilli being self pollinated crop pure lines maintained in the collection can be used as inbred lines. Parents may be selected on the basis of vigour, fruit size and yield.

HYBRID SEED PRODUCTION IN CHILLIES :

A. Emasculation and pollination : Emasculation may be done either early in the morning or in the previous evening before opening of flower and calyx still covering the anther and stigma. With the help of a pair of forceps., the petals are easily parted and anthers are removed and bagged.

ii) Hybridization technique :

Late in the morning or early in the afternoon fresh flower should be plucked from intended male parents which have been previously bagged and pollen dusted on the stigmas of the emasculated flowers.

To facilitate pollination, petals of the pollen flower may be cut off. Crossing - successful - full bloom During cloudy weather - setting is very low Genic male sterility

ONION AND CARROT

The flower of onion are borne in a simple umbel at the terminal end of the flowering stalk whereas in carrot the inflorescence is a compound umbel.

The flowers of both onion and carrot are portandrous.

 selfing in onion - 2 or 3 umbels of the same plant are enclosed in an ordinary large-sized muslin bag or kraft-paper bag before the buds open. Introduce a few flies inside the bag to do the selfing. Carrot the king or main umbel is enclosed in a muslin bag and a second-order umbel of the same plant is also put in the same bag or bagged separately.

2 umbels are rubbed gently against each other by hand daily to ensure self-pollination.

 The technique of crossing As soon as the first bud in an umbel opens, the whole umbel of the female parent is bagged in a muslin bag.
The buds in this umbel are emasculated as they open and when sufficient buds have been emasculated the remaining young flower buds are removed. The umbel of the pollen parent previously kept covered in a muslin bag is cut off and its stalk placed in a glass bottle filled with water which is fastened to a bamboo stake fixed in the soil close to the female parent.

 In onion it is more convenient to plant 2 bulbs each of the male and female parents at the same hill - before anthesis enclose about 2 or 3 umbels each of the 2 parents together inside the same muslin bag. The bag must be shaken at least once or twice daily for a few days for affecting cross-pollination. However, in carrot it would be better to plant the roots of the male and female parents very close to each other, about 20 cm apart, and enclose a few umbels each of the male and female parents in the same cloth bag before anthesis.

ONION
In onion the use of male-sterile lines is common to produce seeds of F1 hybrids. The male sterility in onion is due to the interaction of cytoplasm and nuclear gene The nuclear gene is single recessive msms while its dominant allele MsMs is for male fertility.

PARENTAL LINES
Three parental lines, namely A, B and C, are used for hybrid seed production. The line A, known as the female parent, is male sterile having the genetic constitution Smsms, which produces hybrid seeds, The line B with the geno-type Nmsms is the malefertile counterpart for the maintenance of male sterility of line A The line C is the fertile pollen parent or inbred which is genetically diverse from the lines A and B.

 The hybrid seed is produced in the open in an isolated field. The bulbs of A (male sterile) and C (pollen parent) lines are planted alternately in a ratio of 4 rows of the A line to 1 row of the C line. Sometimes 8 rows of the A line are alternated with 2 rows of the C line. The flowering in the lines A and C must synchronize. If it does not, it can be accomplished by adjusting planting dates of the lines A and C. Daily rogueing of pollen-bearing plants and other off-types in the line A in the morning before the anthers dehisce is essential. When the seed of the line C is not to be saved its flower stalks may be cut and destroyed as soon as the pollination is completed.

USE OF MALE STERILITY AND MARKER GENES

Brown-seeded inbred line in which the brown seedcoat colour was linked with male sterility.

The brown seed-coat colour is governed by a single recessive gene, b, while the black seed-coat colour is due to the dominant gene, B. The brown-seeded male-sterile line and the blackseeded pollen parent line can be used for producing hybrid seeds. The hybrid seed harvested from the male-sterile line will be black in colour.

CARROT
Male-sterile lines are used for the production of F1 hybrid seeds. 2 types of male sterility the brown anther type in which the anthers are shriveled and brown without any viable pollen petalloid type - anthers are modified into petalloid structures.

COLE CROPS AND ROOT CROPS

cabbage, cauliflower, knoll-khol, turnip and radish, which are usually self-incompatible, selfing can be accomplished by bud-pollination. The flower buds are pollinated 2 days prior to their opening by their own pollen by applying fresh pollen from previously bagged flowers of the same plant. seed-set is better - buds are pollinated 2 days before opening when there is at least 30% flowering on the plant

 Enclose 1 or 2 flowering branches in a muslin bag and use individual flower buds or flowers for bud-pollination or crossing as and when they become ready for this purpose. 8 to 10 flower buds or flowers are budpollinated or crossed in each inflorescence branch to ensure better seed-set and the unopened young flower buds at the terminal end are removed.

 overcoming self-incompatibility in radish exposure of plants to 3% CO2 for 2 hr, starting 2 hr after pollination, Pollination assisted by passing a direct or alternating current from the stigmatic surface to the flower stalk was better than CO2 treatment in some strongly incompatible lines.
The artificial pollination is done by hand by shaking the pollen over the stigma directly from the freshly opened previously bagged buds of the male parent. The pollen can also be applied to the stigma with the help of a camel-hair brush.

 When a large quantity of crossed seeds is required, the mature heads of cabbage or curds of cauliflower or roots of turnip or radish of the female and male parents are planted in alternate rows spaced 60 cm apart.

Later about 3 to 4 days before opening of buds the plants are covered under an insect-proof wire net or plastic cage of 22 or 24 mesh, as in case of onion and carrot. Usually 2 plants, 1 female and 1 male, are covered under a small cage or sometimes a cage is used to cover 4 plants, 2 plants each of the male and female parents. A small honeybee colony is placed inside the cage, about 3 to 4 days before opening of buds for pollination of flowers by honeybees.

 A wire-net or plastic-net cage of dimensions 3 m (10 ft) length x 3 m (10 ft) width x 2.5 m (8 ft) height with a small door on one side is convenient for this purpose.
Twenty-five plants of cabbage grown under these cages will produce about 675 g seeds. Similarly, 30 plants of radish and turnip under this cage will give 450 g seeds each, whereas in carrot the seed yield of 40 plants grown under this cage will be 900 g. The hand-pollination is not economical for commercial seed production. Free insect-pollination with the aid of self-incompatibility, male sterility and marker genes.

OKRA
As the flowers in okra are large and solitary it is comparatively easy to emasculate and cross-pollinate or self them. A day prior to opening the flower buds are emasculated, preferably in the afternoon, by scraping off the anthers from the staminal column with the help of the blunt edge of a scalpel. As the floral organs are mucilaginous there are chances of anthers adhering to the staminal column or stigma while removing them. Hence, great care is needed in emasculation so that no anther is left sticking to the floral parts.

 The flowers of male and female parents are enclosed separately in butter-paper bags. For pollination the pollen from the freshly opened but previously bagged male parent is dusted directly on to the stigma of the emasculated flowers of the female parent.

 The pollen can also be applied with the help of a camel-hair brush. For selfing it is convenient to cover the individual flowers in butter-paper bags or to tie the corolla tip with a string or cellophane tape. Better seed-set it is useful to pollinate the flowers by hand using the pollen from the same flowers.

CUCURBITS
Which are monoecious and possess large flowers, selfing and crossing are not difficult. Muskmelon -andromonoecious - monoecious.

The staminate flowers in luffa, cucumber and muskmelon occur in clusters whereas in all the other cucurbits, like bottle gourd, pumpkin and squash, they are solitary.

The pistilalte flowers in all cucurbits are borne singly in the leaf axils.
The staminate and pistillate flower buds are covered separately in butter-paper bags about 24 hours prior to their opening. Often the corolla of the male flower bud is tied with string or very thin wire or adhesive cellophane tape to prevent them from opening instead of covering them under bag.

watermelon and luffa a small moistened wad of cottons placed over the tip of the corolla to prevent the opening of the bud.

The following day when the buds open, the male flowers are collected and their pollen dusted directly on to the stigma of the female flowers which had been bagged previously. After pollinating, the female flower is enclosed in the butter-paper bag and tagged with a small cardboard label on which the details of the cross and date of pollination are written in pencil. It is better to remove the paper bag after 4-5 days of pollination for obtaining better fruit-set. In watermelon the fruit-set is better when the pollination is done just after anthesis.

Increasing fruit-set
Cutting the length of the vine on which the crossed or selfed pistillate flower is situated,

removing other female flower buds or other set fruits from the vine on which the hand-pollinated flower is placed and applying growth-regulating substances.

Cucumber
The following methods may be followed in cucumber: (1) Artificial Pollination Alone : monoeciousemasculation is not required. The female flowers of the seed-parent (female parent) can be artificially pollinated with pollen collected from the male flowers of the pollen parent. A single pollination produces a large number of seeds and, therefore, the cost of production of hybrid seeds is not high.

(2) Removal of Male Buds and Use of Insect-Pollinators

In this method the male buds in the monoecious parent line are removed before they open and only female flowers are left on the plant for pollination by insects in nature.
The seed is collected from the female parent line only. Usually the rows of female and pollen parent lines are planted alternately in an isolated field.

(3) Use of Gynomonoecious Lines

The gynomonoecious and monoecious (pollen parent) lines are planted alternately and at the 10node stage, all the monoecious and other intermediate-sex types are removed from the gynomonoecious line and the hybrid seed is collected from the gynoecious plants which are left for seed production. The monoecious and other intermediate plants from the gynomonoecious line must be removed at an early stage, preferably before the pistillate buds on gynoecious plants open.

 Hybrid seed production in cucumber hybrid Pusa Sanyog is produced by crossing Japanese gynoecious line (E.C. 5082) and Green Long . The planting is done in the channels at a distance of 2 to 2.5 m. seed are sown in the hills at a distance of 90-100 cm in such a way that three hills are made on one side of the channel and two in between these three hills but on the other side the channel. Seeds 7-8 in numbers are sown each hill and are thinned to 2-3 plants per hill.

 Planting ratio of female to male parents is kept 4:1 by adjusting in such a way that 4 hills of female plant surrounds one hill with male parent and is being situated at the centre of each three hills
Field observations - done daily as at many times staminate flower appears in the female line which are to be removed before it flowers. - every day visit to the field should be done to avoid selfing.

 The other most important aspects is maintenance of parental lines.

The male parent can be maintained by selfing and rouging out undesirable plants before they come into flowering. gynoecous female parent is maintained by inducing staminate flowers by spray of silver nitrate @ 20 ppm at two true leaf stage and selfing is carried out it has been observed that 10-11 male flowers appears per 100 nodes.

Although different workers have recommended different concentrations of AgNO3 ranging from 50 to 5000 ppm. Also some workers used GA, 1500 to 500 ppm, but silver nitrate was found more effective.

isolation of atleast one kilometer - with other varieties of cucumber and also C. hardwickii.

Fruits are harvested when they starts turning brown. Fully matured fruit from female line are harvest and seeds are collected by scooping from the splitted fruits. Fermentation by keeping the seeds overnight is commonly used to separate the seeds from the seed jelly. Seeds - washed - clean water - spreaded thinly under sunlight, followed by drying in shade by using drying trays.

6 to 7 fruits /vine producing 300-500 Kg/ha if population is 4:1 female and male,

MUSKMELON
25-30oC for seed germination and vegetative growth a 20-25oC for fruit bud formation, pollination and fruit set. Responds to transplanting, pruning and training (vertical and also horizontal). Significant heterosis is available for all the desired characters in favourable direction.

 The flowering starts in around 40-45 days and it takes30-32 days from flowering to fruit harvest. Flowers open around 6 A.M. Stigma is receptive from earlier evening to the day of anthesis. Pollination could be done from early morning to 9.00 A.M. Bud pollination using stored pollen (at high R.H.) has also given appreciable success in monoecious types), some cantaloupes (eg. Campo) have slow pollen germination and tube growth and need to be pollinated early i.e. around 5.00 to 6.00 A.M. the pollination (cross) is through insects and different types of honey bees do the job.

PRODUCTION OF HYBRID SEED

Hand pollination, - seed parent and pollen parents in isolated plots to avoid intercross.

Collect the male flowers from pollen parent, in the early morning, take out pollen using brush and pollinate the bagged buds (flowers). Monoecious line as seed parent- grow parents side by side (4 female: 1 male) in adjacent rows. Spray 250 ppm Ethrel (2-CPA) at 2 leaf stage repeat at 4 leaf stage and again once before flowering on female parent to avoid any appearance of male flower at initial nodes and harvest initial 3-4 fruits, appearing on 6 to 10 node, from female parent for seed extraction In isolation blocks allowing fee intercross and collecting seed from female parent could be done.

 Using genetic male sterile female parents Female parent could be sown in alkathene bags (nursery) and at the initiation of flowering, only male sterile sibs (50% plants) after testing could be planted in actual seed production block alongside of a suitable pollen parent and seed collected from female (male sterile) plants will give 100% crossed seed. In another method female and male both parents, could be sown side by side and only tested male sterile plants (50%) could be left in field and rest uprooted and destroyed.

 seed extraction
Healthy fruits at fruit to half slip stag are cut open to take out seed jelly and keep it to fermentation in water for 24 hours and then rub it and clean it 25-30 pollinations to produce 3-4 fruits 400 Kg seed/ ha - 15-20% area for male parent. An investment of around 25,000 rupees - return of around 1.0-1.5 lakh / Rs.1200 / Kg seed price.

WATERMELON

Artificial Pollination Alone : Same as in cucumber. Removal of Male Buds and Use of Insect-Pollination. Same as in cucumber.

Use of Marker Genes : The non-lobbing leaf character governed by a single recessive gene- expressed in the seedling stage. The F1 hybrids having lobed leaves can be easily distinguished in the seedling stage. Sowing about 6 to 8 seeds per hill, as on an average only one-third of the plants will be F1 hybrids. This method may be quite easy and economical for production of hybrid seeds.

Triploid Watermelon

F1 hybrids of the cross made between tetraploid and diploid

For producing tetraploids the young seedlings of the diploid variety are treated with an aqueous solution of colchicine (0.2-0.4%). A wad of cotton-wool is placed over the growing point and on it is applied a drop of colchicines solution every morning and evening for about 4 to 6 days. The tetraploids can be maintained by selfing. The tetraploid is pollinated with pollen from the diploid to produce triploid seed.

 The triploid is highly sterile and seedless produces white rudimentary seeds or in some cases coloured empty seed-coats.
The dark-green faintly striped rind colour of the diploid cultivar Sugar Baby was dominant over light-green, striped rind of the tetraploid parent, W 687. In the triploid the rind was medium striped of intermediate colour. Thus the diploid, tetraploid and triploid could be easily identified on the basis of their rind pattern and colour.

Squash and Pumpkin

The same methods as used in watermelon can also be used in squash and pumpkin. The methods suggested are as follows : (1) Artificial Pollination Alone : Same as in cucumber and watermelon. (2) Removal of Male Buds and Use of InsectPollination : Same as in cucumber and watermelon.

 3.Male sterility in Cucurbita pepo has been reported. The male sterility is governed by a single recessive gene.

4. Use of chemicals The production of male flowers on monoecious plants in Cucurbita pepo can be - suppressed for 2-3 weeks by repeated sprays of ethephon (2chloroethylphosphonic acid) at 250 ppm on young plants at the first true-leaf stage. Ethephon is being used for commercial production of hybrid seeds in squash