I am sorry, the question is hard to understand. What embryo etc.

Actually I am trying to develop haploids of Hevea brasiliensis, the rubber tree, through in vitro techniques. In order to ensure the haploid nature of the explant, I am planning to isolate the intact embryo sacs from the un pollinated ovules for furher culturing. In this context, I have to ensure that embryo sac doesn't have any diploid cells. I am not sure how the wall of embryo sac is composed of. Theoretically this sac is a single cell containing 8 haploid nuclei, isn't it? In that case the sac wall should be made of only plant cell wall components. I just want to confirm it. If so, what are the main components ? Cellulose or pectin or something else?

Dear Sushama,

This question interested me when I was developing doubled haploids and have a EndNote library. I have listed some very relevant papers below. If you need PDF please let me know [email protected]

Cao YJ, Russell SD (1997) Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica. Sexual Plant Reproduction 10:368-373

Chen SH, Ang YH, Liao JR, Kuang AX, Tian HQ (2008) Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge. Zygote 16:179-186

De Storme N, Geelen D (2011) The Arabidopsis Mutant jason Produces Unreduced First Division Restitution Male Gametes through a Parallel/Fused Spindle Mechanism in Meiosis II. Plant Physiology 155:1403-1415

Dresselhaus T, Marton ML (2009) Micropylar pollen tube guidance and burst: adapted from defense mechanisms? Current Opinion in Plant Biology 12:773-780

Dumas C, Mogensen HL (1993) Gametes and fertilization - maize as a model system for experimental embryogenesis in flowering plants. Plant Cell 5:1337-1348

Fabian A, Jager K, Darko E, Barnabas B (2008) Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification. Acta Physiologiae Plantarum 30:737-744

Imre K, Kristof Z (1999) Isolation and osmotic relations of developing megagametophytes of Torenia fournieri. Sexual Plant Reproduction 12:152-157

Kaegi C, Gross-Hardt R (2010) Analyzing female gametophyte development and function: There is more than one way to crack an egg. European Journal of Cell Biology 89:258-261

Khalequzzaman M, Haq N (2005) Isolation and in vitro fusion of egg and sperm cells in Oryza sativa. Plant Physiology and Biochemistry 43:69-75

Kranz E, Dresselhaus T (1996) In vitro fertilization with isolated higher plant gametes. Trends in Plant Science 1:82-89

Kranz E, Kumlehn J (1999) Angiosperm fertilisation, embryo and endosperm development in vitro. Plant Science 142:183-197

Matthys-Rochon E, Mol R, Faure JE, Digonnet C, Dumas C (1997) Isolation and micromanipulation of the embryo sac and egg cell in maize

Mohammad K, Nazmul H (2005) Isolation and in vitro fusion of egg and sperm cells in Oryza sativa. Plant Physiology and Biochemistry 43:69-75

Sangthong R, Chin DP, Supaibulwatana K, Mii M (2009) Gametosomatic hybridization between egg cell protoplast and mesophyll protoplast of Petunia hybrida. Plant Biotechnology 26:377-383

Scholten S, Kranz E (2001) In vitro fertilization and expression of transgenes in gametes and zygotes. Sexual Plant Reproduction 14:35-40

Theunis CH, Pierson ES, Cresti M (1991) Isolation of male and female gametes in higher-plants. Sexual Plant Reproduction 4:145-154

Tian HQ, Russell SD (1997) Micromanipulation of male and female gametes of Nicotiana tabacum .1. Isolation of gametes. Plant Cell Reports 16:555-560

Wagner VT, Song YC, Matthys-Rochon E, Dumas C (1989) Observations on the isolated embryo sac of Zea mays L. Plant Science, Irish Republic 59:127-132

Wang YY, Kuang A, Russell SD, Tian HQ (2006) In vitro fertilization as a tool for investigating sexual reproduction of angiosperms. Sexual Plant Reproduction 19:103-115

Wu Y, Zhou C, Koop HU (1993) Enzymatic isolation of viable nucelli at the megaspore mother cell stage and in developing embryo sacs in nicotiana-tabacum. Sexual Plant Reproduction 6:171-175

Younis A, Hwang Y-J, Lim K-B (2014) Exploitation of induced 2n-gametes for plant breeding. Plant Cell Reports 33:215-223

Zhang YN, Wei DM, He EM, Miao S, Tian HQ, Russell SD (2010) Isolation of Male and Female Gametes of Rice. Crop Science 50:2457-2463

Good luck!

Ranjith