Xishi LI,Chunying BA*,Hounan CAO*,Chengwen ZONG,Liu QU,Jizi ZHANG

1.College of Agronomy,Yanbian University,Yanbian 133002,China;

2.Academy of Agricultural Sciences of Yanbian,Yanbian 133002,China

Interspecific Cross Compatibility of Rhododendron in Changbai Mountain

Xishi LI1,Chunying BA1*,Hounan CAO1*,Chengwen ZONG1,Liu QU1,Jizi ZHANG2

1.College of Agronomy,Yanbian University,Yanbian 133002,China;

2.Academy of Agricultural Sciences of Yanbian,Yanbian 133002,China

In order to explore the interspecific cross compatibility of Rhododendron and the feasibility of interspecific cross in germplasm innovation,reciprocal crosses among five species of Rhododendron in Changbai Mountain were made.In addition, the hybrid embryos were cultured in vitro.The results showed that the species of Rhododendron in Changbai Mountain had significant differences in reciprocal cross compatibility.When Rh.chrysanthum Pall.was used as female parent,it showed high cross compatibilities with Rh.mucronulatum Turcz.(92.55%),Rh.mucronulamm Turcz.f.album Nakai(94.14%)and Yabuli Rhododendron(92.42%),and no significant differences were found in fruit setting rate by self-pollination.The germination rates of hybrid embryos were 13.60%,11.59%and 4.12%,respectively.The hybrid seedlings had weak growth vigor and albino status and were trended to be aborted. After 2-3 generations of subculture,the growth vigor of hybrid seedlings were recovered gradually,and their differentiation rate was also significantly higher than that of Rh.chrysanthum Pall.seedlings.But the hybrid seedlings were still yellowish green.In reciprocal crosses,the fruit setting rates were significantly reduced.The combination with the highest fruit setting rate(36.51%)was Yabuli Rhododendron× Rh.chrysanthum Pall.The fruit setting rates of reciprocal crosses between Rh.chrysanthum Pall.and Rh.mucronulatum Turcz.and Rh.chrysanthum Pall.and Rh.schlippenbachii Maxim were 6.52%and 0,respectively.However,the hybrid embryo’s germination rate (39.81%)of Yabuli Rhododendron×Rh.chrysanthum Pall.was significantly higher than that of their original cross.Moreover,the hybrid seedlings grew strongly and could be directly transplanted without subculture.

Rh.chrysanthum Pall.;Interspecific cross compatibility;Embryo culture

R hododendron chrysanthum Pall.is a small green shrub, and it has funnel-shaped corollas and light yellow or white flowers.Rh.chrysanthum Pall.is very beautiful,and can be developed as flower,leaf and fruit ornamentals.It is a rare broadleaf evergreen in Northeast with cold and wind resistance and water-and soil-maintaining ecological functions[1].However,Rh.chrysanthum Pall.is narrowly distributed in China,and it is only distributed in the National Nature Reserve of Changbai Mountain.Due to excessive digging, Rh.chrysanthum Pall.has been listed as a protected and endangered species of China[2-3].Rh.chrysanthum Pall.has low seed germination rate and low transplanting survival rate.In addition,Rh.chrysanthum Pall.is a cold loving plant,and it can grow and develop in the low temperature (above 0℃)environment.Under conditions of high temperature and low humidity, Rh. chrysanthum Pall. grows slowly,and is also vulnerable to infestations of pests and diseases, greatly limiting the exploitation and u tilization of Rh.chrysanthum Pall.

Currently,the studies on Rh.chrysanthum Pall.in China only focuses on tissue culture[4-5],medicinal ingredients[6-8],anatomical structure[9],and so on.There have been almost no reports on domestication of Rh.chrysanthum Pall.In this study,some species of Rhododendron in ChangbaiMountain were used as materials,and the interspecific cross compatibilities were discussed.A number of hybrid seedlings were obtained though mature embryo culture,laying material foundation for germplasm innovation of Rhododendron plants in Changbai Mountain.

Materials and Methods

Materials

The test materials included Rh. chrysanthum Pall.,Rh.mucronulatum Turcz.,Rh.mucronulamm Turcz.f.album Nakai,Yabuli rhododendron and Rh. schlippenbachii Maxim.

Methods

Collection of pollenAt the big bud stage,the flower buds that were about to open were collected and brought back to laboratory.The anthers were isolated and dried at room temperature.Subsequently,the pollen were collected and preserved in pollen bottles at-20℃.

Reciprocal crosses amongRhododendronspp.in Changbai MountainThe interspecific crosses of Rh.chrysanthum Pall.(♀)×Rh.mucronulatum Turcz.(♂),Rh.chrysanthum Pall. (♀)×Rh.mucronulamm Turcz.f.album Nakai(♂)and Rh.chrysanthum Pall.(♀)×Yabuli rhododendron (♀) were made by artificial emasculation method in SmallTianchiLake in Changbai Mountain in June,2011.After hybridization,the plants were all bagged for isolation and marked.All the bags were removed 10 d later.The fruit setting and development situations were observed once a week.Before the cracking of ripen fruits(late August),the fruit setting rates were investigated,and the fruits were harvested for seed separation at room temperature.The separated seeds were preserved at 4℃for use.

The back crosses of Rh.mucronulatum Turcz.(♀)×Rh.chrysanthum Pall.(♀),Yabuli rhododendron (♀)×Rh.chrysanthum Pall.(♀)and Rh.schlippenbachii Maxim (♀)× Rh.chrysanthum Pall.(♀)were made in the Botanical Garden of Yanbian Academy of Agricultural Sciences in late April,2012.The test methods were as described above.

Fruit setting rate =Setting-fruit number/Pollinated flowernumber× 100%.

In vitroculture of mature embryos of different hybrid combinationsReferring to previous test results[10],the mature embryos of the control(naturally pollinated Rh.chrysanthum Pall.) and all the hybrid combinations were all disinfected in sterile room.The seeds were first washed with sterile water twice.They were soaked in 75%alcohol for 30 s.Subsequently, 0.1%mercuric chloride was mixed with the 75%alcohol,and the seeds were further soaked for 5 min.After rinsed with sterile water 4-5 times,the seeds were inoculated in the medium (pH 5.4)composed of Read,GA(2.0 mg/L),sucrose (30 g/L)and agar(7 g/L)and cultured on a culture shelf. The culture conditions were as follows: 25℃ at day,20℃ at night,1 500-2 000 lx of light intensity,12 h/d of photoperiod.The seed germination and seedling growth situations were observed 20 d later,and the germination rates were investigated 60 d later.

Germination rate =Germinated embryo number/Inoculated embryo number×100%.

The embryo seedlings of hybrid combinations were very thin and weak. To get all the hybrid individuals after seedling transplanting,the obtained embryo seedlings were inoculated in the propagation culture medium composed of modified MS,ZT (0.2 mg/L) and IBA (0.5 mg/L).More than 10 generations of subculture were carried out for each seedling plant.The culture conditions were as described above.

Results and Analysis

Fruit setting rates of different hybridcombinationsatharvesting stage

As shown in Table 1,the fruit setting rates of hybrid combinations with Rh.chrysanthum Pall.as the female parents were 92.55%,94.14%and 92.42%,respectively,which showed no significant differences with that of natural pollination,indicating high interspecific cross compatibilities of Rhododendron.The fruits of all the hybrid combinations began to grow large 2 weeks after the pollination,which was a litter earlier than that of fruits of the natural hybrid combination.No significant differences were found in development speed and state between artificial and natural hybrid combinations in later period.Till maturity stage, a lot of capsules were obtained(Fig.1). The fruit setting rates of all the hybrid combinations with Rh.chrysanthum Pall.as the male parents were significantly lower than those of the original crosses.The fruit setting rates of the three hybrid combinations were 6.52%,36.51%and 0,respectively.At the harvesting stage,the fruit setting rate of Rh.schlippenbachii Maxim by self pollination was also found to be 0.

In vitro culture of mature embryos and subculture of embryo seedlings of different hybrid combinations In vitro culture of mature embryos

Table 1 Fruit setting rates of different hybrid combinations at harvesting stage

Before the in vitro culture of hybrid embryos,their morphology was first observed under an optical microscope (Fig.2).The seeds of Rh.chrysanthum Pall.by natural pollination were most plump,followed by those of N×H,and the seeds of N×B and N×Y were smaller.The in vitro culture of mature hybrid embryos showed that there were significant differences in embryo germination rate between interspecific original cross and back cross.Table 2 showed that the embryos of N (♀)×H (♂)germinated earliest,and the embryos began to germinate as early as 20 d after the inoculation.After a 60-d culture,the germination rate of embryos of N(♀)×H(♂)reached 13.6%, followed by that of N (♀)×B (♂) (11.59%),which was close to that of Rh.chrysanthum Pall.by natural pollination,and the germination rate of embryos of N(♀)×Y(♂)was relatively low(4.12%).The growth of embryos of N (♀)×Y (♂)was weaker,and some of the seedlings were transparent(Fig.3).In back crosses,the germination rate of embryos of H(♀)×N(♂) was 0.The germination rate of embryos of Y(♀)×N(♂)(39.81%)was significantly higher than that of their original cross,and with the proceeding of subculture,the leaves of embryo seedlings became greener and the embryo roots became more developed.The growth of embryo seedlings of Y(♀)×N(♂)was better than those of all the reciprocal hybrid combinations,and it was even better than that of embryo seedlings of Rh.chrysanthum Pall.by natural pollination.

Subculture of embryo seedlings

The hybrid seedlings obtained by embryo culture had weak growth and lightyellow leaves.To avoid loss of material,the obtained embryo seedlings were inoculated into the subculture medium.Among all the hybrid combinations,the growth of hybrid seedlings of N×Y was weakest,and some of the hybrid seedlings were almost transparent.With the increased generations of subculture,the leaf color of hybrid seedlings was restored to some extent,but most of them were just yellowish green.In the hybrid seedlings, most of the buds were light yellow or albino,but their differentiation capacity was significantly higher than that of embryo seedlings of Rh.chrysanthum Pall.by natural pollination.The significantly enhanced differentiation capacity might be caused by strong heterosis in hybrids (Fig.4).After propagation culture,only leaf color and leaf area ofembryo seedlingsofRh. chrysanthum Pall.were improved,but none new adventitious buds were observed.The differentiation capacities of embryo seedlings of the other three hybrid combinations were relatively high.It was showed that the differentiation capacity of the embryo seedlings was significantly higher than their elongation capacity,so the overall plant height was relatively low (Fig.5).

Table 2 Germination situations of mature embryos of different hybrid combinations after a 60-d in vitro culture

Discussion

The test results showed that the fruit setting rates of the three hybrid combinations with Rh.chrysanthum Pall.as female parents were all higher than 92.00%.Moreover,large numbers of capsules were obtained from the three reciprocal hybrid combinations. It is suggested that Rh. chrysanthum Pall.has high interspecific cross compatibilities with Rh.mucronulatum Turcz.,Yabuli rhododendron and Rh.schlippenbachii Maxim. But in back crosses,there were great differences in fruit setting rate among all the hybrid combinations.In addition, there were also great differences in fruit setting rate between original and back crosses.In cross breeding,embryo culture is an effective way to overcome the sterility of distant hybridization and to grain interspecific hybrid embryos[11].This test proves that the interspecific cross of Rhododendron is feasible,and it provides a new pathway for germplasm innovation. Interspecific cross of Rhododendron can notonly take advantage of Rhododendron resources in Changbai Mountain but also get a lot of hybrid rhododendrons,which will not only enrich,as well as protecting Rhododendron germplasm resources in Changbai Mountain,but also lay a foundation for future studies on germplasm resources and detection of genetic diversity.

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Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

長(zhǎng)白山杜鵑花屬種間雜交親和性的研究

李西時(shí)1，巴春影1*，曹后男1*，宗成文1，曲柳1,張吉子2
（1.延邊大學(xué)農(nóng)學(xué)院，吉林延邊133002;2.延邊農(nóng)業(yè)科學(xué)院，吉林延邊 133002）

為了探索長(zhǎng)白山杜鵑花屬種間雜交的親和性及種間雜交在種質(zhì)創(chuàng)新中的可行性，以5種長(zhǎng)白山杜鵑花屬植物為試材，進(jìn)行種間正反雜交，并對(duì)雜種胚進(jìn)行離體培養(yǎng)。結(jié)果表明，杜鵑花屬種間正反雜交親和力有顯著差異，以牛皮杜鵑為母本，與迎紅杜鵑、白花迎紅杜鵑、亞布力杜鵑雜交有較高的親和性，與母本的自然授粉坐果率無顯著差異，分別為92.55%、94.14%、92.42%，雜種胚的萌發(fā)率分別為13.60%、11.59%、4.12%；雜種苗生長(zhǎng)勢(shì)微弱，呈白化狀態(tài)，有敗育跡象，經(jīng)繼代培養(yǎng)2～3次后，生長(zhǎng)勢(shì)得到一定的恢復(fù)，且分化率明顯高于牛皮杜鵑種胚苗，但植株的顏色仍是黃綠色。而反交時(shí)，坐果率明顯降低，坐果率最高的組合為亞布力杜鵑×牛皮杜鵑，為36.51%；與迎紅杜鵑的雜交坐果率為6.52%；而與大字杜鵑的雜交坐果率為0，但反交時(shí)亞布力×牛皮杜鵑的種胚萌發(fā)率極顯著地高于正交，為39.81%，而且種胚苗生長(zhǎng)健壯，無需繼代，可直接進(jìn)行煉苗移栽。

牛皮杜鵑（Rhododendron chrysanthum Pall.）；種間雜交親和性；種胚培養(yǎng)

延邊大學(xué)2015年大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目，暨第七屆本科生科研項(xiàng)目資助（ydbksky2015252）。

李西時(shí)（1993-），男，山東日照人，在讀本科生，專業(yè):園藝，E-mail：1028620689@qq.com。*通訊作者，E-mail：15144589828@163.com。

2015-04-08

修回日期 2015-06-29

Supported by Students’Innovation and Entrepreneurship Training Program of Yanbian University in 2015(ydbksky2015252).

*Corresponding author.E-mail:15144589828@163.com

Received:April 8,2015 Accepted:June 29,2015