Growth and Development of Staminate Inflorescence and Anatomic Observation of Male Chestnut Flower

Juan CHEN,Yuting NIE,Jin LIU,Hongyan YAO,Shuiyuan CHENG*

1.College of Life Science/Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization/Hubei Collaborative Innovation Center for the exploitation of Dabie Mountains Featuring Resources,Huanggang Normal University,Huanggang 438000,China;

2.Postdoctoral Research Station of Biology of Yangzhou University,Yangzhou University,Yangzhou 225009,China

Growth and Development of Staminate Inflorescence and Anatomic Observation of Male Chestnut Flower

Juan CHEN1,2,Yuting NIE1,Jin LIU1,Hongyan YAO1,Shuiyuan CHENG1*

1.College of Life Science/Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization/Hubei Collaborative Innovation Center for the exploitation of Dabie Mountains Featuring Resources,Huanggang Normal University,Huanggang 438000,China;

2.Postdoctoral Research Station of Biology of Yangzhou University,Yangzhou University,Yangzhou 225009,China

The growth and development of staminate inflorescence and anatomic structure of male chestnut flower were observed.Results showed that staminate inflorescence on the base of branch formed first,then upward successively.About 50 days were needed from the formation of staminate inflorescence on the base of branch to fully develop the staminate inflorescence on the top of the branch.On the same staminate inflorescence,male flower clusters of the base formed first, then upward successively.About 20 days were needed from the formation of staminate inflorescence on the base of the male flower cluster to fully develop the staminate inflorescence on the top of the branch.5-7 male flowers forming a cluster,the flower number in a cluster was odd number usually,and there was one on the top and each two parallelly arranged downward.The flower on the top came into bloom first,and then downward successively.The flowers parallelly arranged came into bloom at the same time.Sporangium of male flower of chestnut was monolocular. There were a large number of pollen grains in the sporangium.There were large differences between the development process of different sporangium in one male flower.Chestnut had larger quantity of male flowers and pollen and long period of pollination compared with female flower.It is remained to be further studied whether it was necessary for anemophilous pollination.

Chestnut(Castanea mollissima Blume);Staminate inflorescence;Male flower;Anatomic structure;Microsporangium

C hestnut(Castanea mollissima Blume)is a member of the genus Castanea Family Fagaceae,and a species of chestnus native to China.With extremely strong adaptability,it has strong drought resistance and barren resistance,and it can develop in large amount in the barren hills and beaches,promising high nutritional and medical value. Therefore,honored as the“iron crop”, it has become one of the preferred e conomic useful tree varieties to carry out the plant industry construction adjustment in some mountainous area to increase the income of farmers in China a lot of development in the barren hills,beaches,and has high nutritional and medicinal value,make it become the structural adjustment of planting in dustry in many mountainous areas in China[1].In production,it is believed that chestnut is abundant in male flowers,while there are relative fewer

Materials and Methods

Test materials

Chestnut came from the wellgrown chestnut trees in the tangerine gardens of No.3 Xingang Road, Huangzhou City.The growth and development rules of the staminate inflorescence were observed and recorded regularly.In the meantime, the staminate inflorescences grown to a certain time were dissected directly to observe its structure under the stereoscopic microscope.

Observation of the structure of male flowers and sporangium

Paraffin section method was used to observe the structure of male flowers and sporangium.A male flower cluster with the staminate inflorescence at the base not fully flowering was selected and picked out from the inflorescence axis using tweezers.After fixing for 24 h using FAA stationary liquid,the following steps were carried out one by one:ethanol was used to do the gradient elution,xylene to transparent step by step,paraffin to embed,Leica RM2145 semithin microtome to cut into pieces,hematoxylin eosin to stain,Nikon eclipse E200 optical microscope to observe the inner structure of the anther,and Tiger ProMedia camera was used to take photos.

Determination of absolution spectroscopy of pigment in anthers

At first,100 anthers wrapped inside the flowers were selected and picked out using tweezers carefully. And then the 80%acetone extraction was used to do the extraction,and, AgilentCary 100 UV-Vis spectrophotometry was used to determine its absolution spectroscopy.

Results and Analysis

Growth and development of staminate inflorescence of chestnut

The observation results showed that the buds to differentiate the staminate inflorescence was wrapped in the multilayer bud scales to live through the winter(Fig.1a),and began to germinate in early March when the bud scales began to fall off gradually.The stipules of the blades on the base appeared first,which were very large, making it wrap the outer primordium tightly(Fig.1b).Leaves appeared in the middle of March(Fig.1c),and small protuberate structure could be seen at the axils after stripping off the stipules and blades(Fig.1d),which might be the primordium of staminate inflorescence.The staminate inflorescence on the base began to stick out leaves in early April(Fig.1f,1g),and with time passing by,then upward successively.On around May 10,filaments began to show on some of the flower clusters on the bases of some of the branches(Fig.1h).On around May 20,the male flowers on the 3-4 staminate inflorescence on the base all flowered,and the filament of fullyflowered male flowers straightened, and the anther was located at the top of the filament,at the very outside of the whole inflorescence(Fig.1i).On each bearing branch,there were 5-13 staminate inflorescences with the axial length of the staminate inflorescence about 5-25 cm.Generally speaking,the staminate inflorescence axis at the middle was longer than that at the base and top,and some of bases the staminate inflorescence on the top could differentiate female flower clusters(Fig.1i).On one bearing branch, 50 d were needed from the formation of staminate inflorescence on the base of branch to fully develop the staminate inflorescence on the top of the branch,and about 20 d were needed from the formation staminate inflorescence on the base of the male flower cluster to fully develop the staminate inflorescence on the top of the branch.

Growth rules and flowering order of chestnut male flowers on the staminate inflorescence

The test statistics showed that 5-7 male flowers formed a cluster, forming a cyme,and 50-200 cymes grew on the same floral axis,forming catkin(Fig.1h).The number of male flowers was the smallest on the base and top of the same flower clusters at the inflorescence axis,and relative larger in the middle,and there were about 500 male flowers in the same inflorescence axis on average.On the same staminate inflorescence,male flower clusters of the base formed first, then upward successively,and the male flower clusters at the top bloomed at last,belonging to indefinite inflorescence(Fig.2 a,2b,2c).However,there were a few flowers at the top showed filament in advance(as shown with the arrow in Fig.2c).In the same male flower clusters,the flower at the top was the biggest,and flowers in the middle and base were in symmetrical arrangement,with the two flowers in the middle larger than that in the base.Just the very opposite to that of the clusters in the catkin,the flowers at the top of the male flower clusters flowered first,and the two flowers in the middle bloomed almost at the same time,which were all prior to the flowering of the two flowers at the base(Fig.3a).

Anatomic structure of male chestnut flower

Based on the anatomy of single male flower,it could find that there were 6 sepals in each male flower with no petal,and the sepals were green with thick tomentum on them(Fig.3b, 3c).The filament was long and thin and clear with thick tomentum at the base,and the anther presented faint yellow color(Fig.3d).The measured absorption spectrum of the organic solvent extract of the anther showed that there was a small amount of chlorophyll inside(Fig.4).

Paraffin section was conducted to the female chestnut flower clusters tomake sections,and cross sections paralleled to the inflorescence axis were also made.The results showed that female chestnut flowers were simple perianth flower,which was consistent with the above observed results under the anatomical lens.Due to squeezing,the bordered flowers in the same flower clusters presented asymmetrical structure,and the anther of the top male flower was all exposed outside the flower.There were no micro-sporangium inside the perianth, but there were still some micro-sporangium that had not been fully differentiated shown at the base of the flower.Therefore,the number of sporangium in each flower was uncertain (Fig.5).

There were some micro-sporangium(anthers or pollen sac)found in the middle of a few male flowers that had the anthers stuck out of the flowers.With two chamers,the sporangium presented the shape similar to oval structure(Fig.5).In the meantime,of all the 6 observed samples (the sections of 6 different male flowers),there was on flower with a sporangium of 4 chambers(as shown with the arrow in Fig.5).

Conclusion and Discussion

A bud of chestnut before winter could gradually grow into a long branch the next spring when the weather turns into warmer,and there would be a lot of leaves on the branch. Many staminate inflorescence would stick out of the top of the branch,which would gradually differentiate into male flowers,which would further differentiate into many sporangium and pollen grains.And at the base of some staminate inflorescence,there would differentiate male flower clusters(Fig.1). The appearance of every new organ is bound to relate with inner structure of its bud.REN et al.[4]pointed out that the primordium of staminate inflorescence usually began to breed in early June to middle August of the first year, and the morphodifferentiation could last about more than 10 months.However,LI et al.[13]divided the chestnut once inflorescence into the pre-winter inflorescence and post-winter inflorescence,and pre-winter inflorescence primordium formed from the middle June and ended at the middle August, while the post-winter inflorescence primordium originated behind the formation of pre-winter inflorescence in the middle of August of the last year. The shoot apical meristem differentiates into auxillary bud growth cone shaped primordium.Therefore,the corresponding relationship between the inner structure of bud and the differentiation and growth of inflorescence development needs further study.

Chestnut flower has complex organ structures.Both GUO et al.[14]as well as REN et al.[4]noticed that there was a flat or slightly raised structure in the center of chestnut staminate primordium,which might be the pistillode. However,female chestnut flower also contained male flowers but below the pistil stigma,which avoided the selfpollination[4].However,there are relatively fewer study on the inner structure of mature male flowers,such as the morphology of sporangium.BAI et al.[5]believed that there were a large amount of male chestnut flowers, which had different differentiation progress,resulting in the long duration of its differentiation.The observation found that there were a large number of microsporangium in the chestnut male flower,presenting the trend of infinite differentiation,which was consistent with the results of BAI et al[5].

It is widely believed that one of the key factors restricting the production of chestnuts is the fact that chestnut has too many male flowers while few female flowers.As a matter of fact,the growth of chestnut staminate inflorescence lasted for long time,and different branches or the staminate inflorescences at the different positions of the same branch showed inconsistent flowering time, and the flowering time of the male flowers in different positions of the same inflorescence was also different.Moreover,the formation time of its inflorescence primordium was also different[13]. This kind of growth rules of staminate inflorescence of chestnut finally resulted in the inconsistent in the flowering time of different male flowers,and the long period of pollination might be a mechanism for the chestnut to adapt to the inconsistent growth progress of female flowers.

Previous study has conducted the scanning electron microscopy observation on the mature pollen grains of chestnut,but there are few study on the structure of sporangium as well as the number of pollen grains inside the sporangium.Through the anatomy of the male chestnut flowers,we found that the male chestnut flowers had green sepals but no petals,and the pollen grains inside the sporangium were small in size but large in number. Many scholars believed that it was a specific character to adapt to anemophilous pollination.However, the stigma of female chestnut flower cluster was in the shape of needle, and only the top of it,which was very small in size could accept pollen[15], making it very hard to achieve fully fertilization by insect pollination.Moreover,the study using fly net to cover the pistillate flower of chestnut showed that the isolation of insect pollination greatly decreased the number of pollen on the top of the stigma[16]. Moreover,other scholars also pointed out that brown glands were found at the base of male chestnut flowers, which spilt out fragrance during the flowering period,which could lure insects,mainly including beetles,beetles,gold is mainly insects,bees. Therefore,it carried with the characteristics of entomophilous flower.In this study,we also found that when the staminate inflorescence of chestnut began to flower,there were strong fragrance around,and many kinds of insects could be found on the staminate inflorescence,indicating that chestnut also had the characteristics of insect pollination.

To sum up,chestnut had larger quantity of male flowers and pollen and long period of pollination compared with female flower.It is remained to be further studied whether it was necessary for anemophilous pollination.

In a word,Sakaguri Oka,pollen number,pollen blossom time,relative to the number of female flowers few, whether to meet the requirements of wind pollination,remains to be further studied.

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Responsible editor:Na LI

Responsible proofreader:Xiaoyan WU

板栗雄花序生长发育规律及雄花结构解剖观察

陈娟1，2，聂玉婷1，刘津1，姚红艳1，程水源1*
（1.黄冈师范学院生命科学学院/经济林木种质改良与资源综合利用湖北省重点实验室/大别山特色资源开发湖北省协同创新中心，湖北黄冈438000；2.扬州大学生物学博士后流动站，江苏扬州225009）

通过对板栗（Castanea mollissima Blume)雄花序的生长发育规律和雄花解剖结构观察，结果表明，同一枝条上基部雄花序首先形成，依次向上进行，同一枝条上从基部雄花序开始生长至最顶部一个雄花序发育完全需要50 d左右；同一个雄花序上，基部雄花簇首先开放，依次向上进行，而从基部雄花序开始开花至顶部雄花序完全开花需要20 d左右；一个雄花簇有雄花5～7朵，一般为单数，顶部一朵，下方四朵为两朵并列排列，同一雄花簇中最顶部一朵雄花最先开放，依次向下进行，并列排列的两朵同时开花；板栗雄花小孢子囊数量多，且发育进程有较大差异，表现为无限分化的趋势，小孢子囊两室，偶而有四室出现，内含大量花粉粒。总之，板栗雄花多，花粉数量大，开花散粉时间长，对于相对数量较少的雌花而言，是否为适应风媒传粉所需，有待进一步研究。

板栗（Castanea mollissima Blume)；雄花序；雄花；解剖结构；小孢子囊amounts of female flowers with the ratio of female flowers to males reaching up to 1∶2 000-1∶3 000[2].Moreover,the formation of staminate inflorescence is prior to that of the female flowers,making it unfavorable for the development and growth of female flowers in nutrient allocation and conduction,which is the key factor limiting the yield of chestnut.Therefore,it is of great significance for the improvement of yield to clarify the regulations and mechanisms of the differentiation and development of the male flowers and female flowers and regulate the formation of the relative large amount of male flowers[3].There have been some studies on the process of lower bud differentiation of chestnut.For example,REN et al.[4]pointed out that the bud induction period of chestnut fell on middle of June to the middle of August,and the morphodifferentiation of male flowers could last about more than 10 months, while that of the female flowers only 2-3 months.BAI et al.[5]found that there were degraded pistils in the male flowers and thought that measures should be taken during the primordium initiation of bud to make the development towards hermaphrodite flowers,which could produce more seed producing flowers.ZHANG et al.[6]observed the differentiation of male flower clusters, and there are still many researchers studying the effects of fertilization and spraying plant growth regulators on the ratio of female flowers to male flowers[7-12].However,due to the complex process of bud formation of chestnut,it is still not clear how the external shape changes as well as the corresponding relationship of the internal structure during the process from the formation of bud before winter to the formation of inflorescence of the following year.Meanwhile,although there are some research on the differentiation process of the male chestnut flowers[5],there are relative few reports on the structure of mature male flowers,especially the structure of the inner sporangium.Therefore,through the observation of the development and growth of staminate inflorescence and using paraffin sections to learn the morphological structure of the inner sporangium of mature male flowers, this study was aimed to provide basis for the study on the relationship of the growth and development of male flowers and the formation of female flower clusters.

湖北省自然科学基金重点项目（2010CBB03901)；湖北省教育厅高校产学研合作资助重点项目（C2010060)；2011中央财政林业科技推广示范项目（2011BH0032)；经济林木种质改良与资源综合利用湖北省重点实验室开放基金项目（2013000503)；黄冈师范学院产学研合作项目（2012025703），江苏省博士后基金项目（1402115C）。

陈娟（1978-)，女，江苏盐城人，副教授，博士，主要从事植物生理学的教学和研究工作，E-mail：breezy02008@163.com。*通讯作者，教授，E-mail：s_y_cheng@sina.com。

2015-03-10

修回日期 2015-05-05

Supported by the Major Project of the Natural Science Foundation of Hubei Province (2010CBB03901);the Key Project of the Industry-University-Research Cooperation Program of the Education Department of Hubei Province(C2010060);the Demonstration Project of the Forestry Scientific and Technical Extension of the Central Finance in 2011(2011BH0032);the Open Fund Project of the Key Laboratory of Economic Forest Germplasm Improvement Resources Comprehensive Utilization of Hubei Province(2013000503);the Industry-University-Research Cooperation Program of Huanggang Normal University（2012025703）;the Postdoctoral Fund of Jiangsu Province(1402115C).

*Corresponding author.E-mail:s_y_cheng@sina.com

Received:March 10,2015 Accepted:May 5,2015