UNIVERSITY OF HORTICULTURAL SCIENCES , BAGALKOT.

PRESENTATION ON

Tissue culture and Biotechnological approaches in Plantation crops

BY:Mr. CH. LokeshUHS14PGM433

KITTUR RANI CHANNAMMA COLLEGE OF HORTICULTURE, ARABHAVI

Tissue Culture Tissue culture is a technique of growing plant cells by

culturing ex-plant aseptically on a suitable nutrient medium.

Tissue Culture technique relies on: Ex-plant Aseptic environment Nutrient media.

Modes of Culture:

(i)Callus culture

(ii)Cell suspension culture

(iii)Single cell culture.

Biotechnology:Biotechnology is name given to the methods and

techniques that involve the use of living organisms like bacteria, yeast, plant cells etc or their parts or products as tools (for example, genes and enzymes)

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology.

Applications of biotechnology:

Micro propagation of biomass energy production.

Production of disease free, disease resistant, insect resistant, herbicide resistant plants.

Induction and selection of mutants.

Production of somatic hybrids.

Production of somatic plants.

Industrial biotechnology.

Coconut

S.N: Cocos nucifera Family: Palmae Chromosome no:2n=32. Is a versatile palm with varied uses. It is also called as Kalpa vriksha, Tree of heaven.

Micro propagation: Current production of quality planting materials meets only

20% of estimated annual requirement of planting material to replace senile and disease free plantation.

In vitro vegetative multiplication through somatic embryogenesis of high performance individuals thus offers the only hope for production of homogeneous planting materials and for substantial improvement in plantation productivity.

Unfortunately coconut is highly recalcitrant sp with respect to tissue culture.

success in this area has been limited and only a few clonal plants have been ever established in the field.

Various problems encountered during in vitro propagation of coconut are….

1.Intensive tissue browning (due to oxidation of polyphenols),2. Slow in vitro response, 3.low rate of somatic embryogenesis, 4.variation in tissue response due to heterogeneity of explants

taken from different individuals.

A variety of protocols have been developed using a range of explants:

• Immature inflorescence, • Immature and mature zygotic embryos, • Young tender leaflets, leaf bases from unopened spindle

and plumular tissue.

Embryo culture

• Embryo culture has become an important tool for safe Germplasm movement.

• A simple protocol of culturing coconut embryos of 8 to 11month-old nuts was standardized and successfully utilized in Germplasm expeditions.

Protocol broadly consisted of four components: I. Field collection of embryos.II. In vitro active storage.III. In vitro retrieval and IV. Ex vitro establishment of plantlets.

• Embryo, along with a portion of endosperm, is scooped out, surface-sterilized, washed and directly inoculated.

• In vitro active conservation (short-term storage) of coconut zygotic embryos is ideal for enabling embryo storage (for up to 2 months in sterile water).

• For ex vitro studies, protocol uses a potting mixture consisting of sterile soil, sand and coir dust in equal proportions.

• Before transplanting to pots, leaves are trimmed and seedlings are treated with Bavistin (1 g/L) and thereafter with IBA solution (1000 ppm) as a precautionary measure.

• To adjust humidity conditions, plantlets are covered with polythene bags that are gradually perforated.

• Zygotic embryo culture was utilized for Germplasm expeditions of coconut.

• Percent retrieval of embryos varied among location and among accessions.

• A major cause of differential germination was contamination of cultures.

• Treatment of cultures with tetracycline (2 ppm) is effective in treatment of cultures with mild bacterial contamination.

• Germination (%) varied between 54 to 82.2.

• Observations on in vitro retrieval of embryos and their ex vitro establishment suggest that, about 300 to 400 embryos per accession needed to be collected for field establishment of 100 plants in a gene bank.

In vitro conservation• Coconut genetic resources are traditionally conserved ex

situ in gene banks. • Use of in vitro culture techniques including slow growth

and cryopreservation, represents an important additional option for safe medium and long term conservation of coconut Germplasm.

• A medium containing 2g/l of activated charcoal could store embryos for 6 months and gave 77% germination.

Immature embryos from nuts of 7-8 months after pollination could be successfully cryo preserved and retrieved.

Embryos were desiccated for 4 h in air current of laminar flow cabinet, pre-treated for 11-20 hrs on a medium containing 600 g /liter sucrose and 15% glycerol and then rapidly immersed in liquid nitrogen.

Freezing and thawing with recovery rates between 33 and 93% of frozen embryos, depending on the variety was reported.

Subsequently optimal conditions for the medium-term conservation of zygotic embryos were developed.

• Immature embryos collected from 10 and 11 months old nuts of two exotic Germplasm viz., Cochin China Tall (CCNT) and Malayan Yellow Dwarf (MYD) cultivars of coconut were utilized for study.

• Extracted embryos were stored (30 days) individually in 5 ml screw capped bottles containing 2 ml of storage medium.

• Germination at 60 days after inoculation was significant with type and age factor.

• Regarding type, germination was more Dwarf type (93.7%) whereas for CCNT it was 85.6%.

(Brian, 2006.)

What makes clonal propagation of coconut difficult?

S.C. Fernando*, V.R.M. Vidhanaarachchi, L.K. Weerakoon and E.S. Santha

Tissue Culture Division, Coconut Research Institute, Lunuwila, Sri

LankaProceedings Asia Pacific Conference on Plant Tissue

and Agribiotechnology (APaCPA) 17-21 June 2007

Some of the major constraints are…

1

2

Heterogeneous response of various explants due to the influence of ex-plant maturity and genotype.

Undefined culture conditions with regard to the hormonal composition of the culture medium due to the presence of activated charcoal.

3

4

5

Production of highly heterogeneous and compact colloids due to partial de-differentiation of cells of explants.

Poor plant regeneration efficiency due to marked influence of genotype and variable culture conditions.

Slow growth of tissue-cultured plants under in vitro and early ex vitro conditions.

How to overcome these limitations?

1 Selection of the most responsive ex plant

2

3

4

5 Application of stress conditions to improve somatic embryogenesis and plant regeneration

Development of a charcoal-free medium

Establishment of cell suspensions

Repeated multiplication of embryogenic cell masses

Activated charcoal is an essential component of coconut tissue culture medium.

It has strong adsorptive properties and its beneficial effects are attributed to the adsorption of phenols and other growth inhibitory substances.

However, a major disadvantage of using activated charcoal is that it also can adsorb plant regulators (hormones, vitamins) and some minerals (Cu and Zn) (Pan and Staden, 1998).

Effect of activated charcoal

As a solution, a charcoal-free medium was developed…

Addition of PVP (20 gr/l) and ascorbic acid to culture

medium in place of

charcoal gave rise to 60 % callus.

However, the embryogenic potential of those calli was low (Diyasena,

1998).

Use of a single type of charcoal under controlled conditions could be a

way to overcome the problem.

Rubber • S.N: Hevea brasiliensis., • Family: Euphorbiaceae• Origin :Amazon river basin of South America• The principal source of natural rubber. • It is intensively cultivated in South East Asia.

Micro propagation

• Propagation of rubber is possible through tissue culture also.

• Studies on tissue culture of rubber plants were started in 1966.

• Different parts of the plant such as embryo, anther, shoot tip

and integument can be used for tissue culture.

• Rubber Research Institute of India has developed a technique for the production of tissue culture plants from shoot tips as well as somatic embryogenesis of different tissues.

• Rubber plants were developed by the somatic embryogenesis of anther tissue, Integumental tissue, immature Inflorescence etc.

• Attempts are being made for the tissue culture of other plant parts like leaf, floral buds, ovules and micro spores.

Key steps in tissue culture of Hevea include : collection of the explant. sterilization, inoculation of the explant in a nutrient media

supplemented with growth hormones and sucrose. The cultures were kept under optimum light and

temperature conditions for the required period. Plants were formed in about eight months. They were then transferred to small poly bags and kept in a

green house for hardening. Even for the same clone the culture conditions vary with the

physiological stage of the explant, seasons, part of the plant used etc.

Somatic embryogenesis:

• Somatic embryogenesis is one of the powerful tissue culture techniques for mass propagation of elite Hevea clones.

• High frequency somatic embryo induction and plant regeneration were achieved from immature anthers and inflorescence.

• Optimum callus induction was obtained in modified MS medium supplemented with 2.0 mg/L 2,4-D and 0.5 mg/L Kinetin.

• Immature anthers pre-cultured in liquid medium for 10 days followed by 25 days culture in solid medium promoted callus induction.

• Embryo induction efficiency was promoted by supplementing 200 mg glutamine and 400 mg casein hydrolysate in embryo induction medium.

• Incorporation of GA3 up to 2.0 mg increased embryo induction frequency.

• Good callus induction and high frequency somatic embryogenesis were obtained using leaf and ovule as explants.

• Maturation, germination and bipolar differentiation of embryos were achieved in MS medium devoid of growth regulators.

Callus initiation from immature anther explants.

Embryogeniccallus formation

Different stages of somatic embryos

Somatic embryo derived plantlets

Embryo germinationand plantlet development

Somatic embryo derived plants

Anther culture• Rubber being a perennial tree crop with a long life span

and the available varieties of the rubber trees are all highly heterozygous.

• Homozygous diploid pollen plantlets could be directly

utilized in crop improvement programmes.

• The possession of haploid and double haploid plants, on which both dominant and recessive characters would be expressed, considerably shortens the process of selection of desirable characters.

• The heterosis of good combinations of pure lines is much higher than that of crosses between two different varieties.

• Since 4-5 years are required for Hevea from sowing to flowering and the fruit set success on selfing is very low, it is highly impossible to obtain pure lines by conventional breeding.

• By the establishment of anther culture and production of haploid plants one can develop pure lines of different genotypes on a short time and thus pave the way to produce uniform sexual lines combining desirable characters (Chen, 1984; Asokan et al., 1992)

• Das et al. (1994) studied the effect of low temperature a on somatic embryogenesis from anther-derived calli.

• Anthers of various clones were cultured on different media.

• High sucrose (6% ) in the medium and cold treatment of anthers at 8-10° C for 24 hours had positive effects on callus induction.

• Supplementation of the medium with benzyl adenine(BA) along with combination of IAA or NAA produced the highest number of somatic embryos.

Protocol for anther culture

The protocol reported by Chen (1984) is as follows for the induction of pollen plantlets.

It is carried out in three steps :

I. Anther is inoculated on primary medium to induce callus formation in which the microspores grow and develop into multi-cellular masses, haploid embryos or calli.

II. The pollen-derived callus is transferred to differentiation medium in which they develop into minute embryos which subsequently develop into the embryos visible to the naked eye.

• At this point, about 70% of the cells have the haploid chromosome number.

III. The well developed microspore embryos are transferred to plantlet forming medium on which they further develop into intact plantlets.

• For callus induction a medium containing macronutrients and iron salts of MS medium with reduced nitrate and micronutrients, vitamins and organic supplements as suggested by Bourgin and Nitsch (1967) with 5% coconut water containing 9.3 M Kinetin and 9.6 M 2,4-D.

• During the process of embryogenesis, different morphological types, such as round, trumpet, rod and cotyledon types were observed of which the trumpet and cotyledon types are found to be more normal producing majority of plantlets.

• Once the microscopic embryos are formed in the differentiation medium, it will take about 2-3 months to develop terminal buds.

• The embryos should be transferred to plantlet formation medium only after development of terminal buds.

• Otherwise, the embryos form roots but not shoot

Invitro Conservation

• Conservation of genetic resources of Hevea is an urgent need of the time.

• International Plant Genetic Resources Institute (IPGRI), formerly International Bureau of Plant Genetic Resources, (IBPGR) has included rubber as one of the highest priority crops for conservation of the entire gene pool (IBPGR, 1984).

• Both in situ conservation of genotypes in their original habitats and ex situ conservation in special nurseries or fields are feasible in Hevea, of which the latter is widely adopted due to practical considerations.

The storage of Hevea seeds to prolong seed viability is important because seeds are used for the production of seedling rootstocks and as planting material.

In vitro conservation of Hevea accessions, through cryopreservation has been suggested as a viable option for the long term storage of germplasm.

Though cryopreservation protocols have been developed for various field crops, attempts in this direction are also in Hevea.

• Recently two efficient cryopreservation protocols, were developed for embryogenic calli of a commercial clone of Hevea, (PB 260).

Using a classical freezing process Simplified freezing process.

• After pre culture with 1% sucrose and 10% di methyl sulphoxide, embryogenic calli were frozen in a programmable freezer at 0.2°C down to -40°C.

• So that High survival and rapid re growth, as well as production of somatic embryos, were observed.

Oil palm

S.N: Elaeis gueineensisFamily : Palmae Origin: West AfricaChromosome no:2n=32Cheapest and major oil yielding crop.

Biotechnology Objectives:• Producing good tenera palms for commercial plantings.• To multiply parents (both dura and pisifera) for seed

production.• To exploitation of genetic potential of progenies from

interspesific E. oleifera X E. gueninsis crosses • To salvage diseased palms .

Tissue culture process :

• Leaves, Inflorescenses , and roots are used as explants for oil palm tissue culture.

• Young leaf spears are preferred in most laboratories.

• Leaf explants can be surface sterilized easily, and give higher clonability rates.

The process of oil palm tissue culture can be divided into several different stages.

1.Callus is initiated from the explant.2. Embryogenesis 3 shoot and root regeneration, 4.hardening of plantlets5.Field evaluation

• The regeneration process through oil palm takes 2 or 4 years depending on genotype.

• Growth conditions for different stages are typically at 28°C ± 2°C with equal light and dark.

• Explants are placed in media containing either 2, 4-D or NAA for 12 to 14 weeks callus formation.

• The calli are maintained in media containing lower concentrations of 2, 4-D or NAA for up to 12 months for multiplication and embryogenesis.

• The rates of callusing and embryogenesis in oil palm were demonstrated to be genotype dependent .

• A few oil palm clones produce embryoids after one month, whereas others may take as long as 24 months.

• Small clumps of poly embryoids are transferred onto a

basal nutrient medium and kept for at least three months for shoot induction.

• Shoots obtained are separated from poly embryoids and placed onto solid shoot development media containing low concentrations of NAA in culture tubes (takes two to three shoots) or flasks (about 15 shoots).

• When the shoot height reaches 5 Cm, they are transferred into liquid root-initiation media.

Zamzuri .,(1998), introduced double-layer rooting technique for oil palm.

• In this technique, solid shoot development medium is over layed with liquid root initiation media.

• The plantlets are transplanted into small polybags containing 1: 1 ratio of soil and sand and kept for three to four months under shade with relative humidity of more than 70% for acclimatization before transferring to field nursery.

• In the nursery, plantlets are look like seed derived plants.

• In india plantlet develepment was reported from leaf explants of 18 months old dura and 6 months old tenera oil palm seedlings.

Cryopreservation:

The need to conserve oil palm in in vitro arises because of the large area which would be needed for ex vitro field conservation.

Very little work has been done on this aspect inspite of its importance.

Engelmann et al. (1988) were the first to standardize the technique of cryopreservation.

They were able to store embryoids for 15 months in liquid nitrogen and then could regenerate plantlets from the frozen materials.

CASHEW NUT

• S.N: Anacardium occidentalae• Family: Anacardiaceae • Origin: Brazil• Chromosome No:24 to 42.• Is an export oriented crop grown for its nuts.• It is also called as dollar earning, plough crop.

Biotechnology

• Areas where biotechnology would be of importance in cashew are somatic embryogenesis and plantlet regeneration.

• It is useful for genetic transformation to introduce genes for resistance to tea mosquito bug, stem and root borers.

• Standardization of micro-grafting technique, developing haploids and isogenic lines and molecular characterization of existing genetic diversity.

micro-propagation• Micro propagation using seedling explants was

standardized. • Cotyledon explants produced 10-12 shoots/ culture and

shoots were rooted by exposing to IBA (20-60 ppm) for 2 hours.

• Regeneration from mature tree explants has been difficult due to high rate of contamination, browning, slow growth and poor rooting of micro shoots.

• plantlets were regenerated from shoot explants taken from 2-3 year old grafted plants, which were regularly sprayed with Bavistin (0.1%) at weekly intervals.

• Among different surface sterilization treatments used, a combination of antioxidants, antibiotics and fungicides was better with around 40 % contamination free cultures being obtained.

• MS medium containing full strength macro elements supplemented with Kinetin (5 mg /L), NAA (1 mg /L) and Brassinolide (0.1 mg/L) was the best for shoot proliferation and shoot elongation.

• Micro shoots were rooted in vitro at a frequency of 70-80 %, when cultured for 4-8 days in quarter strength MS liquid medium supplemented with IBA (1 mg/L) .

• About 50-60 % of rooted micro shoots survived and produced healthy and vigorously growing plants, which were transferred successfully to the field.

Micro grafting :

• A successful micro grafting technique to rejuvenate mature cashew tree explant material was developed using in vitro germinated seedling as root stock and shoots from stage II cultures as scions.

• Grafting success depended on method of grafting, size of scion and age of rootstock.

• Among methods tried, side grafting preferred, due to ease of operation and faster union.

• High levels of graft union were obtained when 2-3 cm long shoots from stage II cultures were grafted on to 8-9 days old in vitro raised root stocks in full strength MS medium supplemented with Kinetin (2.5 mg/L), NAA (0.5 mg /L) and Brassinolide (1 mg/L).

• Micro grafts after a process of hardening could be successfully transplanted into soil.

Embryo culture :

• Embryos from immature nuts of cashew were cultured in vitro to regenerate improved hybrid plantlets.

• Explants were surface-sterilized.

• Aseptically dissected.

• Cultured into pure basal MS agar medium and MS medium supplemented with 1 micro M each of NAA, benzyl adenine (BA) and Gibberellic acid (GA3).

• Subsequently observed for germination and survival rates until successful ones were transferred to field.

• Explants 6 weeks old or older show better germination rate and the highest survival percentage.

Factors affecting germination Of embryo: Medium composition, Age of embryo and Genotype .

• Germinated explants simultaneously produced shoots and roots, and were ready for transfer to field and acclimatization between 90 and 112 days after inoculation.

Somatic embryogenesis and plantletregeneration from leaf and

inflorescence explants of Arecanut(Areca catechu L.)

Anitha Karun*, E. A. Siril, E. Radha andV. A. Parthasarathy

Biotechnology Section, Central Plantation Crops Research Institute,Kudlu P.O., Kasargod 671 124, India.

• Application of tissue culture technology in clonal multiplication of oil palm, date palm, and coconut has been reported, but not in Arecanut except for a report on adventitious shoot development from mature embryos.

• In vitro retrieval of Arecanut embryos was reported to be Successful.

In vitro propagation of Arecanut through inflorescence explant(var. Sumangala)

A: Callus development from inflorescence segmentcultured in MS medium containing Picloram.(Picloram was found to be the most suitablecallogenic agent)

B:Embryogenic callus developed in Picloram-added medium.

C: Fully developed somatic embryos.

D: Germination of somatic embryos and meristemoid formation. E: Plantlet development through meristemoids in BA-addedmedium.

F: Somatic embryo-derived plantlet growing in half-strengthMS liquid medium containing BA (5 mM).

G: Hardening of in vitro raised areca plantlet.

H: Somatic embryo-derived plant growing inshade net-house.

Anitha Karun et., 2004

Limitations in tissue culture teqnique….1. Soma clonal variations,2. Difficulties in rooting, browning of media due to phenolic

compounds,3. Fungi, bacteria prasent with in the plant tissues,4. Requirement of highly equipped labs, skilled persons..

Venkatachalam, P., et al. 2007 , Current Perspectives on Application of Biotechnology to Assist the Genetic Improvement of Rubber Tree (Hevea brasiliensis Muell. Arg.)

REFERENCES:

Armendariz, B.H.C., Oropeza, C., Chan, J. L., Brian, M., Aguilar C.C.C., and Saenz, L., 2006, Pollen Fertility and Female Flower Anatomy of micropropagated coconut palms. Vol:29.004, Mexico; 373-8.

Anitha Karun*, E. A., Siril , E., Radha and V. A. Parthasarathy, 2004, Somatic embryogenesis and plantlet regeneration from leaf and inflorescence explants of arecanut (Areca catechu L.). CURRENT SCIENCE, VOL. 86, NO. 12, 25.

Bavappa, K. V. A., Nair, M. K. and Premkumar, T., 1982,The Arecapalm (Areca catechu Linn.). Central Plantation Crops Research Institute, Kasargod,

Rajesh, M. K., Radha, E., Anitha Karun and Parthasarathy, V. A., 2003,Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines. Plant Cell Tiss. Org. Cult.,75, 41–47.

Rethinam, P. and Sivaraman, K., Arecanut (Areca catechu L.):present status and future strategies. 2000 ,Indian J. Arecanut, SpicesMed. Plants,, 3, 35–50.

Karun, A. and Sajini, K. K., 1996, Plant regeneration from leaf explant of oil palm. Curr. Sci.,, 71, 922–926.