Chapter6

Germ plasm Preservation of Populus Through In Vitro Culture Systems1

Sung Ho Son, Young Goo Park, Young Woo Chun, and Richard B. Hall

Introduction

Demand for germplasm preservation of various plant species has recently increased due to air pollution, climate change, acid rain, natural destruction, illegal collection, and human impact to ecosystem biodiversity. Forested land in the world is rapidly declining at an annual rate of 9.9 mil­lion ha (Singh and Janz 1995). As a result, there has been a decrease in the genetic pool of forest tree species, includ­ing wild genotypes that could serve as future breeding sources.

Seed storage is common for germplasm preservation of most plant species. For Populus species, seed longevity under natural conditions is from 2 weeks to 1 month, de­pending on the species and/ or clone, time of collection, and environmental conditions (Graham et al. 1964; Trappe 1964). The longevity of seed storage can be greater than 2 years by drying the seeds soon after collection, adjusting seed moisture content to 8 percent, using sealed contain­ers in cold storage, or storing vacuum-packed seeds un­der freezing conditions (Trappe 1964). Even then, seed prppagation of selected tree genotypes may not guarantee the preservation of genetic traits such as fast growth and . Because of the vast land area requirement and the diffi­culty in controlling pests and/ or disease, in situ and ex situ conservation of vegetatively propagated plants is chal­lenging.

In this review we: 1) suggest an alternative approach for germplasm preservation through an in vitro culture

1 Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Re­search Station. 326 p.

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system; 2) discuss methods of germ plasm preservation; 3) discuss the ·currently available teclu,ology; and 4) suggest future strategies.

Poplars as a Model System for Germplasm Preservation In Vitro

The genus Populus includes about 30 species that are widely distributed throughout the North Temperate Zone (between the Arctic Circle and the Tropic of Cancer). This wide distribution represents considerable species' adaptabil­ity and demonstrates its potential over a wide geographic region. Among the woody plants, poplar is one of the most intensively studied species for breeding because of the enor­mous genetic variation that exists. Thousands of poplar clones are being tested and hundreds of these are commer­cially propagated throughout the world (Sa to 1959). Populus spp. are fast growers and have modest nutritional require­ments, therefore, they are an economically important source of pulp for paper industries and are considered an energy crop. Also, cryopreservation of cells/tissues requires rela­tively little space because regeneration of Populus plantlets from single-cell/protoplast cultures is possible. Commercial production of selected individuals does not depend on seed reproduction because vegetative macro and micro­propagation methods without adulteration or dilution of their genetic potential have been intensively studied (Ahuja 1987; Park and Son 1988). Problems controlling open polli­nation under natural conditions makes conservation of taxo­nomically diverse and newly created clones by in situ or ex situ preservation programs for poplar germplasm difficult.

Development of reliable micropropagation systems is essential for germ plasm preservation in vitro. Many reports of in vitro propagation technology of poplars have further developed this species as a model system for germplasm preservation (Aitken-Christie and Singh 1987; Ahuja 1987; Moon et al. 1987; Park and Son 1988; Son et al. 1991; Stoehr

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and Zsuffa 1990). In many clones with a high regeneration capacity in a microenvironment, plantlets from shoot-tip cultures were used in large-scale production and their pro­ductivity was found superior to conventionally propagated plants (Chen and Huang 1980; Lester and Berbee 1977; Mehra and Cheema 1980). Most tissues and organs can serve as initial explant materials for in vitro culture of Populus spp. Germplasm preservation through in vitro culture is ad­vantageous because: 1) entire sets of genetic materials can be copied through regeneration schemes; 2) rapid prolifera­tion is achievable; 3) minimal space is required; 4) disease­free plants can be produced and maintained; and 5) risks related to environmental changes are avoided.

Low-Temperature Storage

Low temperature (about 4 °C) has been widely applied for short-term, minimal-growth storage of cultured plant cells, tissues, and organs. To prepare for nursery planting during the optimal growing season, low-temperature stor­age was used successfully for large-scale synchronization of transplanting propagules (Son et al. 1991 ). If suitable methods are developed, valuable genotypes can be selected at the in vitro level.

For low-temperature storage in vitro, many techniques can be applied individually or in combination. For ex­ample, methods related to maintenance of plant materials under conditions of a mineral oil overlay (Caplin 1959), low-level illumination (Preil and Hoffmann 1985), com­plete darkness (Marino et al. 1985), reduced temperature (Bhojwani 1981 ), various osmotica or plant growth regu­lators (Henshaw et al. 1978), modified culture atmosphere (Moon and Kim 1987), and modified subculturing sched­ules (Withers 1985) have been described.

Although nonfrozen storage systems for germplasm preservation have been reported, such studies focus on herbaceous and/ or crop species and may not apply di­rectly to woody plants. During low-temperature poplar storage, the duration, previous subculturing, and various culture media affect the survival rate (Son et al. 1991 ). With hybrid aspen (Populus alba x P. grandidentata), the previous subculturing period and medium composition are critical variables for the success of germplasm preservation un­der low-temperature storage (Son et al. 1991). In our ex­periment, after 9-months of storage at low temperature, hybrid aspen shoots exhibited slow growth, yet contin­ued to possess some healthy, green leaves and stems (fig­ure 1b). After 20-months of cold storage, shoot cultures had nearly stopped incremental height growth and had thin stems and small, chlorotic leaves. According to one report, explant size can greatly influence survival during

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

Germplasm Preservation of Populus Through In Vitro Culture SY.stems

cold storage (Aitken-Christie and Singh 1987). In our ex­periment, no significant difference in cold-storage survival was observed among the five shoot sizes {1, 3, 5, 7, and 9 em) tested; although 3 em shoots did exhibit a slightly higher survival rate. Under low-temperature storage, sur­vival rates after 2-years and 5-years cold storage were 75 percent and 25 percent, respectively. From 2,000 plantlets transplanted in the greenhouse and I or nursery, less than 1 percent displayed phenotypic variation related to growth performance or pigment accumulation.

Cryopreservation

Cryopreserved plant cells, tissues, and organs stopped their metabolic functions when exposed to ultra-low tem­perature ( -196 °C); yet they retained viability. In this con­dition, plant cells, tissues, and organs are preserved for a long period of time. Cryopreservation systems can reduce the cost of conventional labor-intensive nursery practices and minimize genetic changes, such as point mutation and I or ploidy level change, which may occur during long­term subculture. However, developing a reliable cryopreservation system involves several complicated steps. In many examples of cryopreservation, only a few species were successfully preserved (Bajaj 1977; Kartha et al. 1979; Kartha et al. 1980; Uemura and Sakai 1980). In other examples, further investigations are needed to opti­mize treatments at general stages of pregrowth, cryoprotection, freezing, thawing, and recovery (Chen and Kartha 1987). Preconditioning, such as cold treatment dur­ing culture or applying osmotically active chemicals to the medium before cryopreservation, was beneficial for viabii­ity (Fuchigami et al. 1981). Plant samples require sufficient desiccation before cooling in liquid nitrogen. Without such care, most samples produce intracellular ice crystals that eventually cause cell mortality.

When poplar callus was used for ultra low-temperature storage, optimal results were obtained with the cells from the end of lag phase or exponential growth phase, which occurs approximately 1 week after subculture (data is not shown). This may relate to lower water content and smaller vacuole size of optimal source cells. Pretreatment with cryoprotective chemicals, dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone (PVP), and dextran were useful separately or in combination. Although its mode of action is not clearly understood, DMSO may form mul­tiple hydrogen bonds that prevent water from crystalliz­ing at intracellular levels. Although cryoprotective chemicals were harmful to plant cells in our experiment, DMSO treatments produced 62 percent survival after long­term preservation; until now, calli were stored for 5 years and survival tests were conducted annually.

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Section l .fn Vitro Culture

Protocols

Species used for low-tempera ture s torage and cryopreservation were Populus alba x P. grandide11 tata and P. glandulosa (Suwon poplar), respectively. Protocols de­scribed here are based on preliminary experiments; sup­porting data are not included.

Low-Temperature Storage

a) Shoot cultures were established by bud cultures from greenhouse-grown stock plants. Shoot mul­tip lica tion, elonga tion, and root induction med ia were Murashige and Skoog (MS) (Murashige and Skoog 1962) med ium containing 1.33 ~1M 6-benzylad enine (BA), no p lant growth regu lators, and 0.98 JIM indole-3-butyric acid (IBA), respec­tively. Cultures were mainta ined under a 16-h light regime (photosynthetic photon flux rate of 40 to 60 1-1M m·2 s·1 from cool-white, fluorescent tubes) at 26±1 °C.

b) Shoots approximately 3 em long were used (figure 1a).

c) Shoots were maintained on shoot multiplication me­dium for 1 month before cold s torage.

d) Magenta GA-7 culture vessels (7.6 x 7.6 x 10.2 em, Magenta Corp., Chicago, IL) containing 50 ml me­dium and test tubes containing 15 ml of medium were used for cold s torage.

c) To prevent drying, culture vessels were double sealed with esco film (Banda Chemical Ind., Ltd., Kobe, japan) and s tored within two transpa rent plas­tic bags.

f) During cold storage, cultures were maintained at 4 °C without light.

g) After 4-weeks subculture of cold-stored plants on fresh MS medium, each shoot was excised from its root system and transferred to a Poly terra 1~ p lug styrofoam tray (M40045, Techniculture Co., Sa linas, CA) and eventually to pots containing artificial soil mix (vermiculite: perlite : peat at 1:1:1) (figure lf).

Cryopreservation

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a) Callus was induced from cambial tissues isola ted from i11 vitro-, greenhouse-, and field-grown d onor plants.

Figure 1. Cold storage of in vitro shoot cultures of hybrid poplar (Populus alba x P grandidentata). a) Shoot cultures before storage. b) Shoot cultures after 9-months storage at 4 oc. c) Multiplied shoot cultures on shoot proliferation medium after 5-years storage. d) Shoot regeneration from the cold-stored materials. e) Regenerated plantlets derived from cold-stored shoots. f) Plants estab­lished in pots containing artificial soil mix.

b) Call us proliferation was cond ucted on MS medium with 2.26 p M 2,4-dichlorophenoxyacetic acid (2,4-D).

c) Cold hardening was performed at 10 oc for 1 week before placing the ca llus into cryogenic plastic ves­sels and adding DMSO (callus: DMSO at 1:5 weight/ volume) as a cryoprotectant solution.

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

c

d) Further cooling was initiated by incubating the cal­lus on ice for 1 to 2 h .

e) A freezing temperature ranging from -25 to -45 oc was obtained using a programmable freezer control­ler to drop the cooling temperature by 0.5 to 1 oc per minute, followed by liquid nitrogen immersion.

f) For regrowth of stored callus, temperature was in­creased in reverse of the freezing procedures but quicker. Before culturing, calli were rinsed several times with MS liquid medium containing no plant growth regulators.

g) After subculturing on MS basal medium supple­mented with auxin at the appropriate levels, newly grown callus was collected for use as shoot induc­tion material.

h) For regeneration, selected callus was subdivided into small pieces (5 to 10 mm in diameter) and cultured on a shoot induction medium supplemented with optimal levels of zeatin (figure 2c).

b

Germ plasm Preservation of Populus Through In Vitro Culture Systems

Limitations

Plant cell and tissue culture techniques have developed rapidly and the knowledge associated with culturing sys­tems has increased significantly in comparison to other plant science fields. Based on the theory of totipotency, any cells, tissues, and organs should be regenerable; however, successful establishment of reliable regeneration systems is limited, especially for woody plants. This restriction fre­quently confines the application of tissue culture methods and restricts plant biotechnological applications for many valuable species.

A basic premise of i11 vitro conservation techniques for plant germplasm is that genetic materials must be pre­served without genetic alteration at any level. Most tissue cu lture work is based on adventitious shoot regeneration systems. These systems a re typically associated with a pro­longed undifferentiated phase before regeneration. How­ever, such culture systems have generated phenotypic, genotypic, and I or biochemical variation (Bebeli and Kaltsikes 1990; D' Amato 1978; Whelan 1990). Although this variation frequently occurs at negligible levels, ' the poten­

tial fo r genetic variation should be addressed.

There is also potential for genetic alteration associated with low and ultra-low temperature s torage meth­ods. In a nonfrozen storage system, for example, phenotypic variation was reported for trai ts such as color accumulation in leaves (Son et al. 1991 ), organ shape, seed yield, and photoperiodic response (Grout 1990). During cryopreservation at -196 oc, most cellular chemical reactions can­not occur because the energy levels are too low to allow sufficient mo­lecu lar motion to complete enzy­matic reactions. Nevertheless, certain chemical reactions that affect genetic alteration by damaging nucleic acids via ionizing radiation can accrue at unacceptable levels. Although this kind of chromosome damage is ob­served in plants and microorganisms under storage conditions, OJ A re­pair mechanisms to overcome dam­age from freezing or low temperature conditions are not fu lly understood.

Figure 2. Cryopreservation of in vitro shoot cultures of poplar (Populus glandulosa). a) Cryopreserved callus recently subcultured to proliferation medium. b) Regrowth of selected callus. c) Shoot regeneration from callus. d) Plants established in pots containing artificial soil mix.

Most storage systems have not pro­vided 100 percent survival and are not adaptable for diverse species. In­tensive studies are needed to develop

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Section I In Vitro Culture

methodology and accumulate relevant knowledge for suc­cessful germplasm storage of target plant materials.

Conclusions and Prospects

Due to rapid environmental destruction and associated declines in biological diversity, genetic resource conser­vation has become critical. Recently, major approaches to genetic resource conservation such as ex situ and in situ preservation programs were established for forestry. Al­though these approaches have merit, a successful genetic conservation program will require modification and/ or a combination of existing methods with new technology to meet current needs.

Advances in plant tissue culture systems are widely applicable throughout plant science. This technology is also a valuable tool for tree germplasm conservation through low- or ultra-low temperature storage. Recent progress in somatic embryogenesis may provide other opportunities for storage material. In particular, somatic embryos exhibit high genetic stability and are easily manipulated. Above all, relatively simple desiccation methods that reflect the physiology and development of zygotic embryos offer additional potential for extending the longevity of germplasm storage in vitro. ·

Because poplar has demonstrated totipotency at diverse cell levels, it is a model species for mass clonal propagation, genetic transformation, and other biological research of woody plants. For the conservation and preservation of tree germplasm, intensive studies are needed to refine associ­ated methods and to provide reliable and reproducible results.

Acknowledgments

The authors are grateful to Dr. Gebhart, a senior re­searcher at Institute of Rapid Growth Tree Species, HannMuunden, Germany, for helpful advice and contri­butions to cryopreservation studies.

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