Groundnut

Hybridization

• Artificial hybridization between parental lines to bring together a desirable combination of genes is an integral component of any crop improvement program.
• The cultivated groundnut, a native of South America, is regarded as a self-pollinated leguminous crop (normally with less than 1% cross- pollination), but cross-pollination up to a maximum of 10% has been reported ,resulting in natural hybridization.
• This outcrossing is related to the level of bee activity prevalent on a genotype in a season and at a specific location.
• Bees of the genera Apis, Bombus, act as cross-pollination agents .
• The genus Arachis is morphologically well defined and is clearly delimited from its closest related genera by the development of a 'peg' and geocarpy.
• The genus Arachis has more than 70 wild species existing in nature, of which only A. hypogaea is commonly cultivated . A. hypogaea is a segmental amphidiploid (2n = 4x = 40) .
• Cultivated ground nuts are classified as follows:

• The virginia group consists of plants that have spreading (runner), spreading bunch, or upright (erect bunch) growth habits and an alternate branching pattern; they lack flowers on the main stem leaf axils, possess fresh seed dormancy, flower longer, and mature later than those of the valencia or spanish groups.
• Both the valencia and spanish groups consist of plants that are mostly upright in growth habit, have sequential branching patterns, and have flowers on the main stem leaf axils, and possess no or very limited fresh seed dormancy.
• In the valencia group, secondary branches are absent or occur on distal nodes, while in the spanish group, they are occasionally found irregularly located on primary branches.
• The first branch on the cotyledonary lateral is always vegetative in sub- species hypogaea, while in sub species fastigiata, it is reproductive.
• How- ever, with extensive inter- and intra-subspecific hybridizations being carried out in breeding programs, true breeding intermediate forms have been established due to selection.

• For hybridization in the field, rainy season is the best season as atmospheric humidity is high.
• In greenhouses or growth chambers where it is possible to control light, humidity, and temperature, hybridization can be carried out at any time of the year.
• Hybridization should be restricted to the early phase of flowering because of higher success rates in the production of mature pods from early-formed flowers.
• The skill of the operator and environmental conditions influence the success rate.
• Before beginning the hybridization, it is very important to ensure that all the plants to be used are true-to-type.
• Plants that are vigorous and healthy are selected for hybridization.
• At the onset of flowering, for 1-2 days, all the flowers on the female parents should be removed to help stimulate profuse flowering.

• The internode just above the emasculated bud is then marked with a date-coded colored nylon thread.
• A thread of a different color is used every day to help identify the buds for pollination the next day.
• Though cross-pollination is reported to occur in groundnut, there is generally no need to portect the emasculated bud from unwanted pollen if insects are properly controlled.
• It may take about 40-60 days from fertilization to pod maturity.
• There fore, emasculations should be stopped at least 60 days before the normal harvest time to permit full development and maturity of hybrid pods from the last-pollinated flowers.

• If the style is fresh and of normal length, the flower should be pollinated. If the style has withered or turned brown due to injury during emasculation, the flower should be rejected.
• For pollination, a healthy flower from a pre identified male parent plant is removed by breaking the hypanthium.
• The calyx, standard, and wing petals are detached for ease in operation. The keel petal is gently pressed between the thumb and index finger to squeeze the sticky pollen mass out from the anthers.
• The sticky lump of pollen is deposited on the tip of the stigma of the emasculated flower.
• It is possible to pollinate up to 15 female flowers with 1 male flower, depending on the environmental conditions at the time of pollination.
• The pollinated flower should not be disturbed for some time after pollination to avoid dislodging the pollen from one male parent to another to avoid contamination with unwanted pollen.
• All flowers except those that are artificially pollinated, should be removed every day soon after pollination from the base of the hypanthium.
• This helps in prolonging the duration of flowering of the female parent plant.
• This flower-removal operation should be carried on for at least 2 weeks after completing the last pollination of the season.
• This reduces competition for the development of hybrid pods.

• In this method, a bud about 14-16 mm long, having a hypanthium about 10mm long, is selected and marked by a coloured thread tied around the hypanthium.
• A superficial circular incision is made with a razor blade in the bud at about tow-thirds down from the top or 2mm above the base.
• With forceps, the cone-like incised top, consisting of parts of the calyx and standard, is pulled out.
• This exposes the keel wrapped in the wing petals.
• The wing petals are then turned backwards with the help of the forceps to expose the keel.
• A gentle incision is made with a pointed needle from the bulged base towards the tip of the keel petal.
• This exposes the anthers, which are then carefully pulled out with the forceps.
• Pollination is carried out either by directly brushing extruded pollen from the male flower on the stigma, or by collecting the pollen on the tip of the forceps or a brush, and dusting this on the stigma.
• The hybrid pegs, identified by the thread around the dried hypanthium, are inserted into small aluminum rings before entering the soil.

• Square plastic pots of 30 cm x 30 cm x 27 cm (l x b x h) With 8-10 holes at the base are used for growing groundnut plants in the greenhouse.
• Alternatively, any container big enough to support the normal growth of two groundnut plants can be used.
• A soil medium is prepared by mixing four parts of soil, three parts of sand, and one part of well-decomposed farmyard manure.
• The soil medium is steam sterilized at 62 kpa pressure and 820C for 1 hour.
• Approximately 22-24Kg of the soil mixture is required per pot.
• Stone pieces are first placed around the drainage holes and the pots are then filled with the soil mixture.
• This prevents clogging of the holes and water logging.
• Diammonium phosphate is applied to the soil in the pots at a rate of 10-15g/pot .
• As an added precaution to avoid soil borne fungal pathogens, the soil is drenched with Carbendazim ,In addition, Carbofuran granules are also applied to control damage by insect pests.
• Benches are arranged within the greenhouse so as to provide easy access to the pots.
• Four healthy seeds of a parental line are sown in each pot to be used for female or male parents.
• Two healthy and vigorous seedlings in each female parent pot and three in each male parent pot are then retained after germination.
• The number of pots for each female parental line depends on the number of hybrid seeds required.
• Normally 10 hybrid pods are obtained from each plant.
• For every two to three pots of the female parental line, one pot of the male parental line is sufficient to produce enough flowers for pollination.
• Immediately after pollination, humidifiers should be operated for 2 - 3 h to raise the humidity.
• During the day, if temperatures are high, coolers in the greenhouse should be operated.
• After the last pollination of the season is completed, about 6 - 8g gypsum should be added to each pot including the small pots .
• All the pots should be watered regularly and plants protected from insect pests and diseases.

• However, with careful planning, good crop husbandry, well-trained operators, and by raising the humidity level in the field, it is possible to get high success rates.

• The field selected for hybridization should have a high fertility level to support a good groundnut crop, be well drained, have a reliable irrigation facility, and be easily accessible.
  
  
• It should not have had a groundnut crop for at least 2 years so as to avoid the problem of volunteer plants from previous crops.

• In a crossing block, the row length for the female parent varies according to the number of hybrid seeds desired; the larger the number, the longer will be the row.
  
  
• A row length of 4 m provides about 40 female plants to carry out 200-250 pollinations per cross.
  
  
• Generally the female and male rows are alternated for ease of operation.
  
  
• Row-to-row spacing adopted is usually 120-150 cm, and plant-to-plant spacing is usually 10 cm (for the spanish and the valencia types) or 15 cm (for the virginia types).
  
  
• The wide row spacing provides ample space for the operators during emasculation and pollination and they can work without disturbing the plants.
  
  
• For ease of operation, male and female parental rows are identified with different colored labels.
  
  
• For those crosses where one of the parents may flower early or late, staggered planting may be necessary to ensure synchronization of their flowering periods.

• To obtain high success rates in hybridization, it is essential to grow vigorous and healthy plants free from any biotic or abiotic stresses.
  
  
• Care is to be taken to select only good, healthy seeds free from internal and external seed borne pathogens.
  
  
• These are treated with appropriate chemicals against seed and seedling pests and diseases.
  
  
• Subsequently, the hybridization field is hand weeded. Gypsum is applied as a band on both sides of the plant row at a rate of 400 kg ha –1 soon after the last pollination is completed, and a light earthing up is done along the side of the plants, with manual removal of weeds.
  
  
• Standard schedules for pest and disease control are followed to provide an intensive protection to the crop.
  
  
• High humidity around the plants soon after pollination helps to increase the success rate of hybridization .
  
  
• Humidity is generally high during the early morning hours when pollinations are carried out. It can be further raised by giving a light perfo-irrigation late during the previous evening.
  
  
• The soil stays wet until the next morning, resulting in raised humidity levels.
  
  
• Furrow irrigation can also be given, but this often results in excess wetting of the soil, causing inconvenience to operators.
  
  
• The crop should be visited regularly and precautions should be taken to avoid biotic or abiotic stresses.
  
  
• The top layer of the soil should be kept wet to facilitate the entry of pegs into the soil.

• The female parent plants are harvested at the onset of maturity or at least 60 days after the last pollination.
• The female plants are watered to soften the ground, and the plants are carefully lifted to avoid any pod loss.
• The pods originating from the nodes having the marker nylon thread in their upper internode are the hybrid pods.
• There should be only one pod arising from each such node.
• If more than one pod is seen at the node, then, both the pods are discarded.
• These hybrid pods are removed from the plant and kept in a muslin bag with a label, indicating the pedigree of the cross and season of crossing.
• These pods are dried inside the cloth bag to avoid direct exposure to the sun.
• After drying, the mature hybrid pods are counted and recorded.
• The pods are then stored in a cold room until the next growing season.
• If needed, selfed pods from the female plants are also harvested and retained.

• Flower number per inflorescence Varies with the cultivar.
• A flowering branch never occurs at the same node as a vegetative branch, although they may appear to do so because of the shortness of the internode below the first cataphyll of a branch.
• In variety hypogaea, the inflorescences are simple and expand slightly in length during maturation.
• In variety vulgaris, the inflorescences are compound and expand moderately.
• In variety fastigiata, the inflorescences are simple, but may elongate to form a conspicuous long branch that may ocassionally terminate in leaves.
• The length of the inflorescence is also dependent on the cultivar and may exceed 10 cm in some varieties.

• It has a central area called the stadard crescent which has darker lines radiating from the base to the periphery of the standard in most cultivars.
• The wings are generally yellow wrap around the keel. The keel is pale yellow, and closely wraps around the stamens and the upper parts of the style and stigma.
• There are 10 monadelphous stamens of which two are staminodes (sterile) represented only by filaments. The remaining eight are dimorphic.
• Of these, four are with globose, dorsifixed and monothecous anthers, alternating with four having adnate, introrse, oblong anthers, three of which are bitheeous and one, opposite the standard, monotheeous.
• The basal two-thirds length of the filaments are fused. The filaments of the globose anthers are initially shorter than those of the oblong ones, but they elongate the become equal to or longer than them a few hours after pollination.
• The ovary is situated at the base of the hypanthium; it is superior, about 1.5 mm long, and normally has two tofour ovules, ocasionally five, and rarely six.
• The style is long and filiform with two bends; the first bend is close to the upper end of the hypanthium, and the second one occurs along with the bend in the keel petal.
• The style has upward-slanting hairs on its distal portion. The stigma is club-shaped and usually on level with, or slightly above, the anthers.

• The flowering pattern varies among and within botanical varieties.
• Generally the valencias flower before the other types and have a short time of flowering.
• The spanish types also flower early, but the first flowering peak may be broader than that in the valencias and some varieties have multiple peaks.
• The virginia types take more time than the other two types to start flowering and have multiple flowering peaks.
• However, cultivars within a subspecies vary in their flowering patterns.
• One bud per inflorescence usually reaches anthesis on a given day, but ocasionally tow or more buds per inflorescence may open on the same day.
• The bud is 6-10 mm long 24 hours before anthesis and, during the day, the hypanthium elongates slowly and the bud attains a length of 10-20 mm.
• During the night, elongation of the hypanthium is faster. The flower reaches a maximum length of 50-70mm at the time of anthesis.
• Flower opening is normally at sunrise, but may be delayed by low temperatures.
• Anthers may dehisce 7-8 h before flowers open in some varieties whereas in others they may not do so even at flower opening.
• Stigma becomes receptive about 24 h before anthesis and its receptivity presists for about 12 hours after anthesis.
• Pollen grains are smooth, oval, and sticky, Fertilization occurs about 6h after pollination. In general, self-pollination is the rule.
• However, in situations where the stigma and anthers are exerted from the keel and the stigma is still receptive, the flower is more vulnerable to cross-pollination by bees.
• Fertilization of the egg activates the growth and elongation of the intercalary meristem which is located at the base of the ovary.
• As a result, a stalk-like structure or peg becomes visible within 4-6 days after fertilization under normal environmental conditions.
• A peg, other wise known as carpophore is, at this stage, a small pinkish, purplish, or greenish growth depending upon the cultivar.
• When temperatures are low, appearance of the peg may be delayed by up to 14 days.
• The peg bears the ovary with the fertilized ovules at its tip. It has a positively geotropic growth behavious and can reach a length of up to 15 cm.
• Once the peg enters the soil and penetrates to a depth of 2-4cm, the tip becomes diageotropic and the ovary develos into a pod.

• Distinct rows for female parent, F1, and male parent should be planted.
• Alternatively, they could be planted in continuation, starting with a few seeds of the female parent, crossed seed, and then a few seeds of male parent, with gaps between them.
• All F1 plants should be carefully examined for various morphological traits and compared with both parents to confirm their hybridity.
• All selfs should be uprooted and discarded.
• After harvest, pod and seed characters should also be compared to reconfirm hybridity.