TISSUE CULTURE TECHNIQUES FOR TREE SPECIES

INTRODUCTION
CLONAL PROPAGATION
ISOLATION OF DISEASE-FREE PLANTS
EMBRYO CULTURE
ANTHER CULTURE
EARLY INDUCTION OF FLOWERING TO SHORTEN TO BREEDING CYCLE
SOMATIC HYBRIDS THROUGH PROTOPLAST FUSION
TRANSFORMATION THROUGH UPTAKE OF FOREIGN GENOMES
CRYOPRESERVATION OF GERMPLASM OF TREES
CLONAL REPOSITIES & GERMPLASM BANKS
TYPES OF CULTURES
MEDIA FOR CULTURE
STAGES INVOLVED IN MICROPROPAGATION
FACILITIES REQUIRED FOR MASS MULTIPLICATION
PROBLEMS IN MICROPROPAGATION OF TREES

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Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be made to evolve new methods for mass propagation and production of short duration trees with a rapid turnover of biomass and induction of genetic variability for the production of novel fruit and forest trees which are high yielding, resistant to pest and disease associated with increased photosynthetic efficiency. This required genetic manipulation to evolve vigorous and fast growing trees with a short reproductive cycle which can be mass propagated. It is envisaged that the technology of tissue culture is competent to meet this challenge. Tissue culture techniques have already revolutionized the mass scale propagation of many horticultural crops and several commercial laboratories have been set up in many parts of world for mass production of elite, cloned plant material. However, its exploitation for forest tree species has started only recently. The following are some of the areas of tissue culture which are of prime interest in forestry. They have the biotechnological potential not only from the basic fundamental research point of view, but also for direct application for the immediate improvement of trees and increased biomass production.

Conventional methods of asexual propagationv(vegetative propagation) like grafting, budding, layering etc. for many plants and trees are often too slow or fail completely. Microvegetative propagation using tissue culture allows much greater control and manipulation of the development of tissues within the culture tube than conventional methods. In normal cuttings, each cuttings can result in only one plant, whereas by micropropagation thousands of plants can be produced from a single piece of plant tissue explant. Not only is the rate of multiplication increased, but the mean generation time is also decreased because the process can continue all round the year under controlled laboratory conditions.
            This is of particular importance to forest and fruit tree species which have long generation cycles complicated with the problem of heterozygosity as result of wide crossing. For example forest trees like the eucalyptus, teak and fruit trees like cashew, coconut etc. never breed true to type. Methods of tissue culture are now available for rapidly multiplying “elite” teak and eucalyptus trees, growing in the forests of chandrapur and Tamil Nadu respectively. There are also other reports where tissue culture methods have been developed in India for forests trees Dalbergia sissoo, D.latifolia, Albizia lebbeck, tamarind, sandal, rubber etc.

Virus infection is a major problem with vegetatively propagated species. Conventional methods of elimination of virus from stock by heat treatment are useful only with some varieties. The growing tip (meristem) is usually uninvaded by viruses and plants obtained by the culture of these meristem tips remains virus free. Morel and Martin (1952) made an first attempt to demonstrate that virus free plants could be produced by culture of apical meristems of dahlia plants. This technique has been exploited to produce virus free plants of fruit trees like citrus, apples, etc. In general, meristem culture results in the production of completely disease free root stocks and other plants. The plants produced have been found to be healthier, more vigorous and to produce higher yields.

In traditional plant breeding, hydrid embryos of many interspecific crosses fail to grow to maturity mainly due to the degeneration of the endosperm or an abortion of the embryos has now found wide utilization in the fruit trees. It has been successfully used for peach, plum, pear and apple cultivars. Another application of embryo culture is to overcome seed dormancy which with many trees take several years for germination under natural condition.

Haploid plants being gametophytic in origin possess only half the normal number of chromosomes as present in the parent. They can be used to produce homozygous lines which are invaluable for any breeding programmes and also for various other genetic manipulation. After the first successful report on regeneration of haploid plants from pollen grains of the cultured anthers of datura this technique has been demonstrated in a large number of herbaceous species. However, the technique of culturing anthers and pollen has found only limited success when applied to forest species. Haploid callus has been obtained from cultural anthers of forest tree species of Pinus, Vitis, Citrus, etc.

Trees unlike agricultural crops, take years to attain sexual maturity and to flower. Thus, tree breeders have to wait up to twenty years or even more. This problem is further aggrevated in some trees such as bamboo, which may flower once in forty years. Thus the early induction of flowering by the application of growth regulators in vivo or their use in in vitro cultures would help to reduce the breeding cycle.

Somatic hybridization through protoplast fusion opens an avenue for synthesizing characteristics which were not possible hitherto. Even in wide crosses through embryo cultures, the degree of variability is low. Somatic hybridization is an alternative to sex in order to combine the entire genomes from incompatible parents and is expected to result in hybrids.

Genetic transformation using Agrobacterium mediated transfer is another important technique in which a gene or group of genes encoding for a specific trait can be isolated and cloned.

The rapid rate of diminishing of the genetic resources has caused concern of great magnitude for the conservation of important and elite germplasm. For this, cryopreservation techniques have been evolved which involves freeze preservation of cells, tissue and organs as a meaningfull tool for long term conservation (-196ºC), establishment of gene banks and international exchange of germplasm.

Meristem cultures and in vitro plantlets can be packed in cardboard and foam boxes transported to international destinations. This distributions and the exchange of desirable germplasm can also be effectively carried out by the transfer of frozen cells and tissues in portable liquied nitrogen cylinders and can be transported by air. Hence it is highly desirable to set up “Germplasm banks” and clonal repositories of the rare, elite and other desirable genetic stock of trees.  Such banks should be responsible for the storage, maintenance and the exchange of germplasm of trees both at national and international levels. 

Plant tissue cultures can be divided in to five classes based primarily on the type of material used on the medium.

a) Meristem Culture
            This term is often used loosely to refer to very small shoot apices dissected from terminal or lateral buds.  Strictly speaking, it refers to the microscopic apical dome with only the smallest leaf primordial evident, usually less than 2 mm across.  The advantage of using shoot meristem is that they are most likely to be free of internal pathogens.

b) Callus Culture
            Sometimes explants produce callus rather than new shoot growth particularly where high levels of hormones are applied.  In other cases, callus may be induced intentionally because of its potential for mass production of new plantlets.  The limiting factors are the difficulty in inducing the initiation of new shoot apices, especially in woody species.

c.Cell Suspension Culture
            This is essentially product of callus culture, i.e. callus usually refers to a mass of undifferentiated cells.  Once these are separated in liquid culture, it  becomes a cell suspension.  This culture may be used to produce a product directly from these cells without regenerating new plants.  These cells may be genetically engineered to increase the synthesis of different secondary metabolites.

d) Protoplast Culture
            This is a next step beyond  cell suspension culture were the cell walls of suspended cells are removed using exzymes to digest the cellulose to leave the isolated protoplast.  With the cell wall removed, it is possible to insert or remove foreign materials including the basic genetic materials DNA and RNA or to fuse together cells from entirely different species.

e) Organ Culture
The culture of embryos, anthers, shoots, roots or other organs on a medium is called organ culture.

Media is one of the important component of tissue culture. The media is the source of macronutrients, micronutrients, vitamins, growth regulators, carbon etc., which are needed by cell for their growth and differentiation. The various media composition that are used commonly in tissue culture are presented (Table 1)

Table 1. Standard Basal Media Formulations Commonly used in Plant Tissue Culture

The sequential stages recognized in any micropropagation systems involved are (i) establishment, (ii) multiplication, (iii) pre-transplant and (iv) transplantion. The medium used for micropropagation has two major functions, a) to supply basic ingredients for continued growth of the isolated explant and subsequent propagation and (b) to direct growth and development through hormonal control viz., auxins, cytokinins, gibberellins and abscisic acid. The hormonal control exercised by (i) kind of hormone or growth regulator, (ii) its concentration, and (iii) sequence in which they are applied.

a) Establishment Stage
The factors that affect establishment stage are (i) choice of explant, (ii) elimination of contamination of the explant, and (iii) culture conditions. In general younger tissue such as shoot tips or terminal buds will regenerate better than older and mature tissues of the same stem. In general, ingredients of the culture medium in the first stage are determined by kind of response needed. The culture room conditions involve a temperature range of 25 ± 2º C and with a photoperiod of 16 hrs. light and 8 hrs. darkness. This stage lasts for 4 to 8 weeks which depends on species.

b) Multiplication Stage
The function of this stage is to increase the number of propagules for later rooting to planting stage. The propagules are cut and separated to be grown into plantlets in a new medium. Number of multiplications may vary from 5 to 50 depending on species and method. Generally cytokinins are used for multiplication of shoots.

c) Pre-Transplant Stage
The function of this stage is to prepare the plantlet for transplanting and establishment outside the artificial, closed or open environment. In this stage, prolific root initiation is the main objective. This is achieved by reducing cytokinin concentration, and increasing auxin supplies. Other conditions required are reduction in inorganic salt concentration and addition of pholoroglucinol to improve rooting and to reduce callusing.

d) Transplant Stage
This stage involves the transfer of the plantlet from the aseptic cultural environment to the free living environment of the green house and ultimately required hardening process to the final field location. The plantlet must not only root adequately but also must undergo a period of acclamation to enable it to survive and establishment.

Any full fledged tissue culture laboratory should need the following infrastructural facilities.

a) General Working Laboratory
This laboratory is meant for general purpose working which should contain the following

b) Media Preparation Room
This room should contain

c) Inoculation Room
A small dust tree preferably airconditioned room equipped with an overhead UV lamp and a laminar flow chamber will serve as the inoculation area. A compound microscope with photographic accessories is an optional requirement. A small anti room provided with overhead airshower/air curtain before entry in to the inoculation and culture area should be of added advantage.

d) Culture Room
All types of tissue culture are to be incubated under well controlled temperature, humidity, air circulation, light quality and duration. The temperature should be maintained around 25 ± 2º C with air conditioners controlled by temperature controller. The culture room should be provided with racks with florescent lamps ranging from 1000-10,000 lux with facility to adjust the photoperiod. A lux meter to regularly monitor the intensity of the light is necessary. Shakers and dark chambers may also be required for some species. A generator to maintain uninterrupted power supply is optional requirement.

e) Hardening Chamber
A mist house with controlled fogging system will help in gradual weaning of in vitro raised plants. Then, plantlets are exposed to different light regimes under shade house conditions.

Tissue culture of trees unlike other horticultural plants is beset with very special problems. Some of these include the physiological nature of the material (Juvenile and mature phases), general recalcitrant response of the explants vis a vis medium, inadequate rooting of the regenerated shoots and the associated problems of poor transfer ratio of established plants into soil. Most of the problems arising at tissue culture level can indeed be sorted out but commercial exploitation of techniques developed for tree tissue culture into a technology calls for concerted efforts of tissue culturists, foresters and tree breeders.

Reference: Surendran, C., Parthiban, K.T., Vanangamudi, K., and Balaji, S., 2000. Vegetative Propagation of Trees, Principles and Practices.1-154, TNAU press,