CONSERVATION IN VITRO OF FOREST TREE GENETIC RESOURCES

F. ENGELMANN

Lecture given during the Training Course in Management of Forest Genetic Resources

and Agroforestry Areas

L l / I l

r

23 march 1990

BIOTROP - BOGOR - INDONESIA

Fonds Documenfaire BWST 1

CONSERVATION IN VITRO OF FOREST TREE GENETIC RESOURCES

F. ENGELMANN - ORSTOM - BP 5045 - 34032 MONTPELLIER - FRANCE

1 - INTRODUCTION

As regards preservation possibilities, plant species have been divided into 2 categories (Roberts,l973) :

- orthodox seeds, which can withstand dehydration to 5 O/O or less (dryweight basis) without damage. When dry, the viability of these seeds can be prolongated by keeping them at the lowest temperature and moisture possible.

- recalcitrant seeds, which are high in moisture and are unable to withstand much desiccation. They are predominantly seeds from tropical or subtropical trees or shrubs. They can be stored only in wet medium to avoid dehydration injury and in relatively warm conditions because chilling injury is very common among these species. They remain viable only for a short time (weeks or months), even if kept in required moist conditions. This group comprizes many species of great economic importance (Tabl.1).

Moreover, long-term seed storage cannot be applied to most long-lived forest trees, including gymnosperms and angiosperms, since their juvenile period is very long and they do not produce seeds for several years. The conservation of plants which are vegetatively propagated , such as cassava, potato, yam, etc,,., poses also considerable problems,

In situ conservation has been made almost impossible due to the disappearance of large wild areas, Conservation ex situ is very difficult to carry out due to the following problems :

an adequate sample has to be determinated for the conservation of genetic diversity. It varies from 20 to 30 plants for a singlb population, to several hundreds for gene pool conservation and to 5,000-20,000 plants, depending on the species, for the maintenance of heterozygosity. Thus, land space requirement is very important, particularly in the case of forest trees, which are oftenly of very large size, whereas land availability drastically decreases. Moreover, as most forest trees are genetically heterozygous, it is thus necessary to preserve a larger sample to maintain as much as possible of the genetic variation within a population. Moreover, labour costs and trained personnel requirement are very important, Moreover, material in natural conditions remains exposed to natural disasters, pests and pathogens and is submitted to threats from changing government policies and urban development. Finally, for many climax forest trees, we do not possess even the rudiments of knowledge of the biology of the species.

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The use of in vitro tissue culture techniques can be of great interest for germplasm storage of recalcitrant and vegetatively propagated species. In this aim, tissue culture systems present advantages which are listed below :

(1) very high multiplication rates (2) aseptic system :

(3) reduction of space requirement (4) genetic erosion reduced to zero (5) possibility of producing haploid plants (6) rescue and culture of zygotic embryos which normally abort (7) reduction of the expenses in labour costs and financial terms

- free from fungi, bacteria, viruses and insect pests - production of pathogen-free stocks

In vitro culture is commonly used for medium-term storage, For long term storage, in vitro culture is used jointly with cryopreservation,

2 - MEDIUM-TERM CONSERVATION

2.1 - METHODOLOGY/RESULTS

2.1 -1- CLASSICAL TECHNIQUES

For medium term storage of germplasm, the various techniques listed below can be used :

(1) Alterations to the physical conditions : - temperature - light intensity and photoperiod

(2) Alterations to the basic medium : 2 nutrients - factor essential for normal growth

(3) Addition of: - growth retardants (ABA,CCC) - compounds with osmotic effects (mannitol)

The most commonly used methods concern the decrease of the culture temperature and light intensity, as illustrated in Table 2, It is important to stress the fact that, when setting up a medium-term storage method, the aim is not to search the longest subculture period possible, as it is oftenly the case in the literature, but to determine a frequency which ensures the preservation of the material in the best possible conditions, with the minimum of losses. However, many tropical species are cold-sensitive and cannot be stored at low temperatures. For example, oil palm somatic embryo cultures show after a few weeks at 12-18°C severe injuries which drastically reduce their possibilities of proliferation recovery when the material is replaced in optimal conditions. It is thus very important to develop alternatives methods, more adaptated to tropical species.

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2-12 - ALTERNATIVE METHODS

In this case,the aim is to modify other parameters than temperature. Thus, three methods will be tested :

- modification of the gaseous environment - desiccation - encapsulation

2.1.2.1 - Modification of the gaseous environment

Several methods exist in order to decrease the quantity of oxygen available to the tissues :

- Use of a mineral oil overlay covering the tissues. This technique was developed first by Caplin (1959) and ,tested recently by

Augereau et a/. (1986) and Moriguchi ef a/. (1988) for the storage of callus lines of various species. The results are presented in Table 3b

- Modification of 0 2 partial pressure (Bridgen and Staby,l981) The cultures are placed in an air-tight box. The oxygen pressure is lowered by

using an air-ejector connected to the box, thus decreasing the quantity of oxygen available to the tissues, or by making up a mixture of oxygen and nitrogen, which is injected in the culture box. This method was successfully applied to chrysanthemum and tobacco plants using 0 2 concentrations of 1.3 and 8%. No effect could be observed on the growth of the plants after 6 weeks of storage. This technique was reutilized by Engelmann (unpublished results) for the storage of oil ' palm somatic embryos. After 4 months of storage in an atmosphere containing 1% 02, reproliferation could be obtained very rapidly from the whole culture, whereas control ' cultivated in standard conditions was almost completely necrosed.

2.1.2.2 - Desiccation

This technique was firstly used by Nitzche (1980) on carrot calluses. They could be stored for one year and revived after having undergone drying for one night in the air stream of a laminar flow cabinet.

2.1.2.3 - Encapsulation

This technique is now commonly used for the production of "synthetic seeds" by coating somatic embryos in alginate beads. This technique seems very promising in a near future for preservation purposes. The protection of the material provided by encapsulation could increase its resistance to dehydration and low temperature, thus opening new possibilities for medium-term storage.

Some preliminary experiments were carried out by Bapat ef a/,(1987), Bapat and Rao (1988). Encapsulated mulberry buds could be stored for 45 days at + 4 O C and germinate easily. In a second experiment, sandalwood embryoids could withstand the same storage conditions and duration and give rise afterwards to secondary embryoids.

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2.2 - EXAMPLES OF APPLICATION OF MEDIUM-TERM STORAGE

2.2.1 - FOREST TREE SPECIES

Until now, medium-term storage has been applied mainly to species originating from temperate climates, as listed in Table 4.

For the storage

2.2.1 -1 - Materials

- Type of culture : shoot

of a given species, the following parameters have to be defined

cultures, embryos, etc.., depending on the propagation process.

- Physiological state : shoot size, juvenility, development of the root system..bVery oftenly, already developed plants survive better.

- Substrate : the most suitable culture medium has to be defined. It is advised to chose a medium which is as close as possible to the standard culture medium.

- Container : test-tube, Erlenmeyer flasks, jars..,The choice will depend on the size of the explant, quantity of medium, number of replicates required.

2.2.1.2 - Conditions for cold storage

- Equipment and design : the choice will depend on the mode of conservation. As a standard rule, it is better for safety reasons to have 2 replicates of the collection in 2 separate rooms, so as to further reduce the risks of accidental loss.

- Temperature : the choice will depend on the species and the type of conservation. .It is recalled that most of tropical species are temperature-sensitive, thus the temperature cannot be drastically lowered without taking important risks.

- Light : it is oftenly necessary, but low intensity are sufficient, so as to reduce growth and avoid cultures to turn white and become etiolated.

- Humidity and free water : it is important to avoid excess of water which could lead to vitrification,

- Space ; in viho storage will lead to a drastic space reduction, when compared to the land surface which would be required for the storage in vivo of the same quantity of material (Tabl.5).

2.2.1.3 - Factors affecting cold storage

- Requirement for subculture, length of time in cold storage : these parameters will be defined so as to ensure the maximal survival, The size of the sample to be preserved could thus be reduced.

- Stability : it can be assessed only by comparison with the control. The fact that the material regrowths after storage does not necessarily mean that stored material is free of genetic instability. However, the results in the literature are very encouraging.

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2.2.1.4 - Uses for cold storage

- Short-term (less than 1 year) : in commercial laboratories, in order to smooth out production peaks.

- Long-term (more than 1 year) : for preservation of germplasm collections and commercial laboratories, use for long-term evaluation trials (Aitken-Christie and Singh, 1987)

2.2.2 - COCONUT ZYGOTIC EMBRYOS

Coconut seeds are typically heavy and large ; they are not dormant and germinate quickly. Husk complicates phytosanitary treatments. Storage and seed exchanges are very complicated, particularly when prospecting. In vifro culture of zygotic embryos represents an apt and economic solution to these problems. IRHO has developed techniques for embryo field collection and in vitro cultivation. This method, which has been tested on more than 20 varieties, is very simple and efficient (Assy Bah, 1986; Assy Bah ef d., 1987, 1989). Its performances are summarized in Table 6.

Medium-term storage techniques have been set up for mature and immature embryos, which are very efficient, since embryos can be stored for 6 months without decrease in the germination rate.

2.3 - CONCLUSION

Medium-term storage techniques are now routinely used in many laboratories and international centers, e.g. Cassava at the ClAT (Cali, Colombia), potato at the CIP (Lima, Perou).

Most of the work has been carried out on species from temperate climates. Researches have to be focused now on tropical plants, in order to develop storage techniques adaptated to the biology of these species.

Medium-term storage is very important for the conservation of plant germplasm. However, if long-term preservation is required, even extended subculture intervals cannot be considered sufficient. Moreover, as the risks of genetic variation increase with in vitro culture duration, as illustrated in the case of oil palm (Corley et al., 1986) and can lead to the loss of trueness-to-iype, which is one of the aims of preservation, alternative methods have to be sought.

3 - CRYOPRESERVATION

3.1 METHODOLOGY

Today, cryopreservation, ¡.estorage at a very low temperature, usually that of liquid nitrogen, -196OC, is the only technique which is appliable for long-term storage. The main advantages of cryopreservation, compared with other techniques are listed below :

- storage at a very low temperature (liquid nitrogen, -196°C) - all biological and metabolic processes stopped - preservation possible for a theoretically unlimited period of time - subcultures suppressed, contaminations avoided - space requirement limited - maintenance and labour costs drastically reduced.

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3.1.1 - CRYOPRESERVATION PROCESS

A cryopreservation process comprizes successive steps which have to be defined for every species :

- choice and obtainment of material - pretreatment - freezing - storage - thawing - post-treatment

3.1b1.1 - Choice and obtainment of material

As a general rule, the material will be chosen as young and as meristematic as possible. Indeed, the cells of this type of material are the most likely to withstand freezing: they are small, contain only a few vacuoles, i.e. only a small amount of water, their cytoplasm is dense, their nucleo-cytoplasmic balance is high,

The material can be sampled on in vivo or in vitro plants, In vitro material is generally preferable, since the explants are already miniaturised and free of contaminations.

The physiological stage of the material is very important. In the case of cell suspensions, only material at the exponential stage of growth can successfuly whistand freezing (Fig.l), With carnation meristems, survival depends on their rank on the shoot axis (Fig.2).

It is sometimes necessary to set up a special culture medium in order to obtain starting material in sufficient quantities. It is the case with oil palm embryoids : only a special type of embryoids, shiny white, finger-like shaped, which are oftenly grouped into clumps, are likely to withstand freezing. These particular embryoids are very rarely observed in standard culture conditions. Their frequency is increased by a two- month culture on a medium containing 0.3M sucrose instead of O.lM, which is used in the standard culture medium (Engelmann et a/., 1985).

3.1.1.2 - Pretreatment

The pretreatment corresponds to the culture of the material during a certain period of time (several minutes to a few days) in conditions which prepare it to the freezing process. It is carried out using cryoprotective substances. Some of these compounds are listed in Table 7. They differ greatly one from the other by their molecular weight and their structure. The exact mode of action of these substances is unclear : they have an osmotic role and act thus by dehydrating the cells but they may act also by protecting membranes, enzymatic binding sites from freezing injury. They are sometimes classified in penetrating and non-penetrating compounds, the first ones having both above cited effects, the second ones acting only as osmoticums.

For every species, one will have to determine the nature of cryoprotectants, their concentration as well as the duration of the pretreatment, In some cases, the pretreatment will have to be adaptated to different clones or varieties for the same material (Tabl.8).

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3.1.1.3 - Freezing

Different types of freezing processes can be carried-out : ultra-rapid, rapid, or slow freezing (Fig,3). In the later case, a prçogrammable freezing apparatus will be needed in order to obtain precise and reproducible freezing conditions.

At the cellular level, the different freezing processes described above correspond to different mechanisms as regards water fluxes and cristallisation. During slow freezing, cristallisation occurs firstly in the external medium. The water of the cells flows out to the external ice. The cells will have to be at the same time sufficiently dehydrated so as the cristallisation of the residual water will cause no damage and not too much in order to avoid toxicity due to the concentration of the internal solutes, which increases with dehydration. During rapid freezing, intracellular ice cristallizes in microcristals of a size which is unharmful to the integrity of the components of the cells,

For every material, the following criteria will be determined :

- freezing rate : it can be very precise, as in the case of pea and strawberry meristems (Fig.41, or comprize a much broader range, as in the case of oil palm somatic embryos (Fig.5). - starting and prefreezing temperature : ¡.e, the temperatures of beginning and end of programmed freezing, These parameters are oftenly very important : in the case of cassava meristems, a prefreezing temperature of -20°C ensures 91% of survival ; only 3,3 O/O are observed if the controlled freezing stops at -40°C (Fig.6).

3,l. 1.4 - Storage

The maximal storage duration is theoretically unlimited, provided that the samples are permanently kept at the temperature of liquid nitrogen. The material remains exposed to natural radiations. The following calculation has been made on animal cells : the level of mutations caused by natural radiations during storage will reach an irreparable level after thawing of the stored material only after a minimum of 10,000 years.

3,1.1.5 - Thawing

In the majority of the cases, thawing is carried out rapidly by immersing the cryotubes containing the samples in a water-bath thermostated at around +4OoC. The aim is to avoid the fusion during thawing of the ice microcristals formed during freezing to larger cristals of a size which would be domageable to cellular integrity.

3.1,1.6 - Post-treatment

Post-treatment consists in culturing the material in conditions ensuring its recovery in the best conditions possible. Cryoprotective substances are progressively eliminated by rinsing , dilution, diffusion, for they are toxic if kept too long in contact with the material.

The osmotic shock caused by an immediate transfer on a medium with low osmotic potential has to be attenuated by successive transfers on progressively less concentrated media. In some cases, the nature of the medium must be changed (solid versus liquid, and vice et versa), in order to better the regrowth.

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Recovery can eventualy take place in the dark, in order to avoid photooxidation phenomena which can be harmful for the recovery of the material.

Finally, the hormonal content of the culture medium can be transitorily modified. It is the case with oil palm embryoids, for which an auxin has to be added during a few days after thawing, in order to stimulate the recovery of the proliferation.

3.1,2 - VIABILITY ASSESSMENT

The only definitive assessment of viability is regrowth of the material after thawing. However, it is very important to know as soon as possible if the material is living after freezing, whereas, in many cases, the regrowth is very slow. Two main tests exist in order to measure the viability of the material, which can be applied very rapidly after thawing. However, their major disadvantage is that they are destructive. These tests are :

- FDA (fluorescein diacetate) : FDA is absorbed by the living cells and transformed into fluorescein, whose fluorescence is measured in UV, This test is quantitative (Widholm, 1977),

- TTC (Triphenyl tetrazolium chloride) : TTC is reduced into formazan, colored in red, in the mitochondria of the living cells. It is quantitative for cell suspensions (measurment of O/O of the control), but is only qualitative for large tissues and organs (Steponkus and Lanphear, 1967).

3.2 - RERSULTS

3.2.1 - VARIOUS TYPES OF CULTURES

Today, cryopreservation has been applied to more than 70 different species, However, in many cases, resistance to freezing in LN has been proved in the laboratory, but it does not necessarly mean that the technique is effectively used for germplasm storage of many species. Table 9 presents the list of the species which have been frozen as cell suspensions, calluses, protoplasts, meristems and embryos

Most of the research on trees concerns cell suspensions or calluses, which have been used more as experimental models than for practical purposes, or fruit trees. Moreover, due to the difficulty of in vifro culture of tree species, many of these results are uncomplete. The most promising results, particularly in the frame of plant genetic resources conservation, concern the cryopreservation of zygotic embryos.

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3.2.2 - STORAGE DURATION, TRUENESS-TO-TYPE

The possible variations of the material due to cryopreservation have been principally checked on the production of particular compounds by cell strains (e.g. steroids from Dioscoreu delfoideu, alcaloids from Cufhurunfus roseus)b Until now, no modifications, after thawing, of the properties of the stored material have been observed. Plants obtained from frozen meristems or embryoids of several species (groundnut, potato, oil palm) appeared to be normal.

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As concerns storage duration, the experience is very limited with plant material, Indeed, the maximal storage duration experimented is 4 years, in the case of cassava and potato meristems. Until now, all storage experiments led to the obtainment of true-to- type material. Although, theoretically, there is no difference between 1 minute and many years of storage, since the material does not evolve when it is at -196"C, it was nevertheless important to prove the feasibility of this method of storage.

3.2.3 - EXAMPLES

3.2.3.1 - Carnation meristem cryopreservation

Carnations are routinely propagated in vitro using microcuttings (Fig.7) It is very interesting to multiply on a large scale material which has been made virus-free using thermotherapy. It is thus necessary to store for the long term all these collections of virus- free material,

For cryopreservation, the following conditions have been set up for the different steps of the cryopreservation process which is presented on Fig.7).

The following results have been obtained :

- Terminal buds : recovery ranging from 29.7 O/O to 98.9 %, depending on the variety.

- Axillary buds : 29,7% recovery.

Entire plants were obtained, which set normal flowers, compared to the non- frozen controls. These results allow cryopreservation to be used for the storage of carnation germplasm.

3.2.3.2 - Oil palm somatic embryo cryopreservation

The oil palm vegetative propagation process set up by ORSTOM and IRHO uses somatic embryogenesis. The present development of the process is as follows :

It is now applied in 5 different laboratories in France, Côte d'Ivoire, Malaysia and Indonesia. It has been applied to 850 ortets and 456 embryoid clones have been obtained. About 500,000 ramets have been produced and 138.5 hectares have been planted for trials in Côte d'Ivoire including 79 clones. Abnormalities have been observed on less than 5% of the total of the material.

Researches for setting up a cryopreservation process have started in France in 1982, in order to face the following problems :

- on the one hand, the risks of obtaining abnormal material which increase with in vitro culture duration, as it was shown in the case of oil palm (Corley et o%, 1986). Storing the embryoids as early as possible after they have been obtained should increase our chances of storing true-to type material.

- on the other hand, the continuous production of new clones induces laboratory management problems. Cryopreservation allows to store the clones which are not used for commercial production, thus reducing the quantity of material which has to be regularly subcultured.

in

The cryopreservation process which has been set up is presented in Figure 8. The following conditions have been defined :

- Choice and obtainment of material : only young embryoids , shining white , finger-like shaped, oftenly grouped into clumps, are likely to withstand freezing. They are obtained in sufficient numbers after a two-month culture on a medium enriched with sucrose (1-2).

- Pretreatment : the clumps of embryoids are placed for 7 days on a medium containing 0.75M sucrose. Their water content decreases from 80% to around 60% (3),

- Freezing : the clumps are placed in sterile cryotubes and frozen rapidly by direct immersion in liquid nitrogen (-20O0C,min-1) (4a). A two-step freezing can be carried out using a programmable freezing apparatus : the cryotubes are frozen from +20°C to - 100°C at a rate which can vary from 5 to 4O0Cbmin-l, then plunged in liquid nitrogen (4b).

- Thawing : the cryotubes are plunged in a water-bath thermostated at +40°C for 1 minute (6).

- Post-treatment : the embryoid clumps are cultured for 3 weeks on media added with 2,4-D and containing progressively less sucrose. Afterwards, they are transferred on the standard medium devoid of growth regulators (7a-b).

The technique described above has been successfully applied to 27 different clones, with an average recovery rate of 12.5%. It has been checked with 2 clones that the extension of the storage duration to respectively 12 and 15 months in liquid nitrogen did not modify the recovery rate, Finally, ramets from two cryopreserved clones have been produced and planted in the field at the IRHO La Mé research station in Côte d'Ivoire. No difference was observed when compared with non frozen controls. The first male inflorescences, which were observed {ecently on cryopreserved material, are: perfectly normal. New ramets coming from frozen material are now in the nursery. They will soon be planted in order to confirm these first results.

These results have been judged sufficient to decide to apply the technique in the laboratories producing oil palm embryoids through the ORSTOM-IRHO process. The experiments have started simultaneously in France, Côte d'Ivoire, Malaysia and Indonesia in 1989, on approximatively 155 clones. The results will be available in 1990. We will then have more informations on the possibility of using cryopreservation as a routine technique for the long-term storage of oil palm embryoids.

4 - CONCLUSION

In conclusion, tissue culture techniques, together with cryopreservation techniques, are of great interest for the medium and long-term conservation of many plant species, particularly for that of tropical woody species. For that purpose, the use of zygotic embryos may present many advantages, as it is shown by the growing interest for this type of propagules in cryopreservation,

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However, before routine procedures can be foreseen using in vitro techniques for germplasm preservation, various problems have to be faced. The germplasm has to be evaluated in order to store a representative sample of the variability of the species. Moreover, a minimal knowledge of the biology and physiology of the species is needed. The in vitro culture conditions have to be determined for the species which has to be conserved. Finally, trials must be carried out in order to precise the conditions for slow growth storage as well as cryopreservation.

Practical problems exist as well, particularly in the developing countries, which have been previously mentioned : existence of minimal tissue culture facilities, lack of funds, of trained personnel, of regular and reliable liquid nitrogen supply, In this context, the development of "alternative" techniques, which must be as simple as possible, is of great interest,

The development of such processes nevertheless require sophisticated equipments as well as an important technical background in the research areas concerned, These conditions are presently found only in important centers of multidisciplinary research institutes like ORSTOM, for example. Success can be obtained only through a very close cooperation between the local and the overseas research institutes. This way of cooperation fits perfectly with the aims of ORSTOM. Indeed, ORSTOM has already a very large experience of the various aspects of plant germplasm preservation and is developing this research area,

5 - REFERENCES

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BRAUN A. 1988 - Cryopreservation of sugarbeet germplasm, Plant Cell, Tissue and Organ Culture, 14 : 161-168.

BRIDGEN M.P. and STABY G.L., 1981 - Low pressure and low oxygen storage of Nicotiana fabacum and Chrysanthemum x Morifolium tissue cultures. Plant Science Letters, 22, 177- 186.

BOUMAN H. and de KLERK G.J. 1989 - Cryopreservation of lilly meristems. Abstr. Intl. Conf,, The Impact of Biotechnology in Agriculture, Amiens, 10-12 July, PO CP 3.

BUTENKO R.G., POPOV AS., VOLKOVA L.A, CHERNIAK D.N. and NOSOV A.M. 1984 - Recovery of cell cultures and their biosynthetic capacity after storage of Dioscoreu deltoidea and cells in liquid nitrogen. Plant Sci. Lett., 33 : 285-292.

CAPLIN S., 1959 - Mineral oil overlay for conservation of plant tissue cultures. Amer, J. Bot., 46, 324-329,

CARUSO M.,CRESPI-PERELLINO N,,GAROFANO L.and GUlCClARDl A. 1987 - Long term storage of Vinca minor and Panax ginseng cell cultures. Proc. Adv. Stud. Inst. on Plant Cell Biot., Albufeira (Algave), Portugal, 29 march-1 O april, 1-4.

CHEN H.H., KARTHA K.K., LEUNG N.L., KURZ W.G.Wn, CHATSON K.B. and CONSTABEL F. 1984 - Cryopreservation of alkaloid-producing cell cultures of periwinkle (Catharantus roseus). Plant Physiol,, 75 : 726-731.

CHIN H.F., KRISHNAPILLAY B. and ALANG Z.C. 1988 - Cryopreservation 'of Veitchiu and Howea palm embryos : non-development of the haustorium. Cryo-Lett., 9 : 372-379.

CORLEY R.H.V,, LEE C.H., LAW I.H. and WONG C.I#, 1986 - Abnormal flower development in oil palm clones. Planter, 62, 233-240,

COULIBALY Y. and DEMARLY Y. 1979 - Sur les conditions de développement des microspores de Nicotiana tabacum et d'Oryza sativa soumises à la température de l'azote liquide (-196°C). C. R.Acad. Sci, Paris, Serie D, 286 : 1065-1068.

14

? ,

DELVALLEE 1.1987 - Androgenèse chez le maïs : programme mâle et cryoconservation. Thèse d'université, Lyon.

DEREUDDRE J,,FABRE J. and BASSAGLIA C. 1988 - Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. Var, Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets. Plant Cell Rep., 7 : 170-173.

DEREUDDRE J., SCOTTEZ C., ARNAUD Y. and DURON M. 1990 - Effets d'un endurcissement au froid des vitroplants de Poirier (Pyrus communis L. cv Beurré Hardy) sur la résistance des apex axillaires Ò une congélation dans l'azote liquide. C.R. Acad, Sci. Paris, Ser. 111, 310 : 265-2 72.

DIETTRICH B., POPOV A.S., PFEIFFER B., NEUMANN D,, BUTENKO R. and LUCKNER Mu 1982 - Cryopreservation of Digitdis Ianata cell cultures. Planta medica, 46 : 82-87,

DIETTRICH B., WOLF T., BORMANN A,, POPOV AS,, BUTENKO R.G. and LUCKNER M. 1987 - Cryopreservation of Digitalis lunata shoot tips. Planta medica, 53 : 359-363.

DOUGALL D.K. and WETHERELL D,F, 1974. - Storage of wild carrot cultures in the frozen state, Cryobiology, 11 : 410-415.

DOUGALL D.K, and WHITTEN D.F. 1980 - The ability of wild carrot cell cultures to retain their capacity for anthocyanin synthesis after storage at -140°C. Planta Medica, suppl. : 129- 135.

ENGELMANN F., 1986 - Cryoconservation des embryons somatiques de palmier Ò huile (€beis guineensis Jacq,). Mise au point des conditions de survie et de reprise, Thèse de Doctorat de l'université Paris 6, 228 p.

ENGELMANN F. and BAUBAULT C. 1986 - La cryoconservation des embryons somatiques, polliniques et zygotiques. Bull. Soc. Bot. Fr,, 133 : 89-103.

ENGELMANN F. and DEREUDDRE J. 1988b - Cryopreservation of oil palm somatic embryos : importance of the freezing process, Cryo-Lett,, 7 : 220-235.

ENGELMANN F., DUVAL Y, and DEREUDDRE J, 1985 - Survie et prolifération d'embryons somatiques de Palmier à huile (Hueis guineensis Jacq,) après congelation dans l'azote liquide. C. R. Acad. Sci. Paris,301, Sér. 111, 3 : 111-116.

FABRE J. 1986 - Effets du prétraitement sur'la résistance Ò la congélation dans l'azote liquide (-1 96°C) des méristèmes terminaux et axillaires d'oeillets (Dianthus caryophyI/us L., var. Eolo) cultivés in vitro.

FINKLE B,J. and ULRICH J.M. 1982 - Cryoprotectant removal as a factor in the survival of frozen rice and sugarcane cells. Cryobiology, 19 : 329-335.

FINKLE B.J., ULRICH J.M., RAINS D.W. and STAVAREK S,J. 1985 - Growth and regeneration of alfalfa callus lines after freezing in liquid nitrogen. Plant Sci., 42 : 133-140.

GALERNE M. 1985 - Effets du saccharose sur la résistance Ò la congélation dans l'azote liquide (-1 96°C) des méristèmes d'oeillet (Dianthus caryophy//us) cultivés in vitro. Mémoire de DEA, Univ.Paris 6, 74p.

GALERNE M. and DEREUDDRE J. 1987 - Survie de cals embryogenes d'épicéa après congélation b -196°C. Ann. AFOCEL : 8-33.

GAZEAU CI, and DEREUDDRE J., 1986 - Effet des substances cryoprotectrices au niveau

15

?

cellulaire.Bul1. Soc. Bot. Fr., 133, Actual, Bot,, 3, 41-64.

GROUT B.W.W. and HENSHAW G.G. 1978 - -Freeze-preservation of potato shoot-tips cultures. Ann, Bot.,42 : 1227-1229.

GROUT B,W.W,, SHELTON K. and PRITCHARD H.W, 1983 - Orthodox behaviour of oil palm seed and cryopreservation of the excised embryo for genetic conservation. Ann. Bot., 52 : 38 1-384.

GROUT B.W.W., WESTCOTT R.J. and HENSHAW GOG, 1978 - Survival of shoot meristems of tomato seedlings frozen in liquid nitrogen. Cryobiology, 15 : 478-483.

GUPTA P.K. and DURZAN D.J. 1987 - Somatic polyembryogenesis in cell masses of Picea abies (Norway spruce) and Pinus tueda (loblolly pine) after thawing from liquid nitrogen. Can. J; Bot.,l7 : 1130-1 134.

HAFFNER V. 1985 - Mise au point d'un protocole adapté aux cellules de My~illocactus geometrizans (Mart.) Cons. ( T J Mémoire de DEA, Univ. Aix-Marseille 111, 32p.

HAHNE G. and LORZ H. 1987 - Cryopreservation of embryogenic callus cultures from barley (Hordeum vulgare Lo)# Plant Breed., 99 : 330-332,

HARADA T., INABA A., YAKUWA T. and TAMURA T. 1985 - Freeze-preservation of apices isolated from small heads of Brussels sprouts. HortSci.,20 : 678-680.

HAUPTMAN R.H.and WIDHOLM J.M, 1982 - Cryostorage of cloned amino acid analog- resistant carrot and tobacco suspension cultures, Plant PhysioL70 : 30-34.

KADZlMlN S.B. 1988 - Germplasm preservation of orchid through tissue culture. In The Application of Tissue Culture Techniques in Economically Important Tropical Species, Umaly R.C.,Umboh I,, Halos S. and Normah MuN. Eds,, SAMEO-Biotrop, Bogor, Indonesia, Biotrop Special Publ., 35 : 167-179.

KARTHA K.K. 1985 - Cryopreservation of plant cells and organs. IAPTC Newslett.,45 : 2-14.

KARTHA K.K., LEUNG N.L. and GAMBORG O.L. 1979 - Freeze-preservation of pea meristems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett., 15 : 7-16.

KARTHA K.K., LEUNG N.L. and MROGlNSKl L,A. 1982 - In vitro growth responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crank). 2. Pflanzenphysiol., 107 : 133-140.

KARTHA K.K., LEUNG NUL, and PAHL K, 1980 - Cryopreservation of strawberry meristems and mass propagation of plantlets. J. Amer. Soc. Hort. Sci.,lO5 : 481-484.

KARTHA K.K., LEUNG NUL., GAUDET-LAPRAIRIE P. and CONSTABEL F. 1982 - Cryopreservation of periwinkle (Catharantus roseus) cells cultured in vitro. Plant Cell Rep., 1 : 135-138.

KARTHA K.K., FOWKE L.C. LEUNG N.L., CASWELL K,L. and HAKMAN I. 1988 - Induction of somatic embryos from cryopreserved cell cultures of white spruce (Picea glauca). J. Plant PhYsid., 132 : 529-539.

KATANO M,, ISHIHARA A. and SAKAI A. 1983 - Survival of dormant apple shoot-tips after immersion in liquid nitrogen. HortSci,, 18 : 707-708.

KUMU Y., HARADA T. and YAKUWA T. 1985 - Development of a whole plant from a shoot- tip of Asparagus officinalis L. frozen down to -196°C. J. Fac. Hokkaido Univ., 61 : 285-294.

16

K U 0 C,C. and LINBERGER R.D. 1985 - Survival of in vitro cultured tissue of Jonathan apples exposed to -196°C. HortSci., 20 : 764-767.

LANGIS R., SCHNABEL B.,EARLE E.D.and STEPONKUS P.L. 1989 - Cryopreservation of Brassica campestris L. by vitrification. Cryo-Lett., 1 O :421-428.

LATTA R. 1971 - Preservation of suspension cultures of plant cells by freezing. Can. J. Bot,, 49- : 1253-1254.

MADDOX A.D., GONSALVES FnG. and SHIELDS R, 1983 - Successful cryopreservation of suspension cultures of three Nicotiana species at the temperature of liquid nitrogen. Plant Sci. Lett,, 28 : 157-162.

MARIN M.L. and DURAN-VILA N. 1988 - Survival of somatic embryos and recovery of plants of sweet orange (Citrus sinensis L. Osb,) after immersion in liquid nitrogen. Plant, Cell and Organ Culture, 14 : 51-57.

MAZUR R.A. and HARTMANN J,X. 1978 - Freezing of plant protoplasts. In Plant Cell and Tissue Culture, Principles and Applications, Sharp W.R,, Larsen P.O.,Paddox E.F. and Raghavan V, Eds., Ohio State Univ. Press, Colombus, 876.

MONNIER C. and LEDDET Cb 1980 - Action du saccharose sur la résistance au gel des embryons immatures de capselle. Bull. Soc. Bot, Fr,, 127 : 71-77.

MORlGUCHl T., AKIMA T. and KOZAKI I, 1985 - Freeze-preservation of dormant pear shoot apices. Jpn, J. Breed., 35 : 196-199.

MORlGUCHl T., KOZAKI I., MATSUTA N. and YAMAKI S,, 1988 - Plant regeneration from grape callus stored under a combination of low temperature and silicone treatmentplant Cell, Tissue and Organ Culture, 15, 67-71.

NAG K.K. and STREET H.E. 1973 - Carrot embryogenesis from frozen cultured cells. Nature, 245 : 270-272.

NAG K.K. and STREET H.E, 1975a - Freeze-preservation of cultured plant cells. II The pretreatment phase. Physiol.plant., 34 : 254-260.

NAG K.K, and STREET H.E. 1975b - Freeze-preservation of cultured plant cells. II. The freezing and thawing phase. Physiol. Plant., 34 : 261-265.

NITZSCHE W., 1980 - One year storage of dried carrot callus. Z. Pflanzenphysiol., 100, 269- 271.

NORMAH M.N., CHIN H.F. and HOR Y.L, 1986 - Desiccation and cryopreservation of embryonic axes of Hevea brusiliensis Muell. - Arg. Pertanika,9 : 299-303.

PENCE V.C. and DRESSER B.L. 1988 - Embryo cryostorage as a technique for germplasm preservation of several large-seeded tree species. Abst. Beltsville Symp. in Agric. Res. XIII, Biotic Diversity and Germplasm Preservation - Global Imperatives, may 9-1 1, 24.

PRITCHARD H.W. and PRENDERGAST F.G. 1986 - Effects of desiccation on the in vitro viability of embryos of the recalcitrant seed species Araucuria hunsteinii K. Schum. J. Plant Physiol.,

QUATRANO R.S. 1968 - Freeze-preservation of cultured flax cells utilizing dimethyl sulfoxide. Plant Physiol,, 43 : 2057-2061,

REED B.M. 1989 - The effect of cold hardening and cooling rate on the survival of apical

37 1388-1397.

17

meristems of Vaccinium species frozen in liquid nitrogen. Cryolett,, 10 : 315-322.

REED B.M. and LAGERSTEDT H,B. 1987 - Freeze preservation of apical meristems of Rubus in liquid nitrogen. HortSci., 22 : 302-303.

REUFF I and REINHARD E. 1986 -Long term storage of Coleus blumei suspension cultures and cryopreservation of callus, Abst. 34th Ann. Cong. on Med. Plant Res., Hamburg, sept. 86, 1.

ROBERTS E.H., 1973 - Predicting the viability of seeds@ Seed Sci.Technol,, 1, 499-514.

SAKAI A. and NISHIYAMA Y. 1978 - Cryopreservation of winter-vegetative buds of hardy fruit trees in liquid nitrogen. HortSci., 13 : 225-227,

SAKAI A. and SUGARAWA Y. 1973 - Survival of poplar callus at super-low temperatures after cold acclimation, Plant Cell Physiol,, 14 : 1201-1204.

SAKAI A., YAMAKAWA M., SAUATA D., HARADA T, and YAKUWA T, 1978 - Development of a strawberry runner apex frozen to -196°C. Low TempSci. Ser. Bull., 36 : 31.

SALA F., CELLA R. and ROLLO F. 1979 - Freeze-preservation of rice cells, Plant Physiol.,45 : 170-1 76.

SEIBERT M, 1976 - Shoot initiation from carnation shoot apices frozen to -196°C Science, 191 : 1178-1179.

SEITZ U,, ALFERMANN A.W. and REINHARD Eb 1982 - Stability of biotransformation capacity in Digitalis lunata cell cultures after cryogenic storage. Plant Cell Rep., 2 : 273-276.

SHANQIANG Ku 1986 - Studies on cryopreservation and plant regeneration of strawberry callus. J, Wuhan Bot. Res., 4 : 231-238.

SIMINOVITCH D. 1979 - Protoplasts surviving freezing to -196°C and osmotic dehydration in 5 molar salt solutions prepared from the bark of black locust trees. Plant Physiol., 63 : 722- 725.

STANDKE K.H.C. 1978 - Tiefgefrierung nodaler segmente von Kartoffeln mittels flüssigen Stickstoff. Landbaufirschung Völkenrode, 28 : 77-78.

STEPONKUS P.L. and LANPHEAR F.O., 1967 - Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol., 42, 1432-1436.

TAKEUCHI M., MATSUSHIMA H. and SUGARAWA Y. 1980 - Long-term preservation of protoplasts of carrot and Marchantia. Cryo-Lett,, 1 : 519-524.

TAKEUCHI M.,MATSUSHIMA H. and SUGARAWA Y. 1982 - Totipotency and viability of protoplasts after long-term freeze preservation. Proc. 5th Intl. Cong. Plant tissue and Cell Culture, Fujiwara Ed., Tokyo, 797-798.

TANlGUCHl K., TANAKA R., ASHITANI N. and MIYAGAWA H. 1988 - Freeze preservation of tissue-cultured shoot primordia of the annual Haplopapus gracilis (2n = 24). Jpn. J. Genet., 63 : 267-272.

TISSERAT B., ULRICH J.M. and FINKLE B.J. 1981 - Cryogenic preservation and regeneration of date palm tissue. HortSci.,l6 : 47-48.

18

TOIVONEN P.M.A. and KARTHA K.K. 1989 - Cryopreservation of cotyledons of nongerminated white spruce (Picea glauca Moench Voss) embryos and subsequent plant regeneration. J. Plant Physiol., 134 : 766-768.

TOWILL L.E. 1981 - Solanum etuberosum : a model for studying the cryobiology of shoot- tips in the tuber-bearing Solanum species. Plant Sci. Lett,, 20 : 315-324.

TOWILL L.E. 1988 - Survival of shoot tips from mint species after short term exposure to cryogenic conditions. HortSci., 23 : 839-841.

TOWILL L.E. and MAZUR P. 1974 - Effect of cooling and warming rates on survival of frozen Acer saccharum tissue cultures. Plant Physiol,, 54 : 1 O-XX

ULRICH J,M. and FINKLE B.J. 1979 - Effect of a mixture of cryoprotectants in attaining liquid nitrogen survival of callus of a tropical plant. Cryobiology, 16 : 550-556.

ULRICH J,M., MICKLER R.A., FINKLE B.J. and KARNOSKY D.F. 1984 - Survival and regeneration of American elm callus cultures after being frozen in liquid nitrogen. Can, J. Forest. Res., 14 : 750-753.

ULRICH J.M., FINKLE B.J., MACKEY B.E., SCHAEFFER G.W, and SHARPE F. Jr. 1984 - Responses of six rice callus cultures to deep-frozen temperatures. Crop Sci,, 24 : 84-85.

URAGAMI A., SAKAI A., NAGAI M. and TAKAHASHI Tb 1989 - Survival of cultured cells and somatic embryos of Asparagus offkindis cryopreserved by vitrification. Plant Cell Rep., 8 : 418-421,

WATANABE K., MITSUDA H. and YAMADA Y. 1983 - Retention of metabolic and differentiation potentials of green Lavandula vera callus after freeze-preservation. Plant Cell PhysioL24 : 119-122.

WEBER G., ROTH E.J. and SCHWEIGER H.G. 1983 - Storage of cell suspensions and protoplasts of Glycine max (L.) Merr., Brassica napus (L.), Datura innoxicr (Mill,) and Daucus carota (L.) by freezing. Zu Pflanzenphysiol., 109 : 29-39.

WIDHOLM J.M., 1977 - The use of fluorescein diacetateand phenosafranine for determining viability of cultured pant cells. Stain Technol,, 47, 189-194,

WITHERS L.A. 1979 - Freeze preservation of somatic embryos and clonal plantlets of carrot (Daucus carota). Plant Physiol.,63 : 460-467.

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WITHERS L.A. 1980b - Preservation of germplasm. Int. Rev. Cytol., Suppl. II B : 101-136.

WITHERS L.A. 1982 - The development of cryopreservation techniques for plant cell, tissue and organ culture. Proc. 5th Intl. Congr. Plant Cell Tissue Culture, Fujiwara A. Ed., Tokyo, 793-794. ‘i

WITHERS L.A. 1985 - Cryopreservation of cultured plant cells and protoplasts. In Cryopreservation of Plant Cells and Organs, Kartha K.K. Ed,, Boca Raton, CRC Press, 243- 267.

WITHERS L.A. and KING P.J. 1980 - A simple freezing unit and routine cryopreservation method for plant cell cultures. Cryo-Lett., 1 : 213-220.

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WITHERS L.A. and STREET HbEb 1977 - Freeze-preservation pregrowth phase. Plant Physiol., 171-1 78.

of cultured plant cells. 111, The

YAKUWA H.and OKA S. 1988 - Plant regeneration through meristem culture from vegetative buds of mulberry (Morus bombycis Koidz.) stored in liquid nitrogen. Annb Bot., 62 : 79-82,

ZANDVOORT E.A. 1987 - In vitro germplam conservation of tropical aroids, Acta Bot. Neerl,, 36 : 150,

ZAVALA M.E. and SUSSEX LM. 1986 - Survival of developing wheat embryos and bean axes following cryoprotection and freezing in liquid nitrogen. J. Plant Physiol., 122 : 193- 197.

20

7 . .

Table 1 : Economically important plants with recalcitrant seeds ( After Kartha, 1985)

Family

Anacardiacene Boinbacaceae Diptcrocarpaceae Ery t hroxy laceae Euphorbiaceae Fafnceae Fagaceae Gramincae Guttiferae Juglandaceae Juglandaceae Lau race3e Lauraceae Mcliaceae Moracear Palniaccae Rubiaceae Rubiaccae Ruraccae

Stcrculiaccac Thcaccx Zingihcraccac

Scicntilic name Common name

Mango Durian Borneo camphor Cocaine Rubber Chest nut Oak Wild rice Mangowen Pecan Walnut Cinnamon Avocado blahopny Jackfruit Coconut Coffee COl'fW Orange. lemon. grapcfmit. ctc.

Cacao TC3 Cardamcini

21

Table 2 : In vitro storage strategies for short- to medium-term storage of plant genetic material (After Kartha, 1985).

Species In vitro storage methods

Shoot-tips of apples cultured on basal medium + 5 mg/( benzylaniinopurine and stored at 1°C or 4°C

Nodal cuttings from meristem-derived plantlets maintained in large vessels on nutrient medium with low osmotic concentration and activated charcoal

Meristem-derived plants on filter paper bridges in tuhes containing nutrient medium. at 20°C. I6 hr photoperiod. 100--200 Ix inrensiiy

hlcriateni-dcri\ul plants o n li2 Ils nicdium laclinp in sucrosc. plantlets kept in tuhcs at 26°C. Ih hr plioiciperiodb at -1000 I\ intensity

Plantlcts maintained ai 2 1 ° C

Shoot-tip plantlets storcd at 1 1 ° C . X H I Is. 3 hr photoperiod

f'lantlcts in viiro stored at 1 4 ° C Plantlets in vitro slored at 1 4 T

Maintenance of shocit-tip cultures at 9'C Stortlpr of meristem-derived plants at reduced

tcnipcraturc in thc range of 6-IO'C with a nican sunival 01' 61Ci at 6°C: survival further increased to 83% whrn stored at I X day1h"C night

Mcristcm-Jcrivcd plantlets stored ;II 4'C in dark with addition of I or 2 drops of culture nicdia evcry 3 months

Cultures stored at 12'C

hlcri~tc.ni-dcrived plantlets o n XIS medium with I .O p.\/ each of B A and SAA t S? euch of sucrose and mannitol. at 26'C. I6 hr photopcr- iods and -1000 Ix inrensitv

Storage duration

I year

2 years

1 year

2'1, years

CA. I year

I year

15-18 months 15-18 months

I ycar I ycar

6 years

18 \vecks

Drnstic growth reduction within 3 months

. '

22

Table 3 : Summary of results concerning the medium term storage of various plant species, using mineral overlay.

Caplin, S.M., 1959 Carrot calluses : 157 days in the dark under a 45 mm mineral oil overlay, Results : 1/4 of the control

Augereau et a/,, 1986 -Low temperature storage C. roseus, V, fhouarsii, C. arabica : 4 months in the dark at 15°C Results : regrowth

-Mineral oil preservation Amsonia tabunaemon fana, Atropa belladona, Catharantus roseus, Coffea, Glycine max, Papaver somniferum, Rosmarinus officinalis, Vinca minor, etc.,, : 6 months, 26"C, 16h photoperiod, under a30 mm mineral oil overlay. Results : regrowth 34/37 strains

Moriguchi et al,, 1988 Grape callus : 360 days in the dark at 15°C under a 90 mm silicone oil overlay. Results : 95% regrowth

Table 4 : List of woody species conserved using low temperature storage (, ..a Christie and Singh, 1987).

Shoot/plantlet cultures

Cinchona ledgeriana €uculyptus dalrympleane Eucalypfus gunii Eucalyptus spp. Malus domestica Malus spp. Pinus radiafa Prunus amygdalus

Prunus spp. Punica granatum Pyrus communis Pyrus pashia Salix babylonica Sequoia sempervirens Vitis rupestris Vitis spp. and hybrids

cen-

Ce1 I /ca I I us cu I tu res

Citrus spp. Mallotus japonicus Coffea spp. Pinus sylvestris

23

Table 5 : Comparison between the surfaces required for the storage in vivo or in vitro of various species

Malus domestica : 2,000 cultures = 0.28 m3 (57 ha)

Pinus radiata : 2,000 test tubes = 3,42 m2 (1 ha)

Vitis vinifera : 800 cultivars x 6 test tubes = 2 m2 (1 ha)

Manihot esculenta : 6,000 accessions (5vessels/accession) = 5 x 6 x 3 room (16 ha)

Table 6 : Schematical representation and performances of the coconut mature embryo in vitro culture process (After Assy Bah et a/., 1989).

100 mature embryos cultured on medium containing 60 g/l sucrose

5-20% con tam ¡na ti on

70-90% germination

suppression of the haustorium

1 month

2 months

3 months

5 months transfer to the nursery on sand 73-90% weanable plantlets

7 months prenursery and nursery 92-98% plantlets

24

Table 7 : Main cryoprotective substances used for cryopreservation of plant cells, tissues and organs (After Gazeau and Dereuddre, 1986)

NATURE - ALCOOL

ETHYLENE GLYCOL GLYCEROL PROPANE DIOL SOR BIT0 L MANNITOL - SULFURED COMPOUND

DIMETHYLSULFOXIDE (DMSO) - AMINO ACID

PROLINE - GLUCIDE

GLUCOSE TREHALOSE SACCHAROSE - POLYMERE

POLYETHYLENE GLYCOL (PEG) POLYLYNILPYRROLIDONE (PVP) HYDROXYETHYLE STARCH

MOLECULAR WEIGHT

62 92 75

182 182

78

115

180 342 342

HIGH

CHEMICAL FORMULA

CH20H-CH20H CH20H-CHOH-CH20H CH3-CHOH-CH20H CH2OH-(CHOH)4-CH2OH CH20H-(CHOH)4-CH20H

CH3-SO-CH3

CHO-(CHOH)4-CHOH C12 H22 O11 C12 H22 O11

25

. ?

Table 8 : The influence of the prefreeze incubation period on the survival of shoot tips from several potato genotypes, after ultrarapid freezing in liquid nitrogen (After Kartha, 1985)

% Survival of genotypes (total frozen)

Incubation period Ihr)

O 18.5 24.0 42.0 4s.o 65.5 72.5 96.0 I 17.0 I lX.0 IY2.0

S. tuberosutn ssp. fuberosum

S. goniocaiyy S. tuberosutn ssp. andigenah cv. Desirée' CY. klajestir

O( 15) O(27) O( 'O) 6.7(17)

50.0( IS) 33.3(20) O(18)

50.0( 16) 7.1(20) 20.0(20)

35.0( 17) O(18) 6.2(19) 23.0(22) 29.7(27) O(20) O( 19)

24.0(75)

15.0(20)

17.2(29)

28.6(21)

I5.1(26) 5.0(20)

Cr!oproteci;lnl 10% DhlSO: culture nicdium hlS t 3% sucrose + I mg ( B A (scored after 6 weeks). Cr!ciprciieciant 10% DMSO: culiurc medium hlS + 3% ucrose + I mg ( - I BA (scorcd aficr 22 days). Cqiiproiectani 5% DMSO: culture medium hlS t 3% sucrose -i 0.25 mg ( ' NAA + 2.5 mg C " ' B A (hocIrcd arier 3 week\).

L

26

Table 9 : List of plant species cryopreserved as cell suspensions (a), calluses (b), protoplasts (c), meristems (d), somatic (e), pollinic (f) and zygotic (g) embryos,

(a) cell suspensions

Acer pseudoplatanus

Acer saccharum Atropa belladonna Berberis dicfyophilla Brassica napus Brassica campestris Capsicum annuum Catharantus roseus

Coleus blumei Corydallis sempewirens Datura innoxia Datura stramonium Daucus carota

Digitalis Ianata

Dioscorea deltoidea Glaucium flavium Glycine max

Hordeum vulgare Hyosciamus muticus Linum usitatissimum Medicago sativa Myrtillocactus geometrizans Nicotiana plombaginifolia Nicotiana sylvestris Nicotiana tabacum

Onobrychis viciifolia Oryza sativa

Panax ginseng Pennisetum americanum Populus euramericana

Pseudotsuga menziesii Rhazia orientalis Rhazia stricta Rosa Paul's scarlet Saccharum officinalis

Nag and Street, 1975a and b Withers and Street, 1977 Withers and King, 1980 Towill and Mazur, 1974 Nag and Street, 1975a and b Withers, 1985 Weber et al,, 1983 Langis et al., 1989 Withers and Street, 1977 Kartha et al., 1982 Chen et al., 1984 Withers, 1985 Reuff and Reinhard, 1986 Withers, 1985 Weber et al., 1983 Bajaj, 1976a Latta, 1971 Nag and Street, 1973, 1975a and b Dougall and Wetherell, 1974 Bajaj, 1976a Withers and Street, 1977 Dougall and Whitten, 1980 ,

Weber et al., 1983 Diettrich et a/,, 1982 Seitz et al., 1982 Butenko ef al., 1984 Withers, 1985 Bajaj, 1976a Weber et al., 1983 Withers, 1980b, 1982 Withers, 1985 Quatrano, 1968 Kartha, 1985 Haffner, 1985 Maddox et al., 1983 Maddox et al., 1983 Withers, 1985 Bajaj, 1976a Hauptman and Widholm, 1982 Withers, 1985 Sala et al., 1979 Finkle and Ulrich, 1982 Ulrich et al,, 1984 Butenko et al., 1984 Withers, 1985 Sakai and Sugarawa, 1973 Binder and Zaerr, 1980a Binder and Zaerr, 1980b Withers, 1985 Withers, 1985 Withers and King, 1980 Finkle and Ulrich, 1982

27

(a) cell suspensions (continue) Solanum melongena Sorghum bicolor Triticum monococcum Vinca minor Zea mays

(b) callus

Coleus blumei Frugaria ananassa Gossypium arboreum Hordeum volgare Lavandula vera Medicago sativa Phoenix dacfylifera fopulus americana

Saccharum spp. Triticum aesfivum Ulmus americana

(c) protoplasts

Afropa belladonna Bromus inermis Datura innoxia Daucus carota

Glycine max

Hordeum vulgare Marchanfía polymorpha Nicotiana tabacum Olyzcr x fisum Trificum x fisum Trificum aesfivum Zea mays

(d) meristems

Arachis hypogeaea Asparagus officinalis Beta vulgaris Brassica napus Brassica oleacera (d) meristems (continue)

Withers, 1985 Withers and King, 1980 Withers, 1985 Caruso et al., 1987 Withers and King, 1980

Reuff and Reinhard, 1986 Shanqiang, 1986 Bajaj, 1982 Hahne and Lon, 1987 Watanabe ef a/,, 1983 Finkle ef al., 1985 Tisserat et al., 1981 Sakai and Sugarawa, 1973 Binder and Zaerr, 1980b Ulrich and Finkle, 1979 Bajaj, 1980 Ulrich et al,, 1984a

Bajaj, 1988 Mazur and Hartmann, 1978 Bajaj, 1988 Maur and Hartmann, 1978 Weber et al., 1983 Takeuchi ef a/., 1982 Weber et al., 1983 Takeuchi et a/,, 1982 Takeuchi et al., 1980, 1982 Bajaj, 1988 Bajaj, 1983d Bajaj, 1983d Takeuchi et al., 1982 Withers, 1980

Bajaj, 1979 Kumu et al., 1985 Braun, 1988 Benson et al,, 1984 Harada ef al.,1985

28

b

Cicer arietinum Dianthus caryophyllus

Digitalis lanata

Fragaria ananassa

Haplopapus gracilis Lilium multiflorum Lycopersicon esculentum Malus domestica

Malus spp. Manihot esculenta

Mentha spp. Morus bombycis Pisum sativum Pyrus communis Pyrus serotina

Rubus spp. Solanum etuberosum Solanum goniocalk Solanum tuberosum

Xanthosoma Vanda hookeriana Vaccinium spp.

(e) somatic embryos

Asparagus officinalis Citrus sinensis Coffea arabica Daucus carota

Elaeis guineensis

Picea abies

Picea glauca Pinus taeda Xanthosoma

Bajaj, 1979 Seibert, 1976 Galerne, 1985 Fabre, 1986 Diettrich et a/,, 1987

Sakai et al., 1978 Kartha et al., 1980 Taniguchi et al,, 1988 Bouman and de Klerk, 1989 Grout et al,, 1978 Sakai and Nishiyama, 1978 Kuo and Linberger, 1985 Katano et al., 1983 Bajaj, 1977a, 1983a, 1985 Kartha et a/., 1982 Towill, 1988 Yakuwa and Oka, 1988 Kartha et al., 1979 Dereuddre et al., 1990 Moriguchi et al,, 1985 Arnaud et al,, 1989 Reed and Lagerstedt, 1987 Towill, 1981 Grout and Henshaw, 1978 Standke, 1978 Bajaj, 1985 Benson et al., 1984, 1989 Zandvoort, 1987 Kadzimi, 1988 Reed, 1989

Uragami et al., 1989 Marin and Duran-Vila, 1988 Bertrand-Desbrunais et al., 1988 Withers, 1979 Blandin, 1988 Engelmann et al,, 1985 Engelmann and Duval, 1986 Engelmann and Dereuddre, 1988 Galerne and Dereuddre, 1987 Gupta and Durzan, 1987 Kartha et al,, 1988 Gupta and Durzan, 1987 Zandvoort, 1987

29

(f) pollen embryos

Arachis hypogea Arachis villosa Atropa belladonna Brassica campestris Brassica napus

Citrus spp. Gossypium arboreum Nicotiana tabacum

Oryza sativa Petunia hybrida Primula obconica Triticum aestivum

(9) zygotic embryos

Aesculus hypocasfanea Araraucaria excelsa Brassica napus Capsella bursa pasforis Catva Castanea Cocos nucifera Elaeis guineensis Fagus Hevea brasiliensis Hordeum vulgare Howea fosferiana Juglans Phaseolus vulgaris Picea glauca Quercus Triticum aestivum

Triticale Veitchia merrillii Zea mays

Bajaj, 1983b Bajaj, 1983a Bajaj, 197613, 1977b, 1978 Bajaj, 1983b Bajaj, 1983b Engelmann and Baubault, 1986 Bajaj, 1984 Bajaj, 1982 Bajaj, 1977b, 1978 Coulibaly and Demarly, 1979 Bajaj, 1981a Bajaj, 1978 Bajaj, 1981b Bajaj, 1983b

Pence and Dresser, 1988 Pritchard and Prendergast, 1986 Withers, 1982 Monnier and Leddet, 1980 Pence and Dresser, 1988 Pence and Dresser, 1988 Bajaj, 1984 Grout et al., 1983 Pence and Dresser, 1988 Normah et al., 1986 Withers, 1982 Chin ef al., 1988 Pence and Dresser, 1988 Zavala and Sussex, 1986 Toivonen and Kartha, 1989 Pence and Dresser, 1988 Bajaj, 1983b Zavala and Sussex, 1986 Bajaj, 1983c Chin et al., 1988 Delvallée, 1987

30

timt: (d3~s;

Ficlure 1

.. . .

surv ¡val(%) 100

50

O i 1 i i 1 1 . 1 1 I 1 1

O 5 10 range of ax i l la ry shoot tips

(O=apical shoot t i p )

Fiaure 2

Ficlure 1 : Recovery growth, (estimated by packed cell volume) of suspension cultured cells of Daucus carota sampled during a complete growth cycle (L = lag phase ; E = exponential phase ; DS = decline of growth followed by stationary phase), frozen and thawed and returned to culture at a uniform cell density (After Kartha, 1985),

Ficlure 2 : Changes in the resistance of axillary shoot tips of carnation as a funciion of their range along the stem (range O corresponded to apical shoot tip). DMSO and sucrose concentrations were respectively 5OEO and 0.75M (After Dereuddre et al., 1988).

3 1

-50-

n

o o, -100 - al

J L.

+ 2 -150- al z

1 / 1

al I-

-200 O 10 20 30 40

7 ' 8 /

i /

10 20 Time ( m i n )

Fiaure 3 : 'Typical freeze-thaw profiles (After Withers, 1980)

1, Rapid freezing obtained by immersing specimen/ampoule in liquid nitrogen 2. Relatively rapid freezing produced by suspending specimen over liquid nitrogen ; slope

of curve depends upon distance between specimen and liquid nitrogen. 3. Step-wise or prefreezing ; transition from one step to next may steep as shown here or

more gentle as in the case of larger specimen. 4. Slow freezing at 2°C /min. 5. Slow freezing at l"C/min. 6. Slow freezing interrupted by a period at a holding temperature. 7. Rapid thawing as in warm water at 35-40°C. 8. Slow thawing as in air at room temperature. The arrows indicate the termination of the cooling by transfer to liquid nitrogen.

Fiaure 4 : Effect of cooling rates on the survival and plant regeneration from cryopreserved meristems of pea and strawberry. The meristems were precultured for 2 days on nutrient medium supplemented with 5 Ob DMSO and frozen using 5 Oh DMSO to -40°C followed by storage in liquid nitrogen (From Kartha, 1985).

32

cooling rate PCmin-1)

Fiaure 5 : Effect of cooling rate on survival and recovery of oil palm embryoid clumps from clone BC 068 and BC 156 (From Engelmann et al., 1988).

5 0 -:o - io -ao/* TERMINAL FREEZING TEMPERATURE PC1

Fiaure 6 : Effect of terminal freezing temperature and liquid nitrogen storage on the survival and morphogenetic responses of cassava meristems. The meristems were frozen using 15% DMSO + 3% sucrose by droplet-freezing method at a cooling rate of OS"C/min to the indicated temperatures and stored in liquid nitrogen for 1 hr (After Kartha, 1985).

33

A : MICROPROPAGATION

:

B : CRY OC ON S E RVATl O N

o

Ficlure 7. : Schematical representation of the carnation meristem cryopreservation process (After Fabre, 1986).

- starting material : axillary or terminal buds - pretreatment : 24 hours at 25°C on a medium containing 0.75M sucrose (a-b), followed by 1 hour at 0°C in liquid medium containing 0.75M sucrose + 5% DMSO (cl - freezing : O to -40°C at 05°C min-1, followed by immersion in LN (d-g) - storage : 4.5 months in liquid nitrogen successfully tested (g) - thawing : rapid, by immersion of the samples in a water-bath thermostated at +4OoC (h) - post-treatment : successive transfers on media with progressively decreased sucrose concentrations +I).

34

1 . 2 I 3

u /a .

I 6 ‘I I’

a 7 b 8 9 .

Fiaure 8 : Schematical representation of the oil palm somatic embryo cryopreservation process (After Engelmann, 1986)-

35