Use of androgenesis in intersubspecific hybrid rice breeding

Significant differences of callus induction, green plant regeneration and culture efficiency were observed among restorer lines and their hybrids in rice. Average callus induction percentage of F₁, hybrids was about twice that of their parents. Twenty doubled haploid (DH) lines that showed normal fertility with both indica and japonica CMS lines, were selected as widely compatible restorers (WCRs). TG8 derived from CPSLO17/Minghui 63 not only showed normal fertility to both indica and japonica test varieties and CMS lines, but also exhibited superior agronomic traits, in particular short plant, desired plant type, high tillerability, large panicles, good grain quality and resistance to rice blast disease. Strong heterosis for yield was observed in crosses between indica or japonica CMS lines and TG8. TG8 possessed a dominant, widely compatible gene (WCG) that was inherited from the variety CPSLO17. Results confirmed that the anther culture technique is a quick and effective way of developing widely compatible restorers in rice and that it is applicable to the direct use of intersubspecific heterosis with the three-line method.     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

广亲和粳稻恢复系选育方法

广亲和粳稻恢复系的选育除采用ＷＡ型恢复与广亲和粳稻杂交选育和不同广亲和恢复系间杂交选育外,本研究提出以粳稻为父本杂交选育的４种办法：１、以型杂交稻Ｆ１为母本转育同质广亲和恢复系;２、以ＷＡ型下育系为母本筛选同质广亲和恢复系;３、以ＷＡ型广亲和不育系为母本,筛选同质广亲和恢复系;４、以同质广亲和恢复系为母本转育新的恢复系。用上述方法已育成０２４２８等广亲和恢复系,并可同时恢复ＷＡ型和ＢＴ型胞质不育系     

Inheritance of photoperiod sensitive genic male sterility and breeding of photoperiod sensitive genic male-sterile lines in rice (Oryza sativa L.) through anther culture

The fertility segregations of F₁, F₂, BCF₁ descended from crosses between PSGMR and japonica varieties, and F₁'s anther cultured homozygous diploid pollen plant populations (H₂) were studied to reveal the genetic mechanism of photoperiod sensitive genic male sterility in PSGMR under natural daylight length at Shanghai. Rate of bagged seed-setting was used as an indicator of fertility. Fifteen F₁ showed complete fertility similar to their parents. The ratio of completely sterile plants to fertile plants in fifteen F₂ and four BCF₁ was 1:15 and 1:3, respectively. The ratio of completely sterile to fertile diploid pollen plants in nine diploid populations (H₂) was 1:3. These results demonstrated that the photoperiod sensitive genic male sterility in PSGMR was governed by two pairs of independent major recessive genes. There were no significant fertility segregations in hybrids F₁ and selfed F₂ between Nongken 58S and its derivatives 7001S, 5088S, 5047S and M105-9S, indicating that the photoperiod sensitive genic male-sterile genes in Nongken 58S were allelic to those in its derivatives. Several photoperiod sensitive genic male-sterile diploid pollen lines were bred from anther cultured homozygous diploid populations (H₂) in about a three-year period. Most of these diploid lines showed significant fertility transformation and stable complete sterility from 5 August to 5 September, excellent agronomic traits and high resistance to blast and bacterial leaf blight. This revised version was published online in July 2006 with corrections to the Cover Date.     

从T461的育成探讨广谱广亲和系的选育

T461是以籼粳中间型品种轮回422为广亲和、野败恢复基因供体,与意大利粳稻Cripto杂交选育成的具有良好一般配合力、广谱广亲和的偏粳型恢复系。T461能恢野败、矮败、印尼水田谷、滇型和BT型等核-质互作型籼、粳三系不育系,与籼型和粳型测验种杂交的结实率非常接近,小穗育性平均在90%以上,表现亲和力强、亲和谱广。在T461的改良过程中,选用多个不同的三系不育系在分离世代检测恢复性和配合力的同时,还能起到鉴定广亲和性的作用,达到获得广谱广亲和的目的。     

水稻体细胞无性系雄性不育突变

对5个类型的水稻不育系进行了幼穗培养, 在红源A、 包源A和W6154s中, 共获得了10例雄 性不育变异株, 水稻花粉败育可分为无花粉、 典败、 圆败和染败四种类型。 发现了不育 花粉败育类型之间可以相互转换现象。 对雄性不育变异株用一批现有CMS不育系的保持系和恢复系进行测交和回交, 有的变异株其 恢保关系发生了变化,     

水稻广亲和广谱型恢复系SWR78的选育

利用野败（WA）型、红莲（HL）型、包台（BT型）3种细胞质的同核异质粳稻广亲和不育系苏秋A与19个籼、粳恢复系测交,筛选出对以上3种细胞质不育系均具正常恢复力的广谱型恢复系6078。通过回交,将广亲和品种Lemont的广亲和基因导入6078,育成广亲和广谱型恢复系SWR78。经测交鉴定,SWR78对WA型、印水（ID）型、HL型、BT型4种细胞质的10个籼、粳不育系均具正常恢复力。     

Characterization of CMS and maintainer lines in indica rice (Oryza sativa L.) based on RAPD marker analysis

Total DNA from WA type CMS lines: Zhenshan 97 A, Longtepu A and theirmaintainers Zhenshan 97 B, Longtepu B wasextracted by CTAB method. One hundredprimers were used for screening RAPDmarkers to distinguish CMS line (A) andmaintainer (B) plants at seedling stage.Results showed that under the conditions of37 ^°C annealing temperature and1.5 mM MgCl₂ concentration, in Zhenshan97 A, Longtepu A there was a 1600 bp DNAfragment in product amplified by primerOPA12, while in Zhenshan 97 B, and LongtepuB no 1600 bp fragment was found. The 1600 bpfragment was also found in DA type CMS lineXieqingzao A, but was absent in XieqingzaoB. Also in the restorer line, Minghui 63the 1600 bp fragment was absent. In F₁and F₂ generation of Zhenshan97A/Minghui 63, all plants investigated hadthe 1600 bp fragment. When mitochondrial DNA(mtDNA) was isolated from the three CMS (A)and their B lines and amplified by OPA12,results showed that the 1600 fragment wasfound in all the three A lines and wasabsent in the three B lines. In DA typeXieqingzao A, two other fragments (700 bp,1000 bp) were also found except the 1600 bp.These results indicate that the 1600 bpfragment derived from CMS mitochondrial DNAcan be used as a RAPD marker to distinguishA and B plants at seedling stage, and thefragments (700 bp, 1000 bp) can be used todistinguish WA and DA cytoplasms.     

四种细胞质六千辛A粳稻不育系育种利用特性的比较研究

对由粳稻品系六千辛转育成的4种细胞质的一套同核异质不育系的不育性、开花习性、异交特性及可恢复性进行了鉴定。结果表明:(1)包台(BT)型、红莲(HL)型、茶野(CL)型六千辛A花粉以染败为主,WA型六千辛A花粉100%典败;BT型六千辛A平均自交结实率为0.60%,生产应用的安全性较差;HL型、CL型六千辛A无自交结实现象,不育性较BT型六千辛A稳定。(2)不育系的花粉败育程度影响倒一节间长度,花粉败育越彻底,倒一节间越短,从而使穗伸出度和株高相应缩短;因此,WA型六千辛A表现包颈,而BT型、HL型、CL型六千辛A均无包颈。(3)WA型六千辛A开花不集中,花时明显滞后,花期明显拉长,异交结实率低;HL型、CL型六千辛A的开花习性及异交结实率与BT型六千辛A无显著差异,明显好于WA型六千辛A。(4)BT型六千辛A的可恢复性最好,HL型、CL型六千辛A次之,WA型六千辛A最难恢复;从BT型粳稻恢复系中可以筛选到HL型或CL型粳稻不育系的恢复系。HL型、CL型粳稻不育系较好地协调了育性稳定性、开花习性和可恢复性几方面的矛盾。因此,选育HL型、CL型粳稻不育系及其相应的恢复系,可能是解决目前生产上杂交粳稻BT型不育系育性不够稳定及细胞质单一问题的突破口。     

Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media

The production of microspore-derived green plants from anther culture of indica rice is generally very low compared with japonica cultivars. A modified anther culture medium, consisting of a higher KNO₃ content (31 mm ) and casein hydrolysate (CH, 500 mg/1) but without ammonium salts, was tested in comparison with a medium consisting of the widely-used N6 medium nitrogen background, using four indica × indica F₁ hybrids as test materials. Green plant regeneration frequency was at least three-fold higher in the microspore-calli derived from the former medium than in those derived from the modified N6 medium. More than 700 microspore-derived plants were raised in the field. Another study was carried out using indica × japonica and indica × javanica F₁ hybrids. The results indicated that a medium with higher (3.5 mm ) ammonium sulphate may induce a higher frequency of anthers with microspore-calli but not necessarily lead to a larger number of green-plant regenerating calli. Subsequently, using the indica cv. ‘IR-43’ as the test material, use of a lower level (1.75 mm ) of (NH₄)₂SO₄, in addition to KNO₃ (31 mm ), was found to be better than CH (500 mg/l) for anther-response as well as green plant regenerability of the derived microspore-calli. Nitrate-nitrogen or ammonium-nitrogen alone elicited poor response. Twenty-five media involving combinations of KNO₃ (20–34 mm ) and (NH₄)₂SO₄ (1–3 mm ) were tested for their effects on anther response. Combinations involving KNO₃ (31–34 mM) and (NH₄)₂SO₄ (2.0–2.5 mm ) were found superior not only for achieving greater anther response but also, for subsequent green-plant regeneration. This contrasts with the 28 mm of KNO₃ and 3.5 mm of (NH₄)₂SO₄ in the widely-used N6 medium developed for japonica rice. Other than potassium nitrate and ammonium sulphate, and potassium phosphate to some extent, the levels of other inorganic salts tested did not make any significant difference to the process of anther response. Based on these results, modified media with three levels of ammonium sulphate were tested for anther culture efficiency of indica × japonica and indica × javanica derivatives (F₃s). Microspore-calli derived from a medium of a lower (1.75 mm ) level of (NH₄)₂SO₄ showed a higher regeneration potential overall than those derived from a higher (3.5 mm ) level. A revised medium has been suggested, on the basis of these results, for the realization of improved anther culture efficiency and, consequently, improved feasibility of using doubled haploids in genetic and breeding research with indica rice.     

一个新的水稻花粉半不育性位点的定位分析

利用一套以籼稻珍汕97B为背景的粳稻日本晴染色体片段代换系,鉴定发现1个半不育的代换系。全基因组基因型分析表明,该代换系仅含3个粳稻导入片段,而其他遗传背景与珍汕97B相同。在湖北武汉和海南分别种植其衍生的F₂和F₃分离群体,采用单标记分析和区间作图法分析花粉育性和小穗育性的数量性状位点(QTL),结果表明,该代换系的半不育性是第2染色体上的粳稻导入片段引起的,该片段RM262~RM475区间存在1个新的影响花粉育性的QTL,其贡献率为13.9%。研究结果将为进一步精细定位水稻育性QTL以及鉴定相关功能基因提供重要的试验基础。     

Phenylacetic acid stimulation of direct shoot formation in anther and somatic tissue cultures of rice (Oryza saliva L.)

The effect of phenylacetic acid (PAA) on rice (Oryza saliva L.) anther culture was investigated with six genotypes, using 2,4-D as control. In the two-step culture protocol, replacing 2, 4-D with PAA in the induction medium did not influence callus induction but significantly improved the shoot differentiation from callus, particularly in the indica cultivar Teqing. The anther-derived calli of all genotypes regenerated shoots directly on the callus induction medium containing PAA. Most of the directly-regenerated plantlets had well-developed root systems and were therefore readily transplanted into soil. The improved shoot differentiation potential and the frequency of direct regeneration depended on genotype, basal medium and PAA concentration. The one-step green shoot regeneration frequencies obtained were 1.98% with the indica cultivar ‘129’, 1.5% with the indica × japonica hybrid ‘Teqing/02428’ (F₁), and 1.98% with the indica × indica hybrid ‘Waiyin 2/C.B.’ (F₁). The PAA-based one-step method was most effective on the anther culture of indica genotypes. Three DH populations have been constructed from hybrids (F₁) via one-step culture. PAA also enhanced the one-step plantlet formation in rice somatic tissue culture.     

粳稻品种4502与广亲和品种间杂种F1不育性的遗传分析

利用９个籼、粳稻品种，７个广亲和品种，配制单交Ｆ１、回交及三交组合，研究了粳稻品种４５０２与广亲和品种间Ｆ１不育性的遗传。根据与品种４５０２杂交Ｆ１的亲和性，可将参试的广亲和品种分为两组，Ｉ组是４５０２亲和的品种，如Ｄｕｌａｒ和０２４２８；Ⅱ组是与４５０２不亲和的品种，如热研１号、Ｍｏｒｏｂｅｒｅｋａｎ和ＣＰＳＬＯ１７。回交和三交组合的育性分离表明，４５０２和Ⅱ组广亲和品种间杂种Ｆ１花粉不育性受单     

Anther culture of tropical japonica × indica hybrids of rice (Oryza sativa L.)

Summary Nine japonica × indica F1 hybrids of rice involving 6 indica and 3 japonica tropical varieties, were large scale anther cultured. The frequency of callusing anthers averaged 18.7%. The microspore-derived calli produced green plants with a mean frequency of 8.7%. Albino plants represented 61% of the shoot forming calli. Monitoring of the green and albino plant regenerating capabilities of calli arising between week 4 and week 8 of incubation of the anthers showed no increase of the albino/green ratio and a slow decrease of the shoot forming ability of the transferred calli after the sixth week of culture. Spontaneous doubled haploids (SDH) represented 46% of the regenerated green plants in 4 hybrids. However, a high frequency of partially sterile regenerants was noticed among 132 SDH plants generated from a hybrid.     

籼粳交加倍单倍体后代性状遗传的研究

对一个典型的籼/粳组合、窄叶青8号/京系17的F1花药进行培养,所获二倍体花粉植株除约20%自然结实率为0外,其余植株的结实率在1%至正常结实之间,65%株系在75%以上,并稳定地传至自交后代。对130多个加倍单倍体(DH)多种性状的遗传进行了研究与分析,结果表明,花药培养可以克服籼/粳后代包括育性在内的各种性状长期分离不易     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. III. Ability of winter wheat F₁ populations with the two heterozygous 1AL–IAS/1AL–IRS and 1BL–1BS/1BL–IRS chromosome pairs Application of anther culture to four F₁ hybrids between the IBL–IRS (‘Amigo’) and several 1BL–IRS wheat-rye translocation forms yielded 129 green pollen plants in an average embryo induction frequency of 17.6 %. A total of 2632 anthers was inoculated. 25 % and 42 % of the regenerated plants were haploid and spontaneously doubled haploid, and 33 % had abnormal chromosomal structure. After chromosome doubling treatment 87% of all pollen plants set seeds. By means of multiple peroxidases and Giemsa C-banding patterns, the anther culture progeny could be further classified into 16 plants without the short arm of IR-chromosome of rye, 21 IAL–IRS and 50 1BL–IRS translocation lines and into 16 IAL–IRS, IBL–IRS double translocation lines according to the four possible characteristic types of F₂ gametes of the tested F₁ hybrids. Advantages of the haploid technique for the selection of desirable traits and the meaning of the IRS genes in wheat are discussed.     

A new dominant earliness character of CMS line Zaoxian A and its application value in rice (Oryza sativa L.)

Summary A distant cross between a maintainer Zhenshan 97B and an early maturing F₂ palnt of O. longistaminata x O. rufipogon was made in 1985, and a new CMS line Zaoxian A was bred in 1989 through continuous backcross of new maintainer plants derived from the distant cross to the leading CMS line Zhenshan 97A. Several experiments were carried out in Sichuan during 1990–1992, to analyze the inheritance of earliness of Zaoxian A and its application value to hybrid rice production. The results showed that Zaoxian A was as early-maturing as Zhenshan 97A, the earliness of Zaoxian A was incompletely dominant to the lateness of restorer lines, and was different from the earliness of Zhenshan 97A which was suppressed by the lateness of restorer lines. The F₁ hybrids between Zaoxian A and late maturing restorers combined the early maturity and high yielding capacity, and significantly outyielded the provincial and national checks with same or longer growth duration, because the larger genetic distance between early and late maturing ecotypes remained unchanged and the new earliness could express in F₁ generation. Therefore, the new dominant earliness character of Zaoxian A could enhance the regional and seasonal adaptability of rice. It is also valuable to hybrid rice production and the formation of new cropping systems with high yielding capacity and high economical profit.     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.     

水稻同核异质广亲和不育系的选育及主要农艺性状分析

通过杂交、花药培养和籼粳属性分子标记检测, 育成广亲和保持系国广B. 以生产上广为应用的6种不育质源野败型、矮败型、冈型、 D型、印水型和K型为不育细胞质供亲, 以广亲和保持系国广B为细胞核供体, 进行同核异质广亲和不育系的转育, 经过连续7代回交作核置换育成一套同核异质广亲和不育系, 并对该套不育系的主要性状进行分     

Expression of resistance to Atherigona soccata in F1 hybrids involving shoot fly-resistant and susceptible cytoplasmic male-sterile and restorer lines of sorghum

In recent years, cytoplasmic male‐sterility (CMS) has been recognized as a potential danger to the stability of crop production and resistance to insect pests in sorghum. Therefore, the influence of CMS on the expression of resistance to sorghum shoot fly was studied at the ICRISAT, Patancheru, India using the interlard fishmeal technique. The experimental material consisted of 12 restorer, 12 CMS and the maintainer lines, and their 144 F₁ hybrids. Shoot fly‐resistant CMS lines were preferred for oviposition and had more damage because of deadhearts than the corresponding maintainer lines. The hybrids based on shoot fly‐resistant CMS × resistant restorer lines were significantly less preferred for oviposition than the hybrids based on other cross combinations and exhibited the highest frequency (69.1%) of shoot fly‐resistant hybrids. The hybrids based on glossy and trichomed parents had the highest frequency (>90%) of hybrids with glossy and trichome traits, emphasizing the need to transfer these traits into both parents for better expression in the F₁ hybrids. The expression pattern of trichome density, leaf glossiness and leaf sheath pigmentation in the F₁ hybrids and their parents suggested that the interactions between cytoplasmic and nuclear genes possibly control the expression of traits associated with resistance to sorghum shoot fly in the F₁ hybrids.