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Recent Methods of Citrus Multiplication


Availability of quality planting material is of utmost importance in citrus cultivation as non-availability of disease free planting material of citrus has been the basic reason for such a low production, short productive life and gradual decline of citrus trees. Citrus trees are propagated by seed and by vegetative means. Vegetative propagation is preferred because it ensures true to type plants, uniform quality, regular and early bearing etc. In most areas, a citrus tree is produced by budding the desired scion variety budded onto the chosen seedling rootstocks. Citrus plants are very sensitive to various biotic and abiotic stresses. Therefore selection of an ideal rootstock is a continuing challenge for the citrus industry of India. In process of bud selection, it is required to ensure that clonal purity, good physiological vigour and yield potential are maintained and bud material should be free from transmissible diseases like viruses. Citrus is highly susceptible to both biotic and a biotic stresses but virus and virus- like pathogens are the main biotic agents responsible for poor tree health and reduced yield. For growing healthy citrus trees it is essential to have mandatory bud-wood certification programme. Unless the orchards are planted with disease-free nursery stock, none of the potential of the improved practices can be fully realized. Therefore production of disease-free planting material should made mandatory for bright and healthy future citrus industry of India. Various aspects starting from selection of healthy and resistant root stock to the use of certified bud wood are considered for raising healthy disease-free planting material of citrus. The recent techniques adopted in production of disease free planting material are briefed here. 

Containerized Nursery System

 In India most of the cases citrus plants are raised as field nursery. In field nurseries, the eradication of soil borne pathogens like Phytophthora once introduced becomes very difficult. To avoid this problem, concept of containerized nursery system is to be adopted. The infrastructure required for such nurseries includes shade net houses (50 % shade), sterilized plastic trays, UV stabilized black polybags (100 µ), UV stabilized transparent polythene for solarization, fumigation of potting mixture and a separate set of nursery equipments. 

Potting mixture

The potting mixture, consisting one part of  fertile soil + sand + FYM (sterilized), should be used in plastic trays for seed sowing in primary nursery. The same sterilized mixture has to be used for filling the polybags which is be used in secondary nursery. 

Soil solarization

The potting mixture is first taken onto a concrete floor and spread in the form of flat bed of 1.5’ thick layer. These beds were completely drenched with water before covering it with 100 µ UV stabilized transparent polythene sheets in summer months (April- May) when atmospheric temperature goes up to 45 - 460C. The edges of polythene sheet should be completely sealed with soil to avoid vapour loss, which allowed the inside temperature to rise up to 540C. This process of soil solarization completes within 4 - 6 weeks. 

Soil fumigation

The solarized soil is further fumigated with Basamid (Dazomet) granules, a soil fumigant, which releases methyl isocynide gas and thereby, completely eliminates soil born fungi like Phytophthora spp., Pythium spp., Rhizoctonia spp. and Fusarium spp. from the soil. Solarized and fumigated potting mixture was used to fill nursery pro-trays for raising seedlings &poly-bags. 

Steam sterilization

This is one of the quickest methods of potting mixture sterilization. In this process steam are passed through potting mixture which kills all pathogen and weeds. First mixture is filled in jeep trolly which has nozzel in bottom of trolley. Then steam which is generated in boiler passed in  this covered trolley for about 20 minute. 

Selection of seed

Seed should be collected from healthy fruits of recommended rootstocks. Only selected trees free from diseases are used as seed source. Seeds are extracted from fully ripened fruits by making a shallow cut through the rind round the centre of the fruit and twisting the two halves of fruit apart. Seeds are then washed into cold water with rubbing in ash to make free from pulp and dried under shade condition. The sound seeds, being of greater density are separated from the underdeveloped seeds. Seeds should be sown as early as possible after extraction, since citrus seeds give the highest germination if planted immediately after extraction. 

Raising of seedling in primary nursery

For raising disease- free nursery it is required to grow primary nursery in tray. Plastic tray 60x40x12 cm size is preferably used for primary nursery. After making 6 holes in bottom of trays to drain out excess water, trays are filled with sterilized potting mixture and trays are kept at 1.5 feet height from the ground on the bricks or on cement platform to check the contamination. Seeds are sown at depth of 1- 1.5 cm with spacing 2.5 to 3.0cm in the row and after sowing light shower irrigation should be done with watercan. 

Transplanting of seedling in secondary nursery

Seedling when 10 to15 cm tall having 8 to 10 leaves are transplanted to black polythene bag of 12” to 6” size having 3-4 holes at the bottom to drain out excess water. The polythene bags are filled with sterilized potting mixture and arranged in shade net. To ensure uniform nucellar seedlings, smaller or too taller ones at the time of transplanting should be discarded. Seedlings from primary nursery from trays should be uprooted with fork carefully to minimize root damage. The hook-necked bent or twisted taproot seedlings should be avoided. Selected seedling should be treated with Ridomil (2.75 g/l) solution before transplanting to check the contamination at the time of transplanting.

Selection of mother plants and budwood

Selection of mother plants for budwood is the most critical parts of production of disease-free planting material. Mother plants should be selected from authentic sources with known pedigree with regard to health, vigour , regular bearing and high yield with good quality fruit. Selected plants should be indexed against viruses and Greening bacterium and only disease-free plants should be used as bud source. Such disease-free plants should be marked and bud stick should be carefully taken to avoid infection. Bud wood should be always taken from fairly well matured wood of current season growth. Round twigs having longitudinal white streak on the bark, swollen buds ready to grow should be selected. Bud wood should be kept in moist sphagnum moss and gunny bag to avoid exposure desiccation.

Diagnosis of Viral diseases

Various virus detection tests can be performed for knowing the disease status of mother plants. 

·       Serological diagnosis: Serological indexing was done in DAS-ELISA by using monoclonal (CTV) and polyclonal (CTV, RS, mosaic) antibodies.

·       Bio-diagnosis: The biological indexing was also performed simultaneously using indicator plants like acid lime for tristeza, sweet orange for greening bacterium and mosaic virus, Etrong citron for exocortis and sweet orange and Chenopodium quinoa for ring spot etc. under insect proof controlled conditions.

Budding and maintenance of budlings

Budding is usually done when seedling stems are 3-3.5 in diameter (about the diameter of a pencil). Budding can be done anytime on suitable stock on which the bark is slipping and when suitable budwood is available. Usually, the bark is slipping from April to November, depending on location. The area to be budded should be pruned clean of thorns and twigs. The preferred budding height is 9 inches above ground level following the ’T’ or shield budding method. The budded portion should be wrapped with 100 guage 1.2-1.8 cm wide polythene strips. 

Plant protection measures

In citrus nurseries, Phytophthora diseases may appear any time of plant growth through contaminated water, soil and even through nursery workers and implements. Therefore a regular monitoring should be done for Phytophthora infection. In case of infection, the infected/contaminated plants should immediately uprooted and destroyed to keep the nursery totally free from Phytophthora and other diseases. The nursery plants are sprayed with Bavistin @ 1 g/lit water at monthly interval as a prophylactic measure and spray Ridomil MZ 72 @ 2.75 g or Aliette @ 2.5 g/lit. Nursery implements should be disinfected regularly with sodium hypochloride solution and at the entry of nursery. The arrangement should be made to disinfect the shoe of workers and visitors with copper sulphate and lime dust. Floor should be regularly sprayed with copper fungicides and at the entrance of nursery. The insect pests and mites in nursery should be managed with the regular application of recommended insecticides. 

Production of disease free citrus plants by shoot tip grafting

In vitro shoot tip grafting (STG) is a miniature grafting which involves grafting a minute shoot tip (0.1 to 0.3 mm) on two week old seedling rootstock performed under aseptic conditions. It produces true to type, non juvenile disease free plants unlike nucellar embryony in vitro or in vivo. This technique is a prerequisite for cleaning the citrus cultivars or indigenous collections from virus like diseases and greening bacteria,since absolutely there is no control measure once the virus enters the plant system except shoot tip grafting (STG). Standardized age, type of rootstocks, size and source of shoot tip and growth regulator used influence the efficiency of grafting.
The highest percentage of successful grafts was obtained when seedling were raised in NAA supplementation (0.5 mg/l) followed by BAP. The frequency and successful graft was high if the root stock seedling were 15 days old. It indicates that age of seedling also played an important role. Among the grafting height above 2 cm showed less success.

Micro Budding

Micro budding is a miniature budding on young citrus root stocks measuring 3 mm diameter in which the bud is inserted on the detopped root stock in a wedge cut and immediately protected by covering with a micropipette tip. After a week, micro buds are observed and then micro tips are removed after their sprouting within 12-14 days. Micro budding facilitates faster propagation with reduced cost. This has a tremendous scope in commercial propagation and research. It reduces labour cost and maintenance during the commercial propagation in low cost green house. It can be utilized in biological indexing of virus, viroids, greening bacterium and other disease inoculation and expression studies at much faster pace, enable year round multiplication and shortens the nursery phase.

In-vitro regeneration of citrus Scions and rootstocks through Somatic embryogenesis

In citrus, nucellar embryony has been commercially exploited for breeding and genetic studies. Nucellar embryos, like shoot tip are free of virus and can be used for raising virus free clones, especially in citrus where shoot tip culture has not been successful. Hence technology was developed at NRCC for whole plant regeneration via somatic embryogenesis. Ovules were excised from 8-10 weeks old fruits of Citrus reticulata cv. Nagpur mandarin and C. limonia cv. Rangpur lime Brazilian and cultured in MS - medium supplemented with various organic compounds. Cotyledonary embryoids obtained were subcultured and MS medium supplemented with various growth regulators kinetin in combination with IAA promoted plantlet formation.

In- vitro regeneration of Citrus Rootstocks

The in vivo clonal propagation of plants is an often difficult, expensive and season-specific . Tissue culture methods offer an alternative means of vegetative propagation in short time, space and throughout the year. Multiple shoots obtained at NRCC from mature axillary bud explant / single node of mature trees (>10 years old) of Citrus limonia Osbeck cultivars Rangpur lime Gonicoppal and Brazillian Rangpur lime, when cultured in MS medium supplemented with BAP, kinetin and NAA. In vitro proliferated shoots rooted when shoots were cultured on MS supplemented with IBA. Multiple shoots obtained in modified MS media fortified with BA from the nodal explants of complete in vitro regenerated plants. Rooted complete plantlets transferred to micro pots having sterilized soil mixture with 60% survival. Hardened and acclimatized plants were transferred to the green house & successfully survived in open condition.
Author- Prof. N.K. Mishra

Department of Horticulture, College of Agriculture, G. B. Pant University of Agriculture and Technology, Pantnagar-263 145 (US Nagar, Uttarakhand)

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