Applications of genetic transformation to tree biotechnology …nopr.niscair.res.in/bitstream/123456789/19096/1/IJEB 37… ·  · 2016-07-20Applications of genetic transformation

Indian Journal of Experimental Biology Vol. 37, July 1999, pp. 627-638

Review Paper

Applications of genetic transformation to tree biotechnology

Paramjit Khurana & Jigyasa Khurana

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India

The developments in the field of biotechnology have advanced to a stage where its benefits can be apparent in the field of forestry. The ability to manipulate forest tree species at the cellular and molecular levels holds promise for circumventing inherent limitations in tree improvement programs, e.g., long generation cycles, space for large segregating populations, and the lack of genetically pure lines. Genetic engineering methods complement plant breeding efforts by increasing the diversity of genes and gerrnplasm available for incorporation into desirable plant species, and shortening the time period required for the production of new varieties and hybrids. Future developments in the area!\ of molecular markers and efficient regeneration methods would help in ameliorating the dilemmas of several existing silvicultural practices. The . present article focusses on the first generation applications of genetic engineering of trees, i.e., pest resistance, disease resistance and herbicide tolerance, and discusses the significant progress made in the fields of lignin modification and other traits currently being prospected for genetic manipulation.

The present day global awareness has led to the realization of the unique contribution of trees to the well-being of this planet. Parallel research and developmental efforts are therefore being undertaken to increase the forest area worldwide and to replace plantations being destroyed by industrialization and urbanization. Trees have thus become increasingly important to restore the lost forest stands and to meet the escalating needs of the world economy.

Tree improvement involves not only managing genetic resources but also includes conservation, selection, breeding and propagation of select genotypes'. Traditional breeding methods involve sets of genes being introduced through sexual hybridization and is restricted by problems of sexual incompatibility manifested at the interspecific and intergeneric level, and related problems like sferility and apomixis. In addition, these methods have proved to be extremely time consuming due to the long generation cycles of trees. Recent developments in molecular biology have, however, opened new vistas for broadening gene pools and have provided impetus to various crop improvement programs. Genetic engineering methods thus complement traditional breeding methods and hasten the process of introduction of the desirable traits into \cultivars/ varieties of commercial interest.

Success with genetic engineering is not easy and is

*Corresponding author: Fax: 91-11 -6885270, 91-11-6886427 E-mail: [email protected]

often limited by the n<?n-availability of suitable regenerating systems. Another major prerequisite for the application of recombinant DNA technology to tree improvement is the development of gene transfer systems. In addition to Agrobacterium-mediated transfer, the other promising delivery systems available are through electroporation and microprojectile bombardment. Also, the ultimate regeneration of transgenic plants from transformed cells, continues to remain a major challenge.

The intensive research in the field of crop biotechnology has enabled the identification and isolation of a number of genes responsible for varied characters. Based on these identified traits, tree biotechnologists are concentrating on genetic manipulation of important . tree species. Some of the target traits which are currently being prospected for genetic engineering are:

Category

I. Hybrid production

2. Plant growth

3. Altering inputs

4. Products

Target Character

Self incompatibility Male sterility Structure and architecture (height, branching, leaves, roots) Flowers (structure, color, timing) Herbicide resistance Improved nutrient uptake Improved photosynthetic efficiency Sugar and starch (composition and content) Oils (composition and content) Storage proteins (composition and content)

628 INDIAN J EXP BIOL, JULY 1999

5. Environment

Flavors and fragrances Pharmaceuticals Fibres (textiles) Fruit (ripening and quality) Biotic factors (pest, bacteria, viral and fungal resistance) Abiotic factors (drought, salinity, temperature, heavy metal tolerance)

Cell, Tissue and Protoplast Culture

Owing to rapid deforestation, depletion of genetic stocks and escalating product demand, mass propagation and production of short duration trees with rapid turnover of biomass is the need of the hour. It is therefore, envisaged that in comparison to the traditional time consuming breeding programs, tissue culture techniques are competent to meet the demand for increased biomass production.

During the last two decades, dramatic progress has been made in developing and refining various tissue culture techniques. As a result, methods are now available to culture and regenerate plants from somatic cells, pollen, and protoplasts of a large number of plant species. Considerable progress has been made since plantlets were obtained via organogenesis from PopuLus tremuLoidei. It is now possible to regenerate successfully many of the woody species in vitro which were initially considered to be recalcitrant3.4. There are a number of advantages of micropropagation of trees over sexual propagation. Some of these are:

1. Cloning superior trees, such as hybrids or selected specimens from field populations.

2. Due to the long breeding phase of trees, improvement of planting stock by sexual means is slow, whereas with cloning it is immediate.

3. Quite often the juvenile phase of development can be bypassed.

4. The genetic uniformity of a clone is generally an asset.

5. Elite trees, such as hybrids and polyploids, can be propagated vegetatively.

The availability of protoplast-to-plant technology for various tree species has initiated the exploitation of this technology for various aspects of ttee biology. Biotechnological procedures with direct application to tree improvement include somatic hybridization and in vitro selection using both somaclonal and gametoclonal variation. Somatic hybridization is a prime objective for protoplast manipulation and

certain intergeneric and interspecific protoplast fusions have been performed in which hybrid callus has been produced. Somatic hybrids have been produced by the fusion of protoplasts of sexually incompatible rootstock genotypes of Pyrus communis var. pyraster and Prunus avium x pseudocerasus5

.

The somatic status of the regenerated plantlets was confirmed through chromosome counts, isozyme assessments, and morphological markers.

The existence of an inverse relationship between cell wall regeneration and tolerance to salinity has been demonstrated6

• Salt and drought tolerant plandets have been obtained from colt cherry protoplasts, Prullus avium x pseudocerasus, as a result of protoclonal variation and recurrent in vitro selection strategies? The exploitation of protoplast technology has thus opened up the possibility of creating agronomically useful genetic novelties. Plant regeneration from protoplasts can be a source of spontaneous somaclonal variatIOn for certain genotypes and the same is true for in vitro selection based on protoplasts. By placing particular emphasis on selection of monogenic and allelogenic traits such as disease resistance and herbicide tolerance, extremely desirable phenotypes can be obtained.

Homozygous plants obtained by somatic hybridization (homofusion) of protoplasts of haploid genotypes will prove helpful in the study of inheritance of horticultural traits. Heterofusions between different haploid clones might serve to create genetic novelties of great value for rootstock breeding and might be of interest as novel scion cultivars. Finally, protoplasts are an ideal system for the production of novel transgenic trees and genetic transformation of protoplasts is the ultimate goal of various tree breeders.

Genetic Engineering and Tree Biotechnology

Genetic engineering methods complement plant breeding efforts by not only increasing the diversity of genes and germplasm available for incorporation into crops but also by shortening the time period required for the production of new varieties and

·hybrids. The ability to manipulate forest tree species at the cellular and molecular level also holds promise for ~i,rcumventing inhere?t limitations in tree improiement programs, e.g., long generation cycles, space for large segregating populations, and the lack of genetically pure lines.

KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY 629

Since the production of the first transgenic plants of tobacc08

, the use of genetic engineering methods has increased by leaps and bounds. Although a number of transgenic crop species have been produced, success with tree species has been limited primarily due to lack of efficient in vitro regeneration protocols and suitable gene transfer mechanisms. However, with the growing focus on regeneration and transformation of tree species, it is likely that within few years, the economically important tree species will be amenable to genetic manipulation. !

Methods for Foreign Gene Introduction

The Natural Gene Vector-Agrobacterium Recognition of the ability of the soil bacterium

Agrobacterium tumefaciens to transfer a portion of its DNA to plants was perhaps the most important milestone in plant biotechnolog/,IO. Major advances contributing to the popularity of Agrobacterium­based transformation systems include the development of disarmed strains where the oncogenes which result in tumorigenesis are deleted from the plasmid; the development of binary vectors, in which the T -DNA borders are located on a wide host range plasmid and the virulence genes of the Ti plasmid are located on an independent plasmid and act in trans; were crucial in expanding the use of Agrobacterium­based vectors in plant transformation studies.

The ability to transform tissues and to regenerate transgenic woody trees successfully using the natural gene transfer system of Agrobacterium is dependent primarily on three factors:

1. Virulence of Agrobacterium cells, 2. Efficiency of selection that would allow

growth of transformed cells, and 3. Frequency of regeneration among the

transformed cell population.

Besides the above, quite a number of other biological and physical parameters influence the transformation efficiency of Agrobacterium, the foremost being the susceptibility of the host to infection by Agrobacterium. The host range of Agrobacterium is quite extensive. Populus is known as a natural host for A. tumefaciens ll and various species of Populus have been investigated for their susceptibility to Agrobacterium. A wide variety of trees have also been shown to be transformed by various wild type strains of A. tumefaciens -and A. rhizogenes I2

.15

• Of the several recombinant strains of

A. tumefaciens that have been evaluated for their ability to transform different tree species, LBA 4404 has proved to be most successfuI12.16.19. Besides Agrobacterium cell densitlo.21 and the period of co­cultivation22.27, the transfer of T-DNA is significantly influenced by the plant phenolics secreted in response to wounding and high levels of acetosyringone have resulted in increased foreign gene expression l5 .

Trees often show unusual sensitivity to kanamycin and low concentrations of 25 mgIL have proved to be . sufficient for selection, although a high of 100 mgIL in Malus has also been reported27. Preculturing of explants before co-cultivation, so as to enhance the susceptibility of cells to infection and T-DNA transfer, has also been reportedI5.28.3o. Preculturing of Malus for 1-8 days significantly increased the transient expression of the gus marker gene, although it was found to be detrimental for stable gene expression and subsequent regeneration29

.

Electroporation

Electroporation of protoplasts could be an efficient means of introducing DNA into plant cells, especially in those species not susceptible to Agrobacterium infection. However, for electroporation to be successful, one of the major requirements is a functional protoplast-to-plant regeneration system. With the few exceptions that have been published so far this approach is certainly not easy. Some of the major problems with transformation of protoplasts and subsequent plant regeneration are the possibilities of somaclonal variation among the regenerants, the presence of multiple copies of inserted DNA, rearrangements, instability of inserted DNA, etc . It is thus not surprising that although transient gene expression via electroporation has been reported in Alnus incana31

, Eucalyptus citriodora32 and E. gunnii33

, no report of stable transformation via this technique has been published so far.

Particle bombardment

Particle bombardment, which uses explosive, high speed acceleration to deliver biologically active DNA into a large range of target tissues and cells has become the second most widely used technique in plant genetic engineering. This potential to transfer foreign DNA in regenerable cells, tissues or organs provides the best method for genotype-independent transformation, surpassing Agrobacterium host specificity and tissue culture related regeneration

/

630 INDIAN J EXP BIOL, JULY 1999

difficulties. The first report on the production of a transgenic Allium plant using high velocity microprojectiles34

, stimulated the use of this versatile technique for the production of transgenics from recalcitrant tree species. It took three years before the first transgenic trees of a hybrid poplar, Populus alba x P. grandidentata35 and Carica papaya36 could be produced. Since then transgenics have been produced in Liriodendron tulipijera37 and Citrus reticulata x C. paradisPs, and transient expression has been reported in Elaeis guineensii9

, Hevea brasiliensis40 and Ulmus procera41 and many others.

Like any other transformation method aimed at obtaining high frequency stable transformation, particle bombardment also demands the optimization of DNA delivery conditions. Various biological (type of target tissue, osmotic treatment) and physical (target distance, rupture disc pressure, type of microparticles, DNA and relative particle concentration, chamber vaccum) parameters · of bombardrpent, availability of suitable promoters, select~ble markers and a sensitive selection agent significantly influence gene transfer. Embryos and embryogenic cell lines are the most preferred target tissues. The enrichment of the bombardment medium with sucrose considerably increases the transfor­mation efficiency of zygotic embryos42. The gun powder system has been reported to be as reliable as the helium device for DNA transfer into zygotic embryos of Eucalyptus globulus, and a relatively higher efficiency of the gold particles as compared to the tungsten projectiles is also reported42.

The first transformation of a tree species (Carica papaya) via particle bombardment with an agronomically useful gene (prv cp - coat protein gene of papaya ringspot virus) resulted in virus resistant plants36

• Herbicide tolerant papaya transgenic plants have also been produced following the delivery of bar gene, which confers resistance to phosphinothricin43. The BT gene confering insect resistance has also been introduced in Populus alba x P.grandidentata35

.

In addition to producing agronomically useful transgenic trees, particle bombardment has also been used for studying gene expression and regulation, and identification of plant promoters to ensure strong and constitutive expression of the foreign gene. Transformation of Coffea arabica by the ~-glucuronidase gene using different promoter sequences resulted in significant variations44

• EFla.. At promoter of Arabidposis thaliana was found to be

most effective. In a similar study, on the bombarded embryogenic callus of ELaeis guineensis, Emu promoter was found to be most efficient as compared tt four other promoters39

.

#~tications to Tree Improvement For the application of genetic engineering methods

to tree improvement, initial research had focused on engineering of traits that relate directly to the traditional roles of industry in farming, such as pest resistance, disease resistance and herbicide tolerance (also see, Tzfira et al.45

). Progress is now being observed in the areas of drought and cold tolerance, altering lignin composition, improving fruit quality, reducing juvenility phases and alteration of tree form and architecture.

Herbicide tolerance

Engineering herbicide tolerance into crop plants represents a new alternative for conferring selectivity and enhancing crop safety. Work has been largely concentrated on herbicides with properties of high unit activity, low toxicity, low soil mobility, rapid biodegradability and with a broad spectrum activity against various weeds. The development of crop plants that are tolerant to such herbicides would provide more effective, less costly and environment friendly weed control.

Two general approaches have been employed in engineering herbicide tolerance:

1. Altering the level and sensitivity of the target enzyme for the herbicide, and

2. Incorporating a gene that will detoxify the herbicide.

An example of the first approach is glyphosate, the active ingredient of "Roundup" herbicide, which acts by' specifically inhibiting the enzyme 5-enolpyruvyl­shikimate-3-phosphate synthase (EPSP). Some level of tolerance has been engineered into Populus (P.

alba x P. grandidentata) by the introduction of the mutant aroA gene, which encodes for EPSP less sensitive to glyphosate by Fillati et al.21

• Transgenic trees of hybrid aspen (Populus alba x P. tremula) and poplar (Populus trichocarpa x P. deltoides), resistant to phosphinothricin, have been produced by Agrobacterium-mediated transfer of the bar gene46. Employing particle bombardment, herbicide (phosphinothricin) resistant plants of Carica papaya

KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY 631

have also been produced, which were able to withstand 3-5 times higher concentration of pJ>"r3.

Insect resistance Trees are susceptible to many insect pests and

breeding for resistance is a difficult task in long lived trees. The production of insect resistant plants is another application of genetic engineering with important implications for crop improvement. In plants, resistance to insect pests has been achieved by employing two types of gene products:

1. Protease inhibitors, and

2. Insecticidal crystalline proteins (lCP from Bacillus thuringiensis).

Protease inhibitors, first characterized by Ryan47,

are synthesized in a variety of plants in response to insect predation and have been found to be toxic to diverse groups of insect species. Many plant species possess the genes coding for these protease inhibitors. The promoter for one of these genes, pin2, is inducible. Klopfenstein et a1.48

, introduced the chloroamphenicol acetyl transferase (cat) gene fused to the pin2 promoter into hybrid poplar using Agrobacterium. In another instance, the introduction of a gene encoding the cysteine protenase inhibitor, OCI, from rice into a poplar hybrid clone (Populus tremula x P. tremuloides) has resulted in tolerance

49 I towards Chrysomela tremulae, a coleoptera . nsects feeding on these transgenic poplars exhibit reduced growth, altered development and increased mortality.

Progress in engineering insect resistance in transgenic trees has also been achieved through the use of insect toxin protein genes of Bacillus thuringiensis (BT). B. thuringiensis is an entomocidal bacterium that produces a crystalline protein which is lethal to select pests. Most strains of Bacillus thuringiensis are toxic to lepidopteran (moth and butterfly) larvae, although some strains with toxicity to coleopteran (beetle) or dipteran (fly) larvae have been described. The insect toxicity of B. thurillgiensis resides in a large protein and has no toxicity to beneficial insects, other animals or humans. The mode of action of the BT crystalline protein is thought to be exerted at the level of disruption of ion transport across brush border membranes of susceptible insects.

A lepidopteran specific BT gene has been introduced into hybrid pioneer elm employing particle gun and Agrobacterium-mediated gene transfer

methods50. Transgenic poplars (Populus alba x P.

grandidentata) exhibiting insect resistance via BT gene expression have been produced35

. The transgenic poplars obtained were highly resistant to feeding of lepidopteran insects. Transformed sweetgum (Liquidambar styraciflua) was generated which contained a chimeric BT toxic gene and conferred resistance to the two known defoliators of sweetgum,

?'i . i.e., fall web worm and the gypsy moth--. Transgenic poplar (Populus tremuLa x P. tremuLoides) expressing B. thuringiensis endotoxin cry ilIA, known to be

d49 active against coleopteran has also been produce _ Insects feeding on this transgenic poplar exhibited increased survival mortality.

Viral disease resistance

The family of potyviruses, the largest and most widely distributed group of plant viruses, is known for its ability to severely damage many important crop species51

• This is also the case for Plum Pox Virus (PPV) associated with plums, apricots and peaches. Originally described in Bulgaria, the Sharka disease has spread over great parts of Central and Southern Europe as well as over many Mediterranean countries. In crop plants, disease resistance has been approached through viral cross protection using chimeric genes specifying viral coat protein as a way of protecting plants from viral diseases52

. This approach has been successfully used against a number of different viruses, for example, Tobacco Mosaic i

Virus (TMV), Alfalfa Mosaic Virus (AMV), etc. This approach has resulted in the successful production of

. 5l de' 36.54 transgenic Prunus armemaca- - an arlca papaya resistant to attacks of Plum Pox virus and Papaya Ringspot virus, respecti vel y.

Fungal disease resistance

A number of tree species serve as useful hosts for the fungal pathogens. Although no major progress has been made in the genetic engineering against fungal diseases in trees, efforts are underway to characterize the fungal disease resistance genes. Chitinase genes from hybrid poplars, win6 and Will8, have been characterized20

• Two major fungal di~eases requiring immediate attention are: Chestnut blight and Dutch elm disease. These diseases have devastated both natural populations and planted specimens of chestnuts and elms.

A novel method to control chestnut blight caused by Cryphonectria parasitica has been described by

/' ;'

632 INDIAN J EXP BIOL, JULY 1999

Choi and NUSS55

. The introduction of a cloned dsRNA genome from a less virulent strain into virulent strains converts the virulent strain into a less virulent strain. Symptoms of the disease were significantly reduced by the introduction of the genetically altered strains into chestnut trees. The effect of the cloned dsRNA genes in making other fungal pathogens less virulent is under investigation.

Stress tolerance

When plants are subjected to stress, biotic or abiotic, an increased production of potentially dangerous active oxygen species is favoured56

.

Photochemical oxidants, including ozone, are important sources of stress for trees57

• Ozone toxicity has been implicated in decreasing tree growth58

,

increasing tree mortalitl9 and causing intraspecific and interspecific changes in forest stands. Ozone usually overwhelms the plant's antioxidant defense mechanism, resulting in the degradation of chlorophyll and the destruction of cell membranes60

.

Collectively, this results in reduced photosynthesis, premature foliar aging and increased susceptibility to other stress and/or pests. There is strong evidence for the protective role of various antioxidants such as superoxide dismutases. Superoxide dismutases have been implicated in ozone tolerance in plants, including hybrid poplars61

•62

• Another antioxidant, glutathione, may also have a role in scavenging superoxide radicals. In an attempt to produce stress tolerant transgenic poplars, Foyer et al. introduced genes for glutathione reductase (got)63. Over­expression of glutathione reductase (GR) in the chloroplast results in increased antioxidant capacity of leaves and thereby improves the capacity to withstand oxidative stress.

Efforts to improve growth and productivity of forest trees under stress conditions could benefit from an understanding of the expression of specific drought induced proteins which may contribute to drought tolerance. Evidence for the accumulation of a 66 kDa water stress responsive protein (BspA) in shoots of Populus tremula has been presented64

•

Furthermore, a good correlation between the expression of dehydrin like proteins (DSP 16) and sucrose synthase, and the degree of tolerance and ion leakage in Populus clones was found. The N­terminal amino acid sequence of BspA has been determined and it exhibited high homology to wheat germins GF-2.8 and GF_3.865

• Further analysis of the

expression of these proteins and their mode of action will allow a better understanding of their role in drought tolerance.

Lignin modification

Lignins are complex cell wall phenolic heteropolymers which represent the second most abundant organic compound on earth after cellulose. In general, for tree species, lignin makes up about 15-35% of the dry wood weight. During paper pulping, lignin must be eliminated from wood. The engineering of lignin biosynthesis appears to be a promising aspect for improving trees used in the paper/pulp industry. Genetically reducing the quantity or altering the quality of lignin in pulpwood species could improve the efficiency of these pulping procedures. Sense and antisense expression of sequences encoding CAD (cinamyl alcohol dehydrogenase) and COMT (methyl transferases) enzymes involved in lignin biosynthesis have resulted in the production of altered lignin in poplar66

•67

, the most important source of pulp. Lignin is altered both in composition and statistics, although the content is similar to that in the control. Contrasting results with two enzymes in terms of feasibility of delignification have been reported. In the case of down regulated COMT transgenics there is a decrease in S/G ratio (syringyllguaiacyl) and a consequent difficulty in chemical extraction66

• Transformed lines with a down regulated CAD activity show an increased accumulation of cinnamaldehydes leading to improved pulp properties and easier delignification67

,

Coexpression of COMT alongwith ferulate-5-hydro­xylase, such as was cloned from Arabidopsis, will lead to the conversion of softwood lignin to the hardword type68

•69

.

Another enzyme of prime importance involved in the last step of lignin biosynthesis. is the oxidative polymerization by peroxidases, Poplars have a number of different peroxidase isozymes whose patterns of expression are tissue specific, developmentally regulated and influenced by environmental factors7o

. Kajita et al. altered the expression of a peroxidase isozyme by introducing a genomic clone for peroxidase (prxA 1) under the control of the CaMV 35S promoter in a hybrid poplar, Populus kitakamiensis 17

, Transgenic poplars obtained by introducing the chimeric peroxidase gene have been shown to have an increase in total peroxidase activity that has been accounted for by the .

KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY 633

Table I- Foreign gene expression in tissue or cells of angiosperrnous trees-Coll/d

Species Target Ce1Vfissue Mode of gene Foreign geneh TGE" SGEd Reference transfe~

Uquidambar styraeijlua Leaf A.t. anionic peroxidase + 25

Uriodentir(m tulipifera Embryogenic suspension Pb gus, nptl/ + 37

Malus x domestiea Leaf A.t. IIptll, gus + 27 Leaf At. nptll, gus + 29 Shoot A.t. nptl/, gus + 94 Leaf A.t. nptll, gus + 95

M. pumila Leaf A.t. nptll, nos + 19 \

Malus Leaf A.t. nptll, gus + 28 Leaf A.t. gfp + 96

Mangifera indica Somatic embryo At. gus, nptll + 97

Populus deltoides Leaf disc A.t. gU.f , nptll 98

P. euroamerieana Leaf disc A.t. gus, nptll 98

P. nigra Leaf A.t. gus, nptll, hpt + 18

P. tremula Stem A.t. gus, nptll + 99

P. tremuloides Leaf A.t. npt II, gus + 30 Leaf At. COMT + 100

P. alba x P. grandidenta Leaf A.t. nptll, aro A + 21 Leaf suspension culture A.t. np II-ae, hpt, BT + 24 Stem A.t. bar, nptll + 46 Stem Ep nptll, gus, BT + 35 Leaf A.t. cat, np II + 48 Leaf A.t. aroA, nptll + 101

P. sieboldii x P. Leaf A.t. gus, nptl/, pr XAI + 17 grandidentata (P. kitakamiensis)

P. tremula x P. alba Shoot A.t. nptl/, gus + 102 Stem A.t. nptll, gus + II Stem At. COMT, nptll + 66 Stem A.t. CAD, nptll, gus + 67 Stem A.t. gor, gshll, nptll + 63 Stem A.t. FeSOD, nptll + 103

P. tremula x P. Stem A.t. Bt, cry iliA, tremuloides OCI, npt II + 49

Leaf A.t. rolC, Ae-rol C, nptll + 104

Prunus amygdalus Leaf A.t. lip/II, gus + 16

P.ameriealla Embryogenic callus A.t. gus, nptl/ + 105

P. armeniaea Immature embryo A.t. gus, ppv ep + 53

P. domestiea Hypocotyl A.t. nptll, gus + 106 Immature embryo A.t. gus, pPV cp + 53

P. persiea St~m A.t. oes + 107

COlltd

634 INDIAN J EXP mOL. AlLY 10519

KHURANA & KHURANA: GENETIC TR;\ NSFORMATION IN TREE BIOTECHNOLOGY 635

Table I-Foreign gene expression in ti s~l.1e or cells of angiospermous trees-Collld

P. subhinella aulumno Embryogenic callus A.r. gus, l~plll + :n rosa

Pyrus communis Leaf A.I. IIplll, gus + 26

Robinia pseudoacacia Hypocotyl A.I. nptll + 108 Cotyledon A.I. nplll + 13

Theobroma cacao Leaf A.I. nptll + 109

Ulmus procera Leaf Pb gus + 41 Shoot A.I. gus + 110

ABBREVIATIONS

a A.r. Agmbaclerium rhil.ogenes

A.I. Agrobaclerium lumefaciens

Ep Electroporation

PEG Polyethylene glycol

Ph Particle bombardment

b agr agropine

nplll neomycin phosphotransferase

(ICS octopine synthase

nos nopaline synthase

gus ~-glucuronidase

kall kanamycin phosphotransferase

cal chloroamphenicol acetyl transfera.~e

chs chalcone synthase

BT Bacillu.f Ihuringiensis toxin

hpl hygromycin phosphotransferase

OCI cysteine proteinase inhibitor

prXAI peroxidase

specific overproduction of the peroxidase isozyme (Prx Ai). The anionic peroxidase isozyme has been found to have a pI of 4.4 and the tissue specificity and UV inducibility of this isozyme have been characterized.

Reproductive sterility and early flowering

Before genetically engineered trees can be planted in the field for commercial application, the trans genes introduced into them need to be contained. This is because native and wild populations of trees could suffer a high degree of contamination from the transgenic plants71

, or the transgenes could escape and become wild72.

Flower sterility can be achieved through flower " specific expression of cytotoxic structural genes,

COMT caffeic acid-O-methyl transferase

CAD cinnamyl alcohol dehydrogenase

gor glutathione reductase

gshll glutathione synthetase

aroA 5-enolpyruvyl shikimate 3-phosphate synthase

PPVcp Plum pox virus coat protein

Cry III A Bacillus Ihurillgiellsis 5-endotoxin gene

bar phosphinothricin

PRVep Papaya ring spot virus coat protein

Imsllmr Tumor genes

nop nopaline

man mannopine

FeSOD iron superoxide dislllutase

gfp green flourescent protein

c TGE Transient Gene Expression

d SGE Stable Gene Expression

leading to the specific ablation of floral organs or using antisense or promoter suppression of specific homeotic reproductive development genes 73.

While little research has been done on the production of sterile trees, it is likely that it will be relatively easy to transfer sterility genes being isolated from agricultural crops into trees . Male sterility would minimize gene flow via pollen dispersion . Complete sterility would be desirable for species such as Populus and Salix, which have small wind blown seeds.

In the last few years there has been considerable progress towards cloning flower specific genes and these could potentially be used to regulate flowering. Recent work on meristem identity genes with the model plant, Arabidopsis, has led to precocious

636 INDIAN J EXP BIOL, JULY 1999

flowering in aspen. Hybrid aspen transformation with the same Arabidopsis LEAFY gene (LFY) under control of Cauliflower Mosaic Virus 35S promoter produces flowering after only five months of growth in the greenhouse74

• Needless to mention, such investigations have tremendous potential to revolutionize the entire field of tree breeding.

Future Prospects The demand has never been greater for improved

forest trees. Several new methodologies are being used to address problems in forest biology. Besides addressing problems inherent to the current use of trees, genetic manipulation of trees brings with it the potential to create new industries based on novel characteristics, e.g., trees with trans genes to detoxify specific pollutants and for remediation of contaminated and hazardous wastes. The demand for fibers and wood will continue and advances in wood chemistry and biotechnology may also make the trees an ideal feedstock for 'biorefineries' as alternative sources of petrochemical products. With the continuing demand for wood and wood related products, new silvicultural practices will certainly be needed to speed up the growth of trees and consequently, biotechnological practices would be relied upon.

It might be possible to increase biomass production by altering cellulose biosynthesis in transgenic plants. One way to increase fiber yield in trees is to create a sink for photosynthate in xylem cells. Cloning of the UDP-glucose pyrophosphorylase, responsible for the synthesis of UDPG, a high energy substrate for cellulose biosynthesis in both bacteria and plants, would serve the purpose. It has been found that this gene has xylem specific expression and that its overexpression in transgenic tobacco resulted in increased UDPG-Ppase activity, elevated levels of cellulose and increased plant biomass 75.

A major breakthrough in genetic engineering of trees would be the total loss of ability to form reproductive structures in trees. Such trees will obviously be sterile, thereby providing gene containment for environment and commercial protection, but there is a secondary benefit; avoidance of the reproductive burden is estimated to provide vegetative growth increases of up to 15% .. To achieve this goal, recently a tissue ablation approach has been proposed76

, whereby a promoter of an early flower specific gene is used to express a lethal gene such as

Rnase, protease or nuclease, antisense to a central function, a cytokinin etc.

Fundamental to these efforts would be the integration of genetic engineering with marker aided selection. Molecular marker methods will help in identifying superior genotypes as well as in monitoring the products of improved breeding schemes. Markers have significant associations with height and wood volume, indicating a strong influence of specific quantitative trait loci. AFLP markers have been used to study the genetic architecture of rooting ability in eucalyptus. Maps have now been constructed for 4 species of eucalyptus77

• Thus by a judicious amalgamation of traditional breeding practices and the present day techniques of biotechnology, the forest industry is bound to witness a revolution in the practices and products of forestry. References I Cheliak W M & Rogers D L, Call J For Rcs, 20 (1990) 452. 2 Winton L L, Am J Bot, 57 (1970) 904. 3 Haissig B E, Nelson N D & Kidd G H, BiolTcclZllology, 5

(1987) 52. 4

5

6 7 8

9

10

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13 14

15

16

17

18

19

20

21

Zhao Y X, Yoa D Y & Harris P J C, NitrogclI Fixillg Tree Res Rep, 8 (1990) 113. Ochatt S J, Patat-Ochatt E M, Rech E L, Davey M R & Power J B, Theor Appl Gellet, 78 (1989) 35. Ochatt S J & Power J B, Tree Physiol, 5 (1989) 259. Ochatt S J & Power J B, Plallt Cell Reports, 8 (1989) 365. Horsch A, Fraley R T, Rogers S G, Sanders P R, Lloyd A & Hoffman N, Science, 223 (1984) 496. Barton K A, Binns A N, Mattzke A J M & Chilton M D, Cell, 32 (1983) 1033. Caplan A, Herrera-Estrella L, Inze D, Van Haute E, Van Montagu M, Schell J & Zambryski P, Scicllce, 220 (1983) 815. Leple J C, Miranda Brasileiro A C, Michel M F, Delmotte F & Jouanin L, Plant Cell Reports, II (1992) 137. Machado A da C, Katinger H & Machado M L da C, Euphytica, 77 (1994) 129. Davis J M & Keathley D E, Call J For Rcs, 19 (1989) 1118. Pang S Z & Sanford J C, J Allier Soc Hart Sci, 113 (1988) 287. ConfaIonieri M, Balestrazzi A, Bisofli S & Cella R, Plant Cell Tissue Organ Cult, 43 (1995) 215. Archilleti T, Lauri P & Damiano C, Plant Cell Reports, 14 (1995) 267. Kajita S, Osakabe K, Katayama Y, Kawai S. Matsumoto Y, Hata K & Morohoshi N, Plant Sciellcc, 103 (1994) 231 . Confalonieri M, Balestrazzi A & Bisofti S, Plant Cell Reports, 13 (1994) 256. James D J, Passey A J, Barbara D J & Bevan M, Plallt Ccll Reports, 7 (1989) 658. Davis J M, Clarke H R G, Bradshaw H A & Gordon M P, Plant Mol Bio, 17 (1991) 631.

I

Fillati J J, Sell mer J, McCown B. Haissig B & Comai L. Mol Gen Genet, 206 (1987) 192.

KHURANA & KHURANA: GENETIC TRANSFORMATION IN TREE BIOTECHNOLOGY 637

22 Fitch M M M, Manshardt, R M, Gonsalves, D & Slightom J L, Plant Cell Reports, 12 (1993) 245.

23 Machado A da C, Puschmann M, Piihringer H, Kremen R, Katinger H & Machado M L da C, Plant Cell Reports, 14 (1995) 335.

24 Howe G T, Goldfarb B & Strauss S H, Plant Cell Tissue Organ Cult, 36 (1994) 59.

25 Sullivan J & Lagrimini L M, Plant Cell Reports, 12 (1993) 303.

26 Mourages F, Chevreau E, Lambert C & De Bondt A, Plant Cell Reports, 16 (1996) 245.

27 Sriskandarajah S, Goodwin P B & Speirs J, Plant Cell Tissue Organ Cult, 36 (1994) 317.

28 Schaart J G, Puite K J, Kolova L & Progerbnyak N, Euphytica, 85 (1995) 131.

29 De Bondt A, Eggermont K, Druart P, De Vii M, Goderis I, Vanderleyden J & Broekaert W F, Plant Cell Reports, 13 (1994) 587.

30 Tsai C-J, Podila G K & Chiang V L, Plant Cell Reports, 14 (1994) 94.

31 Seguin A & Lalonde M, Plant Cell Reports, 7 (1988) 367.

32 Manders G, Dos Santos A V P, Utra Vaz F B, Davey M R & Power J B, Plant Cell Tissue Organ Cult, 30 (1992) 69.

33 Teulieres C, Grima-Pettenati J, Curie C, Tossie J & Boudet A-M, Plant Cell Tissue Organ Cult, 25 (1991) 125.

34 Klein T M, Wolf E D, Wu R & Sanford J L, Nature, 327 (1987) 70.

35 McCown B H, McCabe D E, Russell D R, Robison D J, Barton K A & Raffa K F, Plant Cell Reports, 9 (1991) 590.

36 Fitch M M M, Manshardt R M, Gonsalves D, Slightom J L & Sanford J C, Plant Cell Reports, 9 (1990) 189.

37 Wilde H D, Meagher R B & Merkle S A, Plant Physiol, 98 (1992) 114.

38 Yao J-L, Wu J-H, Gleave A P, Morris BAM, Plant Sci, 1\3 (1996) 175.

39 Chowdhury M K U, Parvez G K A & Saleh N M, Plant Cell Reports, 16 (1997) 277.

40 Arokiaraj p, Jones H, Cheong K F, Coomber S & CharI wood B V, Plant Cell Reports, 13 (1993) 425.

41 Gartland J S, Gartland K M A, Main G D, Brasier C M & Fenning T M, Biologia Plantarum, 36 (1994) 331 .

42 Rochange F, Serrano L, Marque C, Teulieres C & Boudet A M, Plant Cell Reports, 14 (1995) 674.

43 Cabrera-Ponce J L, Vegas-Garcia A & Herrera-Estrella L, Plant Cell Reports, 15 (1995) 1.

44 Van Boxtel J, Berthouly M, Carasco C, Dufour M & Eskes A, Plant Cell Reports, 14 (1995) 748.

45 Tzfira T, Zuker A & Altman A, TibTech, 16 (1998) 439. 46 De Block M, Plant Physiol, 93 (1990) II. \

47 Ryan C A, Ann Rev Phytopathol, 28 (1990) 425.

48 Klopfenstein N B, Shi N Q, Kernan A, McNabO'H S, Hall R B, Hart E R & Thornburg R W, Can J For Res, 21 (1991) 1321.

49 Cornu D, Leple J C, Bonade-Bottino M, Ross A, Augustin S, Delplanque A, Jouanin L & Pilate G, In: M. R. Ahuja et ai, (eds.) Somatic Cell Genetics and Molecular Genetics of Trees (Kluwer Academic Publishers) 1996, 131.

50 Bolyard M G, Hajela R K & Sticklen M B, J Arboric, 17 (1991) 34.

51 Hollings M L & Brunt A A, In: Kustak E, (ed.) Handbook of plant virus infections: Comparative diagnosis (Elseiver) 1981, p 731.

52 Powell A P, Nelson R S, De B, Hoffman N, Rogers S G, Fraley R T & Beachy R N, Science, 232 (1986) 738.

53 Machado M L da C, Machado A da C, Hanzer V, Weiss H, Regner F, Steinkellner H, Mattanovich D, Plail R, Knapp E, Kalthoff B & Katinger H, Plant Cell Reports, II (1992) 25 .

54 Fitch M M M, Manshardt R M, Gonsalves D, Slightom J L & Sanford J C, Biolfechnology, 10 (1992) 1466.

55 Choi G H & Nuss D L, Science, 257 (1992) 800.

56 Asada K & Takahashi M, In : D T Kyle et ai, (eds.) Photoinhibition (Elsevier) 1987, P 227.

57 Bernard J E et ai, Changes in forest health and productivity in the United States and Canada, NAPAP State of Scienceffechnology Report 16, 1991.

58 Wang D, Karnosky D F & Bormann F H, Call J For Res, 16 (1986) 47.

59 Karnosky D F, Bioscience, I (1981) 14. 60 Podila G K & Karnosky D, Chem & Industry, Dec (1996)

976. 61 Tanaka K & Sugahara K, Plant Cell Physiol, 21 (1980) 60 I. 62 Jager H J, Bender J & Grunhage L, Environ Pull, 26 (1985)

1425. 63 Foyer C H, Souriau N, Perret S, Lelandais M, Kunert K-J,

pruvost C & Jouanin L, Plant Physiol, 109 (1995) 1047. 64 Altman A, Pelan D, Yarnitsky 0, Tzfira T, Yaari A, Wang

W-X, Shoseyov 0, Vainstein A, Huttermann A & Wang S, In: M. R. Ahuja et ai, (eds.) Somatic cell genetics alld molecular genetics of trees (Kluwer Academic Publishers) 1996,47.

65 Altman A, Alegrand T, Pelah D, Tztira T. Vinocur B, Wang W & Yarnitzky 0, Dendrome, 3 (1996) 5.

66 Van Doorsselaere J, Baucher M, Chognot M, Chabbert B, Tollier M-T, Petit-Conil M, Leple J C, Pilate G, Cornu D, Monties B, Van Montagu M, Inze D, Boerjan W & Jouanin I, Plant J, 8(6) (1995) 855.

67 Baucher M, Chabbert B, Van Doorsselaere J, Pilate G. Cornu D, Petit-Conil M, Montis B, Van Montagu M, Inze D, Jouanin L & Boerjan W, In: M. R. Ahuja et ai, (eds.) Somatic cell genetics and molecular genetics of trees (Kluwer Academic Publishers) 1996. 153.

68 Chapple C C S, Plant Cell, 4 (1992) 1413 .

69 Chapple C C S, Plant Physiol, 108 (1995) 875.

70 Imberty A, Goldberg R & Catesson A M, Planta, 164 (1985) 221.

71 Dale P J, Plant Physiol, 100 (1992) 13 .

72 Keeler K, Biolfechnology, 7 (1989) 1134.

73 Strauss S H, Rottmann W H, Brunner A M & Sheppard L A, Mol Breeding, 5 (1995) 26.

74 Weigel D & Nilsson 0 , Nature, 377 (1995) 495.

75 Bao X, In: Meilan R & Strauss S H, Dendml11e, 4 (1997) 5. 76 Teasdale R D, in M. R. Ahuja el ai, (eds.) Somatic cell

genetics and molecular genetics of trees (Kluwer Academic Publishers) 1996, 69.

77 Sederoff R R & McCord S, Dendrollle, 4( I) (1997) 3.

78 Rugini E, Pellegrineschi A, Mencuccini M & Mariotti D, Plant Cell Reports, 10 (199 J) 290.

638 INDIAN J EXP BIOL, JULY 1999

79 Phelep M, Petit A, Martin L, Duhoux E & Tempe J, Bioffechnology, 9 (1991) 461.

80 Franche C, Diouf D, Le Q V, Bogusz D, N'Diaye A, Gherbi H, Gobe C & Duhoux E, Plant J, II (1997) 897.

81 Mackay J, Seguin A & Lalonde M , Plant Cell Reports, 7 (1988) 229.

82 Naina N S, Gupta P K & Mascarenhas A F, Curr Sci, 58 (1989) 184.

83 Keinonen-Mettala K, Pappinen A & Von Weissenberg K, Plant Cell Reports, 17 (1998) 356.

84 Yang J-S, Yu T-A, Chong Y-H, Yeh S-D, Plant Cell Reports, 15 (1995) 459.

85 Seabra R C & Pais M S, Plant Cell Reports, 17 (1998) 177. 86 Le, Q V, Bogusz D, Gherbi H, Lappartient A, Duhoux E, &

Franche C, Plant Science, 118 (1996) 57. 87 Perez-Molphe-Balche & Ochoa-Alejo N, Plant Cell

Reports, 17 (1998) 591. 88 Vardi A, Bleichman S & Aviv D, Plant Science, 69 (1990)

199. 89 Ho C-K, Chang S-H, Tsay J-Y, Tsai C-J, Chiang V L &

Chen Z-Z, Plant Cell Reports, 17 (1998) 675. 90 Machado L de 0 R, de Andrade G M, Cid L P B, Penchel R

M & Brasileiro A C M, Plant Cell Reports, 16 (1997) 299. 91 Arokiaraj P, Yeang H Y, Cheong K F, Hamzah S, Jones H,

Coomber S & Chari wood B V, Plant Cell Reports, 17 (1998) 621.

92 Dandekar A M & Martin L A, J Amer Soc Hort Sci, 113 (1988) 945.

93 Jouanin L, EIEuch C, Pastuglia M, Capelli P, Doumas P & Jay-Allemand C, in M. R. Ahuja et al, (eds.) Somatic cell genetics and molecular genetics of trees (Kluwer Academic Publishers) 1996, 147.

94 Sriskandarajah S & Goodwin P, Plant Cell Tissue Organ Cult, 53 (1998) I .

95 Norelli J L & Aldwinckle H S, J Amer Soc Hort Sci, 118 (1993)311.

96 Maximova S N, Dandekar A M & Guiltinan M J, Plallt Mol Bioi, 37 (1998) 549.

97 Mathews H, Litz R E, Wilde H D, Merkle S A & Wetzstein H Y, III Vitro Cell Dev Bio, 28 (1992) 172.

98 Confalonieri M, Balestrazzi A & Cella R, Plant Cell Tissue Organ Cult, 48 (1997) 53.

99 Tzfira T, Ben-Meir H, Vainstein A & Altman A, Plant Cell Reports, 15 (1996) 566.

100 Tsai C-J, Popko J L, Mielke M R, Hu W-J, Podila G K & Chiang V L, Plant Physiol, I 17 (1998) 101.

101 Donahue R A, Davis T D, Michler C H, Riemenschneider D E, Carter D R, Marquardt P E, Sankhla N, Sankhla D, Haissig B E & Isebrands J G, Can J For Res, 24 (1994) 2377.

102 Brasileiro A C M, Leple J-C, Muzzin J, Ounnoughi D, Michel M-F & Jouanin L, Plallt Mol BioI, 17 (1991) 441 .

103 Arisi A-C M, Comic G, Jouanin L & Foyer C H, Plant Physiol, 117 (1998) 565.

104 Ahuja M R & Fladung M, in M. R. Ahuja et al, (eds.) Somatic cell genetics and molecular genetics of trees (Kluwer Academic Publishers) 1996,89.

105 Cruz-Hernandez A, Witjaksono, Litz R E & Gomez Lim M, Plant Cell Reports, 17 (1998) 497.

106 Mante S, Morgens P H, Scorza R, Cordts J M & Callahan A M, Bioffechnology, 9 (1991) 853.

107 Hammerschlag F A, Owens L D, & Simogocki A C, J Alii Soc Hort Sci, 114 (1991) 508.

108 Han K-H, Keathley D E, Davis J M & Gordon M P, Plant Science, 88 (1993) 149.

109 Sain S L, Oduro K K & Furtek D B, Plant Cell Tissue Organ Cult, 37 (1994) 243.

110 Fenning T M, Tymens S S, Brasier C M, Gartland J S & Gartland K M A, in M. R. Ahuja et ai, (eds.) Somatic cell genetics and molecular gelletics of trees (Kluwer Academic Publishers) 1996, 105.