Study on Pollen Viability and Stigma Receptivity of Very Later-ripening‘Dongxing’Apricot

Shirong ZHAO,Kang LIAO*,Mengxiang DA,Guixiang XU,Le XU,Shengli DONG,Runqing DU

1.Research Center for Xinjiang Characteristic Fruit Tree,Xinjiang Agricultural University,Urumqi 830052,China;

2.Luntai National Fruit Germplasm Resources Garden in Xinjiang,Luntai 841600,China

Responsible editor:Xiaoxue WANG Responsible proofreader:Xiaoyan WU

Armeniaca vulgaris cv.‘Dongxing’ originated from natural bayberry seedlings in Aksu,Xinjiang,which is belongs to Centro-Asiatic ecological[1].It is the special apricot germplasm resources that have great value of utilization on crossbreeding for its fruit developmental period is as long as 180-195 d.The floral organ characteristics,such as pollen quantity and perfect flower ratio of some cultivation or wild apricot in Xinjiang and Armenia vulgaris cv.‘Katy’ have been studied by SUN et al[2-5].The floral form of ‘Dongxing’has been primary described in Apricot Volume in China Fruit Records,but the pollen quantity,pollen viability and stigma receptivity of ‘Dongxing’ were not reported.Therefore,to understand the characteristic of floral organ development of ‘Dongxing’ and analyze the differences with other apricot varieties can lay a foundation for the crossbreeding and utilization of‘Dongxing’.

Material and Methods

Experimental materials

Armeniaca vulgaris cv.‘Dongxing’ (Luntai National Fruit Germplasm Resources Garden in Xinjiang) was main object of research,Armeniaca vulgaris cv.‘Keziaqia’,Armeniaca vulgaris cv.‘Suogejianali’,Armeniaca vulgaris cv.‘Akeyulvke’,Armeniaca vulgaris cv.‘Jiagedamayisang’ were chosen as reference.All apricot varieties (tree age was 26a),which were studied,under normal growth and health condition,were adopted similar cultivation and management ways.

The morphologicobservation of floral organ

The 25 flowers were selected from each direction (East,South,West and North)of outer crown of different apricot varieties.And measured the floral structure and investigated pistil development which includes the numbers of the flowers that pistils are higher than anthers(♀＞♂),are equal to anthers (♀=♂),shorter than anthers(♀＜♂) and degeneration (♀≈0) of different apricot varieties according to Apricot Germplasm Resources Description Criterion and Data Standards[6].

Ratio of medium style and long style (%)=The ratio of the flower that pistils are higher than anthers+ The ratio of flower that pistils equal to anthers.

The measurement of pollen quantity

The 25 flowers were selected from each direction (the East,South,West,and North)of outer crown of different apricot varieties.The anthers of each flower buds were put in a 1.5 ml centrifuge tube respectively after counting of the anther numbers of per flower buds that were striped from healthy balloons flower buds,collected from target tree.Subsequently,the anthers in 1.5 ml centrifuge tube natural were powdered,added with 1 ml distilled water and the centrifuge tube was centrifuged for 3 min by 12 000 r/min to make sure that the pollen grains were distributed in the solution evenly,1 μl solution was absorbed from the centrifuge tube and spited on glass slide to count pollen quantity of single anther under microscope (Motic BA400-102M) that objective lens was amplified 40 times and eyepiece was amplified 4 times,followed by observation and calculation of the pollen amount of single anther.

The pollen amount of single anther (Grain/anther) = (Total number of pollen grains in each 1 μl solution drops × 1 000) / Total numbers of anthers in each 1.5 ml centrifuge tube

Screening the optimum medium for pollen germination

Anthers were striped from healthy balloons flower buds that were collected from target tree,and were shared in a sulfuric acid paper box in indoor to air dried 1-2 d; the pollen collected in 1.5 ml centrifuge tube after it natural powdered had been stored in -20 ℃for experiments using.The 12 culture media were designed to select the optimum medium,involving vitro pollen germination as the sucrose concentrations at 0%,5%,15%,and 20%,boric acid concentrations at 0%,0.01%,and 0.02%,and all 12 mediums were supplemented with 1% agar and distilled water.And the collected pollens were evenly placed over a glass slide with different culture mediums,and cultured in a constant temperature of 25 ℃for 4 h.Then,the numbers of total pollen and germinated pollen in the different culture mediums were observed with a microscope(Motic BA400-102M) that objective lens was amplified 40 times and eyepiece was amplified 4 times; and the pollen germination rates of different culture mediums were calculated by using the formula[7].

Pollen germination rate (% ) =(Numbers of germinated pollen in each horizon / Total number of pollen in each horizon)×100

The measurement of vivo pollen viability

Aday before flowering,the branches were collected and had been cultured in water; took 25 flowers from each samples after 1,2,3,4,5 d of flowering respectively,and the anther were striped from healthy flower buds that were collected from target samples respectively,and were shared in sulfuric acid paper box in indoor to air dried; And the collected pollen evenly placed over a glass slide with culture medium contains 0.01% boric acid +10% sucrose + 0.01% ager after it natural powdered,and was cultured in constant temperature of 25 ℃for 4 h,then the numbers of total pollen and germinated pollen were observed by using microscope (Motic BA400-102M) that objective lens was amplified 40 times and eyepiece was amplified 4 times; and the pollen germination rates of each treatments (samples) were calculated by using the formula[8].

Pollen germination rate (% ) =(Numbers of germinated pollen in each horizon / Total number of pollen in each horizon)×100

The measurement of vitro pollen viability

Aday before flowering,the branches were collected and cultured in water;the anthers were striped from healthy flower buds,and shared in sulfuric acid paper box in indoor to air dried; the pollen was collected in 1.5 ml centrifuge tube after it natural powdered and stored at 25 ℃for experiments using.And the collected pollen was evenly placed over a glass slide with culture medium containing 0.01%boric acid + 10% sucrose + 0.01%ager after 1,2,3,4 and 5 d of storing respectively,and cultured at a constant temperature of 25 ℃for 4 h respectively.Subsequently,the numbers of total pollen and germinated pollen of different storage time pollen were observed by using microscope (Motic BA400-102M) that objective lens was amplified 40 times and eyepiece was amplified 4 times; and the pollen germination rates of each treatments(different storage time pollen)were calculated by using the formula[8].

Pollen germination rate (% ) =(Numbers of germinated pollen in each horizon / Total number of pollen in each horizon)×100

The measurement of stigma receptivity

The flowers were selected from each direction(East,South,West,and North) of outer crown of different apricot varieties,and flowers were bagged after castration,2 d before the flowering.And 30 stigmas were picked from each flower stages (before 24 and 48 h of flowering and after 0.5,1,2,4,6,8,12,24,48 and 96 h of flowering,respectively.Subsequently,the collected stigmas were evenly placed over a concave slide with reaction solution of Benzedrine and Peroxide (1% Benzedrine,3% Peroxide and distilled water mixed in 4:11:22 proportion),respectively.After 20 min,the stigmas were observed with a Shunyu EX-204 microscope and were ranked to 5 levels as weak (A-),normal (A),slightly strong(A+),strong(A++)and extremelystrong (A+++)according to blue-black part of the stigma and the number of bubbles on the stigma.The stigma quantity was recorded at different levels[9].

Table1 Comparison of development of styles in 5 apricot varieties

Data statistics

The data was analyzed using Excel2007 and SPSS20.0.

Results and Analysis

The characteristics of floral organ

The pedicel length,corolla diameter and petal number of ‘Dongxing’were 1.89,22.54 mm,5 respectively,and the ovary length,ovary width,style length,filament length,anther length,anther width were 3.62,1.28,15.10,13.01,0.78,and 0.49 mm,accordingly.Its floral organ shows petal flower,oval shaped white petal,long oval shaped purple calyx with bluntly tip,and superior ovary with pubescence.

As shown in Table1,the average percentage of medium and long-style(♀＞♂,♀=♂)in 5 surveyed varieties was 71.42%,and the malformation ratio of pistil(♀≈0)was 16.0%.According to analysis,it can be concluded that the malformation rates of pistils of 5 surveyed varieties were lower relatively and extremely significant different from each other.Moreover,the differences among percentage of medium and long-style in 5 varieties were extremely significant too.By comparison of result of flow items in‘Dongxing’ with others,the ratio of pistils higher than anthers(♀＞♂)was the highest,equal to anthers (♀=♂)was lowest,medium and long-style was highest (82.4%),and malformation pistil (♀≈0) was lowest among them.

The observation of pollen quantity

As shown in Table2,the average quantity of anther and pollen in 5 surveyed varieties was 34.76 grains per flower and 56 517.98 grains per flower,and the differences of anther and pollen quantity in one flower among 5 varieties were extremely significant.By comparison on the result of the quantity of anther per flower in ‘Dongxing’with others,it can be concluded that it was the highest (39.2 grain/flower),while the minimum quantity of pollen per anther constituted the minimum quantity of pollen per flower.

The selection of optimum medium for pollen germination

As shown in Table3,the optimal sucrose and boric acid concentration in pollen germination medium of‘Dongxing’ were 15.0% and 0.01%respectively.The germination percentage of ‘Dongxing’ in optimum medium was 53.0%and this level was higher than the average value of oth-ers.The germination percentages of 5 varieties were ‘Jiagedamayisang’(79.9% ) ＞‘Akeyulvke’ (64.2% ) ＞‘Dongxing’ (53.0% ) ＞‘Akeyulvke’(52.3%)＞‘Suogejianali’(46.3%).

Table2 Comparison of pollen quantity in 5 apricot varieties

Table3 The optimum medium selection for pollen germination of 5 apricot varieties %

Table4 Receptivity about stigma in different development steps of 5 apricot varieties

The observation of connect-body pollen viability

It can be concluded from Fig.1 that the germination percentage of connect-body pollen in ‘Dongxing’gradually increased from flowering to the 1std after flowering and decreased from the 1stto the 5thd after flowering.Its germination percentage decreased about half and 25%of the highest level on the 3rdand 5thd after flowering.The descending speed of connect-body pollen germination percentage of 5 varieties became slow gradually and this trend of ‘Dongxing’ was slower compared with others.

The observation of stored pollen viability

It can be concluded from Fig.2 that the germination percentage of stored pollen in ‘Dongxing’gradually decreased from 0 to the 5thd during storing period.Its germination percentage decreased about half and 20%of the highest level on the 3rdand 5th d during storing period.The descending speed of stored pollen germination percentage in ‘Dongxing’was slower compared with others from the 2ndto the 4thd during storing period.The comparison result showed that connect-body pollen germination percentage on the 1std after flowering is higher than stored pollen at the big bud stage(0 d during storing period).

The observation of stigma receptivity

As showed in Table4,the receptivity of stigma in ‘Dongxing’ started to increase in 0.5 h after flowering and showed the strongest from the 2ndto 4thh after flowering.Then,its receptivity began to weaken in 6 h after flowering.According to this phenomenon,the optimum artificial pollination period of ‘Dongxing’ was defined between 1 to 8 h after flowering.And the optimum artificial pollination periods of‘Keziaqia’,‘Suogejianali’,‘Akeyulvke’,‘Jiagedamayisang’ were defined between 8 to 12 h,4 to 12 h,1 to 8 h and 0.5 to 12 h respectively.

Discussion

The differences of floral organ characteristics among ‘Dongxing’and other apricot varieties

The ratios between the medium style and long style,and anther quantity of ‘Dongxing’ were higher compared with other apricot varieties,but the pollen quantity per flower was lower relatively[3].The pollen viabilities of the vivo and vitro stored of ‘Dongxing’were intermediate-level compared with others apricot varieties,and the descending speed of pollen germination percentage with extension of the storage time kept relatively lower.The time of highest stigma receptivity of‘Dongxing’ was earlier than that of other varieties,but the persistent period of the highest stigma receptivity of‘Dongxing’ was shorter; the optimum artificial pollination period of ‘Dongxing’ was shorter than the other varieties.There were some differences between this results and the research results of Sun[2]and An[3]et al.

The correlation between connectbody pollen viability and stigma receptivity of‘Dongxing’

The stigma receptivity is going to enhance with the aid of related metabolic reaction stimulated by external environment after flowering[10].The stigma receptivity of ‘Dongxing’got the highest value in the period of 0-4 h after flowering and decreased 4 h after flowering.The persistent period of the high stigma receptivity was 8 h.The vivo pollen germination of ‘Dongxing’ increased gradually during the period of 0 to 24 h after flowering and decreased to the half of the highest pollination rate after 24 of flowering.These results were similar with the research results of Sun[2],Liu[10],Lei[11]et al.This results indicated that the overlap opportunity of the period of the highest pollen viability and highest stigma receptivity of ‘Dongxing’ was very short which caused the decrease of pollination efficiency in‘Dongxing’.Therefore,it can improve fruiting percentage of ‘Dongxing’ by arrangement of optimal pollinizers in orchard.

The selection about optimum artificial pollination period in hybridization of‘Dongxing’

In the production practice,the change rules of the pollen viability and stigma receptivity and its interrelation should be considerable standards for selection of the optimum pollinizer[12].The stigma receptivity of apricot varieties changed from increasing to decreasing.The pollen acceptation and facilitation of pollen germination ability of stigma were higher at the stigma receptivity.Therefore,the artificial pollination of ‘Dongxing’should consider the change rule of stigma receptivity,i.e.‘Dongxing’ should be artificial pollinated after 2 h of castration.

Conclusion

The ratio of pistillode of ‘Dongxing’ was lower than that in the other apricot varieties,but the pollen quantity per flower was relatively lower than the other varieties.The pollen germination of ‘Dongxing’ in optimum medium (1% agar + 15% sucrose +0.01% boric acid) was 53.0%,i.e.its pollen viability was low.The pollen viability increased at first and then decreased following the period of the bud stage to flowering stage,and pollen viability reached maximum value on the 1st d after flowering.Meanwhile,the stored pollen viability continuously decreased with the extension of storage time,and pollen viability of the both vivo or vitro stored decreased to the half of the maximum value on the 3rdd after flowering or storage.The stigma receptivity of ‘Dongxing’ enhanced at first and then weakened,and maximum stigma receptivity was found during the period of 2 to 4 h after flowering.The optimum time for artificial pollination was 1 to 8 h after flowering and it was shorter than the other apricot varieties.

[1]ZHANG JY (张加延),ZHANG Z (张钊).China Fruit Records:Apricot Volume(中国果树志(杏卷))[M].Beijing:Beijing Agricultural University Publishing House(北京:北京农业大学出版社),2003.

[2]SUN HY(孙慧瑛).The research on pollination and fertilization biological characteristics of Xinjiang apricot(新疆杏品种授粉受精生物学特性研究)[D].Urumqi:Xinjiang Agricultural University (乌鲁木齐:新疆农业大学),2013.

[3]AN XQ(安晓芹),LIAO K(廖康),MA Y(马媛),et al.Studies on flowering biological characteristics of Xinjiang apricot varieties(新疆杏品种开花生物学特性研究)[J].Journal of Xinjiang Agricultural University(新疆农业大学学报),2012,35(3):153-156.

[4]DIAO YQ(刁永强),LIAO K(廖康),XU Z( 许正),et al.Preliminary study on pollin -ating fertilization and biological characters of blossom on wild apricot in Xinjiang(新疆野生杏开花生物学特性及授粉受精初步研究)[J].Xinjiang Agricultural Sciences(新疆农业科学),2010,47(5):947-951.

[5]SUN M (孙猛),LIU GC (刘国成).Study on floral organ characteristics and fruit development dynamic of greenhouse Katy (日光温室凯特杏花器官和果实发育动态观察)[J].Shanxi Fruits (山西果树),2003,(6):3-6.

[6]LIU N (刘宁),LIU WS (刘威生).Apricot germplasm resources description criterion and data standards (杏种质资源描述规范和数据标准)[M].Beijing:China Agriculture Press (北京:中国农业出版社),2006.

[7]LI J (李疆).Research report on physiological and molecular mechanism of low bearing rate of almond in Xinjiang (新疆扁桃结实率低的生理与分子机制)[J].Nonwood Forest Research (经济林研究),2011,29(2):1-7.

[8]HU SY (胡适宜).Experimental methods in plant embryology(1) determination of pollen viability(植物胚胎学试验方法(1),花粉生活力的测定)[J].Chinese Bulletin of Botany(植物学通报),1993,70(2):60-62.

[9]PENG WX(彭伟秀),FENG DL(冯大领),ZHENG H(郑辉),et al.(晚熟李花粉生活力及柱头可授性研究)[J].Journal of Agricultural University of Hebei (河北农业大学学报),2008,31(5):57-60.

[10]LIU LM (刘黎明).Primary research on reproductive ecology of Armeniaca vulgaris Lam (新疆野杏生殖生态初步研究)[D].Urumqi:Xinjiang Agricultural University (乌鲁木齐:新疆农业大学),2010.

[11]LEI LL (雷莉莉).Study on the reproductive biology of Prunus domestica×Armeniaca ‘Fengweimeigui’(杏 李 新品种‘风味玫瑰’生殖生物学研究)[D].Beijing:Chinese Academy of Foresty(北京:中国林业科学研究院),2013:43-45.

[12]JI HJ (姬慧娟).Study on floral dynamics,pollen viability and stigma receptivity of Tamarix ramosissima (多枝柽柳的开花动态及花粉活力和柱头可授性研究)[D].Beijing:Graduate University of Chinese Academy of Sciences(北京:中国科学院研究生院),2013.