木薯分子育种中若干基本科学问题的思考与研究进展

木薯作为全球重要的薯类作物,既是热带地区粮食安全的保障,也是重要的淀粉工业原材料,保障其稳产、高产、优质一直是育种家不变的研究主题．当前,木薯品种选育正处在从杂交育种转向分子育种的发展阶段,深入解析木薯特有的经济性状和生物学特点是利用生物技术进行遗传改良的基础．不同于谷物类作物,木薯光合同化物的转运和库源分配的调控机制必有其独特之处;同时,储藏根的“库容”直接影响其产量．作为热带作物,了解木薯对低温和干旱的响应可为改良其抗逆境能力提供理论依据．不同于其他薯类作物,木薯储藏根特有的“采后生理性变质”问题亟待解决,其发生和调控机制的解析对延长木薯货架期意义重大．随着分子生物学的发展,针对上述各方面的研究日益深入,不仅激发了感兴趣的公众对这些问题的认知和思考,也激励了科研人员不断努力寻找解析相关机制的方法,为最终通过分子育种手段改良木薯提供思路和技术方案,揭开木薯的层层“面纱”,推动木薯分子育种的发展．     

影响根癌农杆菌介导的木薯遗传转化因素分析

采用根癌农杆菌介导的叶盘转化法,以我国南方地区主栽木薯品种—华南8号的胚状体子叶为受体,对影响木薯遗传转化效率的主要因素进行了分析。研究结果表明,在木薯的遗传转化中,选用GV3101作为浸染外植体的农杆菌菌株,将感染时间和共培养时间分别控制在30~45 min和3~4 d、菌液浓度（OD₆₀₀）采用0.45、并添加200 μmol·L^-1的乙酰丁香酮（AS）均可明显提高其转化效率,但若对外植体进行预培养反而会降低其转化效率。利用该体系从453块外植体中共转化获得10株抗性再生植株,经PCR和Southern杂交检测,有8株木薯的基因组中已整合进了外源基因glgC336,转化率为1.77%。     

木薯的组织培养和基因转化

木薯是重要的热带作物之一, 其块根富含淀粉, 是热带、亚热带地区近５ 亿人粮食的主要来源。简单介绍近年来运用生物技术改良木薯的研究进展。     

Using microsatellites,isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections

The cassava core collection was selected to represent, with minimum repetitiveness, the potential genetic diversity of the crop. The core (630 accessions) was chosen from the base collection (over 5500 accessions) on the basis of diversity of origin (country and geographic), morphology, isozyme patterns and specific agronomic criteria. To asses the genetic diversity of the core, 521 accessions were typed with four microsatellite loci. Allele diversity and frequency, and size variance of dinucleotide repeats (Rst statistic) were estimated. Microsatellite allele numbers and frequencies varied among countries: Colombia and Brazil had the largest number of different alleles across all loci. Mexico also had a high number, ranking fifth after Peru, Costa Rica and Venezuela (which tied). Unique alleles were present in accessions from Brazil, Colombia, Guatemala, Venezuela and Paraguay. A small number (1.34%) of potential duplicates were identified through isozyme and AFLP profiles. Thus, the present results indicated that traditional markers have been highly effective at selecting unique genotypes for the core. Future selections of cassava germplasm sets can be aided by DNA-based markers to ensure genetically representative, non-redundant samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

植物生长调节物质IP－1号对木薯产量及其生物性状的影响

１９９０和１９９１年在木薯（ＭａｎｉｈｏｔｅｓｃｕｌｅｎｔａＣｒａｎｔｚ）生长期以植物生长调节物质ＩＰ－１号０,２０,３０和４０ｐｐｍ进行叶面喷洒,结果表明：３０ｐｐｍ处理可使木薯块根产量平均增加５４．４４％,块根淀粉含量平均提高２０．８１％。单株最大薯重提高３１．５５％,块根数增加２１．１７％,块根长度增长１７．６２％,地上部鲜重增加３４．３６％,植株高度增加４．３６％,植株收获期保留青叶数增加１９．４２％,主茎直径增加６．２６％,块根直径增加２．５８％,叶片的叶绿素和蛋白质含量分别提高５．５７％和２５．９６％,叶片光合作用强度提高１５．８６％,而对主茎高度、主茎节数没有明显影响。     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

木薯雌雄花分化形态结构观察及生理调控研究

本研究以木薯品种新选048为试验材料,观察和测定木薯花分化各时期的形态结构、内源激素及碳氮化合物含量,并开展木薯雌雄花分化的外源激素调控研究,揭示木薯花分化规律,初步探究木薯花分化的生理机制.试验结果表明:木薯从花芽分化开始到完成开花的全过程需要30 d,分化出的花序主要有全雄花序和雌少雄多花序两种类型,木薯性别分化前...     

Development of polymorphic markers from expressed sequence tags of Manihot esculenta Crantz

In this study, 49 primers were designed from sequences containing di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of four repeats and presence of motif size polymorphisms (insertion/deletion) from cassava (Manihot esculenta Crantz) expressed sequence tags deposited in public sequence database. Each locus was subsequently screened on 29 M. esculenta Crantz obtained from 15 different countries. Cross-amplification was tested with M. esculenta Crantz (ssp. flabellifolia) and four different Manihot species, M. chlorosticta, M. carthaginensis, M. filamentosa and M. tristis. Of these, nine loci showed polymorphic profiles within M. esculenta Crantz, which revealed two to four alleles per locus. The average unbiased and direct count heterozygosities were 0.4901 and 0.5674, respectively.     

Photosynthesis and Leaf Structure in Domesticated Cassava (Euphorbiaceae) and a Close Wild Relative: Have Leaf Photosynthetic Parameters Evolved Under Domestication?

Broad patterns across wild plant species show that leaf composition and morphology vary predictably among habitats, richer habitats favoring resource‐acquisition strategies and poorer habitats favoring resource‐conservation strategies. Domestication is often accompanied by a shift to richer habitats, and might thus be expected to lead to a shift in leaf composition and morphology and hence in photosynthetic parameters. We compared leaf photosynthetic parameters in domesticated cassava (Manihot esculenta) and a close wild relative, using greenhouse‐grown plants. In domesticated cassava, CO₂ exchange rate expressed per unit mass and specific leaf area (SLA, m²/kg dry mass) were greater than in the wild relative, whereas leaf dry matter content (LDMC, dry mass/fresh mass) was lower in the domesticate. These results suggest that SLA and net photosynthetic rates may both have increased in the evolution of cassava under domestication, enabling more rapid growth in relatively resource‐rich and protected agricultural habitats. Previous comparisons of photosynthetic rates in domesticated plants and wild relatives have usually considered only leaf area‐based measures. Here, we discuss the interest of using mass‐based rates to study the evolution of ecological strategies under domestication.     

Production of stably transformed cassava plants via particle bombardment

 A novel protocol, based on biolistics and regeneration via organogenesis, was developed for genetic transformation of cassava (Manihot esculenta Crantz). The in vitro performance of cassava cultivars CMC40, MPer183 and MCol22 was evaluated, and the regeneration protocol was modified to improve shoot production from explants for transformation experiments. Somatic cotyledons were used as a target tissue in the transformation experiments using the Particle Inflow Gun and a plasmid containing the uidA gene in transient assays. The effect of different parameters for particle bombardment efficiency, including the amount of DNA used, the flying distance of the projectiles and the pre- and post-plasmolysis time of the target tissue, was evaluated and the conditions were partially optimised. Stably transformed cassava plants of cvs. MCol22 and TMS60444 were produced using the partially optimised conditions and two different vector constructs carrying the hpt gene as the selectable marker. The selection protocol was optimised further, and a rooting test was developed for screening the regenerants for antibiotic resistance to reduce the number of escapes obtained after primary selection. The production of stably transformed cassava lines and the expression of the transgenes was verified by Southern blot analysis and RT-PCR. Received: 10 December 1999 / Revision received: 12 January 2000 / Accepted: 7 February 2000     

P5CR基因的进化及其在木薯中的表达分析

吡咯啉-5-羧酸还原酶（P5CR）催化脯氨酸合成途径的最后一步反应;在植物逆境胁迫响应中起重要作用。采用生物信息学方法从木薯（Manihot esculenta Crantz）等45个已完成基因组测序的植物中共鉴定出54条P5CR序列;分析其在不同物种的分布和序列特征;用MEGA软件构建Neighbor-joining系统进化树;采用实时荧光定量PCR技术分析MeP5CR在不同组织和胁迫处理下的表达特性。P5CR在内含子长度上差别较大;而在氨基酸长度、外显子数目、等电点和分子量等方面差别不大。在大多数植物（如木薯）中;P5CR有且仅有1个拷贝;表明P5CR在进化上比较保守;且是单基因起源。然而;在某些植物（如大豆、苹果等）中存在2-3个拷贝;说明在物种进化的过程中P5CR发生了多次重复事件;并将它们归纳为3种进化模式。表达分析表明;MeP5CR在叶片、须根和储藏根中表达量最高;而在叶柄和茎中的表达量最低。MeP5CR表达量受低温胁迫诱导、但被干旱胁迫抑制。MeP5CR在转录水平参与木薯干旱和低温胁迫响应。     

PIG-mediated cassava transformation using positive and negative selection

 In order to develop new selection systems for production of transgenic cassava (Manihot esculenta Crantz), two different selection regimes were assessed for their efficiency on regeneration of transgenic cassava plants: positive selection using mannose and negative selection using hygromycin. Explants from somatic cotyledons and embryogenic suspensions were used as target tissues in the transformation experiments and bombarded using the particle inflow gun. Different culture and selection strategies were assessed to optimise the selection protocols. For the first time transgenic plants could be obtained using positive, and in the case of embryogenic suspensions, hygromycin-based negative selection. The stably transformed nature of the regenerated cassava plant lines and the expression of the transgenes were verified with PCR, RT-PCR, Southern and northern analyses. A rooting test for transgenic plants on a medium supplemented with mannose was developed to further improve the efficacy of the positive selection system. Our results demonstrate that it is possible to obtain transgenic cassava plants using non-antibiotic positive selection. Received: 21 February 2000 / Revision received: 2 May 2000 / Accepted: 5 May 2000     

3个木薯品种嫩茎叶中氢氰酸、总黄酮及主要营养成分含量的变化

以木薯(Manihot esculenta Crantz)品种'华南7号'('South China 7')、'华南9号'('South China 9')和'华南205号'('South China 205')为研究对象,分别对种植后90、120、150、180和210 d的3个木薯品种嫩茎叶中氢氰酸、总黄酮及主要营养成分含量的变化进行了分析;在此基础上,明确供试3个品种嫩茎叶作为饲料的最佳采收期.结果表明:随种植后时间延长,3个品种嫩茎叶中的氢氰酸和总黄酮含量变化均呈波动趋势,其中,'华南7号'嫩茎叶的氢氰酸含量在种植后150和210 d显著降低(分别为347843和320507 mg·kg-1),'华南9号'嫩茎叶的氢氰酸含量在种植后150 d最低(313643 mg·kg-1),'华南205号'嫩茎叶的氢氰酸含量在种植后210 d最低(75103 mg·kg-1);'华南7号'和'华南205号'嫩茎叶的总黄酮含量在种植后120 d最高(分别为1963%和1917%),而'华南9号'嫩茎叶的总黄酮含量则在种植后210 d最高(1801%).不同生长期3个木薯品种嫩茎叶的粗蛋白质和粗灰分含量均符合相关动物饲料的标准,而总磷含量均较低.其中,'华南7号'嫩茎叶的粗蛋白质、粗脂肪、粗纤维、粗灰分和总钙含量在种植后150 d相对较高,分别为25273%、7687%、23077%、7157%和1660%,其无氮浸出物含量(26823%)相对较低;'华南9号'嫩茎叶的上述5种营养成分含量在种植后120 d相对较高,分别为28050%、6990%、21557%、8467%和1493%,其无氮浸出物含量(24723%)相对较低;'华南205号'嫩茎叶的粗蛋白质、粗脂肪和粗灰分含量在种植后120 d相对较高,分别为24273%、7080%和7633%,其粗纤维(18470%)、无氮浸出物(32037%)和总钙(1323%)含量相对较低.综合分析结果显示:'华南7号'、'华南9号'和'华南205号'嫩茎叶作为饲料的最佳采收时间分别为种植后150、120和120 d.