The Cultivation of Hybrid Cotton in Vitro to Overcome the Incompatibility of Hybridization between the Species

2—3 days after cross-pollination the hybrid ovules of cotton (Gossypium hirsutum×G. arboreum) were cultivated in vitro. The hybrid zygot in excised ovule started to divide, frequently passing through several stages to develop into cotyledonous embryos. The embryos were inducted into seedlings. The hybrid plants in blossom were obtained as a result. The histological study was done for ovules of self- pollination in vivo and of cross-pollination in vitro. The early abortion of endosperm in hybrid ovules was observed. In the prophase the nucellus was disintegrated slowly, and in the middle and terminal phase it disintegrated more rapidly. These were identified in hybrid F1. The size of corolla is intermadiate form between female and male. The violet-red spots at the base of petals are similar to male, while there are no such spots in female. None of the pollen can fertilize. Hybrid F1 is not fertile after self-pollination or backcross by the pollen of parent. The chromosome number in root tip Cell of hybrid F1 is 2n=3x=39.     

Embryo rescue and plant regeneration in vitro of selfed chickpea (Cicer arietinum L.) and its wild annual relatives

The main constraint to the transfer of desired traits into cultivated chickpea from wild Cicer relatives is the presence of post-zygotic barriers which result in abortion of the immature embryo following interspecific hybridisation. Rescue of hybrid embryos in vitro and regeneration of hybrid plantlets could allow chickpea breeders to transfer desirable traits from wild relatives of chickpea. The development of embryo rescue techniques using selfed chickpea and selfed wild relatives is being used as a first step to protocols for wide hybrids. Optical microscopy studies of embryogenesis, in both selfs and hybrids, identified deleterious changes in the fertilised hybrid seed as early as 2–4 days after pollination in some crosses. These observations suggest that the appropriate time to rescue chickpea × C. bijugum hybrids is at the early globular stage of embryogenesis (2–7 days old), which requires the development of a complex tissue culture medium. In contrast hybrids between chickpea × C. pinnatifidum abort later (up to 15–20 days old) at the heart-shaped or torpedo stages, and are easier to rescue in vitro. Genotype also plays a significant role in the ability of immature selfed ovules to germinate in vitro. In this paper we report on the optimisation of␣protocols for rescueing immature embryos using selfed chickpea and its wild relatives in ovule, and subsequently to regenerate plantlets.     

中棉×戴维逊氏棉胚胎发育的研究

以棉花栽培种中棉作母本,野生种戴维逊氏棉作父本进行杂交试验,并用中棉自交作对照,比较研究了杂交情况下花粉粒的萌发、花粉管的生长、受精作用及胚和胚乳的发育过程,得到以下结果:(1)中棉×戴维逊氏棉花粉粒的萌发及花粉管在异己花柱中的生长基本正常,有花粉管胚珠的频率约20％,为中棉自交的1/4左右;(2)在杂交情况下,有花粉管进入的胚珠基本上能实现受精;(3)杂种胚乳在授粉后7天发育异常,11天开始解体,16天才有部分胚珠的胚乳开始形成细胞壁;(4)杂种胚不分化或畸形分化,在授粉后11—22天坏死。     

鹤顶兰花粉管在子房中的生长途径

运用扫描电镜对鹤顶兰(Phaiustankervilliae(Aiton)Bl.)花粉管在子房内的生长途径进行了观察。结果表明:花粉管在子房中的生长途径可以分为3个阶段:(1)沿子房壁轴向生长阶段,从授粉开始至大孢子母细胞四分体时期,花粉管经过合蕊柱到达子房,经由胎座基部沿子房壁轴向生长;(2)沿子房径向生长阶段,二核胚囊之后,花粉管在胚珠之间穿梭,以径向生长为主;(3)朝珠孔定向生长阶段,胚囊成熟时,花粉管朝珠孔定向生长进入胚囊。实验结果说明花粉管的定向生长受胚珠的分子信号调控。     

Unfertilized ovules of Epilobium obcordatum (Onagraceae) continue to grow in developing fruits

To determine whether unfertilized ovules continue to grow when in an ovary containing fertilized ovules, we measured ovule lengths in developing fruits of Epilobium obcordatum that were harvested 4, 5, 8, and 10 d post pollination. We found that unfertilized ovules that were in the presence of fertilized ovules continued to grow and that there was a broad range of overlap in their sizes at all sampling times. This effect was found for two types of unfertilized ovules that occur throughout the length of the ovary: normal, unfertilized ovules, apparently bypassed by pollen tubes; and sterile ovules lacking an embryo sac. In addition, there is a position effect within developing fruits. Both fertilized and unfertilized ovules are larger at the stylar end. In six samples resulting from pollination with a single pollen tetrad, a total of 18 embryos were found, and the effect on unfertilized ovules, greatest at the stylar end, diminished with distance from the ovules with embryos. Our results are consistent with the interpretation that diffusible hormones produced by developing seeds cause nearby unfertilized ovules to grow. We conclude that caution is necessary when attempting to infer ovule fertilization histories from the appearances of ovules in developing and mature fruits. What are often inferred to be aborted seeds, in many cases, may not be seeds at all. They may be enlarged, unfertilized ovules.     

Intergeneric crossability studies on obtaining hybrids between Salix viminalis and four Populus species

For the first time, hybrid embryos and plantlets were obtained from in vitro-pollinated whole catkins of Salix viminalis with pollen grains of Populus tremula, P. tomentosa and P. alba. Only hybrid embryos were obtained from cross S. viminalis with P. lasiocarpa. Pollen of poplar species germinated on stigmas of willow and some pollen tubes were observed growing into the ovary. After 36–48 h of pollination, several-celled globular embryos were found inside the embryo sacs. During the next 14 days in some ovules, the process of embryogenesis proceeded similarly as in the control material, that is after selfing in vitro. In some other embryo sacs embryos degenerated mainly at the globular stage. Endosperm development was poor or not present. Out of 599 catkins, which jointly included around 80.000 pistils, 141 fully developed embryos were isolated and transferred into MS medium with whole or half concentrations of macroelements. From about 65% of the cultured embryos, plantlets were established. Further investigations are in progress on finding the suitable environmental conditions for culturing plantlets in soil and establishing the hybrid characteristics of the progeny.     

COORDINATED TIMETABLES FOR MEGAGAMETOPHYTE DEVELOPMENT AND POLLEN TUBE GROWTH IN RHODODENDRON NUTTALLII FROM ANTHESIS TO EARLY POSTFERTILIZATION

Rhododendron nuttallii T. W. Booth (Ericaceae) was used to derive concurrent timetables for megagametophyte, pollen tube and early postfertilization development from anthesis through 3 wk after pollination, based on timed collections of self-pollinated pistils. Stages of development were determined for over 33,500 cleared ovules, including, for selected collection dates, stages on different portions of the placenta. Pollen tube information was obtained by fluorescence microscopy of pistil squashes stained with aniline blue. Because of the very large number of ovules observed, it was possible to recognize a much more closely graded series of stages in megagametophyte development than is usually the case. While a range of stages occurred on all days, development progressed steadily from a majority of functional spores and 2-nucleate gametophytes on the day of anthesis to mostly a late zygote-primary endosperm stage at 18 days, and some 2-celled endosperm stages at 21 days, after pollination. At all times the most advanced stages, including first pollen tube entries, occurred on the outer surface of the lower half of the placenta, and the youngest on the inner surface of the uppermost portion. Fertilizable ovules were not found in any frequency until 8 days after pollination (then in only about 34% of the ovules); a few fertilized ones were seen after 10 days but constituted less than 5% until 12 days after pollination, thereafter increasing to about 60%. Fertilization occurred in any one of three morphologically recognizable stages distinguished by position and state of fusion of polar nuclei. Pollen germinated on the stigma 1–2 hr after pollination, and pollen tubes grew at a rate of about 1–1.25 cm/day, reaching the top of the ovary in 8–9 days with the first ovule entries seen after 10 days. There was a close correlation between megagametophyte development and pollen tube growth, with large numbers of functionally mature ovules not being found until pollen tubes had reached the ovary. While nuclei within ovules could not be distinguished in the squashes, three gametophyte stages that could be recognized—unelongated, elongated either without or with a pollen tube—were tallied for almost 29,000 ovules. The progression in these general stages corresponded well with that documented in more detail from cleared ovules. Unpollinated pistils showed a similar progression of gametophyte stages until the time fertilization would start to occur, after which there was continued accumulation of functionally mature ovules. A variety of abnormally developed and/or collapsing(ed) ovules or gametophytes were seen; collectively, they averaged over 8.6% of all ovules.     

Fossil floral and fruit evidence for the evolution of unusual developmental characters in Fagales

Flowers of many living Fagales exhibit unusual developmental characteristics. At anthesis, ovulate flowers have carpels bearing immature orthotropous ovules. After pollination, the ovules increase in size and become anatropous and the ovary enlarges. Simultaneously, the pollen tubes extend from the stigma to the ovules with several phases of growth and quiescence. Finally, after the first fertilization, the remaining ovules abort, resulting in a single‐seeded fruit. Three‐dimensionally preserved potentially fagaceous mesofossil flowers from the Campanian of Massachusetts, USA, provide evidence on the evolution of these characters. The fossils share putative synapomorphies with the Fagales (six tepals, mostly inferior, three‐carpellate ovary with each locule initially containing two pendant ovules, punctate‐rugulate, tricolporate pollen and fruit with a single seed). However, the fossil is bisexual and has nectaries, characters shared with the sister order Cucurbitales, and both lack the fagalean immature orthotropous developmental stage. The fossil shares synapomorphies of an inferior ovary and a single‐seeded indehiscent fruit with both living orders and appears to be transitional. Comparison of ontogenetic changes between the fossil and related fagalean taxa suggests independent stepwise changes in development in which some characters of the modern clades were in place at ～ 75 Myr and others evolved more recently. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 353–376.     

Delayed fertilization and pollen-tube growth in pistils of Fagus japonica (Fagaceae)

In contrast to most angiosperms, in which fertilization occurs 1 or 2 days after pollination, in some plant orders, including the Fagales, fertilization is delayed from 4 days to more than 1 year, raising questions regarding why fertilization is delayed and where and how pollen tubes remain in the pistil during the delay. To answer these questions, we investigated pollen-tube growth in pistils of Fagus japonica (Fagaceae), which are tricarpellate and have six ovules, using light, fluorescence, and scanning electron microscopy. The ovules were immature at the time of pollination and required 5 weeks to become fully developed. During this 5 weeks, pollen tubes grew from the stigma to the embryo sac in association with the development of ovules and intermittently in three steps with two growth-cessation sites, i.e., on the funicle and near the micropyle. The number of pollen tubes was gradually reduced from many to one at the two growth-cessation sites, and fertilization occurred in one ovule that apparently developed earlier than the others in the pistil. Thus, delayed fertilization plays an important role in gametophyte competition and selection leading to nonrandom fertilization. Intermittent pollen-tube growth is also likely widespread in angiosperms because it is known in other Fagales and an unrelated order Garryales.     

Investigation of Sexual Process in Interspecific Crosses Between Gossipium hirsutum and G. arboreum

The present investigation is the first of a series of cytological researches on inter-specific hybridization of cottons. The present report deals with the cytological behavior of sex and related cells revealed in crossing Gossipium hirsutum (female parent) with G. arboreum. Self-pollenated G. hirsutum served as control. The germination of pollen grains on the stigma and the growth of pollen tubes in the style and the entry of pollen tubes into the micropyles in crosses are in a fairly normal' manner when compared with controls. The percentages of pollen tubes entering ovules of the crossed and controls have no significant difference, being 48.2% in crosses and 54.7% in controls. Fertilization proceeds also normal in cross pollinated ovules. About 92.7% of pollinated ovules are found to have completed the fusion of sexual elements. It is thus: shown that the growth of foreign pollen tubes and fusion of female elements with foreign sperms are not the factors which cause failure in hybridization. The development of endosperm in crosses is abnormal. The number of free nuclei of hybrid endosperm is much smaller than that of controls; the formation of hybrid cellular endosperm is 6 or 7 days earlier than that of controls. The hybrid endosperm cells start to abort soon after their formation. About 14 days after pollination they become completely disintegrated. The hybrid embryo which thus far developed normally becomes arrested in its development and aborts also after that time. It may be concluded that the sterility of crosses between G. hirsutum and G. arboreum is primarily attributed to the abortion of hybrid endosperm. The sound development of the hybrids should be sought in the improvement nutrition of the hybrid embryo from resource other than their own endosperm.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.     

Overcoming prefertilization barriers in the crossDiplotaxis siettiana ×Brassica juncea using irradiated mentor pollen

Hybridization between the nearly extinct speciesDiplotaxis siettiana andBrassica juncea is prevented because of strong prefertilization barriers. Use of mentor pollen ofD. siettiana irradiated with 1000 Gy gamma radiation before the incompatible pollination led to fertilization. 5 d after pollination 17% ovules showed entry of pollen tubes, 10 d after pollination 27% ovules showed small globular embryos which grew no further. No embryos were found in control pollinations. Thus , use of irradiated mentor pollen brings about fertilization in this difficult cross and hybrids can be obtained if embryos are rescued.     

Overcoming prefertilization barriers in the crossDiplotaxis siettiana × Brassica juncea using irradiated mentor pollen

Hybridization between the nearly extinct speciesDiplotaxis siettiana andBrassica juncea is prevented because of strong prefertilization barriers. Use of mentor pollen ofD. siettiana irradiated with 1000 Gy gamma radiation before the incompatible pollination led to fertilization. 5 d after pollination 17% ovules showed entry of pollen tubes, 10 d after pollination 27% ovules showed small globular embryos which grew no further. No embryos were found in control pollinations. Thus , use of irradiated mentor pollen brings about fertilization in this difficult cross and hybrids can be obtained if embryos are rescued.     

焦核与大核龙眼雌配子体发育、授粉受精及早期胚胎发育的比较研究

利用人工授粉,采用压片法对大核龙眼‘九月乌’和焦核龙眼‘闽焦64-1’、‘闽焦64-2’、‘白核’等的自交与杂交后花粉管的生长特性进行研究,同时应用常规石蜡切片技术对大核与焦核龙眼的雌配子体以及合子胚早期发育进行观察。结果表明,龙眼胚珠在单核胚囊形成前就开始败育,且焦核品种(系)的败育率显著高于大核品种。不同亲本组合的授粉率存在差异,所有授粉组合在授粉36～48 h后均能观察到1个花粉管生长并进入胚囊受精。焦核品种(系)的胚胎在谢花后10 d开始败育,且败育率明显高于大核品种。受精是龙眼子房发育的首要条件,胚珠败育的雌蕊在谢花后10 d不膨大,不能发育形成焦核果实。谢花后10～30 d的早期胚胎败育是形成焦核龙眼的主要原因。焦核品种‘白核’胚乳具有成胚能力。约有24%的‘闽焦64-1’胚珠在胚胎发育过程中,其助细胞、合点端细胞及胚乳发生异常,这可能与早期胚胎败育有关。     

Fruit harvesting time and corresponding morphological changes of seed integuments influence in vitro seed germination of <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

In vitro asymbiotic seed germination of Dendrobium nobile varied significantly with fruit harvesting time and growth medium used for culturing seeds. Seeds harvested 129 days after pollination (DAP) possessing globular shaped embryos and a discontinuous cuticle layer showed a substantially greater germination on P668 medium. Alternatively, immature seeds harvested 96 and 116 DAP displayed a significantly lower germination response on various growth media. Most of the ovules at 96 DAP are in archesporial and megaspore mother cell stages, whereas the majority of ovules are mature and fertilized at 116 DAP. Mature seeds harvested 158 DAP also germinated at a higher frequency at Stage 5 (emergence of the first leaf) after 8 weeks of culture on different growth media indicating the absence of testa imposed dormancy in this endangered epiphytic orchid.     

Cytological Studies on the Embryogenesis of Hybrids between Oryza sativa L.(Pennisetum sp.

(1) The pollen grains of Pennisetum can germinate normally on the stigma of rice and the pollen tubes can grow into the style and enter the embryo sacs. However, the process of double fertilization is slow and more or less abnormal and phenomenon of simple fertilization often occurs. (2) It has been found that in the majority of cases the development of the embryos is slow and stays long in the stage of globular embryos, thus, the differentiation of the embryos is very difficult and degeneration of the embryos appears many times. Simple differentiation was observed only in some embryos during 16–24 days after pollination. Normal differenting and developing embryos were not observed. The cause of the degeneration of the embryos is related to the state of endosperm development and also to the non-coordination of the genomes of both parents. (3) The development of the endosperm is abnormal. The change from the free nuclei into the cells in the endosperm is delayed as late as the 8th day after pollination. The whole endosperm tissue is composed of the cell masses which are quite different both in shape and function, a part of these endospemn cells lacks the ability to synthesize starch. The disintegration of the endosperm could be frequently observed during their development. (4) A lots of starch are accumulated in the nucellar cells near the antipodals, It is shown that there was some metabolic confusion resulted from the crossing in the embryo sacs. Based on the above mentioued results the authers consider that the failure of producing seeds by crossing is at least related to the nutrient condition which are essential for the development of embryos. If embryo culture technique is employed at the early stage of the embryo development the hybrid seeds could be obtained.     

云南野山茶与金花茶杂交有性过程的观察

作者对云南野山茶与金花茶种间杂交的有性过程进行了观察,得到如下结果: 1.金花茶花粉粒在野山茶柱头上于授粉后4小时开始萌发。花粉管在花柱中的伸长基本正常。 2.双受精于授粉25—30天前后发生。杂种幼胚及胚乳早期的发育较为正常,授粉后第85天杂种胚与对照相比较,在胚的分化程度和胚乳的发育方面都未见明显差异,所观察到的胚有70％以上进入了鱼雷期。 3.授粉第90天以后,幼胚胚芽开始分化,自交胚生长发育极为迅速,而杂种胚在生长和分化方面都显著地迟缓下来。这可能是杂种胚与母体组织遗传和生理上的某些不协调所致。因此适时地进行杂种幼胚的离体培养,对育种工作是十分必要的。     

Immature embryos culture in Italian red chicory

Chicory (Cichorium intybus L.) embryo maturation in vitro was achieved in B5 medium without growth regulators. Immature embryos were collected at different developmental stages 4–168 h after pollination. Only a few plantlets were obtained from ovules collected shortly after pollination since the embryos at these early stages developed abnormal leaves and roots which regenerated shoots ‘via’ organogenesis. Zygotic embryos collected 24–72 h after pollination needed one month of culture in vitro before acclimation. Embryos collected at the heart or early torpedo stage of development (three days after pollination) produced green plantlets after two weeks of in vitro culture and thus they could be transferred to the greenhouse for acclimation. Culture of zygotic embryos in vitro has shown to be a suitable technique to accelerate the breeding process in biennial cultivated Italian red chicory. In fact it is possible to obtain plantlets ready for performing the selection for the desired agronomic traits one month after pollination. Since flowering occurs after vernalization, the selection can be performed in the correct season before flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hybridization between Gossypium herbaceum and Gossypium stocksii through embryo rescue

INTRODUCTIONGOSndumherbaceuminoneofthetwo'A'genomediploidcultivatedspeciesofcotton.ItisgrowninsomepartsofcentralIndia,esDeciallyasarainfedcrop.G-sto~isawiIdspeciesbelongingto'E'ge11omeandisfoundinWestAsia.Thisspecieshasaverygoodrootsystemandistoleranttodrought[1]-Thus,thehybridizationbetweenG.herbaceumandG.stock8iisdesiredtodevelopdrought-tolerantvarietiesinG.herbaceum.Theattemptstoproduceinterspecifichybridsbytheconventionalmethodsareoftenunsuccessfulduetotheformationofunderdevel-op…