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Abstract While being one of the worlds most important crops， maize （Zea mays L.） is still difficult to regenerate in tissue culture which severely limits its improvement by genetic engineering. Currently， immature zygotic embryos provide the predominantly used material for regeneration and transformation. However， the procedures involved are often laborious， time consuming and season dependent. Here， we further improved an efficient tissue culture and plant regeneration system that uses maize leaf segments of young seedlings as an alternative explant source. Embryogenic calli were evaluated by morphology， proliferation and regeneration capacity. All these indicated that seedling derived leaf materials have the potential to replace immature embryos for tissue culture and regeneration.
Key words Maize （Zea mays L.）; Leaf materials; Primary callus; Embryogenic callus; Regeneration
Maize （Zea mays L.） is an important model organism for basic and applied research. While being one of the worlds most important crop germplasms， maize is still difficult to achieve somatic embryogenesis， regeneration and transformation in tissue culture which severely limits its usage in transgenetic research fields.
The first successful regeneration of maize through tissue culture was achieved through the use of immature embryos more than 30 years ago[1]. Since then， various explants have been tried to establish an efficient tissue culture system， such as anthers[2-3]， immature inflorescences[4]， axillary buds[5] and coleoptilar nodes[6]， etc. Nevertheless， immature zygotic embryos remain the most widely used explants for the establishment of competent callus cultures， cell suspensions or protoplasts for genetic transformation of maize[7] although the procedures involved are laborious， time consuming， genotype dependent and usually required greenhouse facilities[8].
Recently， germinated seeds have been used in tissue culture for maize regeneration in order to overcome some of the limitations of immature embryos presented. The major explants derived from germinated seeds are coleoptilar nodes[6]， shoot tips and shoot apical meristems[9-10]， apical buds[9，11] and axillary buds[5]. However， due to the relatively small amounts of each of these explants extracted， high workload is often required to obtain large amounts of calli. In comparison， seedling derived young leaves could provide much more materials. They are also easier to manipulate than mature embryos， apical buds， shoot tips and other explants. Earlier work has confirmed the possibility of inducing somatic embryogenesis from cultured leaf materials[12-13，8]. Recently， Ahmadabadi et al.[8] made some progress in improving plant tissue culture and achieved transgenic plants using leaf materials of maize hybrid Pa91×H99. However， due to large numbers of maize strains， complex genetic background and numerous factors that affect the isolated cultures， the usage of maize leaf materials in tissue culture is still difficult to accomplish in most varieties[14]. It is particularly true for elite inbred lines that adapt to sub tropical and temperate climatic conditions and these are cultivated broadly in China. To harness the benefits of genetic transformation in various breeding programs， it is important to develop an efficient methodology for leaf derived tissue culture and regeneration for these maize inbred lines. Therefore， leaf materials were tested as the alternative explants for an efficient regeneration system for some of the well adapted maize inbred lines in China， aiming at providing evidence for the replacement of immature embryos in maize tissue culture and regeneration prior to genetic transformation. Materials and Methods
Mature dry seeds of six maize elite inbred lines， Qi319， LY92， Mo17， C7 2， 178 and 478， kindly gifted by Dr. Wang Qi bai from Shandong Agriculture University， were used in this study. These lines were the parental lines of many promising maize hybrids cultivated in China. Seeds were surface sterilized with 70% ethanol for 5 min ， followed by immersing in 0.1% mercuric chloride （HgCl 2） for 10 min. The sterilized seeds were rinsed five times with sterilized distilled water and germinated in 1/2 MS medium[15] in an illuminated incubator （16 h light at 26 ℃; 8 h darkness at 24 ℃）. When the seedlings reached 5-10 cm high， the bottom section （about 2 cm） was cut into approximately 1 mm×2 mm fragments. These leaf pieces were subsequently transferred to callus induction medium to induce callus formation （Fig. 1A）.
The callus induction medium contained N6 basal salts[16] and B5 vitamins[17]， 2.0 mg/L of glycine， 25 mM of L proline， 100 mg/L of casein hydrolysate and 20 g/L of sucrose. Various concentrations of 2，4 D （0， 1， 2， 2.5， 3， 3.5 or 4 mg/L） were supplemented to the medium to test the initiation rate of callus. The pH was adjusted to 5.8 prior to the addition of 8 g/L agar， and the medium was sterilized by autoclaving at 121℃ for 18 min. The leaf materials were incubated in the above media at （27± 0.5） ℃ in darkness. After 2-3 weeks， visible primary callus growth occurred on the callus induction medium. The number of primary calli that emerged out of the total number of leaf fragments was calculated. These primary calli were transferred to subculture medium.
The basal composition of the subculture medium was the same as the induction medium except for 6 mM of L proline， 2 mg/L 2，4 D and 10 mg/L silver nitrate （AgNO 3） were supplemented. The pH was adjusted to 5.8 prior to the addition of 8 g/L agar and the medium was sterilized as before. AgNO 3 was filter sterilized and added just before pouring the media into petri dishes. The primary calli were transferred onto fresh subculture medium every 2 weeks. After about 4 weeks， embryogenic calli were evaluated by morphology， proliferation capacity， and regeneration capacity. Usually， calli are divided into two types， embryogenic and non embryogenic. There are two types of embryogenic calli observed， TYPE I and TYPE II， depending on the genotype of maize[18]. TYPE I calli are light yellow， slow growing， hard and compact， whereas TYPE II calli are relatively rapid growing， bright yellow， soft and friable. Many maize genotypes， especially elite varieties， are poor in somatic embryogenesis and readily form non embryogenic callus， and thus only a limited number of genotypes have been efficiently transformed so far[14]. The proliferation capacity is also used as an indicator of callus quality. Embryogenic calli are fragmented and transferred onto fresh subculture medium every 2 weeks. The proliferation capacity is the number of embryogenic calli survived at the end of the second round of subculture out of the initial number of calli. The embryogenic calli were transferred to regeneration medium that contained MS[15] basal salts and B5 vitamins， 1 mg/L 6 benzylaminopurine （6 BA）， 2 mg/L glycine， 0.5 g/L MES and 20 g/L sucrose. The pH of the medium was adjusted to 5.8 before the addition of 8 g/L agar， and the medium was sterilized by autoclaving as before. The calli were incubated in an illumination incubator （16 h light at 26 ℃; 8 h darkness at 24 ℃） for about 2 weeks. The percentage of plant regeneration was calculated based on the number of callus producing plantlets out of the total number of explants inoculated on induction medium. When the heights of the regenerated buds reached 3-5 cm， they were transferred to a 250 ml flask containing the rooting medium to initiate root development. The rooting medium was essentially the same as the regeneration medium where 6 BA and MES were eliminated. When the roots spread to 7-8 strips， the flask was opened to expose the plantlets to air for 1-2 d to acclimatize. They were carefully removed from the flask and transplanted into a mixture of equal parts （v/v） of sterilized soil and vermiculite and grown in a growth room （16 h light at 26 ℃; 8 h darkness at 24 ℃） with moderate humidity for 2 weeks before transferred into a greenhouse and grown to maturity.
Results
Primary calli were induced and appeared from the cut edges of leaf discs derived from young seedlings after 2-3 weeks （Fig. 1B C）. To evaluate the impact of 2，4 D on primary callus induction， concentrations of 0， 1， 2， 2.5， 3， 3.5， 4 mg/L in callus induction medium were tested on two inbred lines， Qi319 and Mo17， respectively. It showed that calli could not be induced at all in the absence of 2，4 D， demonstrating that 2，4 D was indispensable for callus induction （Fig. 2）. Within the range of 0-3 mg/L， the primary callus induction rate increased as 2，4 D concentration increased. The optimum concentration of 2，4 D on callus induction medium for Qi319 and Mo17 was 3 mg/L under which the highest callus induction rate of 58.43% and 41.51%， respectively were achieved. However， when the 2，4 D concentration was higher than 3 mg/L， primary callus induction rate started to decline. The callusing rate decreased to 29.43% （Qi319） and 19.9% （Mo17） when the 2，4 D concentration was 4 mg/L. Therefore， callus induction medium supplemented with 3 mg/L 2，4 D was used to induce primary callus for other four inbred lines.
Primary calli generated on the induction medium described above were transferred to fresh subculture medium. Following two rounds of subculture， the callus morphology from different genotypes was observed and recorded （Fig. 3）. Most calli of inbred lines Qi319， LY92 and Mo10 were embryogenic with those from Qi319 and LY92 exhibiting characteristic morphology of TYPE II calli that were bright yellow， friable granular and rapid growth （Fig. 3A， B）， and those from Mo17 forming TYPE I calli that most were yellowish， compact structure and slow growth （Fig. 3C）. However， most calli from C7 2， 178 and 478 were non embryogenic with brown， waterlogged and stationary growth （Fig. 3D F）. The proliferation capacity of calli from all six genotypes was assessed （Table 1）. As expected， embryogenic calli of Qi319， LY92 and Mo17 with favourable callus morphology also had higher proliferation capacity per callus than the non embryogenic callus lines of C7 2， 178 and 478 with the highest score of 2.33 achieved from inbred line Qi319 and the lowest score of 1.04 from line 478 （Table 1）.
A： Leaf segments of 1 mm×2 mm derived from basal section of seedlings as explants; B C： primary callus emerged from cut edges of maize leaf material; D： embryogenic callus maintained on subculture medium; E： cluster buds regenerated on regeneration medium; F： regenerated plantlet was transplanted to soil in a small pot.
The capacity of regenerating plantlets was correlated with the ability of forming embryogenic calli. However， not all embryogenic calli regenerated plantlets （Table 2）. After the embryogenic calli were transferred to regeneration medium， green shoots were regenerated within one weeks and plantlets regenerated in about two weeks （Fig. 1E）. When the regenerated plantlets grew to 3-5 cm high， they were transferred to rooting medium for developing roots. Plantlets with well developed roots were transferred to small pot and grown in a growing room （Fig. 1F）.
In our research， leaf materials of all six inbred lines could regenerate plantlets. The percentage of callus differentiation ranged from 8.36% to 27.58% （Table 2）. Genotype effects on plant regeneration from maize embryo cultures have been reported previously[19-20]. In this study， inbred lines Qi319， Mo17 and LY92， had higher regeneration frequency （27.58%， 23.49% and 18.29%）. However， other three lines， presented the regeneration frequency ranging from 8.36% to 9.91%.
Disscusion
In this study， we evaluated six inbred lines of maize for their performance in leaf based regeneration in tissue culture. These six lines are well adapted maize inbred lines and have been used widely in various breeding programmes in China. They are also the commonly used parental lines to create many maize hybrids that are cultivated throughout different regions of China today[21]. Therefore， to establish a regeneration protocol for these lines will be of great importance for future genetic transformation studies to produce traits with desired qualities that are readily adapted to cultivation conditions in the region.
We studied the process of callus induction and regeneration from leaf materials from all six lines. Although all six tested could regenerate plantlets from leaf materials， with a frequency of 8.36%-27.58% ， three lines， Qi319， Mo17 and LY 92， have been identified for their overall performance in high primary callus induction rate， favourable callus morphology and high differentiation rate （Table 1， 2 and Fig.3）. The results of our study also showed that the presence of 2，4 D in callus induction medium was critical for callus induction and embryogenic callus formation from maize leaf material and the optimum concentration was 3 mg/L （Fig. 2）. This is in agreement with the immature embryo based regeneration system where studies have showed that the inclusion of 2，4 D in the callus induction medium was also a determinant factor for inducing primary callus formation[22-24]. Earlier work has confirmed the possibility to induce somatic embryogenesis from leaf material of maize[12-13]. Later， callus induction and regeneration from maize leaves was improved and a genetic transformation system for seedling derived leaf material was developed[8]. Because leaf based callus induction and regeneration systems present a great deal of advantages over the immature embryo based tissue culture system， it has attracted a lot of interest in recent years. The method we reported here further proved the feasibilities of using leaf tissue as an alternative explant to immature embryos for the efficient regeneration of maize.
References
[1] GREEN CE， PHILLIPS RL. Plant regeneration from tissue culture of maize[J]. Crop Sci， 1975， 15： 417-421.
[2] TING YC， YU M， ZHENG WZ. Improved anther culture of maize[J]. Plant Sci Lett， 1981， 23： 139-145.
[3] BARLOY D， BECKERT M. Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize[J]. Plant Cell， Tissue and Organ Culture， 1993， 33： 45-50.
[4] PAREDDY DR， PETOLINO JF. Somatic embryogenesis and plant regeneration from immature inflorescences of several elite inbreds of maize[J]. Plant Sci， 1990， 67： 211-219.
[5] ZHANG S， WILLIAMS CARRIER R， JACKSON D， et al. Expression of CDC2ZM and KNOTTED1 during in vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize （Zea mays L.） and barely （Hordeum vulgare L.）[J]. Planta， 1998， 204： 542-549.
[6] SIDOROV V， GILBERTSON L， ADDAE P， et al. Agrobacterium mediated transformation of seedlings derived maize callus[J]. Plant cell rep， 2006， 25： 320-328.
[7] ARMSTRONG CL. The first decade of maize transformation： A review and future perspective[Zea mays L. genetic engineering][J]. Maydica， 1999， 44（1）： 101-109.
[8] AHMADABADI M， RUF S， BOCK R. A leaf based regeneration and transformation system for maize （Zea mays L.）[J]. Transgenic Res， 2007， 16： 437-448.
[9] ZHONG H， SRINIVASAN C， STICKLEN MB. In vitro morphogenesis of corn （Zea mays L.）. I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips[J]. Planta， 1992a， 187： 483-489.
[10] ZHANG S， WILLIAMS CARRIER R， LEMAUX PG. Transformation of recalcitrant maize elite inbreds using in vitro shoot meristematic cultures induced from germinated seedlings[J]. Plant Cell Rep， 2002， 21： 263-270.
[11] ZHONG H， WANG W， STICKLEN MB. In vitro morphogenesis of Sorghum bicolor （L.） moench： efficient plant regeneration from shoot apices[J]. J Plant Physiol， 1998， 153： 719-726. [12] CONGER BV， NOVAK FJ， AFZA R， et al. Somatic embryogenesis from cultured leaf segments of Zea mays[J]. Plant Cell Rep， 1987， 6： 345-347.
[13] RAY SD， GHOSH PD. Somatic embryogenesis and plant regeneration from cultured leaf explants of Zea mays[J]. Ann Bot， 1990， 66： 497-500.
[14] ISHIDA Y， HIEI Y， KOMARI T. Agrobacterium mediated transformation of maize[J]. Nat. Protocols， 2007， 2： 1614-1621.
[15] MURASHIGE T， SKOOG F. A revised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiologia Plant， 1962 15： 473-497.
[16] CHU CC， WANG CC， SUN CS， et al. Establishment of an efficient medium for another culture of rice through comparative experiments on nitrogen source[J]. Sci Sin， 1975， 18： 659-668.
[17] GAMBORG OL， MILLER RA， OJIMA K. Nutrient requirements of suspension cultures of soybean root cells[J]. Exp. Cell Res， 1968， 50： 151-158.
[18] BAJAJ YPS. Plant protoplast and genetic engineering[M]. In： Bajaj YPS （ed） Biotechnology in Agriculture and Forestry， Berlin， Heidelberg， New York： Springer， 1994， 29： 3-23.
[19] DUNCAN DR， WIDHOLM JM. Improved plant regeneration from maize callus cultures using 6 benzylaminopurine[J]. Plant Cell Rep， 1998， 7： 452-455.
[20] TOMES DT， SMITH OS. The effects of parental genotypes on initiation of embryogenic callus from elite maize germplasm[J]. Theor Appl Genet， 1985， 70： 505-509
[21] HUANG XQ， WEI ZM. High frequency plant regeneration through callus initiation from mature embryos of maize[J]. Plant Cell Rep， 2004， 22： 793-800.
[22] ARMSTRONG CL， GREEN CE. Establishment and maintenance of friable， embryogenic maize callus and the involvement of L proline[J]. Planta， 1985， 164： 207-214.
[23] BOHOROVA NE， LUNA B， BRITO RM， et al. Regeneration potential of tropical， sub tropical， midaltitude and highland maize inbreds[J]. Maydica， 1995， 40： 275-281.
[24] CARVALHO CHS， BOHOROVA N， BORDALLO PN， et al. Type II callus production and plant regeneration in tropical maize genotypes[J]. Plant Cell Rep， 1997， 17： 73-76.
Key words Maize （Zea mays L.）; Leaf materials; Primary callus; Embryogenic callus; Regeneration
Maize （Zea mays L.） is an important model organism for basic and applied research. While being one of the worlds most important crop germplasms， maize is still difficult to achieve somatic embryogenesis， regeneration and transformation in tissue culture which severely limits its usage in transgenetic research fields.
The first successful regeneration of maize through tissue culture was achieved through the use of immature embryos more than 30 years ago[1]. Since then， various explants have been tried to establish an efficient tissue culture system， such as anthers[2-3]， immature inflorescences[4]， axillary buds[5] and coleoptilar nodes[6]， etc. Nevertheless， immature zygotic embryos remain the most widely used explants for the establishment of competent callus cultures， cell suspensions or protoplasts for genetic transformation of maize[7] although the procedures involved are laborious， time consuming， genotype dependent and usually required greenhouse facilities[8].
Recently， germinated seeds have been used in tissue culture for maize regeneration in order to overcome some of the limitations of immature embryos presented. The major explants derived from germinated seeds are coleoptilar nodes[6]， shoot tips and shoot apical meristems[9-10]， apical buds[9，11] and axillary buds[5]. However， due to the relatively small amounts of each of these explants extracted， high workload is often required to obtain large amounts of calli. In comparison， seedling derived young leaves could provide much more materials. They are also easier to manipulate than mature embryos， apical buds， shoot tips and other explants. Earlier work has confirmed the possibility of inducing somatic embryogenesis from cultured leaf materials[12-13，8]. Recently， Ahmadabadi et al.[8] made some progress in improving plant tissue culture and achieved transgenic plants using leaf materials of maize hybrid Pa91×H99. However， due to large numbers of maize strains， complex genetic background and numerous factors that affect the isolated cultures， the usage of maize leaf materials in tissue culture is still difficult to accomplish in most varieties[14]. It is particularly true for elite inbred lines that adapt to sub tropical and temperate climatic conditions and these are cultivated broadly in China. To harness the benefits of genetic transformation in various breeding programs， it is important to develop an efficient methodology for leaf derived tissue culture and regeneration for these maize inbred lines. Therefore， leaf materials were tested as the alternative explants for an efficient regeneration system for some of the well adapted maize inbred lines in China， aiming at providing evidence for the replacement of immature embryos in maize tissue culture and regeneration prior to genetic transformation. Materials and Methods
Mature dry seeds of six maize elite inbred lines， Qi319， LY92， Mo17， C7 2， 178 and 478， kindly gifted by Dr. Wang Qi bai from Shandong Agriculture University， were used in this study. These lines were the parental lines of many promising maize hybrids cultivated in China. Seeds were surface sterilized with 70% ethanol for 5 min ， followed by immersing in 0.1% mercuric chloride （HgCl 2） for 10 min. The sterilized seeds were rinsed five times with sterilized distilled water and germinated in 1/2 MS medium[15] in an illuminated incubator （16 h light at 26 ℃; 8 h darkness at 24 ℃）. When the seedlings reached 5-10 cm high， the bottom section （about 2 cm） was cut into approximately 1 mm×2 mm fragments. These leaf pieces were subsequently transferred to callus induction medium to induce callus formation （Fig. 1A）.
The callus induction medium contained N6 basal salts[16] and B5 vitamins[17]， 2.0 mg/L of glycine， 25 mM of L proline， 100 mg/L of casein hydrolysate and 20 g/L of sucrose. Various concentrations of 2，4 D （0， 1， 2， 2.5， 3， 3.5 or 4 mg/L） were supplemented to the medium to test the initiation rate of callus. The pH was adjusted to 5.8 prior to the addition of 8 g/L agar， and the medium was sterilized by autoclaving at 121℃ for 18 min. The leaf materials were incubated in the above media at （27± 0.5） ℃ in darkness. After 2-3 weeks， visible primary callus growth occurred on the callus induction medium. The number of primary calli that emerged out of the total number of leaf fragments was calculated. These primary calli were transferred to subculture medium.
The basal composition of the subculture medium was the same as the induction medium except for 6 mM of L proline， 2 mg/L 2，4 D and 10 mg/L silver nitrate （AgNO 3） were supplemented. The pH was adjusted to 5.8 prior to the addition of 8 g/L agar and the medium was sterilized as before. AgNO 3 was filter sterilized and added just before pouring the media into petri dishes. The primary calli were transferred onto fresh subculture medium every 2 weeks. After about 4 weeks， embryogenic calli were evaluated by morphology， proliferation capacity， and regeneration capacity. Usually， calli are divided into two types， embryogenic and non embryogenic. There are two types of embryogenic calli observed， TYPE I and TYPE II， depending on the genotype of maize[18]. TYPE I calli are light yellow， slow growing， hard and compact， whereas TYPE II calli are relatively rapid growing， bright yellow， soft and friable. Many maize genotypes， especially elite varieties， are poor in somatic embryogenesis and readily form non embryogenic callus， and thus only a limited number of genotypes have been efficiently transformed so far[14]. The proliferation capacity is also used as an indicator of callus quality. Embryogenic calli are fragmented and transferred onto fresh subculture medium every 2 weeks. The proliferation capacity is the number of embryogenic calli survived at the end of the second round of subculture out of the initial number of calli. The embryogenic calli were transferred to regeneration medium that contained MS[15] basal salts and B5 vitamins， 1 mg/L 6 benzylaminopurine （6 BA）， 2 mg/L glycine， 0.5 g/L MES and 20 g/L sucrose. The pH of the medium was adjusted to 5.8 before the addition of 8 g/L agar， and the medium was sterilized by autoclaving as before. The calli were incubated in an illumination incubator （16 h light at 26 ℃; 8 h darkness at 24 ℃） for about 2 weeks. The percentage of plant regeneration was calculated based on the number of callus producing plantlets out of the total number of explants inoculated on induction medium. When the heights of the regenerated buds reached 3-5 cm， they were transferred to a 250 ml flask containing the rooting medium to initiate root development. The rooting medium was essentially the same as the regeneration medium where 6 BA and MES were eliminated. When the roots spread to 7-8 strips， the flask was opened to expose the plantlets to air for 1-2 d to acclimatize. They were carefully removed from the flask and transplanted into a mixture of equal parts （v/v） of sterilized soil and vermiculite and grown in a growth room （16 h light at 26 ℃; 8 h darkness at 24 ℃） with moderate humidity for 2 weeks before transferred into a greenhouse and grown to maturity.
Results
Primary calli were induced and appeared from the cut edges of leaf discs derived from young seedlings after 2-3 weeks （Fig. 1B C）. To evaluate the impact of 2，4 D on primary callus induction， concentrations of 0， 1， 2， 2.5， 3， 3.5， 4 mg/L in callus induction medium were tested on two inbred lines， Qi319 and Mo17， respectively. It showed that calli could not be induced at all in the absence of 2，4 D， demonstrating that 2，4 D was indispensable for callus induction （Fig. 2）. Within the range of 0-3 mg/L， the primary callus induction rate increased as 2，4 D concentration increased. The optimum concentration of 2，4 D on callus induction medium for Qi319 and Mo17 was 3 mg/L under which the highest callus induction rate of 58.43% and 41.51%， respectively were achieved. However， when the 2，4 D concentration was higher than 3 mg/L， primary callus induction rate started to decline. The callusing rate decreased to 29.43% （Qi319） and 19.9% （Mo17） when the 2，4 D concentration was 4 mg/L. Therefore， callus induction medium supplemented with 3 mg/L 2，4 D was used to induce primary callus for other four inbred lines.
Primary calli generated on the induction medium described above were transferred to fresh subculture medium. Following two rounds of subculture， the callus morphology from different genotypes was observed and recorded （Fig. 3）. Most calli of inbred lines Qi319， LY92 and Mo10 were embryogenic with those from Qi319 and LY92 exhibiting characteristic morphology of TYPE II calli that were bright yellow， friable granular and rapid growth （Fig. 3A， B）， and those from Mo17 forming TYPE I calli that most were yellowish， compact structure and slow growth （Fig. 3C）. However， most calli from C7 2， 178 and 478 were non embryogenic with brown， waterlogged and stationary growth （Fig. 3D F）. The proliferation capacity of calli from all six genotypes was assessed （Table 1）. As expected， embryogenic calli of Qi319， LY92 and Mo17 with favourable callus morphology also had higher proliferation capacity per callus than the non embryogenic callus lines of C7 2， 178 and 478 with the highest score of 2.33 achieved from inbred line Qi319 and the lowest score of 1.04 from line 478 （Table 1）.
A： Leaf segments of 1 mm×2 mm derived from basal section of seedlings as explants; B C： primary callus emerged from cut edges of maize leaf material; D： embryogenic callus maintained on subculture medium; E： cluster buds regenerated on regeneration medium; F： regenerated plantlet was transplanted to soil in a small pot.
The capacity of regenerating plantlets was correlated with the ability of forming embryogenic calli. However， not all embryogenic calli regenerated plantlets （Table 2）. After the embryogenic calli were transferred to regeneration medium， green shoots were regenerated within one weeks and plantlets regenerated in about two weeks （Fig. 1E）. When the regenerated plantlets grew to 3-5 cm high， they were transferred to rooting medium for developing roots. Plantlets with well developed roots were transferred to small pot and grown in a growing room （Fig. 1F）.
In our research， leaf materials of all six inbred lines could regenerate plantlets. The percentage of callus differentiation ranged from 8.36% to 27.58% （Table 2）. Genotype effects on plant regeneration from maize embryo cultures have been reported previously[19-20]. In this study， inbred lines Qi319， Mo17 and LY92， had higher regeneration frequency （27.58%， 23.49% and 18.29%）. However， other three lines， presented the regeneration frequency ranging from 8.36% to 9.91%.
Disscusion
In this study， we evaluated six inbred lines of maize for their performance in leaf based regeneration in tissue culture. These six lines are well adapted maize inbred lines and have been used widely in various breeding programmes in China. They are also the commonly used parental lines to create many maize hybrids that are cultivated throughout different regions of China today[21]. Therefore， to establish a regeneration protocol for these lines will be of great importance for future genetic transformation studies to produce traits with desired qualities that are readily adapted to cultivation conditions in the region.
We studied the process of callus induction and regeneration from leaf materials from all six lines. Although all six tested could regenerate plantlets from leaf materials， with a frequency of 8.36%-27.58% ， three lines， Qi319， Mo17 and LY 92， have been identified for their overall performance in high primary callus induction rate， favourable callus morphology and high differentiation rate （Table 1， 2 and Fig.3）. The results of our study also showed that the presence of 2，4 D in callus induction medium was critical for callus induction and embryogenic callus formation from maize leaf material and the optimum concentration was 3 mg/L （Fig. 2）. This is in agreement with the immature embryo based regeneration system where studies have showed that the inclusion of 2，4 D in the callus induction medium was also a determinant factor for inducing primary callus formation[22-24]. Earlier work has confirmed the possibility to induce somatic embryogenesis from leaf material of maize[12-13]. Later， callus induction and regeneration from maize leaves was improved and a genetic transformation system for seedling derived leaf material was developed[8]. Because leaf based callus induction and regeneration systems present a great deal of advantages over the immature embryo based tissue culture system， it has attracted a lot of interest in recent years. The method we reported here further proved the feasibilities of using leaf tissue as an alternative explant to immature embryos for the efficient regeneration of maize.
References
[1] GREEN CE， PHILLIPS RL. Plant regeneration from tissue culture of maize[J]. Crop Sci， 1975， 15： 417-421.
[2] TING YC， YU M， ZHENG WZ. Improved anther culture of maize[J]. Plant Sci Lett， 1981， 23： 139-145.
[3] BARLOY D， BECKERT M. Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize[J]. Plant Cell， Tissue and Organ Culture， 1993， 33： 45-50.
[4] PAREDDY DR， PETOLINO JF. Somatic embryogenesis and plant regeneration from immature inflorescences of several elite inbreds of maize[J]. Plant Sci， 1990， 67： 211-219.
[5] ZHANG S， WILLIAMS CARRIER R， JACKSON D， et al. Expression of CDC2ZM and KNOTTED1 during in vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize （Zea mays L.） and barely （Hordeum vulgare L.）[J]. Planta， 1998， 204： 542-549.
[6] SIDOROV V， GILBERTSON L， ADDAE P， et al. Agrobacterium mediated transformation of seedlings derived maize callus[J]. Plant cell rep， 2006， 25： 320-328.
[7] ARMSTRONG CL. The first decade of maize transformation： A review and future perspective[Zea mays L. genetic engineering][J]. Maydica， 1999， 44（1）： 101-109.
[8] AHMADABADI M， RUF S， BOCK R. A leaf based regeneration and transformation system for maize （Zea mays L.）[J]. Transgenic Res， 2007， 16： 437-448.
[9] ZHONG H， SRINIVASAN C， STICKLEN MB. In vitro morphogenesis of corn （Zea mays L.）. I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips[J]. Planta， 1992a， 187： 483-489.
[10] ZHANG S， WILLIAMS CARRIER R， LEMAUX PG. Transformation of recalcitrant maize elite inbreds using in vitro shoot meristematic cultures induced from germinated seedlings[J]. Plant Cell Rep， 2002， 21： 263-270.
[11] ZHONG H， WANG W， STICKLEN MB. In vitro morphogenesis of Sorghum bicolor （L.） moench： efficient plant regeneration from shoot apices[J]. J Plant Physiol， 1998， 153： 719-726. [12] CONGER BV， NOVAK FJ， AFZA R， et al. Somatic embryogenesis from cultured leaf segments of Zea mays[J]. Plant Cell Rep， 1987， 6： 345-347.
[13] RAY SD， GHOSH PD. Somatic embryogenesis and plant regeneration from cultured leaf explants of Zea mays[J]. Ann Bot， 1990， 66： 497-500.
[14] ISHIDA Y， HIEI Y， KOMARI T. Agrobacterium mediated transformation of maize[J]. Nat. Protocols， 2007， 2： 1614-1621.
[15] MURASHIGE T， SKOOG F. A revised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiologia Plant， 1962 15： 473-497.
[16] CHU CC， WANG CC， SUN CS， et al. Establishment of an efficient medium for another culture of rice through comparative experiments on nitrogen source[J]. Sci Sin， 1975， 18： 659-668.
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