应用胚挽救技术获得三倍体柑橘植株

以3个柑橘异源四倍体体细胞杂种(即四倍体粗柠檬与哈姆林甜橙体细胞杂种,简称"HR";酸柚与粗柠檬体细胞杂种,简称"SR";墨西哥来檬与伏令夏甜橙体细胞杂种,简称"KV")为父本,分别与二倍体单胚性沙田柚进行有性杂交,在有性胚还没有完全败育以前,通过胚抢救技术进行三倍体植株培养(以四季柚花粉亲本为对照)。结果表明,处理的杂交组合直接成苗率极显著低于对照(P0.01),其中又以沙田柚×HR组合成苗率相对较高,达10.1%;较适合瘪籽沙田柚幼胚离体培养的培养基是MT+GA31.0mg/L+蜂皇浆200mg/L+水解乳蛋白250mg/L+蔗糖30g/L+琼脂0.7%;流式细胞分析仪检测结果显示,本试验成功获得了柑橘三倍体植株。     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙+粗柠檬”及其亲本的花粉活力、花器官发育、花器官形态发生与花粉母细胞减数分裂四分体阶段进行了观测和统计.结果发现“哈姆林甜橙+粗柠檬”的花粉染色活力、萌发率、每花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点.但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲.以体细胞杂种“哈姆林甜橙+粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵.     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙＋粗柠檬”及其亲本的花粉活力,花器官发育,花器官形态发生与共母细胞减数分裂四分体阶段进行了观测和统计,结果发现：“哈姆林甜橙＋粗柠檬”的花粉染色活力,萌发率,线花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点,但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲。以体细胞杂种“哈姆林甜橙＋粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵。     

RAPD为基础的柑橘体细胞杂种有性后代遗传研究

筛选31条谱带清晰、多态性强及重复性好的10 bp引物, 运用RAPD技术对以异源四倍体柑橘体细胞杂种(哈姆林甜橙(Citrus sinensis Osbeck)+粗柠檬(C. jambhiri Luss))为父本与二倍体单胚类型宜本杂4号(华农本地早橘(C. reticulataBlanco)×宜昌橙(C. ichangensis Swingle))杂交获得的杂种后代进行遗传分析. χ₂测验表明: 杂交父本与母本的特异标记总体上是随机传递给后代的, 父本即柑橘异源四倍体体细胞杂种融合双亲的单个特异标记也是随机传递给后代的. 通过组群平均数假设测验, 二倍体组群与三倍体组群间各类亲本标记的平均数不存在显著差异. 基于遗传距离的聚类结果显示: 杂种后代聚为3类: 第1类与融合亲本之一哈姆林甜橙聚为一类, 有15株杂种后代; 第2类与母本宜本杂4号聚为一类, 有16株杂种后代; 第3类与父本体细胞杂种(哈姆林甜橙+粗柠檬)聚为一类, 有61株杂种植株, 大多数杂种后代与父本的遗传关系更为密切.     

柑橘体细胞杂种有性后代的创造及杂种鉴定

以异源四倍体柑橘体细胞杂种[哈姆林甜橙(Citrus sinensis Osbeck) 粗柠檬(C.jambhiri Luss)]为父本与二倍体单胚类型宜本杂4号[华农本地早橘(C.reticulata Blanco)×宜昌橙(C.ichangen- sis Swingle)]杂交,采用胚抢救技术获得了110株有性后代植株,通过染色体计数及倍性分析仪鉴定,其中93株植株为3倍体,余下的17林植株为2倍体。RAPD分析表明:获得的有性后代植株均为杂种。     

两类柑橘杂种砧木对脐橙幼树生长及若干生理生化指标的影响

以体细胞杂种红橘+枳、红橘+粗柠檬和有性杂种Swingle枳柚、Troyer枳橙(对照)作砧木的脐橙幼树为试材,以盆栽试验研究砧木对幼树的影响。结果表明,体细胞杂种砧木处理和有性杂种Swingle枳柚的树体生长势总体上强于对照;叶片可溶性糖含量较高的是红橘+粗柠檬处理和对照;叶绿素含量最高的是红橘+粗柠檬处理。两年中叶片POD活性分别与当年树冠体积呈负相关。     

柑桔种间体细胞杂种的花粉特性及贮藏研究

对粗柠檬与“哈姆林”甜橙种间体细胞杂种的花粉特性(形态、花粉量、生活力等)进行了观察、检测,并于-20℃的低温条件下开展了贮藏试验。结果表明：花粉离体萌发的适宜培养基为0．8％琼脂 16％蔗糖 10mg／kg硼酸,萌发率为15．31％,其生活力介于两亲本之间,可作为杂交亲本进一步被利用。贮藏试验结果显示：以真空、干燥(CaCl2或硅胶作干燥剂)贮藏花粉的效果较好。授粉试验证明,贮藏花粉与新鲜花粉的座果率差异不显著,说明低温、真空和干燥是贮藏粗柠檬与“哈姆林”甜橙种间体细胞杂种花粉的较适条件。     

不同砧木对柑橘幼树生长和叶片糖含量的影响

以体细胞杂种红橘 枳和红橘 粗柠檬、有性杂种Troyer枳橙和Swingle枳柚、枳(对照)作砧木的‘国庆4号’温州蜜柑2a生嫁接苗为试材,通过盆栽试验研究了砧木对柑橘幼树生长和叶片糖含量的影响。结果表明,4种处理的生长势比对照旺盛;红橘 枳处理的花量极显著多于对照,其余3种砧木处理的则极显著少于对照。在光合色素含量上,Troyer枳橙处理最高,对照最低。不同砧木处理对叶片可溶性糖含量的年变化动态特征影响明显;不同砧木处理的叶片淀粉含量年变化动态特征相似。在不同时期,不同砧木处理对叶片可溶性糖和淀粉含量有明显的影响。初步评价了体细胞杂种红橘 枳和红橘 粗柠檬用作柑橘砧木的利用价值。     

甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。     

甜橙与酸橙体细胞杂种核质组成鉴定

采用流式细胞术(flow cytometry,FCM)、简单重复序列(simple sequence repeat,SSR)和酶切扩增多型性序列(cleaved amplifiedpolymorphic sequence,CAPS)等技术分析酸橙(Citrus aurantium L.)叶肉原生质体和甜橙(C.sinenis Osbeck cv.Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种.FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体.用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本.在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种.通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样.结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本.讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用.     

以异源四倍体体细胞杂种为父本杂交培育三倍体柑桔植株的研究

以 3个柑桔原生质体融合而来的四倍体体细胞杂种为父本 ,与二倍体单胚性种柚子 (Citrusgrandis)以及单多胚混合型品种“华农本地早”桔 (C.reticulata)有性杂交 ,授粉后 90 d,发现种子干瘪 ,大部分种子的胚败育。将干瘪种子在 MT附加 1mg/L GA3 或 50 0 mg/L麦芽浸出物的培养基中 ,经培养抢救 ,有 2 5.6%的种子萌发成苗或继续进行胚的生长 ,后者进一步诱导能形成丛芽 ,经试管嫁接或诱导生根形成完整植株。共获得 6个组合 73棵完整植株 ,染色体数检查表明 ,2 0株为三倍体 (2 n=3x=2 7) ,32株为二倍体 (2 n=2 x=18) ,8株为非整倍体 ,其它 13株还有待于进一步检查。     

Triploid Citrus Plants Obtained from Crossing the Diploids with Allotetraploid Somatic Hybrids

Hybridizing the diploid monoembryonic pummelo (Citrus grandis) and polyembryonic tangerine (C. reticulata cv. Huanongbendizao) with allotetraploid somatic hybrids from protoplast fusion were conducted. Seeds of pollinated fruits were found to be abortive 90 days after pollination. The aborted seeds were then cultured on media of MT supplement with 1 mg/L GA3 or with 500 mg/L of malt extract. 25.6% of the seeds germinated or developed into embryoids: The entire plants were transplanted into soil after grafting shoots on the root-stocks of trifoliate orange in vitro, if the germinated embryos have poor roots or no root at all. A total of 73 plants from 6 different combinations were obtained, among which 20 were verified as triploids with 2n= 3x=27 chromosomes, 32 diploids 2n= 2x= 18, 8 a- neuploids and the rest 13 unconfirmed.     

‘沙田’柚果生长期套袋对果实品质的影响

‘沙田’柚果生长期进行套袋,是提高果实品质的有效措施之一。在田间生长条件下,研究了套袋对‘沙田’柚果实品质的影响。结果表明,套袋促进了柚果生长发育,提高了商品率,降低了含酸量,较明显地提高其糖酸比,并增加了维生素C的含量,从而提高了柚果品质。     

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Somatic hybridization in citrus: An effective tool to facilitate variety improvement

Summary Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.     

不同贮藏条件对沙田柚果实苦味物质含量的影响

沙田柚原产广西,营养丰富且适合高血压和糖尿病等患者食用,近年来在沙田柚产业中存在品质下降、有苦味、异味生成等问题。为了研究不同贮藏条件下沙田柚苦味物质的含量变化,该文利用高效液相色谱仪,对室温包薄膜袋(A)、室温不包薄膜袋(B)和4℃低温不包薄膜袋(C)三种不同贮藏条件下,沙田柚外果皮、中果皮、囊衣、汁胞和种子中柚皮苷、柠檬苦素和诺米林在贮藏过程中的含量变化进行了测定。结果表明：沙田柚果实苦味物质以柚皮苷为主,主要分布在中果皮和囊衣中,柠檬苦素主要分布在种子和外果皮中,诺米林主要分布在种子中。各处理果实在贮藏过程中苦味物质含量变化不同,其中柚皮苷含量略有上升,柠檬苦素和诺米林含量整体呈下降趋势,略有波动,而汁胞中柠檬苦素和诺米林含量先上升再下降。三种不同贮藏条件相比,4℃低温不包薄膜袋贮藏的果实汁胞中柚皮苷和柠檬苦素的含量最高,在贮藏第30天,A、B、C 三种不同贮藏条件果实汁胞中柚皮苷含量分别为(0.139±0.006)、(0.190±0.009)和(0.194±0.019) mg?g-1,柠檬苦素含量分别为(47.28±1.91)、(33.64±1.90)和(84.19±5.56)μg?g-1。与此相反,外果皮、中果皮、囊衣和种子中柚皮苷、柠檬苦素和诺米林含量最低。该研究结果为采后沙田柚贮藏保鲜方式的选择提供了理论指导。     

细胞壁代谢与琯溪蜜柚果实成熟过程汁胞粒化的关系

以易发生汁胞粒化的老龄树和不易发生汁胞粒化的适龄树的琯溪蜜柚（Citrus grandis（L．）Osbeck‘Guanximiyou’1果实为材料,研究了果实成熟过程中汁胞粒化发生与细胞壁代谢的关系。结果表明：老龄树果实的汁胞粒化指数随着果实成熟而上升。在汁胞粒化发生过程中,汁胞维持较低的细胞壁降解酶[果胶甲酯酶（PE）、多聚半乳糖醛酸酶（PG）、纤维素酶（Cx）]活性,保持较高的细胞壁物质（原果胶、纤维素、半纤维素）含量;尤其在汁胞粒化的起动阶段和加快阶段,纤维素、半纤维素含量极显著增加。相反,适龄树果实的汁胞粒化指数在果实成熟过程中变化不大,汁胞中细胞壁降解酶活性较高,促进原果胶、纤维素、半纤维素等细胞壁物质的降解,保持较低的细胞壁物质含量,使汁胞发育正常、柔软多汁。这说明PE、PG、Cx活性和原果胶、纤维素、半纤维素含量与錧溪蜜柚汁胞粒化密切相关。     

Citrus tissue culture employing vegetative explants

Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth culture of shoots of C grandis without loss of regeneration capacity during 31 years, observed so far, are some other significant results. Plants of C. aurantifolia and C. sinensis raised from shoot meristem and micrografting were grown in a nethouse and those from nodal stem segments in the field along with the in vitro-raised plants of rootstocks, namely, C. jambhiri, C. karna and C. limonia. All the plants showed normal healthy growth. Significantly enough, the meristem regenerated plants of C. aurantifolia attained the reproductive phase just in 1 year of transplantation to soil similar to those raised from nodal stem segments of mature trees, which also produced normal fruits in the subsequent year while growing under field conditions. Thus, a significant fundamental concept of a maturity factor, carried over through as small a shoot meristem as 200 microm in length to cloned plants has been demonstrated. The concept is of far-reaching significance in citrus industry besides production of pathogen-free orchards.