Su Xiao-hua, Zhang Bing-yu, Huang Qin-jun, Huang Lie-jian and Zhang Xiang-hua
This paper introduces the advances in both basic and applied researches in tree genetic engineering in China. Some recommendations are made for prioritizing future studies in tree genetic engineering in order to meet the requirements of environmental improvement and the timber industries.
Researches on transgenic forest trees started in China at the end of 1980s. Since then, Chinese researchers have developed techniques of tissue culture and genetic transformation for many trees species, such as poplar, birch, eucalypt, larch, walnut, apple, citrus, Chinese goosebeery, etc. They successfully applied tissue culture and genetic transformation in many tree species [1,2,4,10,11], and successfully transferred Bt gene, salty tolerance gene, drought tolerance gene, sterile gene, anti-ACC synthetase and oxidase gene, etc, into poplar genome by using leaf-disc, ballistic and other methods with regenerated transgenic plants or varieties obtained.
The application of genetic engineering technology for insect resistance breeding is considered a new approach of great interest to protecting forest trees from insect damages. Some successes have been achieved.
Transgenic Populus nigra with Bt gene resistant to leaf insect was commercialized. The research on transferring Bt gene into Populus nigra started in China in 1989, and transgenic plants were put into field trails in 1994. According to 4-year results of the field trails, the transgenic Populus nigra plants were significantly resistant to target insects. Insect population in both trees and soil were decreased, and both transgenic and non-transgenic plants in the same planting site were protected. In 1998, transgenic Populus nigra plants were approved for environmental release at Manasi in Xinjiang by the Bio-Engineering Security Committee of the Ministry of Agriculture, and approved for environmental release in Beijing, Jilin, Shandong, Jiangsu, Henan, and Shanxi provinces in 1999. Because of their rapid growth and high resistance to the target insect, commercialization of the transgenic Populus nigra plants were approved by the Gene Security Committee of the State Forestry Administration in 2002. Up to now, one million of the transgenic Populus nigra plants have been propagated and the plantations of transgenic plants have been established.
Transgenic hybrid poplar 741 with bi-resistant insect genes was approved for environmental release. In 1996, researchers transferred the bi-vector resistant insect that contained the improved Bt Cry1Ac gene and API gene to hybrid poplar 741 (Populus alba L.¡Á(P. davidiana Dode + P. simonii Carr.)¡ÁP. tomentosa Carr.), and identified that novel genes had transferred into poplar plants with normal growth and development of transgenic plants. Through a 5-year experiment, the Forest Bio-Engineering Security Committee approved the environmental release of 6 clones of transgenic hybrid poplar 741 in 2001. So far, 400,000 plants of transgenic poplar 741 have been propagated and a breeding system and a trail plantation have been established to provide new elite varieties.
Transgenic poplar plants resistant to trunk-insect (longhorn beetle) Researchers have transferred the LcI and Bt genes into poplar. The imago of Anoplophora glabripennis (Motschulsky) was fed with the leaves of transgenic plants. More than 30 clones were obtained. Propagation of some transgenic plants and field trails has been conducted .
Chinese researchers not only successfully transferred the Bt gene into poplar, larch and walnut, but also transferred CpTI, OC-I, and AaIT genes into some varieties of poplar [7,8]. The preliminary study on transferring the insect resistant gene into Betula platyphylla was also started .
Up to now, few researches on genetic engineering targeted at drought and salty tolerance, mainly because the plant response to drought and salty is a multi-component-system, and the physiological mechanism controlled by multi-genes has not been known clearly. Chinese researchers transferred SacB and HVA1 genes into poplar through agrobacterium tumefaciens-mediated method, and obtained regenerated transgenic plants. Zhan et al transferred BetA into Populus simonii × P. nigra by agrobacterium tumefaciens mediated method using sterile seedling leaf as explants, and the results of Southern-blot analysis indicated that the novel gene was integrated into target genome . In addition, the experiments of transferring BADH and GUTD genes into poplar and transferring Ip3 and Ip5 genes into larch are in progress.
Chinese researchers have made some meaningful attempts, although many pathogenies are not isolated and identified, causing difficulties in transgenic engineering for disease resistance. Researchers transferred the rabbit alexin gene NP-1 into Populus tomentosa Carr. by agrobacterium tumefaciens-mediated method, and identified by Southern analysis that the novel gene had integrated into transgenic plant genome, and results from the experiment of inhibitory in vitro indicated that the transgenic plants could restrict the growth of many kinds of microbe, such as Bacillus subtilis, agrobacterium tumefaciens, and Rhizoctonia solani . Researchers also successfully transferred some novel genes into papaya by agrobacterium tumefaciens mediated [15,16,19,20].
For preventing senescence of forest trees, researchers used the anti-RNA technology to control the ethylene biosyntheses. They transferred the anti-fragment of ACC oxidase gene into Populus deltiodes, and the results of Southern analysis showed that a single copy of novel gene was integrated into transgenic plant genome, the ethylene bio-synthesis of transgenic plants was depressed, and the ethylene produced by their seedlings was 28% less than by the control plants . The genetic transformation studies for decreasing the content of lignin in poplar and larch are being conducted, and a few transgenic clones were obtained.
Researchers attempted to study the novel gene escape from transgenic trees, they transferred TA29-BARNASE gene into Populus deltiodes, and identified by PCR, Southern blot and Nouthern blot analysis that the novel gene was integrated into transgenic plant genome .
In abroad, except that the virus resistance transgenic papaya was commercialized, almost all other transgenic trees were under small scale of field trails. In China, the environmental release of transgenic Populus nigra with Bt gene is being conducted at 8 sites in Beijing, Jilin, Henan, Shandong, Xinjiang, Shanxi, and Jiangsu provinces with a total area of 80 hm2.
The research on transgenic safety evaluation started in China. Chinese researchers have studied the effects of Bt gene transgenic Populus nigra on un-targeted insects, microbes in soil, mammals and human beings, and obtained some results. More researches on environmental release of Bt gene related to environmental security are under way. Researches in this field are considered useful for proper application of transgenic forest trees, and security evaluation system and related standards.
Chinese researchers have made great efforts in gene cloning in order to win intellectual property right of our own. Researchers cloned the full length of genome, ABREBP-type transcript factor genes from Caragana korshinskii growing in desert with drought tolerance, and the cDNA full length is being cloned. Liu et al. cloned the mtl-D gene related to salty tolerance from E. coli, inserted it to binary vector and transferred into a poplar cultivar Balizhuangyang by agrobacterium tumefaciens, the transgenic plants grew very well in media containing 0.6% NaCl; PCR and Southern blot analysis indicated that the novel gene was integrated into the target genome and expressed . Wei et al. cloned a COMT cDNA fragment, encoding for methylase that relates to lignin biosynthesis pathway, from Chinese White Poplar (Populus tomentosa) . Researchers are currently cloning CCoAOMT, PAL and CCR genes. They have identified a new species of Bacillus thuringiensis, virulent to Ceram bycidae from the natural cadaver of Ceram bycidae and from the resources of Bacillus thuringiensis; and feeding experiment showed this new Bt could kill 50% imago of Ceram bycidae. The related fragment of this Bt gene was then amplified using the special annex primer, and identified that the fragment was really a kind of Bt gene resistant to Ceram bycidae. According to the results of sequencing, the full length of this gene cloned and the expression vector for transformation is being constructed.
Although many fast-growing tree species have been developed by breeding and introduction in recent 20 years, fast growth has been over emphasized while improvement of wood quality has been overlooked, resulting in high cost of plantation management and largely constraining wood processing and utilization. Overlooking of the resistance to biological or non - biological stresses of trees in ecologically fragile environment in western China, had led to very low survive rate of tree plantings and frequent disasters of Cerambycidae, Apocheima cinerarius (Erschoff), Lymantria dispar L., etc., large area of timber plantations and ecological protective forests are losing their functions, all these indicate clearly that studies on gene transformation of trees must be immediate action with no delay.
Considering the unique climate and nature of China, and new threats from diseases and insects related to global warming, we suggest that the following key fields be included in research programs in the near future.
Among the transgenic studies, research on insect resistance has been best conducted and achieved the best progress. However, all current studies are focused on lepidopterous insects, and inadequate attention has been paid to Cerambycidae, a trunk-insect that damages many tree species, prevails widely and causes the most severe economic lost.
Cerambycidae has a wide range of host trees, including poplar, willow, elm, maple, Sophora japonica, mulberry, etc. Damages to poplar and pine are the most serious. As is well known, Bursaphelenchus nematode has caused fatal damage to pine plantations, with an area of 74000 hm2, and its carrier was Monochamus alternatus Hope. The Three North Shelterbelts Project, which attracts worldwide attention, covers 500 counties, but now Cerambycidae disaster (mainly caused by Anoplophora glabripennis M.) has happened in more than 300 counties, covering an area of 268,000 hm2. More than 17 million cube meters, timber was destroyed, causing a direct economic loss of up to 5 billion RMB. This disaster will spread to natural forests if no effective measure was taken.
Trunk hosting makes Cerambycidae more difficult to control. An important target for tree genetic engineering is to find out and obtain the gene that could kill Cerambycidae effectively. Bacillus thuringiensis is recognized as a kind of pathogeny microbes that are harmless to human or livestock, non-polluting to environment, and protective towards natural enemies. A feasible approach is to screen out and identify the genes that could kill Cerambycidae from Bt, analyze the function and structure of Bt gene and improve it, and conduct the transgenic researches to obtain the tree species resistant to Ceram bycidae so that the goal of controlling Cerambycidae could be reached.
According to incomplete statistic, the occurrence of forest insect and disease in China covered an area of 1 million hm2 in 1950s, 1.4 million hm2 in the 1960s, and up to 11 million hm2 in the 1990s, the annual increase was 25%. The economic loss per year reached more than 5 billion RMB, the disaster affected area was 8.2% of the total forest area and 23.7% of the total plantation area. Disease and insects have already been an important factor restricting the sustainable forestry development in China. Among the diseases, canker and rust are the most serious diseases. Disease decreases the quality of wood from timber plantations and reduces productivity of economic forest, leading to serious damages and economic loss.
The fungi-resistance genes mainly are those genes from the inter- or outer- plant pathogenies, such as antagonistic protein gene and virulent protein gene, the genes encoded motivating factors of pathogeny, protease inhibitor genes of pathogeny and genes that controlling sensitive response of host and pathogeny, etc. But at present, the effective genes resistant to Botryosphaeria ribis and Melampsora larici-populina Kleb have not been cloned. Therefore, we should first conduct the gene clone experiments in genetic engineering program. It is suggested that our government increase investment in this research fields.
The stresses, such as salty, drought, waterlogging, unsuitable temperature, strong sunlight, residue of pesticide, etc, restrict the range of forest plantations, timber productivity and wood quality. For making full use of the existing land, expanding forest plantations and improving timber productivity and wood quality, breeding for stresses resistance is attracting more and more attentions. In traditional breeding project, for lacking of the resources of resistance and the knowledge of resistance mechanism, little progress has been made in this field. However, the recent advance of biotechnology has provided a new opportunity.
Because the mechanisms of drought and salty resistance in plants are very complex and involve a series of variation of phenotype and metabolism, the single gene transformation could only obtain partial resistance in most cases. If the transgenic trees are expected to be able to grow in drought area or near the beach, and to be irrigated with sea water, the followings are needed: first, transferring multi-genes; second, using the advanced methods, such as chloroplast transformation, and enhancing novel gene expression; third, transferring with transcript factors. Transcript regulation factors, through the mediation of related cis-action elements, could induce many gene expressions and enhance plant resistance to drought, salty and cold. Transgenic trees with drought and salty resistance genes will have a broad application in China, but research in this field is still weak and needs further strengthening.
At present, the genetic engineering of lignin biosynthesis appears to be a promising forest biotechnological application to improve wood quality. Because the economy of pulping processes depends almost entirely on the efficiency of removing lignin which encrusts the wood fibers. Thus, one way to improve the efficiency of pulping is to genetically reduce the quantity or to alter the quality of lignin in pulpwood species. Lignin biosynthesis was the most peculiar biological process involving various components of wood. Genetic manipulation of lignin is largely a matter of manipulating genes encoding lignin pathway-specific enzymes. It takes a long time to reach the breeding goals in traditional breeding program, because multi-genes controlled most wood traits. At present, transgenic method is of great expectations. So, the genetic engineering of lignin biosynthesis has bright prospects in its application in tree breeding for pulping and papermaking industries. Research in this field should be strengthened, to breed new varieties for commercial plantations for pulp in Yangtze and Yellow River systems.
The research on transgenic safety evaluation is very important for the extension of transgenic trees in China and in the world, and for setting up the basis for the evaluation systems and standards. Since 2000, Chinese researchers have studied the effects of transferring Bt gene of Populus nigra on un-target insects, microbes in soil, mammals, and human beings, and progress has been made. But because the safety evaluation is a long-term and complex process, support for these activities should be strengthened for keeping the leading position in the world.
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|  Professor, Genetics and
Breeding of Trees. Research Institute of Forestry, CAF, Beijing, 100091.
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