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Abstract [Objectives] The genetic characteristics of wild germplasm resources of Saccharum spontaneum L. were further investigated to provide a scientific theoretical basis for the improved hybridization of sugarcane varieties.
[Methods] The first flowering date data of 112 wild resources were statistically analyzed by broad heritability for 3 consecutive years， and pollen quantity and yield were investigated to provide reference for hybridization.
[Results] The broad heritability of the first flowering date of S. spontaneum was 0.079. The differences in the first flowering date between the S. spontaneum resources were in the range of 4-49 d， with an average of 13.65 d. The inter annual changes within 20 d accounted for 90 %， and only 4.5% exceeded 30 d. The first flowering dates of some S. spontaneum resources were obviously postponed after being treated.
[Conclusions] In the hybridization season， the hybridization plan must be adjusted according to the actual situation of the flowering period to make full use of it.
Key words Saccharum spontaneum L.; First flowering date; Heritability; Pollen
Saccharum spontaneum L. is one of the wild species of sugarcane， which is characterized by good tolerance to drought and poor soil and strong resistance to stress. It is one of the important sources of stress resistance genes for sugarcane variety improvement， and all current sugarcane cultivars are genetically related to S. spontaneum[1-2]. The research on the wild resources of S. spontaneum is a basic work， and it is found after investigation that its flowering habit is a heritable trait. Under natural conditions， the first flowering dates of various lines vary from year to year （some are bring forward， and some are delayed）， and the flowering period differences of individual varieties in different years can exceed one month. Making full use of this inter annual change and timely rationally adjusting the hybridization plan will help to increase the number of the created "noble" populations of sugarcane. S. spontaneum is the wild variety with the widest distribution and the most types， which has a higher sugar content in Saccharum . It is the main source of excellent traits such as stress resistance， perenniality and adaptability in the current sugarcane breeding in the world[3-4]. China has always attached great importance to the collection and conservation of S. spontaneum and its application in the improvement of sugarcane varieties. Qi et al.[5]， Qiu et al.[6]， Zhang et al.[7] and Yang et al.[8] studied the genetic characteristics of S. spontaneum germplasm resources in Guangdong， Yunnan， Guangxi and Sichuan， respectively. S. spontaneum has various types and rich genetic diversity. Intensifying the research on S. spontaneum and making full use of S. spontaneum with different characteristics from different ecological regions can facilitate the creation of high quality sugarcane parents with different resistance. Improving the foundation for sugarcane variety improvement is of great significance to the prospect of sustainable and stable development of the sugarcane industry. Based on the first flowering dates of the complete 112 materials recorded in the three hybridization seasons from 2012/2013 to 2014/2015， the first flowering dates were subjected to broad heritability analysis， and comparison was performed on the inter annual variations in the first flowering date， pollen quantities and pollen development rates， aiming at providing a scientific theoretical basis for the production of "noble" hybrid sugarcane seeds.
Materials and Methods
Experiment time
The experiment was carried out in 2012/2013， 2013/2014， 2014/2015， three hybridization seasons in total.
Experimental location
The experiment was carried out in the Wild Sugarcane Germplasm Resources Garden of Hainan Sugarcane Breeding Farm， Guangzhou Sugarcane Industry Research Institute （Yacheng， Sanya）.
Materials
The 112 germplasm materials of S. spontaneum planted in the cement pens served as the objects of study.
Methods
In the three hybridization seasons， the flowering of each line was investigated and recorded every day， and the day when the first flower cluster of each line bloomed was recorded as the first flowering date of the line. For the convenience of analysis， November 1 was recorded as 1[9]， and the number was increased by 1 with the delay of one day in the first flowering date. For example， November 30 was recorded as 30， December 1 was recorded as 31， and October 31 was recorded as -1. Investigation of the pollen quantity： Every day at about 5：00 pm， the sugarcane plants with the flowering amount reaching 1/3-1/2 were cut and brought back to the greenhouse or laboratory， and the stems were cultured with a sulfite solution until 8：00-9：00 am of the next day when the pollen was taken for quantitative analysis. Development rate： The pollen was stained with tincture of iodine， and then observed under high power lens， and the proportion of the pollen grains that were completely stained in one field of view to all pollen grains in the field of view was calculated as the development rate. Regulation of the flowering period of some early flowering varieties： In mid June， half of the plants in the cement pens were cut at the middle height with a sickle， to re culture new tillers.
Statistical analysis was performed by broad heritability （referring to the method of Wang[10]）. In this study， only lines with complete records served as the objects of analysis， while those who flowered in some years and did not flowered in other years were not analyzed because the data were not complete and brought difficulties in quantification and statistics. The pollen quantity and development rate were for reference only and were not used for analytical studies. Results and Analysis
Broad heritability analysis on the first flowering date of S. spontaneum
The results of the investigation on the first flowering dates of 112 S. spontaneum germplasm resources from the Wild Sugarcane Germplasm Resources Garden of Hainan Sugarcane Breeding Farm are shown in Table 1. In the table， the earliest flowering date was September 13 （Guangdong 5， -49）， and the latest was December 25 （Yunnan 87 35， 55）.
In the variable components of Table 2， the intra annual variable was the genetic variable （Vg） the intra line variable was the environmental variables （Ve）， and the broad heritability of the first flowering date in S. spontaneum was：
h2 b=Vg/（Vg+Ve）= 44.073 1/（44.073 1+514.334 6）=0.079
The above analysis indicates that the first flowering date of S. spontaneum is a heritable trait. In the selection of noble hybrid seeds， this trait can be more strictly selected in the early stage of hybridization selection， so as to select some sugarcane parents with the same flowering date purposefully for hybrid seed production. Populations genetically related to S. spontaneum can be cultivated as much as possible to improve the probability of breeding excellent F 1 generations. Many excellent traits of S. spontaneum are heritable. For example， excellent traits such as stress resistance， perenniality and adaptability can be expressed in the hybrid progeny[11-12]. This feature broadens the field of sugarcane variety improvement， and the prospects are considerable.
Reference for utilization of inter annual variation of the first flowering date in S. spontaneum and its pollen quantity and development rate
The results in Table 1 showed that there was a big inter annual difference in the first flowering date. Among the 112 lines， the smallest inter annual difference was 4 d （Sichuan 79 I 17）， and the largest was 49 d （Guangdong 2）. The inter annual variations within 20 d accounted for 90%， and only 4.5% of them were over 30 d. It seems that there is no necessary relationship between the first flowering date characteristic of a line （early flowering or late flowering） and the inter annual difference in the first flowering date， that is， the inter annual variation of the early flowering line is not necessarily smaller than that of the late flowering line， vice versa. At the same time， in a particular hybrid season， there is no obvious law in whether the flowering periods of different lines are earlier or later than another year. For example， the 2012/13 hybrid season was compared with the 2014/15 hybrid season， the first flowering date of Guangdong 2 was October 10 in the 2012/13 hybrid season and November 27 in the 2014/15 hybrid season， showing a delay of 49 d， while the first flowering date of Sichuan 88 27 was November 21 in the 2012/13 hybrid season and November 10 in the 2014/15 hybrid season， which meant the first flowering data was 11 d earlier （Table 1）. In the hybridization season， it is necessary to adjust the hybridization plan in a timely and flexible manner according to the actual situation. There were six lines with very much pollen， including Hainan 92 51， Hainan 92 52， Sichuan 79 I 11， Yunnan 82 110， Yunnan 82 5 and Yunnan 75 II 21; there were eight lines having very little pollen ， namely Yunnan 6， Yunnan 75 II 4， Yunnan 75 II 6， Sichuan 79 I 7， Hainan 92 70， Hainan 92 41， Guizhou 78 II 09 and Guizhou 78 II 08. In the hybridization plan， the lines with much or very much pollen can be considered as a male parent， and those having little or very little pollen can be considered as a female parent， which is conducive to the breeding of true and false hybrids. The pollen development rates of eight lines reached 100%， namely， Guangdong 47， Guangdong 49， Guangdong 50， Guangdong 75， Guangdong 76， Guangdong 82， Sichuan 79 II 9 and Sichuan 88 1. Such lines can be considered for the combination with sugarcane parents having no high affinity or the hybrid seed production during the creation of complexes. Inter annual variations of some early flowering S. spontaneum after treatment
The results in Table 3 showed that the earliest first flowering date was on October 14 （Guangdong 5）， and the first flowering date was delayed by one month after being treated; and the latest was on November 29 （Guangdong 38）， and after the treatment， the first flowering date was postponed for more than a month. Obviously， the first flowering dates of the treated parents were all significantly delayed. In cross breeding， many lines that are unlikely to hybridize can be crossed by the staged head cutting method. In this way， a large number of hybrid populations can be easily obtained， from which excellent hybrid progenies can be selected.
Conclusions
The first flowering date of S. spontaneum is a heritable trait. In the process of noble seed production， it is possible to make full use of this trait by more strictly selecting it in the early stage of hybridization selection.
The broad heritability of the first flowering date of the S. spontaneum was 0.079. The inter annual differences of the first flowering date of S. spontaneum ranged from 4 to 49 d， with an average of 13.65 d. The lines with an inter annual variation within 20 d accounted for 90%， and those with an inter annual variation over 30 d only accounted for 4.5%. The first flowering dates of some parents were significantly delayed after the head cutting treatment. In cross breeding， many lines that are unlikely to hybridize can be crossed by the staged head cutting method. In the hybridization season， the hybridization plan of the current year must be properly adjusted according to the actual situation of the flowering period， and populations related to S. spontaneum should be bred as much as possible to improve the probability of breeding excellent F 1 generations.
There is no necessary relationship between the first flowering date characteristic of a line （early flowering or late flowering） and the inter annual difference in the first flowering date. In a particular hybridization season， there is no obvious law in whether the flowering periods of different lines are earlier or later than another year.
In the cross breeding program， the lines with much or very much pollen can be considered as the male parent， and those with little or very little can be considered as the female parent， which is beneficial to the breeding of true and false hybrids. For the sugarcane parents having no strong affinity or the hybrid seed production during the creation of complexes， the selection can be performed according to the development rate. References
[1] BREMER. Problems in breeding and cytology of sugar cane[J]. phytica， 1961， 10： 59-78.
[2] WANG YQ， WANG ZL， PAN SM， et al. Study on sugarcane germplasm resources[J]. Acta Agriculturae Jiangxi， 2002， 14（1）： 21-26. （in Chinese）
[3] Ministry of Agriculture， the Peoples Republic of China. Chinese crop genetic resources[M]. Beijing： China Agriculture Press， 1998： 499-518. （in Chinese）
[4] CHEN H， FAN YH， SHI XW， et al. research on genetic diversity and systemic evolution in Saccharum spontaneum L.[J]. Acta Agronomica Sinica， 2001， 27（5）： 645-652. （in Chinese）
[5] QI YW， FAN LN， HE HY， et al. Genetic diversity assessment of Saccharum spontaneum L. native to Guangdong area with agronomic traits[J]. Sugarcane and Canesugar， 2009（3）： 7-10. （in Chinese）
[6] QIU CL， HE SC， YANG SQ. Studies on Saccharum spontaneum L. in Yunnan II： Studies on esterase isoenzymes[J]. Journal of Yunnan Agricultural University， 1987（2）： 80-83. （in Chinese）
[7] ZHANG GM， LIAO JX， HUANG HT. Genetic diversity of Saccharum spontaneum L. with high sugar content in guangxi based on phenotypic traits and RAPD markers[J]. Journal of Southwest University， 2007， 29（8）： 83-88. （in Chinese）
[8] YANG RZ， WU WC， HUANG JK. Clustering classification of Sichuan Saccharum spontaneum[J]. Sugarcane， 2001， 8（2）： 4-9. （in Chinese）
[9] HUANG JF， DENG HH. Initial Flowering time and its heritability of the commonly used sugarcane parents in hainan sugarcane breeding station[J]. Sugarcane and Canesugar， 2004（4）： 6-10. （in Chinese）
[10] WANG JM. Selected papers on sugarcane selection[J]. Nanning： Guangxi Nationalities Publishing House， 1991， 314-329. （in Chinese）
[11] LI QW. Modern sugarcane improvement technology[M]. Guangdong： South China University of Technology Press， 2000. （in Chinese）
[12] WANG LP， MA L， XIA HM， et al. Application of Saccharum spontaneum in sugarcane cross breeding[J]. Sugar Crops of China， 2006（1）： 1-4. （in Chinese）
[Methods] The first flowering date data of 112 wild resources were statistically analyzed by broad heritability for 3 consecutive years， and pollen quantity and yield were investigated to provide reference for hybridization.
[Results] The broad heritability of the first flowering date of S. spontaneum was 0.079. The differences in the first flowering date between the S. spontaneum resources were in the range of 4-49 d， with an average of 13.65 d. The inter annual changes within 20 d accounted for 90 %， and only 4.5% exceeded 30 d. The first flowering dates of some S. spontaneum resources were obviously postponed after being treated.
[Conclusions] In the hybridization season， the hybridization plan must be adjusted according to the actual situation of the flowering period to make full use of it.
Key words Saccharum spontaneum L.; First flowering date; Heritability; Pollen
Saccharum spontaneum L. is one of the wild species of sugarcane， which is characterized by good tolerance to drought and poor soil and strong resistance to stress. It is one of the important sources of stress resistance genes for sugarcane variety improvement， and all current sugarcane cultivars are genetically related to S. spontaneum[1-2]. The research on the wild resources of S. spontaneum is a basic work， and it is found after investigation that its flowering habit is a heritable trait. Under natural conditions， the first flowering dates of various lines vary from year to year （some are bring forward， and some are delayed）， and the flowering period differences of individual varieties in different years can exceed one month. Making full use of this inter annual change and timely rationally adjusting the hybridization plan will help to increase the number of the created "noble" populations of sugarcane. S. spontaneum is the wild variety with the widest distribution and the most types， which has a higher sugar content in Saccharum . It is the main source of excellent traits such as stress resistance， perenniality and adaptability in the current sugarcane breeding in the world[3-4]. China has always attached great importance to the collection and conservation of S. spontaneum and its application in the improvement of sugarcane varieties. Qi et al.[5]， Qiu et al.[6]， Zhang et al.[7] and Yang et al.[8] studied the genetic characteristics of S. spontaneum germplasm resources in Guangdong， Yunnan， Guangxi and Sichuan， respectively. S. spontaneum has various types and rich genetic diversity. Intensifying the research on S. spontaneum and making full use of S. spontaneum with different characteristics from different ecological regions can facilitate the creation of high quality sugarcane parents with different resistance. Improving the foundation for sugarcane variety improvement is of great significance to the prospect of sustainable and stable development of the sugarcane industry. Based on the first flowering dates of the complete 112 materials recorded in the three hybridization seasons from 2012/2013 to 2014/2015， the first flowering dates were subjected to broad heritability analysis， and comparison was performed on the inter annual variations in the first flowering date， pollen quantities and pollen development rates， aiming at providing a scientific theoretical basis for the production of "noble" hybrid sugarcane seeds.
Materials and Methods
Experiment time
The experiment was carried out in 2012/2013， 2013/2014， 2014/2015， three hybridization seasons in total.
Experimental location
The experiment was carried out in the Wild Sugarcane Germplasm Resources Garden of Hainan Sugarcane Breeding Farm， Guangzhou Sugarcane Industry Research Institute （Yacheng， Sanya）.
Materials
The 112 germplasm materials of S. spontaneum planted in the cement pens served as the objects of study.
Methods
In the three hybridization seasons， the flowering of each line was investigated and recorded every day， and the day when the first flower cluster of each line bloomed was recorded as the first flowering date of the line. For the convenience of analysis， November 1 was recorded as 1[9]， and the number was increased by 1 with the delay of one day in the first flowering date. For example， November 30 was recorded as 30， December 1 was recorded as 31， and October 31 was recorded as -1. Investigation of the pollen quantity： Every day at about 5：00 pm， the sugarcane plants with the flowering amount reaching 1/3-1/2 were cut and brought back to the greenhouse or laboratory， and the stems were cultured with a sulfite solution until 8：00-9：00 am of the next day when the pollen was taken for quantitative analysis. Development rate： The pollen was stained with tincture of iodine， and then observed under high power lens， and the proportion of the pollen grains that were completely stained in one field of view to all pollen grains in the field of view was calculated as the development rate. Regulation of the flowering period of some early flowering varieties： In mid June， half of the plants in the cement pens were cut at the middle height with a sickle， to re culture new tillers.
Statistical analysis was performed by broad heritability （referring to the method of Wang[10]）. In this study， only lines with complete records served as the objects of analysis， while those who flowered in some years and did not flowered in other years were not analyzed because the data were not complete and brought difficulties in quantification and statistics. The pollen quantity and development rate were for reference only and were not used for analytical studies. Results and Analysis
Broad heritability analysis on the first flowering date of S. spontaneum
The results of the investigation on the first flowering dates of 112 S. spontaneum germplasm resources from the Wild Sugarcane Germplasm Resources Garden of Hainan Sugarcane Breeding Farm are shown in Table 1. In the table， the earliest flowering date was September 13 （Guangdong 5， -49）， and the latest was December 25 （Yunnan 87 35， 55）.
In the variable components of Table 2， the intra annual variable was the genetic variable （Vg） the intra line variable was the environmental variables （Ve）， and the broad heritability of the first flowering date in S. spontaneum was：
h2 b=Vg/（Vg+Ve）= 44.073 1/（44.073 1+514.334 6）=0.079
The above analysis indicates that the first flowering date of S. spontaneum is a heritable trait. In the selection of noble hybrid seeds， this trait can be more strictly selected in the early stage of hybridization selection， so as to select some sugarcane parents with the same flowering date purposefully for hybrid seed production. Populations genetically related to S. spontaneum can be cultivated as much as possible to improve the probability of breeding excellent F 1 generations. Many excellent traits of S. spontaneum are heritable. For example， excellent traits such as stress resistance， perenniality and adaptability can be expressed in the hybrid progeny[11-12]. This feature broadens the field of sugarcane variety improvement， and the prospects are considerable.
Reference for utilization of inter annual variation of the first flowering date in S. spontaneum and its pollen quantity and development rate
The results in Table 1 showed that there was a big inter annual difference in the first flowering date. Among the 112 lines， the smallest inter annual difference was 4 d （Sichuan 79 I 17）， and the largest was 49 d （Guangdong 2）. The inter annual variations within 20 d accounted for 90%， and only 4.5% of them were over 30 d. It seems that there is no necessary relationship between the first flowering date characteristic of a line （early flowering or late flowering） and the inter annual difference in the first flowering date， that is， the inter annual variation of the early flowering line is not necessarily smaller than that of the late flowering line， vice versa. At the same time， in a particular hybrid season， there is no obvious law in whether the flowering periods of different lines are earlier or later than another year. For example， the 2012/13 hybrid season was compared with the 2014/15 hybrid season， the first flowering date of Guangdong 2 was October 10 in the 2012/13 hybrid season and November 27 in the 2014/15 hybrid season， showing a delay of 49 d， while the first flowering date of Sichuan 88 27 was November 21 in the 2012/13 hybrid season and November 10 in the 2014/15 hybrid season， which meant the first flowering data was 11 d earlier （Table 1）. In the hybridization season， it is necessary to adjust the hybridization plan in a timely and flexible manner according to the actual situation. There were six lines with very much pollen， including Hainan 92 51， Hainan 92 52， Sichuan 79 I 11， Yunnan 82 110， Yunnan 82 5 and Yunnan 75 II 21; there were eight lines having very little pollen ， namely Yunnan 6， Yunnan 75 II 4， Yunnan 75 II 6， Sichuan 79 I 7， Hainan 92 70， Hainan 92 41， Guizhou 78 II 09 and Guizhou 78 II 08. In the hybridization plan， the lines with much or very much pollen can be considered as a male parent， and those having little or very little pollen can be considered as a female parent， which is conducive to the breeding of true and false hybrids. The pollen development rates of eight lines reached 100%， namely， Guangdong 47， Guangdong 49， Guangdong 50， Guangdong 75， Guangdong 76， Guangdong 82， Sichuan 79 II 9 and Sichuan 88 1. Such lines can be considered for the combination with sugarcane parents having no high affinity or the hybrid seed production during the creation of complexes. Inter annual variations of some early flowering S. spontaneum after treatment
The results in Table 3 showed that the earliest first flowering date was on October 14 （Guangdong 5）， and the first flowering date was delayed by one month after being treated; and the latest was on November 29 （Guangdong 38）， and after the treatment， the first flowering date was postponed for more than a month. Obviously， the first flowering dates of the treated parents were all significantly delayed. In cross breeding， many lines that are unlikely to hybridize can be crossed by the staged head cutting method. In this way， a large number of hybrid populations can be easily obtained， from which excellent hybrid progenies can be selected.
Conclusions
The first flowering date of S. spontaneum is a heritable trait. In the process of noble seed production， it is possible to make full use of this trait by more strictly selecting it in the early stage of hybridization selection.
The broad heritability of the first flowering date of the S. spontaneum was 0.079. The inter annual differences of the first flowering date of S. spontaneum ranged from 4 to 49 d， with an average of 13.65 d. The lines with an inter annual variation within 20 d accounted for 90%， and those with an inter annual variation over 30 d only accounted for 4.5%. The first flowering dates of some parents were significantly delayed after the head cutting treatment. In cross breeding， many lines that are unlikely to hybridize can be crossed by the staged head cutting method. In the hybridization season， the hybridization plan of the current year must be properly adjusted according to the actual situation of the flowering period， and populations related to S. spontaneum should be bred as much as possible to improve the probability of breeding excellent F 1 generations.
There is no necessary relationship between the first flowering date characteristic of a line （early flowering or late flowering） and the inter annual difference in the first flowering date. In a particular hybridization season， there is no obvious law in whether the flowering periods of different lines are earlier or later than another year.
In the cross breeding program， the lines with much or very much pollen can be considered as the male parent， and those with little or very little can be considered as the female parent， which is beneficial to the breeding of true and false hybrids. For the sugarcane parents having no strong affinity or the hybrid seed production during the creation of complexes， the selection can be performed according to the development rate. References
[1] BREMER. Problems in breeding and cytology of sugar cane[J]. phytica， 1961， 10： 59-78.
[2] WANG YQ， WANG ZL， PAN SM， et al. Study on sugarcane germplasm resources[J]. Acta Agriculturae Jiangxi， 2002， 14（1）： 21-26. （in Chinese）
[3] Ministry of Agriculture， the Peoples Republic of China. Chinese crop genetic resources[M]. Beijing： China Agriculture Press， 1998： 499-518. （in Chinese）
[4] CHEN H， FAN YH， SHI XW， et al. research on genetic diversity and systemic evolution in Saccharum spontaneum L.[J]. Acta Agronomica Sinica， 2001， 27（5）： 645-652. （in Chinese）
[5] QI YW， FAN LN， HE HY， et al. Genetic diversity assessment of Saccharum spontaneum L. native to Guangdong area with agronomic traits[J]. Sugarcane and Canesugar， 2009（3）： 7-10. （in Chinese）
[6] QIU CL， HE SC， YANG SQ. Studies on Saccharum spontaneum L. in Yunnan II： Studies on esterase isoenzymes[J]. Journal of Yunnan Agricultural University， 1987（2）： 80-83. （in Chinese）
[7] ZHANG GM， LIAO JX， HUANG HT. Genetic diversity of Saccharum spontaneum L. with high sugar content in guangxi based on phenotypic traits and RAPD markers[J]. Journal of Southwest University， 2007， 29（8）： 83-88. （in Chinese）
[8] YANG RZ， WU WC， HUANG JK. Clustering classification of Sichuan Saccharum spontaneum[J]. Sugarcane， 2001， 8（2）： 4-9. （in Chinese）
[9] HUANG JF， DENG HH. Initial Flowering time and its heritability of the commonly used sugarcane parents in hainan sugarcane breeding station[J]. Sugarcane and Canesugar， 2004（4）： 6-10. （in Chinese）
[10] WANG JM. Selected papers on sugarcane selection[J]. Nanning： Guangxi Nationalities Publishing House， 1991， 314-329. （in Chinese）
[11] LI QW. Modern sugarcane improvement technology[M]. Guangdong： South China University of Technology Press， 2000. （in Chinese）
[12] WANG LP， MA L， XIA HM， et al. Application of Saccharum spontaneum in sugarcane cross breeding[J]. Sugar Crops of China， 2006（1）： 1-4. （in Chinese）