**Plant Cryopreservation**

R.K. Radha, William S. Decruse and P.N. Krishnan

*Plant Biotechnology and Bioinformatics Division Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala, India* 

#### **1. Introduction**

430 Current Frontiers in Cryopreservation

Xin, Z.; Li, P.H. (1993) Relationship between proline and abscisic acid in the induction of

Yoshiba, Y.; Kiyosue, T.; Nakashima, K.; Yamaguchi-Shinozaki, K; Shinozaki, K. (1997)

Zhang, L.P.; Mehta, S.K.; Liu, Z.P.; Yang, Z.M. (2008) Copper-induced proline synthesis is

613.

*Cell and Physiology*, Vol.38,1095–1102.

*Physiology,* Vol.49,411–419.

chilling tolerance in maize suspension- cultured cells. *Plant Physiology*, Vol.103,607–

Regulation of levels of proline as an osmolyte in plants under water stress. *Plant* 

associated with nitric oxide generation in *Chlamydomonas reinhardtii*. *Plant and Cell* 

Two basic approaches to conservation of plant genetic resources are *ex situ* and *in situ* conservation. *Ex situ* conservation includes seed storage, *in vitro* storage, DNA storage, pollen storage, field genebanks and botanical gardens while the *in situ* approach encompasses genetic reserves, on farm and home garden conservation.

Cryopreservation is a part of biotechnology. Biotechnology plays an important role in international plant conservation programs and in preservation of the world's genetic resources (Bajaj, 1995; Benson, 1999). Advances in biotechnology provide new methods for plant genetic resources and evaluation (Paunesca, 2009). Cryopreservation, developed during the last 25 years, is an important and the most valuable method for long-term conservation of biological materials. The main advantages in cryopreservation are simplicity and the applicability to a wide range of genotypes (Engelmann, 2004). This can be achieved using different procedures, such as pre-growth, desiccation, pregrowth-desiccation, vitrification, encapsulationvitrification and droplet-freezing (Engelmann, 2004). Cryopreservation involves storage of plant material (such as seed, shoot tip, zygotic and somatic embryos and pollen) at ultra-low temperatures in LN (-196°C) or its vapor phase (-150°C). To avoid the genetic alterations that may occur in long tissue cultures storage, cryopreservation has been developed (Martin *et al*., 1998). At this temperature, cell division, metabolic, and biochemical activities remain suspended and the material can be stored without changes and deterioration for long time. Walters *et al*. (2009) proposed that this assumption, based on extrapolations of temperaturereaction kinetic relationships, is not completely supported by accumulating evidence that dried seeds can deteriorate during cryogenic storage. After 30 years of cryogenic storage, seeds of some species exhibited quantitatively lower viability and vigor. In cryopreservation method, subcultures are not required and somaclonal variation is reduced. Advantages of cryopreservation are that germplasm can be kept for theoretically indefinite time with low costs and little space. Besides its use for the conservation of genetic resources, cryopreservation can also be applied for the safe storage of plant tissues with specific characteristics. Different types of plant cell, tissues and organs can be cryopreserved. Cryopreservation is the most suitable long-term storage method for genetic resources of vegetatively maintained crops (Kaczmarczyk *et al*., 2008). For vegetatively propagated species, the best organs are shoot apices excised from *in vitro* plants. Shoot apices or meristems cultures are suitable because of virus-free plant production, clonal propagation, improving health status, easier recovery and

Plant Cryopreservation 433

species which are propagated vegetatively the emphasis will be on *ex situ* conservation techniques, including field genebank and *in vitro* storage. However it is essential to recognize that owing to various problems and limitations encountered with both genetic reserves and field genebanks, cryopreservation currently offers the only safe and cost effective option for the long-term conservation of genetic resources of these problem species. Significant progress has been made during the past 10 years in the area of plant cryopreservation with the development of various efficient cryopreservation protocols. An important advantage of these new techniques is their operational simplicity, since they will be applied mainly in developing tropical countries where the largest part of genetic resources of problem species is located. Encouraging results in medicinal plants have been published in recent years which present extensive list of plant species whose embryos and or embryonic axes have been successfully

In comparison with results obtained with vegetatively propagated species, it is clear that research is still at a very preliminary stage for recalcitrant seeds. The desiccation technique is mainly employed for freezing embryos and embryonic axes, the survival achieved are extremely uneven. And also survival is often limited and regeneration often restricted to callusing or incomplete development of plantlets. In only a limited number of cases, the whole plants have been regenerated from cryopreserved material (Chin and Pritchard 1988, Assy Bah and Engelmann 1992). Seeds and embryos of recalcitrant species also display various characteristics which make their cryopreservation difficult. One of the characteristics of recalcitrant seeds is that there is no arrest in their development, as with orthodox seeds. It is very difficult to select seeds at a precise developmental stage, even though this parameter is often of critical importance to achieve successful cryopreservation. Seeds of many species are too large to be frozen directly and embryos or embryonic axes have to be employed. However, embryos are often very complex tissue composition which display differential sensitivity to desiccation and freezing, the root pole seeming more resistant than the shoot pole (Pence 1995). In some species, embryos are extremely sensitive to desiccation and even minor reduction in their moisture content down to levels much too high to obtain survival after freezing leads to irreparable structural damage. It should be emphasized that selecting embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann *et al.,* 1995) However, in these cases basic protocols for disinfection, *in vitro* germination of embryos or embryonic axes, plantlet development and possibly limited propagation will have to be established

Cryostorage of seed was initially developed for the preservation of genetic resources of agriculturally important species for breeding and selection. The development of comparatively simple cryostorage protocols allowed seeds of over 155 agricultural species (Stanwood, 1985) to be stored at low cost, in an environment without obvious problems of seed ageing, genetic variations and predation common to many conventional seed storage methods. With the regular use of cryostorage system for seeds of agri-crops, the same process is now viewed as having important application for preserving seeds of medicinal plants (Decruse *et al.*, 1999), endangered species (Touchel and Dixon 1994) and other native plant species (Pence 1991, Touchel and Dixon 1993, Decruse and Seeni 2002). For the longterm preservation of species producing recalcitrant seeds, zygotic embryos were used for cryopreservation. Incidentally excised zygotic embryos or embryonic axes were successfully employed for the cryopreservation of coconut (Assy-Bah and Engelman, 1992 a,b, Chin *et al.,*

cryopreserved (Kartha and Engelmann 1994, Pence 1995, Engelmann *et al* 1995).

prior to any cryopreservation experiment.

less mutation (Scowcroft, 1984). Seed and field collections have been the only proper for the long-term germplasm conservation of woody species, while a large number of forest angiosperms have recalcitrant seeds with a very limited period of conservability. The species, which are mainly vegetatively propagated, require the conservation of huge number of accessions (Panis and Lambardi, 2005). The storage of this huge number needs large areas of land and high running costs. Preservation of plant germplasm is part of any plant breeding program. The most efficient and economical way of germplasm storage is the form of seeds. However, this kind of storage is not always feasible because 1) some seeds deteriorate due to invasion of pathogens and insects, 2) some plants do not produce seeds and they are propagated vegetatively, 3) some seeds are very heterozygous thus, not proper for maintaining true-to-type genotype, 4) seeds remain viable for a limited time, and 5) clonally propagated crops such as fruit, nut, and many root and tuber vegetables cannot be stored as seed (Chang and Reed, 2001; Bekheet *et al*., 2007). Cryopreservation offers a good method for conservation of the species, especially woody plant germplasm (Panis and Lambardi, 2005). Cryostorage of seeds in LN was initially developed for the conservation of genetic resources of agriculturally important species (Rajasekharan, 2006). The development of simple cryostorage protocols for orthodox seeds has allowed cryopreservation of a large number of species at low cost, significantly reducing seed deterioration in storage (Stanwood, 1987). Only few reports are available on the application of cryopreservation on seeds of wild and endangered species and medicinal plants (Rajasekharan, 2006). New cryobiological studies of plant materials has made cryopreservation a realistic tool for long-term storage, for tropical species, which are not intrinsically tolerant to low temperature and desiccation, has been less extensively investigated (Rajasekharan, 2006). Cryopreservation has been applied to more than 80 plant species (Zhao *et al*., 2005). Number of species, which can be cryopreserved has rapidly increased over the last several years because of the new techniques and progress of cryopreservation research (Rajasekharan, 2006). The vitrification/one-step freezing and encapsulation dehydration methods have been applied to an increasing number of species (Panis and Lambardi, 2005). A new method, named encapsulation- vitrification is noteworthy (Sakai, 2000). These techniques have produced high levels of post-thaw and minor modifications (Rajasekharan, 2006). In cryopreservation, information recording such as type and size of explants, pretreatment and the correct type and concentration of cryoprotectants, explants water content, cryopreservation method, rate of freezing and thawing, thawing method, recovery medium and incubation conditions is very important (Reed, 2001 ; González-Benito *et al*., 2004; Bekheet *et al*., 2007). All germplasm requires safe storage because even exotic germplasm without obvious economic merit may contain genes or alleles that may be needed as new disease, insect, environmental, or crop production problems arise (Westwood, 1989). It is important to record also the recovery percentage after a short conservation period. A major concern is the genetic stability of conserved material.

For many plant species which produce orthodox seeds, i.e. which can be dehydrated extensively and stored dry at low temperature, the emphasis for genetic resource conservation will be on seed/embryo storage. Recalcitrant seeds cannot tolerate desiccation to moisture content that would permit exposure to low temperature. They are often large with considerable quantities of fleshy endosperm. Therefore, recent investigations have identified species displaying an intermediate form of seed/embryo storage. As regards the balance of techniques employed within complementary strategies developed for conserving the genetic resources of these problems species, the emphasis in the case of non-orthodox (intermediate/ recalcitrant) forest tree species will be on *in situ* conservation in genetic reserves, while for

less mutation (Scowcroft, 1984). Seed and field collections have been the only proper for the long-term germplasm conservation of woody species, while a large number of forest angiosperms have recalcitrant seeds with a very limited period of conservability. The species, which are mainly vegetatively propagated, require the conservation of huge number of accessions (Panis and Lambardi, 2005). The storage of this huge number needs large areas of land and high running costs. Preservation of plant germplasm is part of any plant breeding program. The most efficient and economical way of germplasm storage is the form of seeds. However, this kind of storage is not always feasible because 1) some seeds deteriorate due to invasion of pathogens and insects, 2) some plants do not produce seeds and they are propagated vegetatively, 3) some seeds are very heterozygous thus, not proper for maintaining true-to-type genotype, 4) seeds remain viable for a limited time, and 5) clonally propagated crops such as fruit, nut, and many root and tuber vegetables cannot be stored as seed (Chang and Reed, 2001; Bekheet *et al*., 2007). Cryopreservation offers a good method for conservation of the species, especially woody plant germplasm (Panis and Lambardi, 2005). Cryostorage of seeds in LN was initially developed for the conservation of genetic resources of agriculturally important species (Rajasekharan, 2006). The development of simple cryostorage protocols for orthodox seeds has allowed cryopreservation of a large number of species at low cost, significantly reducing seed deterioration in storage (Stanwood, 1987). Only few reports are available on the application of cryopreservation on seeds of wild and endangered species and medicinal plants (Rajasekharan, 2006). New cryobiological studies of plant materials has made cryopreservation a realistic tool for long-term storage, for tropical species, which are not intrinsically tolerant to low temperature and desiccation, has been less extensively investigated (Rajasekharan, 2006). Cryopreservation has been applied to more than 80 plant species (Zhao *et al*., 2005). Number of species, which can be cryopreserved has rapidly increased over the last several years because of the new techniques and progress of cryopreservation research (Rajasekharan, 2006). The vitrification/one-step freezing and encapsulation dehydration methods have been applied to an increasing number of species (Panis and Lambardi, 2005). A new method, named encapsulation- vitrification is noteworthy (Sakai, 2000). These techniques have produced high levels of post-thaw and minor modifications (Rajasekharan, 2006). In cryopreservation, information recording such as type and size of explants, pretreatment and the correct type and concentration of cryoprotectants, explants water content, cryopreservation method, rate of freezing and thawing, thawing method, recovery medium and incubation conditions is very important (Reed, 2001 ; González-Benito *et al*., 2004; Bekheet *et al*., 2007). All germplasm requires safe storage because even exotic germplasm without obvious economic merit may contain genes or alleles that may be needed as new disease, insect, environmental, or crop production problems arise (Westwood, 1989). It is important to record also the recovery percentage after a short conservation period. A major concern is the genetic stability

For many plant species which produce orthodox seeds, i.e. which can be dehydrated extensively and stored dry at low temperature, the emphasis for genetic resource conservation will be on seed/embryo storage. Recalcitrant seeds cannot tolerate desiccation to moisture content that would permit exposure to low temperature. They are often large with considerable quantities of fleshy endosperm. Therefore, recent investigations have identified species displaying an intermediate form of seed/embryo storage. As regards the balance of techniques employed within complementary strategies developed for conserving the genetic resources of these problems species, the emphasis in the case of non-orthodox (intermediate/ recalcitrant) forest tree species will be on *in situ* conservation in genetic reserves, while for

of conserved material.

species which are propagated vegetatively the emphasis will be on *ex situ* conservation techniques, including field genebank and *in vitro* storage. However it is essential to recognize that owing to various problems and limitations encountered with both genetic reserves and field genebanks, cryopreservation currently offers the only safe and cost effective option for the long-term conservation of genetic resources of these problem species. Significant progress has been made during the past 10 years in the area of plant cryopreservation with the development of various efficient cryopreservation protocols. An important advantage of these new techniques is their operational simplicity, since they will be applied mainly in developing tropical countries where the largest part of genetic resources of problem species is located. Encouraging results in medicinal plants have been published in recent years which present extensive list of plant species whose embryos and or embryonic axes have been successfully cryopreserved (Kartha and Engelmann 1994, Pence 1995, Engelmann *et al* 1995).

In comparison with results obtained with vegetatively propagated species, it is clear that research is still at a very preliminary stage for recalcitrant seeds. The desiccation technique is mainly employed for freezing embryos and embryonic axes, the survival achieved are extremely uneven. And also survival is often limited and regeneration often restricted to callusing or incomplete development of plantlets. In only a limited number of cases, the whole plants have been regenerated from cryopreserved material (Chin and Pritchard 1988, Assy Bah and Engelmann 1992). Seeds and embryos of recalcitrant species also display various characteristics which make their cryopreservation difficult. One of the characteristics of recalcitrant seeds is that there is no arrest in their development, as with orthodox seeds. It is very difficult to select seeds at a precise developmental stage, even though this parameter is often of critical importance to achieve successful cryopreservation. Seeds of many species are too large to be frozen directly and embryos or embryonic axes have to be employed. However, embryos are often very complex tissue composition which display differential sensitivity to desiccation and freezing, the root pole seeming more resistant than the shoot pole (Pence 1995). In some species, embryos are extremely sensitive to desiccation and even minor reduction in their moisture content down to levels much too high to obtain survival after freezing leads to irreparable structural damage. It should be emphasized that selecting embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann *et al.,* 1995) However, in these cases basic protocols for disinfection, *in vitro* germination of embryos or embryonic axes, plantlet development and possibly limited propagation will have to be established prior to any cryopreservation experiment.

Cryostorage of seed was initially developed for the preservation of genetic resources of agriculturally important species for breeding and selection. The development of comparatively simple cryostorage protocols allowed seeds of over 155 agricultural species (Stanwood, 1985) to be stored at low cost, in an environment without obvious problems of seed ageing, genetic variations and predation common to many conventional seed storage methods. With the regular use of cryostorage system for seeds of agri-crops, the same process is now viewed as having important application for preserving seeds of medicinal plants (Decruse *et al.*, 1999), endangered species (Touchel and Dixon 1994) and other native plant species (Pence 1991, Touchel and Dixon 1993, Decruse and Seeni 2002). For the longterm preservation of species producing recalcitrant seeds, zygotic embryos were used for cryopreservation. Incidentally excised zygotic embryos or embryonic axes were successfully employed for the cryopreservation of coconut (Assy-Bah and Engelman, 1992 a,b, Chin *et al.,*

Plant Cryopreservation 435

storage, the vials were retrieved from LN and rewarmed in a water bath at 400C for 1-2 min. The rewarmed embryos were also transferred to germination medium and cultured under

 Observations on the germination of embryos were made after 8 weeks and results analyzed statistically in a completely randomized model. Survival rate was assessed as the percentage of embryonic axes that exhibited any kind of growth, including seedling

*Moisture content determination.* Moisture content (MC) of the embryos was determined by

The embryonic axes with cotyledons (Fig 1a) freshly dissected from the seeds possessed 55.7% MC and exhibited 86.67% germination and normal growth in MS medium devoid of PGRs within a week of culture. Dehydration under laminar airflow reduced the MC to 43.7% after 30min and 31.3% after 60min. without appreciable reduction in viability so that 76-77% of them germinated (Fig.1). Dehydration for 120min reduced MC to 19.6% and germination to 66.67% and was the optimum dehydration period (Fig.2) to get maximum germination (60%) after LN treatment (Fig.2). Root and shoot emergence was observed after one week of culture (Fig.1b) in 60% of the desiccated (120 min.) and LN treated embryonic axes and well developed seedlings were obtained within 20 days of culture (Fig.1c). Dehydration beyond 120 min. gradually reduced MC and drastically reduced viability. The MC came down to 12.1% after 210 min. when none of the embryos survived. Prolonged dehydration (150-180min) not only reduced survival down to 16.67-10% but also caused

abnormal growth with only radicle development in the survived embryos (Fig.1d).

Research in the past two decades has shown that most orthodox seeds remain viable for long periods of storage after attaining appropriate desiccation levels of about 3-5% moisture content (Roberts, 1973). Contrary to this, recalcitrant seeds of several tropical and temperate species are desiccation sensitive, eg. Tea, Cocoa, Citrus, Jack fruit (Chin and Roberts, 1980). There are various options available to improve storage of non-orthodox seeds/embryos. Desiccation is the simplest procedure since it consists of dehydrating explants, and then freezing them rapidly by direct immersion in LN has been applied to embryonic axes extracted from recalcitrant and intermediate seeds (Engelmann, 1997). It should also be noted that selection of embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann *et al*., 1995). The conservation efforts of *N. nimmoniana* are hampered mainly due to relatively large and intermediate type of seeds with desiccation sensitivity. The viability of the embryos was not much affected when the embryos were desiccated from 55.7% to 43.7% (i.e. 30 min. desiccation). Significant loss of viability due to further reduction of moisture content shows the intermediate nature of the embryos is in line with the report of Dussert *et al* (1995). Safe moisture content of the embryonic axes as obtained in the present study is 19.6% (60% survival). Damage to plumule rather than radicle occurred due to excessive dehydration of *N. nimmoniana* embryos is as observed earlier in *Auracaria hunstenni* where desiccation damage is reported to be more serious in the plumule (Pritchard and Prendergast, 1986). The exact causes of embryonic death and its relationship with moisture content are not fully understood. Chin *et al* stated that seed death could be due either to the moisture content falling below a critical value or simply a general physiological deterioration with time. If embryonic axes have been desiccated to around 20% moisture content without loss of viability, it is possible that

stated conditions for recovery. The whole experiment was repeated three times.

development; shoot growth and root growth.

constant temperature oven method (103 0C) for 17h.

1989), cocoa (Pence, 1991, Chandel *et al.,* 1995) oil palm (Chabrillange *et al.,* 1997), walnut (de Boucaud *et al.,* 1991), jack fruit (Chandel *et al.,* 1995, Thammasiri, 1999), rubber (Normann, 1986), tea (Chauduryi *et al.,* 1991) and neem (Berjack and Dumet, 1996).

National Gene bank for Medicinal and Aromatic Plants at Tropical Botanic Garden and Research Institute (TBGRI) is one among the four (CIMAP, Lucknow, NBPGR, New Delhi and RRL, Jammu) having the mandate of conserving the medicinal and aromatic plants (MAPs) of Peninsular India through biotechnological intervention including collection, ex *situ* conservation and characterization of the precious taxa that are rare, endangered, threatened, endemic, vulnerable or over exploited as the case may be. TBGRI has significantly developed cryopreservation protocol on rare and endangered medicinal plants of India (Decruse *et al.,* 1999, Decruse and Seeni, 2002, Radha *et al*., 2006). A cryobank was also established which now holds more than 25 accessions of medcinal and aromatic plants (Decruse *et al.,* 1999b, Decruse and Seeni 2002b, Radha *et al*., 2010).

### **2. Cryopreservation of excised embryonic axes of** *Nothapodytes nimmoniana* **(Graham) Mebberly, a vulnerable medicinal tree species of the Western Ghats**

*Nothapodytes nimmoniana* (Graham) Mebberly, of family Icacinaceae is a small vulnerable medicinal tree distributed in India, Sri Lanka, Myanmar, Thailand, Malaysia and China. In India it is distributed in upper ranges of the Western Ghats particularly in the Nilgiris and Palni hills of southern peninsula. The stem and roots are an important source of the antitumour quinoline alkaloid camptothecin (Hsiang etal.,1985) and also find applications against retrovirus and human immunodeficiency virus. Consequently natural population of this species in the Western Ghats are severely depleted owing to habitat destruction and over exploitation (Cragg *et al*.,1993, Ravikumar and Ved, 2000) and hence conservation efforts are undertaken by certain agencies in the region.

Seeds of N. nimmoniana are large intermediate type showed 100% germination under controlled conditions. Embryonic axes with cotyledons having moisture content of 55.7% presumed to be intermediate in nature, lose their viability within a short period after maturity. Cryopreservaton of zygotic embryos is recognized as an effective tool for the long-term preservation of such plant species those produce recalcitrant/large seeds (Engelmann, 1997).

Desiccation and cryopreservation. The seeds were separated from the fruits (drupe), rinsed in running tap water for one hour to remove the mucilage and washed in commercial detergent (1% Teepol, Godrej, India Ltd., Mumbai) for 10 min. followed by thorough washing in running tap water for 10-20 min. Seeds were then surface decontaminated by immersion in 0.01% (w/v) HgCl2 for 5-10 min. followed by 3-5 rinses in sterile distilled water. Seed coat was broken and embryos with cotyledons were dissected out free of the endosperm in aseptic condition in the laminar air flow cabinet. Immediately after dissection, batches of 20 embryos each were subjected to dehydration under laminar airflow for 30, 60, 90, 120,150,180 and 210 min. period. A sample of 10 embryos was inoculated into MS medium (Murashige and Skoog, 1962) devoid of PGR as fresh control and cultured under 10/14h light/dark periods (30 - 50 μmol m–2 s–2) at 25±2 0C for 8 weeks. After desiccation at 30 min intervals, equally divided samples of 10 embryos were transferred to germination medium and another 10 packed in 2ml cryovial and transferred to LN (at –196 0C) After 24h

1989), cocoa (Pence, 1991, Chandel *et al.,* 1995) oil palm (Chabrillange *et al.,* 1997), walnut (de Boucaud *et al.,* 1991), jack fruit (Chandel *et al.,* 1995, Thammasiri, 1999), rubber (Normann,

National Gene bank for Medicinal and Aromatic Plants at Tropical Botanic Garden and Research Institute (TBGRI) is one among the four (CIMAP, Lucknow, NBPGR, New Delhi and RRL, Jammu) having the mandate of conserving the medicinal and aromatic plants (MAPs) of Peninsular India through biotechnological intervention including collection, ex *situ* conservation and characterization of the precious taxa that are rare, endangered, threatened, endemic, vulnerable or over exploited as the case may be. TBGRI has significantly developed cryopreservation protocol on rare and endangered medicinal plants of India (Decruse *et al.,* 1999, Decruse and Seeni, 2002, Radha *et al*., 2006). A cryobank was also established which now holds more than 25 accessions of medcinal and aromatic plants

**2. Cryopreservation of excised embryonic axes of** *Nothapodytes nimmoniana* **(Graham) Mebberly, a vulnerable medicinal tree species of the Western** 

*Nothapodytes nimmoniana* (Graham) Mebberly, of family Icacinaceae is a small vulnerable medicinal tree distributed in India, Sri Lanka, Myanmar, Thailand, Malaysia and China. In India it is distributed in upper ranges of the Western Ghats particularly in the Nilgiris and Palni hills of southern peninsula. The stem and roots are an important source of the antitumour quinoline alkaloid camptothecin (Hsiang etal.,1985) and also find applications against retrovirus and human immunodeficiency virus. Consequently natural population of this species in the Western Ghats are severely depleted owing to habitat destruction and over exploitation (Cragg *et al*.,1993, Ravikumar and Ved, 2000) and hence conservation

Seeds of N. nimmoniana are large intermediate type showed 100% germination under controlled conditions. Embryonic axes with cotyledons having moisture content of 55.7% presumed to be intermediate in nature, lose their viability within a short period after maturity. Cryopreservaton of zygotic embryos is recognized as an effective tool for the long-term preservation of such plant species those produce recalcitrant/large seeds (Engelmann, 1997). Desiccation and cryopreservation. The seeds were separated from the fruits (drupe), rinsed in running tap water for one hour to remove the mucilage and washed in commercial detergent (1% Teepol, Godrej, India Ltd., Mumbai) for 10 min. followed by thorough washing in running tap water for 10-20 min. Seeds were then surface decontaminated by immersion in 0.01% (w/v) HgCl2 for 5-10 min. followed by 3-5 rinses in sterile distilled water. Seed coat was broken and embryos with cotyledons were dissected out free of the endosperm in aseptic condition in the laminar air flow cabinet. Immediately after dissection, batches of 20 embryos each were subjected to dehydration under laminar airflow for 30, 60, 90, 120,150,180 and 210 min. period. A sample of 10 embryos was inoculated into MS medium (Murashige and Skoog, 1962) devoid of PGR as fresh control and cultured under 10/14h light/dark periods (30 - 50 μmol m–2 s–2) at 25±2 0C for 8 weeks. After desiccation at 30 min intervals, equally divided samples of 10 embryos were transferred to germination medium and another 10 packed in 2ml cryovial and transferred to LN (at –196 0C) After 24h

1986), tea (Chauduryi *et al.,* 1991) and neem (Berjack and Dumet, 1996).

(Decruse *et al.,* 1999b, Decruse and Seeni 2002b, Radha *et al*., 2010).

efforts are undertaken by certain agencies in the region.

**Ghats** 

storage, the vials were retrieved from LN and rewarmed in a water bath at 400C for 1-2 min. The rewarmed embryos were also transferred to germination medium and cultured under stated conditions for recovery. The whole experiment was repeated three times.

 Observations on the germination of embryos were made after 8 weeks and results analyzed statistically in a completely randomized model. Survival rate was assessed as the percentage of embryonic axes that exhibited any kind of growth, including seedling development; shoot growth and root growth.

*Moisture content determination.* Moisture content (MC) of the embryos was determined by constant temperature oven method (103 0C) for 17h.

The embryonic axes with cotyledons (Fig 1a) freshly dissected from the seeds possessed 55.7% MC and exhibited 86.67% germination and normal growth in MS medium devoid of PGRs within a week of culture. Dehydration under laminar airflow reduced the MC to 43.7% after 30min and 31.3% after 60min. without appreciable reduction in viability so that 76-77% of them germinated (Fig.1). Dehydration for 120min reduced MC to 19.6% and germination to 66.67% and was the optimum dehydration period (Fig.2) to get maximum germination (60%) after LN treatment (Fig.2). Root and shoot emergence was observed after one week of culture (Fig.1b) in 60% of the desiccated (120 min.) and LN treated embryonic axes and well developed seedlings were obtained within 20 days of culture (Fig.1c). Dehydration beyond 120 min. gradually reduced MC and drastically reduced viability. The MC came down to 12.1% after 210 min. when none of the embryos survived. Prolonged dehydration (150-180min) not only reduced survival down to 16.67-10% but also caused abnormal growth with only radicle development in the survived embryos (Fig.1d).

Research in the past two decades has shown that most orthodox seeds remain viable for long periods of storage after attaining appropriate desiccation levels of about 3-5% moisture content (Roberts, 1973). Contrary to this, recalcitrant seeds of several tropical and temperate species are desiccation sensitive, eg. Tea, Cocoa, Citrus, Jack fruit (Chin and Roberts, 1980). There are various options available to improve storage of non-orthodox seeds/embryos. Desiccation is the simplest procedure since it consists of dehydrating explants, and then freezing them rapidly by direct immersion in LN has been applied to embryonic axes extracted from recalcitrant and intermediate seeds (Engelmann, 1997). It should also be noted that selection of embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann *et al*., 1995). The conservation efforts of *N. nimmoniana* are hampered mainly due to relatively large and intermediate type of seeds with desiccation sensitivity. The viability of the embryos was not much affected when the embryos were desiccated from 55.7% to 43.7% (i.e. 30 min. desiccation). Significant loss of viability due to further reduction of moisture content shows the intermediate nature of the embryos is in line with the report of Dussert *et al* (1995). Safe moisture content of the embryonic axes as obtained in the present study is 19.6% (60% survival). Damage to plumule rather than radicle occurred due to excessive dehydration of *N. nimmoniana* embryos is as observed earlier in *Auracaria hunstenni* where desiccation damage is reported to be more serious in the plumule (Pritchard and Prendergast, 1986). The exact causes of embryonic death and its relationship with moisture content are not fully understood. Chin *et al* stated that seed death could be due either to the moisture content falling below a critical value or simply a general physiological deterioration with time. If embryonic axes have been desiccated to around 20% moisture content without loss of viability, it is possible that

Plant Cryopreservation 437

cooling and storage in LN will be progressed more easily. In most of the reports of successful cryopreservation, excised embryos or embryonic axes have been used for desiccation sensitive species, i.e. zygotic embryos of Citrus (Mumford and Grout, 1979) Oil palm (Grout *et al*., 1983) Coconut (Chin *et al*., 1989) Hevea (Normah *et al*.,1986) where the embryos withstand freezing after being subjected to partial desiccation. The desiccated embryonic axes do not lose viability after rapid cooling and storage at the temperature of LN. At such temperature there should be no change in the tissue either genetic or developmental, over a period of decades (Ashwood *et al*., 1977). This situation together with the ease to develop independent plants *in vitro* (Satheeshkumar and Seeni, 2000, Ravishankar Rai, 2002) from embryonic axes suggest cryopreservation is an effective technique for the long-term conservation of *N. nimmoniana,* a medicinal tree species

Ashwood Smith MJ and Grant E (1977). The freezing of mammalian embryos, at *CIBA* 

Bajaj YPS (1995). Cryopreservation of plant cell, tissue and organ culture for the

Benson EE (1999). Cryopreservation. In: Benson EE (ed) *Plant Conservation Biotechnology*,

Chabrillange N, Aberlenc-Bertossi F, Engelman F and Duval Y (1997). *Cryo-Letters* 18: 68-74. Chandel KPS, Chaudhury R, Radhamani J and Malik SK (1995). *Ann.Bot*., 76: 443-450.

Chin HF, Krishnapillay B and Standwood PC (1989). In: *Seed Moisture* (eds. P C Standwood and M B MC Donald). *Crop Science Society of America,* Madison, WI, USA, 15-22.

Chin HF and Pritchard HW (1988). Recalcitrant seeds, A Status Report. *IBPGR*, Rome. Chin HF and Roberts EH (1980). *Recalcitrant crop seeds*, Tropical press SDN, BHD. Cragg GM, Schepartz SA, Suffness M, Grever MR (1993). *J Nat. Prod*. 56:1657-1668.

De Boucaud M, Brison M, Ledoux C, Germain E and Lutz A (1991). *Cryo-Letters* 12:163-166 Decruse SW, Seeni S and Pushpangadan P (1999a) *Seed Sci and Technol.* 27: 501- 505

Engelmann FD, Dumet N, Chabrillange A, Abdelnour Esquivel B, Assy-Bah J Dereuddre

González-Benito ME, Clavero-Ramirez I, López-Aranda JM (2004). *Spanish J Agric Res* 2 (3):

and Duval Y (1995). *Plant Genetic Resources Newsletter* 103: 27-31.

conservation of germplasm and biodiversity. In: Bajaj YPS (ed) *Biotechnology in Agriculture and Forestry Cryopreservation of Plant Germplasm I*, New York, Springer-

*symposium*, (eds) Elliot K, Whelan J, Elsevier, Holland, 251-271.

Bekheet SA, Taha HS, Saker MM, Solliman ME (2007). *J Appl Sci Res.,* 3 (9): 859-866.

Chaudury R, Radhamani J and Chandel KPS (1991). *Cryo-Letters* 12: 31-6 Chin HF, Krishnapillay B and Hor YL (1989). *Pertanika* 12: 183-86

Decruse SW Seeni S and Pushpangadan P (1999b). *Cryo-Letters* 20: 243-250

producing large intermediate type of seeds.

Verlage, pp. 3-18.

Assy-Bah B and Engelman F (1992a). *Cryo-Letters* 13: 67-74 Assy-Bah B and Engelman F (1992b). *Cryo-Letters* 13: 117-26

Taylor and Francis, London, pp. 83-95. Berjack P and Dumet D (1996). *Cryo-Letters* 17: 99-104

Chang Y and Reed BM (2001. *HortSci* 36 (7): 1329-1333.

Decruse SW and Seeni S (2002b). *Cryo-Letters* 23:55-60

341-351.

Decruse SWand Seeni S (2002a). *Seed Sci and Technol* (In Press) Engelmann F (1997). *Plant Genetic Resources Newsletter* 112: 9-18. Engelmann F (2004). *In Vitro Cell Dev Biol Plant* 40: 427-433.

**3. References** 

Fig..1 a. Isolated embryonic axes, b. Cryopreserved embryo showing germination after 30 days of culture on MS basal medium, c. Seedling from Cryopreserved embryo after 60 days of culture on MS basal medium and d. radicle development and degeneration of plumule in embryo subjected to desiccation for 180 min.

Fig. 2. Effect of cryopreservation on germination of *N. nimmoniana* zygotic embryos. Different letter (s) in a data series shows significant difference at 5% level based on LSD multiple 't' test. \*Control and LN treated values differ significantly at 5% level based on Student 't' test. The bars represent SEM.

Fig..1 a. Isolated embryonic axes, b. Cryopreserved embryo showing germination after 30 days of culture on MS basal medium, c. Seedling from Cryopreserved embryo after 60 days of culture on MS basal medium and d. radicle development and degeneration of plumule in

c c

0 30 60 90 120 150 180 210 Duration of dehydration (min)

control LN m.c (%)

Fig. 2. Effect of cryopreservation on germination of *N. nimmoniana* zygotic embryos. Different letter (s) in a data series shows significant difference at 5% level based on LSD multiple 't' test. \*Control and LN treated values differ significantly at 5% level based on

b\*

a

e

d

0

10

20

30

Moisture content (%)

40

50

60

e

d

cd <sup>c</sup>

embryo subjected to desiccation for 180 min.

a

b b

b\*

d\*

d\*

Student 't' test. The bars represent SEM.

% Germination

cooling and storage in LN will be progressed more easily. In most of the reports of successful cryopreservation, excised embryos or embryonic axes have been used for desiccation sensitive species, i.e. zygotic embryos of Citrus (Mumford and Grout, 1979) Oil palm (Grout *et al*., 1983) Coconut (Chin *et al*., 1989) Hevea (Normah *et al*.,1986) where the embryos withstand freezing after being subjected to partial desiccation. The desiccated embryonic axes do not lose viability after rapid cooling and storage at the temperature of LN. At such temperature there should be no change in the tissue either genetic or developmental, over a period of decades (Ashwood *et al*., 1977). This situation together with the ease to develop independent plants *in vitro* (Satheeshkumar and Seeni, 2000, Ravishankar Rai, 2002) from embryonic axes suggest cryopreservation is an effective technique for the long-term conservation of *N. nimmoniana,* a medicinal tree species producing large intermediate type of seeds.

### **3. References**


**Part 6** 

**Equipment and Assays** 

Hsiang YH, Herzberg R, Hecht S and Liu LF (1985). *J Biol Chem* 260: 14873-14878.


**Part 6** 

**Equipment and Assays** 

438 Current Frontiers in Cryopreservation

Kaczmarczyk A, Shvachko N, Lupysheva Y, Hajirezaei MR, Keller ERJ (2008). *Plant Cell Rep*

Kartha KK, Engelmann F (1994). Cryopreservation and germplasm storage. In: Vasil, I.K.

Martin C, Iridono JM, Benito-Gonzales E, Perez C (1998). *Agro-Food-Ind Hi-Tech* 9 (1): 37-40.

Panis B, Lambardi M (2005). Status of cryopreservation technologies in plants (Crops and Forest trees).At: *The Role of Biotechnology*, Villa Gualino, Turin, Italy.

Pence VC (1995). In: Bajaj YPS (ed) *Cryopreservation of Plant Germplasm, Biotechnology in* 

Radha RK, Decruse SW, Seeni S (2006). Cryopreservation of embryonic axes of recalcitrant seed

Rajasekharan PE (2006). Prospects of new cryopreservation techniques for conservation

Ravikumar K, and Ved,DK (2000). Illustrated field guide of 100 Red listed medicinal plants

Sakai A (2000). Development of cryopreservation techniques. In: Engelmann F Takagi H

Stanwood PC (1985). In: Kartha KK ed. *Cryopreservation of plant cells and organs* (Boca Raton,

Scowcroft WR (1984). Genetic variability in tissue culture: Impact on germplasm conservation and utilization. *International Board for Plant Genetic Resources* Secretariat, Rome, p. 42.

Walters C, Volk GM, Towill LE, Forsline P (2009). Survival of cryogenically-stored dormant

Zhao MA, Dhital SP, Fang YL, Khu DM, Song YS, Park EJ, Kang CW and Lim HT (2005). *J* 

apple buds: a 20 year assessment. Paper presented at the *1st International Symposium on Cryopreservation in Horticultural Species, Leuven, Belgium,* 5-9 April.

Ravishankar Rai V (2002). *In Vitro Cellular & Developmental Biology. Plant* 38 (4): 347-351.

species *Myristica malabarica* Lam., a rare medicinal plant of the Southern Western Ghats. Presented in the *National Seminar on Plant Resources of the Western Ghats* organized by Karnataka Biodiversity Board at IISc. Bangalore on 7th and 8th December.

oftropical horticultural species. Paper presented at the *ICAR Short Course on In Vitro Conservation and Cryopreservation-New Options to Conserve Horticultural Genetic* 

of Conservation concern in Southern India. Foundation for Revitalization of Local

(eds) *Cryopreservation of tropical plant germplasm*. International of Plant Genetic

*Agriculture and Forestry*, Springer, Berlin Heidelberg, pp. 29-50.

Radha RK, Decruse SW, Krishnan PN (2010). *Ind. J. Biotechnology* 9: 435-437.

Hsiang YH, Herzberg R, Hecht S and Liu LF (1985). *J Biol Chem* 260: 14873-14878.

and Thorpe, T.A. (eds) *Plant Cell Tiss Cult* Springer 195-230.

Mumford PM, Grout BWW (1979). *Seed Science Tech* 7: 407-410. Murashige T and Skoog F (1962). *Physiol Plant* 15: 473-497. Normah MN, Chin HF and Hor LY (1986). *Pertanika* 9: 299-303.

Paunesca A (2009). *Romanian Biotech Letters* 14 (1): 4095-4104.

Pritchard HW, and Prendergast, FG (1986). *J. Exp. Bot* 37: 1388-1397

*Resources*, Banglore, India, 21-30 September.

Health traditions (FRLHT), Bangalore, pp 465.

Satheeshkumar K and Seeni S (2000). *Ind J Expt Biol* 38: 273-277.

Touchel DH and Dixon KW (1994). *Annals of Bot*. 74: 541-546

Touchel DH and Dixon KW (1993). *Biodiversity and Conservation* 2:594-602

27: 1551-1558.

Pence VC (1991). *Plant Cell Rep*. 10: 144-7

Reed BM (2001). *Cryo-Letters* 22:97-104 Roberts EH (1973). *Seed Sci Tech,* 1: 499-514

Resources Institute, Rome, pp. 1-7.

Florida: CRC press) pp 199-226 Stanwood PC (1987). *Crop Sci* 27: 327-331.

Westwood MN (1989). *Plant Breeding Rev* 7: 111-128.

*Plant Biotech* 7 (3):183-186

Pence VC (1991). *Seed Sci. and Technol.* 19: 235-251

**22** 

**X Ray Diffraction: An Approach to** 

**Preserved Tissues in Tissue Banks** 

*Instituto Nacional de Donación y Trasplante (INDT), Ministerio de Salud Pública -* 

The purpose of this chapter is to introduce new methods of analysis, to evaluate the final quality of human origin bio therapeutics products generated in Tissue Banks (TB), using well developed and known techniques in various fields of Physics, Chemistry and Biology

Cryopreservation techniques are fundamental supports in the conservation procedures of biological materials in TB work. However, controversial views remain on the effects at the molecular level that cryogenic temperatures and thawing could produce on the functional structures of tissues. The same concept can be sustained to glycerolized tissue preservation. Taking into account this scope, we implemented a methodological scheme to analyze tissue specimens before and after programmed cryopreservation, or glycerolization in order to find structural differences in the basic material constitutive collagen, using the techniques

It is noteworthy that both methods of analysis can be applied to any type of tissue preserved for the aforesaid purposes, with other conservation techniques, such as freeze drying or un

The TB are technical establishments whose main institutional objectives are collection, preservation, storage, release and distribution of biological tissues for therapeutic use in transplantation medicine. These objectives is met according to scientific criteria from agreed international protocols (Spanish Association of Tissue Banks: AEBT, International Atomic Energy Agency, IAEA, European Association of Tissue Banks: EATB; American Association of Tissue Banks: AATB) and according to the legal frameworks of the different countries and

*1 Instituto Nacional de Donación y Trasplante (INDT), Ministerio de Salud Pública - Fac. de Medicina, Uruguay* 

**2. The tissue banks and the "viability" of it's therapeutically products** 

Mc. Saldias1, G. Sanchez1, P. Martucci1, Mc. Acosta1, I. Alvarez1, R. Faccio2, L. Suescun2,

*2 Laboratorio de Cristalografía, Estado Sólido y Materiales (DETEMA) Fac. de Química, Uruguay*

as applied X – Ray diffraction (XRD), and Raman Scattering (RS).

formerly mentioned: diffractive and scattering.

**1. Introduction** 

programmed freezing.

M. Romero2 and A. Mombru2

 \* **Structural Quality of Biological** 

H. Perez Campos et al.\*

*Fac. de Medicina,* 

*Uruguay* 
